10,000 Matching Annotations
  1. May 2025
    1. Reviewer #1 (Public review):

      In this study, the authors conducted a single-cell RNA sequencing analysis of the cellular and transcriptional landscape of the gastric cancer tumor microenvironment, stratifying patients according to their H. pylori status into currently infected, previously infected and non-infected patients. The authors comprehensively dissect various cellular compartments, including epithelial, stromal and immune cells and describe specific cell types and signatures to be associated with H. pylori infection, including i) inflammatory and EMT signatures in malignant epithelial cells, ii) inflammatory CAFs in stromal cells, iii) Angio-TAMs, TREM2+ TAMs, exhausted and suppressive T cells in immune cells. Looking at ligand-receptor interactions as well as correlations between cell type abundances, they suggest that iCAFs interact with immunosuppressive T cells via a NECTIN2-TIGIT axis, as well as Angio-TAMs through a VEGFA/B-VEGFR1 axis and thereby promote immune escape, tumor angiogenesis and resistance to immunotherapy.

      The authors conduct a comprehensive and thorough analysis of the complex tumor microenvironment of gastric cancer, both single-cell RNA sequencing data as well as the analysis seem of high quality and according to best practices. The authors validate their findings using external datasets and include some prognostic value of the identified signatures and cell types. Furthermore, they validate some of their findings using immunofluorescence. While the authors confirm key transcriptional signatures in external cohorts comparing HP infected and non-infected cases, the main conclusions drawn from their own patient cohort are based on the comparison between HPGC and healthy controls. This approach does not fully resolve which signatures and cell types are specifically driven by H. pylori infection. As the authors also acknowledge in the limitations of their studies, their conclusions would benefit from functional validation.

      In summary, this study provides a valuable resource of the cellular and transcriptional heterogeneity of the tumor microenvironment in gastric cancers, distinguishing between positive, negative and previously positive HP infected gastric cancer patients. Given that HP is the main risk factor for gastric cancer development, the study provides valuable insights into potential HP driven transcriptional signatures and how these might contribute to this increased risk. However, the study would highly benefit from a clearer and more systematic comparison between HPGC and non-HPGC to better delineate infection-specific effects.

    2. Reviewer #2 (Public review):

      Summary:

      This study aims the describe the single-cell transcriptomes of H pylori-associated (Hp) gastric cancers and tumour microenvironment (TME), as a starting point to understand TME diversity stratified by Hp status.<br /> RNAseq was performed for gastric cancers with current Hp+ (from N=9 people), ex-Hp+ (N=6), non-Hp (N=6), and healthy gastric tissue (N=6).<br /> The study expands on previous single-cell transcriptomic studies of gastric cancers and was motivated by previous observations about the effect of H pylori status on therapeutic outcomes. The study includes a brief review of previous work and provides valuable context for this study.

      Strengths:

      The observations are supported by solid RNAseq study design and analysis. The authors describe correlations between Hp status and inferred molecular characteristics including cell lineages, enrichment for cell subclusters identifed as tumour-infiltrating lyphocyte cell types, tumour-infiltrating myeloid cells and cancer-associated fibroblasts.<br /> The observed correlations between Hp status and enrichment of cell subclusters were broadly corroborated using comparisons to deconvolved bulk RNAseq from publicly available gastric cancer data, providing a convincing starting point for understanding the diversity of tumour microenvironment by Hp-status.

      Weaknesses:

      The authors acknowledge several limitations of this study.<br /> The correlations with HP-status are based on a small number of participants per Hp category (N=9 with current Hp+; N=6 for ex-HP+ and non-HP), and would benefit from further validation to establish reproducibility in other cohorts.<br /> The ligand-receptor cross-talk analysis and the suggestion that suppressive T cells could interact with the malignant epithelium through TIGIT-NECTIN2/PVR pairs, are preliminary findings based on transcriptomic analysis and immunostaining and will require further validation.

    1. Reviewer #1 (Public review):

      Summary:

      The manuscript discusses the role of phosphorylated ubiquitin (pUb) by PINK1 kinase in neurodegenerative diseases. It reveals that elevated levels of pUb are observed in aged human brains and those affected by Parkinson's disease (PD), as well as in Alzheimer's disease (AD), aging, and ischemic injury. The study shows that increased pUb impairs proteasomal degradation, leading to protein aggregation and neurodegeneration. The authors also demonstrate that PINK1 knockout can mitigate protein aggregation in aging and ischemic mouse brains, as well as in cells treated with a proteasome inhibitor. While this study provided some interesting data, several important points should be addressed before being further consideration.

      Strengths:

      (1) Reveals a novel pathological mechanism of neurodegeneration mediated by pUb, providing a new perspective on understanding neurodegenerative diseases.

      (2) The study covers not only a single disease model but also various neurodegenerative diseases such as Alzheimer's disease, aging, and ischemic injury, enhancing the breadth and applicability of the research findings.

      Comments on revisions:

      This study, through a systematic experimental design, reveals the crucial role of pUb in forming a positive feedback loop by inhibiting proteasome activity in neurodegenerative diseases. The data are comprehensive and highly innovative. However, some of the results are not entirely convincing, particularly the staining results in Figure 1.

      In Figure 1A, the density of DAPI staining differs significantly between the control patient and the AD patient, making it difficult to conclusively demonstrate a clear increase in PINK1 in AD patients. Quantitative analysis is needed. In Fig 1C, the PINK1 staining in the mouse brain appears to resemble non-specific staining.

    1. Reviewer #1 (Public review):

      Summary:

      The manuscript entitled "Phosphodiesterase 1A Physically Interacts with YTHDF2 and Reinforces the Progression of Non-Small Cell Lung Cancer" explores the role of PDE1A in promoting NSCLC progression by binding to the m6A reader YTHDF2 and regulating the mRNA stability of several novel target genes, consequently activating the STAT3 pathway and leading to metastasis and drug resistance.

      Strengths:

      The study addresses a novel mechanism involving PDE1A and YTHDF2 interaction in NSCLC, contributing to our understanding of cancer progression.

    2. Reviewer #2 (Public review):

      Summary:

      This revised manuscript investigates the role and the mechanism by which PDE1 impacts NSCLC progression, providing solid data to demonstrate that PDE1 binds to m6A reader YTHDF2, in turn, regulating STAT3 signaling pathway through its interaction, promoting metastasis and angiogenesis. The study provides a valuable information to lung cancer field.

      Strength:

      The study uncovers a novel PDE1A/YTHDF2/SOCS2/STAT3 pathway in NSCLC progression and the findings provide a potential treatment strategy for NSCLC patients with metastasis.

      Weakness:

      Given that physical interaction of PDE1A and YTHDF2 plays a critical role in PDE1A-mediated NSCLC metastasis, the in vivo data to show that YTHDF2 mimics the effect of PDE1A in metastasis will strength the manuscript although this point was mentioned in the revised manuscript.

    1. Reviewer #1 (Public review):

      IKK is the key signaling node for inflammatory signaling. Despite the availability of molecular structures, how the kinase achieves its specificity remains unclear. This paper describes a dynamic sequence of events in which autophosphorylation of a tyrosine near the activate site facilitates phosphorylation of the serine on the substrate via a phosphor-transfer reaction. The proposed mechanism is conceptually novel in several ways, suggesting that the kinase is dual specificity (tyrosine and serine) and that it mediates a phospho-transfer reaction. While bacteria contain phosphorylation-transfer enzymes, this is unheard of for mammalian kinases. However, what the functional significance of this enzymatic activity might remain unaddressed.

      The revised manuscript adequately addresses all the points I suggested in the review of the first submission.

    2. Reviewer #2 (Public review):

      The authors investigate the phosphotransfer capacity of Ser/Thr kinase IκB kinase (IKK), a mediator of cellular inflammation signaling. Canonically, IKK activity is promoted by activation loop phosphorylation at Ser177/Ser181. Active IKK can then unleash NF-κB signaling by phosphorylating repressor IκBα at residues Ser32/Ser26. Noting the reports of other IKK phosphorylation sites, the authors explore the extent of autophosphorylation.

      Semi-phosphorylated IKK purified from Sf9 cells, exhibits the capacity for further autophosphorylation. Anti-phosphotyrosine immunoblotting indicated unexpected tyrosine phosphorylation. Contaminating kinase activity was tested by generating a kinase-dead K44M variant, supporting the notion that the unexpected phosphorylation was IKK-dependent. In addition, the observed phosphotyrosine signal required phosphorylated IKK activation loop serines.

      Two candidate IKK tyrosines were examined as the source of the phosphotyrosine immunoblotting signal. Activation loop residues Tyr169 and Tyr188 were each rendered non-phosphorylatable by mutation to Phe. The Tyr variants decreased both autophosphorylation and phosphotransfer to IκBα. Likewise, Y169F and Y188F IKK2 variants immunoprecipitated from TNFa-stimulated cells also exhibited reduced activity in vitro.

      The authors further focus on Tyr169 phosphorylation, proposing a role as a phospho-sink capable of phosphotransfer to IκBα substrate. This model is reminiscent of the bacterial two-component signaling phosphotransfer from phosphohistidine to aspartate. Efforts are made to phosphorylate IKK2 and remove ATP to assess the capacity for phosphotransfer. Phosphorylation of IκBα is observed after ATP removal, although there are ambiguous requirements for ADP.

      Strengths:

      Ultimately, the authors draw together the lines of evidence for IKK2 phosphotyrosine and ATP-independent phosphotransfer to develop a novel model for IKK2-mediated phosphorylation of IκBα. The model suggests that IKK activation loop Ser phosphorylation primes the kinase for tyrosine autophosphorylation. With the assumption that IKK retains the bound ADP, the phosphotyrosine is conformationally available to relay the phosphate to IκBα substrate. The authors are clearly aware of the high burden of evidence required for this unusual proposed mechanism. Indeed, many possible artifacts (e.g., contaminating kinases or ATP) are anticipated and control experiments are included to address many of these concerns. The analysis hinges on the fidelity of pan-specific phosphotyrosine antibodies, and the authors have probed with two different anti-phosphotyrosine antibody clones. Taken together, the observations are thought-provoking, and I look forward to seeing this model tested in a cellular system.

      Weaknesses:

      Multiple phosphorylated tyrosines in IKK2 were apparently identified by mass spectrometric analyses. LC-MS/MS spectra are presented, but fragments supporting phospho-Y188 and Y325 are difficult to distinguish from noise. It is common to find non-physiological post-translational modifications in over-expressed proteins from recombinant sources. Are these IKK2 phosphotyrosines evident by MS in IKK2 immunoprecipitated from TNFa-stimulated cells? Identifying IKK2 phosphotyrosine sites from cells would be especially helpful in supporting the proposed model.

    3. Reviewer #3 (Public review):

      Summary:

      The authors investigate the kinase activity of IKK2, a crucial regulator of inflammatory cell signaling. They describe a novel tyrosine kinase activity of this well-studied enzyme and a highly unusual phosphotransfer from phosphorylated IKK2 onto substrate proteins in the absence of ATP as a substrate.

      Strengths:

      The authors provide an extensive biochemical characterization of the processes with recombinant protein, western blot, autoradiography, protein engineering and provide MS data now.

      Weaknesses:

      The identity and purity of the used proteins has improved in the revised work. Since the findings are so unexpected and potentially of wide-reaching interest - this is important. Similar specific detection of phospho-Ser/Thr vs phospho-Tyr relies largely on antibodies which can have varying degrees of specificity. Using multiple antibodies and MS improves the quality of the data.

    1. Reviewer #1 (Public review):

      In this study, Brickwedde et al. leveraged a cross-modal task where visual cues indicated whether upcoming targets required visual or auditory discrimination. Visual and auditory targets were paired with auditory and visual distractors, respectively. The authors found that during the cue-to-target interval, posterior alpha activity increased along with auditory and visual frequency-tagged activity when subjects were anticipating auditory targets. The authors conclude that their results disprove the alpha inhibition hypothesis, and instead implies that alpha "regulates downstream information transfer." However, as I detail below, I do not think the presented data irrefutably disproves the alpha inhibition hypothesis. Moreover, the evidence for the alternative hypothesis of alpha as an orchestrator for downstream signal transmission is weak. Their data serves to refute only the most extreme and physiologically implausible version of the alpha inhibition hypothesis, which assumes that alpha completely disengages the entire brain area, inhibiting all neuronal activity.

      (1) Authors assign specific meanings to specific frequencies (8-12 Hz alpha, 4 Hz intermodulation frequency, 36 Hz visual tagging activity, 40 Hz auditory tagging activity), but the results show that spectral power increases in all of these frequencies towards the end of the cue-to-target interval. This result is consistent with a broadband increase, which could simply be due to additional attention required when anticipating auditory target (since behavioral performance was lower with auditory targets, we can say auditory discrimination was more difficult). To rule this out, authors will need to show a power spectral density curve with specific increases around each frequency band of interest. In addition, it would be more convincing if there was a bump in the alpha band, and distinct bumps for 4 vs 36 vs 40 Hz band.<br /> (2) For visual target discrimination, behavioral performance with and without the distractor is not statistically different. Moreover, the reaction time is faster with distractor. Is there any evidence that the added auditory signal was actually distracting?<br /> (3) It is possible that alpha does suppress task-irrelevant stimuli, but only when it is distracting. In other words, perhaps alpha only suppresses distractors that are presented simultaneously with the target. Since the authors did not test this, they cannot irrefutably reject the alpha inhibition hypothesis.<br /> (4) In the abstract and Figure 1, the authors claim an alternative function for alpha oscillations; that alpha "orchestrates signal transmission to later stages of the processing stream." In support, the authors cite their result showing that increased alpha activity originating from early visual cortex is related to enhanced visual processing in higher visual areas and association areas. This does not constitute a strong support for the alternative hypothesis. The correlation between posterior alpha power and frequency-tagged activity was not specific in any way; Fig. 10 shows that the correlation appeared on both 1) anticipating-auditory and anticipating-visual trials, 2) the visual tagged frequency and the auditory tagged activity, and 3) was not specific to the visual processing stream. Thus, the data is more parsimonious with a correlation than a causal relationship between posterior alpha and visual processing.

    2. Reviewer #2 (Public review):

      Brickwedde et al. investigate the role of alpha oscillations in allocating intermodal attention. A first EEG study is followed up with an MEG study that largely replicates the pattern of results (with small to be expected differences). They conclude that a brief increase in the amplitude of auditory and visual stimulus-driven continuous (steady-state) brain responses prior to the presentation of an auditory - but not visual - target speaks to the modulating role of alpha that leads them to revise a prevalent model of gating-by-inhibition.

      Overall, this is an interesting study on a timely question, conducted with methods and analysis that are state-of-the-art. I am particularly impressed by the author's decision to replicate the earlier EEG experiment in MEG following the reviewer's comments on the original submission. Evidently, great care was taken to accommodate the reviewers suggestions.

      In an earlier version, I was struggling with the report for two main reasons: It was difficult to follow the rationale of the study, due to structural issues with the narrative and missing information or justifications for design and analysis decisions, and I was not convinced that the evidence is strong, or even relevant enough for revising the mentioned alpha inhibition theory.

      The authors have addressed my concerns through extensive revisions, and I find that it is now easier to follow, and makes a better case for rethinking how alpha may influence sensory processing through a clearer presentation of results and additional arguments.

    3. Reviewer #3 (Public review):

      Brickwedde et al. attempt to clarify the role of alpha in sensory gain modulation by exploring the relationship between attention-related changes in alpha and attention-related changes in sensory-evoked responses, which surprisingly few studies have explicitly examined. The authors find evidence against the alpha-inhibition account, at least in early sensory processing, adding valuable data to the field to support our understanding of the alpha-inhibition hypothesis.

      Due to task and measurement considerations, the EEG task is not sufficiently compelling to support the authors' claims that alpha inhibition does not occur in early sensory processing. However, the findings are bolstered by the additional MEG study which included changes in task design and a source-localization analysis. Importantly, the MEG results are aligned with the EEG study's key findings and support the authors' initial results, making a stronger case for their claims.

      It is important to note that task designs can have great implications for the assessment of alpha inhibition, particularly with the use of stimuli that evoke a steady-state response, and the authors review these considerations during their discussion and interpretation of the theory. Overall, this paper is an excellent contribution to the alpha-inhibition literature and will hopefully motivate additional research on the specific relationship between these attention-related changes using both frequency-tagged and non-frequency-tagged stimuli in different task contexts.

    1. Reviewer #1 (Public review):

      Hearing and balance rely on specialized ribbon synapses that transmit sensory stimuli between hair cells and afferent neurons. Synaptic adhesion molecules that form and regulate transsynaptic interactions between inner hair cells (IHCs) and spiral ganglion neurons (SGNs) are crucial for maintaining auditory synaptic integrity and, consequently, for auditory signaling. Synaptic adhesion molecules such as neurexin-3 and neuroligin-1 and -3 have recently been shown to play vital roles in establishing and maintaining these synaptic connections ( doi: 10.1242/dev.202723 and DOI: 10.1016/j.isci.2022.104803). However, the full set of molecules required for synapse assembly remains unclear.

      Karagulan et al. highlight the critical role of the synaptic adhesion molecule RTN4RL2 in the development and function of auditory afferent synapses between IHCs and SGNs, particularly regarding how RTN4RL2 may influence synaptic integrity and receptor localization. Their study shows that deletion of RTN4RL2 in mice leads to enlarged presynaptic ribbons and smaller postsynaptic densities (PSDs) in SGNs, indicating that RTN4RL2 is vital for synaptic structure. Additionally, the presence of "orphan" PSDs-those not directly associated with IHCs-in RTN4RL2 knockout mice suggests a developmental defect in which some SGN neurites fail to form appropriate synaptic contacts, highlighting potential issues in synaptic pruning or guidance. The study also observed a depolarized shift in the activation of CaV1.3 calcium channels in IHCs, indicating altered presynaptic functionality that may lead to impaired neurotransmitter release. Furthermore, postsynaptic SGNs exhibited a deficiency in GluA2/3 AMPA receptor subunits, despite normal Gria2 mRNA levels, pointing to a disruption in receptor localization that could compromise synaptic transmission. Auditory brainstem responses showed increased sound thresholds in RTN4RL2 knockout mice, indicating impaired hearing related to these synaptic dysfunctions.

      The findings reported here significantly enhance our understanding of synaptic organization in the auditory system, particularly concerning the molecular mechanisms underlying IHC-SGN connectivity. The implications are far-reaching, as they not only inform auditory neuroscience but also provide insights into potential therapeutic targets for hearing loss related to synaptic dysfunction.

      Comments on the Latest Version:

      In the revised manuscript, the authors have addressed my previous comments and incorporated my recommendations by adding missing experimental details, using color-blind-friendly figure colors, and discussing the differences between GluA3 KO and RTN4RL2 KO phenotypes. They also clarified why the animals needed for additional experiments are no longer available. Although these specific animals are unavailable, the authors made an effort to address my concerns by performing

    2. Reviewer #3 (Public review):

      In this study, the authors used RNAscope to explore the expression of RTN4RL2 RNA in hair cells and spiral ganglia. Through RTN4RL2 gene knockout mice, they demonstrated that the absence of RTN4RL2 leads to pre-synaptic changes of an increase in the size of presynaptic ribbons and a depolarized shift in the activation of calcium channels in inner hair cells. Additionally, they observed a post-synaptic reduction in GluA2-4 AMPA receptors and identified additional "orphan PSDs" not paired with presynaptic ribbons via immunostaining and an increased number of type I SGNs that are not connected with a ribbon synapse via serial block face imaging. These synaptic alterations ultimately resulted in an increased hearing threshold in mice, confirming that the RTN4RL2 gene is essential for normal hearing. These data are intriguing as they suggest that RTN4RL2 contributes to the proper formation and function of auditory afferent synapses and is critical for normal hearing. Most strikingly, the post-synaptic changes and hearing threshold changes are similar to recently published results by Carlton et al, 2024 on a mutation in Bai1, which is a potential binding partner for RTN4RL2. Overall this work provides some clues to the function of RTN4RL2 in the cochlea, but further studies are required to elucidate the function.

      A few points would improve the manuscript and the strength of the data presented.

      (1) A quantitative assessment is necessary in Figure 1 when discussing RNA scope data. It would be beneficial to show that expression levels are quantitatively reduced in KO mice compared to wild-type mice. This suggestion also applies to Figure 3D, which examines expression levels of Gria2. Data is provided for KO reduction in SGN, but not showing that hair cell labeling is specific. If slides are not available for the young ages, showing hair cell expression at P40 would be sufficient along with a loss of labeling at in the KO at P40.

      (2) In Figure 2, the authors present a morphological analysis of synapses and discuss the presence of "orphan PSDs." I agree that Homer1 not juxtaposed with Ctbp2 is increased in KO mice compared to the control group. However, in quantifying this, they opted to measure the number of Ctbp2 puncta with Homer 1 juxtaposed, which indicates the percentages of orphan ribbons rather than directly quantifying the number of Homer1 not juxtaposed with Ctbp2. Quantifying the number of Homer1 not juxtaposed with Ctbp2 would more clearly represent "orphan PSDs" and provide stronger support for the discussion surrounding their presence. A measurement of these was provided in the rebuttal letter, and while this number much more clearly demonstrates the increase in the number of orphan puncta, this analysis is not provided in the manuscript. This number also suggests the number of orphan receptors may be quite high, outnumbering ribbons 2:1.

      (3) In Figure 3, the authors discuss GluA2/3 puncta reduction and note that Gria2 RNA expression remains unchanged. However, the GluA2/3 labeling is done at 1-1.5 months, whereas the Gria2 RNAscope is done at P4. Additionally, there is a lack of quantification for Gria2 RNA expression due to their tissue being processed separately. RNA scope at a comparable age to the GluA2/3 would be stronger support for their statement that Gria2 expression is comparable despite a reduction in GluA2/3 puncta.

      (4) In Figure 4, the authors indicate that RTN4RL2 deficiency reduces the number of type 1 SGNs connected to ribbons. Given that the number of ribbons remains unchanged (Figure 2), it is important to clearly explain the implications of this finding. It is already known that each type I SGN forms a single synaptic contact with a single IHC. The fact that the number of ribbons remains constant while additional "orphan PSDs" are present suggests that the overall number of SGNs might need to increase to account for these findings, however, the authors noted no change in the number of SGN soma. This discrepancy is important to point out.

    1. Reviewer #1 (Public review):

      Summary:

      In this study, the authors used a multi-alternative decision task and a multidimensional signal-detection model to gain further insight into the cause of perceptual impairments during the attentional blink. The model-based analyses of behavioural and EEG data show that such perceptual failures can be unpacked into distinct deficits in visual detection and discrimination, with visual detection being linked to the amplitude of late ERP components (N2P and P3) and discrimination being linked to coherence of fronto-parietal brain activity.

      Strengths:

      The strength of this paper lies in the fact that it presents a novel perspective on the cause of perceptual failures during the attentional blink. The multidimensional signal-detection modelling approach is explained clearly, and the results of the study show that this approach offers a powerful method to unpack behavioural and EEG data into distinct processes of detection and discrimination. The discussion of the paper addresses how the findings of separable neural processes involved in detection and discrimination might be linked to extant findings on object recognition and the question of whether the attentional blink involves an all-or-none or gradual impairment in perception.

      Weakness:

      A minor, unnecessary weakness of the paper is that the authors introduce their study with the aim of determining whether the attentional blink might be due to a criterion shift or to reduced sensitivity in the perceptual process. The criterion shift account remains to be no more than a strawman as the argumentation for this account is weak and easily refuted based on many previous findings. Specifically, the authors suggest that criterion shift might explain the lag-dependent AB effect because participants might be able to infer the lag of a specific trial, thus raising their criterion in case of a short-lag trial, based on factors such as the length of the trial sequence. Importantly, however, attentional blinks have also been observed in many studies in which the sequence length was not indicative of the T1-T2 lag, including - for instance - the many experiments reported in the seminal study by Chun and Potter (1995). The criterion shift account was and remains, therefore, highly implausible and should not have deserved such a prominent role in describing the theoretical motivation for the study.

    2. Reviewer #2 (Public review):

      Summary:

      The authors had two aims: First, to decompose the attentional blink (AB) deficit into the two components of signal detection theory: sensitivity and bias. Second, the authors aimed to assess the two subcomponents of sensitivity: detection and discrimination. They observed that the AB is only expressed in sensitivity. Furthermore, detection and discrimination were doubly dissociated. Detection modulated N2p and P3 ERP amplitude, but not frontoparietal beta-band coherence, whereas this pattern was reversed for discrimination.

      Strengths:

      The experiment is elegantly designed, and the data -both behavioral and electrophysiological- are aptly analyzed. The outcomes, in particular the dissociation between detection and discrimination blinks, are very consistently and clearly supported by the results. The discussion of the results is also appropriately balanced.

      Weaknesses:

      The lack of an effect of stimulus contrast does not seem very surprising from what we know of the nature of AB already. Low-level perceptual factors are not thought to cause the AB. This is fine, as there are also other, novel findings reported. In their revision, the authors have bolstered the importance of these (null) findings by referring to AB-specific papers that would have predicted different outcomes in this regard.

      The ERP analyses are extended in the revised manuscript, including those of the N1 component, which is now more appropriately analyzed at more lateral electrode sites.

      Impact & Context:<br /> The results of this study will likely influence how we think about selective attention in the context of the AB phenomenon. In their revision, the authors have further extended their theoretical framing by referring to recent work on the nature of the AB deficit, showing that it can be discrete (all-or-none) and gradual.

    3. Reviewer #3 (Public review):

      In the present study, the authors aimed to achieve a better understanding of the mechanisms underlying the attentional blink, that is, a deficit in processing the second of two target stimuli when they appear in rapid succession. Specifically, they used a concurrent detection and identification task in- and outside of the attentional blink and decoupled effects of perceptual sensitivity and response bias using a novel signal detection model. They conclude that the attentional blink selectively impairs perceptual sensitivity but not response bias, and link established EEG markers of the attentional blink to deficits in stimulus detection (N2p, P3) and discrimination (fronto-parietal high-beta coherence), respectively. Taken together, their study suggests distinct mechanisms mediating detection and discrimination deficits in the attentional blink.

      This innovative study appears to have been carefully conducted and the overall conclusions seem warranted given the results. In my opinion, the manuscript is a valuable contribution to the current literature on the attentional blink. Moreover, the novel paradigm and signal detection model are likely to stimulate future research.

      Major strengths of the present study include its innovative approach to investigating the mechanisms underlying the attentional blink, an elegant, carefully calibrated experimental paradigm, a novel signal detection model, multifaceted data analyses using state-of-the-art model comparisons and robust statistical tests, and an interesting discussion on the neural mechanisms underlying detection versus identification.

      Weaknesses concern a lack of clarity regarding specific statistical hypotheses and correction for multiple comparisons (e.g., across or within the multiple classes of tests) in the Methods, relatively low statistical power (N = 24/18 for behavioral/ERP data, respectively), unusual and heavy EEG filtering (0.5-18 Hz bandpass and 9-11 Hz bandstop), data-driven analyses (e.g., pooling of lag 1 and 3 trials a posteriori), and the absence of a discussion of limitations.

    1. Reviewer #2 (Public review):

      Summary:

      Chromosomal inversions have been predicted to play a role in adaptive evolution and speciation because of their ability to "lock" together adaptive alleles in genomic regions of low recombination. In this study, the authors use a combination of cutting-edge genomic methods, including BioNano and PacBio HiFi sequencing, to identify six large chromosomal inversions segregating in over 100 species of Lake Malawi cichlids, a classic example of adaptive radiation and rapid speciation. By examining the frequencies of these inversions present in species from six different linages, the authors show that there is an association between the presence of specific inversions with specific lineages/habitats. Using a combination of phylogenetic analyses and sequencing data, they demonstrate that three of the inversions have been introduced to one lineage via hybridization. Finally, genotyping of laboratory crosses suggests that two inversions are associated with XY sex determination systems in a subset of species. The data add to a growing number of systems in which inversions have been associated with adaptation to divergent environments. However, like most of the other recent studies in the field, this study does not go beyond describing the presence of the inversions to demonstrate that the inversions are under sexual or natural selection or that they contribute to adaptation or speciation in this system.

      Strengths:

      All analyses are very well done, and the conclusions about the presence of the six inversions in Lake Malawi cichlids, the frequencies of the inversions in different species, and the presence of three inversions in the benthic lineages due to hybridization are well-supported. Genotyping of 48 individuals resulting from laboratory crosses provides strong support that the chromosome 10 inversion is associated with a sex-determination locus.

      Weaknesses:

      The evidence supporting a role for the chromosome 11 inversion is based on relatively few individuals and therefore remains suggestive. The authors are mostly cautious in their interpretations of the data, although there are places where the language is imprecise and therefore a little misleading.

    1. Reviewer #1 (Public review):

      Summary:

      The current work explored the link between the pulvinar intrinsic organisation and its functional and structural connectivity patterns of the cortex using different dimensional reduction techniques. Overall they find relationships between pulvinar-cortical organization and cortico-cortical organization, and little evidence for clustered organization. Moreover they investigate PET maps to understand how neurotransmitter/receptor distributions vary within the pulvinar and along its structural and functional connectivity axes.

      Strengths:

      (1) There is a replication dataset and different modalities are compared against each other to understand the structural and functional organisation of the pulvinar complex

      In their revision, the authors further detailed the motivation of their study and performed various robustness checks, answering my concerns. Nevertheless, further work is needed to fully understand the role of the pulvinar nuclei and the rest of the thalamic nuclei as well as the rest of the brain, including more diverse datasets and techniques.

    2. Reviewer #2 (Public review):

      Summary:

      The authors aimed to explore and better understand the complex topographical organization of the human pulvinar, a brain region crucial for various high-order functions such as perception and attention. They sought to move beyond traditional histological subdivisions by investigating continuous 'gradients' of cortical connections along the dorsoventral and mediolateral axes. Using advanced imaging techniques and a comprehensive PET atlas of neurotransmitter receptors, the study aimed to identify and characterize these gradients in terms of structural connections, functional coactivation, and molecular binding patterns. Ultimately, the authors targeted to provide a more nuanced understanding of pulvinar anatomy and its implications for brain function in both healthy and diseased states.

      Strengths:

      A key strength of this study lies in the authors' effort to comprehensively combine multimodal data, encompassing both functional and structural connectomics, alongside the analysis of major neurotransmitter distributions. This approach enabled a more nuanced understanding of the overarching organizational principles of the pulvinar nucleus within the broader context of whole-brain connectivity. By employing cortex-wide correlation analyses of multimodal embedding patterns derived from 'gradients,' which provide spatial maps reflecting the underlying connectomic and molecular similarities across voxels, the study offers a thorough characterization of the functional neuroanatomy of the pulvinar.

      Weaknesses:

      Despite its strengths, the current manuscript falls short in presenting the authors' unique perspectives on integrating the diverse biological principles derived from the various neuroimaging modalities. The findings are predominantly reported as correlations between different gradient maps, without providing the in-depth interpretations that would allow for a more comprehensive understanding of the pulvinar's role as a central hub in the brain's network.

    1. Reviewer #1 (Public review):

      In the revision of their paper, N'Guessan et al have improved the report of their study of expression QTL (eQTL) mapping in yeast using single cells. The authors make use of advances in single cell RNAseq (scRNAseq) in yeast to increase the efficiency with which this type of analysis can be undertaken. Building on prior research led by the senior author that entailed genotyping and fitness profiling of almost 100,000 cells derived from a cross between two yeast strains (BY and RM) they performed scRNAseq on a subset of ~5% (n = 4,489) individual cells. To address the sparsity of genotype data in the expression profiling they used a Hidden Markov Model (HMM) to infer genotypes and then identify the most likely known lineage genotype from the original dataset. To address the relationship between variance in fitness and gene expression the authors partition the variance to investigate the sources of variation. They then perform eQTL mapping and study the relationship between eQTL and fitness QTL identified in the earlier study.

      This paper seeks to address the question of how quantitative trait variation and expression variation are related. scRNAseq represents an appealing approach to eQTL mapping as it is possible to simultaneously genotype individual cells and measure expression in the same cell. As eQTL mapping requires large sample sizes to identify statistical relationships, the use of scRNAseq is likely to dramatically increase the statistical power of such studies. However, there are several technical challenges associated with scRNAseq and the authors' study is focused on addressing those challenges. My main suggestion from my review of the revised version of the manuscript has been addressed in the revised figure 3. I agree with the authors that they have successfully demonstrated their stated goal of developing, and illustrating the benefit of, a one-pot scRNA-seq experiment and analysis for eQTL mapping.

    2. Reviewer #2 (Public review):

      This work describes the single-cell expression profiling of thousands of cells of recombinant genotypes from a model natural-variation system, a cross between two divergent yeast strains.

      I appreciate the addition of lines 282-291, which now makes the authors' point about one advantage of the single-cell technique for eQTL mapping clearly: the authors don't need to normalize for culture-to-culture variation the way standard bulk methods do (e.g. in Albert et al., 2018 for the current yeast cross), and without this normalization, they can integrate analyses of expression with those of estimates of growth behaviors from the abundance of a genotype in the pool. The main question the manuscript addresses with the latter, in Figure 3, is how much variation in growth appears to have nothing to do with expression, for which the answer the authors given is 30%. I agree that this represents a novel finding. The caveats are (1) the particular point will perhaps only be interesting to a small slice of the eQTL research community; (2) the authors provide no statistical controls/error estimate or independent validation of the variance partitioning analysis in Figure 3, and (3) the authors don't seem to use the single-cell growth/fitness estimates for anything else, as Figure 4 uses loci mapped to growth from a previously published, standard culture-by-culture approach. It would be appropriate for the manuscript to mention these caveats.

      I also think it is not appropriate for the manuscript to avoid a comparison between the current work and Boocock et al., which reports single-cell eQTL mapping in the same yeast system. I recommend a citation and statement of the similarities and differences between the papers.

      I appreciate the new statement about the single-cell technique affording better power in eQTL mapping (lines 445-453).

    1. Reviewer #1 (Public review):

      This work provides a new Python toolkit for combining generative modeling of neural dynamics and inversion methods to infer likely model parameters that explain empirical neuroimaging data. The authors provided tests to show the toolkit's broad applicability and accuracy; hence, it will be very useful for people interested in using computational approaches to better understand the brain.

      Strengths:

      The work's primary strength is the tool's integrative nature, which seamlessly combines forward modelling with backward inference. This is important as available tools in the literature can only do one and not the other, which limits their accessibility to neuroscientists with limited computational expertise. Another strength of the paper is the demonstration of how the tool can be applied to a broad range of computational models popularly used in the field to interrogate diverse neuroimaging data, ensuring that the methodology is not optimal to only one model. Moreover, through extensive in-silico testing, the work provided evidence that the tool can accurately infer ground-truth parameters, which is important to ensure results from future hypothesis testing are meaningful.

      Weaknesses:

      Although the tool itself is the main strength of the work, the paper lacked a thorough analysis of issues concerning robustness and benchmarking relative to existing tools.

      The first issue is the robustness to the choice of features to be included in the objective function. This choice significantly affects the training and changes the results, as the authors even acknowledged themselves multiple times (e.g., Page 17 last sentence of first paragraph or Page 19 first sentence of second paragraph). This brings the question of whether the accurate results found in the various demonstrations are due to the biased selection of features (possibly from priors on what worked in previous works). The robustness of the neural estimator and the inference method to noise was also not demonstrated. This is important as most neuroimaging measurements are inherently noisy to various degrees.

      The second issue is on benchmarking. Because the tool developed is, in principle, only a combination of existing tools specific to modeling or Bayesian inference, the work failed to provide a more compelling demonstration of its added value. This could have been demonstrated through appropriate benchmarking relative to existing methodologies, specifically in terms of accuracy and computational efficiency.

    2. Reviewer #2 (Public review):

      Summary:

      Whole-brain network modeling is a common type of dynamical systems-based method to create individualized models of brain activity incorporating subject-specific structural connectome inferred from diffusion imaging data. This type of model has often been used to infer biophysical parameters of the individual brain that cannot be directly measured using neuroimaging but may be relevant to specific cognitive functions or diseases. Here, Ziaeemehr et al introduce a new toolkit, named "Virtual Brain Inference" (VBI), offering a new computational approach for estimating these parameters using Bayesian inference powered by artificial neural networks. The basic idea is to use simulated data, given known parameters, to train artificial neural networks to solve the inverse problem, namely, to infer the posterior distribution over the parameter space given data-derived features. The authors have demonstrated the utility of the toolkit using simulated data from several commonly used whole-brain network models in case studies.

      Strengths:

      (1) Model inversion is an important problem in whole-brain network modeling. The toolkit presents a significant methodological step up from common practices, with the potential to broadly impact how the community infers model parameters.

      (2) Notably, the method allows the estimation of the posterior distribution of parameters instead of a point estimation, which provides information about the uncertainty of the estimation, which is generally lacking in existing methods.

      (3) The case studies were able to demonstrate the detection of degeneracy in the parameters, which is important. Degeneracy is quite common in this type of model. If not handled mindfully, they may lead to spurious or stable parameter estimation. Thus, the toolkit can potentially be used to improve feature selection or to simply indicate the uncertainty.

      (4) In principle, the posterior distribution can be directly computed given new data without doing any additional simulation, which could improve the efficiency of parameter inference on the artificial neural network if well-trained.

      Weaknesses:

      (1) While the posterior estimator was trained with a large quantity of simulated data, the testing/validation is only demonstrated with a single case study (one point in parameter space) per model. This is not sufficient to demonstrate the method's accuracy and reliability, but only its feasibility. Demonstrating the accuracy and reliability of the posterior estimation in large test sets would inspire more confidence.

      (2) The authors have only demonstrated validation of the method using simulated data, but not features derived from actual EEG/MEG or fMRI data. So, it is unclear if the posterior estimator, when applied to real data, would produce results as sensible as using simulated data. Human data can often look quite different from the simulated data, which may be considered out of distribution. Thus, the authors should consider using simulated test data with out-of-distribution parameters to validate the method and using real human data to demonstrate, e.g., the reliability of the method across sessions.

      (3) The z-scores used to measure prediction error are generally between 1-3, which seems quite large to me. It would give readers a better sense of the utility of the method if comparisons to simpler methods, such as k-nearest neighbor methods, are provided in terms of accuracy.

      (4) A lot of simulations are required to train the posterior estimator, which seems much more than existing approaches. Inferring from Figure S1, at the required order of magnitudes of the number of simulations, the simulation time could range from days to years, depending on the hardware. Although once the estimator is well-trained, the parameter inverse given new data will be very fast, it is not clear to me how often such use cases would be encountered. Because the estimator is trained based on an individual connectome, it can only be used to do parameter inversion for the same subject. Typically, we only have one session of resting state data from each participant, while longitudinal resting state data where we can assume the structural connectome remains constant, is rare. Thus, the cost-efficiency and practical utility of training such a posterior estimator remains unclear.

    1. Reviewer #1 (Public review):

      Summary:

      In this study, Donofrio et al. investigated cerebellar Purkinje cell (PC) degeneration during normal aging using both mouse and human samples. They found that PC loss followed a stripe pattern rather than occurring randomly. Although this pattern resembled the pattern of zebrin II expression in the anterior cerebellum, the overall pattern was different from zebrin II expression. Surviving PCs exhibited severe degeneration, including thickened axons, axonal torpedoes, and shrunken dendrites. These structural changes were accompanied by functional deficits in motor coordination and tremor. Understanding why certain PC subpopulations are more vulnerable than others may provide insight into regional susceptibility (or resilience) to aging and inform potential therapeutic strategies for age-related neurological disorders. Overall, the findings are novel and significant, supported by compelling evidence from structural and functional analyses. However, I have several concerns about the results and hope that my comments will help improve the clarity and impact of this paper.

      Strengths:

      The cerebellum is often overlooked in aging research, despite its increasingly recognized role in motor and non-motor functions. This study, which examines the pattern of PC loss during normal aging, offers a new perspective on the aging process.

      The finding that PC loss follows a stripe pattern is a major conceptual advance, challenging the previous assumption that PC loss occurs uniformly in the cerebellum.

      The analyses using wholemount immunohistochemistry, PC-specific reporter mice, and light-sheet imaging of cleared brain tissue are meticulous. By visualizing PCs in three dimensions, this study provides strong evidence for the patterned loss of PCs across different cerebellar subdivisions during aging.

      The inclusion of human samples along with the animal model strengthens the impact and translational relevance of these findings.

      The data are clearly presented, and the manuscript is very well written.

      Weaknesses:

      While the authors have largely ruled out zebrin II as the key protein underlying PC vulnerability or resistance to age-related loss, the molecular basis of this phenomenon remains unidentified. This reviewer acknowledges the complexity of this investigation and considers it a minor issue, as the manuscript thoughtfully discusses the gap and highlights it as a future direction.

      In cases where no PC loss is observed in aged mice (Figure 1F), it is unclear whether these PCs undergo morphological degeneration, such as thickened axons and shrunken dendrites. Further characterization of these resilient PCs would help understand why the aged mice without PC loss still exhibit motor deficits (Figure 7).

      The histologic analysis is based on mice with different genetic backgrounds. For example, the PC-specific reporter mice include two strains: Pcp2-Cre; Ai32 and Pcp2-Cre; Ai40D. These genetic variations may contribute to the heterogeneity of PC loss (Figure 1). To improve clarity, please add the genetic background details to Table 1.

      Please indicate from which lobule in the anterior or posterior human cerebellum the images in Figure 8 were taken.

    2. Reviewer #2 (Public review):

      Summary:

      The cerebellum is known to be vulnerable to aging, yet specific cell type vulnerability remains understudied. This important study convincingly demonstrates that the normal aged mouse cerebellum exhibits Purkinje cell loss, and that the vulnerable PCs to age are arranged on the basis of the known zebrin stripe pattern that represents a particular subtype of the PCs. Although the patterns of PC loss were analyzed qualitatively, the phenotype is robust enough to clearly appreciate that PC loss occurs predominantly in zebrin-negative regions when combined with zebrin immunohistochemistry. Interestingly, the authors demonstrate that this phenotype appears stochastically even within the inbred C57BL/6J mouse strain examined, though the mechanisms behind this individual variability remain unexplored. In contrast to the expectation that the PC loss could account for age-related motor decline, the authors did not find any correlation between them. While the authors attempt to draw parallels with normal human aging, the human phenotypes have not been conclusively shown to match those in mice beyond the occurrence of potentially age-related PC loss. Future studies should investigate why this PC loss phenotype occurs stochastically across the population and whether these findings parallel human cerebellar aging.

      Strength:

      (1) Banding pattern of PC loss is very clearly demonstrated by combining immunostaining for zebrin.

      (2) A critical methodological concern that a standard PC marker, calbindin, could be compromised in aging has been addressed by performing control experiments with appropriate counterstaining.

      (3) Parallels with neurodegenerative phenotype would be helpful to understand the mechanisms of PC loss in the future.

      Weakness:

      (1) Limited strain diversity: The study exclusively uses C57BL/6J mice despite known genetic and motor differences even the closely related strains like C57BL/6N.

      (2) No correlation quantified between the degree of PC loss, aging, and motor performance.

      (3) It has not been demonstrated whether the neurodegenerative changes are indeed observed in zebrin-negative PCs.

      (4) The mechanisms of why only a subset of mice show PC loss remain unexplored and not discussed.

      (5) Linkages with normal human aging and cerebellar function are not well supported. While motor behavioral assays captured phenotypes that mimic aged people, correlation with PC loss is demonstrated to be absent in mice. It remains unclear whether this PC loss phenomenon is universal or specific to a particular individual; and whether specific to a human PC subtype.

      (6) Analyses in the paraflocculus are currently not easy to understand. This lobule has heterogeneous PC subtypes, developmentally or molecularly. Zebrin-weak and Zebrin-intense PCs are known to be arranged in stripes, which resembles the pattern of developmentally defined PC subsets (Fujita et al., 2014, Plos one; Fujita et al., 2012, J Neurosci). In the data presented, it is hard to appreciate whether the viewing angle is consistent relative to the angle of the paraflocculus. This may be a limitation of the analysis of the paraflocculus in general, that the orientation of this lobule is so susceptible to fixation and dissection. Discrepancy between PC loss stripe and zebrin pattern may be an overstatement, because appropriate analyses on the paraflocculus would require a rigorously standardized analytic method.

    3. Reviewer #3 (Public review):

      Summary:

      Donofrio et al. report a new observation that in normal aging mice, anti-calbindin wholemount staining and coronal immunohistochemistry in the cerebellum often show a sagittally patterned loss of Purkinje cells with age. The authors address a central concern that calbindin antibody staining alone is not sufficient to definitively assess Purkinje cell loss, and corroborate their antibody staining data with transgenic Pcp2-CRE x flox-GFP reporter mice and Neutral Red staining. The authors then investigate whether this patterned Purkinje loss correlates with the known parasagittal expression of zebrin-II, finding a strong but imperfect correlation with zebrin-II antibody staining. They next draw a connection between this age-related Purkinje loss to the age-related decline in motor function in mice, with a trending but non-significant statistical association between the severity/patterning of Purkinje loss and motor phenotypes within cohorts of aged mice. Finally, the authors look at post-mortem human cerebellar tissues from deceased healthy donors between 21 and 74 years of age, finding a positive correlation between Purkinje degeneration and age, but with unknown spatial patterning.

      Strengths:

      The conclusions drawn from this study are well supported by the data provided. The authors highlight several examples of parasagittal patterning of Purkinje cell degeneration in disease, and show that proper methodologies must be used to account for these patterns to avoid highly variable data in the sagittal plane. The authors aptly point out that additional work is needed to investigate the spatial patterns of Purkinje cell loss in the human cerebellum.

      Weaknesses:

      (1) In Figure 3, the authors use Pcp2-CRE mice to drive GFP expression in Purkinje cells in order to avoid the confounding variable of loss of calbindin expression in aging Purkinje cells. The authors go on to say, "we argue that calbindin expression alone is not a reliable, sufficient indicator of Purkinje cell loss". However, in Figure 4, the authors return to calbindin staining alone to assess the correlation of Purkinje cell loss with zebrin-II expression. Could the authors comment on why zebrin-II co-staining experiments were not performed in GFP reporter mice to avoid potential confounds of calbindin expression? Without this experiment, should readers accept the data presented in Figure 4 as a "reliable, sufficient indicator of Purkinje cell loss", given the author's prior claim?

      (2) Throughout the manuscript, there is a considerable reliance on the authors' interpretation of imaging data with no accompanying quantification (categorization of "striped" or "non-striped" PC loss, correlation of GFP/calbindin/zebrin-II staining, etc.). While this may be difficult to obtain, the results would be much stronger with a quantitative approach to support the stated categorizations/observations.

    1. Reviewer #1 (Public review):

      Summary:

      The authors note that there is a large corpus of research establishing the importance of LC-NE projections to the medial prefrontal cortex (mPFC) of rats and mice in attentional set or 'rule' shifting behaviours. However, this is complex behavior, and the authors were attempting to gain an understanding of how locus coeruleus modulation of the mPFC contributes to set shifting.

      The authors replicated the ED-shift impairment following NE denervation of mPFC by chemogenetic inhibition of the LC. They further showed that LC inhibition changed the way neurons in mPFC responded to the cues, with a greater proportion of individual neurons responsive to 'switching', but the individual neurons also had broader tuning, responding to other aspects of the task (i.e., response choice and response history). The population dynamics were also changed by LC inhibition, with reduced separation of population vectors between early-post-switch trials, when responding was at chance, and later trials when responding was correct. This was what they set out to demonstrate, and so one can conclude they achieved their aims.

      The authors concluded that LC inhibition disrupted mPFC "encoding capacity for switching" and suggest that this "underlie the behavioral deficits."

      Strengths:

      The principal strength is the combination of inactivation of LC with calcium imaging in the mPFC. This enabled detailed consideration of the change in behavior (i.e., defining epochs of learning, with an 'early phase' when responding is at chance being compared to a 'later phase' when the behavioral switch has occurred) and how these are reflected in neuronal activity in the mPFC, with and without LC-NE input.

      Weaknesses:

      Methodologically, some improvement could be made in terms of the statistical descriptions. Supplementary Figure 2: For the peripheral CNO, the 'control group' (saline) was n=4 and the test group (CNO), n=5. For the central CNO, the test group was n = 8 and the control was n = 7. The authors explain that the group sizes were not statistically determined and mice were assigned to groups 'arbitrarily', but why did they not at least make the group sizes equal?

      In Figure 1 (e), given the small sample size, it would be helpful if all the data points were included on the bar charts. As a t-test was performed on only the ED stage of the test, seeing all the data points would reassure that there would not have been a statistically significant 'improvement' in the ID stage in the group given mPFC CNO. It would also be helpful to give effect sizes for all statistical tests.

    2. Reviewer #2 (Public review):

      Summary:

      The authors were building on prior research linking cortical norepinephrine in a test of attentional set shifting. They extended prior research by assessing the effects of excitatory or inhibitory DREADDs prior to the test of attentional set shifting.

      Strengths:

      The use of DREADDs in the previously validated test of attentional set shifting improves temporal control of corticopetal, noradrenergic pathways during behavior. While mice typically require multiple intradimensional shifts to form an attentional set, the subjects in the current study perform a variant of the task similar to that used in humans, improving the translational validity of the work.

      Weaknesses:

      A critical piece of evidence needed to support the behavioral claim that mice form an attentional set is a statistically significant difference between the number of trials to reach criterion at the intradimensional vs. the extradimensional stage of the task. Based on prior literature, this could be done as a planned comparison, which would improve the power to detect differences beyond that found using an HSD test. An additional methodological ambiguity is the amount of time between the administration of CNO and the performance of the ED. As reported, it seems likely that the DREADDS were impacting performance at multiple stages of the test.

      Overall, the authors seem to have achieved their aims, but have failed to provide critical statistical support for claims made.

      The work is likely to be of interest to the burgeoning number of scientists investigating the role of cortical norepinephrine in cognitive flexibility.

    3. Reviewer #3 (Public review):

      Summary:

      Nigro et al examine how the locus coeruleus (LC) influences the medial prefrontal cortex (mPFC) during attentional shifts required for behavioral flexibility. Specifically, they propose that LC-mPFC inputs enable mice to shift attention effectively from texture to odor cues to optimize behavior. The LC and its noradrenergic projections to the mPFC have previously been implicated in this behavior. The authors further establish this by using chemogenetics to inhibit LC terminals in mPFC and show a selective deficit in extradimensional set-shifting behavior. However, the study's primary innovation is the simultaneous inhibition of LC while recording multineuron patterns of activity in mPFC. Analysis at the single neuron and population levels revealed broadened tuning properties, less distinct population dynamics, and disrupted predictive encoding when LC is inhibited. These findings add to our understanding of how neuromodulatory inputs shape attentional encoding in mPFC. However, several issues somewhat limit the overall impact and interpretation of the results.

      Strengths:

      The more naturalistic set-shifting task used in the study is a major strength, and its implementation in freely-moving animals is very useful. The inclusion of localized suppression of LC-mPFC terminals is also a strength that builds confidence in the specificity of their behavioral effect. Moreover, the combination of chemogenetic inhibition of LC while simultaneously recording neural activity in mPFC with miniscopes is state-of-the-art. The authors apply analyses to population dynamics, in particular, that can advance our understanding of how the LC modifies patterns of mPFC neural activity. The authors show that neural encoding at both the single-cell level and the population level is disrupted when LC is inhibited. They also show that activity is less able to predict key aspects of the behavior when the influence of LC is disrupted. This is quite interesting and adds to a growing understanding of how neuromodulatory systems sharpen the tuning of mPFC activity.

      Weaknesses:

      There are some concerns about tying the results to noradrenergic circuit activity. The authors use a DBH-Cre mouse line, but the histology images provided are low resolution, and surprisingly, there appears to be little overlap between HM4Di expression and TH immunostaining. It is unclear what explains this, but without further confirmation, it is hard to be sure whether the manipulation selectively impacts a specific LC population. While the authors are generally conservative in relating their findings to norepinephrine (NE) signaling, it is still implied that this is likely. But even if HM4Di is expressed specifically in DBH+ LC neurons, there is no confirmation that NE release is suppressed, and these neurons may release other neurotransmitters, including glutamate and dopamine. In the absence of careful controls, it is important to recognize that effects may or may not be due to LC-mPFC NE.

      Another weakness is that the behavior of miniscope mice is not shown. These experiments make up the bulk of the study, including the most significant results (Figures 2-4). Interpreting the chemogenetics + imaging results without this data is more challenging and relies on the assumption that they were affected similarly to an animal from Figure 1. More fundamentally, the imaging analyses are entirely from the extradimensional shift session. Showing similar analyses from the intradimensional shift (IDS) session would confirm that test group mice do not exhibit broadened tuning prior to injecting CNO and would help to establish whether the observed changes are to some feature of activity that is specific to extradimensional shifts. The ideal experiment would also include a separate group of animals with LC suppression during the IDS, which would show whether the observed effects are specific to an extradimensional shift and might explain behavioral effects.

      There are also some weaknesses in how the single neuron encoding data is analyzed and presented. First, the corresponding methods section is insufficient to fully understand how selectively tuned neurons were classified. The authors perform ROC analysis for the period 0 - 5s before choice to reveal choice-tuned neurons. It would be useful to know what proportion of the total neurons this represents, and whether this includes neurons with activity that is significantly increased, decreased, or both. Further, insufficient detail is provided to be able to understand how neurons are further classified into 'choice', 'history', and 'switch' categories, or what percentage of ROC-identified neurons fall into each category (only % of total neurons is provided).

      Finally, there are some concerns about lumping all the identified neurons together (as in Figure 2F). The miniscope experiments include very few mice (n=4 controls, n=5 test), and effects may be driven by only 1 or 2 subjects. Also, plotting the data on a per-animal basis would help to better understand the effects in greater detail. Overall, the results are interesting, but these weaknesses limit the strength and specificity of the claims that can be made.

    1. 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.

      Comments on revisions:

      The author provided satisfactory responses to my queries, offering clarifications and additional explanations to address potential points of confusion. The supplementary experimental data further corroborate the author's conclusions. Although a more in-depth and detailed analysis did not yield significant results, this does not undermine the overall integrity of the article's structure or the reliability of its conclusions. Based on the content and the supporting evidence presented, I believe this article meets the necessary criteria for publication.

    2. Reviewer #2 (Public review):

      Summary:

      This Phase II clinical trial investigates the combination of Gamma Knife Stereotactic Body Radiation Therapy (SBRT) with Tislelizumab for the treatment of metastatic colorectal cancer (mCRC) in patients with proficient mismatch repair (pMMR). The study addresses a critical clinical challenge in the management of pMMR CRC, focusing on the selection of appropriate candidates. The results suggest that the combination of Gamma Knife SBRT and Tislelizumab provides a safe and potent treatment option for patients with pMMR/MSS/MSI-L mCRC who have become refractory to first- and second-line chemotherapy. The study design is rigorous, and the outcomes are promising.

      Advantage:

      The trial design was meticulously structured, and appropriate statistical methods were employed to rigorously analyze the results. Bioinformatics approaches were utilized to further elucidate alterations in the patient's tumor microenvironment and to explore the underlying factors contributing to the observed differences in treatment efficacy. The conclusions drawn from this trial offer valuable insights for managing advanced colorectal cancer in patients who have not responded to first- and second-line therapies.

      Weakness:

      (1) Clarity and Structure of the Abstract<br /> - Results Section: The results section should contain important data, I suggest some important sequencing data should be shown to enhance understanding.<br /> (2) As the author using the NanoString assay for transcriptome analysis, more detail should be shown such as the version of R, and the bioinformatics analysis methods.<br /> (3) It is interesting for included patients that PD-L1 increase expression after Gamma Knife Stereotactic Body Radiation Therapy (SBRT) treatment, How to explain it?<br /> (4) It would be helpful to include a brief discussion of the limitations of the study, such as sample size constraints and their impact on the generalizability of the results. This will give readers a more comprehensive understanding of the findings.<br /> (5) Language Accuracy: There are a few instances where wording could be more professional or precise.

      Revision comment:

      The author had responded to all questions and improved the manuscript. The author's answers and revisions are very satisfactory to me. I believe it is an important study for the immunotherapy of colorectal cancer.

    1. 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 support the conclusions drawn in the discussion.

      Comments on latest version:

      The third version of this paper has been substantially improved. The English is significantly better, there are only few paragraphs and sentences which are hard to understand (see my comments and suggestions below). Almost all of my suggestions were incorporated.

      Remaining minor concerns:<br /> About epileptic and non-epileptic (non-affected) tissue. I am aware that temporal lobe neocortical tissue derived from epileptic patients is regarded as non-affected by many groups, and they are quite similar to the cortex of non-epileptic (tumour) patients in their electrophysiological properties and synaptic physiology. But please, note, that one paper you cited did not use samples from epileptic patients, but only tissue from non-epileptic tumor patients (Molnár et al. PLOS 2008).<br /> When you look deeper, and make thorough comparison of tissues derived from epileptic and non-epileptic patients, there are differences in the fine structure, as well as in several electrophysiological features. See for example Tóth et al., J Physiol, 2018, where higher density of excitatory synapses were found in L2 of neocortical samples derived from epileptic patients compared to non-epileptic (tumor) patients. Furthermore, the appearance of population bursts is similar, but their occurrence is more frequent and their amplitude is higher in tissue from epileptic compared to non-epileptic patients. So, I still cannot agree, that temporal neocortex of epileptic patients with the seizure focus in the hippocampus would be non-affected. Therefore I suggested to use the term biopsy tissue.

      It is still not emphasized in the first paragraph of the Discussion, that only excitatory axon terminals were investigated.

      The text in the Results and the Discussion are somewhat inconsistent.<br /> The last two paragraphs of the Results section ends with several sentences which should be part of the discussion, such as line 328: This finding strongly supports multivesicular release... or line 344: --- pointing towards a layer-specific regulation of the putative RRP. Moreover, the results suggest that... and line 370: ... it is most likely... Please, correct this.<br /> The first paragraph of the Discussion summarizes the work of the quantitative EM work and gives one conclusion about the astrocytic coverage. This last sentence is inconsistent with the other parts of the paragraph. I would either write that "astrocytic coverage was also investigated" (or something similar), or move this sentence to the paragraph which discusses the astrocytic coverage.<br /> Results line 180-183. "Special connections" between astrocytic processes and synaptic boutons are mentioned, but not shown. Either show these (but then prove with staining!), or leave out this paragraph.

    2. Reviewer #2 (Public review):

      Summary:

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

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

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

      Strengths:

      The MS is well written easy to read. Result section gives a detailed set of figures showing many morphological parameters of synaptic boutons and surrounding glial elements. The authors provide comparative data of all the layers examined by them so far in the Discussion. Given that anatomical data in human brain are still very limited, the current MS has substantial relevance.<br /> The work appears to be generally well done, the EM and EM tomography images are of very good quality. The analyses is clear and precise.

      Weaknesses:

      The authors made all the corrections required and answered all of my concerns, included additional data sets, and clarified statements where needed.

    1. Reviewer #1 (Public review):

      Summary:

      Fecal virome transfer (FVT) has the potential to take advantage of microbiome-associated phages to treat diseases such as NEC. However, FVT is also associated with toxicity due to the presence of eukaryotic viruses in the mixture, which are difficult to filter out. The authors use a chemostat propagation system to reduce the presence of eukaryotic viruses (these become lost over time during culture). They show in pig models of NEC that chemostat propagation reduces the incidence of diarrhea induced by FVTs.

      Strengths:

      The authors report an innovative yet simple approach that has the potential to be useful for future applications. Most of the experiments are easy to follow and are performed well.

      Weaknesses:

      The biggest weakness is that the authors show that their technique addresses safety, but they are unable to demonstrate that they retain efficacy in their NEC model. This could be due to technical issues or perhaps the efficacy of FVT reported in the literature is not robust. If they cannot demonstrate the efficacy of the chemostat-propagated virome mixture, the value of the study is compromised.

      The above issue is especially concerning because the chemostat propagation selected for bacteria that may not necessarily be the ones that harbor the beneficial phages. Without an understanding of exactly how FVT works, is it possible to make any conclusion about the usefulness of the chemostat approach?

      Finally, can the authors rule out that their observations in THP-1 cells are driven by LPS or some other bacterial product in the media?

    2. Reviewer #2 (Public review):

      The authors hypothesized that chemostat propagated viromes could modulate the GM and reduce NEC lesions while avoiding potential side effects, such as the earlier onset of diarrhea. This is interesting.

      Major Comments:

      (1) As the authors state that the aim of the research is 'We hypothesized that chemostat propagated viromes could modulate the GM and reduce NEC lesions while avoiding potential side effects, such as earlier onset of diarrhea'.<br /> a) For the efficacy, in Figure 5, there is no significance in stomach pathology and enterocolitis between groups, even between the control group and experimental groups, is it because of the low incidence of NEC? This may affect the statistical power of the conclusions. Therefore, it is unclear how one can draw the conclusion that chemostat can reduce NEC lesions?<br /> b) Convincing pathology images would be helpful.<br /> c) For the safety, such as body weight development, FVT had no statistical significance difference from control, CVT, and CVT-MO. So how can the authors draw the conclusion that chemostat can avoid potential side effects?<br /> d) There is a lack of evidence to convince the reader that there is a decrease in eukaryotic viruses. More quantitative data here would be useful.

      (2) Questions regarding Figure 3F:<br /> a) How can the medium have 'the baseline viral content'?<br /> b) What is the statistical significance of the relative abundance of specific eukaryotic viruses?<br /> c) The hosts for some of the listed eukaryotic viruses are neither pigs or humans, as such, the significance of a decrease in these viruses to humans is unclear.

      (3) In this study, pH 6.5 was selected as the pH value for chemostat cultivation, but considering the different adaptability of different bacteria to pH, it is recommended to further explore the effect of pH on bacteria and virus groups. In particular, it was optimized to maintain the growth of beneficial bacteria such as Lactobacillaceae and Bacteroides in order to improve the effect of chemostat cultivation.

      (4) Please improve the quality of the images, charts, error bars, and statistical significance markers throughout and mark the n's. used in each experiment.

    3. Reviewer #3 (Public review):

      This study investigated the in vitro amplification of donor fecal virus using chemostat culturing technology, aiming to reduce eukaryotic virus load while preserving bacteriophage community diversity, thereby optimizing the safety and efficacy of FVT. The research employed a preterm pig model to evaluate the effects of chemostat-propagated viromes (CVT) in preventing necrotizing enterocolitis (NEC) and mitigating adverse effects such as diarrhea.

      Strengths:

      (1) Enhanced Safety Profile:<br /> Chemostat cultivation effectively reduced eukaryotic virus load, thereby minimizing the potential infection risks associated with virome transplantation and offering a safer virome preparation method for clinical applications.

      (2) Process Reproducibility:<br /> The chemostat system achieved stable amplification of bacteriophage communities (Bray-Curtis similarity >70%), mitigating the impact of donor fecal variability on therapeutic efficacy.

      Weaknesses:

      (1) Loss of Phage Functionality:<br /> The chemostat cultivation resulted in a reduction in phage diversity (e.g., the loss of Lactobacillaceae phages), which may compromise their protective effects against NEC (potentially linked to the immunomodulatory functions of Lactobacilli). The authors should explicitly address this limitation in the discussion section, particularly if additional experiments cannot be conducted to resolve it within the current study.

      (2) Limitations in Experimental Design:<br /> The low incidence of NEC lesions in the control group reduced the statistical power of the study. This limitation undermines the ability to conclusively evaluate the efficacy and safety of the chemostat-propagated virome as a novel intervention for NEC. Future studies should optimize experimental conditions (e.g., using a more NEC-susceptible model or diet) to ensure adequate disease incidence for robust statistical comparisons.

    1. 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 the asymmetric response to NH4Cl.

      Weaknesses:

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

      Comments on revisions: Looks good - all the best

    1. Reviewer #1 (Public review):

      Summary:

      In this study, the authors sequenced emm89 serotype genomes of clinical isolates from patients in Japan, where the number of invasive Group A Streptococcus (GAS), especially those of the emm89 serotype, has drastically increased over the past 10-15 years. The sequences from this cohort were compared against a large collection of publicly available global isolates, yielding a total of almost 1000 genomes in the analysis. Because the researchers focused on the emm89 serotype, they could construct a common core genome, with subsequent ability to analyze genomic differences in accessory genes and intergenic regions that contributed to the invasive phenotype using multiple types of GWAS analysis (SNP, k-mer). Their analysis demonstrates some mutations responsible for invasiveness are specific to the Japanese strains, and that multiple independent virulence factors can contribute to invasiveness. None of the invasive phenotypes were correlated with new gene acquisition. Together, the data support that synergy between bacterial survival and upregulation of virulence factors contribute to the development of severe infection.

      Strengths:

      • The authors verify their analysis by confirming that covS is one of the more frequently mutated genes in invasive strains of GAS, as has been shown in other publications.

      • A mutation in one of the SNPs attributed to invasiveness (SNP fhuB) was introduced into an invasive strain. The authors demonstrate that this mutant strain survives less well in human blood. Therefore, the authors have experimental data to support their claims that their analysis uncovered a new mutation/SNP that contributed to invasiveness.

      Weaknesses:

      • It would be helpful for the authors to highlight why their technique (large scale analysis of one emm type) can yield more information than a typical GWAS analysis of invasive vs. non-invasive strains. Are SNPs easier to identify using a large-scale core genome? Is it more likely evolutionarily to find mutations in non-coding regions as opposed to the core genome and accessory genes, and this is what this technique allows? Did the analysis yield unexpected genes or new genes that had not been previously identified in other GWAS analyses? These points may need to be made more apparent in the results and deserves some thought in the discussion section.

      • The Alpha-fold data does not demonstrate why the mutations the authors identified could contribute to the invasive phenotype. It would be helpful to show an overlay of the predicted structures containing the different SNPs to demonstrate the potential structural differences that can occur due to the SNP. This would make the data more convincing that the SNP has a potential impact on the function of the protein. Similarly, the authors discuss modification of the hydrophobicity of the side chain in the ferrichrome transporter (lines 317-318) due to a SNP, but this is not immediately obvious in the figure (Fig. 5).

      Comments on revisions:

      The authors have addressed the concerns from reviewers. The implemented revisions have improved the manuscript's clarity.

    1. Reviewer #1 (Public review):

      The authors introduces DIPx, a deep learning framework for predicting synergistic drug combinations for cancer treatment using the AstraZeneca-Sanger (AZS) DREAM Challenge dataset. While the approach is innovative, I have following concerns and comments, and hopefully will improve the study's rigor and applicability, making it a more powerful tool in real clinical world.

      (1) The model struggles with predicting synergies for drug combinations not included in its training data (showing only Spearman correlation 0.26 in Test Set 2). This limits its potential for discovering new therapeutic strategies. Utilizing techniques such as transfer learning or expanding the training dataset to encompass a wider range of drug pairs could help to address this issue.

      (2) The use of pan-cancer datasets, while offering broad applicability, may not be optimal for specific cancer subtypes with distinct biological mechanisms. Developing subtype-specific models or adjusting the current model to account for these differences could improve prediction accuracy for individual cancer types.

      (3) Line 127, "Since DIPx uses only molecular data, to make a fair comparison, we trained TAJI using only molecular features and referred to it as TAJI-M.". TAJI was designed to use both monotherapy drug-response and molecular data, and likely won't be able to reach maximum potential if removing monotherapy drug-response from the training model. It would be critical to use the same training datasets and then compare the performances. From Figure 6 of TAJI's paper (Li et al., 2018, PMID: 30054332) , i.e., the mean Pearson correlation for breast cancer and lung cancer are around 0.5 - 0.6.

      The following 2 concerns have been included in the Discussion section which are great:

      (1) Training and validating the model using cell lines may not fully capture the heterogeneity and complexity of in vivo tumors. To increase clinical relevance, it would be beneficial to validate the model using primary tumor samples or patient-derived xenografts.

      (2) The Pathway Activation Score (PAS) is derived exclusively from primary target genes, potentially overlooking critical interactions involving non-primary targets. Including these secondary effects could enhance the model's predictive accuracy and comprehensiveness.

    2. Reviewer #2 (Public review):

      Trac, Huang, et al used the AZ Drug Combination Prediction DREAM challenge data to make a new random forest-based model for drug synergy. They make comparisons to the winning method and also show that their model has some predictive capacity for a completely different dataset. They highlight the ability of the model to be interpretable in terms of pathway and target interactions for synergistic effects.

      In their revised manuscript and response, the authors have tried to address all points. I do not fully agree with them about the definition of overfitting still. If the objective it to identify synergies given any 2 drugs, not just those in a dataset at different doses, then the results certainly appear overfit to the training set given the performance degradation. However, at this time, I cannot add any useful suggestions to improve performance.

    3. Reviewer #3 (Public review):

      Summary:

      Predicting how two different drugs act together by looking at their specific gene targets and pathways is crucial for understanding the biological significance of drug combinations. This study incorporates drug-specific pathway activation scores (PASs) to estimate synergy scores as one of the key advancements for synergy prediction. The new algorithm, Drug synergy Interaction Prediction (DIPx), developed in this study, uses gene expression, mutation profiles, and drug synergy data to train the model and predict synergy between two drugs. Comprehensive comparisons with another best-performing algorithm, TAIJI-M, highlight the potential of its capabilities.

      Strengths:

      DIPx uses target and driver genes to elucidate pathway activation scores (PASs) to predict drug synergy. Its performance was tested using the AstraZeneca-Sanger (AZS) DREAM Challenge dataset, especially in Test Set 1, where the Spearman correlation coefficient between predicted and observed drug synergy was 0.50 (95% CI: 0.47-0.53). DIPx's ability to handle novel combinations, as evidenced by its performance in test set 2, indicates the potential for predicting new and untested drug combinations, even though it's lower than that of the test set 1.

      Weaknesses:

      While the DIPx algorithm shows promise in predicting drug synergy based on pathway activation scores, it's essential to consider its limitations. One limitation is that the availability of training data for specific drug combinations may influence its predictive capability. Further testing and experimental validation of the predictions in future studies would be necessary to assess the algorithm's generalizability and robustness.

    1. Reviewer #1 (Public review):

      Summary:

      The authors performed bidirectional two-sample Mendelian randomization using publicly available GWAS summary data to assess the directional causal association between atherosclerosis and intracranial aneurysms. They have used a similar strategy to identify the role of matrix metalloproteinases (MMP), especially MMP12, in mediating the above causal association. They finally substantiated these results by measuring and comparing the MMP12 levels in the plasma samples collected from carotid atherosclerosis and intracranial aneurysm patients with those of healthy controls. Local tissue levels of MMP12 were also measured in experimental mouse models.

      Strengths:

      The authors have chosen to address an important problem that could be of interest to many researchers and clinicians in the subfield.

      Weaknesses:

      Mendelian Randomization (MR) is a powerful approach to explore the directional causal relationship between comorbid conditions using genetic variants as instrumental variables. The validity of causal inference derived from MR strongly depends on genetic instruments satisfying the three core assumptions- relevance, independence, and exclusion restriction. The violation of these assumptions is hard to verify in many real-world situations and may result in spurious results. Rigorous sensitivity analysis is essential to ensure the robustness of the results. The sensitivity analysis presented in the current manuscript is incomplete. The key points are as follows:

      (1) The GWAS summary datasets used by the authors for assessing the causal relationship between atherosclerosis and intracranial aneurysms were all from the FinnGen study and thus may have overlapping samples which is known to introduce bias into the causal estimates and inflate type 1 error rates.

      (2) Both atherosclerosis and aneurysms share common risk factors (mentioned by the authors as well) such as hypertension, cholesterol, diabetes, smoking, etc., which could lead to correlated pleiotropy while performing Mendelian randomization. MR-PRESSO may not effectively account for the same.

      (3) The authors explored the role of matrix metalloproteinases as intermediate biomarkers mediating the risk of atherosclerosis in the intracranial aneurysms. Separating the exposure to biomarker MR from biomarker to outcome MR limits the interpretation of the results. The effect size of the indirect effect cannot be assessed.

      (4) The scatter plots presented in Supplementary Figures 1-3 are neither cited nor discussed in the manuscript. Some of the plots show variability in the direction and magnitude of the causal estimates from MR-Egger and MR-IVW methods, indicating either masking of the causal estimates or directional pleiotropy. Discussing these results is crucial to inform the readers of the limitations of the derived causal estimates.

      (5) When there is substantial evidence available for the frequent coexistence of atherosclerosis and aneurysms, the additional value of the cross-sectional data showing the increased prevalence of atherosclerosis in patients with intracranial aneurysms without adjusting for confounding risk factors is not clear.

      (6) It is also not clear from the manuscript whether the authors are projecting the MMP12 as a shared biomarker or as a mediator between atherosclerosis and intracranial aneurysms. As also noted by the authors, assessment of plasma MMP12 levels in a cross-sectional sample is not sufficient to substantiate the role of MMP12 as an intermediate biomarker connecting atherosclerosis to the increased risk of intracranial aneurysms.

      Impact:

      The findings from this study can form the basis for a more systematic analysis towards identifying molecular intermediates mediating the risk of atherosclerosis in patients with intracranial aneurysms or vice versa, which in turn helps develop novel strategies to manage these comorbid conditions.

    2. Reviewer #2 (Public review):

      The manuscript by Liu and colleagues applied Mendelian Randomization (MR) techniques to study the causal relationship of atherosclerosis (categorized into four subtypes) and intracranial aneurysms (classified as unruptured or ruptured), as well as the potential mediation by 12 plasma matrix metalloproteinase (MMP) levels. The authors have followed rigorous MR analysis guidelines by using multiple analytical approaches, implementing strict selection criteria, and employing comprehensive sensitivity analyses. One of the strengths is the lack of overlapping samples in their two-sample MR analysis. This approach helps mitigate potential biases and increases the reliability of their causal inference. The analysis is fundamentally sound, but there are still several nuanced areas where the methodology could be strengthened. Given that most of the identified causal associations do not hold after correcting for multiple tests, the conclusions should be carefully reviewed to be fully supported by the results.

      The recommendations below are meant to enhance the already robust approach.

      (1) The selection of 12 MMPs lacks a clear, explicit rationale in the provided excerpt. A more detailed explanation of why these specific MMPs were chosen would strengthen the methodological rigor.

      (2) Adjusting p-value for multiple testing using Bonferroni correction needs to be elucidated better.

      (3) The authors should provide a more robust explanation of why they shifted from 5×10-9 to 5×10-6 to select genomic instruments.

      (4) Egger's intercept may be a more robust approach for this study to test horizontal pleiotropy rather than MR-PRESSO.

    1. Reviewer #1 (Public review):

      Summary:

      The authors, Dalal, et. al., determined cryo-EM structures of open, closed, and desensitized states of the pentameric ligand-gated ion channel ELIC reconstituted in liposomes, and compared them to structures determined in varying nanodisc diameters. They argue that the liposomal reconstitution method is more representative of functional ELIC channels, as they were able to test and recapitulate channel kinetics through stopped-flow thallium flux liposomal assay. The authors and others have described channel interactions with membrane scaffold proteins (MSP), initially thought to be in a size-dependent manner. However, the authors reported that their cryo-EM ELIC structure interacts with the large nanodisc spNW25, contrary to their original hypotheses. This suggests that the channel's interactions with MSPs might alter its structure, possibly not accurately representing/reflecting functional states of the channel.

      Strengths:

      Cryo-EM structural determination from proteoliposomes is a promising methodology within the ion channel field due to their large surface area and lack of MSP or other membrane mimetics that could alter channel structure. Comparing liposomal ELIC to structures in various-sized nanodiscs gives rise to important discussions for other membrane protein structural studies when deciding the best method for individual circumstances.

      Weaknesses:

      The overarching goal of the study was to determine structural differences of ELIC in detergent nanodiscs and liposomes. Including comparisons of the results to the native bacterial lipid environment would provide a more encompassing discussion of how the determined liposome structures might or might not relate to the native receptor in its native environment. The authors stated they determined open, closed, and desensitized states of ELIC reconstituted in liposomes and suggest the desensitization gate is at the 9' region of the pore. However, no functional studies were performed to validate this statement.

    2. Reviewer #2 (Public review):

      Summary

      The report by Dalas and colleagues introduces a significant novelty in the field of pentameric ligand-gated ion channels (pLGICs). Within this family of receptors, numerous structures are available, but a widely recognised problem remains in assigning structures to functional states observed in biological membranes. Here, the authors obtain both structural and functional information of a pLGIC in a liposome environment. The model receptor ELIC is captured in the resting, desensitized, and open states. Structures in large nanodiscs, possibly biased by receptor-scaffold protein interactions, are also reported. Altogether, these results set the stage for the adoption of liposomes as a proxy for the biological membranes, for cryoEM studies of pLGICs and membrane proteins in general.

      Strengths

      The structural data is comprehensive, with structures in liposomes in the 3 main states (and for each, both inward-facing and outward-facing), and an agonist-bound structure in the large spNW25 nanodisc (and a retreatment of previous data obtained in a smaller disc). It adds up to a series of work from the same team that constitutes a much-needed exploration of various types of environment for the transmembrane domain of pLGICs. The structural analysis is thorough.

      The tone of the report is particularly pleasant, in the sense that the authors' claims are not inflated. For instance, a sentence such as "By performing structural and functional characterization under the same reconstitution conditions, we increase our confidence in the functional annotation of these structures." is exemplary.

      Weaknesses

      Core parts of the method are not described and/or discussed in enough detail. While I do believe that liposomes will be, in most cases, better than, say, nanodiscs, the process that leads from the protein in its membrane down to the liposome will play a big role in preserving the native structure, and should be an integral part of the report. Therefore, I strongly felt that biochemistry should be better described and discussed. The results section starts with "Optimal reconstitution of ELIC in liposomes [...] was achieved by dialysis". There is no information on why dialysis is optimal, what it was compared to, the distribution of liposome sizes using different preparation techniques, etc... Reading the title, I would have expected a couple of paragraphs and figure panels on liposome reconstitution. Similarly, potential biochemical challenges are not discussed. The methods section mentions that the sample was "dialyzed [...] over 5-7 days". In such a time window, most of the members of this protein family would aggregate, and it is therefore a protocol that can not be directly generalised. This has to be mentioned explicitly, and a discussion on why this can't be done in two days, what else the authors tested (biobeads? ... ?) would strengthen the manuscript.

      To a lesser extent, the relative lack of both technical details and of a broad discussion also pertains to the cryoEM and thallium flux results. Regarding the cryoEM part, the authors focus their analysis on reconstructions from outward-facing particles on the basis of their better resolutions, yet there was little discussion about it. Is it common for liposome-based structures? Are inward-facing reconstructions worse because of the increased background due to electrons going through two membranes? Are there often impurities inside the liposomes (we see some in the figures)? The influence of the membrane mimetics on conformation could be discussed by referring to other families of proteins where it has been explored (for instance, ABC transporters, but I'm sure there are many other examples). If there are studies in other families of channels in liposomes that were inspirational, those could be mentioned. Regarding thallium flux assays, one argument is that they give access to kinetics and set the stage for time-resolved cryoEM, but if I did not miss it, no comparison of kinetics with other techniques, such as electrophysiology, nor references to eventual pioneer time-resolved studies are provided.

      Altogether, in my view, an updated version would benefit from insisting on every aspect of the methodological development. I may well be wrong, but I see this paper more like a milestone on sample prep for cryoEM imaging than being about the details of the ELIC conformations.

    1. 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.

    2. Reviewer #2 (Public review):

      Summary:

      This Phase II clinical trial investigates the combination of Gamma Knife Stereotactic Body Radiation Therapy (SBRT) with Tislelizumab for the treatment of metastatic colorectal cancer (mCRC) in patients with proficient mismatch repair (pMMR). The study addresses a critical clinical challenge in the management of pMMR CRC, focusing on the selection of appropriate candidates. The results suggest that the combination of Gamma Knife SBRT and Tislelizumab provides a safe and potent treatment option for patients with pMMR/MSS/MSI-L mCRC who have become refractory to first- and second-line chemotherapy. The study design is rigorous, and the outcomes are promising.

      Advantage:

      The trial design was meticulously structured, and appropriate statistical methods were employed to rigorously analyze the results. Bioinformatics approaches were utilized to further elucidate alterations in the patient's tumor microenvironment and to explore the underlying factors contributing to the observed differences in treatment efficacy. The conclusions drawn from this trial offer valuable insights for managing advanced colorectal cancer in patients who have not responded to first- and second-line therapies.

      Weakness:

      (1) Clarity and Structure of the Abstract<br /> - Results Section: The results section should contain important data, I suggest some important sequencing data should be shown to enhance understanding.<br /> (2) As the author using the NanoString assay for transcriptome analysis, more detail should be shown such as the version of R, and the bioinformatics analysis methods.<br /> (3) It is interesting for included patients that PD-L1 increase expression after Gamma Knife Stereotactic Body Radiation Therapy (SBRT) treatment, How to explain it?<br /> (4) It would be helpful to include a brief discussion of the limitations of the study, such as sample size constraints and their impact on the generalizability of the results. This will give readers a more comprehensive understanding of the findings.<br /> (5) Language Accuracy: There are a few instances where wording could be more professional or precise.

    1. Reviewer #1 (Public review):

      Summary:

      In this manuscript, Ning et al. reported that Bcas2 played an indispensable role in zebrafish primitive hematopoiesis via sequestering β-catenin in the nucleus. The authors showed that loss of Bcas2 caused primitive hematopoietic defects in zebrafish. They unraveled that Bcas2 deficiency promoted β-catenin nuclear export via a CRM1-dependent manner in vivo and in vitro. They further validated that BCAS2 directly interacted with β-catenin in the nucleus and enhanced β-catenin accumulation through its CC domains. They unveil a novel insight into Bcas2, which is critical for zebrafish primitive hematopoiesis via regulating nuclear β-catenin stabilization rather than its canonical pre-mRNA splicing functions. Overall, the study is impressive and well-performed, although there are also some issues to address.

      Strengths:

      The study unveils a novel function of Bcas2, which is critical for zebrafish primitive hematopoiesis by sequestering β-catenin. The authors validated the results in vivo and in vitro. Most of the figures are clear and convincing. This study nicely complements the function of Bcas2 in primitive hematopoiesis.

      Comments on revisions:

      The authors have nicely answered all my questions, I have no problem.

    2. Reviewer #2 (Public review):

      Summary:

      Ning and colleagues present studies supporting a role for breast carcinoma amplified sequence 2 (Bcas2) in positively regulating primitive wave hematopoiesis through amplification of beta-catenin-dependent (canonical) Wnt signaling. The authors present compelling evidence that zebrafish bcas2 is expressed at the right time and place to be involved in primitive hematopoiesis, that there are primitive hematopoietic defects in hetero- and homozygous mutant and knockdown embryos, that Bcas2 mechanistically positively regulates canonical Wnt signaling, and that Bcas2 is required for nuclear retention of B-cat through physical interaction involving armadillo repeats 9-12 of B-cat and the coiled-coil domains of Bcas2. Overall, the data and writing are clean, clear, and compelling. This study is a first rate analysis of a strong phenotype with highly supportive mechanistic data. The findings shed light on the controversial question of whether, when, and how canonical Wnt signaling may be involved in hematopoietic development.

      In the revised version of their previous work, they have included responses to some of our suggestions for minor experiments and edits. We previously suggested they examine the structural compatibility of a Bcas2/beta-catenin dimer with binding to the DNA-binding protein Tcf7l1 (previously Tcf3), which would be expected for a beta-catenin nuclear-retention factor that potentiates canonical Wnt signaling responses. Although the authors did not test compatibility of Bcas2 with Tcf3 binding to beta-catenin, they show that a three-way complex with the family member Tcf4 is possible (Fig. S12), which suggests that Lef/Tcf family binding in general is plausible.

      The authors' acceptance of our suggestion to evaluate cdx and hox gene expression is welcome, as these genes have previously been defined as canonical Wnt targets (Lengerke et al., 2009) that regionalize the lateral plate mesoderm (LPM) and confer pre-hematopoietic identity there (Davidson et al., 2003; Davidson and Zon, 2004). The authors' finding that cdx4 and hoxa9a are diminished in the bcas2 mutants (Fig. S7) validates this suggestion and seem to imply that the primary defect here is specification of the early hematopoietic field in the LPM, however the results are a little confusing or surprising given that scl - which is unaffected in the bcas2 mutant (Fig. 2A) - is a downstream target of Cdx4 (Davidson et al., 2003, Fig. 1b, 3d). The results in the current submission imply that early maintenance of pre-hematopoietic competence in the LPM is a canonical-Wnt-directed phenomenon separable from the earliest specification of the hematopoietic field. We believe it would be of value to further evaluate regulation of cdx1, which has been shown to cooperate with cdx4 in regulation of the LPM hematopoietic field, as well as analyze some of the putative downstream hox family targets.

      We previously reviewed the article as suitable for publication and we continue to support our prior assessment. The authors have presented strong data supporting a role for Bcas2 in hematopoietic development across phyla and a mechanistic involvement in promoting canonical Wnt signaling.

      Strengths:

      (1) The study features clear and compelling phenotypes and results.<br /> (2) The manuscript narrative exposition and writing are clear and compelling.<br /> (3) The authors have attended to important technical nuances sometimes overlooked, for example, focusing on different pools of cytosolic or nuclear b-catenin.<br /> (4) The study sheds light on a controversial subject: regulation of hematopoietic development by canonical Wnt signaling and presents clear evidence of a role.<br /> (5) The authors present evidence of phylogenetic conservation of the pathway.

    3. Reviewer #3 (Public review):

      Summary:

      This manuscript utilized zebrafish bcas2 mutants to study the role of bcas2 in primitive hematopoiesis, and further confirms that it has a similar function in mice. Moreover, they showed that bcas2 regulates the transition of hematopoietic differentiation from angioblasts via activating Wnt signaling. By performing a series of biochemical experiments, they also showed that bcas2 accomplishes this by sequestering b-catenin within the nucleus, rather than through its known function in pre-mRNA splicing.

      Strengths:

      The work is well-performed, and the manuscript is well-written.

      Comments on revisions:

      The revised manuscript is substantially improved, and all my previous questions are now well addressed.

    1. Reviewer #1 (Public review):

      Du et al. address the cell cycle-dependent clearance of misfolded protein aggregates mediated by the endoplasmic reticulum (ER) associated Hsp70 chaperone family and ER reorganisation. The observations are interesting and impactful to the field.

      Strength:

      The manuscript addresses the connection between the clearance of misfolded protein aggregates and the cell cycle using a proteostasis reporter targeted to ER in multiple cell lines. Through imaging and some biochemical assays, they establish the role of BiP, an Hsp70 family chaperone, and Cdk1 inactivation in aggregate clearance upon mitotic exit. Furthermore, the authors present an initial analysis of the role of ER reorganisation in this clearance. These are important correlations and could have implications for ageing-associated pathologies. Overall, the results are convincing and impactful to the field.

      Weakness:

      The manuscript still lacks a mechanistic understanding of aggregate clearance. Even though the authors have provided the role of different cellular components, such as BiP, Cdk1 and ATL2/3 through specific inhibitors, at least an outline establishing the sequence of events leading to clearance is missing. Moreover, the authors show that the levels of ER-FlucDM-eGFP do not change significantly throughout the cell cycle, indicating that protein degradation is not in play. Therefore, addressing/elaborating on the mechanism of disassembly can add value to the work. Also, the physiological relevance of aggregate clearance upon mitotic exit has not been tested, nor have the cellular targets of this mode of clearance been identified or discussed.

    2. Reviewer #2 (Public review):

      This paper describes an interesting observation that ER-targeted misfolded proteins are trapped within vesicles inside nucleus to facilitate quality control during cell division. This work supports the concept that transient sequestration of misfolded proteins is a fundamental mechanism of protein quality control. The authors satisfactorily addressed several points asked in the review of first submission. The manuscript is improved but still unable to fully address the mechanisms.

      Strengths:

      The observations in this manuscript are very interesting and open up many questions on proteostasis biology.

      Weaknesses:

      Despite inclusions of several protein-level experiments, the manuscript remained a microscopy-driven work and missed the opportunity to work out the mechanisms behind the observations.

    3. Reviewer #3 (Public review):

      This paper describes a new mechanism for the clearance of protein aggregates associated to endoplasmic reticulum re-organization that occurs during mitosis.

      Experimental data showing clearance of protein aggregates during mitosis is solid, statistically significant, and very interesting. The authors made several new experiments included in the revised version to address the concerns raised by reviewers. A new proteomic analysis, co-localization of the aggregates with the ER membrane Sec61beta protein, expression of the aggregate-prone protein in the nucleus does not result in accumulation of aggregates, detection of protein aggregates in the insoluble faction after cell disruption and mostly importantly knockdown of ATL proteins involved in the organization of ER shape and structure impaired the clearance mechanism. This last observation addresses one of the weakest points of the original version which was the lack of experimental correlation between ER structure capability to re-shape and the clearance mechanism.

      In conclusion, this new mechanism of protein aggregate clearance from the ER was not completely understood in this work but the manuscript presented, particularly in the revised version, an ensemble of solid observations and mechanistic information to scaffold future studies that clarify more details of this mechanism. As stated by the authors: "How protein aggregates are targeted and assembled into the intranuclear membranous structure waits for future investigation". This new mechanism of aggregate clearance from the ER is not expected to be fully understood in a single work but this paper may constitute one step to better comprehend the cell capability to resolve protein aggregates in different cell compartments.

      [Editors' note: The authors have appropriately addressed the previous reviewers' concerns.]

    1. Reviewer #1 (Public review):

      Summary:

      The authors quantified information in gesture and speech, and investigated the neural processing of speech and gestures in pMTG and LIFG, depending on their informational content, in 8 different time-windows, and using three different methods (EEG, HD-tDCS and TMS). They found that there is a time-sensitive and staged progression of neural engagement that is correlated with the informational content of the signal (speech/gesture).

      Strengths:

      A strength of the paper is that the authors attempted to combine three different methods to investigate speech-gesture processing.

      Comments on revisions:

      I thank the authors for their careful responses to my comments. However, I remain not convinced by their argumentation regarding the specificity of their spatial targeting and the time-windows that they used.

      The authors write that since they included a sham TMS condition, that the TMS selectively disrupted the IFG-pMTG interaction during specific time windows of the task related to gesture-speech semantic congruency. This to me does not show anything about the specificity of the time-windows itself, nor the selectivity of targeting in the TMS condition.

      It could still equally well be the case that other regions or networks relevant for gesture-speech integration are targeted, and it can still be the case that these timewindows are not specific, and effects bleed into other time periods. There seems to be no experimental evidence here that this is not the case.

      To be more specific, the authors write that double-pulse TMS has been widely used in previous studies (as found in their table). However, the studies cited in the table do not necessarily demonstrate the level of spatial and temporal specificity required to disentangle the contributions of tightly-coupled brain regions like the IFG and pMTG during the speech-gesture integration process. pMTG and IFG are located in very close proximity, and are known to be functionally and structurally interconnected, something that is not necessarily the case for the relatively large and/or anatomically distinct areas that the authors mention in their table.

      But also more in general: The mere fact that these methods have been used in other contexts does not necessarily mean they are appropriate or sufficient for investigating the current research question. Likewise, the cognitive processes involved in these studies are quite different from the complex, multimodal integration of gesture and speech. The authors have not provided a strong theoretical justification for why the temporal dynamics observed in these previous studies should generalize to the specific mechanisms of gesture-speech integration.

      Moreover, the studies cited in the table provided by the authors have used a wide range of interpulse intervals, from 20 ms to 100 ms, suggesting that the temporal precision required to capture the dynamics of gesture-speech integration (which is believed to occur within 200-300 ms; Obermeier & Gunter, 2015) may not even be achievable with their 40 ms time windows.

      I do appreciate the extra analyses that the authors mention. However, my 5th comment is still unanswered: why not use entropy scores as a continous measure?

      In light of these concerns, I do not believe the authors have adequately demonstrated the spatial and temporal specificity required to disentangle the contributions of the IFG and pMTG during the gesture-speech integration process. While the authors have made a sincere effort to address the concerns raised by the reviewers, and have done so with a lot of new analyses, I remain doubtful that the current methodological approach is sufficient to draw conclusions about the causal roles of the IFG and pMTG in gesture-speech integration.

      Reference:<br /> Obermeier, C., & Gunter, T. C. (2015). Multisensory Integration: The Case of a Time Window of Gesture-Speech Integration. Journal of Cognitive Neuroscience, 27(2), 292-307. https://doi.org/10.1162/jocn_a_00688

    2. Reviewer #2 (Public review):

      Summary

      The study is an innovative and fundamental study that clarified important aspects of brain processes for integration of information from speech and iconic gesture (i.e., gesture that depicts action, movement, and shape), based on tDCS, TMS and EEG experiments. They evaluated their speech and gesture stimuli in information-theoretic ways and calculated how informative speech is (i.e., entropy), how informative gesture is, and how much shared information speech and gesture encode. The tDCS and TMS studies found that the left IFG and pMTG, the two areas that were activated in fMRI studies on speech-gesture integration in the previous literature, are causally implicated in speech-gesture integration. The size of tDC and TMS effects are correlated with entropy of the stimuli or mutual information, which indicates that the effects stems from the modulation of information decoding/integration processes. The EEG study showed that various ERP (event-related potential, e.g., N1-P2, N400, LPC) effects that have been observed in speech-gesture integration experiments in the previous literature are modulated by the entropy of speech/gesture and mutual information. This makes it clear that these effects are related to information decoding processes. The authors propose a model of how speech-gesture integration process unfolds in time, and how IFG and pMTG interact with each other in that process.

      Strengths

      The key strength of this study is that the authors used information-theoretic measures of their stimuli (i.e., entropy and mutual information between speech and gesture) in all of their analyses. This made it clear that the neuro-modulation (tDCS, TMS) affected information decoding/integration and ERP effects reflect information decoding/integration. This study used tDCS and TMS methods to demonstrate that left IFG and pMTG are causally involved in speech-gesture integration. The size of tDCS and TMS effects are correlated with information-theoretic measures of the stimuli, which indicate that the effects indeed stem from disruption/facilitation of information decoding/integration process (rather than generic excitation/inhibition). The authors' results also showed correlation between information-theoretic measures of stimuli with various ERP effects. This indicates that these ERP effects reflect the information decoding/integration process.

      Weakness

      The "mutual information" cannot capture all types of interplay of the meaning of speech and gesture. The mutual information is calculated based on what information can be decoded from speech alone and what information can be decoded from gesture alone. However, when speech and gesture are combined, a novel meaning can emerge, which cannot be decoded from a single modality alone. When example, a person produce a gesture of writing something with a pen, while saying "He paid". The speech-gesture combination can be interpreted as "paying by signing a cheque". It is highly unlikely that this meaning is decoded when people hear speech only or see gestures only. The current study cannot address how such speech-gesture integration occur in the brain, and what ERP effects may reflect such a process. The future studies can classify different types of speech-gesture integration and investigate neural processes that underlie each type. Another important topic for future studies is to investigate how the neural processes of speech-gesture integration change when the relative timing between the speech stimulus and the gesture stimulus changes.

      Comments on revisions: The authors addressed my concerns well.

    1. Reviewer #1 (Public review):

      Summary:

      This is a significant study because it adapts current methods to develop an approach for identifying promising targets for therapeutics in viral genomic RNA. The authors provide a wide array of data from different methods to help support their findings.

      Strengths:

      There are a number of strengths to highlight in this manuscript.

      (1) The study uses a sophisticated technique (SHAPE-MaP) to analyze the PEDV RNA genome in situ, providing valuable insights into its structural features.

      (2) The authors provide a strong rationale for targeting specific RNA structures for antiviral development.

      (3) The study includes a range of experiments, including structural analysis, compound screening, siRNA design, and viral proliferation assays, to support their conclusions.

      (4) Finally, the findings have potential implications for the development of new antiviral therapies against PEDV and other RNA viruses.

      Overall, this interesting study highlights the importance of considering RNA structure when designing antiviral therapies and provides a compelling strategy for identifying promising RNA targets in viral genomes.

    2. Reviewer #2 (Public review):

      Summary:

      Luo et. al. use SHAPE-MaP to find suitable RNA targets in Porcine Epidemic Diarrhoea Virus. Results show that dynamic and transient structures are good targets for small molecules, and that exposed strand regions are adequate targets for siRNA. This work is important to segment the RNA targeting.

      Strengths:

      This work is well done and the data supports its findings and conclusions. When possible, more than one technique was used to confirm some of the findings.

      Weaknesses:

      The study uses a cell line that is not porcine (not the natural target of the virus). That being said, authors used a widely used cell line that has been used in similar studies.

    3. Reviewer #3 (Public review):

      Summary:

      This manuscript by Luo et al. applied SHAPE-Map to analyze the secondary structure of the Porcine Epidemic Diarrhoea Virus (PEDV) RNA genome in infected cells. By combining SHAPE reactivity and Shannon entropy, the study indicated that the folding of the PEDV genomic RNA was nonuniform, with the 5' and 3' untranslated regions being more compactly structured, which revealed potentially antiviral targetable RNA regions. Interestingly, the study also suggested that compounds bound to well-folded RNA structures in vitro did not necessarily exhibit antiviral activity in cells, because the binding of these compounds did not necessarily alter the functions of the well-folded RNA regions. Later in the manuscript, the authors focus on guanine-rich regions, which may form G-quadruplexes and be potential targets for small interfering RNA (siRNA). The manuscript shows the binding effect of Braco-19 (a G-quadruplex-binding ligand) to a predicted G4 region in vitro, along with the inhibition of PEDV proliferation in cells. This suggests that targeting high SHAPE-high Shannon G4 regions could be a promising approach against RNA viruses. Lastly, the manuscript identifies 73 single-stranded regions with high SHAPE and low Shannon entropy, which demonstrated high success in antiviral siRNA targeting.

      Strengths:

      The paper presents valuable data for the community. Additionally, the experimental design and data analysis are well documented.

      Weaknesses:

      I have no further comments after the authors validated their concept by adding the ThT fluorescence assay in the revised version.

    1. Reviewer #1 (Public review):

      This is a very interesting paper addressing the hierarchical nature of the mammalian auditory system. The authors use an unconventional technique to assess brain responses -- functional ultrasound imaging (fUSI). This measures blood volume in the cortex at a relatively high spatial resolution. They present dynamic and stationary sounds in isolation and together, and show that the effect of the stationary sounds (relative to the dynamic sounds) on blood volume measurements decreases as one ascends the auditory hierarchy. Since the dynamic/stationary nature of sounds is related to their perception as foreground/background sounds (see below for more details), this suggests that neurons in higher levels of the cortex may be increasingly invariant to background sounds.

      The study is interesting, well conducted, and well written. I am broadly convinced by the results. However, I do have some concerns about the validity of the results, given the unconventional technique. fUSI is convenient because it is much less invasive than electrophysiology, and can image a large region of the cortex in one go. However, the relationship between blood volume and neuronal activity is unclear, and blood volume measurements are heavily temporally averaged relative to the underlying neuronal responses. I am particularly concerned about the implications of this for a study on dynamic/stationary stimuli in auditory cortical hierarchy, because the time scale of the dynamic sounds is such that much of the dynamic structure may be affected by this temporal averaging. Also, there is a well-known decrease in temporal following rate that is exhibited by neurons at higher levels of the auditory system. This means that results in different areas will be differently affected by the temporal averaging. I would like to see additional control models to investigate the impact of this.

      I also think that the authors should address several caveats: the fact that their measurements heavily spatially average neuronal responses, and therefore may not accurately reflect the underlying neuronal coding; that the perceptual background/foreground distinction is not identical to the dynamic/stationary distinction used here; and that ferret background/foreground perception may be very different from that in humans.

      Major points

      (1) Changes in blood volume due to brain activity are indirectly related to neuronal responses. The exact relationship is not clear, however, we do know two things for certain: (a) each measurable unit of blood volume change depends on the response of hundreds or thousands of neurons, and (b) the time course of the volume changes are are slow compared to the potential time course of the underlying neuronal responses. Both of these mean that important variability in neuronal responses will be averaged out when measuring blood changes. For example, if two neighbouring neurons have opposite responses to a given stimulus, this will produce opposite changes in blood volume, which will cancel each other out in the blood volume measurement due to (a). This is important in the present study because blood volume changes are implicitly being used as a measure of coding in the underlying neuronal population. The authors need to acknowledge that this is a coarse measure of neuronal responses and that important aspects of neuronal responses may be missing from the blood volume measure.

      (2) More importantly for the present study, however, the effect of (b) is that any rapid changes in the response of a single neuron will be cancelled out by temporal averaging. Imagine a neuron whose response is transient, consisting of rapid excitation followed by rapid inhibition. Temporal averaging of these two responses will tend to cancel out both of them. As a result, blood volume measurements will tend to smooth out any fast, dynamic responses in the underlying neuronal population. In the present study, this temporal averaging is likely to be particularly important because the authors are comparing responses to dynamic (nonstationary) stimuli with responses to more constant stimuli. To a first approximation, neuronal responses to dynamic stimuli are themselves dynamic, and responses to constant stimuli are themselves constant. Therefore, the averaging will mean that the responses to dynamic stimuli are suppressed relative to the real responses in the underlying neurons, whereas the responses to constant stimuli are more veridical. On top of this, temporal following rates tend to decrease as one ascends the auditory hierarchy, meaning that the comparison between dynamic and stationary responses will be differently affected in different brain areas. As a result, the dynamic/stationary balance is expected to change as you ascend the hierarchy, and I would expect this to directly affect the results observed in this study.

      It is not trivial to extrapolate from what we know about temporal following in the cortex to know exactly what the expected effect would be on the authors' results. As a first-pass control, I would strongly suggest incorporating into the authors' filterbank model a range of realistic temporal following rates (decreasing at higher levels), and spatially and temporally average these responses to get modelled cerebral blood flow measurements. I would want to know whether this model showed similar effects as in Figure 2. From my guess about what this model would show, I think it would not predict the effects shown by the authors in Figure 2. Nevertheless, this is an important issue to address and to provide control for.

      (3) I do not agree with the equivalence that the authors draw between the statistical stationarity of sounds and their classification as foreground or background sounds. It is true that, in a common foreground/background situation - speech against a background of white noise - the foreground is non-stationary and the background is stationary. However, it is easy to come up with examples where this relationship is reversed. For example, a continuous pure tone is perfectly stationary, but will be perceived as a foreground sound if played loudly. Background music may be very non-stationary but still easily ignored as a background sound when listening to overlaid speech. Ultimately, the foreground/background distinction is a perceptual one that is not exclusively determined by physical characteristics of the sounds, and certainly not by a simple measure of stationarity. I understand that the use of foreground/background in the present study increases the likely reach of the paper, but I don't think it is appropriate to use this subjective/imprecise terminology in the results section of the paper.

      (4) Related to the above, I think further caveats need to be acknowledged in the study. We do not know what sounds are perceived as foreground or background sounds by ferrets, or indeed whether they make this distinction reliably to the degree that humans do. Furthermore, the individual sounds used here have not been tested for their foreground/background-ness. Thus, the analysis relies on two logical jumps - first, that the stationarity of these sounds predicts their foreground/background perception in humans, and second, that this perceptual distinction is similar in ferrets and humans. I don't think it is known to what degree these jumps are justified. These issues do not directly affect the results, but I think it is essential to address these issues in the Discussion, because they are potentially major caveats to our understanding of the work.

    2. Reviewer #2 (Public review):

      Summary:

      Noise invariance is an essential computation in sensory systems for stable perception across a wide range of contexts. In this paper, Landemard et al. perform functional ultrasound imaging across primary, secondary, and tertiary auditory cortex in ferrets to uncover the mesoscale organization of background invariance in auditory cortex. Consistent with previous work, they find that background invariance increases throughout the cortical hierarchy. Importantly, they find that background invariance is largely explained by progressive changes in spectrotemporal tuning across cortical stations, which are biased towards foreground sound features. To test if these results are broadly relevant, they then re-analyze human fMRI data and find that spectro-temporal tuning fails to explain background invariance in human auditory cortex.

      Strengths:

      (1) Novelty of approach: Though the authors have published on this technique previously, functional ultrasound imaging offers unprecedented temporal and spatial resolution in a species where large-scale calcium imaging is not possible and electrophysiological mapping would take weeks or months. Combining mesoscale imaging with a clever stimulus paradigm, they address a fundamental question in sensory coding.

      (2) Quantification and execution: The results are generally clear and well supported by statistical quantification.

      (3) Elegance of modeling: The spectrotemporal model presented here is explained clearly and, most importantly, provides a compelling framework for understanding differences in background invariance across cortical areas.

      Weaknesses:

      (1) Interpretation of the cerebral blood volume signal: While the results are compelling, more caution should be exercised by the authors in framing their results, given that they are measuring an indirect measure of neural activity, this is the difference between stating "CBV in area MEG was less background invariant than in higher areas" vs. saying "MEG was less background invariant than other areas". Beyond framing, the basic properties of the CBV signal should be better explored:

      a) Cortical vasculature is highly structured (e.g. Kirst et al.( 2020) Cell). One potential explanation for the results is simply differences in vasculature and blood flow between primary and secondary areas of auditory cortex, even if fUS is sensitive to changes in blood flow, changes in capillary beds, etc (Mace et al., 2011) Nat. Methods.. This concern could be addressed by either analyzing spontaneous fluctuations in the CBV signal during silent periods or computing a signal-to-noise ratio of voxels across areas across all sound types. This is especially important given the complex 3D geometry of gyri and sulci in the ferret brain.

      b) Figure 1 leaves the reader uncertain what exactly is being encoded by the CBV signal, as temporal responses to different stimuli look very similar in the examples shown. One possibility is that the CBV is an acoustic change signal. In that case, sounds that are farther apart in acoustic space from previous sounds would elicit larger responses, which is straightforward to test. Another possibility is that the fUS signal reflects time-varying features in the acoustic signal (e.g. the low-frequency envelope). This could be addressed by cross-correlating the stimulus envelope with fUS waveform. The third possibility, which the authors argue, is that the magnitude of the fUS signal encodes the stimulus ID. A better understanding of the justification for only looking at the fUS magnitude in a short time window (2-4.8 s re: stimulus onset) would increase my confidence in the results.

      (2) Interpretation of the human data: The authors acknowledge in the discussion that there are several differences between fMRI and fUS. The results would be more compelling if they performed a control analysis where they downsampled the Ferret fUS data spatially and temporally to match the resolution of fMRI and demonstrated that their ferret results hold with lower spatiotemporal resolution.

    3. Reviewer #3 (Public review):

      This paper investigates invariance to natural background noise in the auditory cortex of ferrets and humans. The authors first replicate, in ferrets, a finding from human neuroimaging showing that invariance to background noise increases along the cortical hierarchy (i.e., from primary to non-primary auditory cortex). Next, the authors ask whether this pattern of invariance could be explained by differences in tuning to low-level acoustic features across primary and non-primary regions. The authors conclude that this tuning can explain the spatial organization of background invariance in ferrets, but not in humans. The conclusions of the paper are generally well supported by the data, but additional control analyses are needed to fully substantiate the paper's claims. Finally, additional discussion and potentially analysis, are needed to reconcile these findings with similar work in the literature (particularly that of Hamersky et al. 2025 J. Neurosci.).

      The paper is very straightforwardly written, with a generally clear presentation including well-designed and visually appealing figures. Not only does this paper provide an important replication in a non-human animal model commonly used in auditory neuroscience, but it also extends the original findings in three ways. First, the authors reveal a more fine-grained gradient of background invariance by showing that background invariance increases across primary, secondary, and tertiary cortical regions. Second, the authors address a potential mechanism that might underlie this pattern of invariance by considering whether differences in tuning to frequency and spectrotemporal modulations across regions could account for the observed pattern of invariance. The spectrotemporal modulation encoding model used here is a well-established approach in auditory neuroscience and seems appropriate for exploring potential mechanisms underlying invariance in auditory cortex, particularly in ferrets. However, as discussed below, the analyses based on this simple encoding model are only informative to the extent that the model accurately captures neural responses. Thus, its limitations in modeling non-primary human auditory cortex should be considered when interpreting cross-species comparisons. Third, the authors provide a more complete picture of invariance by additionally analyzing foreground invariance, a complementary measure not explored in the original study. While this analysis feels like a natural extension and its inclusion is appreciated, the interpretation of these foreground invariance findings remains somewhat unclear, as the authors offer limited discussion of their significance or relation to existing literature.

      As mentioned above, interpretation of the invariance analyses using predictions from the spectrotemporal modulation encoding model hinges on the model's ability to accurately predict neural responses. Although Figure S5 suggests the encoding model was generally able to predict voxel responses accurately, the authors note in the introduction that, in human auditory cortex, this kind of tuning can explain responses in primary areas but not in non-primary areas (Norman-Haignere & McDermott, PLOS Biol. 2018). Indeed, the prediction accuracy histograms in Figure S5C suggest a slight difference in the model's ability to predict responses in primary versus non-primary voxels. Additional analyses should be done to a) determine whether the prediction accuracies are meaningfully different across regions and b) examine whether controlling for prediction accuracy across regions (i.e., sub-selecting voxels across regions with matched prediction accuracy) affects the outcomes of the invariance analyses.

      A related concern is the procedure used to train the encoding model. From the methods, it appears that the model may have been fit using responses to both isolated and mixture sounds. If so, this raises questions about the interpretability of the invariance analyses. In particular, fitting the model to all stimuli, including mixtures, may inflate the apparent ability of the model to "explain" invariance, since it is effectively trained on the phenomenon it is later evaluated on. Put another way, if a voxel exhibits invariance, and the model is trained to predict the voxel's responses to all types of stimuli (both isolated sounds and mixtures), then the model must also show invariance to the extent it can accurately predict voxel responses, making the result somewhat circular. A more informative approach would be to train the encoding model only on responses to isolated sounds (or even better, a completely independent set of sounds), as this would help clarify whether any observed invariance is emergent from the model (i.e., truly a result of low-level tuning to spectrotemporal features) or simply reflects what it was trained to reproduce.

      Finally, the interpretation of the foreground invariance results remains somewhat unclear. In ferrets (Figure 2I), the authors report relatively little foreground invariance, whereas in humans (Figure 5G), most participants appear to show relatively high levels of foreground invariance in primary auditory cortex (around 0.6 or greater). However, the paper does not explicitly address these apparent cross-species differences. Moreover, the findings in ferrets seem at odds with other recent work in ferrets (Hamersky et al. 2025 J. Neurosci.), which shows that background sounds tend to dominate responses to mixtures, suggesting a prevalence of foreground invariance at the neuronal level. Although this comparison comes with the caveat that the methods differ substantially from those used in the current study, given the contrast with the findings of this paper, further discussion would nonetheless be valuable to help contextualize the current findings and clarify how they relate to prior work.

    1. Reviewer #1 (Public review):

      The ventral nerve cord (VNC) of organisms like Drosophila is an invaluable model for studying neural development and organisation in more complex organisms. Its well-defined structure allows researchers to investigate how neurons develop, differentiate, and organise into functional circuits. As a critical central nervous system component, the VNC plays a key role in controlling motor functions, reflexes, and sensory integration.

      Particularly relevant to this work, the VNC provides a unique opportunity to explore neuronal hemilineages-groups of neurons that share molecular, genetic, and functional identities. Understanding these hemilineages is crucial for elucidating how neurons cooperate to form specialized circuits, essential for comprehending normal brain function and dysfunction.

      A significant challenge in the field has been the lack of developmentally stable, hemilineage-specific driver lines that enable precise tracking and measurement of individual VNC hemilineages. The authors address this need by generating and validating a comprehensive, lineage-specific split-GAL4 driver library.

      Strengths and weaknesses:

      The authors select new marker genes for hemilineages from previously published single-cell data of the VNC. They generate and validate specific and temporally stable lines for almost all the hemilineages in the VNC. They successfully achieved their aims, and their results support their conclusions. This will be a valuable resource for investigating neural circuit formation and function.

      Comments on revisions:

      The manuscript has been amended, and the points raised by the reviewers have been addressed.

    2. Reviewer #2 (Public review):

      It is my pleasure to review this manuscript from Stoffers, Lacin, and colleagues, in which they identify pairs of transcription factors unique to (almost) every ventral nerve cord hemilineage in Drosophila and use these pairs to create reagents to label and manipulate these cells. The advance is sold as largely technical-as a pipeline for identifying durably expressed transcription factor codes in postmitotic neurons from single cell RNAseq data, generating knock-in alleles in the relevant genes, using these to match transcriptional cell types to anatomic cell types, and then using the alleles as a genetic handle on the cells for downstream explication of their function. Yet I think the work is gorgeous in linking expression of genes that are causal for neuron-type-specific characteristics to the anatomic instantiations of those neurons. It is astounding that the authors are able to use their deep collective knowledge of hemilineage anatomy and gene expression to match 33 of 34 to transcriptional profiles. Together with other recent studies, this work drives a major course correction in developmental biology, away from empirically identified cell type "markers" (in Drosophila neuroscience, often genomic DNA fragments that contain enhancers found to be expressed in specific neurons at specific times), and towards methods in which the genes that generate neuronal type identity are actually used to study those neurons. Because the relationship between fate and form/function are built into the tools, I believe that this approach will be a trojan horse to integrate the fields of neural development and systems neuroscience.

      Comments on revisions:

      The authors have addressed my (minor) suggestions.

    3. Reviewer #3 (Public review):

      Summary:

      Soffers et al. developed a comprehensive genetic toolkit that enables researchers to access neuronal hemilineages during developmental and adult time points using scRNA-seq analysis to guide gene cassette exchange-based or CRISPR-based tool building. Currently, research groups studying neural circuit development are challenged with tying together findings in the development and mature circuit function of hemilineage related neurons. Here, authors leverage publicly available scRNA-seq datasets to inform the development of a split-Gal4 library that targets 32 of 34 hemilineages in development and adult stages. The authors demonstrated that the split-Gal4 library, or genetic toolkit, can be used to assess the functional roles, neurotransmitter identity, and morphological changes in targeted cells. The tools presented in this study should prove to be incredibly useful to Drosophila neurobiologists seeking to link neural developmental changes to circuit assembly and mature circuit function. Additionally, some hemilineages have more than one split-Gal4 combination that will be advantageous for studies seeking to disrupt associated upstream genes.

      Strengths:

      Informing genetic tool development with publicly available scRNA-seq datasets is a powerful approach to creating specific driver lines. Additionally, this approach can be easily replicated by other researchers looking to generate similar driver lines for more specific subpopulations of cells, as mentioned in the Discussion.

      The unification of optogenetic stimulation data of 8B neurons and connectomic analysis of the Giant-Fiber-induced take-off circuit was an excellent example of the utility of this study. The link between hemilineage-specific functional assays and circuit assembly has been limited by insufficient genetic tools. The tools and data present in this study will help better understand how collections of hemilineages develop in a genetically constrained manner to form circuits amongst each other selectively.

    1. Reviewer #1 (Public review):

      Summary:

      Oor et al. report the potentially independent effects of the spatial and feature-based selection history on visuomotor choices. They outline compelling evidence, tracking the dynamic history effects based on their clever experimental design (urgent version of the search task). Their finding broadens the framework to identify variables contributing to choice behavior and their neural correlates in future studies.

      Strengths:

      In their urgent search task, the variable processing time of the visual cue leads to a dichotomy in choice performance-uninformed guesses vs. informed choices. Oor et al. did rigorous analyses to find a stronger influence of the location-based selection history on the uninformed guesses and a stronger influence of the feature-based selection history on the informed choices. It is a fundamental finding that contributes to understanding the drivers of behavioral variance. The results are clear, and the authors convincingly addressed all previously raised concerns, strengthening their conclusions.

    2. Reviewer #2 (Public review):

      Summary:

      This is a clear and systematic study on trial history influences on the performance of monkeys in a target selection paradigm. The primary contribution of the paper is to add a twist in which the target information is revealed after, rather than before, the cue to make a foveating eye movement. This twist results in a kind of countermanding of an earlier "uninformed" saccade plan by a new one occurring right after the visual information is provided. As with countermanding tasks in general, time now plays a key factor in success in this task, and it is time that allows the authors to quantitatively assess the parametric influences of things like previous target location, previous target identity, and previous correctness rate on choice performance. The results are logical and consistent with the prior literature, but the authors also highlight novelties in the interpretation of prior-trial effects that they argue are enabled by the use of their paradigm.

      Strengths:

      Careful analysis of a multitude of variables influencing behavior

      Weaknesses:

      Results appear largely confirmatory

      Comments on revisions:

      The authors have addressed the previous comments.

    1. Reviewer #3 (Public review):

      Summary:

      The manuscript by Guo and colleagues features the documentation and interpretation of three successions of continental to marginal marine deposits spanning the P/T transition and their respective ichnofaunas. Based on these new data inferences concerning end-Permian mass extinction and Triassic recovery in the tropical realm are discussed.

      Strengths:

      The manuscript is well written and organized and includes a large amount of new lithological and ichnological data that illuminate ecosystem evolution in a time of large scale transition. The lithological documentations, facies interpretations and ichnotaxonomic assignments look alright (with few exceptions).

      Weaknesses: [all eliminated in revision]

    2. Reviewer #2 (Public review):

      Summary:

      The authors made a thorough revision of the manuscript, strengthening the message. They also considered all the comments made by the reviewers and provided appropriate and convincing arguments.

      Strengths:

      The revised manuscript clarifies all the major points raised by the reviewers, and the way the information is presented (in the text, figures and tables) is clear.

      Weaknesses:

      The authors provided an appropriate and convincing rebuttal regarding the potential weakness I pointed out in the first review of the manuscript. Therefore, I do not see any major issue in their work.

    3. Reviewer #1 (Public review):

      Summary:

      This is a very well-written paper presenting interesting findings related to the recovery following the end-Permian event in continental settings, from N China. The finding is timely as the topic is actively discussed in the scientific community. The data provides additional insights into the faunal, and partly, floral global recovery following the EPE, adding to the global picture.

      Strengths: The conclusions are supported by an impressive amount of sedimentological and paleontological data (mainly trace fossils) and illustrations.

      Weaknesses: [eliminated in revision]

    1. Reviewer #1 (Public review):

      Summary:

      The paper by Papagiannakis et al is an elegant, mostly observational work detailing observations that polysome accumulation appears to drive nucleoid splitting and segregation. Overall I think this is an insightful work with solid observations.

      Strengths:

      The strengths of this paper are the careful and rigorous observational work that leads to their hypothesis. They find the accumulation of polysomes correlates with nucleoid splitting, and that the nucleoid segregation occurring right after splitting correlates with polysome segregation. These correlations are also backed up by other observations:

      (1) Faster polysome accumulation and DNA segregation at faster growth rates.<br /> (2) Polysome distribution negatively correlating with DNA positioning near asymmetric nucleoids.<br /> (3) Polysomes form in regions inaccessible to similarly sized particles.

      These above points are observational, I have no comments on these observations leading to their hypothesis.

      Comments on revisions:

      The authors have satisfied all of my concerns.

    2. Reviewer #2 (Public review):

      Summary:

      The authors perform a remarkably comprehensive, rigorous, and extensive investigation into the spatiotemporal dynamics between ribosomal accumulation, nucleoid segregation, and cell division. Using detailed experimental characterization and rigorous physical models, they offer a compelling argument that nucleoid segregation rates are determined at least in part by the accumulation of ribosomes in the center of the cell, exerting a steric force to drive nucleoid segregation prior to cell division. This evolutionarily ingenious mechanism means cells can rely on ribosomal biogenesis as the sole determinant for the growth rate and cell division rate, avoiding the need for two separate 'sensors,' which would require careful coupling.

      Strengths:

      In terms of strengths; the paper is very well written, the data are of extremely high quality, and the work is of fundamental importance to the field of cell growth and division. This is an important and innovative discovery enabled through the combination of rigorous experimental work and innovative conceptual, statistical, and physical modeling.

      Weaknesses:

      The authors have reasonably addressed by minor weaknesses raised in the first round of reviews, and I see no other weaknesses at this point worth raising.

    3. Reviewer #3 (Public review):

      Summary:

      Papagiannakis et al. present a detailed study exploring the relationship between DNA/polysome phase separation and nucleoid segregation in Escherichia coli. Using a combination of experiments and modelling, the authors aim to link physical principles with biological processes to better understand nucleoid organisation and segregation during cell growth.

      Strengths:

      The authors have a conducted a large number of experiments under different growth conditions and physiological perturbations (using antibiotics) to analyse the biophysical factors underlying the spatial organisation of nucleoids within growing E. coli cells. A simple model of ribosome-nucleoid segregation has been developed to explain the observations and tested with cleverly designed perturbation experiments.

      The model and explanation presented in the original version have been strengthened with additional results and consideration of new factors. In particular, the radial attachment of the nucleoid, supported by previous studies and the A22 treatment data in this study, provides a plausible mechanism that prevents ribosomes from diffusing between and around the nucleoid lobes through the radial shells surrounding the nucleoid. The revised version of the paper incorporates this effect, resulting in model predictions that align well with the drug treatment outcomes and the observed mid-cell accumulation and confinement of ribosomes.

      Furthermore, experiments involving plasmid-based gene expression, designed to redirect transcription away from chromosomal loci, offer compelling validation of the model's predictions. Overall, this is a robust and insightful study that will be of significant value to the quantitative microbiology community.

    1. Reviewer #1 (Public review):

      Summary:

      This manuscript is a focused investigation of the phosphor-regulation of a C. elegans kinesin-2 motor protein, OSM-3. In C-elegans sensory ciliary, kinesin-2 motor proteins Kinesin-II complex and OSM-3 homodimer transport IFT trains anterogradely to the ciliary tip. Kinesin-II carries OSM-3 as an inactive passenger from the ciliary base to the middle segment, where kinesin-II dissociates from IFT trains and OSM-3 gets activated and transports IFT trains to the distal segment. Therefore, activation/inactivation of OSM-3 plays an essential role in its ciliary function.

      Strengths:

      In this study, using mass spectrometry, the authors have shown that the NEKL-3 kinase phosphorylates a serine/threonine patch at the hinge region between coiled coils 1 and 2 of an OSM-3 dimer, referred to as the elbow region in ubiquitous kinesin-1. Phosphomimic mutants of these sites inhibit OSM-3 motility both in vitro and in vivo, suggesting that this phosphorylation is critical for the autoinhibition of the motor. Conversely, phospho-dead mutants of these sites hyperactivate OSM-3 motility in vitro and affect the localization of OSM3 in C. elegans. The authors also showed that Alanine to Tyrosine mutation of one of the phosphorylation rescues OS-3 function in live worms.

      Weaknesses:

      Collectively, this study presents evidence for the physiological role of OSM-3 elbow phosphorylation in its autoregulation, which affects ciliary localization and function of this motor. Overall, the work is well performed, and the results mostly support the conclusions of this manuscript. During revision, the authors further supported conclusions and ruled out alternative explanations by filling some logical gaps with new experimental evidence and in-text clarifications.

      Comments on revisions: I have no additional comments or concerns.

    2. Reviewer #2 (Public review):

      Summary:

      The regulation of kinesin is fundamental to cellular morphogenesis. Previously, it has been shown that OSM-3, a kinesin required for intraflagellar transport (IFT), is regulated by autoinhibition. However, it remains totally elusive how the autoinhibition of OSM-3 is released. In this study, the authors have shown that NEKL-3 phosphorylates OSM-3 and release its autoinhibition.

      The authors found NEKL-3 directly phosphorylates OSM-3 (Figure 1). The phophorylated residue is the "elbow" of OSM-3. The authors introduced phospho-dead (PD) and phospho-mimic (PM) mutations by genome editing and found that the OSM-3(PD) protein does not form cilia, and instead, accumulates to the axonal tips. The phenotype is similar to another constitutive active mutant of OSM-3, OSM-3(G444A) (Imanishi et al., 2006; Xie et al., 2024). osm-3(PM) has shorter cilia, which resembles with loss of function mutants of osm-3 (Figure 2). The authors did structural prediction and shows that G444E and PD mutations change the conformation of OSM-3 protein (Figure 3). In the single molecule assays G444E and PD mutations exhibited increased landing rate (Figure 4). By unbiased genetic screening, the authors identified a suppressor mutant of osm-3(PD), in which A489T occurs. The result confirms the importance of this residue. Based on these results, the authors suggest that NEKL-3 induces phosphorylation of the elbow domain and inactivates OSM-3 motor when the motor is synthesized in the cell body. This regulation is essential for the proper cilia formation.

      Strengths:

      The finding is interesting and gives new insight into how IFT motor is regulated.

      Comments on revisions: In the revised manuscript, the authors describe why they focused on NEKL-3 and detailed experimental procedures are presented.

      My only minor concern is the title, which appears to be too general. Researchers in the motor protein field may firstly assume this paper focuses on kinesin-1, because the "elbow" domain was originally suggested in kinesin-1. This paper newly determines the elbow region of OSM-3 and shows its crucial role in autoinhibition. Therefore, a more specific title, "Kinesin-2 Autoinhibition Requires Elbow Phosphorylation" or "OSM-3 Autoinhibition Requires Elbow phosphorylation" may be better.

    1. Reviewer #1 (Public review):

      Summary:

      Ma & Yang et al. report a new investigation aimed at elucidating one of the key nutrients S. Typhimurium (STM) utilizes with the nutrient-poor intracellular niche within macrophage, focusing on the amino acid beta-alanine. From these data, the authors report that beta-alanine plays important roles in mediating STM infection and virulence. The authors employ a multidisciplinary approach that includes some mouse studies, and ultimately propose a mechanism by which panD, involved in B-Ala synthesis, mediates regulation of zinc homeostasis in Salmonella.

      Strengths and weaknesses:

      The results and model are adequately supported by the authors' data. Further work will need to be performed to learn whether the Zn2+ functions as proposed in their mechanism. By performing a small set of confirmatory experiments in S. Typhi, the authors provide some evidence of relevance to human infections.

      Impact:

      This work adds to the body of literature on the metabolic flexibility of Salmonella during infection that enable pathogenesis.

    2. Reviewer #3 (Public review):

      Salmonella is interesting due to its life within a compact compartment, which we call SCV or Salmonella containing vacuole in the field of Salmonella. SCV is a tight-fitting vacuole where the acquisition of nutrients is a key factor by Salmonella. The authors among many nutrients, focused on beta-alanine. It is also known that Salmonella requires beta-alanine from many other studies. The authors have done in vitro RAW macrophage infection assays and In vivo mouse infection assays to see the life of Salmonella in the presence of beta-alanine. They concluded by comprehending that beta-alanine modulates the expression of many genes including zinc transporters which is required for pathogenesis.

      [Editors' note: The authors have appropriately addressed the previous reviewers' concerns.]

    1. Reviewer #2 (Public review):

      Summary:

      In contrast to the recent findings reported by Schuster S et al., this brief paper presents evidence suggesting that the stumpy form of T. brucei is likely the most pre-adapted form to progress through the life cycle of this parasite in the tsetse vector.

      Strengths:

      One significant experimental point is that all fly infection experiments are conducted in the absence of "boosting" metabolites like GlcNAc or S-glutathione. As a result, flies infected with slender trypanosomes present very low or nonexistent infection rates. This provides important experimental evidence that the findings of Schuster S and colleagues may need to be revisited.

      In the revised submission the authors also compared trypanosome midgut infection levels in tsetse flies when either young (teneral) or mature adult flies received infected bloodmeals, with or without 60 mM GlcNAc. The data clearly show that, unlike in teneral flies, the addition of GlcNAc to the trypanosome-infected bloodmeal does not enhance midgut infection in mature adult flies. This is now convincingly demonstrated in Figure 2 and provides strong experimental support for the suggestion that the effect reported by Schuster S. et al. may have been influenced by both fly age and the inclusion of metabolic "boosters" in the bloodmeal.

    2. Reviewer #3 (Public review):

      The dogma in the Trypanosome field is that transmission by Tsetse flies is ensured by stumpy forms. This has been recently challenged by the Engstler lab (Schuster et al. ), who showed that slender forms can also be transmitted by teneral flies. In this work, the authors aimed to test whether transmission by slender forms is possible and frequent. The authors observed that most stumpy forms infections with teneral and adult flies were successful while only 1 out of 24 slender form infections were successful.

      The comparison of midgut infection in adult vs teneral flies was significant in most of the conditions. However, the critical comparison is still missing: within each type of fly (adult or teneral), was the MG infection significantly different between slender and stumpy forms?

      Figure 2 convincingly demonstrates the effect of the metabolite N-acetylglucosamine on Tsetse infection. This addition helps better integrate the study with previous work. I thank the authors for their effort in performing this experiment.

      It is still remains unknown why this work and Schuster et al. reached different conclusions. As a result it remains unclear in which conditions slender forms could be important for transmission. Several variables could explain differences between the two groups: the strain used, the presence or absence of glutathione, how Tsetse colonies were maintained, thorough molecular and cellular characterisation of slender and stumpy forms (to avoid using intermediate forms as slender forms), comparison to recent field parasite strains.

    1. Reviewer #1 (Public review):

      Summary:

      In this manuscript, the authors performed an integration of 48 scRNA-seq public datasets and created a single-cell transcriptomic atlas for AML (222 samples comprising 748,679 cells). This is important since most AML scRNA-seq studies suffer from small sample size coupled with high heterogeneity. They used this atlas to further dissect AML with t(8;21) (AML-ETO/RUNX1-RUNX1T1), which is one of the most frequent AML subtypes in young people. In particular, they were able to predict Gene Regulatory Networks in this AML subtype using pySCENIC, which identified the paediatric regulon defined by a distinct group of hematopoietic transcription factors (TFs) and the adult regulon for t(8;21). They further validated this in bulk RNA-seq with AUCell algorithm and inferred prenatal signature to 5 key TFs (KDM5A, REST, BCLAF1, YY1, and RAD21), and the postnatal signature to 9 TFs (ENO1, TFDP1, MYBL2, KLF1, TAGLN2, KLF2, IRF7, SPI1, and YXB1). They also used SCENIC+ to identify enhancer-driven regulons (eRegulons), forming an eGRN, and found that prenatal origin shows a specific HSC eRegulon profile, while a postnatal origin shows a GMP profile. They also did an in silico perturbation and found AP-1 complex (JUN, ATF4, FOSL2), P300, and BCLAF1 as important TFs to induce differentiation. Overall, I found this study very important in creating a comprehensive resource for AML research.

      Strengths:

      (1) The generation of an AML atlas integrating multiple datasets with almost 750K cells will further support the community working on AML.

      (2) Characterisation of t(8;21) AML proposes new interesting leads.

      Weaknesses:

      Were these t(8;21) TFs/regulons identified from any of the single datasets? For example, if the authors apply pySCENIC to any dataset, would they find the same TFs, or is it the increase in the number of cells that allows identification of these?

    2. Reviewer #2 (Public review):

      Summary:

      The authors assemble 222 publicly available bone marrow single-cell RNA sequencing samples from healthy donors and primary AML, including pediatric, adolescent, and adult patients at diagnosis. Focusing on one specific subtype, t(8;21), which, despite affecting all age classes, is associated with better prognosis and drug response for younger patients, the authors investigate if this difference is reflected also in the transcriptomic signal. Specifically, they hypothesize that the pediatric and part of the young population acquires leukemic mutations in utero, which leads to a different leukemogenic transformation and ultimately to differently regulated leukemic stem cells with respect to the adult counterpart. The analysis in this work heavily relies on regulatory network inference and clustering (via SCENIC tools), which identifies regulatory modules believed to distinguish the pre-, respectively, post-natal leukemic transformation. Bulk RNA-seq and scATAC-seq datasets displaying the same signatures are subsequently used for extending the pool of putative signature-specific TFs and enhancer elements. Through gene set enrichment, ontology, and perturbation simulation, the authors aim to interpret the regulatory signatures and translate them into potential onset-specific therapeutic targets. The putative pre-natal signature is associated with increased chemosensitivity, RNA splicing, histone modification, stem-ness marker SMARCA2, and potentially maintained by EP300 and BCLAF1.

      Strengths:

      The main strength of this work is the compilation of a pediatric AML atlas using the efficient Cellxgene interface. Also, the idea of identifying markers for different disease onsets, interpreting them from a developmental angle, and connecting this to the different therapy and relapse observations, is interesting. The results obtained, the set of putative up-regulated TFs, are biologically coherent with the mechanisms and the conclusions drawn. I also appreciate that the analysis code was made available and is well documented.

      Weaknesses:

      There were fundamental flaws in how methods and samples were applied, a general lack of critical examination of both the results and the appropriateness of the methods for the data at hand, and in how results were presented. In particular:

      (1) Cell type annotation:

      a) The 2-phase cell type annotation process employed for the scRNA-seq sample collection raised concerns. Initially annotated cells are re-labeled after a second round with the same cell types from the initial label pool (Figure 1E). The automatic annotation tools were used without specifying the database and tissue atlases used as a reference, and no information was shown regarding the consensus across these tools.

      b) Expression of the CD34 marker is only reported as a selection method for HSPCs, which is not in line with common practice. The use of only is admitted as a surface marker, while robust annotation of HSPCs should be done on the basis of expression of gene sets.

      c) During several analyses, the cell types used were either not well defined or contradictory, such as in Figure 2D, where it is not clear if pySCENIC and AUC scores were computed on HSPCs alone or merged with CMPs. In other cases, different cell type populations are compared and used interchangeably: comparing the HSPC-derived regulons with bulk (probably not enriched for CD34+ cells) RNA samples could be an issue if there are no valid assumptions on the cell composition of the bulk sample.

      (2) Method selection:

      a) The authors should explain why they use pySCENIC and not any other approach. They should briefly explain how pySCENIC works and what they get out in the main text. In addition they should explain the AUCell algorithm and motivate its usage.

      b) The obtained GRN signatures were not critically challenged on an external dataset. Therefore, the evidence that supports these signatures to be reliable and significant to the investigated setting is weak.

      (3) There are some issues with the analysis & visualization of the data.

      (4) Discussion:

      a) What exactly is the 'regulon signature' that the authors infer? How can it be useful for insights into disease mechanisms?

      b) The authors write 'Together this indicates that EP300 inhibition may be particularly effective in t(8;21) AML, and that BCLAF1 may present a new therapeutic target for t(8;21) AML, particularly in children with inferred pre-natal origin of the driver translocation.' I am missing a critical discussion of what is needed to further test the two targets. Put differently: Would the authors take the risk of a clinical study given the evidence from their analysis?

    1. Reviewer #1 (Public review):

      Summary:

      In this manuscript, the authors use gene functional analysis, pharmacology and live imaging to develop a proposed model of diverse G protein family signalling that takes place in the papillae during the ascidian Ciona larval adhesion to regulate the timing of initiation of the morphological changes of metamorphosis. Their experiments provide solid evidence that antagonistic G protein signalling regulates cAMP levels in the papillae, which provides a threshold for triggering metamorphosis that is reflective of a larva keeping a strong and sustained level of contact with a substrate for a minimum period of approximately half an hour. The authors discuss their reasoning and address different specific aspects of their proposed timing mechanism to provide a logical flow to the manuscript. The results are nicely linked to the ecology of Ciona larval settlement and will be of interest to developmental biologists, neurobiologists, molecular biologists, marine biologists as well as provide information relevant to antifouling and aquaculture sectors.

      First, the authors knock down the G proteins Gaq and Gas to show that these genes are important for Ciona larval metamorphosis. They then provide evidence that the Gaq protein acts through a Ca2+ pathway mediated by phospholipase C and inositol triphosphate by showing that inositol phosphate and phospholipase C gene knockdown also inhibits metamorphosis, while overexpression of Gaq or phospholipase C allows larvae to undergo metamorphosis even in the absence of their mechanosensory cue, which is deprived by removing the posterior half of the tail and culturing the larvae on agar-coated dishes. The authors used calcium imaging with a genetically encoded fluorescent calcium sensor to show that Gq knockdown larvae lack a Ca2+ spike in their papillae after mechanostimulation, confirming that Gaq acts through a Ca2+ pathway. Similarly the authors show that overexpression of Gas also enables larvae to metamorphose in the absence of mechanostimulation, suggesting a role for both Gaq and Gas in this process.

      To confirm that Gas acts through cAMP signalling, the authors use pharmacological treatment or overexpression of a photoactivating adenylate cyclase to increase cAMP, and show that this also enables larvae to metamorphose in the absence of mechanostimulation, but only when their adhesive papillae are still present. Transcriptome data indicate that both Gs and Gq pathway genes are expressed in the adhesive papillae of the Ciona larva. The authors use a fluorescent cAMP indicator, Pink Flamindo, to show that cAMP increases in the papillae upon adhesion to a substrate, and this increase is lost in Gs and Gq knockdown larvae. Complementary to this, larvae that fail to undergo metamorphosis lack a cAMP increase in papillae.

      The authors then provide evidence that GABA signalling within the papillae is acting downstream of the G proteins to induce metamorphosis. Transcriptome data shows that the genes for the GABA-producing enzyme (GAD), and for GABAb receptors, are both expressed in papillae. Pharmacological experiments show that GABA induces metamorphosis in the absence of mechanosensory cues, but only in larvae that retain their papillae. To show that GABA signalling within the papillae, rather than from the brain of the larva is important, the authors also demonstrate that anterior segments of larvae lacking the brain, can also be stimulated to metamorphose by GABA, and show changes in gene expression caused by GABA.

      The authors then use a combination of pharmacology and knockdown experiments in the presence or absence of mechanosensory cues to show that Gq/Ca2+ signalling acts upstream of Gs/cAMP signalling. As elevation of cAMP by pharmacology or photoactivating adenylate cyclase rescued GABA pathway mutant larvae, the Gq and Gs pathways were concluded to be downstream of GABA signaling. However, as GABA treatment could still induce Gaq- and Gas-knockdown larvae to metamorphose, suggesting an alternative pathway to metamorphosis, which the authors deduce to be through a third G protein, Gai. They identify an unusual Gai protein that based on transcriptome data is strongly expressed in the papillae. Gai knockdown larvae fail to metamorphose but are rescued by GABA treatment, which can be explained by a potential additional Gai protein being still present (this is confirmed experimentally with MO knockdown experiments). The authors then use overexpression and knockdown experiments to show that the Gai protein acts through Gβγi complex to activate phospholipase C. Their experiments also indicate potential for a complementary or compensatory role for Gai and Gaq signalling through Gβγi. By inhibiting the potassium channel GIRK through knockdown, and the MAPK pathway gene MEK1/2 by pharmacology, the authors also establish a role for these in their proposed model of signalling, allowing GABA and cAMP to compensate or interact with each other.

      The strength of this paper is the meticulous and extensive experiments, which are carefully designed to be able to precisely target specific genes in the putative signalling pathway to build step by step a complex model that can demonstrate how metamorphosis of the ascidian larva is timed so as to only occur when strongly attached to a suitable substrate. The unique possibility of inhibiting mechanosensory-induced metamorphosis by removing some of the tail and smoothing the attachment substrate allows the authors to investigate potential effects on both activation and inhibition of metamorphosis, and to confirm that specific signalling pathways are clearly downstream of the initial mechanosensory stimulation. The study is also clear about which aspects of the model still remain unknown, such as which ligands and receptors may be responsible for the binding and activation of Gaq and Gas. Experiments testing metamorphosis of just the anterior region of the larvae nicely demonstrate the need for signalling in the region of the papillae, as do experiments where the papillae are removed, which then block metamorphosis in treatments that would otherwise stimulate it. The final model makes a clear summary of how the extensive experiments all fit together into a cohesive potential signalling network, which can be built upon in the future to potentially integrate the role of sensory cues additional to mechanosensation.

    2. Reviewer #2 (Public review):

      Summary:

      This work aims to characterize the neural signaling cascade underlying the initiation of metamorphosis in Ciona larvae. Combining gene-specific functional analyses, pharmacological experiments, and live imaging approaches, the authors identify the molecular players downstream of GABA to initiate Ciona metamorphosis. The results of this study will serve as a useful framework for future research on animal metamorphosis.

      Strengths:

      Taking advantage of the Ciona model system, the authors meticulously conducted genetic manipulation and pharmacological experiments to test the epistatic relationships among the signaling players controlling the initiation of Ciona metamorphosis. The experiments were well designed, and the results were convincing. Based on the experimental data, the final working model proposed by the authors will server as an important foundation for further investigation on metamorphosis controls in Ciona and other marine invertebrate larvae.

      Weaknesses:

      In this revised manuscript, the authors have greatly improved the descriptions of their experimental results, and have clarified my previous concerns. I do not have further comments on "weaknesses".

    1. 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 a study this small is not powered to fully characterize efficacy or safety of a treatment approach, and can, at best, can 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 readers 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.

    1. Reviewer #1 (Public review):

      Summary

      In their paper Zhan et al. have used Pf genetic data from simulated data and Ghanaian field samples to elucidate a relationship between multiplicity of infection (MOI) (the number of distinct parasite clones in a single host infection) and force of infection (FOI). Specifically, they use sequencing data from the var genes of Pf along with Bayesian modeling to estimate MOI individual infections and use these values along with methods from queueing theory that rely on various assumptions to estimate FOI. They compare these estimates to known FOIs in a simulated scenario and describe the relationship between these estimated FOI values and another commonly used metric of transmission EIR (entomological inoculation rate).

      This approach does fill an important gap in malaria epidemiology, namely estimating force of infection, which is currently complicated by several factors including superinfection, unknown duration of infection, and highly genetically diverse parasite populations. The authors use a new approach borrowing from other fields of statistics and modeling and make extensive efforts to evaluate their approach under a range of realistic sampling scenarios. However, the write-up would greatly benefit from added clarity both in the description of methods, and in the presentation of the results. Without these clarifications, rigorously evaluating whether the author's proposed method of estimating FOI is sound remains difficult. Additionally, there are several limitations that call into question the stated generalizability of this method that should at minimum be further discussed by authors and in some cases require a more thorough evaluation.

      Major comments:

      (1) Description and evaluation of FOI estimation procedure.

      a. The methods section describing the two-moment approximation and accompanying appendix is lacking several important details. Equations on line 891 and 892 are only a small part of the equations in Choi et al. and do not adequately describe the procedure notably several quantities in those equations are never defined some of them are important to understand the method (e.g. A, S as the main random variables for inter-arrival times and service times, aR and bR which are the known time average quantities, and these also rely on the squared coefficient of variation of the random variable which is also never introduced in the paper). Without going back to the Choi paper to understand these quantities, and to understand the assumptions of this method it was not possible to follow how this works in the paper. At minimum, all variables used in the equations should be clearly defined.

      b. Additionally, the description in the main text of how queueing procedure can be used to describe malaria infections would benefit from a diagram currently as written it's very difficult to follow.

      c. Just observing the box plots of mean and 95% CI on a plot with the FOI estimate (Figures 1, 2 and 10-14) is not sufficient to adequately assess the performance of this estimator. First, it is not clear whether authors are displaying the bootstrapped 95%Cis or whether they are just showing the distribution of the mean FOI taken over multiple simulations, and then it seems that they are also estimating mean FOI per host on an annual basis. Showing a distribution of those per host estimates would also be helpful. Second, a more quantitative assessment of the ability of the estimator to recover the truth across simulations (e.g. proportion of simulations where the truth is captured in the 95% CI or something like this) is important in many cases it seems that the estimator is always underestimating the true FOI and may not even contain the true value in the FOI distribution (e.g. figure 10, figure 1 under the mid IRS panel). But it's not possible to conclude on way or the other based on this visualization. This is a major issue since it calls into question whether there is in fact data to support that these methods give good and consistent FOI estimates.

      d. Furthermore authors state in the methods that the choice of mean and variance (and thus second moment) parameters for inter arrival times are varied widely, however, it's not clear what those ranges are there needs to be a clear table or figure caption showing what combinations of values were tested and which results are produced from them, this is an essential component of the method and it's impossible to fully evaluate its performance without this information. This relates to the issue of selecting the mean and variance values that maximize the likelihood of observing a given distribution of MOI estimates, this is very unclear since no likelihoods have been written down in the methods section of the main text, which likelihood are the authors referring to, is this the probability distribution of the steady state queue length distribution? At other places the authors refer to these quantities as Maximum Likelihood estimators, how do they know they have found the MLE? There are no derivations in the manuscript to support this. The authors should specify and likelihood and include in an appendix why their estimation procedure is in fact maximizing this likelihood preferably with evidence of the shape of the likelihood, and how fine the grid of values they tested are for their mean and variance since this could influence the overall quality of the estimation procedure.

      (2) Limitation of FOI estimation procedure.

      a. The authors discuss the importance of duration of infection to this problem. While I agree that empirically estimating this is not possible, there are other options besides assuming that all 1-5 year olds have the same duration of infection distribution as naïve adults co-infected with syphilis. E.g. it would be useful to test a wide range of assumed infection duration and assess their impact on the estimation procedure. Furthermore, if the authors are going to stick to the described method for duration of infection, the potentially limited generalizability of this method needs to be further highlighted in both the introduction, and the discussion. In particular, for an estimated mean FOI of about 5 per host per year in the pre-IRS season as estimated in Ghana (Figure 3) it seems that this would not translate to 4 year old being immune naïve, and certainly this would not necessarily generalize well to a school-aged child population or an adult population.

      b. The evaluation of the capacity parameter c seems to be quite important, and is set at 30, however, the authors only describe trying values of 25 and 30, and claim that this does not impact FOI inference, however it is not clear that this is the case. What happens if carrying capacity is increased substantially? Alternatively, this would be more convincing if the authors provided a mathematical explanation of why the carrying capacity increasing will not influence the FOI inference, but absent that, this should be mentioned and discussed as a limitation.

      Comments on revisions:

      The authors have adequately responded to all comments.

    2. Reviewer #2 (Public review):

      Summary:

      The authors combine a clever use of historical clinical data on infection duration in immunologically naive individuals and queuing theory to infer the force of infection (FOI) from measured multiplicity of infection (MOI) in a sparsely sampled setting. They conduct extensive simulations using agent based modeling to recapitulate realistic population dynamics and successfully apply their method to recover FOI from measured MOI. They then go on to apply their method to real world data from Ghana before and after an indoor residual spraying campaign.

      Strengths:

      - The use of historical clinical data is very clever in this context<br /> - The simulations are very sophisticated with respect to trying to capture realistic population dynamics<br /> - The mathematical approach is simple and elegant, and thus easy to understand

      Weaknesses:

      - The assumptions of the approach are quite strong, and the authors have made clear that applicability is constrained to individuals with immune profiles that are similar to malaria naive patients with neurosyphilis. While the historical clinical data is a unique resource and likely directionally correct, it remains somewhat dubious to use the exact estimated values as inputs to other models without extensive sensitivity analysis.

    3. Reviewer #3 (Public review):

      Summary:

      It has been proposed that the FOI is a method of using parasite genetics to determine changes in transmission in areas with high asymptomatic infection. The manuscript attempts to use queuing theory to convert multiplicity of infection estimates (MOI) into estimates of the force of infection (FOI), which they define as the number of genetically distinct blood-stage strains. They look to validate the method by applying them to simulated results from a previously published agent based model. They then apply these queuing theory methods to previously published and analysed genetic data from Ghana. They then compare their results to previous estimates of FOI.

      Strengths:

      It would be great to be able to infer FOI from cross sectional surveys which are easier and cheaper than current FOI estimates which require longitudinal studies. This work proposes a method to convert MOI to FOI for cross sectional studies. They attempt to validate this process using a previously published agent based model which helps us understand the complexity of parasite population genetics.

      Weaknesses:

      (1) I fear that the work could be easily over-interpreted as no true validation was done as no field estimates of FOI (I think considered true validation) were measured. You have developed a method of estimating FOI from MOI which makes a number of biological and structural assumptions. I would not call being able to recreate model results that were generated using a model that makes its own (probably similar) defined set of biological and structural assumptions acts as a validation of what is going on in the field. The authors claim this at times (for example, Line 153 ) and I feel it would be appropriate to differentiate this in the discussion.

      (2) Another aspect of the paper is adding greater realism to the previous agent based model, by including assumptions on missing data and under sampling. This takes prominence in the figures and results section, but I would imagine is generally not as interesting to the less specialised reader. The apparent lack of impact of drug treatment on MOI is interesting and counterintuitive, though it is not really mentioned in the results or discussion sufficiently to allay my confusion. I would have been interested in understanding the relationship between MOI and FOI as generated by your queuing theory method and the model. It isn't clear to me why these more standard results are not presented, as I would imagine they are outputs of the model (though happy to stand corrected - it isn't entirely clear to me what the model is doing in this manuscript alone).

      (3) I would suggest that outside of malaria geneticists, the force of infection is considered to be the entomological inoculation rate, not the number of genetically distinct blood-stage strains. I appreciate that FOI has been used to explain the later before by others, though the authors could avoid confusion by stating this clearly throughout the manuscript. For example, the abstract says FOI is "the number of new infections acquired by an individual host over a given time interval" which suggests the former, please consider clarifying.

      (4) Line 319 says "Nevertheless, overall, our paired EIR (directly measured by the entomological team in Ghana (Tiedje et al., 2022)) and FOI values are reasonably consistent with the data points from previous studies, suggesting the robustness of our proposed methods". I would agree that the results are consistent, given that there is huge variation in Figure 4 despite the transformed scales, but I would not say this suggests a robustness of the method.

      (5) The text is a little difficult to follow at times, and sometimes requires multiple reads to understand. Greater precision is needed with the language in a few situations and some of the assumptions made in the modelling process are not referenced, making it unclear whether it is a true representation of the biology.

      Comments on revisions:

      I think the authors gave a robust but thorough response to our reviews and made some important changes to the manuscript which certainly clarify things for me.

    1. Reviewer #1 (Public review):

      Cellulose is the major component of the plant cell wall and as such is a major component of all plant biomass on the planet. It is made at the cell surface by a large membrane-bound complex known as the cellular synthase complex. It is the structure of the cellulose synthase complex that determines the structure of the cellulose microfibril, the unit of cellulose found in nature. Consequently, while understanding the molecular structure of individual catalytic subunits that synthesise individual beta 1-4 glucose chains is important, to really understand cellulose synthesis it is necessary to understand the structure of the entire complex.

      In higher plants cellulose is synthesised by a large membrane-bound complex composed of three different CESA proteins. During cellulose synthesis in the primary cell wall this is composed of members of groups CESA1, CESA3 and CESA6. While the authors have previously presented structural data on CESA8, required for cellulose synthesis in the secondary cell wall, here they provide structural and enzymatic analysis of CESA1, CESA3 and CESA6 from soybean.

      The authors have utilised their established protocol to purify trimers for all three classes of CESA proteins and obtain structural information using electron microscopy. The structures reveal some subtle, but interesting differences between the structures obtained in this study and that previously obtained for CESA8. In particular, they identify a change in the position of transmembrane helices 7 that in previous structures formed part of the transmembrane channel. In the structure of CESA1 TM7 is shifted laterally to a position more towards the periphery of the protomer where is stabilised by inter protomer interactions. This creates a large lipid exposed channel opening that is likely encountered by the growing cellulose chain. In the discussion the authors speculate this channel might facilitate lateral movement of cellulose chains in the membrane what would allow them to associate to form the microfibril. There is, however, no explanation for why this might be different for CESA proteins involved in primary and secondary cell wall CESA proteins.

      Interactions within the trimer as stabilised by the plant conserved regions (PCR), while in common with previous studies that class-specific regions (CSR) is not resolved, likely of it being highly disordered as has been suggested in previous studies. As the name suggests these regions are likely to be important for determining how different CESA proteins interact, but it remains to be seen how they achieve this. Similarly, the N-terminal domain (NTD) remains rather intriguing. In the CESA3 structure, the NTD forms a stalk that protrudes into the cytoplasm that was previously observed for CESA8, while it remains unresolved in CESA1 and CESA6. The authors suggest the inability to resolve this region is likely the result of the NTD being able to form multiple conformations. Loss of the NTD does not prevent the formation of trimers and CESA1 and CESA3 are still able to interact. Previous bioinformatic studies suggest that the CSR part of the NTD is also highly class-specific (Carrol et al. 2011 Frontiers in Plant Science 2, 5-5) suggesting it is also likely to participate in interactions between different CESA proteins. This analysis provides little new information on the structure of the NTD or how it functions as part of the cellulose synthase complex.

      The other important point regarding cellulose synthesis is how the different CESA trimers function during cellulose synthesis and complex assembly. The authors provide biochemical evidence that mixed complexes of two different CESA proteins are able to synergistically increase the rate of cellulose synthesis. This increase is not dramatic, around 2-fold as it is unclear what brings about this increase and whether it results from the ability to form larger complexes favouring greater rates of cellulose synthesis.

      It is clear however from electron microscopy that mixing of CESA proteins can lead to the formation of large aggregates not seen with single CESA proteins. The aggregates observed do not form rosette type shapes but appear to be much more random aggregates of different CESA trimers. The authors suggest that this is likely a result of the fact that the complexes are not constrained in two dimensions by the membrane, however if these are biologically relevant interactions that form aggregates is somewhat surprising that they do not form hexameric structures, particularly since that are essentially forming as a single layer.

      Overall the study provides some important data and raises a number of important questions.

    2. Reviewer #3 (Public review):

      Cellulose is a major component of the primary cell wall of growing cells and it is made by cellulose synthases (CESAs) organized into multi-subunit complexes in the plasma membrane. Previous results have resolved the structure of secondary cell wall CESAs, which are only active in a subset of cells. Here, the authors evaluate the structure of CESAs from soybean (Glycine max, Gm) via cryo-EM and compare these structures to secondary cell wall CESAs. First, they express a select member of the GmCESA1, GmCESA3, or GmCESA6 families in insect cells, purified these proteins as both monomers and homotrimers, and demonstrated their capacity to incorporate 3H-labelled glucose into cellulase-sensitive product in a pH and divalent cation (e.g., Mg2+) -dependant fashion (Figure 2). Although CESA1, CESA3, and a CESA6-like isoforms are essential for cellulose synthesis in Arabidopsis, in this study, monomers and homotrimers both showed catalytic activity, and there was more variation between individual isoforms than between their oligomerization states (i.e., CESA3 monomers and trimers showed similar activities, which were substantially different from CESA1 monomers or trimers).

      They next use cryo-EM to solve the structure of each homotrimer to ~3.0 to 3.3 A (Figure 3). They compare this with PttCESA8 and find important similarities, such as the unidentified density at a positively-charged region near Arg449, Lys452, and Arg453; and differences, such as the position and relatively low resolution (suggesting higher flexibility) of TM7, which presumably creates a large lateral lipid-exposed channel opening, rather than the transmembrane pore in PttCESA8. Like PttCESA8, an oligosaccharide in the translocation channel was co-resolved with the protein structure. Neither the N-terminal domains nor the CSRs (a plant-specific insert into the cytosolic loop between TM2 and TM3) are resolved well.

      Several previous models have proposed that the cellulose synthase complexes may be composed of multiple heterotrimers, but since the authors were able to isolate beta-glucan-synthesizing homotrimers, their results challenge this model. Using the purified trimers, the authors investigated how the CESA homotrimers might assemble into higher order complexes. They detected interactions between each pair of CESA homotrimers via pull down assays (Figure 4), although these same interactions were also detected among monomers (Supplemental Figure 4). Neither catalytic activity nor these inter-homotrimer interactions required the N-terminal domain (Figure 5). When populations of homotrimers were mixed, they formed larger aggregations in vitro (Figure 6) and displayed increased activity, compared to the predicted additive activity of each enzyme alone (Figure 7). Intriguingly, this synergistic behavior is observed even when one trimer is chemically inactivated before mixing (supplemental figure 7), suggesting that the synergistic effects are due to structural interactions.

      The main strength of this manuscript is its detailed characterization of the structure of multiple CESAs implicated in primary cell wall synthesis, which complements previous studies of secondary cell wall CESAs. They provide a comprehensive comparison of these new structures with previously resolved CESA structures and discuss several intriguing similarities and differences. The synergistic activity observed when different homotrimers are mixed is a particularly interesting result. These results provide fundamental in vitro support for a cellulose synthase complex comprised of a hexamer of CESA homotrimers.

      The main weakness of the manuscript is that the authors' evidence that these proteins make cellulose in vitro is limited to beta-glucanase-sensitive digestion of the product. Previous reports characterizing CESA structures have used multiple independent methods: sensitivity and resistance of the product to various enzymes, linkage analysis, and importantly, TEM of the product to ensure that it makes genuine cellulose microfibrils, rather than amorphous beta-glucan.

    1. Reviewer #1 (Public review):

      Summary:

      Using lineage tracing and single-cell RNA sequencing, Li et al. reported brain ECs can differentiate into pericytes after stroke. This finding is novel and important to the field.

      Strengths:

      Detailed characterization of each time point and genetic manipulation of genes for study role of ECs and E-pericyte.

      Weaknesses:

      Genetic evidence for lineage tracing of ECs and E-pericytes requires more convincing data that include staining, FACS, and scRNA-seq analysis.

      Comments on revisions:

      Authors have addressed some of my concerns and questions, and also plan to include more convincing data to support the conclusion. Some unpublished data should be included in the online supporting files.

    2. Reviewer #2 (Public review):

      Summary:

      In this manuscript, Li and colleagues study the fate of endothelial cells in a mouse model of ischemic stroke. Using genetic lineage tracing approaches, they find that endothelial cells give rise to non-endothelial cells, which they term "E-pericytes." They further show that depleting these cells exacerbates blood-brain barrier leakage and worsens functional recovery. The authors also provide evidence that endothelial-to-mesenchymal transition, myeloid cell-derived TGFβ1, and endothelial TGFβRII are involved in this process. These are potentially interesting findings, however, the experimental evidence that endothelial cells undergo transdifferentiation to non-endothelial cells is weak, as is the evidence that these cells are pericytes. Addressing this foundational weakness will facilitate interpretation of the other findings.

      In this revised manuscript, the authors corrected labeling errors and included negative controls for flow cytometry and immunohistochemistry data. They did not, however, substantively address the major weaknesses below related to rigorously demonstrating the cellular origin and identity of "E-pericytes."

      Strengths:

      (1) The authors address an important question about blood vessel function and plasticity in the context of stroke.

      (2) The authors use a variety of genetic approaches to understand cell fate in the context of stroke. Particularly commendable is the use of several complementary lineage tracing strategies, including an intersectional strategy requiring both endothelial Cre activity and subsequent mural cell NG2 promoter activity.

      (3) The authors address upstream cellular and molecular mechanisms, including roles for myeloid-derived TGFβ.

      Weaknesses:

      (1) The authors use Cdh5-CreERT2; Ai47 mice to permanently label endothelial cells and their progeny with eGFP. They then isolate eGFP+ cells from control and MCAO RP7D and RP34D brains, and use single cell RNA-seq to identify the resulting cell types. Theoretically, all eGFP+ cells should be endothelial cells or their progeny. This is a very powerful and well-conceived experiment. The authors use the presence of a pericyte cluster as evidence that endothelial to pericyte transdifferentiation occurs. However, pericytes are also present in the scRNA-seq data from sham mice, as are several other cell types such as fibroblasts and microglia. This suggests that pericytes and these other cell types might have been co-purified (e.g., as doublets) with eGFP+ endothelial cells during FACS and may not themselves be eGFP+. Pericyte-endothelial doublets are common in scRNA-seq given that these cell types are closely and tightly associated. Additionally, tight association (e.g., via peg-socket junctions) can cause fragments of endothelial cells to be retained on pericytes (and vice-versa) during dissociation. Finally, it is possible that after stroke or during the dissociation process, endothelial cells lyse and release eGFP that could be taken up by other cell types. All of these scenarios could lead to purification of cells that were not derived (transdifferentiated) from endothelial cells. Authors note that the proportion of pericytes increased in the stroke groups, but it does not appear this experiment was replicated and thus this conclusion is not supported by statistical analysis. The results of pseudotime and trajectory analyses rely on the foundation that the pericytes in this dataset are endothelial-derived, which, as discussed above, has not been rigorously demonstrated.

      (2) I have the same concern regarding inadvertent purification of cells that were not derived from endothelial cells in the context of the bulk RNA-seq experiment (Fig. S4), especially given the sample-to-sample variability in gene expression in the RP34D, eGFP+ non-ECs group (e.g., only 2/5 samples are enriched for mesenchymal transcription factor Tbx18, only 1/5 samples are enriched for mural cell TF Heyl). If the sorted eGFP+ non-ECs were pericytes, I would expect a strong and consistent pericyte-like gene expression profile.

      (3) Authors use immunohistochemistry to understand localization, morphology, and marker expression of eGFP+ cells in situ. The representative "E-pericytes" shown in Fig. 3A-D are not associated with blood vessels, and the authors' quantification also shows that the majority of such cells are not vessel-associated ("avascular"). By definition, pericytes are a component of blood vessels and are embedded within the vascular basement membrane. Thus, concluding that these cells are pericytes ("E-pericytes") may be erroneous.

      (4) CD13 flow cytometry and immunohistochemistry are used extensively to identify pericytes. In the context of several complementary lineage tracing strategies noted in Strength #2, CD13 immunohistochemistry is the only marker used to identify putative pericytes (Fig. S3J-M). In stroke, CD13 is not specific to pericytes; dendritic cells and other monocyte-derived cells express CD13 (Anpep) in mouse brain after stroke (PMID: 38177281, https://anratherlab.shinyapps.io/strokevis/).

      (5) Authors conclude that "EC-specific overexpression of the Tgfbr2 protein by a virus (Tgfbr2) decreases Evans blue leakage, promotes CBF recovery, alleviates neurological deficits and facilitates spontaneous behavioral recovery after stroke by increasing the number of E-pericytes." All data in Fig. 10, however, compare endothelial Tgfbr2 overexpression to a DsRed overexpression control. There is no group in which Tgfbr2 is overexpressed but "E-pericytes" are eliminated with DTA (this is done in Fig. 9B, but this experiment lacks the Tgfbr2 overexpression-only control). Thus, the observed functional outcomes cannot be ascribed to "E-pericytes"; it remains possible that endothelial Tgfbr2 overexpression affects EB leakage, CBF, and behavior through alternative mechanisms.

      In response to this comment, authors wrote: "in Figures 9A-B, we observed no significant difference in Evans blue leakage between the Tgfbr2 overexpression group and the Tgfbr2 overexpression + DTA group (P=0.8153), this suggests that the impact of Tgfbr2 overexpression on the blood-brain barrier (BBB) is primarily attributed from the E-pericytes generated by Tgfbr2 expression."

      I do not see data from a Tgfbr2 overexpression-only group in Fig. 9B. Further, I do not understand authors' logic: If the mechanism by which EC Tgfbr2 overexpression acts to reduce BBB leakage is by increasing the number of "E-pericytes," depleting "E-pericytes" with DTA in this context should increase BBB leakage.

      (6) Single-cell and bulk RNA-seq data are not available in a public repository (such as GEO). Depositing these data would facilitate their independent reevaluation and reuse.

      In response to this comment, authors indicated they submitted data to GEO, but did not provide an accession number.

    3. Reviewer #3 (Public review):

      Summary:

      The data and experiments presented in that study convincingly show that a subpopulation of endothelial cells undergo transformation into pericyte-like cells after stroke in mice. These so-called "E-pericytes" are protective and might present a new target for stroke recovery. The authors used a huge battery of different techniques and modified signaling pathways and cellular interactions using several genetic and pharmacological tools to show that TGFbeta and EndoMT are causes of this transformation.

      Strengths:

      The amount of different genetic and pharmacological approaches in combination with sophisticated techniques such as single-cell RNAseq is impressive and convincing. The results support their conclusions and the authors achieved their aims. The findings will strongly impact the field of cerebrovascular recovery after stroke and might open up new therapeutic targets.

      Weaknesses:

      In addition to improving the written and graphical presentation of the results, there is only one point I would like to see clarified: the inclusion of additional experiments, even if they have already been performed but are not applicable due to methodological difficulties regarding the role of Procr+ cells. Negative results also help the scientific community avoid unnecessary experiments and advance understanding.

    1. Reviewer #1 (Public review):

      Summary:

      The paper by Tolossa et al. presents classification studies that aim to predict the anatomical location of a neuron from the statistics of its in-vivo firing pattern. They study two types of statistics (ISI distribution, PSTH) and try to predict the location at different resolutions (region, subregion, cortical layer).

      Strengths:

      This paper provides a systematic quantification of the single-neuron firing vs location relationship.

      The quality of the classification setup seems high.

      The paper uncovers that, at the single neuron level, the firing pattern of a neuron carries some information on the neuron's anatomical location, although the predictive accuracy is not high enough to rely on this relationship in most cases.

      Weaknesses:

      As the authors mention in the Discussion, it is not clear whether the observed differences in firing is epiphenomenal. If the anatomical location information is useful to the neuron, to what extent can this be inferred from the vicinity of the synaptic site, based on the neurotransmitter and neuromodulator identities? Why would the neuron need to dynamically update its prediction of the anatomical location of its pre-synaptic partner based on activity when that location is static, and if that information is genetically encoded in synaptic proteins, etc (e.g., the type of the synaptic site)? Note that the neuron does not need to classify all possible locations to guess the location of its pre-synaptic partner because it may only receive input from a subset of locations. Ultimately, the inability to dissect whether the paper's findings point to a mechanism utilized by neurons or merely represent an epiphenomenon is the main weakness of the curious, though somewhat weak, observations described in this paper.

    2. Reviewer #2 (Public review):

      Summary:

      In this manuscript, Tolossa et al. analyze Inter-spike intervals from various freely available datasets from the Allen Institute and from a dataset from Steinmetz et al.. They show that they can modestly decode between gross brain regions (Visual vs. Hippocampus vs. Thalamus), and modestly separate sub areas within brain regions (DG vs. CA1 or various visual brain areas). The core result is that a multi-layer perceptron trained on the ISI distributions can modestly classify different brain areas and perhaps in a reasonably compelling way generalize across animals. The result is interesting but the exact problem formulation still feels a tad murky to me because I am worried the null is a strawman and I'm unsure if anyone has ever argued for this null hypothesis ("the impact of anatomy on a neuron's activity is either nonexistent or unremarkable"). Given the patterns of inputs to different brain areas and the existence of different developmental origin and different cell types within these areas, I am unclear why this would be a good null hypothesis. Nevertheless, the machine learning is reasonable, and the authors demonstrate that a nonlinear population based classifier can pull out reasonable information about the brain area and layer.

      Strengths:

      The paper is reasonably well written, and the definitions are quite well done. For example, the authors clearly explained transductive vs. inductive inference in their decoders. E.g., transductive learning allows the decoder to learn features from each animal, whereas inductive inference focuses on withheld animals and prioritizes the learning of generalizable features. The authors walk the reader through various analyses starting as simply as PCA, then finally showing a MLP trained on ISI distributions and PSTHs performs modestly well in decoding brain area. The key is ISI distributions work well in inductive settings for generalizing from one mouse to the other.

      Weaknesses:

      As articulated in my overall summary, I still found the null hypothesis a tad underwhelming. I am not sure this is really a valid null hypothesis ("the impact of anatomy on a neuron's activity is either nonexistent or unremarkable"), although in the statistical sense it is fine. The authors took on board some of the advice from the first review and clarified the paper but there are portions that are unnecessarily verbose (e.g., "Beyond fundamental scientific insight, our findings may be of benefit in various practical applications, such as the continued development of brain-machine interfaces and neuroprosthetics"). Also, given that ISIs cannot separate between visual areas, why is the statement that these are conserved. I still find it somewhat underwhelming that the thalamus, hippocampus , and visual cortex have different ISI distributions. Multiple researchers have reported similar things in cortex perhaps without the focus on decoding area from these ISI distributions.

      All in all, it is an interesting paper with the notion that ISI distributions can modestly predict brain area and layer. It could have some potential for a tool for neuropixels, although this needs to be developed further for this use case.

    1. Joint Public Review:

      This study presents novel insights into the formation and characterization of a penetration ring during host infection by Magnaporthe oryzae. Based on the solid genetic evidence and localization data, the authors demonstrate the structural presence of the penetration ring and the contribution of Ppe1 to fungal virulence. Nevertheless, the mechanisms through which the penetration ring influences host-pathogen interaction, including its potential function in effector translocation, remain only partially resolved. Further work using higher-resolution imaging and functional assays will help address this knowledge gap. Overall, the findings are valuable for advancing our understanding of plant-pathogen interactions, though important mechanistic questions remain open.

    1. Reviewer #1 (Public review):

      Summary:

      This paper proposes a neural mechanism underlying the perception of ambiguous images: neuromodulation changes the gain of neural circuits promoting a switch between two possible percepts. Converging evidence for this is provided by indirect measurements of neuromodulatory activity and large-scale brain dynamics which are linked by a neural network model. However, both the data analysis as well as the computational modeling are incomplete and would benefit from a more rigorous approach.

      This is a revised version of the manuscript which, in my view, is a considerable step forward compared to the original submission.

      In particular, the authors now model phasic gain changes in the RNN, based on the network's uncertainty. This is original and much closer to what is suggested by the phasic pupil responses. They also show that switching is actually a network effect because switching times depend on network configuration (Fig 2). This resolves my main comments 1 and 2 about the model.

      The mechanism, as I understand it, is different from what the authors described before in the RNN with tonic gain changes. As uncertainty increases, the network enters a regime in which the two excitatory populations start to oscillate. My intuition is that this oscillation arises from the feedback loop created by the new gain control mechanism. If my intuition is correct, I think it would be worth to explain this mechanism in the paper more explicitly.

      Comments on revisions:

      This is a second revision. I have no further comments. The authors have not answered the question that I had in the previous round (about the origin of oscillations in the RNN). I think this topic deserves to be explored in more detail but perhaps that is beyond the scope of the current paper.

    2. Reviewer #2 (Public review):

      This paper tests the hypothesis that perceptual switches during the presentation of ambiguous stimuli are accompanied by changes in neuromodulation that alter neural gain and trigger abrupt changes in brain activity. To test this hypothesis, the study combines pupillometry, artificial recurrent network (RNN) analysis and fMRI recording. In particular, the study uses methods of energy landscape analysis inspired by physics, which is particularly interesting.

      Strengths<br /> - The authors should be commended for combining different methods (pupillometry, RNNs, fMRI) to test their hypothesis. This combination provides a mechanistic insight into perceptual switches in the brain and artificial neural networks.<br /> - The study combines different viewpoints and fields of scientific literature, including neuroscience, psychology, physics, dynamical systems. In order to make this combination more accessible to the reader, the different aspects are presented in a pedagogical way to be accessible to all fields.<br /> - This combination of methods and viewpoints is rarely done, so it is very useful.<br /> - The authors introduce dynamic gain modulation in their recurrent neural network, which is novel. They devote a section of the paper to studying the dynamics, fixed points and convergence of this type of network.

      Weaknesses<br /> - The study may not be specific to perceptual switches. This is because the study relies on a paradigm in which participants report when they identify a switch in the item category. Therefore, it is unclear whether the effects reported in the paper are related to the perceptual switch itself, to attention, or to the detection of behaviourally relevant events. The authors are cautious and explicitly acknowledge this point in their study.<br /> - The demonstration of the causal role of gain modulation in perceptual switches is partial. This causality is clearly demonstrated in the simulation work with the RNN. However, it is not fully demonstrated in the pupil analysis and the fMRI analysis. One reason is that this work is correlative (which is already very informative).<br /> - Some effects may reflect the expectation of a perceptual switch rather than the perceptual switch itself. To mitigate this risk, the design of the fMRI task included catch trials, in which no switch occurs, to reduce the expectation of a switch. The pupil study, however, did not include such catch trials.<br /> - The paper uses RNN-based modelling to provide mechanistic insight into the role of gain modulation in perceptual switches. However, the RNN solves a task that differs from that performed by human participants, which may limit the explanatory value of the model. The RNN is provided with two inputs characterising the sensory evidence supporting the first and last image category in the sequence (e.g. plane and shark). In contrast, observers in the task don't know in advance the identity of the last image at the beginning of the sequence. The brain first receives sensory evidence about the image category (e.g. plane) with which the sequence begins, which is very easy to recognise, then it sees a sequence of morphed images and has to discover what the final image category will be. To discover the final image category, the brain considers several possibilities for the second images (it is a shark?, a frog?, a bird?, etc.), rather than comparing the likelihood of just two categories. This search process among many alternatives and the perceptual switch in the task is therefore different from the competition between only two inputs in the RNN.<br /> - Another aspect of the motivation for the RNN model remains unclear. The authors introduce dynamic gain modulation in the RNN, but it is not clear what the added value of dynamic gain modulation is. Both static (Fig. S1) and dynamic (Fig. 2F) gain modulation lead to the predicted effect: faster switching when the gain is larger.<br /> - The authors are to be commended for addressing their research questions with multiple tools and approaches. There are links between the different parts of the study. The RNN and the pupil are linked by the notion of gain modulation, the RNN and the fMRI analysis are linked by the study of the energy landscape, the fMRI study and the pupil study are indirectly linked by previous work for this group showing that the peak in LC fMRI activity precedes a flattening of the energy landscape. These links are very interesting but could have been stronger and more complete.

      Comments on revisions:

      I thank the authors for their responses.<br /> My review presents points that the authors themselves present as weaknesses or limitations. It also includes points that cannot be addressed in a revision (e.g. causality).<br /> Regarding the fact that the RNN only considers two categories, whereas subjects consider more categories (because they don't know the final image), I have toned down my remark (removing "markedly" different, removing the fact that the hypothesis space is vast given that participants have some priors). I also removed the qualifier "mechanistically" different, because it can be understood in different ways. The point remains that the proposed model has 2 inputs, the corresponding network in the brain has >2 inputs (because it considers more categories than the RNN), which is different, and which is the point of my remark. I think it may limit the value of the model, but I don't think it is not "sensible".

    1. Reviewer #1 (Public review):

      Summary:

      The manuscript reports that expression of the E. coli operon topAI/yjhQ/yjhP is controlled by the translation status of a small open reading frame, that authors have discovered and named toiL, located in the leader region upstream of the operon. Authors propose the following model for topAI activation: Under normal conditions, toiL is translated but topAI is not expressed because of Rho-dependent transcription termination within the topAI ORF and because its ribosome binding site and start codon are trapped in an mRNA hairpin. Ribosome stalling at various codons of the toiL ORF, prompted in this work by some ribosome-targeting antibiotics, triggers an mRNA conformational switch which allows translation of topAI and, in addition, activation of the operon's transcription because presence of translating ribosomes at the topAI ORF blocks Rho from terminating transcription. The model is appealing and several of the experimental data mainly support it. However, it remains unanswered what is the true trigger of the translation arrest at toiL and what is the physiological role of the induced expression of the topAI/yjhQ/yjhP operon.

    2. Reviewer #2 (Public review):

      Summary:

      Baniulyte and Wade describe how translation of an 8-codon uORF denoted toiL upstream of the topAI-yjhQP operon is responsive to different ribosome-targeting antibiotics, consequently controlling translation of the TopAI toxin as well as Rho-dependent termination with the gene.

      Strengths:

      The authors used multiple different approaches such as a genetic screen to identify factors such as 23S rRNA mutations that affect topA1 expression and ribosome profiling to examine the consequences of various antibiotics on toiL-mediated regulation.

      Weaknesses: Future experiments will be needed to better understand the physiological role of the toiL-mediated regulation and elucidate the mechanism of specific antibiotic sensing.

      The results are clearly described, and the revisions have helped to improve the presentation of the data.

    3. Reviewer #3 (Public review):

      The authors provide convincing data to support an elegant model in which ribosome stalling by ToiL promotes downstream topAI translation and prevents premature Rho-dependent transcription termination. However, the physiological consequences of activating topAI-yjhQP expression upon exposure to various ribosome-targeting antibiotics remain unresolved. The authors have satisfactorily addressed all major concerns raised by the reviewers, particularly regarding the SHAPE-seq data. Overall, this study underscores the diversity of regulatory ribosome-stalling peptides in nature, highlighting ToiL's uniqueness in sensing multiple antibiotics and offering significant insights into bacterial gene regulation coordinated by transcription and translation.

      [Editors' note: The earlier public reviews are included. No additional reviews were requested.]

    1. Reviewer #2 (Public review):

      Summary:

      In this manuscript, the authors reveal that GIF/MT-3 regulates the zinc homeostasis depending on the cellular redox status. The manuscript technically sounds, and their data concretely suggest that the recombinant MTs, not only GIF/MT-3 but also canonical MTs such as MT-1 and MT-2, contain sulfane sulfur atoms for the Zn-binding. The scenario proposed by the authors seems to be reasonable to explain the Zn homeostasis by the cellular redox balance.

      Strengths:

      The data presented in the manuscript solidly reveal that recombinant GIF/MT-3 contains sulfane sulfur.

      Weaknesses:

      It remains unclear whether native MTs, in particular induced MTs in vivo contain sulfane sulfur or not.

      Comments on revisions:

      Although the authors have revealed the sulfane sulfur content in native MT-3, my question, namely, whether canonical MT-1 and MT-2 contained sulfane sulfur after the induction has been left.<br /> The authors argue that the biological significance of sulfane sulfur in MTs lies in its ability to contribute to metal binding affinity, provide a sensing mechanism against oxidative stress, and aid in the regulation of the protein. Due to their biological roles, induced MT-1 and MT-2 could contain sulfane sulfur in their molecules. Thus, I expect the authors to evaluate or explain the sulfane sulfur content in induced MT-1 and MT-2.

    2. Reviewer #3 (Public review):

      Summary:

      The authors were trying to show that a novel neuronal metallothionein of poorly defined function, GIF/MT3, is actually heavily persulfidated in both the Zn-bound and apo (metal-free) forms of the molecule as purified from a heterologous (bacterial) or native host. Evidence in support of this conclusion is strong, with both spectroscopic and mass spectrometry evidence strongly consistent with this general conclusion. The authors would appear to have achieved their aims.

      Strengths:

      The analytical data in support of the author's primary conclusions are strong. The authors also provide some modeling evidence that supports the contention that MT3 (and other MTs) can readily accommodate a sulfane sulfur on each of the 20 cysteines in the Zn-bound structure, with little perturbation of the overall structure. This is not the case with Cys trisulfides, which suggests that the persulfide-metallated state is clearly positioned at lower energy relative to the immediately adjacent thiolate- or trisulfidated metal coordination complexes.

      Weaknesses:

      The biological significance of the findings is not entirely clear. On the one hand, the analytical data are solid (albeit using a protein derived from a bacterial over-expression experiment), and yes, it's true that sulfane S can protect Cys from overoxidation, but everything shown in the summary figure (Fig. 9D) can be done with Zn release from a thiol by ROS, and subsequent reduction by the Trx/TR system. In addition, it's long been known that Zn itself can protect Cys from oxidation. I view this as a minor shortcoming that will motivate follow-up studies.

      Impact:

      The impact will be high since the finding is potentially disruptive to the MT field for sure. The sulfane sulfur counting experiment (the HPE-IAM electrophile trapping experiment) may well be widely adopted by the field. Those in the metals field always knew that this was a possibility, and it will interesting to see the extent to which metal binding thiolates broadly incorporate sulfane sulfur into their first coordination shells.

      Comments on revisions:

      The revised manuscript is only slightly changed from the original, with the inclusion of a supplementary figure (Fig. S2) and minor changes in the text. The authors did not choose to carry out the quantitative Zn binding experiment (which I really wanted to see), but given the complexities of the experiment, I'll let it go.

    1. Reviewer #1 (Public review):

      Summary:

      Laura Morano and colleagues have performed a screen to identify compounds that interfere with the formation of TopBP1 condensates. TopBP1 plays a crucial role in the DNA damage response, and specifically the activation of ATR. They found that the GSK-3b inhibitor AZD2858 reduced the formation of TopBP1 condensates and activation of ATR and its downstream target CHK1 in colorectal cancer cell lines treated with the clinically relevant irinotecan active metabolite SN-38. This inhibition of TopBP1 condensates by AZD2858 was independent from its effect on GSK-3b enzymatic activity. Mechanistically, they show that AZD2858 thus can interfere with intra-S-phase checkpoint signaling, resulting in enhanced cytostatic and cytotoxic effects of SN-38 (or SN-38+Fluoracil aka FOLFIRI) in vitro in colorectal carcinoma cell lines.

      Major comments from the first round of peer review:

      Overall the work is rigorous and the main conclusions are convincing. However, they only show the effects of their combination treatments on colorectal cancer cell lines. I'm worried that blocking the formation of TopB1 condensates will also be detrimental in non-transformed cells. Furthermore it is somewhat disappointing that it remains unclear how AZD2858 blocks self-assembly of TopBP1 condensates, although I understand that unraveling this would be complex and somewhat out-of-reach for now. Here are some specific points for improvement:

      1) The authors conclude that "These data supports [sic] the feasibility of targeting condensates formed in response to DNA damage to improve chemotherapy-based cancer treatments". To support this conclusion the authors need to show that proliferating non-transformed cells (e.g. primary cell cultures or organoids) can tolerate the combination of AZD2858 + SN-38 (or FOLFIRI) better than colorectal cancer cells.

      2) Page 19 "This suggests that the combination... arrests the cell cycle before mitosis in a DNA-PKsc-dependent manner." I find the remark that this arrest would be DNA-PKcs-dependent too speculative. I suppose that the authors base this claim on reference 55 but if they want to support this claim they need to prove this by adding DNA-PKcs inhibitors to their treated cells.

      3) When discussing Figure S5B the authors claim that SN-38 + AZD2858 progressively increases the fractions of BrdU positive cells, but this is not supported by statistical analysis. The fractions are still very small, so I would like to see statistics on these data. Alternatively, the authors could take out this conclusion.

      Comments on revised version:

      I have reviewed the revised manuscript and read the rebuttal. The authors have carefully addressed my concerns. There is however one point that needs further work:

      This follows up on my major point #1 in my initial review. I had I asked the authors to demonstrate that FOLFIRI + AZD are less toxic to untransformed colorectal cells than colorectal cancer cell lines.

      It is good to see that the authors took my advice and show effects of the drug treatments on the untransformed colorectal cell line CCD841. It seems to be less sensitive to AZD and FOLFIRI in the figure in the rebuttal. What surprises me is that I cannot find these new figures anywhere in the revised manuscript. Also, the data seem preliminary, because I do not see any standard errors in the graphs, and I cannot find a description of the time of drug incubation. I ask the authors to make sure that the CCD841 data are reproducible, and make sure they incorporate the data in the revised manuscript.

    2. Reviewer #2 (Public review):

      Summary:

      In 2021 (PMID: 33503405) and 2024 (PMID: 38578830) Constantinou and colleagues published two elegant papers in which they demonstrated that the Topbp1 checkpoint adaptor protein could assemble into mesoscale phase-separated condensates that were essential to amplify activation of the PIKK, ATR, and its downstream effector kinase, Chk1, during DNA damage signalling. A key tool that made these studies possible was the use of a chimeric Topbp1 protein bearing a cryptochrome domain, Cry2, which triggered condensation of the chimeric Topbp1 protein, and thus activation of ATR and Chk1, in response to irradiation with blue light without the myriad complications associated with actually exposing cells to DNA damage.

      In this current report Morano and co-workers utilise the same optogenetic Topbp1 system to investigate a different question, namely whether Topbp1 phase-condensation can be inhibited pharmacologically to manipulate downstream ATR-Chk1 signalling. This is of interest, as the therapeutic potential of the ATR-Chk1 pathway is an area of active investigation, albeit generally using more conventional kinase inhibitor approaches.

      The starting point is a high throughput screen of 4730 existing or candidate small molecule anti-cancer drugs for compounds capable of inhibiting the condensation of the Topbp1-Cry2-mCherry reporter molecule in vivo. A surprisingly large number of putative hits (>300) were recorded, from which 131 of the most potent were selected for secondary screening using activation of Chk1 in response to DNA damage induced by SN-38, a topoisomerase inhibitor, as a surrogate marker for Topbp1 condensation. From this the 10 most potent compounds were tested for interactions with a clinically used combination of SN-38 and 5-FU (FOLFIRI) in terms of cytotoxicity in HCT116 cells. The compound that synergised most potently with FOLFIRI, the GSK3-beta inhibitor drug AZD2858, was selected for all subsequent experiments.

      AZD2858 is shown to suppress the formation of Topbp1 (endogenous) condensates in cells exposed to SN-38, and to inhibit activation of Chk1 without interfering with activation of ATM or other endpoints of damage signalling such as formation of gamma-H2AX or activation of Chk2 (generally considered to be downstream of ATM). AZD2858 therefore seems to selectively inhibit the Topbp1-ATR-Chk1 pathway without interfering with parallel branches of the DNA damage signalling system, consistent with Topbp1 condensation being the primary target. Importantly, neither siRNA depletion of GSK3-beta, or other GSK3-beta inhibitors were able to recapitulate this effect, suggesting it was a specific non-canonical effect of AZD2858 and not a consequence of GSK3-beta inhibition per se.

      To understand the basis for synergism between AZD2858 and SN-38 in terms of cell killing, the effect of AZD2858 on the replication checkpoint was assessed. This is a response, mediated via ATR-Chk1, that modulates replication origin firing and fork progression in S-phase cell under conditions of DNA damage or when replication is impeded. SN-38 treatment of HCT116 cells markedly suppresses DNA replication, however this was partially reversed by co-treatment with AZD2858, consistent with the failure to activate ATR-Chk1 conferring a defect in replication checkpoint function.

      Figures 4 and 5 demonstrate that AZD2858 can markedly enhance the cytotoxic and cytostatic effects of SN-38 and FOLFIRI through a combination of increased apoptosis and growth arrest according to dosage and treatment conditions. Figure 6 extends this analysis to cells cultured as spheroids, sometimes considered to better represent tumor responses compared to single cell cultures.

      Significance:

      Liquid phase separation of protein complexes is increasingly recognised as a fundamental mechanism in signal transduction and other cellular processes. One recent and important example was that of Topbp1, whose condensation in response to DNA damage is required for efficient activation of the ATR-Chk1 pathway. The current study asks a related but distinct question; can protein condensation be targeted by drugs to manipulate signalling pathways which in the main rely on protein kinase cascades?

      Here, the authors identify an inhibitor of GSK3-beta as a novel inhibitor of DNA damage-induced Topbp1 condensation and thus of ATR-Chk1 signalling.

      This work will be of interest to researchers in the fields of DNA damage signalling, biophysics of protein condensation, and cancer chemotherapy.

      Comments on latest version:

      Morano et al. have revised their manuscript in response to the points raised by reviewer #3 as follows.

      1) Fig. 2E: Correcting the previously erroneous labelling of this Fig. makes it match the textual description.

      2) Figs 3A and B: The revised textual description of the flow cytometry BrdU incorporation is now precise.

      3) Fig. 3E: Removing the suspect WB images is a pragmatic decision that does not significantly affect the overall conclusions of the paper.

      4) Fig. 3D: Despite its puzzling appearance this data is now described accurately in the text as "DSBs remained elevated after the combined treatment" rather than "increased after the combined treatment. A more convincing increase in the presumed damaged DNA band is evident in Fig. 4D when AZD2858 is combined with a much lower concentration of SN38 (1.5nM) which could mean that the concentration used in Fig. 3D (300nM) induced maximal damage that could not be further enhanced.

    3. Reviewer #3 (Public review):

      Summary:

      The authors have extended their previous research to develop TOPBP1 as a potential drug target for colorectal cancer by inhibiting its condensation. Utilizing an optogenetic approach, they identified the small molecule AZD2858, which inhibits TOPBP1 condensation and works synergistically with first-line chemotherapy to suppress colorectal cancer cell growth. The authors investigated the mechanism and discovered that disrupting TOPBP1 assembly inhibits the ATR/Chk1 signaling pathway, leading to increased DNA damage and apoptosis, even in drug-resistant colorectal cancer cell lines.

      Comments on latest version:

      The authors have addressed most of the concerns that I raised in the first round of revision and I have no further questions. I appreciate the authors's efforts in carrying out an preliminary in vivo work, although as the authors pointed out the compound seems to be not effective in vivo. Future work is desired to address this to clarify the significance of the work.

    1. Reviewer #1 (Public review):

      Summary:

      In this study, the authors present a thorough mechanistic study of the J-domain protein Apj1 in Saccharomyces cerevisiae, establishing it as a key repressor of Hsf1 during the attenuation phase of the heat shock response (HSR). The authors integrate genetic, transcriptomic (ribosome profiling), biochemical (ChIP, Western), and imaging data to dissect how Apj1, Ydj1, and Sis1 modulate Hsf1 activity under stress and non-stress conditions. The work proposes a model where Apj1 specifically promotes displacement of Hsf1 from DNA-bound heat shock elements, linking nuclear PQC to transcriptional control.

      Strengths:

      Overall, the work is highly novel - this is the first detailed functional dissection of Apj1 in Hsf1 attenuation. It fills an important gap in our understanding of how Hsf1 activity is fine-tuned after stress induction, with implications for broader eukaryotic systems. I really appreciate the use of innovative techniques, including ribosome profiling and time-resolved localization of proteins (and tagged loci) to probe the Hsf1 mechanism. The overall proposed mechanism is compelling and clear - the discussion proposes a phased control model for Hsf1 by distinct JDPs, with Apj1 acting post-activation, while Sis1 and Ydj1 suppress basal activity.

      The manuscript is well-written and will be exciting for the proteostasis field and beyond.

    2. Reviewer #2 (Public review):

      Despite over 50 years of investigation, our understanding of how the ubiquitous heat shock response, governed by the transcription factor HSF1, was regulated was minimal. In recent years, a coordinated yet simple negative feedback circuit has been elucidated in high detail that centers on the chaperone Hsp70 as a direct-binding inhibitor of HSF1 transcriptional activation. However, roles for the obligatory Hsp70 J-domain partner co-chaperones are currently poorly understood. The present study applies several orthogonal techniques to the question and uncovers an unexpected role for the nuclear JDP Apj1 in attenuation of the heat shock response (HSR) via removal of Hsf1 from HSEs in heat shock gene promoter regions. Interestingly, Apj1 appears to play no role in initiating repression of Hsf1, as null mutants do not exhibit constitutive derepression of the HSR. This role is likely filled by the general nucleo/cytoplasmic JDP Ydj1, as previously reported. These results enhance understanding of HSR regulation and underscore the pivotal role that chaperones play in controlling pro-survival gene expression.

      Overall, the work is exceptionally well done and controlled, and the results are properly and appropriately interpreted. Several of the approaches, while powerful, are somewhat indirect (i.e., following gene expression via ribosomal profiling) but ultimately provide a compelling answer to the main question being asked. However, at the end of the day, there is really only one major finding here: Apj1 regulates Hsf1 attenuation via Hsp70. That finding is strongly supported by the experimental data but lacks the one piece of mechanistic evidence found in other recent papers - differential binding of Ssa1/2 to Hsf1 at either the N- or C-terminal binding sites.

    3. Reviewer #3 (Public review):

      Summary:

      The heat shock response (HSR) is an inducible transcriptional program that has provided paradigmatic insight into how stress cues feed information into the control of gene expression. The recent elucidation that the chaperone Hsp70 controls the DNA binding activity of the central HSR transcription factor Hsf1 by direct binding has spurred the question of how such a general chaperone obtains specificity. This study has addressed the next logical question: how J-domain proteins execute this task in budding yeast, the leading cell model for studying the HSR. While an involvement and in part overlapping function of general class A and B J-domain proteins, Ydj1 and Sis1 are indicated by the genetic analysis, a highly specific role for the class A Apj1 in displacing Hsf1 from the promoters is found, unveiling specificity in the system.

      Strengths

      The central strong point of the paper is the identification of class A J-domain protein Apj1 as a specific regulator of the attenuation of the HSR by removing Hsf1 from HSEs at the promoters. The genetic evidence and the ChIP data strongly support this claim. This identification of a specific role for a lowly expressed nuclear J-domain protein changes how the wiring of the HSR should be viewed. It also raises important questions regarding the model of chaperone titration, the concept that a chaperone with limited availability is involved in a tug of war involving competing interactions with misfolded protein substrates and regulatory interactions with Hsf1. Perhaps Apj1, with its low levels and interactions with misfolded and aggregated proteins in the nucleus, is the titrated Hsp70 (co)chaperone that determines the extent of the HSR? This would mean that Apj1 is at the nexus of the chaperone titration mechanism. Although Apj1 is not a highly conserved J domain protein among eukaryotes the strength of the study is that is provides a conceptual framework for what may be required for chaperone titration in other eukaryotes: One or more nuclear J-domain proteins with low nuclear levels that has an affinity for Hsf1 and that can become limiting due to interactions with misfolded Hsp70 proteins. The provides a pathway for how these may be identified using, for example, ChIP-seq.

      Weaknesses

      A built-in challenge when studying the mechanism of the HSR is the general role of the Hsp70 chaperone system and its J domain proteins. Indeed, a weakness of the study is that it is unclear which of the phenotypic effects have to do with directly recruiting Hsp70 to Hsf1 dependent on a J domain protein and what instead is an indirect effect of protein misfolding caused by the mutation. This interpretation problem is clearly and appropriately dealt with in the manuscript text and in experiments, but is of such fundamental nature that it cannot easily be fully ruled out. One way forward is a reconstituted biochemical system that monitors how Hsf1 DNA binding is affected by the Hsp70 system, misfolded proteins, and the various J domain proteins. Yet this approach is clearly beyond the scope of this study.

    1. 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. With 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 lack of background information. In comparison to the Methods, which are incredibly detailed, the Results in the main section read 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.

      The authors have done a good job of responding to the reviewer's comments, and the paper is now much improved.

    2. Reviewer #2 (Public review):

      I would like to thank the authors for the revision and the input they invested in this study.

      With the revised text of the study, my earlier criticism holds, and your arguments about the counterfactual approach are irrelevant to that. The recent rise of the counterfactual approach might likely mirror the fact that there are too many scientists behind their computers, and few go into the field to collect in situ data. Studies like the one presented here are a good intellectual exercise but the real impact is questionable. All your main conclusions are inferred from published studies on 7! bird species. In addition, spatial sampling in those seven species was not ideal in relation to your target questions. Thus, no matter how fancy your findings look, the basic fact remains that your input data were for 7 bird species only! Your conclusion, „our study provides a novel understanding of how QTP shapes migration patterns of birds, " is simply overstretching.

      The way you respond to my criticism on L 81-93 is something different than what you admit in the rebuttal letter. The text of the ms is silent about the drawbacks and instead highlights your perspective. I understand you; you are trying to sell the story in a nice wrapper. In the rebuttal you state: „we assume species' responses to environments are conservative and their evolution should not discount our findings." But I do not see that clearly stated in the main text.

      In your rebuttal, you respond to my criticism of "No matter how good the data eBird provides is, you do not know population-specific connections between wintering and breeding sites" when you responded: ... "we can track the movement of species every week, and capture the breeding and wintering areas for specific populations" I am having a feeling that you either play with words with me or do not understand that from eBird data nobody will be ever able to estimate population-specific teleconnections between breeding and wintering areas. It is simply impossible as you do not track individuals. eBird gives you a global picture per species but not for particular populations. You cannot resolve this critical drawback of your study. I am sorry that you invested so much energy into this study, but I see it as a very limited contribution to understanding the role of a major barrier in shaping migration.

      My modest suggestion for you is: go into the field. Ideally use bird radars along the plateau to document whether the birds shift the directions when facing the barrier.

    1. Reviewer #1 (Public review):

      Summary:

      This study considers learning with brain-computer interfaces (BCIs) in nonhuman primates, and in particular, the high speed and flexibility with which subjects learn to control these BCIs.

      The authors raise the hypothesis that such learning is based on controlling a small number of input or control variables, rather than directly adapting neural connectivity within the network of neurons that drive the BCI. Adapting a small number of input variables would circumvent the issue of credit assignment in high dimensions and allow for quick learning, potentially using cognitive strategies ("re-aiming"). Based on a computational model, the authors show that such a strategy is viable in a number of experimental settings and reproduces previous experimental observations:

      (1) Differences in learning with decoders either within or outside of the neural manifold (the space spanned by the dominant modes of neural activity).

      (2) A novel, theory-based prediction on biases in BCI learning due to the positivity of neural firing rates, which is then confirmed in data from previous experiments.

      (3) An example of "illusory credit assignment": Changes in neurons' tuning curves depending on whether these neurons are affected by changes in the BCI decoder, even though learning only happens on the level of low-dimensional control variables.

      (4) A reproduction of results from operant conditioning of individual neurons, in particular, the observation that it is difficult to change the firing rates of neurons strongly correlated before learning in different directions (up vs down).

      Taken together, these observations yield strong evidence for the plausibility that subjects use such a learning strategy, at least during short-term learning.

      Strengths:

      Text and figures are clearly structured and allow readers to understand the main concepts well. The study presents a very clear and simple model that explains a number of seemingly disparate or even contradictory observations (neuron-specific credit assignment vs. low-dimensional, cognitive control). The predicted and tested bias due to positivity of firing rates provides a neat example of how such a theory can help understand experimental results. The idea that subjects first use a small number of command variables (those sufficient in the calibration task) and later, during learning, add more variables provides a nice illustration of the idea that learning takes place on multiple time scales, potentially with different mechanisms at play. On a more detailed level, the study is a nice example of closely matching the theory to the experiment, in particular regarding the modeling of BCI perturbations.

      Weaknesses:

      Overall, I find only two minor weaknesses. First, the insights of this study are, first and foremost, of feed-forward nature, and a feed-forward network would have been enough (and the more parsimonious model) to illustrate the results. While using a recurrent neural network (RNN) shows that the results are, in general, compatible with recurrent dynamics, the specific limitations imposed by RNNs (e.g., dynamical stability, low-dimensional internal dynamics) are not the focus of this study. Indeed, the additional RNN models in the supplementary material show that under more constrained conditions for the RNN (low-dimensional dynamics), using the input control alone runs into difficulties.

      Second, explaining the quantitative differences between the model and data for shifts in tuning curves seems to take the model a bit too literally. The model serves greatly for qualitative observations. I assume, however, that many of the unconstrained aspects of the model would yield quantitatively different results.

    2. Reviewer #2 (Public review):

      Summary :

      The paper proposes a model to explain the learning that occurs in brain-computer interface (BCI) tasks when animals need to adapt to novel BCI decoders. The model consists of a network formulation of the "re-aiming" learning strategy, which assumes that BCI learning does not modify the underlying neural circuitry, but instead occurs through a reorganization of existing neural activity patterns.

      The authors formalize this in a recurrent neural network (RNN) model, driven by upstream inputs that live in a low-dimensional space.

      They show that modelling BCI learning as reorganization of these upstream inputs can explain several experimental findings, such as the difference in the ability of animals to adapt to within vs outside-manifold perturbations, biases in the decoded behaviour after within-manifold perturbations, or qualitative changes in the neural responses observed during credit assignment rotation perturbations or operant conditioning of individual neurons.

      Overall, while the idea of re-aiming as a learning strategy has previously been proposed in the literature, the authors show how it can be formalized in a network model, which allows for more direct comparisons to experimental data.

      Strengths:

      The paper is very well written. The presentation of the model is clear, and the use of vanilla RNN dynamics driven by upstream inputs that are constant in time is consistent with the broader RNN modeling literature.

      The main value of the paper lies in the fact that it proposes a network implementation for a learning strategy that had been proposed previously. The network model has a simple form, but the optimization problem is performed in the space of inputs, which requires the authors to solve a nonlinear optimization problem in that space.

      While some of the results (eg the fact that the model can adapt to within but not outside-manifold perturbations) are to be expected based on the model assumptions, having a network model allows to make more direct and quantitative comparisons to experiments, to investigate analytically how much the dimension of the output is constrained by the input, and to make predictions that can be tested in data.

      The authors perform such comparisons across three different experiments. The results are clearly presented, and the authors show that they hold for various RNN connectivities.

      Weaknesses :

      The authors mention alternative models (eg, based on synaptic plasticity in the RNN and/or input weights) that can explain the same experimental data that they do, they do not provide any direct comparisons to those models.

      Thus, the main argument that the authors have in favor of their model is the fact that it is more plausible because it relies on performing the optimization in a low-dimensional space. It would be nice to see more quantitative arguments for why the re-aiming strategy may be more plausible than synaptic plasticity (either by showing that it explains data better, or explaining why it may be more optimal in the context of fast learning).

      In particular, the authors model the adaptation to outside-manifold perturbations (OMPs) through a "generalized re-aiming strategy". This assumes the existence of additional command variables, which are not used in the original decoding task, but can then be exploited to adapt to these OMPs. While this model is meant to capture the fact that optimization is occurring in a low-dimensional subspace, the fact that animals take longer to adapt to OMPs suggests that WMPs and OMPs may rely on different learning mechanisms, and that synaptic plasticity may actually be a better model of adaptation to OMPs. It would be important to discuss how exactly generalized re-aiming would differ from allowing plasticity in the input weights, or in all weights in the network. Do those models make different predictions, and could they be differentiated in future experiments?

    1. Reviewer #1 (Public review):

      In this manuscript, Kerlin et al. introduce a novel and conceptually important framework for analyzing allelic transcriptional heterogeneity using single-molecule microscopy. The authors aim to distinguish regulatory interactions occurring in cis-between genes on the same allele-from those in trans, between alleles, thereby extending classical models of transcriptional noise into the spatial and allelic domain. They apply this approach to three genes within the FOS locus in MCF7 cells, under both basal and estrogen-induced conditions, and report distinct patterns of transcriptional coordination that depend on gene proximity and chromatin insulation.

      A major strength of this work lies in its innovative methodology and the clarity with which the analytical framework is described. The authors effectively build on foundational ideas in gene expression variability and adapt them to resolve a previously underexplored question - how nearby genes on the same allele may influence each other's transcriptional activity. The imaging data are of high quality, the mathematical derivation is comprehensive, and the overall presentation is strong. The study makes a compelling argument for the value of allele-resolved analysis, highlighting that failure to account for allelic and chromatin context may lead to inaccurate or incomplete interpretations of regulatory mechanisms.

      That said, the scope of the data is currently limited to a single locus in one cell type. As such, some of the general conclusions, particularly those in the abstract and discussion, may be overstated. The evidence supports the findings within the FOS locus, but it remains unclear whether the observed patterns apply broadly across the genome. The utility and generality of the method would be significantly strengthened by additional validation.

      One specific area where the analysis could be improved is through the inclusion of randomized control comparisons. For example, the results presented in Figure 2D and analyzed in Figure 3 could be compared against randomized datasets to establish a baseline of what would be expected by chance. This would help determine the significance of the observed correlations and strengthen confidence in the model's specificity.

      Additionally, the framework should be tested on simulated datasets with a known ground truth to evaluate the robustness of its assumptions and the reliability of its outputs. Testing the approach against existing allele-specific single-cell datasets from other studies would also help assess its generalizability. While the authors suggest the framework could be extended to transcriptomics and spatial omics, these possibilities are not explored in the current study, and future work in this direction should be clearly marked as such.

      In summary, this manuscript presents a methodologically rigorous and biologically significant advance in the study of gene regulation. The approach fills an important gap by enabling allele-resolved, locus-specific analysis of transcriptional coordination, with implications for both basic science and clinical applications. The conclusions are well supported within the studied context, but further validation - particularly through randomized data comparison, simulations, and broader application - would be valuable in assessing the broader utility of the framework.

    2. Reviewer #2 (Public review):

      Summary:

      I am not familiar with mathematical modeling of gene expression, so I will evaluate this manuscript solely from a biological point of view.

      Kerlin et al. combined single-molecule RNA FISH and mathematical modeling approaches to quantitatively characterize changes in the transcriptional dynamics of three neighboring genes at the FOS locus in response to estradiol (E2) stimulation. They showed that the neighboring JDP2 and BATF genes, located on the same side of the TAD boundary, exhibit highly coordinated bursting dynamics. While FOS and JDP2/BATF are strongly insulated (~7:1 intra-to-inter-domain contact ratio) by the TAD boundary, correlated bursting dynamics were still observed between these gene pairs, suggesting that enhancers can bypass strong insulation sites. The authors proposed that burst co-occurrence arises from the activity of ERα-bound enhancers at the locus. They also proposed that the burst size correlation between two neighboring genes located on the same side of the TAD boundary results from local spreading of histone marks.

      Strengths:

      The direct visualization of coordinated transcriptional bursting across a strong insulation site is novel. This finding was carefully analyzed using the mathematical framework developed by the authors.

      Weaknesses:

      Several models were proposed based on single-molecule RNA FISH analysis of the FOS locus, but the generality of these findings remains uncertain. The proposed models were not directly tested through follow-up experiments, leaving the authors' conclusions largely speculative.

    3. Reviewer #3 (Public review):

      Summary

      Kerlin et.al combined single-molecule RNA FISH with oligonucleotide-based DNA FISH to directly examine the transcriptional activities of three adjacent genes at individual alleles in MCF7 cells. Importantly, they provided quantitative methods to resolve allele-specific (cis) and cell-to-cell (trans) variation and quantified the contribution of burst co-occurrence and burst size, which may help to more accurately analyze transcription coregulation. They found that transcriptional variability is largely gene-autonomous, and by disentangling burst co-occurrence and burst size after E2 induction, they proposed two distinct mechanisms of local gene regulation.

      Strengths:

      (1) Innovative Research Methods: Successfully integrates single-molecule RNA FISH with oligonucleotide-based DNA FISH to directly image the transcriptional activities of three adjacent genes at individual alleles. This enables the observation of transcriptional dynamics more precisely and provides a powerful tool for studying gene regulation.

      (2) Novel Data Analysis Approaches: Develops two new analysis methods to dissect the sources of gene activity (co)variation. One approach separates allele-extrinsic, allele-intrinsic, and gene-autonomous components, and the other quantifies the contributions of burst co-occurrence and burst size correlations. These methods help to more accurately analyze transcriptional correlations between genes and reveal potential regulatory mechanisms.

      Weaknesses:

      Biological Insights: The findings challenge the traditional view of contact insulation sites as strict regulators of gene coregulation and suggest two distinct coregulatory mechanisms influenced by local chromosome folding. However, expression activity of multiple genes is differentially correlated at the population-level or cell-level versus single-allele-level. More in-depth analysis is needed for further biological insights.

    1. Reviewer #1 (Public review):

      Summary:

      The authors hypothesized that the lung immune landscape in mice with diabetes and TB comorbidity is different from that of mice with DM-only or TB-only, or healthy mice. Systematically, the authors established the 'basal' lung immune landscape in DM or healthy animals before infection with Mycobacterium tuberculosis, allowing them to tease out changes in cell types with TB infection and focused subsequent studies on DM-TB and TB comparisons. The authors chose day 21 post-Mtb infection as the point of analysis since this is the peak of immune responses to Mtb infection as per an earlier study (Das et al. 2021). As expected, the authors found differences in the cellular composition of the DM mice with or without TB or TB-only mice, including reduced IFNg response, elevated Th17 cells, increased IL-16 signaling, and altered naive CD4+ and naive CD8+ T cell numbers. The authors have used a series of techniques for methodological and analytical approaches to identify potential pathways that can be targeted for therapies against DM-TB. However, the authors have failed to propose a model that could explain their observations at the time point tested, lowering enthusiasm for the conclusions of the study.

      Strengths:

      The strength of the study is the use of a validated model of mouse DM-TB and a meticulous approach to establish and define a 'baseline" lung cellular landscape in DM and healthy mice before Mtb infection. The use of an up-to-date analytical pipeline by the authors is commendable.

      The literature review is exhaustive, and the authors have put considerable effort into borrowing from other conditions where the identified genes of pathways have been implicated.

      Weaknesses:

      The key limitations of the study include:

      (1) The authors have failed to link a specific cell type, that is, Th17 cell activation, to or with IL-16 signaling as the drivers regulating conditions that contribute significantly to the dysregulated immune responses in DM-TB. For context, naive CD4+ and naive CD8+ T cells cannot be considered "specific cell types" because they have no determined cell fate; they could mature to any other cell type - cytotoxic T cells, Th1, or even Th17 or Tc17 cells.

      (2) Since day 21 post-Mtb infection is an earlier timepoint, the authors should have provided data on cellular composition in the experiments in Figure 7. From the work of Kornfeld and colleagues, there is delayed cell recruitment in DM-TB, but it is likely that later on, due to persistent inflammation (from chronic hyperglycemia), DM-TB mice have more or equal cell numbers in the lung. Anecdotally, the authors found differences in CFU at a later time point but not at 21 days post-infection. This fits with human studies where there is a higher prevalence of cavities in DM-TB compared to TB-only patients. The authors missed the opportunity to clarify this important point by excluding cellular data from the 56-day post-infection experiments.

      (3) The power of the study would be improved by the direct comparisons of three groups: DM vs DM-TB vs TB to recapitulate the human populations and allow the authors to address the question of 'why does DM worsen TB outcome?'. The current analysis of DM-TB vs TB is not fit for this because the TB is on a healthy background, while DM-TB is a result of two conditions that independently perturb immune homeostasis.

    2. Reviewer #2 (Public review):

      Summary:

      While immune cell distribution in tuberculosis (TB) is well documented, research on its disruption in diabetes-tuberculosis (DM-TB) comorbidity remains limited. In this study, Chaudhary et al. explore immune cell perturbations in DM-TB using single-cell RNA sequencing (scRNA-seq), providing key insights into the impaired host immune response. By elucidating the molecular mechanisms underlying immune dysfunction in DM-TB, this study addresses an important knowledge gap. The study demonstrates that diabetes impairs lung immune cell infiltration and contributes to a dampened immune response against Mycobacterium tuberculosis. Reduced Th1 and M1 macrophage populations indicate a compromised ability to mount an effective pro-inflammatory response, which is essential for TB control. The observed increase in IL-16 signaling and reduction in TNF and IFN-II responses suggest a shift toward a more immunosuppressive or dysregulated inflammatory state. The interplay between chronic inflammation, hyperglycemia, and dyslipidemia in diabetes further exacerbates immune dysfunction, reinforcing the idea that metabolic disorders significantly impact TB pathogenesis.

      Strengths:

      This well-designed study employs robust methodology, well-executed experiments, and a well-written manuscript. The use of scRNA-seq is a notable strength, offering high-resolution analysis of immune cell heterogeneity in the lung environment. Additionally, the study corroborates its findings in a long-term infection model, demonstrating that chronic M. tuberculosis (H37Rv) infection in diabetic mice leads to increased bacterial burden and worsened tissue pathology.

      Weaknesses:

      (1) The study focuses on CD3⁺ and CD11c⁺ cells but does not extensively examine other key immune players that may contribute to DM-TB pathogenesis. Given that diabetes affects multiple immune compartments, a broader immune profiling approach would provide a more comprehensive understanding.

      (2) While the study identifies increased IL-16 signaling and reduced TNF/IFN-II responses, the precise molecular mechanisms driving these changes remain unclear. Further investigation into metabolic-immune crosstalk (e.g., how hyperglycemia affects immune cell differentiation and cytokine secretion) would strengthen the mechanistic depth of the findings.

      (3) The study suggests targeting IL-16 and Th17 cells as potential therapeutic strategies; however, no experimental validation (e.g., testing IL-16 inhibitors in DM-TB models) is provided. Validating these interventions would enhance their translational relevance.

      (4) Incorporating clinical samples (e.g., PBMCs from DM-TB patients) could help bridge the gap between murine and human studies, offering more translational insights into disease mechanisms.

      Overall, this study provides valuable findings, but addressing these concerns would further strengthen its impact on understanding DM-TB immunopathogenesis.

    1. Reviewer #1 (Public review):

      Summary:

      The paper is well written and investigates the cross-species insemination of fish eggs with mouse sperm. I have a few major and minor comments.

      Strengths:

      The experiments are well executed and could provide valuable insights into the complex mechanisms of fertilization in both species. I found the information presented to be very interesting,

      Weaknesses:

      The rationale of some of the experiments is not well defined.

      Major Comments:

      (1) Figure 5<br /> I do not understand the rationale for performing experiments using CatSper-null sperm and CD9-null oocytes. It is well established that CatSper-null sperm are unable to penetrate the zona pellucida (ZP), so the relevance of this approach is unclear.

      (2) Micropyle penetration and sperm motility<br /> CatSper-null sperm are reportedly unable to cross the micropyle, but this could be due to their reduced motility rather than a lack of hyperactivation per se. Were these experiments conducted using capacitated or non-capacitated spermatozoa? What was the observed motility of CatSper-null sperm during these assays? Clarifying these conditions is essential to avoid drawing incorrect conclusions from the results.

      (3) Rheotaxis and micropyle navigation<br /> Previous studies have shown that CatSper-null sperm fail to undergo rheotaxis. Could this defect be related to their inability to locate and penetrate the micropyle? Exploring a potential shared mechanism could be informative.

      (4) Lines 61-74<br /> This paragraph omits important information regarding acrosomal exocytosis, which occurs prior to sperm-egg fusion. Including this detail would strengthen the discussion.

    2. Reviewer #2 (Public review):

      Summary:

      Garibova et al. investigated the conservation of sperm recognition and interaction with the egg envelope in two groups of distantly related animals: mammals (mouse) and fish (zebrafish). Previous work and key physiological differences between these two animal groups strongly suggest that mouse sperm would be incapable of interaction with the zebrafish egg envelope (chorion) and its constituent proteins, though homologous to the mammalian zona pellucida (ZP). Indeed, the authors showed that mouse sperm do not bind recombinant zebrafish ZP proteins nor the intact chorion. Surprisingly, however, mouse sperm are able to locate and bind to the zebrafish micropyle, a specialized canal within the chorion that serves as the egg's entry point for sperm. This study suggests that sperm attraction to the egg might be highly conserved from fish to mammals and depends on the presence of a still unknown glycosylated protein within the micropyle. The authors further demonstrate that mouse sperm are able to enter the micropyle and accumulate within the intrachorionic space, potentially through a CatSper-dependent mechanism.

      Strengths:

      The authors convincingly demonstrate that mouse sperm do not bind zebrafish ZP proteins or the chorion. Furthermore, they make the interesting observation that mouse sperm are able to locate and enter the zebrafish micropyle in an MP-dependent manner, which is quite unexpected given the large evolutionary distance between these species, the many physiological differences between mouse and zebrafish gametes, and the largely different modes of both fertilization and reproduction in these species. This may indicate that the sperm chemoattractant in the egg is conserved between mammals and fish; however, whether zebrafish sperm are attracted to mouse eggs was not tested.

      Weaknesses:

      The key weakness of this study lies in the rationale behind the overall investigation. In mammals, the zona pellucida (ZP) has been implicated in binding sperm in a taxon-specific manner, such that human sperm are incapable of binding the mouse ZP. Indeed, work by the corresponding author showed that this specificity is mediated by the N-terminal region of the ZP protein ZP2 (Avella et al., 2014). The N-termini of human and mouse ZP2 share 48% identity, which is higher than the overall identity between mouse and zebrafish ZP2, with the latter ortholog entirely lacking the N-terminal domain that is essential for sperm binding to the ZP. Given this known specificity for mouse vs. human sperm-ZP binding, it does not follow that mouse sperm would bind ZP proteins from not only a species that is much more distantly related, but also one that is not even a mammal, the zebrafish. Furthermore, the fish chorion does not play a role in sperm binding at all, while the mammalian ZP can bind sperm at any location. On the contrary, the zebrafish chorion prevents polyspermy by limiting sperm entry to the single micropyle.

      In addition, though able to provide some information regarding the broad conservation of sperm-egg interaction mechanisms, the biological relevance of these findings is difficult to describe. Fish and mammals are not only two very distinct and distantly related animal groups, but also employ opposite modes of fertilization and reproduction (external vs. internal, oviparous vs viviparous). Fish gametes interact in a very different environment compared to mammals and lack many typically mammalian features of fertilization (e.g., sperm capacitation, presence of an acrosome, interaction with the female reproductive tract), making it difficult to make any physiologically relevant claims from this study. While this study may indicate conserved mechanisms of sperm attraction to the egg, the identity of the molecular players involved is not investigated. With this knowledge, the reader is forced to question the motivation behind much of the study.

      During fertilization in fish, the sperm enters the micropyle and subsequently, the egg, as it is simultaneously activated by exposure to water. During egg activation, the chorion lifts as it separates from the egg and fills with water. This mechanism prevents supernumerary sperm from entering the egg after the successfully fertilizing sperm has bound and fused. In this study, the authors show that mouse sperm enter the micropyle and accumulate in the intrachorionic space. Whether any sperm successfully entered the egg is not addressed, and the status of egg activation is not reported. In Supplementary Videos 3-4, the egg shown has been activated for some time, as evident by the separation of yolk and cytoplasm, yet the chorion is only partially expanded (likely due to mouse IVF conditions). How multiple sperm were able to enter the micropyle but presumably not the egg is not addressed, yet this suggests that the zebrafish mechanism of blocking polyspermy (fertilization by multiple sperm) is not effective for mouse sperm or is rendered ineffective due to mouse IVF conditions. The authors do not discuss these observations in the context of either species' physiological process of fertilization, highlighting the lack of biological context in interpreting the results.

      The authors further show that the zebrafish micropyle does not trigger the acrosome reaction in mouse sperm. Whether the acrosome reacts is not correlated with a sperm's ability to cross the micropyle opening, as both acrosome-intact and acrosome-reacted sperm were observed within the intrachorionic space. While the acrosome reaction is a key event during mammalian fertilization and is required for sperm to fertilize the egg, zebrafish sperm do not contain an acrosome. Thus, these results are particularly difficult to interpret biologically, bringing into question whether this observation has biological relevance or is a byproduct of egg activation/chorion lifting that indirectly draws sperm into the chorion.

      The final experiments regarding CatSper1's role in mediating mouse sperm entry into the micropyle/chorion are not convincing. As no molecular interactions are described or perturbed, the reader cannot be sure whether the sperm's failure to enter is due to signaling via CatSper1 or whether the overall failure to undergo hyperactivation limits sperm motility such that the mutant sperm can no longer find and enter the zebrafish micropyle. Indeed, in Figure 5E, no CatSper1 mutant sperm are visible near any part of the egg, suggesting that overall motility is impaired, and this is not a phenotype specific to interactions with the micropyle.

    1. Reviewer #1 (Public review):

      Summary:

      This study investigates how chronic stress may contribute to LC dysfunction in AD by examining the mechanisms underlying NA accumulation and α2A-AR internalization. Using electrophysiological recordings and molecular analyses, the authors propose that stress-induced receptor internalization impairs autoinhibition, leading to excessive NA accumulation and increased MAO-A activity. The findings have potential implications for understanding the progression of AD-related neurodegeneration and targeting noradrenergic dysfunction as a therapeutic strategy.

      Strengths:

      (1) The study integrates electrophysiology and molecular approaches to explore the mechanistic effects of chronic stress on LC neurons.

      (2) The evidence supporting NA accumulation and α2A-AR internalization as contributing factors to LC dysfunction is novel and relevant to AD pathology.

      (3) The electrophysiological findings, particularly the loss of spike-frequency adaptation and reduction in GIRK currents, provide functional insights into stress-induced changes in LC activity.

      Weaknesses:

      (1) The manuscript's logical flow is challenging and hard to follow, and key arguments could be more clearly structured, particularly in transitions between mechanistic components.

      (2) The causality between stress-induced α2A-AR internalization and the enhanced MAO-A remains unclear. Direct experimental evidence is needed to determine whether α2A-AR internalization itself or Ca2+ drives MAO-A activation, and how they activate MAO-A should be considered.

      (3) The connection between α2A-AR internalization and increased cytosolic NA levels lacks direct quantification, which is necessary to validate the proposed mechanism.

      (4) The chronic stress model needs further validation, including measurements of stress-induced physiological changes (e.g., corticosterone levels) to rule out systemic effects that may influence LC activity. Additional behavioral assays for spatial memory impairment should also be included, as a single behavioral test is insufficient to confirm memory dysfunction.

      (5) Beyond b-arrestin binding, the role of alternative internalization pathways (e.g., phosphorylation, ubiquitination) in α2A-AR desensitization should be considered, as current evidence is insufficient to establish a purely Ca²⁺-dependent mechanism.

      (6) NA leakage for free NA accumulation is also influenced by NAT or VMAT2. Please discuss the potential role of VMAT2 in NA accumulation within the LC in AD.

      (7) Since the LC is a small brain region, proper staining is required to differentiate it from surrounding areas. Please provide a detailed explanation of the methodology used to define LC regions and how LC neurons were selected among different cell types in brain slices for whole-cell recordings.

      Impact:

      This study provides valuable insights into the impact of chronic stress on LC function and its relevance to AD pathogenesis. The proposed mechanism linking NA dysregulation and receptor internalization may have implications for developing therapeutic strategies targeting the noradrenergic system in neurodegenerative diseases. However, additional validation is needed to strengthen the mechanistic claims before the findings can be fully integrated into the field.

    2. Reviewer #2 (Public review):

      Summary:

      This manuscript investigates the mechanism by which chronic stress induces locus coeruleus (LC) neuron degeneration. The authors demonstrate that chronic stress leads to internalization of α2A-adrenergic receptors (α2A-ARs) on LC-neurons, causing increased cytosolic noradrenaline (NA) accumulation and subsequent production of the neurotoxic metabolite DOPEGAL via monoamine oxidase A (MAO-A). The study suggests a mechanistic link between stress-induced α2A-AR internalization, disrupted autoinhibition, elevated NA metabolism, asparagine endopeptidase (AEP) activation, and Tau pathology relevant to Alzheimer's disease (AD). The conclusions of this paper are mostly well supported by data, but some aspects of image acquisition need to be extended.

      Strengths:

      This study clearly demonstrates the effects of chronic stimulation on the excitability of LC neurons using electrophysiological techniques. It also elucidates the role of α2-adrenergic receptor (α2-AR) internalization and the associated upstream and downstream signaling pathways of GIRK1 using a range of pharmacological agents, highlighting the innovative nature of the work.

      Additionally, the study identifies the involvement of the MAO-A-DOPEGAL-AEP pathway in this process. The topic is timely, the proposed mechanistic pathway is compelling, and the findings have translational relevance, particularly regarding therapeutic strategies targeting α2A-AR internalization in neurodegenerative diseases.

      Weaknesses:

      (1) The manuscript reports that chronic stress for 5 days increases MAO-A levels in LC neurons, leading to the production of DOPEGAL, activation of AEP, and subsequent tau cleavage into the tau N368 fragment, ultimately contributing to neuronal damage. However, the authors used wild-type C57BL/6 mice, and previous literature has indicated that AEP-mediated tau cleavage in wild-type mice is minimal and generally insufficient to cause significant behavioral alterations. Please clarify and discuss this apparent discrepancy.

      (2) It is recommended that the authors include additional experiments to examine the effects of different durations and intensities of stress on MAO-A expression and AEP activity. This would strengthen the understanding of stress-induced biochemical changes and their thresholds.

      (3) Please clarify the rationale for the inconsistent stress durations used across Figures 3, 4, and 5. In some cases, a 3-day stress protocol is used, while in others, a 5-day protocol is applied. This discrepancy should be addressed to ensure clarity and experimental consistency.

      (4) The abbreviation "vMAT2" is incorrectly formatted. It should be "VMAT2," and the full name (vesicular monoamine transporter 2) should be provided at first mention.

    3. Reviewer #3 (Public review):

      Summary:

      The authors present a technically impressive data set showing that repeated excitation or restraint stress internalises somato dendritic α2A adrenergic autoreceptors (α2A ARs) in locus coeruleus (LC) neurons. Loss of these receptors weakens GIRK-dependent autoinhibition, raises neuronal excitability, and is accompanied by higher MAO-A, DOPEGAL, AEP, and tau N368 levels. The work combines rigorous whole-cell electrophysiology with barbadin-based trafficking assays, qPCR, Western blotting, and immunohistochemistry. The final schematic is appealing and could, in principle, explain early LC hyperactivity followed by degeneration in ageing and Alzheimer's disease.

      Strengths:

      (1) Multi-level approach - The study integrates electrophysiology, pharmacology, mRNA quantification, and protein-level analysis.

      (2) The use of barbadin to block β-arrestin/AP-2-dependent internalisation is both technically precise and mechanistically informative.

      (3) Well-executed electrophysiology.

      (4) Translation relevance - converges to a model that can be discussed by peers (scientists can only discuss models - not data!).

      Weaknesses:

      Nevertheless, the manuscript currently reads as a sequence of discrete experiments rather than a single causal chain. Below, I outline the key points that should be addressed to make the model convincing.

    1. Reviewer #1 (Public review):

      Summary:

      The manuscript by Kostanjevec et al. investigates the mechanism behind spiral pattern formation in the cornea. The authors demonstrate that the spiral motion pattern on the mammalian corneal surface emerges from the interaction between the limbus position, cell division, extrusion, and collective cell migration. Using LacZ mosaic murine corneas, they reveal a tightening spiral flow pattern and show that their cell-based, in silico model accurately reproduces these patterns without global guidance cues. Additionally, they present a continuum model that extends the XYZ hypothesis to describe cell flux on the cornea, offering a quantitative explanation for tissue-scale processes on curved surfaces.

      Strengths:

      The manuscript is well-written, with a systematic approach that clearly explains experimental setups, model construction, assumptions, parameter selection, and predictions. The discussion also provides insightful perspectives on the broader implications of the results for both physics and biology.

      Weaknesses:

      The central premise of the manuscript, that the spiral patterning of epithelial corneal cells occurs without guidance cues, is not fully supported. The authors overlook the potential role of axons in guiding epithelial cells, despite clear evidence of spiral axon patterns in their own Fig. 1b. Previous literature indicates that axon patterning precedes epithelial cell patterning, suggesting that epithelial migration might be influenced by pre-existing neural structures (e.g., Leiper et al. 2002, IOVS 2013). The authors need to address this point, possibly by exploring whether axonal patterns serve as a template for epithelial cell migration, or by providing experimental evidence to rule out axon-based guidance.

      While the model is well-constructed, it currently falls short of its stated goal of elucidating the mechanisms of spiral formation. Key questions remain unanswered:<br /> Is the curvature of the cornea necessary for spiral formation, or would a simpler disk geometry suffice?<br /> What role do boundary conditions play?<br /> How well do the model's predictions quantitatively match experimental data?<br /> The current comparisons in Fig. 4c-f lack quantitative agreement, and this discrepancy should be discussed with possible explanations.

      The authors emphasize polar alignment as a key feature of the spiral pattern based on simulation results. However, they do not provide experimental evidence for this polar alignment. The manuscript includes discussions of polar and nematic symmetries that, without supporting data, feel somewhat distracting. If direct experimental evidence for polar alignment is not available, the authors could instead quantify nematic alignment as the spiral forms. This would also allow them to explore potential crosstalk between nematic cell orientation and the polar alignment of self-propulsion, especially considering recent studies showing alternative mechanisms for vortex formation in similar systems.

    2. Reviewer #2 (Public review):

      In K. Kostanjevec et.al, the authors study a possible mechanism for the formation of spiral patterns in the cornea. First the authors analyze an inferred velocity field, which is deduced from images of fixed corneas, and then determine the position-dependent spiral angle of this velocity fields. Next, the authors analysed two possible markers of cell polarity: the direction of the centrosome-nuclei and the axis of mitosis. Then the authors introduce a stochastic agent-based model of self-propelled particles with over-damped dynamics and with aligning interactions to the orientation of the nearest neighbors and to the particle's velocity. The authors claim to be able to reproduce the equal-time autocorrelation function and the velocity Fourier spectrum. Then the authors introduce the geometry of the cornea by constraining the dynamics on a spherical cap and show that their model can reproduce a typical trajectory in experiments. Finally, the authors produce a phase diagram of the states at a fixed time point as a function of the spherical cap radius and the strength of the coupling aligning constant. Finally, the authors propose an interpretation of the cell fluxes based on the equation of mass conservation.

    1. Reviewer #1 (Public review):

      Summary:

      This manuscript from Azeroglu et al. presents the application of END-Seq to examine the sequence composition of chromosome termini, i.e., telomeres. END-seq is a powerful genome sequencing strategy developed in Andre Nussesweig's lab to examine the sequences at DNA break sites. Here, END-Seq is applied to explore the nucleotide sequences at telomeres and to ascertain (i) whether the terminal end sequence is conserved in cells that activate the ALT telomere elongation mechanism and (ii) whether the processes responsible for telomere end sequence regulation are conserved. With these aims clearly articulated, the authors convincingly show the power of this technique to examine telomere end-processing.

      Strengths:

      (1) The authors effectively demonstrate the application of END-seq for these purposes. They verify prior data that 5'terminal sequences of telomeres in HeLa and RPE cells end in a canonical ATC sequence motif. They verify that the same sequence is present at the 5' ends of telomeres by performing END-seq across a panel of ALT cancer cells. As in non-ALT cells, the established role of POT1, a ssDNA telomere binding protein, in coordinating the mechanism that maintains the canonical ATC motif is likewise verified. However, by performing END-Seq in mouse cells lacking POT1 isoforms, POT1a and POT1b, the authors uncover that POT1b is dispensable for this process. This reveals a novel, important insight relating to the evolution of POT1 as a telomere regulatory factor.

      (2) The authors then demonstrate the utility of S1-END-seq, a variation of END-Seq, to explore the purported abundance of single-stranded DNA at telomeres within telomeres of ALT cancer cells. Here, they demonstrate that ssDNA abundance is an intrinsic aspect of ALT telomeres and is dependent on the activity of BLM, a crucial mediator of ALT.

      Overall, the authors have effectively shown that END-seq can be applied to examine processes maintaining telomeres in normal and cancerous cells across multiple species. Using END-Seq, the authors confirm prior cell biological and sequencing data and the role of POT1 and BLM in regulating telomere termini sequences and ssDNA abundance. The study is nice and well-written, with the experimental rationale and outcomes clearly explained.

      Weaknesses:

      This reviewer finds little to argue with in this study. It is timely and highly valuable for the telomere field. One minor question would be whether the authors could expand more on the application of END-Seq to examine the processive steps of the ALT mechanism? Can they speculate if the ssDNA detected in ALT cells might be an intermediate generated during BIR (i.e., is the ssDNA displaced strand during BIR) or a lesion? Furthermore, have the authors assessed whether ssDNA lesions are due to the loss of ATRX or DAXX, either of which can be mutated in the ALT setting?

    2. Reviewer #2 (Public review):

      This is a short yet very clear manuscript demonstrating that two methods (END-seq and S1-END-seq), previously developed in the Nussenzweig laboratory to study DSBs in the genome, can also be applied to the 5' ends of mammalian telomeres and the accumulation of telomeric single-stranded DNA.

      The authors first validate the applicability of END-seq using different approaches and confirm that mammalian telomeres preferentially end with an ATC 5' end through a mechanism that requires intact POT1 (POT1a in mice). They then extend their analysis to cells that maintain telomeres through the ALT mechanism and demonstrate that, in these cells as well, telomeres frequently end in an ATC 5' sequence via a POT1-dependent mechanism. Using S1-END-seq, the authors further show that ALT telomeres contain single-stranded DNA and estimate that each telomere in ALT cells harbors at least five regions of ssDNA.

      I find this work very interesting and incisive. It clearly demonstrates that END-seq can be applied with unprecedented depth and precision to the study of telomeric features such as the 5' end and ssDNA. The data are very clear and thoroughly interpreted, and the manuscript is well written. The results are carefully analyzed and effectively presented. Overall, I find this manuscript worthy of publication, as the optimized END-seq methods described here will likely be widely utilized in the telomere field.

      I only have a few minor suggestions:

      How can we be sure that all telomeres are equally represented? The authors seem to assume that END-seq captures all chromosome ends equally, but can we be certain of this? While I do not see an obvious way to resolve this experimentally, I recommend discussing this potential bias more extensively in the manuscript.

      I believe Figures 1 and 2 should be merged.

      Scale bars should be added to all microscopy figures.

    3. Reviewer #3 (Public review):

      Summary:

      A subset of cancer cells attain replicative immortality by activating the ALT mechanism of telomere maintenance, which is currently the subject of intense research due to its potential for novel targeted therapies. Key questions remain in the field, such as whether ALT telomeres adhere to the same end-protection rules as telomeres in telomerase-expressing cells, or if ALT telomeres possess unique properties that could be targeted with new, less toxic cancer therapies. Both questions, along with the approaches developed by the authors to address them, are highly relevant.

      Strengths:

      Since chromosome ends resemble one-ended DSBs, the authors hypothesized that the previously described END-SEQ protocol could be used to accurately sequence the 5' end of telomeres on the C-rich strand. As expected, most reads corresponded to the C-rich strand and, confirming a previous observation by de Lange's group, most chromosomes end with the ATC-5' sequence, a feature that was found to be dependent on POT1 and to be conserved in both human ALT cells and mouse cells. Through a complementary method, S1-END-SEQ, the authors further explored ssDNA regions at telomeres, providing new insights into the characteristics of ALT telomeres. The study is original, the experiments were well-controlled and excellently executed.

      Weaknesses:

      Overall, the discussion section is lacking depth and should be expanded and a few additional experiments should be performed to clarify the results.

      (1) The finding that the abundance of variant telomeric repeats (VTRs) within the final 30 nucleotides of the telomeric 5' ends is similar in both telomerase-expressing and ALT cells is intriguing, but the authors do not address this result. Could the authors provide more insight into this observation and suggest potential explanations? As the frequency of VTRs does not seem to be upregulated in POT1-depleted cells, what then drives the appearance of VTRs on the C-strand at the very end of telomeres? Is CST-Pola complex responsible?

      (2) The authors also note that, in ALT cells, the frequency of VTRs in the first 30 nucleotides of the S1-END-SEQ reads is higher compared to END-SEQ, but this finding is not discussed either. Do the authors think that the presence of ssDNA regions is associated with the VTRs? Along this line, what is the frequency of VTRs in the END-SEQ analysis of TRF1-FokI-expressing ALT cells? Is it also increased? Has TRF1-FokI been applied to telomerase-expressing cells to compare VTR frequencies at internal sites between ALT and telomerase-expressing cells?

      Finally, in these experiments (S1-END-SEQ or END-SEQ in TRF1-Fok1), is the frequency of VTRs the same on both the C- and the G-rich strands? It is possible that the sequences are not fully complementary in regions where G4 structures form.

      (3) Based on the ratio of C-rich to G-rich reads in the S1-END-SEQ experiment, the authors estimate that ALT cells contain at least 3-5 ssDNA regions per chromosome end. While the calculation is understandable, this number could be discussed further to consider the possibility that the observed ratios (of roughly 0.5) might result from the presence of extrachromosomal DNA species, such as C-circles. The observed increase in the ratio of C-rich to G-rich reads in BLM-depleted cells supports this hypothesis, as BLM depletion suppresses C-circle formation in U2OS cells. To test this, the authors should examine the impact of POLD3 depletion on the C-rich/G-rich read ratio. Alternatively, they could separate high-molecular-weight (HMW) DNA from low-molecular-weight DNA in ALT cells and repeat the S1-END-SEQ in the HMW fraction.

      (4) What is the authors' perspective on the presence of ssDNA at ALT telomeres? Do they attribute this to replication stress? It would be helpful for the authors to repeat the S1-END-SEQ in telomerase-expressing cells with very long telomeres, such as HeLa1.3 cells, to determine if ssDNA is a specific feature of ALT cells or a result of replication stress. The increased abundance of G4 structures at telomeres in HeLa1.3 cells (as shown in J. Wong's lab) may indicate that replication stress is a factor. Similar to Wong's work, it would be valuable to compare the C-rich/G-rich read ratios in HeLa1.3 cells to those in ALT cells with similar telomeric DNA content.

      Minor Points:

      (1) The Y-axes of Figure 4 should be relabeled to account for the G-strand reads. Additionally, statistical analyses are absent in Figure 4 and Figure S3.

      (2) A careful proofreading of the manuscript is necessary.

    1. Reviewer #1 (Public review):

      Summary:

      The study investigates the role of asymptomatic pertussis carriage in transmission between mothers and their infants, in particular. The authors used a longitudinal cohort study that involved 1,315 mother-infant dyads in Lusaka, Zambia, and they utilized qPCR-based detection of IS481 to track Bordetella pertussis transmission over time. Insights from the study suggest that minimally symptomatic or asymptomatic mothers may act as a reservoir for B. pertussis transmission in the infants, thus challenging the traditional surveillance methods that focus on symptomatic cases. Additionally, the study also identified a subgroup of persistently colonized individuals where mothers were majorly asymptomatic despite sustained bacterial presence.

      The authors aimed to improve comprehension of pertussis transmission dynamics in high-burden low-resource settings, and they advocated for enhanced molecular surveillance strategies to capture full pertussis infection, including those that might have gone undetected.

      Strengths:

      The strengths are the use of innovative study design, especially the longitudinal approach and routine sampling, rather than symptom-driven testing that minimizes bias in the study. The methodology was also rigorous and transparent by evaluating the IS481 signal strength to classify pertussis detection and conducting retesting to assess qPCR reliability. There were also important epidemiological insights, and the findings challenge the traditional wisdom by suggesting that pertussis transmission may frequently occur outside of symptomatic cases. The findings also showed its relevance to global health and policy by arguing for the incorporation of molecular tools like qPCR for surveillance of pertussis in low-resource settings.

      Weaknesses:

      These include reliability on qPCR-based detection without additional validation measures like confirmatory culture or serology. There are also potential alternate explanations for transmission patterns observed in the study such as shared environmental exposure or household transmission. Additionally, there is limited generalizability as the study was done in a single urban site in Zambia. There is also a lack of functional immune data.

    2. Reviewer #2 (Public review):

      Summary:

      In this paper, the authors describe the results of a longitudinal study of pertussis infection in mother/infant dyads in Lusaka, Zambia. Unlike many past studies, the authors assessed the infection status of individuals independently of whether they were symptomatic for a respiratory infection. As a result, this work represents one of the first studies specifically designed to assess asymptomatic transmission of pertussis. Using qPCR, the authors find strong evidence for the role of asymptomatic transmission from mothers to infants and also evidence for long-term bacterial carriage. This work represents an important contribution to our understanding of the global burden of pertussis. Also, it highlights the still under-appreciated role of asymptomatic transmission across many infectious diseases (including vaccine-preventable ones).

      Strengths:

      Unlike many past studies, the authors assessed the infection status of individuals independently of whether they were symptomatic for a respiratory infection. As a result, this work represents one of the first studies specifically designed to assess asymptomatic transmission of pertussis. Using qPCR, the authors find strong evidence for the role of asymptomatic transmission from mothers to infants and also evidence for long-term bacterial carriage.

      Weaknesses:

      While I am quite enthusiastic about the work, I am concerned that a number of likely relevant confounders were not discussed and that the broader implications of their findings were not well grounded in the existing literature. For example, I could not find information on the vaccination status of the mothers in the study. Given the conclusions about asymptomatic transmission and the durability of immunity, it is important to know the vaccination status of the mothers. Moreover, did the authors have other metadata on the mother/infant dyads, e.g., household size, vaccination status of household members, etc.? Given the potential implications of more widespread asymptomatic transmission associated with pertussis infection, I believe the authors should better couch their results in the context of the broader debate around asymptomatic transmission.

    1. Reviewer #1 (Public review):

      This study uses structural and functional approaches to investigate regulation of the Na/Ca exchanger NCX1 by an activator, PIP2 and an inhibitor, SEA0400. Previous functional studies suggest both of these compounds interact with the Na-dependent inactivation process to mediate their effects.

      State of the art methods are employed here, and the data are of high quality and presented very clearly. While there is merit in combining structural studies on both compounds as they relate to Na-dependent activation, in the end it is somewhat disappointing that neither is explored in further depth.

      The novel aspect of this work is the study on PIP2. Unfortunately, technical limitations precluded structural data on binding of the native PIP2, and so an unnatural short-chained analog, di-C8 PIP2, was used instead. This raises the question of whether these two molecules, which have similar but very distinctly different profiles of activation, actually share the same binding pocket and mode of action. The authors conduct a "competition" experiment, arguing the effect of di-C8-PIP2 addition subsequent to PIP2 suggests competition for a single binding site. In this scenario, PIP2 would need to vacate the binding site prior to di-C8-PIP2 occupying it. However, the lack of an effect of washout alone, suggests PIP2 does not easily unbind. This raises the possibility (probability?) of a non-competitive effect of di-C8-PIP2 at a different site. An additionally informative experiment would be to determine if a saturating concentration of di-C8-PIP2 could prevent the full activation induced by subsequent PIP2 addition. However, the relative affinities of the two ligands might make such an experiment challenging in practice.

      In an effort to address the binding site directly, the authors mutate key residues predicted to be important in liganding the phosphorylated head group of PIP2. However, the only mutations that have a significant effect in PIP2 activation also influence the Na-dependent inactivation process independently of PIP2. While these data are consistent with altering PIP2 binding (which cannot be easily untangled from its functional effect on Na-dependent inactivation), a primary effect on Na-inactivation, rather than PIP2 binding, cannot be fully ruled out. A more extensive mutagenic study, based on other regions of the di-C8 PIP2 binding site, would have given more depth to this work and might have been more revealing mechanistically.

      The SEA0400 aspect of the work does not integrate particularly well with the rest of the manuscript. This study confirms the previously reported structure and binding site for SEA0400 but provides little further information. While interesting speculation is presented regarding the connection between SEA0400 inhibition and Na-dependent inactivation, further experiments to test this idea are not included here.

      Comments on revisions:

      (1) The competition assay data for di-C8-PIP2 and PIP2 is a nice addition, but in its description in the text, the authors should be a bit more circumspect about their conclusions, based on the possibility/probability that the effect observed is actually non-competitive (as detailed above).<br /> (2) The authors should acknowledge the formal possibility that the functional effects of the mutations studies are a consequence of a direct effect on Na-dependent inactivation, independent of PIP2 binding.<br /> (3) The authors might strengthen their arguments for combining studies on PIP2 and SEA0400.<br /> (4) The authors could be clearer where their work on SEA0400 extends beyond the previously published observations.

    2. Reviewer #3 (Public review):

      NCXs are key Ca2+ transporters located on the plasma membrane, essential for maintaining cellular Ca2+ homeostasis and signaling. The activities of NCX are tightly regulated in response to cellular conditions, ensuring precise control of intracellular Ca2+ levels, with profound physiological implications. Building upon their recent breakthrough in determining the structure of human NCX1, the authors obtained cryo-EM structures of NCX1 in complex with its modulators, including the cellular activator PIP2 and the small molecule inhibitor SEA0400. Structural analyses revealed mechanistically informative conformational changes induced by PIP2 and elucidated the molecular basis of inhibition by SEA0400. These findings underscore the critical role of the interface between the transmembrane and cytosolic domains in NCX regulation and small molecule modulation. Overall, the results provide key insights into NCX regulation, with important implications for cellular Ca2+ homeostasis.

      Comments on revisions:

      The authors have adequately addressed my previous comments.

    1. Reviewer #2 (Public review):

      Summary:

      In this manuscript, the authors present the repurposing of cipargamin (CIP), a known drug against plasmodium and toxoplasma against babesia. They proved the efficacy of CIP on babesia in the nanomolar range. In silico analyses revealed the drug resistance mechanism through a single amino acid mutation at amino acid position 921 on the ATP4 gene of babesia. Overall, the conclusions drawn by the authors are well justified by the data presented. I believe this study opens up a novel therapeutic strategy against babesiosis.

      Strengths:

      The authors have carried out a comprehensive study. All the experiments performed were carried out methodically and logically.

    2. Reviewer #3 (Public review):

      Summary:

      The authors aim to establish that cipargamin can be used for the treatment of infection caused by Babesia organisms.

      Strengths:

      The study provides strong evidence that cipargamin is effective against various Babesia species. In vitro growth assays were used to establish that cipargamin is effective against Babesia bovis and Babesia gibsoni. Infection of mice with Babesia microti demonstrated that cipargamin is as effective as the combination of atovaquone plus azithromycin. Cipargamin protected mice from lethal infection with Babesia rodhaini. Mutations that confer resistance to cipargamin were identified in the gene encoding ATP4, a P-type Na ATPase that is found in other apicomplexan parasites, thereby validating ATP4 as the target of cipargamin. A 7-day treatment of cipagarmin, when combined with a single dose of tafenoquine, was sufficient to eradicate Babesia microti in a mouse model of severe babesiosis caused by a lack of adaptive immunity.

      Weaknesses:

      Cipargamin was tested in vivo at a single dose administered daily for 7 days. Despite the prospect of using cipargamin for the treatment of human babesiosis, there was no attempt to identify the lowest dose of cipagarmin that protects mice from Babesia microti infection.

      Comments on revisions:

      The authors have edited the manuscript and, in doing so, have addressed all queries pertaining to experimental design. The authors have decided to keep the discussion unchanged, but have replied to this reviewer regarding comments on interpretation of some data. The reader could have benefited from the authors' explanation. Nonetheless, the manuscript in its present form describes a valuable and significant body of work.

    1. Reviewer #1 (Public review):

      This study examined the interaction between two key cortical regions in the mouse brain involved in goal-directed movements, the rostral forelimb area (RFA) - considered a premotor region involved in movement planning, and the caudal forelimb area (CFA) - considered a primary motor region that more directly influences movement execution. The authors ask whether there exists a hierarchical interaction between these regions, as previously hypothesized, and focus on a specific definition of hierarchy - examining whether the neural activity in the premotor region exerts a larger functional influence on the activity in the primary motor area, than vice versa. They examine this question using advanced experimental and analytical methods, including localized optogenetic manipulation of neural activity in either region while measuring both the neural activity in the other region and EMG signals from several muscles involved in the reaching movement, as well as simultaneous electrophysiology recordings from both regions in a separate cohort of animals.

      The findings presented show that localized optogenetic manipulation of neural activity in either RFA or CFA resulted in similarly short-latency changes of the muscle output and in firing rate changes in the other region. However, perturbation of RFA led to a larger absolute change in the neural activity of CFA neurons. The authors interpret these findings as evidence for reciprocal, but asymmetrical, influence between the regions, suggesting some degree of hierarchy in which RFA has a greater effect on the neural activity in CFA. They go on to examine whether this asymmetry can also be observed in simultaneously recorded neural activity patterns from both regions. They use multiple advanced analysis methods that either identify latent components in the population level or measure the predictability of firing rates of single neurons in one region using firing rates of single neurons in the other region. Interestingly, the main finding across these analyses seems to be that both regions share highly similar components that capture a high degree of the variability of the neural activity patterns in each region. Single units' activity from either region could be predicted to a similar degree from the activity of single units in the other region, without a clear division into a leading area and a lagging area, as one might expect to find in a simple hierarchical interaction. However, the authors find some evidence showing a slight bias towards leading activity in RFA. Using a two-region neural network model that is fit to the summed neural activity recorded in the different experiments and to the summed muscle output, the authors show that a network with constrained (balanced) weights between the regions can still output the observed measured activities and the observed asymmetrical effects of the optogenetic manipulations, by having different within-region local weights. These results emphasize the challenges in studying interactions between brain regions with reciprocal interactions, multiple external inputs, and recurrent within-region connections.

      Strengths:

      The experiments and analyses performed in this study are comprehensive and provide a detailed examination and comparison of neural activity recorded simultaneously using dense electrophysiology probes from two main motor regions that have been the focus of studies examining goal-directed movements. The findings showing reciprocal effects from each region to the other, similar short-latency modulation of muscle output by both regions, and similarity of neural activity patterns, are convincing and add to the growing body of evidence that highlight the complexity of the interactions between multiple regions in the motor system and go against a simple feedforward-like hierarchy.

      The neural network model complements these findings and adds an important demonstration that the observed asymmetry can, in theory, also arise from differences in local recurrent connections and not necessarily from different input projections from one region to the other. This sheds an important light on the multiple factors that should be considered when studying the interaction between any two brain regions, with a specific emphasis on the role of local recurrent connections, that should be of interest to the general neuroscience community.

      Weaknesses:

      While the reciprocal interaction and similarity in neural activity across RFA and CFA is an important observation that is supported by the authors' findings, the evidence for a hierarchical interaction between the two regions appears to be weaker. The primary evidence for a hierarchical interaction comes from a causal optogenetic manipulation, carried out at the onset of the reaching movement and conducted with n = 3 in each experimental group, which shows an effect in both regions, yet the effect is greater when silencing the activity in RFA and examining the resulting change in CFA, than vice versa. Analysis of the simultaneously recorded neural activity, on the other hand, reveals mostly no clear hierarchy with leading or lagging dynamics between the regions. The findings of the optogenetic manipulation might be more compelling if similar effects were observed when the same manipulation was applied at different stages of movement preparation and execution, indicating a consistent interaction that is independent from the movement phase.

      The methods used to investigate hierarchical interactions through analysis of simultaneously recorded activity yielded inconsistent results. For instance, CCA and PLS showed no clear lead-lag relationship, while DLAG provided some evidence suggesting RFA leads CFA. Overall, these methods largely failed to demonstrate a clear hierarchical interaction. Assuming a partial hierarchy exists, this inconsistency may indicate that the hierarchy is not reflected in the activity patterns or that these analytical methods are inadequate for detecting such interactions within complex neural networks that are influenced by multiple external inputs, reciprocal inter-regional connections, and dominant intra-regional recurrent activity.

      As is also argued by the authors, these inconsistent findings underscore the need for caution when interpreting results from similar analyses used to infer inter-regional interactions from neural activity patterns alone. However, the study lacks sufficient explanation for why different methods yielded different results and more elaborate clarification is needed for the findings presented. For example, in the population-level analyses using CCA and PLS, the authors show that both techniques reveal components that are highly similar across regions and explain a substantial portion of each region's variance. Yet, shifting the activity of one region relative to the other to explore potential lead-lag relationships does not alter the results of these analyses. If the regions' activities were better aligned at some unknown true lead-lag time (or aligned at zero), one would expect a peak in alignment within the tested range, as is observed when these same analyses are applied to activity within a single region. It is thus unclear why shifting one region's activity relative to the other does not change the outcome. The interpretation of these results therefore, remains ambiguous and would benefit from further clarification.

    2. Reviewer #2 (Public review):

      Summary:

      While technical advances have enabled large-scale, multi-site neural recordings, characterizing inter-regional communication and its behavioral relevance remains challenging due to intrinsic properties of the brain such as shared inputs, network complexity, and external noise. This work by Saiki-Ishkawa et al. examines the functional hierarchy between premotor (PM) and primary motor (M1) cortices in mice during a directional reaching task. The authors find some evidence consistent with an asymmetric reciprocal influence between the regions, but overall, activity patterns were highly similar and equally predictive of one another. These results suggest that motor cortical hierarchy, though present, is not fully reflected in firing patterns alone.

      Strengths:

      Inferring functional hierarchies between brain regions, given the complexity of reciprocal and local connectivity, dynamic interactions, and the influence of both shared and independent external inputs, is a challenging task. It requires careful analysis of simultaneous recording data, combined with cross-validation across multiple metrics, to accurately assess the functional relationships between regions. The authors have generated a valuable dataset simultaneously recording from both regions at scale from mice performing a cortex-dependent directional reaching task.

      Using electrophysiological and silencing data, the authors found evidence supporting the traditionally assumed asymmetric influence from PM to M1. While earlier studies inferred a functional hierarchy based on partial temporal relationships in firing patterns, the authors applied a series of complementary analyses to rigorously test this hierarchy at both individual neuron and population levels, with robust statistical validation of significance.

      In addition, recording combined with brief optogenetic silencing of the other region allowed authors to infer the asymmetric functional influence in a more causal manner. This experiment is well designed to focus on the effect of inactivation manifesting through oligosynaptic connections to support the existence of a premotor to primary motor functional hierarchy.

      Subsequent analyses revealed a more complex picture. CCA, PLS, and three measures of predictivity (Granger causality, transfer entropy, and convergent cross mapping) emphasized similarities in firing patterns and cross-region predictability. However, DLAG suggested an imbalance, with RFA capturing CFA variance at a negative time lag, indicating that RFA 'leads' CFA. Taken together these results provide useful insights for current studies of functional hierarchy about potential limitations in inferring hierarchy solely based on firing rates.

      While I would detail some questions and issues on specifics of data analyses and modeling below, I appreciate the authors' effort in training RNNs that match some behavioral and recorded neural activity patterns including the inactivation result. The authors point out two components that can determine the across-region influence - 1) the amount of inputs received and 2) the dependence on across-region input, i.e., relative importance of local dynamics, providing useful insights in inferring functional relationships across regions.

      Weaknesses:

      (1) Trial-averaging was applied in CCA and PLS analyses. While trial-averaging can be appropriate in certain cases, it leads to the loss of trial-to-trial variance, potentially inflating the perceived similarities between the activity in the two regions (Figure 4). Do authors observe comparable degrees of similarity, e.g., variance explained by canonical variables? Also, the authors report conflicting findings regarding the temporal relationship between RFA and CFA when using CCA/PLS versus DLAG. Could this discrepancy be due to the use of trial-averaging in former analyses but not in the latter?

      (2) A key strength of the current study is the precise tracking of forelimb muscle activity during a complex motor task involving reaching for four different targets. This rich behavioral data is rarely collected in mice and offers a valuable opportunity to investigate the behavioral relevance of the PM-M1 functional interaction, yet little has been done to explore this aspect in depth. For example, single-trial time courses of inter-regional latent variables acquired from DLAG analysis can be correlated with single-trial muscle activity and/or reach trajectories to examine the behavioral relevance of inter-regional dynamics. Namely, can trial-by-trial change in inter-regional dynamics explain behavioral variability across trials and/or targets? Does the inter-areal interaction change in error trials? Furthermore, the authors could quantify the relative contribution of across-area versus within area dynamics to behavioral variability. It would also be interesting to assess the degree to which across-area and within-area dynamics are correlated. Specifically, can across-area dynamics vary independently from within-area dynamics across trials, potentially operating through a distinct communication subspace?

      (3) While network modeling of RFA and CFA activity captured some aspects of behavioral and neural data, I wonder if certain findings such as the connection weight distribution (Figure 7C), across-region input (Figure 7F), and the within-region weights (Figure 7G), primarily resulted from fitting the different overall firing rates between the two regions with CFA exhibiting higher average firing rates. Did the authors account for this firing rate disparity when training the RNNs?

      (4) Another way to assess the functional hierarchy is by comparing the time courses of movement representation between the two regions. For example, a linear decoder could be used to compare the amount of information about muscle activity and/or target location as well as time courses thereof between the two regions. This approach is advantageous because it incorporates behavior rather than focusing solely on neural activity. Since one of the main claims of this study is the limitation of inferring functional hierarchy from firing rate data alone, the authors should use the behavior as a lens for examining inter-areal interactions.

      Comments on revisions:

      I appreciate the authors' thoughtful revisions in response to prior reviews, which I believe have substantially improved the manuscript. In particular, I found the addition of the new section "Manifestations of hierarchy in firing patterns" to be valuable, as it begins to address some of the more complex and potentially conflicting observations

    3. Reviewer #3 (Public review):

      This study investigates how two cortical regions which are central to the study of rodent motor control (rostral forelimb area, RFA, and caudal forelimb area, CFA) interact during directional forelimb reaching in mice. The authors investigate this interaction using (1) optogenetic manipulations in one area while recording extracellularly from the other, (2) statistical analyses of simultaneous CFA/RFA extracellular recordings, and (3) network modeling. The authors provide solid evidence that asymmetry between RFA and CFA can be observed, although such asymmetry is only observed in certain experimental and analytical contexts.

      The authors find asymmetry when applying optogenetic perturbations, reporting a greater impact of RFA inactivation on CFA activity than vice-versa. The authors then investigate asymmetry in endogenous activity during forelimb movements and find asymmetry with some analytical methods but not others. Asymmetry was observed in the onset timing of movement-related deviations of local latent components with RFA leading CFA (computed with PCA) and in a relatively higher proportion and importance of cross-area latent components with RFA leading than CFA leading (computed with DLAG). However, no asymmetry was observed using several other methods that compute cross-area latent dynamics, nor with methods computed on individual neuron pairs across regions. The authors follow up this experimental work by developing a two-area model with asymmetric dependence on cross-area input. This model is used to show that differences in local connectivity can drive asymmetry between two areas with equal amounts of across-region input.

      Overall, this work provides a useful demonstration that different cross-area analysis methods result in different conclusions regarding asymmetric interactions between brain areas and suggests careful consideration of methods when analyzing such networks is critical. A deeper examination of why different analytical methods result in observed asymmetry or no asymmetry, analyses that specifically examine neural dynamics informative about details of the movement, or a biological investigation of the hypothesis provided by the model would provide greater clarity regarding the interaction between RFA and CFA.

      Strengths:

      The authors are rigorous in their experimental and analytical methods, carefully monitoring the impact of their perturbations with simultaneous recordings and providing valid controls for their analytical methods. They cite relevant previous literature that largely agrees with the current work, highlighting the continued ambiguity regarding the extent to which there exists an asymmetry in endogenous activity between RFA and CFA.

      A strength of the paper is the evidence for asymmetry provided by optogenetic manipulation. They show that RFA inactivation causes a greater absolute difference in muscle activity than CFA interaction (deviations begin 25-50 ms after laser onset, Figure 1) and that RFA inactivation causes a relatively larger decrease in CFA firing rate than CFA inactivation causes in RFA (deviations begin <25ms after laser onset, Figure 3). The timescales of these changes provide solid evidence for an asymmetry in impact of inactivating RFA/CFA on the other region that could not be driven by differences in feedback from disrupted movement (which would appear with a ~50ms delay).

      The authors also utilize a range of different analytical methods, showing an interesting difference between some population-based methods (PCA, DLAG) that observe asymmetry, and single neuron pair methods (granger causality, transfer entropy, and convergent cross mapping) that do not. Moreover, the modeling work presents an interesting potential cause of "hierarchy" or "asymmetry" between brain areas: local connectivity that impacts dependence on across-region input, rather than the amount of across-region input actually present.

      Weaknesses:

      There is no attempt to examine neural dynamics that are specifically relevant/informative about the details of the ongoing forelimb movement (e.g., kinematics, reach direction). Thus, it may be preemptive to claim that firing patterns alone do not reflect functional influence between RFA/CFA. For example, given evidence that the largest component of motor cortical activity doesn't reflect details of ongoing movement (reach direction or path; Kaufman, et al. PMID: 27761519) and that the analytical tools the authors use likely include this component (PCA, CCA), it may not be surprising that CFA and RFA do not show asymmetry if such asymmetry is related to control of movement details. An asymmetry may still exist in the components of neural activity that encode information about movement details, and thus it may be necessary to isolate and examine the interaction of behaviorally-relevant dynamics (e.g., Sani, et al. PMID: 33169030).

      The idea that local circuit dynamics play a central role in determining the asymmetry between RFA and CFA is not supported by experimental data in this paper. The plausibility of this hypothesis is supported by the model but is not explored in any analyses of the experimental data collected. Further experimental investigation is needed to separate this hypothesis from other possibilities.

      Comments on revisions:

      The authors have improved the manuscript by reviewing several aspects of the text and the addition of supplemental materials. I believe these revisions have clarified some important aspects of the results.

    1. Reviewer #1 (Public review):

      In this paper, the authors reveal that the MK2 inhibitor CMPD1 can inhibit the growth, migration and invasion of breast cancer cells both in vitro and in vivo by inducing microtubule depolymerization, preferentially at the microtubule plus-end, leading to cell division arrest, mitotic defects, and apoptotic cell death. They also showed that CMPD1 treatment upregulates genes associated with cell migration and cell death, and downregulates genes related to mitosis and chromosome segregation in breast cancer cells, suggesting a potential mechanism of CMPD1 inhibition in breast cancer. Besides, they used the combination of an MK2-specific inhibitor, MK2-IN-3, with the microtubule depolymerizer vinblastine to simultaneously disrupt both the MK2 signaling pathway and microtubule dynamics, and they claim that inhibiting the p38-MK2 pathway may help to enhance the efficacy of MTAs in the treatment of breast cancer.

    2. Reviewer #2 (Public review):

      Summary:

      This study explores the potential of inhibiting the p38-MK2 signaling pathway to enhance the efficacy of microtubule-targeting agents (MTAs) in breast cancer treatment using a dual-target inhibitor.

      Strengths:

      The study identifies the p38-MK2 pathway as a promising target to enhance the efficacy of microtubule-targeting agents (MTAs), offering a novel therapeutic strategy for breast cancer treatment. The study also employs a wide range of techniques, especially live-cell imaging, to assess the microtubule dynamics in TNBC cells. The revised manuscript added new in vitro and in vivo evidence that furtherly supported the conclusions.

      Comments on revisions:

      The authors have appropriately addressed all of my comments and concerns. Specifically, they performed additional in vitro experiments using MCF10A cells and p53 knockout cells to determine the IC50 of CMPD1. They also repeated the in vivo treatment experiment and evaluated the toxicity of the drug treatment in the CAL-51 model. Furthermore, they provided genetic evidence for the combination treatment. I'm satisfied with the revision and have no further major comments. Minor comment: make sure the name of the chemo drug shown in Fig. 3 is consistent.

    3. Reviewer #3 (Public review):

      Summary:

      The authors demonstrated MK2i could enhance the therapeutic efficacy of MTAs. With the tumour xenograft and migration assay, the author suggested that the p38-MK2 pathway may serve as a promising therapeutic target in combination with MTAs in cancer treatment.

      Strengths:

      The authors provided a potential treatment for breast cancer.

      Comments on revisions:

      A xenograft experiment should be included to evaluate the synergistic effect of MK2i and vinblastine.

    1. Reviewer #1 (Public review):

      In this study, Marocco and colleages perform a deep characterization of the complex molecular mechanism guiding the recognition of a particular CELLmotif previously identified in hepatocytes in another publication. Having miR-155-3p with or without this CELLmotif as initial focus, authors identify 21 proteins differentially binding to these two miRNA versions. From these, they decided to focus on PCBP2. They elegantly demonstrate PCBP2 binding to miR-155-3p WT version but not to CELLmotif-mutated version. miR-155-3p contains a hEXOmotif identified in a different report, whose recognition is largely mediated by another RNA-binding protein called SYNCRIP. Interestingly, mutation of the hEXOmotif contained in miR-155-3p did not only blunt SYNCRIP binding, but also PCBP2 binding despite the maintenance of the CELLmotif. This indicates that somehow SYNCRIP binding is a pre-requisite for PCBP2 binding. EMSA assay confirms that SYNCRIP is necessary for PCBP2 binding to miR-155-3p, while PCBP2 is not needed for SYNCRIP binding. Then authors aim to extend these finding to other miRNAs containing both motifs. For that, they perform a small-RNA-Seq of EVs released from cells knockdown for PCBP2 versus control cells, identifying a subset of miRNAs whose expression either increases or decreases. The assumption is that those miRNAs containing PCBP2-binding CELLmotif should now be less retained in the cell and go more to extracellular vesicles, thus reflecting a higher EV expression. The specific subset of miRNAs having both the CELLmotif and hEXOmotif (9 miRNAs) whose expressions increase in EVs due to PCBP2 reduction is also affected by knocking-down SYNCRIP in the sense that reduction of SYNCRIP leads to lower EV sorting. Further experiments confirm that PCBP2 and SYNCRIP bind to these 9 miRNAs and that knocking down SYNCRIP impairs their EV sorting.

      In the revised manuscript, the authors have addressed most of my concerns and questions. I believe the new experiments provide stronger support for their claims. My only remaining concern is the lack of clarity in the replicates for the EMSA experiment. The one shown in the manuscript is clear; however, the other three replicates hardly show that knocking down SYNCRIP has an effect on PCBP2 binding. Even worse is the fact that these replicates do not support at all that PCBP2 silencing has no effect on SYNCRIP binding, as the bands for those types of samples are, in most of the cases, not visible. I think the authors should work on repeating a couple of times EMSA experiment.

    2. Reviewer #2 (Public review):

      Summary:

      The author of this manuscript aimed to uncover the mechanisms behind miRNA retention within cells. They identified PCBP2 as a crucial factor in this process, revealing a novel role for RNA-binding proteins. Additionally, the study discovered that SYNCRIP is essential for PCBP2's function, demonstrating the cooperative interaction between these two proteins. This research not only sheds light on the intricate dynamics of miRNA retention but also emphasizes the importance of protein interactions in regulating miRNA behavior within cells.

      Strengths:

      This paper makes important progress in understanding how miRNAs are kept inside cells. It identifies PCBP2 as a key player in this process, showing a new role for proteins that bind RNA. The study also finds that SYNCRIP is needed for PCBP2 to work, highlighting how these proteins work together. These discoveries not only improve our knowledge of miRNA behavior but also suggest new ways to develop treatments by controlling miRNA locations to influence cell communication in diseases. The use of liver cell models and thorough experiments ensures the results are reliable and show their potential for RNA-based therapies

      Weaknesses:

      The manuscript is well-structured and presents compelling data, but I noticed a few minor corrections that could further enhance its clarity:

      Figure References: In the response to Reviewer 1, the comment states, "It's not Panel C, it's Panel A of Figure 1"-this should be cross-checked for consistency.<br /> Supplementary Figure 2 is labeled as "Panel A"-please verify if additional panels (B, C, etc.) are intended.

      Western Blot Quality: The Alix WB shows some background noise. A repeat with optimized conditions (or inclusion of a cleaner replicate) would strengthen the data. Adding statistical analysis for all WBs would also reinforce robustness.

      These are relatively small refinements, and the manuscript is already in excellent shape. With these adjustments, it will be even stronger.

    1. Reviewer #1 (Public review):

      Summary:

      This fundamental work employed multidisciplinary approaches and conducted rigorous experiments to study how a specific subset of neurons in the dorsal striatum (i.e., "patchy" striatal neurons) modulates locomotion speed depending on the valence of the naturalistic context.

      Strengths:

      The scientific findings are novel and original and significantly advance our understanding of how the striatal circuit regulates spontaneous movement in various contexts.

      Weaknesses:

      This is extensive research involving various circuit manipulation approaches. Some of these circuit manipulations are not physiological. A balanced discussion of the technical strengths and limitations of the present work would be helpful and beneficial to the field. Minor issues in data presentation were also noted.

    2. Reviewer #2 (Public review):

      Hawes et al. investigated the role of striatal neurons in the patch compartment of the dorsal striatum. Using Sepw1-Cre line, the authors combined a modified version of the light/dark transition box test that allows them to examine locomotor activity in different environmental valence with a variety of approaches, including cell-type-specific ablation, miniscope calcium imaging, fiber photometry, and opto-/chemogenetics. First, they found ablation of patchy striatal neurons resulted in an increase in movement vigor when mice stayed in a safe area or when they moved back from more anxiogenic to safe environments. The following miniscope imaging experiment revealed that a larger fraction of striatal patchy neurons was negatively correlated with movement speed, particularly in an anxiogenic area. Next, the authors investigated differential activity patterns of patchy neurons' axon terminals, focusing on those in GPe, GPi, and SNr, showing that the patchy axons in SNr reflect movement speed/vigor. Chemogenetic and optogenetic activation of these patchy striatal neurons suppressed the locomotor vigor, thus demonstrating their causal role in the modulation of locomotor vigor when exposed to valence differentials. Unlike the activation of striatal patches, such a suppressive effect on locomotion was absent when optogenetically activating matrix neurons by using the Calb1-Cre line, indicating distinctive roles in the control of locomotor vigor by striatal patch and matrix neurons. Together, they have concluded that nigrostriatal neurons within striatal patches negatively regulate movement vigor, dependent on behavioral contexts where motivational valence differs.

      In my view, this study will add to the important literature by demonstrating how patch (striosomal) neurons in the striatum control movement vigor. This study has applied multiple approaches to investigate their functionality in locomotor behavior, and the obtained data largely support their conclusions. Nevertheless I have some suggestions for improvements in the manuscript and figures regarding their data interpretation, accuracy, and efficacy of data presentation.

      (1) The authors found that the activation of the striatonigral pathway in the patch compartment suppresses locomotor speed, which contradicts with canonical roles of the direct pathway. It would be great if the authors could provide mechanistic explanations in the Discussion section. One possibility is that striatal D1R patch neurons directly inhibit dopaminergic cells that regulate movement vigor (Nadal et al., Sci. Rep., 2021; Okunomiya et al., J Neurosci., 2025). Providing plausible explanations will help readers infer possible physiological processes and give them ideas for future follow-up studies.

      (2) On page 14, Line 301, the authors stated that "Cre-dependent mCheery signals were colocalized with the patch marker (MOR1) in the dorsal striatum (Fig. 1B)". But I could not find any mCherry on that panel, so please modify it.

      (3) From data shown in Figure 1, I've got the impression that mice ablated with striatal patch neurons were generally hyperactive, but this is probably not the case, as two separate experiments using LLbox and DDbox showed no difference in locomotor vigor between control and ablated mice. For the sake of better interpretation, it may be good to add a statement in Lines 365-366 that these experiments suggest the absence of hyperactive locomotion in general by ablating these specific neurons.

      (4) In Line 536, where Figure 5A was cited, the author mentioned that they used inhibitory DREADDs (AAV-DIO-hM4Di-mCherrry), but I could not find associated data on Figure 5. Please cite Figure S3, accordingly.

      (5) Personally, the Figure panel labels of "Hi" and "ii" were confusing at first glance. It would be better to have alternatives.

      (6) There is a typo on Figure 4A: tdTomata → tdTomato

    3. Reviewer #3 (Public review):

      Hawes et al. combined behavioral, optical imaging, and activity manipulation techniques to investigate the role of striatal patch SPNs in locomotion regulation. Using Sepw1-Cre transgenic mice, they found that patch SPNs encode locomotion deceleration in a light-dark box procedure through optical imaging techniques. Moreover, genetic ablation of patch SPNs increased locomotion speed, while chemogenetic activation of these neurons decreased it. The authors concluded that a subtype of patch striatonigral neurons modulates locomotion speed based on external environmental cues. Below are some major concerns:

      The study concludes that patch striatonigral neurons regulate locomotion speed. However, unless I missed something, very little evidence is presented to support the idea that it is specifically striatonigral neurons, rather than striatopallidal neurons, that mediate these effects. In fact, the optogenetic experiments shown in Fig. 6 suggest otherwise. What about the behavioral effects of optogenetic stimulation of striatonigral versus striatopallidal neuron somas in Sepw1-Cre mice?

      In the abstract, the authors state that patch SPNs control speed without affecting valence. This claim seems to lack sufficient data to support it. Additionally, speed, velocity, and acceleration are very distinct qualities. It is necessary to clarify precisely what patch neurons encode and control in the current study.

      One of the major results relies on chemogenetic manipulation (Figure 5). It would be helpful to demonstrate through slice electrophysiology that hM3Dq and hM4Di indeed cause changes in the activity of dorsal striatal SPNs, as intended by the DREADD system. This would support both the positive (Gq) and negative (Gi) findings, where no effects on behavior were observed.

      Finally, could the behavioral effects observed in the current study, resulting from various manipulations of patch SPNs, be due to alterations in nigrostriatal dopamine release within the dorsal striatum?

    1. Reviewer #1 (Public review):

      Summary:

      The authors use the teleost medaka as an animal model to study the effect of seasonal changes in day-length on feeding behaviour and oocyte production. They report a careful analysis how day-length affects female medakas and a thorough molecular genetic analysis of genes potentially involved in this process. They show a detailed analysis of two genes and include a mutant analysis of one gene to support their conclusions

      Strengths:

      The authors pick their animal model well and exploit the possibilities to examine in this laboratory model the effect of a key environmental influence, namely the seasonal changes of day-length. The phenotypic changes are carefully analysed and well controlled. The mutational analysis of the agrp1 by a ko-mutant provides important evidence to support the conclusions. Thus this report exceeds previous findings on the function of agrp1 and npyb as regulators of food-intake and shows how in medaka these genes are involved in regulating the organismal response to an environmental change. It thus furthers our understanding on how animals react to key exogenous stimuli for adaptation.

      Weaknesses:

      The authors are too modest when it comes to underscoring the importance of their findings. Previous animal models used to study the effect of these neuropeptides on feeding behaviour have either lost or were most likely never sensitive to seasonal changes of day-length. Considering the key importance of this parameter on many aspects of plant and animal life it could be better emphasised that a suitable animal model is at hand that permits this.<br /> The molecular characterization of the agrp1 ko-mutant that the authors have generated lacks some details that would help to appreciate the validity of the mutant phenotype. Additional data would help in this respect.

      Comments on revisions:

      The authors dealt adequately with the comments and suggestions of this reviewer.

    2. Reviewer #2 (Public review):

      Summary:

      The authors investigated the mechanisms behind breeding season-dependent feeding behavior using medaka, a well-known photoperiodic species, as a model. Through a combination of molecular, cellular, and behavioral analyses, including tests with mutants, they concluded that AgRP1 plays a central role in feeding behavior, mediated by ovarian estrogenic signals.

      Strengths:

      This study offers valuable insights into the neuroendocrine mechanisms that govern breeding season-dependent feeding behavior in medaka. The multidisciplinary approach, which includes molecular and physiological analyses, enhances the scientific contribution of the research.

      Comments on revised version:

      My concerns from the first review have been addressed. The manuscript's key points are clearly presented, and the conclusions are readily comprehensible

    3. Reviewer #3 (Public review):

      Summary:

      Understanding the mechanisms whereby animals restrict the timing of their reproduction according to day length is a critical challenge given that many of the most relevant species for agriculture are strongly photoperiodic. However, the principal animal models capable of detailed genetic analysis do not respond to photoperiod so this has inevitably limited progress in this field. The fish model medaka occupies a uniquely powerful position since it's reproduction is strictly restricted to long days and it also offers a wide range of genetic tools for exploring, in depth, various molecular and cellular control mechanisms.

      For these reasons, this manuscript by Tagui and colleagues is particularly valuable. It uses the medaka to explore links bridging photoperiod, feeding behaviour and reproduction. The authors demonstrate that in female, but not male medaka, photoperiod-induced reproduction is associated with an increase in feeding, presumably explained by the high metabolic cost of producing eggs on a daily basis during the reproductive period. Using RNAseq analysis of the brain, they reveal that the expression of the neuropeptides agrp and npy that have been previously implicated in the regulation of feeding behaviour in mice, are upregulated in the medaka brain during exposure to long photoperiod conditions. Unlike the situation in mouse, these two neuropeptides are not coexpressed in medaka neurons and food deprivation in medaka led to increases in agrp but also a decrease in npy expression. Furthermore, the situation in fish may be more complicated than in mouse due to the presence of multiple gene paralogs for each neuropeptide. Exposure to long day conditions increases agrp1 expression in medaka as the result of increases in the number of neurons expressing this neuropeptide, while the increase in npyb levels results from increased levels of expression in the same population of cells. Using ovariectomized medaka and in situ hybridization assays, the authors reveal that the regulation of agrp1 involves estrogen acting via the estrogen receptor esr2a. Finally, a loss of agrp1 function mutant is generated where the female mutants fail to show the characteristic increase in feeding associated with long day enhanced reproduction as well as yielding reduced numbers of eggs during spawning.

      Strengths:

      This manuscript provides important foundational work for future investigations aiming to elucidate the coordination of photoperiod sensing, feeding activity and reproduction function. The authors have used a combination of approaches with a genetic model that is particularly well suited to studying photoperiodic dependent physiology and behaviour. The data are clear and the results are convincing and support the main conclusions drawn. The findings are relevant not only for understanding photopriodic responses but also provide more general insight into links between reproduction and feeding behaviour control.

      The manuscript has been further strengthened by the inclusion of additional information according to my advice: The analysis of ovariectomized female fish and juvenille fish has now been reported in terms of their feeding behaviour and so provide a complete view of the position of this feeding regulatory mechanism in the context of reproduction status. Furthermore, the discussion section has been expanded to speculate on the functional significance of linking feeding behaviour control with reproductive function. Modifications made in order to address technical concerns of the other 2 reviewers have also significantly strengthened the presentation of this work.

      Weaknesses:

      These have now been addressed in the revised version.

    1. Reviewer #1 (Public review):

      This paper presents a computational model of the evolution of two different kinds of helping ("work," presumably denoting provisioning, and defense tasks) in a model inspired by cooperatively breeding vertebrates. The helpers in this model are a mix of previous offspring of the breeder and floaters that might have joined the group, and can either transition between the tasks as they age or not. The two types of help have differential costs: "work" reduces "dominance value," (DV), a measure of competitiveness for breeding spots, which otherwise goes up linearly with age, but defense reduces survival probability. Both eventually might preclude the helper from becoming a breeder and reproducing. How much the helpers help, and which tasks (and whether they transition or not), as well as their propensity to disperse, are all evolving quantities. The authors consider three main scenarios: one where relatedness emerges from the model, but there is no benefit to living in groups, one where there is no relatedness, but living in larger groups gives a survival benefit (group augmentation, GA), and one where both effects operate. The main claim is that evolving defensive help or division of labor requires the group augmentation; it doesn't evolve through kin selection alone in the authors' simulations.

      This is an interesting model, and there is much to like about the complexity that is built in. Individual-based simulations like this can be a valuable tool to explore the complex interaction of life history and social traits. Yet, models like this also have to take care of both being very clear on their construction and exploring how some of the ancillary but potentially consequential assumptions affect the results, including robust exploration of the parameter space. I think the current manuscript falls short in these areas, and therefore, I am not yet convinced of the results. Much of this is a matter of clearer and more complete writing: the Materials and Methods section in particular is incomplete or vague in some important junctions. However, there are also some issues with the assumptions that are described clearly.

      Below, I describe my main issues, mostly having to do with model features that are unclear, poorly motivated (as they stand), or potentially unrealistic or underexplored.

      One of the main issues I have is that there is almost no information on what happens to dispersers in the model. Line 369-67 states dispersers might join another group or remain as floaters, but gives no further information on how this is determined. Poring through the notation table also comes up empty as there is no apparent parameter affecting this consequential life history event. At some point, I convinced myself that dispersers remain floaters until they die or become breeders, but several points in the text contradict this directly (e.g., l 107). Clearly this is a hugely important model feature since it determines fitness cost and benefits of dispersal and group size (which also affects relatedness and/or fitness depending on the model). There just isn't enough information to understand this crucial component of the model, and without it, it is hard to make sense of the model output.

      Related to that, it seems to be implied (but never stated explicitly) that floaters do no work, and therefore their DV increases linearly with age (H_work in eq.2 is zero). That means any floaters that manage to stick around long enough would have higher success in competition for breeding spots relative to existing group members. How realistic is this? I think this might be driving the kin selection-only results that defense doesn't evolve without group augmentation (one of the two main ways). Any subordinates (which are mainly zero in the no GA, according to the SI tables; this assumes N=breeder+subordinates, but this isn't explicit anywhere) would be outcompeted by floaters after a short time (since they evolve high H and floaters don't), which in turn increases the benefit of dispersal, explaining why it is so high. Is this parameter regime reasonable? My understanding is that floaters often aren't usually high resource holding potential individuals (either b/c high RHP ones would get selected out of the floater population by establishing territories or b/c floating isn't typically a thriving strategy, given that many resources are tied to territories). In this case, the assumption seems to bias things towards the floaters and against subordinates to inherit territories. This should be explored either with a higher mortality rate for floaters and/or a lower DV increase, or both.

      When it comes to floaters replacing dead breeders, the authors say a bit more, but again, the actual equation for the scramble competition (which only appears as "scramble context" in the notation table) is not given. Is it simply proportional to R_i/\sum_j R_j ? Or is there some other function used? What are the actual numbers of floaters per breeding territory that emerge under different parameter values? These are all very important quantities that have to be described clearly.

      I also think the asexual reproduction with small mutations assumption is a fairly strong one that also seems to bias the model outcomes in a particular way. I appreciate that the authors actually measured relatedness within groups (though if most groups under KS have no subordinates, that relatedness becomes a bit moot), and also eliminated it with their ingenious swapping-out-subordinates procedure. The fact remains that unless they eliminate relatedness completely, average relatedness, by design, will be very high. (Again, this is also affected by how the fate of the dispersers is determined, but clearly there isn't a lot of joining happening, just judging from mean group sizes under KS only.) This is, of course, why there is so much helping evolving (even if it's not defensive) unless they completely cut out relatedness.

      Finally, the "need for division of labor" section is also unclear, and its construction also would seem to bias things against division of labor evolving. For starters, I don't understand the rationale for the convoluted way the authors create an incentive for division of labor. Why not implement something much simpler, like a law of minimum (i.e., the total effect of helping is whatever the help amount for the lowest value task is) or more intuitively: the fecundity is simply a function of "work" help (draw Poisson number of offspring) and survival of offspring (draw binomial from the fecundity) is a function of the "defense" help. As it is, even though the authors say they require division of labor, in fact, they only make a single type of help marginally less beneficial (basically by half) if it is done more than the other. That's a fairly weak selection for division of labor, and to me it seems hard to justify. I suspect either of the alternative assumptions above would actually impose enough selection to make division of labor evolve even without group augmentation.

      Overall, this is an interesting model, but the simulation is not adequately described or explored to have confidence in the main conclusions yet. Better exposition and more exploration of alternative assumptions and parameter space are needed.

    2. Reviewer #2 (Public review):

      Summary:

      This paper formulates an individual-based model to understand the evolution of division of labor in vertebrates. A main conclusion of the paper is that direct fitness benefits are the primary factor causing the evolution of vertebrate division of labor, rather than indirect fitness benefits.

      Strengths:

      The paper formulates an individual-based model that is inspired by vertebrate life history. The model incorporates numerous biologically realistic details, including the possibility to evolve age polytheism where individuals switch from work to defence tasks as they age or vice versa, as well as the possibility of comparing the action of group augmentation alone with that of kin selection alone.

      Weaknesses:

      The model makes assumptions that restrict the possibility that kin selection leads to the evolution of helping. In particular, the model assumes that in the absence of group augmentation, subordinates can only help breeders but cannot help non-breeders or increase the survival of breeders, whereas with group augmentation, subordinates can help both breeders and non-breeders and increase the survival of breeders. This is unrealistic as subordinates in real organisms can help other subordinates and increase the survival of non-breeders, even in the absence of group augmentation, for instance, with targeted helping to dominants or allies. This restriction artificially limits the ability of kin selection alone to lead to the evolution of helping, and potentially to division of labor. Hence, the conclusion that group augmentation is the primary driving factor driving vertebrate division of labor appears forced by the imposed restrictions on kin selection. The model used is also quite particular, and so the claimed generality across vertebrates is not warranted.

      I describe some suggestions for improving the paper below, more or less in the paper's order.

      First, the introduction goes to great lengths trying to convince the reader that this model is the first in this or another way, particularly in being only for vertebrates, as illustrated in the abstract where it is stated that "we lack a theoretical framework to explore the conditions under which division of labor is likely to evolve" (line 13). However, this is a risky and unnecessary motivation. There are many models of division of labor and some of them are likely to be abstract enough to apply to vertebrates even if they are not tailored to vertebrates, so the claims for being first are not only likely to be wrong but will put many readers in an antagonistic position right from the start, which will make it harder to communicate the results. Instead of claiming to be the first or that there is a lack of theoretical frameworks for vertebrate division of labor, I think it is enough and sufficiently interesting to say that the paper formulates an individual-based model motivated by the life history of vertebrates to understand the evolution of vertebrate division of labor. You could then describe the life history properties that the model incorporates (subordinates can become reproductive, low relatedness, age polyethism, etc.) without saying this has never been done or that it is exclusive to vertebrates; indeed, the paper states that these features do not occur in eusocial insects, which is surprising as some "primitively" eusocial insects show them. So, in short, I think the introduction should be extensively revised to avoid claims of being the first and to make it focused on the question being addressed and how it is addressed. I think this could be done in 2-3 paragraphs without the rather extensive review of the literature in the current introduction.

      Second, the description of the model and results should be clarified substantially. I will give specific suggestions later, but for now, I will just say that it is unclear what the figures show. First, it is unclear what the axes in Figure 2 show, particularly for the vertical one. According to the text in the figure axis, it presumably refers to T, but T is a function of age t, so it is unclear what is being plotted. The legend explaining the triangle and circle symbols is unintelligible (lines 227-230), so again it is unclear what is being plotted; part of the reason for this unintelligibility is that the procedure that presumably underlies it (section starting on line 493) is poorly explained and not understandable (I detail why below). Second, the axes in Figure 3 are similarly unclear. The text in the vertical axis in panel A suggests this is T, however, T is a function of t and gamma_t, so something else must be being done to plot this. Similarly, in panel B, the horizontal axis is presumably R, but R is a function of t and of the helping genotype, so again some explanation is lacking. In all figures, the symbol of what is being plotted should be included.

      Third, the conclusions sound stronger than the results are. A main conclusion of the paper is that "kin selection alone is unlikely to select for the evolution of defensive tasks and division of labor in vertebrates" (lines 194-195). This conclusion is drawn from the left column in Figure 2, where only kin selection is at play, and the helping that evolves only involves work rather than defense tasks. This conclusion follows because the model assumes that without group augmentation (i.e., xn=0, the kin selection scenario), subordinates can only help breeders to reproduce but cannot help breeders or other subordinates to survive, so the only form of help that evolves is the least costly, not the most beneficial as there is no difference in the benefits given among forms of helping. This assumption is unrealistic, particularly for vertebrates where subordinates can help other group members survive even in the absence of group augmentation (e.g., with targeted help to certain group members, because of dominance hierarchies where the helping would go to the breeder, or because of alliances where the helping would go to other subordinates). I go into further details below, but in short, the model forces a narrow scope for the kin selection scenario, and then the paper concludes that kin selection alone is unlikely to be of relevance for the evolution of vertebrate division of labor. This conclusion is particular to the model used, and it is misleading to suggest that this is a general feature of such a particular model.

      Overall, I think the paper should be revised extensively to clarify its aims, model, results, and scope of its conclusions.

    1. Reviewer #1 (Public review):

      Summary:

      This manuscript presents a practical modification of the orthogonal hybridization chain reaction (HCR) technique, a promising yet underutilized method with broad potential for future applications across various fields. The authors advance this technique by integrating peptide ligation technology and nanobody-based antibody mimetics - cost-effective and scalable alternatives to conventional antibodies - into a DNA-immunoassay framework that merges oligonucleotide-based detection with immunoassay methodologies. Notably, they demonstrate that this approach facilitates a modified ELISA platform capable of simultaneously quantifying multiple target protein expression levels within a single protein mixture sample.

      Strengths:

      The hybridization chain reaction (HCR) technique was initially developed to enable the simultaneous detection of multiple mRNA expression levels within the same tissue. This method has since evolved into immuno-HCR, which extends its application to protein detection by utilizing antibodies. A key requirement of immuno-HCR is the coupling of oligonucleotides to antibodies, a process that can be challenging due to the inherent difficulties in expressing and purifying conventional antibodies.

      In this study, the authors present an innovative approach that circumvents these limitations by employing nanobody-based antibody mimetics, which recognize antibodies, instead of directly coupling oligonucleotides to conventional antibodies. This strategy facilitates oligonucleotide conjugation - designed to target the initiator hairpin oligonucleotide of HCR -through peptide ligation and click chemistry.

      Weaknesses:

      The sandwich-format technique presented in this study, which employs a nanobody that recognizes primary IgG antibodies, may have limited scalability compared to existing methods that directly couple oligonucleotides to primary antibodies. This limitation arises because the C-region types of primary antibodies are relatively restricted, meaning that the use of nanobody-based detection may constrain the number of target proteins that can be analyzed simultaneously. In contrast, the conventional approach of directly conjugating oligonucleotides to primary antibodies allows for a broader range of protein targets to be analyzed in parallel.

      Additionally, in the context of HCR-based protein detection, the number of proteins that can be analyzed simultaneously is inherently constrained by fluorescence wavelength overlap in microscopy, which limits its multiplexing capability. By comparison, direct coupling of oligonucleotides to primary antibodies can facilitate the simultaneous measurement of a significantly greater number of protein targets than the sandwich-based nanobody approach in the barcode-ELISA/NGS-based technique.

    1. Reviewer #1 (Public review):

      Summary:

      This study puts forth the model that under IFN-B stimulation, liquid-phase WTAP coordinates with the transcription factor STAT1 to recruit MTC to the promoter region of interferon stimulated genes (ISGs), mediating the installation of m6A on newly synthesized ISG mRNAs. This model is supported by strong evidence that the phosphorylation state of WTAP, regulated by PPP4, is regulated by IFN-B stimulation, and that this results in interactions between WTAP, the m6A methyltransferase complex, and STAT1, a transcription factor that mediates activation of ISGs. This was demonstrated via a combination of microscopy, immunoprecipitations, m6A sequencing, and ChIP. These experiments converge on a set of experiments that nicely demonstrate that IFN-B stimulation increases the interaction between WTAP, METTL3, and STAT1, that this interaction is lost with knockdown of WTAP (even in the presence of IFN-B), and that this IFN-B stimulation also induces METTL3-ISG interactions.

      Strengths:

      The evidence for the IFN-B stimulated interaction between METTL3 and STAT1, mediated by WTAP, is quite strong. Removal of WTAP in this system seems to be sufficient to reduce these interactions and the concomitant m6A methylation of ISGs. The conclusion that the phosphorylation state of WTAP is important in this process is also quite well supported. The authors have now also provided substantial evidence that phase separation of WTAP upon interferon stimulation facilitates m6A-methylation of multiple interferon stimulated genes.

    2. Reviewer #2 (Public review):

      In this study, Cai and colleagues investigate how one component of the m6A methyltransferase complex, the WTAP protein, responds to IFNb stimulation. They find that viral infection or IFNb stimulation induces the transition of WTAP from aggregates to liquid droplets through dephosphorylation by PPP4. This process affects the m6A modification levels of ISG mRNAs and modulates their stability. In addition, the WTAP droplets interact with the transcription factor STAT1 to recruit the methyltransferase complex to ISG promoters and enhance m6A modification during transcription. The investigation dives into a previously unexplored area of how viral infection or IFNb stimulation affects m6A modification on ISGs. The observation that WTAP undergoes a phase transition is significant in our understanding of the mechanisms underlying m6A's function in immunity. However, there are still key gaps that should be addressed to fully accept the model presented.

      Major points:<br /> (1) More detailed analyses on the effects of WTAPsgRNA on the m6A modification of ISGs:<br /> a. A comprehensive summary of the ISGs, including the percentage of ISGs that are m6A-modified,<br /> b. The distribution of m6A modification across the ISGs, and<br /> c. A comparison of the m6A modification distribution in ISGs with non-ISGs.<br /> In addition, since the authors propose a novel mechanism where the interaction between phosphorylated STAT1 and WTAP direct the MTC to the promoter regions of ISGs to facilitate co-transcriptional m6A modification, it is critical to analyze whether the m6A modification distribution holds true in the data.

      (2) Since a key part of the model includes the cytosol-localized STAT1 protein undergoing phosphorylation to translocate to the nucleus to mediate gene expression, the authors should focus on the interaction between phosphorylated STAT1 and WTAP in Figure 4, rather than the unphosphorylated STAT1. Only phosphorylated STAT1 localizes to the nucleus, so the presence of pSTAT1 in the immunoprecipitate is critical for establishing a functional link between STAT1 activation and its interaction with WTAP.

      (3) The authors should include pSTAT1 ChIP-seq and WTAP ChIP-seq on IFNb-treated samples in Figure 5 to allow for a comprehensive and unbiased genomic analysis for comparing the overlaps of peaks from both ChIP-seq datasets. These results should further support for their hypothesis that WTAP interacts with pSTAT1 to enhance m6A modifications on ISGs.

      Minor points:<br /> (1) Since IFNb is primarily known for modulating biological processes through gene transcription, it would be informative if the authors discussed the mechanism of how IFNb would induce the interaction between WTAP and PPP4.

      (2) The authors should include mCherry alone controls in Figure 1D to demonstrate that mCherry does not contribute to the phase separation of WTAP. Does mCherry have or lack a PLD?

      (3) The authors should clarify the immunoprecipitation assays in the methods. For example, the labeling in Fig. 2A suggests that antibodies against WTAP and pan-p were used for two immunoprecipitations. Is that accurate?

      (4) The authors should include overall m6A modification levels quantified of GFPsgRNA and WTAPsgRNA cells, either by mass spectrometry (preferably) or dot blot.

      Comments on revisions:

      The authors thoroughly addressed the aforementioned points during the review process.

    3. Reviewer #3 (Public review):

      Summary:

      This study presents a valuable finding on the mechanism used by WTAP to modulate the IFN-β stimulation. It describes the phase transition of WTAP driven by IFN-β-induced dephosphorylation. The evidence supporting the claims of the authors is solid.

      Strength:

      The key finding is the revelation that WTAP undergoes phase separation during virus infection or IFN-β treatment. The authors conducted a series of precise experiments to uncover the mechanism behind WTAP phase separation and identified the regulatory role of 5 phosphorylation sites. They also succeeded in pinpointing the phosphatase involved.

    1. Reviewer #2 (Public review):

      Summary:

      TDP-43 mislocalization occurs in nearly all of ALS, roughly half of FTD, and as a co-pathology in roughly half of AD cases. Both gain of function and loss of function mechanisms associated with this mislocalization likely contribute to disease pathogeneisis.

      Here, the authors describe a new method to induce TDP-43 mislocalization in cellular models. They endogenously-tagged TDP-43 with a C-terminal GFP tag in human iPSCs. They then expressed an intrabody - fused with a nuclear export signal (NES) - that targeted GFP to the cytosol. Expression of this intrabody-NES in human iPSC derived neurons induced nuclear depletion of homozygous TDP-43-GFP, caused its mislocalization to the cytosol, and at least in some cells appeared to cause cytosolic aggregates. This mislocalization was accompanied by induction of cryptic exons in well characterized transcripts known to be regulated by TDP-43, a hallmark of functional TDP-43 loss and consistent with pathological nuclear TDP-43 depletion. Interestingly, in heterozygous TDP-43-GFP neurons, expression of intrabody-NES appeared to also induce the mislocalization of untagged TDP-43 in roughly half of the neurons, suggesting that this system can also be used to study effects on untagged endogenous TDP-43 as well as TDP-43-GFP fusion protein.

      Strengths:

      A clearer understanding of how TDP-43 mislocalization alters cellular function, as well as pathways that mitigate clearance of TDP-43 aggregates, is critical. But modeling TDP-43 mislocalization in disease-relevant cellular systems has proven to be challenging. High levels of overexpression of TDP-43 lacking an NES can drive endogenous TDP-43 mislocalization, but such overexpression has direct and artificial consequences on certain cellular features (e.g. altered exon skipping) not seen in diseased patients. Toxic small molecules such as MG132 and arsenite can induce TDP-43 mislocalization, but co-induce myriad additional cellular dysfunctions unrelated to TDP-43 or ALS. TDP-43 binding oligonucleotides can cause cytosolic mislocalization as well. Each system has pros and cons, and additional ways to induce TDP-43 mislocalization would be useful for the field. The method described in this manuscript could provide researchers with a powerful way to study the combined biology of cytosolic TDP-43 mislocalization and nuclear TDP-43 depletion, with additional temporal control that is lacking in current method. Indeed, the author see some evidence of differences in RNA splicing caused by pure TDP-43 depletion versus their induced mislocalization model. Finally, their method may be especially useful in determining how TDP-43 aggregates are cleared by cells, potentially revealing new biological pathways that could be therapeutically targeted.

      Weaknesses:

      The method and supporting data have some limitations.

      • Tagging of TDP-43 with a bulky GFP tag may alter its normal physiological functions, for example, phase separation properties and functions within complex ribonucleoprotein complexes. The authors show that normal splicing function of GFP-TDP-43 is maintained, suggesting that physiology is largely preserved, but other functions and properties of TDP-43 that were not directly tested could be altered.

      • Potential differences in splicing and micro RNAs between TDP-43 knockdown and TDP-43 mislocalization are potentially interesting. However, different patterns of dysregulated RNA splicing can occur at different levels of TDP-knockdown and can differ in different batches of experiments, thus it is difficult to asses whether the changes observed in this paper are due to mislocalization per se, or rather just reflect differences in nuclear TDP-43 abundance or batch effects.

    1. Reviewer #1 (Public review):

      Summary:

      This paper reports an intracranial SEEG study of speech coordination, where participants synchronize their speech output with a virtual partner that is designed to vary its synchronization behavior. This allows the authors to identify electrodes throughout the left hemisphere of the brain that have activity (both power and phase) that correlates with the degree of synchronization behavior. They find that high-frequency activity in secondary auditory cortex (superior temporal gyrus) is correlated to synchronization, in contrast to primary auditory regions. Furthermore, activity in inferior frontal gyrus shows a significant phase-amplitude coupling relationship that is interpreted as compensation for deviation from synchronized behavior with the virtual partner.

      Strengths:<br /> (1) The development of a virtual partner model trained for each individual participant, which can dynamically vary its synchronization to the participant's behavior in real time, is novel and exciting.<br /> (2) Understanding real-time temporal coordination for behaviors like speech is a critical and understudied area.<br /> (3) The use of SEEG provides the spatial and temporal resolution necessary to address the complex dynamics associated with the behavior.<br /> (4) The paper provides some results that suggest a role for regions like IFG and STG in the dynamic temporal coordination of behavior both within an individual speaker and across speakers performing a coordination task.

      Weaknesses:

      (1) The main weakness of the paper is that the results are presented in a largely descriptive and vague manner. For instance, while the interpretation about predictive coding and error correction is interesting, it is not clear how the experimental design or analyses specifically support such a model, or how they differentiate that model from the alternatives. It's possible that some greater specificity could be achieved by a more detailed examination of this rich dataset, for example by characterizing the specific phase relationships (e.g., positive vs negative lags) in areas that show correlations with synchronization behavior. However, as written, it is difficult to understand what these results tell us about how coordination behavior arises.<br /> (2) In the results section, there's a general lack of quantification. While some of the statistics reported in the figures are helpful, there are also claims that are stated without any statistical test. For example, in the paragraph starting on line 342, it is claimed that there is an inverse relationship between rho-value and frequency band, "possibly due to the reversed desynchronization/synchronization process in low and high frequency bands". Based on Figure 3, the first part of this statement appears to be true qualitatively, but is not quantified, and is therefore impossible to assess in relation to the second part of the claim. Similarly, the next paragraph on line 348 describes optimal clustering, but statistics of the clustering algorithm and silhouette metric are not provided. More importantly, it's not entirely clear what is being clustered - is the point to identify activity patterns that are similar within/across brain regions? Or to interpret the meaning of the specific patterns? If the latter, this is not explained or explored in the paper.<br /> (3) Given the design of the stimuli, it would be useful to know more about how coordination relates to specific speech units. The authors focus on the syllabic level, which is understandable. But as far as the results relate to speech planning (an explicit point in the paper), the claims could be strengthened by determining whether the coordination signal (whether error correction or otherwise) is specifically timed to e.g., the consonant vs the vowel. If the mechanism is a phase reset, does it tend to occur on one part of the syllable?<br /> (4) In the discussion the results are related to a previously described speech-induced suppression effect. However, it's not clear what the current results have to do with SIS, since the speaker's own voice is present and predictable from the forward model on every trial. Statements such as "Moreover, when the two speech signals come close enough in time, the patient possibly perceives them as its own voice" are highly speculative and apparently not supported by the data.<br /> (5) There are some seemingly arbitrary decisions made in the design and analysis that, while likely justified, need to be explained. For example, how were the cutoffs for moderate coupling vs phase-shifted coupling (k ~0.09) determined? This is noted as "rather weak" (line 212), but it's not clear where this comes from. Similarly, the ROI-based analyses are only done on regions "recorded in at least 7 patients" - how was this number chosen? How many electrodes total does this correspond to? Is there heterogeneity within each ROI?

      Comments on revisions:

      The authors have generally responded to the critiques from the first round of review, and have provided additional details that help readers to understand what was done.

      In my opinion, the paper still suffers from a lack of clarity about the interpretation, which is partly due to the fact that the results themselves are not straightforward. For example, the heterogeneity across individual electrodes that is obvious from Fig 3 makes it hard to justify the ROI-based approach. And even the electrode clustering, while more data-driven, does not substantially help the fact that the effects appear to be less spatially-organized than the authors may want to claim.

      I recognize the value of introducing this new mutual adaptation paradigm, which is the main strength of the paper. However, the conclusions that can be drawn from the data presented here seem incomplete at best.

    2. Reviewer #2 (Public review):

      Summary:

      This paper investigates the neural underpinnings of an interactive speech task requiring verbal coordination with another speaker. To achieve this, the authors recorded intracranial brain activity from the left (and to a lesser extent, the right) hemisphere in a group of drug-resistant epilepsy patients while they synchronised their speech with a 'virtual partner'. Crucially, the authors were able to manipulate the degree of success of this synchronisation by programming the virtual partner to either actively synchronise or desynchronise their speech with the participant, or else to not vary its speech in response to the participant (making the synchronisation task purely one-way). Using such a paradigm, the authors identified different brain regions that were either more sensitive to the speech of the virtual partner (primary auditory cortex), or more sensitive to the degree of verbal coordination (i.e. synchronisation success) with the virtual partner (left secondary auditory cortex and bilateral IFG). Such sensitivity was measured by (1) calculating the correlation between the index of verbal coordination and mean power within a range of frequency bands across trials, and (2) calculating the phase-amplitude coupling between the behavioural and brain signals within single trials (using the power of high-frequency neural activity only). Overall, the findings help to elucidate some of the brain areas involved in interactive speaking behaviours, particularly highlighting high-frequency activity of the bilateral IFG as a potential candidate supporting verbal coordination.

      Strengths:

      This study provides the field with a convincing demonstration of how to investigate speaking behaviours in more complex situations that share many features with real-world speaking contexts e.g. simultaneous engagement of speech perception and production processes, the presence of an interlocutor and the need for inter-speaker coordination. The findings thus go beyond previous work that has typically studied solo speech production in isolation, and represent a significant advance in our understanding of speech as a social and communicative behaviour. It is further an impressive feat to develop a paradigm in which the degree of cooperativity of the synchronisation partner can be so tightly controlled; in this way, this study combines the benefits of using pre-recorded stimuli (namely, the high degree of experimental control) with the benefits of using a live synchronisation partner (allowing the task to be truly two-way interactive, an important criticism of other work using pre-recorded stimuli). A further key strength of the study lies in its employment of stereotactic EEG to measure brain responses with both high temporal and spatial resolution, an ideal method for studying the unfolding relationship between neural processing and this dynamic coordination behaviour.

      Weaknesses:

      One limitation of the current study is the relatively sparse coverage of the right hemisphere by the implanted electrodes (91 electrodes in the right compared to 145 in the left). Of course, electrode location is solely clinically motivated, and so the authors did not have control over this. In a previous version of this article, the authors therefore chose not to include data from the right hemisphere in reported analyses. However, after highlighting previous literature suggesting that the right hemisphere likely has high relevance to verbal coordination behaviours such as those under investigation here, the authors have now added analyses of the right hemisphere data to the results. These confirm an involvement of the right hemisphere in this task, largely replicating left hemisphere results. Some hemispheric differences were found in responses within the STG; however, interpretation should be tempered by an awareness of the relatively sparse coverage of the right hemisphere meaning that some regions have very few electrodes, resulting in reduced statistical power.

    1. Reviewer #1 (Public review):

      Summary:

      The authors set out to explore the role of upstream open reading frames (uORFs) in stabilizing protein levels during Drosophila development and evolution. By utilizing a modified ICIER model for ribosome translation simulations and conducting experimental validations in Drosophila species, the study investigates how uORFs buffer translational variability of downstream coding sequences. The findings reveal that uORFs significantly reduce translational variability, which contributes to gene expression stability across different biological contexts and evolutionary timeframes.

      Strengths:

      (1) The study introduces a sophisticated adaptation of the ICIER model, enabling detailed simulation of ribosomal traffic and its implications for translation efficiency.<br /> (2) The integration of computational predictions with empirical data through knockout experiments and translatome analysis in Drosophila provides a compelling validation of the model's predictions.<br /> (3) By demonstrating the evolutionary conservation of uORFs' buffering effects, the study provides insights that are likely applicable to a wide range of eukaryotes.

      Weaknesses:

      (1) Although the study is technically sound, it does not clearly articulate the mechanisms through which uORFs buffer translational variability. A clearer hypothesis detailing the potential molecular interactions or regulatory pathways by which uORFs influence translational stability would enhance the comprehension and impact of the findings.<br /> (2) The study could be further improved by a discussion regarding the evolutionary selection of uORFs. Specifically, it would be beneficial to explore whether uORFs are favored evolutionarily primarily for their role in reducing translation efficiency or for their capability to stabilize translation variability. Such a discussion would provide deeper insights into the evolutionary dynamics and functional significance of uORFs in genetic regulation.

      Comments on revisions:

      The authors have adequately addressed my previous concerns.

    2. Reviewer #2 (Public review):

      uORFs, short open reading frames located in the 5' UTR, are pervasive in genomes. However, their roles in maintaining protein abundance are not clear. In this study, the authors propose that uORFs act as "molecular dam", limiting the fluctuation of the translation of downstream coding sequences. First, they performed in silico simulations using an improved ICIER model, and demonstrated that uORF translation reduces CDS translational variability, with buffering capacity increasing in proportion to uORF efficiency, length, and number. Next, they analysed the translatome between two related Drosophila species, revealing that genes with uORFs exhibit smaller fluctuations in translation between the two species and across different developmental stages within the same species. Moreover, they identified that bicoid, a critical gene for Drosophila development, contains a uORF with substantial changes in translation efficiency. Deleting this uORF in Drosophila melanogaster significantly affected its gene expression, hatching rates, and survival under stress conditions. Lastly, by leveraging public Ribo-seq data, the authors showed that the buffering effect of uORFs is also evident between primates and within human populations. Collectively, the study significantly advances our understanding of how uORFs regulate the translation of downstream coding sequences at the genome-wide scale, as well as during development and evolution. It would be particularly interesting to explore whether similar buffering functions are conserved in other organisms, and whether their regulatory effects could be harnessed for practical applications, such as improving crop traits or benefiting human health.

      Comments on revisions:

      The authors have fully addressed all of my concerns, and the revisions have substantially improved the manuscript. I have no further comments.

    1. Reviewer #1 (Public review):

      Summary:

      This study aimed at replicating two previous findings that showed (1) a link between prediction tendencies and neural speech tracking, and (2) that eye movements track speech. The main findings were replicated which supports the robustness of these results. The authors also investigated interactions between prediction tendencies and ocular speech tracking, but the data did not reveal clear relationships. The authors propose a framework that integrates the findings of the study and proposes how eye movements and prediction tendencies shape perception.

      Strengths:

      This is a well-written paper that addresses interesting research questions, bringing together two subfields that are usually studied in separation: auditory speech and eye movements. The authors aimed at replicating findings from two of their previous studies, which was overall successful and speaks for the robustness of the findings. The overall approach is convincing, methods and analyses appear to be thorough, and results are compelling.

      Weaknesses:

      Eye movement behavior could have presented in more detail and the authors could have attempted to understand whether there is a particular component in eye movement behavior (e.g., blinks, microsaccades) that drives the observed effects.

    2. Reviewer #2 (Public review):

      Summary

      Schubert et al. recorded MEG and eye tracking activity while participants were listening to stories in single-speaker or multi-speaker speech. In a separate task, MEG was recorded while the same participants were listening to four types of pure tones in either structured (75% predictable) or random (25%) sequences. The MEG data from this task was used to quantify individual 'prediction tendency': the amount by which the neural signal is modulated by whether or not a repeated tone was (un)predictable, given the context. In a replication of earlier work, this prediction tendency was found to correlate with 'neural speech tracking' during the main task. Neural speech tracking is quantified as the multivariate relationship between MEG activity and speech amplitude envelope. Prediction tendency did not correlate with 'ocular speech tracking' during the main task. Neural speech tracking was further modulated by local semantic violations in the speech material and by whether or not a distracting speaker was present. The authors suggest that part of the neural speech tracking is mediated by ocular speech tracking. Story comprehension was negatively related with ocular speech tracking.

      Strengths

      This is an ambitious study, and the authors' attempt to integrate the many reported findings related to prediction and attention in one framework is laudable. The data acquisition and analyses appear to be done with great attention to methodological detail. Furthermore, the experimental paradigm used is more naturalistic than was previously done in similar setups (i.e.: stories instead of sentences).

      Weaknesses

      While the analysis pipeline is outlined in much detail, some analysis choices appear ad-hoc and could have been more uniform and/or better motivated (other than: this is what was done before).

    3. Reviewer #3 (Public review):

      I thank the authors for their extensive revision of this paper, and I found some elements greatly improved.<br /> In particular, the authors do embrace a somewhat more speculative tone in the current version, which I think is fitting for this work, as the data seem (to me) to be not fully conclusive. The data set collected here is clearly valuable and unique (and I would encourage the authors to make it publicly available!), however, my overall impression is that the specific analyses reported here might not fully

      Despite the revised description of methods, results and figures, I still have trouble understanding many of the results and the authors conclusive interpretation of them. These are my main reservations:

      (1) Regarding "individual prediction tendency" - thank you for adding clarifying methodological details and showing the data in a new Figure (#2). Honestly, however, I still can't say that I fully understand the result. For example, why is there also a significant response in the random condition as well? And how do you interpret the interesting time-course (with a peak ~200ms prior to the stimulus, and a reduction overtime from there?<br /> Also (I may have missed this, but..) what neural data was used to train the classifier and derive the "prediction tendency" index? Was it just the broadband neural response? Is there a way to know which sensors contributed to this metric (e.g., are they predominantly auditory? Frontal?)? And is there a way to establish the statistical significance of this metric (e.g., how good the decoder actually was in predicting behavioral sensitivity?). I don't see any statistics in the results section describing the individual prediction tendency.

      (2) Regarding the TRF analysis - Thanks for clarifying the approach used to obtain 2-second long "segments" of speech tracking. This is an interesting approach, however I think quite new(?) , and for me it raises a whole new set of questions, as well as additional controls and data that I would have liked to see, to be convinced that results are significant. I will elaborate:

      - Do I understand correctly that you segment the real and predicted neural response into 2-second long segments and then calculate the Pearsons' correlation between them to assess the goodness of the model? This is very unclear, since in the methods section you state only that "the same" analysis was performed as for the full data - but what exactly? Clearly, values will be very different when using such short segments. I feel that additional details are still required (and perhaps data shown) to fully understand the "semantic violation" analysis of TRFs.

      - I would like to reiterate my previous comment regarding the use of permutation tests to verify the validity of TRF-based measures derived. This would be especially important when using new approaches (such as the segmentation used here). The authors argue that this is not needed since this was not done in their previously published study. However, this sounds a bit like "two wrongs make a right" argument... why not just do it, and let us know that this 2-second segmentation approach allows estimating reliable speech tracking?

      - Following up on my previous comment that defining "clusters" as at least two neighboring channels (Figure 3) - the fact that this is a default in Fieldtrip is by no means sufficient justification!. This seems quite liberal to me, especially given the many comparisons performed. Here too, permutations can help to determine the necessary data-driven threshold for corrections. This is of course critical for interpreting the result shown in Figures 3E&G that are critical "take home messages" of the paper - i.e., that the prediction-index from the first part of the experiment is related to speech tracking in the second part of the experiment. To my eyes, this does not look extremely convincing, but perhaps the authors can show more conclusive data to support this (e.g., scatter plots of the betas across participant?).<br /> - A similar point can be made for the effect of semantic violations (though here the scalp-level result is somewhat more clustered). The authors point out that the semantic effect is a "replication" of their result reported in Schubert et al. 2023, but if I am not mistaken the results there were somewhat different (as was the manipulation). It would be nice to explicitly discuss the similarity/difference between these effects.

      (3) Regarding the ocular-TRFs -

      - Maybe this is just me, but I believe that effects that are robust should be clearly visible in the data, without the need for fancy "black-box" statistical models. In the case of the ocular TRFs, it is hard for me to see how these time-courses are not just noise (and, again, a permutation test would have helped to convince me..). The inconsistent results for horizontal and vertical eye-movements vis a vis the experimental conditions (single vs. multi-speaker conditions) don't help either, despite the authors argument that these are "independent" - but why should this be the case, especially if there is nothing really to look at in this task?<br /> - I remain with this scepticism for the mediation-portion of the analysis as well... But perhaps replications from other groups or making the data public will help shed further light on this in the future.

      Minor<br /> - Thanks for adding information about the creation of semantic-violation stimuli. Since the violations and lexical-controls were taken from different audio recordings, it would have been nice to verify that differences between neural responses cannot be attributed to differences in articulations (e.g., by comparing their spectro-temporal properties).

    1. Reviewer #1 (Public review):

      Summary:

      The study tests the conservation of imprinting of the ZBDF2 locus across mammals. ZDBF2 is known to be imprinted in mouse, human and rat. The locus has a unique mechanism of imprinting: although imprinting is conferred by a germline DMR methylated in oocytes, the DMR is upstream to ZDBF2 (at GPR1) and monoallelic methylation of the gDMR does not persist beyond early developmental stages. Instead, a lncRNA (GPR1-AS, also known as Liz in mouse) initiating at the gDMR is expressed transiently in embryos and sets up a secondary DMR (by mechanisms not fully elucidated) that then confers monoallelic expression of ZDBF2 in somatic tissues.

      In this study, the authors first interrogate existing placental RNA-seq datasets from multiple mammalian species, and detect GPR1-AS1 candidate transcripts in human, baboon, macaque and mouse, but not in about a dozen other mammals. Because of the varying depth, quality and nature of these RNA-seq libraries, the ability to definitely detect the GPR1-AS1 lncRNA is not guaranteed; therefore, they generate their own deep, directional RNA-seq data from tissues/embryos from five species, finding evidence of GPR1-AS in rabbit, chimpanzee, but not bovine, pig or opossum. From these surveys, the authors conclude that the lncRNA is present only in Euarchontoglires mammals. To test the association between GPR1-AS and ZDBF2 imprinting, they perform RT-PCR and sequencing in tissue from wallabies and cattle, finding biallelic expression of ZDBF2 in these species that also lack a detected GPR1-AS transcript. From inspection of the genomic location of the GPR1-AS first exon, the authors identify an overlap with a solo LTR of the MER21C retrotransposon family in those species in which the lncRNA is observed, except for some rodents, including mouse. However, they do detect a degree of homology (46%) to the MER21C consensus at the first exon on Liz in mouse. Finally, the authors explore public RNA-seq datasets to show that GPR1-AS is expression transiently during human preimplantation development, an expression dynamic that would be consistent with the induction of monoallelic methylation of a somatic DMR at ZDBF2 and consequent monoallelic expression.

      Strengths:

      The analysis uncovers a novel mechanism by which a retrotransposon-derived LTR may be involved in genomic imprinting.<br /> The genomic analysis is very well executed.<br /> New directional and deeply-sequenced RNA-seq datasets from placenta or trophectoderm of five mammalian species and marsupial embryos, which will be of value to the community.

      Weaknesses:

      Although the genomic analysis is very strong, the study remains entirely correlative. All of the data are descriptive, and much of the analysis is performed on RNA-seq and other datasets from the public domain; a small amount of primary data is generated by the authors.<br /> Evidence that the residual LTR in mouse is functionally relevant for Liz lncRNA expression is lacking.

      Comments on revision:

      The authors have responded very constructively to all points raised by me and the other reviewers. For example, the authors have gone to further, extensive efforts in seeking to identify an LTR at the mouse Liz locus - which is not found - but additional multiple genome alignments provide evidence for sequence conservation consistent with retention of a functional relic of the MER21C in rodent genomes. Moreover, they demonstrate the promoter activity of this mouse sequence region in transfections. They have also demonstrated imprinted expression of ZDBF2 in two additional species - rabbit and rhesus macaque - consistent with their model.

    2. Reviewer #2 (Public review):

      Summary:

      This work concerns the evolution of ZDBF2 imprinting in mammalian species via initiation of GPR1 antisense (AS) transcription from a lineage-specific long-terminal repeat (LTR) retrotransposon. It extends previous work describing the mechanism of ZDBF2 imprinting in mice and humans by demonstrating conservation of GPR1-AS transcripts in rabbits and non-human primates. By identifying the origin of GPR1-AS transcription as the LTR MER21C, the authors claim to account for how imprinting evolved in these species but not in those lacking the MER21C insertion. This illustrates the principle of LTR co-option as a means of evolving new gene regulatory mechanisms, specifically to achieve parent-of-origin allele specific expression (imprinting). Examples of this phenomenon have been described previously, but usually involve initiation of transcription during gametogenesis rather than post-fertilization, as in this work. The findings of this paper are therefore relevant to biologists studying imprinted genes or interested more generally in the evolution of gene regulatory mechanisms.

      Strengths:

      (1) The authors convincingly demonstrate the existence of GPR1-AS orthologs in specific mammalian lineages using high quality RNA-seq libraries collected from diverse mammalian species.

      (2) The authors demonstrate imprinting of the ZDBF2 locus in rabbits and Rhesus macaques using allele-specific expression analysis. The transcription of GPR1-AS orthologs therefore correlates with imprinting of the ZDBF2 locus.

      Weaknesses:

      (1) Experimental evidence directly linking GPR1-AS transcription to ZDBF2 imprinting in rabbits and non-human primates is lacking. Consideration should be given to the challenges associated with studying non-model species and manipulating repeat sequences. Further, this mechanism is established in humans and mice, so the authors' model is arguably sufficiently supported merely by the existence of GPR1-AS orthologs in other mammalian lineages.

    3. Reviewer #3 (Public review):

      Kobayashi et al identify MER21C as a common promoter of GPR1-AS/Liz in Euarchontoglires, which establishes a somatic DMR that controls ZFDB2 imprinting. In mice, MER21C appears to have diverged significantly from its primate counterparts and is no longer annotated as such.

      The authors used high-quality cross-species RNA-seq data to characterise GPR1-AS-like transcripts, which included generating new data in five different species. The association between MER21C/B elements and the promoter of GPR1-AS in most species is clear and convincing. The expression pattern of MER21C/B elements overall further supports their role in enabling correct temporal expression of GPR1-AS during embryonic development.

      In the revised version of the manuscript the authors provided additional support for the common evolutionary origin of the GPR1-AS/Liz promoter between primates and rodents. They also showed a more extensive concordance between the presence of GPR1-AS-like transcripts and ZDBF2 imprinting.

      Altogether, these findings robustly support the conclusions of the paper, shedding light into the events underlying the evolution of imprinting at the ZDBF2 locus.

    1. Reviewer #1 (Public Review):

      Summary:

      Glaser et al present ExA-SPIM, a light-sheet microscope platform with large volumetric coverage (Field of view 85mm^2, working distance 35mm ), designed to image expanded mouse brains in their entirety. The authors also present an expansion method optimized for whole mouse brains, and an acquisition software suite. The microscope is employed in imaging an expanded mouse brain, the macaque motor cortex and human brain slices of white matter.

      This is impressive work, and represents a leap over existing light-sheet microscopes. As an example, it offers a ~ fivefold higher resolution than mesoSPIM (https://mesospim.org/), a popular platform for imaging large cleared samples. Thus while this work is rooted in optical engineering, it manifests a huge step forward and has the potential to become an important tool in the neurosciences.

      Strengths:

      -ExA-SPIM features an exceptional combination of field of view, working distance, resolution and throughput.

      -An expanded mouse brain can be acquired with only 15 tiles, lowering the burden on computational stitching. That the brain does not need to be mechanically sectioned is also seen as an important capability.

      -The image data is compelling, and tracing of neurons has been performed. This demonstrates the potential of the microscope platform.

      Review of the revised manuscript:

      The authors have carefully addressed my previous concerns and suggestions.

    2. Reviewer #2 (Public Review):

      In this manuscript, Glaser et al. describe a new selective plane illumination microscope designed to image a large field of view that is optimized for expanded and cleared tissue samples. For the most part, the microscope design follows a standard formula that is common among many systems (e.g. Keller PJ et al Science 2008, Pitrone PG et al. Nature Methods 2013, Dean KM et al. Biophys J 2015, and Voigt FF et al. Nature Methods 2019). The primary conceptual and technical novelty is to use a detection objective from the metrology industry that has a large field of view and a large area camera. The authors characterize the system resolution, field curvature, and chromatic focal shift by measuring fluorescent beads in a hydrogel and then show example images of expanded samples from mouse, macaque, and human brain tissue.

      Glaser et al. have responded to the reviewer comments by removing some of the overstated claims from the prior manuscript and editing portions of the manuscript text to enhance the clarity. Although the manuscript would be stronger if the authors had been able to provide data that justified the original high-impact claims from the initial publication (e.g. that the images could be used for robust and automated neuronal tracing across large volumes), the amended manuscript text now more closely matches the supporting data. As with the initial submission, I believe that the microscope design and characterization is a useful contribution to the field and the data are quite stunning.

    1. Reviewer #1 (Public review):

      In this paper Weber et al. investigate the role of 4 dopaminergic neurons of the Drosophila larva in mediating the association between an aversive high-salt stimulus and a neutral odor. The 4 DANs belong to the DL1 cluster and innervate non-overlapping compartments of the mushroom body, distinct from those involved in appetitive associative learning. Using specific driver lines for individual neurons, the authors show that activation of the DAN-g1 is sufficient to mimic an aversive memory and it is also necessary to form a high-salt memory of full strength, although optogenetic silencing of this neuron has only a partial phenotype. The authors use calcium imaging to show that the DAN-g1 is not the only DAN responding to salt. DAN-c1 and d1 also respond to salt, but they seem to play no role for the associative memory. DAN-f1, which does not respond to salt, is able to lead to the formation of a memory (if optogenetically activated), but it is not necessary for the salt-odor memory formation in normal conditions. However, when silenced together with DAN-g1, it enhances the memory deficit of DAN-g1. Overall, this work brings evidence of a complex interaction between DL1 DANs in both the encoding of salt signals and their teaching role in associative learning, with none of them being individually necessary and sufficient for both functions.

      Overall, the manuscript contributes interesting results that are useful to understand the organization and function of the dopaminergic system. The behavioral role of the specific DANs is accessed using specific driver lines which allow to test their function individually and in pairs. Moreover, the authors perform calcium imaging to test whether DANs are activated by salt, a prerequisite for inducing a negative association to it. Proper genetic controls are carried across the manuscript.

    2. Reviewer #2 (Public review):

      Summary:

      In this work the authors show that dopaminergic neurons (DANs) from the DL1 cluster in Drosophila larvae are required for the formation of aversive memories. DL1 DANs complement pPAM cluster neurons which are required for the formation of attractive memories. This shows the compartmentalized network organization of how an insect learning center (the mushroom body) encodes memory by integrating olfactory stimuli with aversive or attractive teaching signals. Interestingly, the authors found that the 4 main dopaminergic DL1 neurons act partially redundant, and that single cell ablation did not result in aversive memory defects. However, ablation or silencing of a specific DL1 subset (DAN-f1,g1) resulted in reduced salt aversion learning, which was specific to salt but no other aversive teaching stimuli tested. Importantly, activation of these DANs using an optogenetic approach was also sufficient to induce aversive learning in the presence of high salt. Together with the functional imaging of salt and fructose responses of the individual DANs and the implemented connectome analysis of sensory (and other) inputs to DL1/pPAM DANs this represents a very comprehensive study linking the structural, functional and behavioral role of DL1 DANs. This provides fundamental insight into the function of a simple yet efficiently organized learning center which displays highly conserved features of integrating teaching signals with other sensory cues via dopaminergic signaling.

      Strengths:

      This is a very careful, precise and meticulous study identifying the main larval DANs involved in aversive learning using high salt as a teaching signal. This is highly interesting because it allows to define the cellular substrates and pathways of aversive learning down to the single cell level in a system without much redundancy. It therefore sets the basis to conduct even more sophisticated experiments and together with the neat connectome analysis opens the possibility to unravel different sensory processing pathways within the DL1 cluster and integration with the higher order circuit elements (Kenyon cells and MBONs). The authors' claims are well substantiated by the data and balanced, putting their data in the appropriate context. The authors also implemented neat pathway analyses using the larval connectome data to its full advantage, thus providing network pathways that contribute towards explaining the obtained results.

      Weaknesses:

      Previous comments were fully addressed by the authors.

    3. Reviewer #3 (Public review):

      The study of Weber et al. provides a thorough investigation of the roles of four individual dopamine neurons for aversive associative learning in the Drosophila larva. They focus on the neurons of the DL-1 cluster which already have been shown to signal aversive teaching signals. But the authors go beyond the previous publications and test whether each of these dopamine neurons responds to salt or sugar, is necessary for learning about salt, bitter, or sugar, and is sufficient to induce a memory when optogenetically activated. In addition, previously published connectomic data is used to analyze the synaptic input to each of these dopamine neurons. The authors conclude that the aversive teaching signal induced by salt is distributed across the four DL-1 dopamine neurons, with two of them, DAN-f1 and DAN-g1, being particularly important. Overall, the experiments are well designed and performed, support the authors' conclusions, and deepen our understanding of the dopaminergic punishment system.

      Strengths:

      (1) This study provides, at least to my knowledge, the first in vivo imaging of larval dopamine neurons in response to tastants. Although the selection of tastants is limited, the results close an important gap in our understanding of the function of these neurons.

      (2) The authors performed a large number of experiments to probe for the necessity of each individual dopamine neuron, as well as combinations of neurons, for associative learning. This includes two different training regimen (1 or 3 trials), three different tastants (salt, quinine and fructose) and two different effectors, one ablating the neuron, the other one acutely silencing it. This thorough work is highly commendable, and the results prove that it was worth it. The authors find that only one neuron, DAN-g1, is partially necessary for salt learning when acutely silenced, whereas a combination of two neurons, DAN-f1 and DAN-g1, are necessary for salt learning when either being ablated or silenced.

      (3) In addition, the authors probe whether any of the DL-1 neurons is sufficient for inducing an aversive memory. They found this to be the case for two of the neurons, largely confirming previous results obtained by a different learning paradigm, parameters and effector.

      (4) This study also takes into account connectomic data to analyze the sensory input that each of the dopamine neurons receives. This analysis provides a welcome addition to previous studies and helps to gain a more complete understanding. The authors find large differences in inputs that each neuron receives, and little overlap in input that the dopamine neurons of the "aversive" DL-1 cluster and the "appetitive" pPAM cluster seem to receive.

      (5) Finally, the authors try to link all the gathered information in order to describe an updated working model of how aversive teaching signals are carried by dopamine neurons to the larva's memory center. This includes important comparisons both between two different aversive stimuli (salt and nociception) and between the larval and adult stages.

    1. Reviewer #1 (Public review):

      Summary:

      In this study, Setogawa et al. employ an auditory discrimination task in freely moving rats, coupled with small animal imaging, electrophysiological recordings, and pharmacological inhibition/lesioning experiments to better understand the role of two striatal subregions: the anterior Dorsal Lateral Striatum (aDLS) and the posterior Ventrolateral Striatum (pVLS), during auditory discrimination learning. Attempting to better understand the contribution of different striatal subregions to sensory discrimination learning strikes me as a highly relevant and timely question, and the data presented in this study are certainly of major interest to the field. The authors have set up a robust behavioral task, systematically tackled the question about a striatal role in learning with multiple observational and manipulative techniques. Additionally, the structured approach the authors take by using neuroimaging to inform their pharmacological manipulation experiments and electrophysiological recordings is a strength.

      Comments on revisions:

      The authors have addressed some concerns raised in the initial review but some remain. In particular it is still unclear what conclusions can be drawn about task-related activity from scans that are performed 30 minutes after the behavioral task. I continue to think that a reorganization/analysis data according to event type would be useful and easier to interpret across the two brain areas, but the authors did not choose to do this. Finally, switching the cue-response association, I am convinced, would help to strengthen this study.

    2. Reviewer #2 (Public review):

      The study by Setogawa et al. aims to understand the role that different striatal subregions belonging to parallel brain circuits have in associative learning and discrimination learning (S-O-R and S-R tasks). Strengths of the study are the use of multiple methodologies to measure and manipulate brain activity in rats, from microPET imaging to excitotoxic lesions and multielectrode recordings across anterior dorsolateral (aDLS), posterior ventral lateral (pVLS)and dorsomedial (DMS) striatum.

      The main conclusions are that the aDLS promotes stimulus-response association and suppresses response-outcome associations. The pVLS is engaged in the formation and maintenance of the stimulus-response association. There is a lot of work done and some interesting findings however, the manuscript can be improved by clarifying the presentation and reasoning. The inclusion of important controls will enhance the rigor of the data interpretation and conclusions.

      Comments on revisions:

      The authors have made important revisions to the manuscript and it has improved in clarity. They also added several figures in the rebuttal letter to answer questions by the reviewers. I would ask that these figures are also made public as part of the authors' response or if not, included in the manuscript.

    1. Reviewer #1 (Public review):

      Summary:

      In Causal associations between plasma proteins and prostate cancer: a Proteome-Wide Mendelian Randomization, the authors present a manuscript which seeks to identify novel markers for prostate cancer through analysis of large biobank-based datasets and to extend this analysis to potential therapeutic targets for drugs. This is an area that is already extensively researched, but remains important, due to the high burden and mortality of prostate cancer globally.

      Strengths:

      The main strengths of the manuscript are the identification and use of large biobank data assets, which provide large numbers of cases and controls, essential for achieving statistical power. The databases used (deCODE, FinnGen, and the UK Biobank) allow for robust numbers of cases and controls. The analytical method chosen, Mendelian Randomization, is appropriate to the problem. Another strength is the integration of multi-omic datasets, here using protein data as well as GWAS sources to integrate genomic and proteomic data.

      Weaknesses:

      The main weaknesses of the manuscript relate to the following areas:

      (1) The failure of the study to analyse the data in the context of other closely related conditions such as benign prostatic hyperplasia (BPH) or lower urinary tract symptoms (LUTS), which have some pathways and biomarkers in common, such as inflammatory pathways (including complement) and specific markers such as KLK3. As a consequence, it is not possible for readers to know whether the findings are specific to prostate cancer or whether they are generic to prostate dysfunction. Given the prevalence of prostate dysfunction (half of men reaching their sixth decade), the potential for false positives and overtreatment from non-specific biomarkers is a major problem, resulting in the evidence presented in this manuscript being weak. Other researchers have addressed this issue using the same data sources as presented here, for example, in this paper, looking at BPH in the UK Biobank population.<br /> https://www.nature.com/articles/s41467-018-06920-9

      (2) There is no discussion of Gleason scores with regard to either biomarkers or therapies, and a general lack of discussion around indolent disease as compared with more aggressive variants. These are crucial issues with regard to the triage and identification of genomically aggressive localized prostate cancers. See, for example, the work set out in: https://doi.org/10.1038/nature20788 .

      (3) An additional issue is that the field of PCa research is fast-moving. The manuscript cites ~80 references, but too few of these are from recent studies, and many important and relevant papers are not included. The manuscript would be much stronger if it compared and contrasted its findings with more recent studies of PCa biomarkers and targets, especially those concerned with multi-omics and those including BPH.

      (4) The Methods section provides no information on how the Controls were selected. There is no Table providing cohort data to allow the reader to know whether there were differences in age, BMI, ethnic grouping, social status or deprivation, or smoking status, between the Cases and Controls. These types of data are generally recorded in Biobank data, so this sort of analysis should be possible, or if not, the authors' inability to construct an appropriately matched set of Controls should be discussed as a Limitation.

      Assessing impact:

      Because of the weaknesses of the approach identified above, without further additions to the manuscript, the likely impact of the work on the field is minimal. There is no significant utility of the methods and data to the community, because the data are pre-existing and are not newly introduced to the community in this work, and Mendelian randomization is a well-described approach in common use, and therefore, the assets and methods described in the manuscript are not novel. With regard to the authors achieving their aims, without assessing specificity and without setting their findings in the context of the latest literature, the authors (and readers) cannot know or assess whether the biomarkers identified or the druggable targets will be useful in the clinic.

      In conclusion, adding additional context and analysis to the manuscript would both help readers interpret and understand the work and would also greatly enhance its significance. For example, the UK Biobank includes data on men with BPH / LUTS, as analysed in this paper, for example, https://doi.org/10.1038/s41467-018-06920-9. By extending this analysis to identify which biomarkers and druggable targets are specific to PCa, and which are generic to prostate dysfunction, the authors would substantially reduce the risks of diagnostic false positives. This would help to manage the risks of inappropriate treatment or overtreatment.

    2. Reviewer #2 (Public review):

      This is potentially interesting work, but the analyses are attempted in a rather scattergun way, with little evident critical thought. The structure of the work (Results before Methods) can work in some manuscripts, but it is not ideal here. The authors discuss results before we know anything about the underlying data that the results come from. It gives the impression that the authors regard data as a resource to be exploited, without really caring where the data comes from. The methods can provide meaningful insights if correctly used, but while I don't have reasons to doubt that the analyses were conducted correctly, findings are presented with little discussion or interpretation. No follow-up analyses are performed.

      In summary, there are likely some gems here, but the whole manuscript is essentially the output from an analytic pipeline.

      Taking the researchers aims in turn:

      (1) Meta-GWAS - while combining two datasets together can provide additional insights, the contribution of this analysis above existing GWAS is not clear. The PRACTICAL consortium has already reported the GWAS of 70% of these data. What additional value does this analysis provide? (Likely some, but it's not clear from the text.) Also, the presentation of results is unclear - authors state that only 5 gene regions contained variants at p<5x10-8, but Figure 1 shows dozens of hits above 5x10-8. Also, the red line in Figure 1 (supposedly at 5x10-8) is misplaced.

      (2) Cross-phenotype analysis. It is not really clear what this analysis is, or why it is done. What is the iCPAGdb? A database? A statistical method? Why would we want to know cross-phenotype associations? What even are these? It seems that the authors have taken data from an online resource and have written a paragraph based on this existing data with little added value.

      (3) PW-MR. I can see the value of this work, but many details are unclear. Was this a two-sample MR using PRACTICAL + FinnGen data for the outcome? How many variants were used in key analyses? Again, the description of results is sparse and gives little added value.

      (4) Colocalization - seems clear to me.

      (5) Additional post-GWAS analyses (pathway + druggability) - again, the analyses seem to be performed appropriately, although little additional insight other than the reporting of output from the methods.

      Minor points:

      (6) The stated motivation for this work is "early detection". But causality isn't necessary for early detection. If the authors are interested in early detection, other analysis approaches are more appropriate.

      (7) The authors state "193 proteins were associated with PCa risk", but they are looking at MR results - these analyses test for disease associations of genetically-predicted levels of proteins, not proteins themselves.

      Strengths:

      The data and methods used are state-of-the-art.

      Weaknesses:

      The reader will have to provide their own translational insight.

    1. Reviewer #1 (Public review):

      Summary:

      This meta-analysis synthesized data from 79 studies across 22 African countries, encompassing over 27,000 breast cancer patients, to estimate 5-year survival rates. The pooled survival rate was 48%, with substantial regional variation, ranging from 64% in Northern Africa to 32% in Western Africa. Survival outcomes were associated with socioeconomic indicators such as education level, Human Development Index (HDI), and Socio-demographic Index (SDI). Although no significant differences in survival were observed between sexes, non-Black Africans had better outcomes. Despite global advances in cancer care, breast cancer survival in Africa has largely stagnated since the early 2010s, underscoring the need for improved healthcare infrastructure, early detection, and equitable access to treatment.

      Strengths:

      The study has several strengths. It features a comprehensive literature search, adherence to the PRISMA reporting guideline, and prospective registration on PROSPERO, including documentation of protocol deviations. The authors employed rigorous meta-analytic techniques, including subgroup analyses and meta-regression, allowing for a nuanced investigation of potential effect modifiers.

      Weaknesses:

      Analyses of crude 5-year survival rates are inherently difficult to interpret, particularly in the absence of key clinical variables such as stage at diagnosis or whether cancers were detected through screening programs. This omission raises concerns about lead time bias, where earlier diagnosis (e.g., via screening) may falsely appear to improve survival without affecting actual mortality. The higher survival seen in North Africa, for example, may reflect earlier diagnosis rather than improved prognosis or care quality. In this context, the age of the study population is another important aspect.

      Relatedly, the representativeness of the included study populations is unclear. The data sources for individual studies - whether from national cancer registries or single tertiary hospitals -are not systematically reported. This distinction is crucial, as survival outcomes differ significantly between population-based and hospital-based cohorts. Without this contextual information, the generalizability of the findings is limited.

      The meta-regression analyses further raise concerns. The authors use study-level covariates (e.g., national HDI, average years of schooling) to explain variation in survival, yet they do not acknowledge the risk of ecological bias. Inferring individual-level effects from aggregated data is methodologically flawed, and the authors' causal interpretation of these associations is inappropriate, especially given the potential for confounding by unmeasured variables at both the individual and study levels.

      The assessment of publication bias is similarly problematic. While funnel plot asymmetry and a significant Egger's test are interpreted as evidence of bias, such methods are unreliable in meta-analyses of observational studies. Smaller studies may differ meaningfully from larger ones, not due to selective reporting, but because they may recruit patients from specialized tertiary centers where outcomes are poorer. The observed relationship between study size and survival may therefore reflect true differences in patient populations, not publication bias.

      Despite claiming to search for gray literature via Google Scholar, no such studies appear in the PRISMA flowchart. This is a missed opportunity. Gray literature - especially reports from cancer registries - could have enhanced the quality and completeness of the data. While cancer registration systems are not available in all African countries, several do exist, and the authors should have made greater efforts to incorporate routine surveillance data where available. Mortality data from vital statistics systems, available in some countries, could also have provided useful context or validation.

      The study's approach to quality assessment is limited. The scoring tool, adapted from Ssentongo et al., conflates completeness of reporting with risk of bias and fails to address key domains such as study population representativeness, selection bias, and lead time bias. Rather than calculating an overall quality score, the authors should have used a structured tool that evaluates risk of bias across defined domains-such as ROBINS-I, ROBINS-E, or tools developed for prevalence studies (e.g., Tonia et al., BMJ Mental Health, 2023). Cochrane guidance and the textbook by Egger, Higgins, and Davey Smith (DOI:10.1002/9781119099369) provide valuable resources for this purpose.

      The cumulative meta-analysis is not particularly informative, considering the massive heterogeneity in survival rates. It would be more meaningful to stratify the analysis by calendar period. In general, with such important heterogeneity, the calculation of an overall estimate does not add much.

      The authors spend quite some time discussing differences in survival between men and women and between the current and the 2018 estimates, despite the fact that the survival rates are similar, with widely overlapping confidence intervals. The use of a Z-test in this context is inappropriate as it ignores the heterogeneity between studies.

      Minor point:

      The terms retrospective and prospective are not particularly helpful - every longitudinal study of survival is retrospective. What the authors mean is whether or not the data were collected within a study designed to address this question, or whether existing data were used that were collected for another purpose. See also DOI: 10.1136/bmj.302.6771.249.

    2. Reviewer #2 (Public review):

      Summary:

      The study provides an updated literature review and meta-analysis for the 5-year survival estimates in breast cancer patients across continental Africa. The findings reveal substantial disparities between regions and other factors, highlighting the disadvantaged areas in Africa and the urgent need to address these inequities across the continent.

      Strengths:

      The main strengths of this study include:<br /> (1) the thorough literature search with an increasing number of included studies that enhances result reliability;<br /> (2) standard and appropriate statistical methods with clear reporting;<br /> (3) a comprehensive discussion.

      Overall, the paper is well-structured, clearly presented, and provides useful insights.

      Weaknesses:

      However, I have a few concerns that I would like the authors to address.

      (1) The conclusion "A country-wise comparison with 2018 estimates suggests a declining survival tendency, with WHO AFRO countries reporting the poorest estimates among other WHO regions." appears to have been drawn from the comparisons across both different regions and different time periods, which is incorrect! As shown in Figure 8, survival in Africa has increased from below 30% (WHO AFRO 2017) to around 50% (AFRICA 2024, presumably the current study). Section 3.5 is confusing and headed in the wrong direction. The key message in Figure 8 is that the current survival estimate in Africa is still lower than that of other WHO regions from a few years ago, highlighting the urgent need to improve survival in Africa.

      (2) The previous review by Ssentongo et al. classified countries into North Africa and sub-Saharan Africa (SSA), regions divided by the Sahara Desert. This classification is not only geographical-based, but also accounts for the significant differences in ethnicity, health system, and socioeconomic factors. North Africa (especially Egypt, Tunisia, Morocco) has better cancer registries, earlier detection, more treatment access, and therefore better survival outcomes (as shown in Figure 2). SSA tends to have worse outcomes, due to later-stage diagnosis, limited pathology, and access barriers. Given that the survival in women with breast cancer is among the lowest for several SSA countries, the study would benefit from an additional comparison between pooled estimates of North African and SSA, and comparisons with previous pooled estimates.

      (3) The authors classified studies under the female group. Females constituted at least 80% of the sample population, and subgroup analysis revealed only a marginal discrepancy in survival rates between the two sexes. However, most of the breast cancer patients and related studies consist predominantly of females. Given the non-negligible differences in various aspects between females and males, sensitivity analyses restricted to studies among females (as in Figure 2-3) would be informative for future research in breast cancer patients.

      (4) Stage at diagnosis and treatment are the strongest prognostic factors for breast cancer survival. Though data regarding these variables are not available for all studies, and it's complicated to compare or pool the results from different studies (as mentioned in the limitation), could the authors perform the regression analyses regarding early vs. late stages, and the percentage of treatment received? These two factors are too significant to overlook in studies on breast cancer survival.

      (5) The authors reported that studies published before 2019 had a higher survival than those conducted from 2019 onwards, which could be misleading and requires further explanation. As the authors noted ─"the year of publication may not be a reliable measure of the effect in question"─ a better approach would be to use the year of inclusion, i.e., the year the studies were conducted.

      (6) Northern and Western Africa both have the highest incidence of breast cancer in Africa, yet their 5-year survival estimates differ substantially. However, the authors have discussed the survival disparities without considering their similarly higher incidence rates. Could this disparity reflect different contributing factors, with the higher incidence rate in Northern Africa resulting from better screening programs (leading to more detections), while that in Western Africa stems from other epidemiological factors despite lower screening participation? Though the incidence rate is not the primary focus of this study, briefly exploring this dichotomy would enhance the discussion and provide valuable insights for readers.

    1. Reviewer #1 (Public review):

      Summary:

      This study uses mesoscale simulations to investigate how membrane geometry regulates the multiphase organization of postsynaptic condensates. It reveals that dimensionality shifts the balance between specific and non-specific interactions, thereby reversing domain morphology observed in vitro versus in vivo.

      Strengths:

      The model is grounded in experimental binding affinities, reproduces key experimental observations in 3D and 2D contexts, and offers mechanistic insight into how geometry and molecular features drive phase behavior.

      Weaknesses:

      The model omits other synaptic components that may influence domain organization and does not extensively explore parameter sensitivity or broader physiological variability.

    2. Reviewer #2 (Public review):

      This is a timely and insightful study aiming to explore the general physical principles for the sub-compartmentalization--or lack thereof--in the phase separation processes underlying the assembly of postsynaptic densities (PSDs), especially the markedly different organizations in three-dimensional (3D) droplets on one hand and the two-dimensional (2D) condensates associated with a cellular membrane on the other. Simulation of a highly simplified model (one bead per protein domain) is carefully executed. Based on a thorough consideration of various control cases, the main conclusion regarding the trade-off between repulsive excluded volume interactions and attractive interactions among protein domains in determining the structures of 3D vs 2D model PSD condensates is quite convincing. The results in this manuscript are novel; however, as it stands, there is substantial room for improvement in the presentation of the background and the findings of this work. In particular, (i) conceptual connections with prior works should be better discussed, (ii) essential details of the model should be clarified, and (iii) the generality and limitations of the authors' approach should be better delineated. Specifically, the following items should be addressed (with the additional references mentioned below cited and discussed):

      (1) Excluded volume effects are referred to throughout the text by various terms and descriptions such as "repulsive force according to the volume" (e.g., in the Introduction), "nonspecific volume interaction", and "volume effects" in this manuscript. This is somewhat curious and not conducive to clarity, because these terms have alternate or connotations of alternate meanings (e.g., in biomolecular modeling, repulsive interactions usually refer to those with longer spatial ranges, such as that between like charges). It will be much clearer if the authors simply refer to excluded volume interactions as excluded volume interactions (or effects).

      (2) Inasmuch as the impact of excluded volume effects on subcompartmentalization of condensates ("multiple phases" in the authors' terminology), it has been demonstrated by both coarse-grained molecular dynamics and field-theoretic simulations that excluded volume is conducive to demixing of molecular species in condensates [Pal et al., Phys Rev E 103:042406 (2021); see especially Figures 4-5 of this reference]. This prior work bears directly on the authors' observation. Its relationship with the present work should be discussed.

      (3) In the present model setup, activation of the CaMKII kinase affects only its binding to GluN2Bc. This approach is reasonable and leads to model predictions that are essentially consistent with the experiment. More broadly, however, do the authors expect activation of the CaMKII kinase to lead to phosphorylation of some of the molecular species involved with PSDs? This may be of interest since biomolecular condensates are known to be modulated by phosphorylation [Kim et al., Science 365:825-829 (2019); Lin et al, eLife 13:RP100284 (2025)].

      (4) The forcefield for confinement of AMPAR/TARP and NMDAR/GluN2Bc to 2D should be specified in the main text. Have the authors explored the sensitivity of their 2D findings on the strength of this confinement?

      (5) Some of the labels in Figure 1 are confusing. In Figure 1A, the structure labeled as AMPAR has the same shape as the structure labeled as TARP in Figure 1B, but TARP is labeled as one of the smaller structures (like small legs) in the lower part of AMPAR in Figure 1A. Does the TARP in Figure 1B correspond to the small structures in the lower part of AMPAR? If so, this should be specified (and better indicated graphically), and in that case, it would be better not to use the same structural drawing for the overall structure and a substructure. The same issue is seen for NMDAR in Figure 1A and GluN2Bc in Figure 1B.

      (6) In addition to clarifying Figure 1, the authors should clarify the usage of AMPAR vs TARP and NMDAR vs GluN2Bc in other parts of the text as well.

      (7) The physics of the authors' model will be much clearer if they provide an easily accessible graphical description of the relative interaction strengths between different domain-representing spheres (beads) in their model. For this purpose, a representation similar to that given by Feric et al., Cell 165:1686-1697 (2016) (especially Figure 6B in this reference) of the pairwise interactions among the beads in the authors' model should be provided as an additional main-text figure. Different interaction schemes corresponding to inactive and activated CAMKII should be given. In this way, the general principles (beyond the PSD system) governing 3D vs 2D multiple-component condensate organization can be made much more apparent.

      (8) Can the authors' rationalization of the observed difference between 3D and 2D model PSD condensates be captured by an intuitive appreciation of the restriction on favorable interactions by steric hindrance and the reduction in interaction cooperativity in 2D vs 3D?

      (9) In the authors' model, the propensity to form 2D condensates is quite weak. Is this prediction consistent with the experiment? Real PSDs do form 2D condensates around synapses.

      (10) More theoretical context should be provided in the Introduction and/or Discussion by drawing connections to pertinent prior works on physical determinants of co-mixing and de-mixing in multiple-component condensates (e.g., amino acid sequence), such as Lin et al., New J Phys 19:115003 (2017) and Lin et al., Biochemistry 57:2499-2508 (2018).

      (11) In the discussion of the physiological/neurological significance of PSD in the Introduction and/or Discussion, for general interest it is useful to point to a recently studied possible connection between the hydrostatic pressure-induced dissolution of model PSD and high-pressure neurological syndrome [Lin et al., Chem Eur J 26:11024-11031 (2020)].

      (12) It is more accurate to use "perpendicular to the membrane" rather than "vertical" in the caption for Figure 3E and other such descriptions of the orientation of the CaMKII hexagonal plane in the text.

    3. Reviewer #3 (Public review):

      Summary:

      In this work, Yamada, Brandani, and Takada have developed a mesoscopic model of the interacting proteins in the postsynaptic density. They have performed simulations, based on this model and using the software ReaDDy, to study the phase separation in this system in 2D (on the membrane) and 3D (in the bulk). They have carefully investigated the reasons behind different morphologies observed in each case, and have looked at differences in valency, specific/non-specific interactions, and interfacial tension.

      Strengths:

      The simulation model is developed very carefully, with strong reliance on binding valency and geometry, experimentally measured affinities, and physical considerations like the hydrodynamic radii. The presented analyses are also thorough, and great effort has been put into investigating different scenarios that might explain the observed effects.

      Weaknesses:

      The biggest weakness of the study, in my opinion, has to do with a lack of more in-depth physical insight about phase separation. For example, the authors express surprise about similar interactions between components resulting in different phase separation in 2D and 3D. This is not surprising at all, as in 3D, higher coordination numbers and more available volume translate to lower free energy, which easily explains phase separation. The role of entropy is also significantly missing from the analyses. When interaction strengths are small, entropic effects play major roles.

      In the introduction, the authors present an oversimplified view of associative and segregative phase transitions based on the attractive and repulsive interactions, and I'm afraid that this view, in which all the observed morphologies should have clear pairwise enthalpic explanations, diffuses throughout the analysis. Meanwhile, I believe the authors correctly identify some relevant effects, where they consider specific/non-specific interactions, or when they investigate the reduced valency of CaMKII in the 2D system.

      Also, I sense some haste in comparing the findings with experimental observations. For example, the authors mention that "For the current four component PSD system, the product of concentrations of each molecule in the dilute phase is in good agreement with that of the experimental concentrations (Table S2)." But the data used here is the dilute phase, which is the remnant of a system prepared at very high concentrations and allowed to phase separate. The errors reported in Table S2 already cast doubt on this comparison. Or while the 2D system is prepared via confining the particles to the vicinity of the membrane, the different diffusive behavior in the membrane, in contrast to the bulk (i.e., the Saffman-Delbrück model), is not considered. This would thus make it difficult to interpret the results of a coupled 2D/3D system and compare them to the actual system.

    1. Reviewer #1 (Public review):

      The authors present a substantial improvement to their existing tool, MorphoNet, intended to facilitate assessment of 3D+t cell segmentation and tracking results, and curation of high-quality analysis for scientific discovery and data sharing. These tools are provided through a user-friendly GUI, making them accessible to biologists who are not experienced coders. Further, the authors have re-developed this tool to be a locally installed piece of software instead of a web interface, making the analysis and rendering of large 3D+t datasets more computationally efficient. The authors evidence the value of this tool with a series of use cases, in which they apply different features of the software to existing datasets and show the improvement to the segmentation and tracking achieved.

      While the computational tools packaged in this software are familiar to readers (e.g., cellpose), the novel contribution of this work is the focus on error correction. The MorphoNet 2.0 software helps users identify where their candidate segmentation and/or tracking may be incorrect. The authors then provide existing tools in a single user-friendly package, lowering the threshold of skill required for users to get maximal value from these existing tools. To help users apply these tools effectively, the authors introduce a number of unsupervised quality metrics that can be applied to a segmentation candidate to identify masks and regions where the segmentation results are noticeably different from the majority of the image.

      This work is valuable to researchers who are working with cell microscopy data that requires high-quality segmentation and tracking, particularly if their data are 3D time-lapse and thus challenging to segment and assess. The MorphoNet 2.0 tool that the authors present is intended to make the iterative process of segmentation, quality assessment, and re-processing easier and more streamlined, combining commonly used tools into a single user interface.

      One of the key contributions of the work is the unsupervised metrics that MorphoNet 2.0 offers for segmentation quality assessment. These metrics are used in the use cases to identify low-quality instances of segmentation in the provided datasets, so that they can be improved with plugins directly in MorphoNet 2.0. However, not enough consideration is given to demonstrating that optimizing these metrics leads to an improvement in segmentation quality. For example, in Use Case 1, the authors report their metrics of interest (Intensity offset, Intensity border variation, and Nuclei volume) for the uncurated silver truth, the partially curated and fully curated datasets, but this does not evidence an improvement in the results. Additional plotting of the distribution of these metrics on the Gold Truth data could help confirm that the distribution of these metrics now better matches the expected distribution.

      Similarly, in Use Case 2, visual inspection leads us to believe that the segmentation generated by the Cellpose + Deli pipeline (shown in Figure 4d) is an improvement, but a direct comparison of agreement between segmented masks and masks in the published data (where the segmentations overlap) would further evidence this.

      We would appreciate the authors addressing the risk of decreasing the quality of the segmentations by applying circular logic with their tool; MorphoNet 2.0 uses unsupervised metrics to identify masks that do not fit the typical distribution. A model such as StarDist can be trained on the "good" masks to generate more masks that match the most common type. This leads to a more homogeneous segmentation quality, without consideration for whether these metrics actually optimize the segmentation

      In Use case 5, the authors include details that the errors were corrected by "264 MorphoNet plugin actions ... in 8 hours actions [sic]". The work would benefit from explaining whether this is 8 hours of human work, trying plugins and iteratively improving, or 8 hours of compute time to apply the selected plugins.

    2. Reviewer #2 (Public review):

      Summary:

      This article presents Morphonet 2.0, a software designed to visualise and curate segmentations of 3D and 3D+t data. The authors demonstrate their capabilities on five published datasets, showcasing how even small segmentation errors can be automatically detected, easily assessed, and corrected by the user. This allows for more reliable ground truths, which will in turn be very much valuable for analysis and training deep learning models. Morphonet 2.0 offers intuitive 3D inspection and functionalities accessible to a non-coding audience, thereby broadening its impact.

      Strengths:

      The work proposed in this article is expected to be of great interest to the community by enabling easy visualisation and correction of complex 3D(+t) datasets. Moreover, the article is clear and well written, making MorphoNet more likely to be used. The goals are clearly defined, addressing an undeniable need in the bioimage analysis community. The authors use a diverse range of datasets, successfully demonstrating the versatility of the software.

      We would also like to highlight the great effort that was made to clearly explain which type of computer configurations are necessary to run the different datasets and how to find the appropriate documentation according to your needs. The authors clearly carefully thought about these two important problems and came up with very satisfactory solutions.

      Weaknesses:

      There is still one concern: the quantification of the improvement of the segmentations in the use cases and, therefore, the quantification of the potential impact of the software. While it appears hard to quantify the quality of the correction, the proposed work would be significantly improved if such metrics could be provided.

      The authors show some distributions of metrics before and after segmentations to highlight the changes. This is a great start, but there seem to be two shortcomings: first, the comparison and interpretation of the different distributions does not appear to be trivial. It is therefore difficult to judge the quality of the improvement from these. Maybe an explanation in the text of how to interpret the differences between the distributions could help. A second shortcoming is that the before/after metrics displayed are the metrics used to guide the correction, so, by design, the scores will improve, but does that accurately represent the improvement of the segmentation? It seems to be the case, but it would be nice to maybe have a better assessment of the improvement of the quality.

    3. Reviewer #3 (Public review):

      Summary:

      A very thorough technical report of a new standalone, open-source software for microscopy image processing and analysis (MorphoNet 2.0), with a particular emphasis on automated segmentation and its curation to obtain accurate results even with very complex 3D stacks, including timelapse experiments.

      Strengths:

      The authors did a good job of explaining the advantages of MorphoNet 2.0, as compared to its previous web-based version and to other software with similar capabilities. What I particularly found more useful to actually envisage these claimed advantages is the five examples used to illustrate the power of the software (based on a combination of Python scripting and the 3D game engine Unity). These examples, from published research, are very varied in both types of information and image quality, and all have their complexities, making them inherently difficult to segment. I strongly recommend the readers to carefully watch the accompanying videos, which show (although not thoroughly) how the software is actually used in these examples.

      Weaknesses:

      Being a technical article, the only possible comments are on how methods are presented, which is generally adequate, as mentioned above. In this regard, and in spite of the presented examples (chosen by the authors, who clearly gave them a deep thought before showing them), the only way in which the presented software will prove valuable is through its use by as many researchers as possible. This is not a weakness per se, of course, but just what is usual in this sort of report. Hence, I encourage readers to download the software and give it time to test it on their own data (which I will also do myself).

      In conclusion, I believe that this report is fundamental because it will be the major way of initially promoting the use of MorphoNet 2.0 by the objective public. The software itself holds the promise of being very impactful for the microscopists' community.

    1. Reviewer #1 (Public review):

      Summary:

      This in situ cryo-ET workflow of selected plant structures provides several detailed strategies using plunge-freezing and the HPF waffle method and lift-out for notoriously difficult samples (compared to cell culture, yeast, and algae, which are far more prevalent in the literature).

      Strengths:

      A very difficult challenge whereby the authors demonstrate successful vitrification of selected plants/structures using waffle and lift-out approaches for cryoET. Because there are relatively few examples of multi-cellular plant cryo-ET in the literature, it is important for the scientific community to be motivated and have demonstrated strategies that it is achievable. This manuscript has a number of very helpful graphics and videos to help guide researchers who would be interested in undertaking that would help shorten the learning curve of admittedly tedious and complex workflows. This is a slow and tedious process, but you have to start somewhere, and I applaud the authors for sharing their experiences with others, and I expect will help other early adopters to come up to speed sooner.

      Weaknesses:

      While important, the specific specimen and cell-types selected that were successful (perhaps other plant specimen and tissues tried were unsuccessful and thus not reported) in this approach did not demonstrate success to broadly applicable to other much more prevalent and interesting and intensive areas plant biology and plant structures (some mentioned in more detail below).

      This manuscript is essentially a protocol paper and in its paragraph form, and even with great graphics, will definitely be difficult to follow and reproduce for a non-expert. Also considering the use of 3 different FIB-SEM platforms and 2 different cryo-FLM platforms, I wonder if a master graphic of the full workflow(s) could be prepared as a supplementary document that walks through the major steps and points to the individual figures at the critical steps to make it more accessible to the broader readership.

      Multiple times in the manuscript, important workflow details seemed to point to and be dependent on two "unpublished" manuscripts:

      (1) Line 583, 755, 790, 847-848, (Poge et al., will soon be published as a protocol).

      (2) Lines 140, 695, 716 (Capitanio et al., will soon be described in a manuscript).

      It is not clear if/when these would be publicly available. It may be important to wait until these papers can be included in published form.

    2. Reviewer #2 (Public review):

      Summary:

      Poge et al. present a workflow for studying plant tissue by combining high-pressure freezing, cryo-fluorescence microscopy, FIB milling, and cryo-electron tomography (cryo-ET). They tested various plant tissues, including Physcomitrium patens, Arabidopsis thaliana, and Limonium bicolor. The authors successfully produce thin lamellae suitable for cryo-ET studies. Using sub-tomogram averaging, they determined the Rubisco structure at subnanometer resolution, demonstrating the potential of this workflow for plant tissue studies.

      Strengths:

      This manuscript is likely the first to systematically apply FIB milling and cryo-ET to plant tissue samples. It provides a detailed methodological description, which is not only valuable for plant tissue studies but also adaptable to a broader range of biological tissue samples. The study compares the plunge freezing method with a high-pressure freezing method, demonstrating that high-pressure freezing can vitrify thick tissues while preserving their native state. Additionally, the authors explore two methods for plant tissue sample preparation, the "waffle" method and in-carrier high-pressure freezing combined with the "lift-out" approach. The "waffle" method is suitable for samples less than 25um, while the in-carrier high-pressure freezing method can process samples up to 100um.

      Weaknesses:

      The described workflow is very complicated and requires special expertise. The success rate of this workflow is not very high, particularly for high-pressure freezing and life-out technology. Further improvements are needed for automation and increasing throughput.

    3. Reviewer #3 (Public review):

      Summary:

      The authors aimed to improve cryo-TEM workflows for plant cells. The authors present details on high-pressure-freezing protocols to vitrify, ion-mill, and image certain plant cell types.

      Strengths:

      Clear step-by-step outline on how to preserve and image cryo samples derived from plants.

      Weaknesses:

      A general current weakness of cryo-TEM is the problem of vitrifying cells that are embedded in tissues. The vast majority of cells in the plant body are currently not accessible to this technology. This is not a weakness of this specific manuscript but a general problem.

      The manuscript is well organized and well written, and the discussion covers practically all questions I had while reading the results section. I only have a few comments, all of which I consider minor.

    1. Reviewer #1 (Public review):

      This is a well-designed and very interesting study examining the impact of imprecise feedback on outcomes in decision-making. I think this is an important addition to the literature, and the results here, which provide a computational account of several decision-making biases, are insightful and interesting.

      I do not believe I have substantive concerns related to the actual results presented; my concerns are more related to the framing of some of the work. My main concern is regarding the assertion that the results prove that non-normative and non-Bayesian learning is taking place. I agree with the authors that their results demonstrate that people will make decisions in ways that demonstrate deviations from what would be optimal for maximizing reward in their task under a strict application of Bayes' rule. I also agree that they have built reinforcement learning models that do a good job of accounting for the observed behavior. However, the Bayesian models included are rather simple, per the author's descriptions, applications of Bayes' rule with either fixed or learned credibility for the feedback agents. In contrast, several versions of the RL models are used, each modified to account for different possible biases. However, more complex Bayes-based models exist, notably active inference, but even the hierarchical Gaussian filter. These formalisms are able to accommodate more complex behavior, such as affect and habits, which might make them more competitive with RL models. I think it is entirely fair to say that these results demonstrate deviations from an idealized and strict Bayesian context; however, the equivalence here of Bayesian and normative is, I think, misleading or at least requires better justification/explanation. This is because a great deal of work has been done to show that Bayes optimal models can generate behavior or other outcomes that are clearly not optimal to an observer within a given context (consider hallucinations for example) but which make sense in the context of how the model is constructed as well as the priors and desired states the model is given.

      As such, I would recommend that the language be adjusted to carefully define what is meant by normative and Bayesian and to recognize that work that is clearly Bayesian could potentially still be competitive with RL models if implemented to model this task. An even better approach would be to directly use one of these more complex modelling approaches, such as active inference, as the comparator to the RL models, though I would understand if the authors would want this to be a subject for future work.

      Abstract:

      The abstract is lacking in some detail about the experiments done, but this may be a limitation of the required word count. If word count is not an issue, I would recommend adding details of the experiments done and the results.<br /> One comment is that there is an appeal to normative learning patterns, but this suggests that learning patterns have a fixed optimal nature, which may not be true in cases where the purpose of the learning (e.g. to confirm the feeling of safety of being in an in-group) may not be about learning accurately to maximize reward. This can be accommodated in a Bayesian framework by modelling priors and desired outcomes. As such, the central premise that biased learning is inherently non-normative or non-Bayesian, I think, would require more justification. This is true in the introduction as well.

      Introduction:

      As noted above, the conceptualization of Bayesian learning being equivalent to normative learning, I think requires further justification. Bayesian belief updating can be biased and non-optimal from an observer perspective, while being optimal within the agent doing the updating if the priors/desired outcomes are set up to advantage these "non-optimal" modes of decision making.

      Results:

      I wonder why the agent was presented before the choice, since the agent is only relevant to the feedback after the choice is made. I wonder if that might have induced any false association between the agent identity and the choice itself. This is by no means a critical point, but it would be interesting to get the authors' thoughts.

      The finding that positive feedback increases learning is one that has been shown before and depends on valence, as the authors note. They expanded their reinforcement learning model to include valence, but they did not modify the Bayesian model in a similar manner. This lack of a valence or recency effect might also explain the failure of the Bayesian models in the preceding section, where the contrast effect is discussed. It is not unreasonable to imagine that if humans do employ Bayesian reasoning that this reasoning system has had parameters tuned based on the real world, where recency of information does matter; affect has also been shown to be incorporable into Bayesian information processing (see the work by Hesp on affective charge and the large body of work by Ryan Smith). It may be that the Bayesian models chosen here require further complexity to capture the situation, just like some of the biases required updates to the RL models. This complexity, rather than being arbitrary, may be well justified by decision-making in the real world.

      The methods mention several symptom scales- it would be interesting to have the results of these and any interesting correlations noted. It is possible that some of the individual variability here could be related to these symptoms, which could introduce precision parameter changes in a Bayesian context and things like reward sensitivity changes in an RL context.

      Discussion:

      (For discussion, not a specific comment on this paper): One wonders also about participants' beliefs about the experiment or the intent of the experimenters. I have often had participants tell me they were trying to "figure out" a task or find patterns even when this was not part of the experiment. This is not specific to this paper, but it may be relevant in the future to try and model participant beliefs about the experiment especially in the context of disinformation, when they might be primed to try and "figure things out".

      As a general comment, in the active inference literature, there has been discussion of state-dependent actions, or "habits", which are learned in order to help agents more rapidly make decisions, based on previous learning. It is also possible that what is being observed is that these habits are at play, and that they represent the cognitive biases. This is likely especially true given, as the authors note, the high cognitive load of the task. It is true that this would mean that full-force Bayesian inference is not being used in each trial, or in each experience an agent might have in the world, but this is likely adaptive on the longer timescale of things, considering resource requirements. I think in this case you could argue that we have a departure from "normative" learning, but that is not necessarily a departure from any possible Bayesian framework, since these biases could potentially be modified by the agent or eschewed in favor of more expensive full-on Bayesian learning when warranted.

      Indeed, in their discussion on the strategy of amplifying credible news sources to drown out low-credibility sources, the authors hint at the possibility of longer-term strategies that may produce optimal outcomes in some contexts, but which were not necessarily appropriate to this task. As such, the performance on this task- and the consideration of true departure from Bayesian processing- should be considered in this wider context.

      Another thing to consider is that Bayesian inference is occurring, but that priors present going in produce the biases, or these biases arise from another source, for example, factoring in epistemic value over rewards when the actual reward is not large. This again would be covered under an active inference approach, depending on how the priors are tuned. Indeed, given the benefit of social cohesion in an evolutionary perspective, some of these "biases" may be the result of adaptation. For example, it might be better to amplify people's good qualities and minimize their bad qualities in order to make it easier to interact with them; this entails a cost (in this case, not adequately learning from feedback and potentially losing out sometimes), but may fulfill a greater imperative (improved cooperation on things that matter). Given the right priors/desired states, this could still be a Bayes-optimal inference at a social level and, as such, may be ingrained as a habit that requires effort to break at the individual level during a task such as this.

      The authors note that this task does not relate to "emotional engagement" or "deep, identity-related issues". While I agree that this is likely mostly true, it is also possible that just being told one is being lied to might elicit an emotional response that could bias responses, even if this is a weak response.

    2. Reviewer #2 (Public review):

      This valuable paper studies the problem of learning from feedback given by sources of varying credibility. The solid combination of experiment and computational modeling helps to pin down properties of learning, although some ambiguity remains in the interpretation of results.

      Summary:

      This paper studies the problem of learning from feedback given by sources of varying credibility. Two bandit-style experiments are conducted in which feedback is provided with uncertainty, but from known sources. Bayesian benchmarks are provided to assess normative facets of learning, and alternative credit assignment models are fit for comparison. Some aspects of normativity appear, in addition to deviations such as asymmetric updating from positive and negative outcomes.

      Strengths:

      The paper tackles an important topic, with a relatively clean cognitive perspective. The construction of the experiment enables the use of computational modeling. This helps to pinpoint quantitatively the properties of learning and formally evaluate their impact and importance. The analyses are generally sensible, and parameter recovery analyses help to provide some confidence in the model estimation and comparison.

      Weaknesses:

      (1) The approach in the paper overlaps somewhat with various papers, such as Diaconescu et al. (2014) and Schulz et al. (forthcoming), which also consider the Bayesian problem of learning and applying source credibility, in terms of theory and experiment. The authors should discuss how these papers are complementary, to better provide an integrative picture for readers.

      Diaconescu, A. O., Mathys, C., Weber, L. A., Daunizeau, J., Kasper, L., Lomakina, E. I., ... & Stephan, K. E. (2014). Inferring the intentions of others by hierarchical Bayesian learning. PLoS computational biology, 10(9), e1003810.<br /> Schulz, L., Schulz, E., Bhui, R., & Dayan, P. Mechanisms of Mistrust: A Bayesian Account of Misinformation Learning. https://doi.org/10.31234/osf.io/8egxh

      (2) It isn't completely clear what the "cross-fitting" procedure accomplishes. Can this be discussed further?

      (3) The Credibility-CA model seems to fit the same as the free-credibility Bayesian model in the first experiment and barely better in the second experiment. Why not use a more standard model comparison metric like the Bayesian Information Criterion (BIC)? Even if there are advantages to the bootstrap method (which should be described if so), the BIC would help for comparability between papers.

      (4) As suggested in the discussion, the updating based on random feedback could be due to the interleaving of trials. If one is used to learning from the source on most trials, the occasional random trial may be hard to resist updating from. The exact interleaving structure should also be clarified (I assume different sources were shown for each bandit pair). This would also relate to work on RL and working memory: Collins, A. G., & Frank, M. J. (2012). How much of reinforcement learning is working memory, not reinforcement learning? A behavioral, computational, and neurogenetic analysis. European Journal of Neuroscience, 35(7), 1024-1035.

      (5) Why does the choice-repetition regression include "only trials for which the last same-pair trial featured the 3-star agent and in which the context trial featured a different bandit pair"? This could be stated more plainly.

      (6) Why apply the "Truth-CA" model and not the Bayesian variant that it was motivated by?

      (7) "Overall, the results from this study support the exact same conclusions (See SI section 1.2) but with one difference. In the discovery study, we found no evidence for learning based on 50%-credibility feedback when examining either the feedback effect on choice repetition or CA in the credibility-CA model (SI 1.2.3)" - this seems like a very salient difference, when the paper reports the feedback effect as a primary finding of interest, though I understand there remains a valence-based difference.

      (8) "Participants were instructed that this feedback would be "a lie 50% of the time but were not explicitly told that this meant it was random and should therefore be disregarded." - I agree that this is a possible explanation for updating from the random source. It is a meaningful caveat.

      (9) "Future studies should investigate conditions that enhance an ability to discard disinformation, such as providing explicit instructions to ignore misleading feedback, manipulations that increase the time available for evaluating information, or interventions that strengthen source memory." - there is work on some of this in the misinformation literature that should be cited, such as the "continued influence effect". For example: Johnson, H. M., & Seifert, C. M. (1994). Sources of the continued influence effect: When misinformation in memory affects later inferences. Journal of experimental psychology: Learning, memory, and cognition, 20(6), 1420.

      (10) Are the authors arguing that choice-confirmation bias may be at play? Work on choice-confirmation bias generally includes counterfactual feedback, which is not present here.

    3. Reviewer #3 (Public review):

      Summary

      This paper investigates how disinformation affects reward learning processes in the context of a two-armed bandit task, where feedback is provided by agents with varying reliability (with lying probability explicitly instructed). They find that people learn more from credible sources, but also deviate systematically from optimal Bayesian learning: They learned from uninformative random feedback, learned more from positive feedback, and updated too quickly from fully credible feedback (especially following low-credibility feedback). Overall, this study highlights how misinformation could distort basic reward learning processes, without appeal to higher-order social constructs like identity.

      Strengths

      (1) The experimental design is simple and well-controlled; in particular, it isolates basic learning processes by abstracting away from social context.

      (2) Modeling and statistics meet or exceed the standards of rigor.

      (3) Limitations are acknowledged where appropriate, especially those regarding external validity.

      (4) The comparison model, Bayes with biased credibility estimates, is strong; deviations are much more compelling than e.g., a purely optimal model.

      (5) The conclusions are interesting, in particular the finding that positivity bias is stronger when learning from less reliable feedback (although I am somewhat uncertain about the validity of this conclusion)

      Weaknesses

      (1) Absolute or relative positivity bias?

      In my view, the biggest weakness in the paper is that the conclusion of greater positivity bias for lower credible feedback (Figure 5) hinges on the specific way in which positivity bias is defined. Specifically, we only see the effect when normalizing the difference in sensitivity to positive vs. negative feedback by the sum. I appreciate that the authors present both and add the caveat whenever they mention the conclusion (with the crucial exception of the abstract). However, what we really need here is an argument that the relative definition is the *right* way to define asymmetry....

      Unfortunately, my intuition is that the absolute difference is a better measure. I understand that the relative version is common in the RL literature; however previous studies have used standard TD models, whereas the current model updates based on the raw reward. The role of the CA parameter is thus importantly different from a traditional learning rate - in particular, it's more like a logistic regression coefficient (as described below) because it scales the feedback but *not* the decay. Under this interpretation, a difference in positivity bias across credibility conditions corresponds to a three-way interaction between the exponentially weighted sum of previous feedback of a given type (e.g., positive from the 75% credible agent), feedback positivity, and condition (dummy coded). This interaction corresponds to the non-normalized, absolute difference.

      Importantly, I'm not terribly confident in this argument, but it does suggest that we need a compelling argument for the relative definition.

      (2) Positivity bias or perseveration?

      A key challenge in interpreting many of the results is dissociating perseveration from other learning biases. In particular, a positivity bias (Figure 5) and perseveration will both predict a stronger correlation between positive feedback and future choice. Crucially, the authors do include a perseveration term, so one would hope that perseveration effects have been controlled for and that the CA parameters reflect true positivity biases. However, with finite data, we cannot be sure that the variance will be correctly allocated to each parameter (c.f. collinearity in regressions). The fact that CA- is fit to be negative for many participants (a pattern shown more strongly in the discovery study) is suggestive that this might be happening. A priori, the idea that you would ever increase your value estimate after negative feedback is highly implausible, which suggests that the parameter might be capturing variance besides that it is intended to capture.

      The best way to resolve this uncertainty would involve running a new study in which feedback was sometimes provided in the absence of a choice - this would isolate positivity bias. Short of that, perhaps one could fit a version of the Bayesian model that also includes perseveration. If the authors can show that this model cannot capture the pattern in Figure 5, that would be fairly convincing.

      (3) Veracity detection or positivity bias?

      The "True feedback elicits greater learning" effect (Figure 6) may be simply a re-description of the positivity bias shown in Figure 5. This figure shows that people have higher CA for trials where the feedback was in fact accurate. But, assuming that people tend to choose more rewarding options, true-feedback cases will tend to also be positive-feedback cases. Accordingly, a positivity bias would yield this effect, even if people are not at all sensitive to trial-level feedback veracity. Of course, the reverse logic also applies, such that the "positivity bias" could actually reflect discounting of feedback that is less likely to be true. This idea has been proposed before as an explanation for confirmation bias (see Pilgrim et al, 2024 https://doi.org/10.1016/j.cognition.2023.105693 and much previous work cited therein). The authors should discuss the ambiguity between the "positivity bias" and "true feedback" effects within the context of this literature....

      The authors get close to this in the discussion, but they characterize their results as differing from the predictions of rational models, the opposite of my intuition. They write:

      Alternative "informational" (motivation-independent) accounts of positivity and confirmation bias predict a contrasting trend (i.e., reduced bias in low- and medium credibility conditions) because in these contexts it is more ambiguous whether feedback confirms one's choice or outcome expectations, as compared to a full-credibility condition.

      I don't follow the reasoning here at all. It seems to me that the possibility for bias will increase with ambiguity (or perhaps will be maximal at intermediate levels). In the extreme case, when feedback is fully reliable, it is impossible to rationally discount it (illustrated in Figure 6A). The authors should clarify their argument or revise their conclusion here.

      (4) Disinformation or less information?

      Zooming out, from a computational/functional perspective, the reliability of feedback is very similar to reward stochasticity (the difference is that reward stochasticity decreases the importance/value of learning in addition to its difficulty). I imagine that many of the effects reported here would be reproduced in that setting. To my surprise, I couldn't quickly find a study asking that precise question, but if the authors know of such work, it would be very useful to draw comparisons. To put a finer point on it, this study does not isolate which (if any) of these effects are specific to *disinformation*, rather than simply _less information._ I don't think the authors need to rigorously address this in the current study, but it would be a helpful discussion point.

      (5) Over-reliance on analyzing model parameters

      Most of the results rely on interpreting model parameters, specifically, the "credit assignment" (CA) parameter. Exacerbating this, many key conclusions rest on a comparison of the CA parameters fit to human data vs. those fit to simulations from a Bayesian model. I've never seen anything like this, and the authors don't justify or even motivate this analysis choice. As a general rule, analyses of model parameters are less convincing than behavioral results because they inevitably depend on arbitrary modeling assumptions that cannot be fully supported. I imagine that most or even all of the results presented here would have behavioral analogues. The paper would benefit greatly from the inclusion of such results. It would also be helpful to provide a description of the model in the main text that makes it very clear what exactly the CA parameter is capturing (see next point).

      (6) RL or regression?

      I was initially very confused by the "RL" model because it doesn't update based on the TD error. Consequently, the "Q values" can go beyond the range of possible reward (SI Figure 5). These values are therefore *not* Q values, which are defined as expectations of future reward ("action values"). Instead, they reflect choice propensities, which are sometimes notated $h$ in the RL literature. This misuse of notation is unfortunately quite common in psychology, so I won't ask the authors to change the variable. However, they should clarify when introducing the model that the Q values are not action values in the technical sense. If there is precedent for this update rule, it should be cited.

      Although the change is subtle, it suggests a very different interpretation of the model.

      Specifically, I think the "RL model" is better understood as a sophisticated logistic regression, rather than a model of value learning. Ignoring the decay term, the CA term is simply the change in log odds of repeating the just-taken action in future trials (the change is negated for negative feedback). The PERS term is the same, but ignoring feedback. The decay captures that the effect of each trial on future choices diminishes with time. Importantly, however, we can re-parameterize the model such that the choice at each trial is a logistic regression where the independent variables are an exponentially decaying sum of feedback of each type (e.g., positive-cred50, positive-cred75, ... negative-cred100). The CA parameters are simply coefficients in this logistic regression.

      Critically, this is not meant to "deflate" the model. Instead, it clarifies that the CA parameter is actually not such an assumption-laden model estimate. It is really quite similar to a regression coefficient, something that is usually considered "model agnostic". It also recasts the non-standard "cross-fitting" approach as a very standard comparison of regression coefficients for model simulations vs. human data. Finally, using different CA parameters for true vs false feedback is no longer a strange and implausible model assumption; it's just another (perfectly valid) regression. This may be a personal thing, but after adopting this view, I found all the results much easier to understand.

    1. Reviewer #1 (Public review):

      The authors identified five complex amacrine cell (CAM) subtypes based on their morphology and synaptic connectivity. It's suggested that the differences in structure may be directly correlated with different functional roles. The authors also describe synaptic compartmentalization in the SFL tract relating to three types of CAM input regions, again implying a specialized role for these cells. The authors also identified neural progenitor cells, which suggests that the octopus's vertical lobe can undergo neurogenesis throughout its life.

      The work presented here is valuable and convincing. Below are some suggestions the authors may wish to incorporate:

      a) Quantitative measurements to define the CAM subtypes<br /> I think the categorization of the CAMs into five subtypes is convincing, however, I wonder how easily these categories could be identified by other researchers. Would it be possible for the authors to include additional quantitative measurements of these cell types to make their categorization less qualitative and more quantitative? For example, density, volume, and orientation of their dendritic fields?

      b) The definition of the neuritic backbone is included in the methods, but I found the term confusing when I first encountered it in the results, so I would suggest adding the definition to the results too.

      c) The authors wrote, 'Note that given the pronounced difference in diameters between the neuritic backbones (208.27 +/-87.95 nm) and axons (121.55 +/- 21.28 nm)'. What figure is this in?

      d) I am slightly confused about how the authors decided on the specific cubes to reflect the different synaptic compartments in the SFL tract. Is this organisation arranged/repeated vertically or horizontally throughout the SFL tract? The location of the cubes looks to me to be chosen at random, so more information here would be helpful.

      e) In Figure 2, could the authors plot the number of synapses per cube to make the result clearer, so that cube 1 has the lowest synaptic density and cube 2 has the highest?

      f) SAMs are ACh and excitatory<br /> The authors refer to SAMs as excitatory cholinergic. They should provide more detailed explanations/citations to back up this claim. Could SAMs be synthesizing any other neurotransmitters? Could there be a subpopulation of inhibitory SAMs?

      g) CAMs are GABA and inhibitory

      The 5 subtypes of CAMs described here have never been directly confirmed to be GABAergic. Could CAMs be synthesizing any other neurotransmitters? Could a subpopulation of CAMs be excitatory? I believe the authors should make this clearer to readers when referring to CAMs, perhaps by saying, 'hypothesized to be inhibitory neurons', or 'putative inhibitory neurons'.

      h) Fast neurotransmitters and neuromodulators<br /> The authors refer to neuromodulatory connections in their summary in Figure 4, however, cephalopod receptors have yet to be extensively functionally characterized, therefore, the role different molecules play as neurotransmitters or neuromodulators is not yet known. For example, many invertebrates are known to have functional diversity in their receptors: C. elegans has both excitatory and inhibitory receptors for a range of neurotransmitters, anionic ACh- and glutamate-gated channels, and cationic peptide-gated channels have also been identified in some molluscs. So, probably the authors should be cautious in speculating about how a particular transmitter/modulator acts in the octopus brain.

      i) In the methods, the authors refer to "an adult Octopus", what age and size was it? I also know this is Octopus vulgaris, but it would be good to specify it here.

      j) A general comment about all figures. All panels should have a letter associated with them to make it easier to refer to them in the text. For example, in Figure 4, please also add letters to the main schematic, the CAM subtypes, and the VL wiring diagram. In addition, D and E are missing boxes on the main schematic. It's also not immediately obvious that A-E are zooms of the larger schematic; perhaps this could be made clearer with colours or arrows. Please also add names to the CAM subtypes.

      a) Typo: 'Additionally, the unique characteristics of LTP in the octopus VL, such as its reliance on a NO-dependent mechanism, independent of de novo protein synthesis, persistent activation of (Turchetti-Maia et al., 2018).'

    2. Reviewer #2 (Public review):

      Summary:

      The paper examines the diversity of complex amacrine neurons in the ventral lobe of the adult octopus brain, a structure involved in learning and memory. The work builds on a recent paper by the authors that described the connectivity of the much larger population of simple amacrine (SAM) interneurons from the same pioneering EM volume.

      Strengths:

      While the EM volume only provides a snapshot of a tiny fraction of an adult octopus' brain, the authors can make specific conclusions and formulate precise hypotheses about neuron function, synaptic pathways, and developmental trajectories. One example is the reconstruction of a putative maturation sequence for the SAM neuronal lineage, based on the correlation of soma position and the number of synapses, uncovering a plausible developmental sequence of cell morphologies, with interesting parallels to vertebrate neurogenesis.

      Weaknesses:

      The weakness of the study is that it is examining a relatively small volume (260 × 390 × 27 µm), and several neurons are only incompletely reconstructed. It also remains unclear approximately how many neurons remain to be reconstructed from this volume.

      To improve the presentation, the authors should consider showing videos with the volumetric reconstructions of the different types with their partners/synapses and their relation to the SFL track and SAMs. Such videos would help the reader to appreciate the morphological differences between the cell types. The authors could also consider carrying out further morphological analyses to strengthen their cell-type classification, including Sholl value, radial density of input and output synapses, the number of branch nodes, and similar measures.

    3. Reviewer #3 (Public review):

      (1) The authors described "the excitatory glutamatergic SFL axons and cholinergic SAM inputs". However, the evidence of their transmitter specificity has not been provided. Compelling evidence was neither provided nor discussed in the context of the study.

      (2) Specific interference for inhibitory or excitatory synapses based on EM or other studies must be detailed and elaborated

      (3) Different local microcircuits (submodules) referred to in the text should be better described and more specifically defined.

      (4) I would recommend incorporating a more detailed description of synapses and, especially, synaptic vesicles, clarifying their diversity and similarity across cell subtypes. Are there any differences between cholinergic and glutamatergic synaptic vesicles, postsynaptic densities, or other features...? It would be good, if possible, to explicitly clarify: how many vesicles per different types of synapses? How many synapses per neuron of different types? How many inputs and outputs per a given neuron?

      (5) Authors discuss retrograde messengers like NO? Is there any identifiable morphological type of neuron(s) or synapses that might be nitrergic?

      (6) It would be good to provide separate illustrations showing the detailed organization of any glial cell or different types of glial cells they identified in this study. Authors mainly discuss glial processes but refer to "recognized glial types, such as radial glia and astrocyte-like glia" without specific illustrations, which can be deciphered from their EM data. What are vesicular organizations within different types of glial cells?

      (7) The authors also discuss "supervising inputs of inhibitory (pain) and neuromodulatory (supervising) signals", without any details. It would be important to provide these details in the discussion. Specifically, I suggest incorporating comments about differences/similarities of transmitters and morphology between pain and modulatory pathways/signaling/circuits.

    4. Reviewer #4 (Public review):

      Summary:

      The authors present a follow-up to their initial publication of a volume EM reconstruction of a part of the Octopus vulgaris vertical lobe (VL) (Bidel, Meirovitch et al., eLife 2023). In their previous study, they presented a swath of novel observations pertaining to the neuron types making up the VL and their synaptic connectivity. Here, the authors present an extension of those findings in which they (1) demonstrate that the Complex Amacrine cells (CAMs), which they identified previously, can be grouped into at least 5 distinct subclasses; (2) show that there appears to be distinct compartments in the SFL tract that contain specific synapse types; and (3) present morphological evidence that there may be a neurogenic niche in the VL. The findings are intriguing, advance our understanding of memory circuitry in octopus and across the phylogenetic tree, and open new avenues for deeper investigation.

      Strengths:

      A deeper dissection of the morphologies of CAMs and their distinct complements of synapse types is valuable. The identification of multiple categories of CAMs makes it clearer how the very simple SFL-to-SAM connectivity is likely enriched by a population of diverse interneurons.

      The observation that synapse types may be compartmentalized in the superior frontal lobe tract is an intriguing one, and invites more extensive segmentation and future anatomical studies to further characterize the precise architecture of these compartments.

      Finally, the evidence of the possibility of a neurogenic niche in the VL is exciting as it suggests that ongoing neurogenesis may be a common feature of memory circuitry, perhaps contributing to keeping the representation space of the circuit flexible and adequately sparse.

      Weaknesses:

      A key weakness is the reconstruction and grouping of the CAMs:

      (1) CAMs are relatively few in number compared to SAMs, and as such, only 53 are reconstructed in this study. Of those 53 cells, 18 were not classified into one of the 5 categories the authors designate, begging the question of how robust those categories are.

      (2) Related to (1), in Figure 1B, the proportions given in the bar graph are given cumulatively across the entire population of each category. The proportions should be presented as means within each category to adequately capture the variability of the small sample sizes.

      (3) While the xy dimensions of the serial section EM volume are adequate to capture relatively whole cells and neuronal arbors, the volume is only 27µm thick. Thus, many neurite branches are likely truncated in the z-dimension. This may have contributed to ~1/3 of CAMs eluding categorization. However, it is hard to estimate the effect this may have had without knowing the extent of the truncation. It may be worth the authors' time to count the proportion of CAM neurites that are cut off at the edges of the volume.

      (4) The authors state that CAMs appear to have axons and dendrites based on neurite widths. This is an interesting finding, given that amacrine cells are generally thought to possess only one type of neurite, which both send and receive synaptic potentials, and therefore deserves more attention. Is the distribution of neurite widths indeed bimodally distributed? Can the axons and dendrites be differentiated by examining the presence and absence of synaptic vesicle pools, respectively?

      In Figure 2, the compartmentalization of synapse types is intriguing; however, due to the 3D nature of the data, it is difficult to appreciate clearly from the panels presented. This is particularly true for the suggestion that glia may be forming a barrier around these compartments. This could be rectified by providing Neuroglancer links for these specific reconstructions (neurites, synapses, and glia).

      Lastly, although the identification of a putative neurogenic niche is tantalizing, morphological data alone is only an initial hint. Although the chances are slim, it would be more convincing if the authors could identify any actively dividing cells in the proposed niche. More likely, further work, for instance, immunofluorescence, which the lab has previously shown to be viable in octopus, will be needed to add weight to the claim.

    1. Reviewer #1 (Public review):

      Summary:

      Parise presents another instantiation of the Multisensory Correlation Detector model that can now accept stimulus-level inputs. This is a valuable development as it removes researcher involvement in the characterization/labeling of features and allows analysis of complex stimuli with a high degree of nuance that was previously unconsidered (i.e., spatial/spectral distributions across time). The author demonstrates the power of the model by fitting data from dozens of previous experiments, including multiple species, tasks, behavioral modalities, and pharmacological interventions.

      Strengths:

      One of the model's biggest strengths, in my opinion, is its ability to extract complex spatiotemporal co-relationships from multisensory stimuli. These relationships have typically been manually computed or assigned based on stimulus condition and often distilled to a single dimension or even a single number (e.g., "-50 ms asynchrony"). Thus, many models of multisensory integration depend heavily on human preprocessing of stimuli, and these models miss out on complex dynamics of stimuli; the lead modality distribution apparent in Figures 3b and c is provocative. I can imagine the model revealing interesting characteristics of the facial distribution of correlation during continuous audiovisual speech that have up to this point been largely described as "present" and almost solely focused on the lip area.

      Another aspect that makes the MCD stand out among other models is the biological inspiration and generalizability across domains. The model was developed to describe a separate process - motion perception - and in a much simpler organism - Drosophila. It could then describe a very basic neural computation that has been conserved across phylogeny (which is further demonstrated in the ability to predict rat, primate, and human data) and brain area. This aspect makes the model likely able to account for much more than what has already been demonstrated with only a few tweaks akin to the modifications described in this and previous articles from Parise.

      What allows this potential is that, as Parise and colleagues have demonstrated in those papers since our (re)introduction of the model in 2016, the MCD model is modular - both in its ability to interface with different inputs/outputs and its ability to chain MCD units in a way that can analyze spatial, spectral, or any other arbitrary dimension of a stimulus. This fact leaves wide open the possibilities for types of data, stimuli, and tasks a simplistic, neutrally inspired model can account for.

      And so it's unsurprising (but impressive!) that Parise has demonstrated the model's ability here to account for such a wide range of empirical data from numerous tasks (synchrony/temporal order judgement, localization, detection, etc.) and behavior types (manual/saccade responses, gaze, etc.) using only the stimulus and a few free parameters. This ability is another of the model's main strengths that I think deserves some emphasis: it represents a kind of validation of those experiments, especially in the context of cross-experiment predictions (but see some criticism of that below).

      Finally, what is perhaps most impressive to me is that the MCD (and the accompanying decision model) does all this with very few (sometimes zero) free parameters. This highlights the utility of the model and the plausibility of its underlying architecture, but also helps to prevent extreme overfitting if fit correctly (but see a related concern below).

      Weaknesses:

      There is an insufficient level of detail in the methods about model fitting. As a result, it's unclear what data the models were fitted and validated on. Were models fit individually or on average group data? Each condition separately? Is the model predictive of unseen data? Was the model cross-validated? Relatedly, the manuscript mentions a randomization test, but the shuffled data produces model responses that are still highly correlated to behavior despite shuffling. Could it be that any stimulus that varies in AV onset asynchrony can produce a psychometric curve that matches any other task with asynchrony judgements baked into the task? Does this mean all SJ or TOJ tasks produce correlated psychometric curves? Or more generally, is Pearson's correlation insensitive to subtle changes here, considering psychometric curves are typically sigmoidal? Curves can be non-overlapping and still highly correlated if one is, for example, scaled differently. Would an error term such as mean-squared or root mean-squared error be more sensitive to subtle changes in psychometric curves? Alternatively, perhaps if the models aren't cross-validated, the high correlation values are due to overfitting?

      While the model boasts incredible versatility across tasks and stimulus configurations, fitting behavioral data well doesn't mean we've captured the underlying neural processes, and thus, we need to be careful when interpreting results. For example, the model produces temporal parameters fitting rat behavior that are 4x faster than when fitting human data. This difference in slope and a difference at the tails were interpreted as differences in perceptual sensitivity related to general processing speeds of the rat, presumably related to brain/body size differences. While rats no doubt have these differences in neural processing speed/integration windows, it seems reasonable that a lot of the differences in human and rat psychometric functions could be explained by the (over)training and motivation of rats to perform on every trial for a reward - increasing attention/sensitivity (slope) - and a tendency to make mistakes (compression evident at the tails). Was there an attempt to fit these data with a lapse parameter built into the decisional model as was done in Equation 21? Likewise, the fitted parameters for the pharmacological manipulations during the SJ task indicated differences in the decisional (but not the perceptual) process and the article makes the claim that "all pharmacologically-induced changes in audiovisual time perception" can be attributed to decisional processes "with no need to postulate changes in low-level temporal processing." However, those papers discuss actual sensory effects of pharmacological manipulation, with one specifically reporting changes to response timing. Moreover, and again contrary to the conclusions drawn from model fits to those data, both papers also report a change in psychometric slope/JND in the TOJ task after pharmacological manipulation, which would presumably be reflected in changes to the perceptual (but not the decisional) parameters.

      The case for the utility of a stimulus-computable model is convincing (as I mentioned above), but its framing as mission-critical for understanding multisensory perception is overstated, I think. The line for what is "stimulus computable" is arbitrary and doesn't seem to be followed in the paper. A strict definition might realistically require inputs to be, e.g., the patterns of light and sound waves available to our eyes and ears, while an even more strict definition might (unrealistically) require those stimuli to be physically present and transduced by the model. A reasonable looser definition might allow an "abstract and low-dimensional representation of the stimulus, such as the stimulus envelope (which was used in the paper), to be an input. Ultimately, some preprocessing of a stimulus does not necessarily confound interpretations about (multi)sensory perception. And on the flip side, the stimulus-computable aspect doesn't necessarily give the model supreme insight into perception. For example, the MCD model was "confused" by the stimuli used in our 2018 paper (Nidiffer et al., 2018; Parise & Ernst, 2025). In each of our stimuli (including catch trials), the onset and offset drove strong AV temporal correlations across all stimulus conditions (including catch trials), but were irrelevant to participants performing an amplitude modulation detection task. The to-be-detected amplitude modulations, set at individual thresholds, were not a salient aspect of the physical stimulus, and thus only marginally affected stimulus correlations. The model was of course, able to fit our data by "ignoring" the on/offsets (i.e., requiring human intervention), again highlighting that the model is tapping into a very basic and ubiquitous computational principle of (multi)sensory perception. But it does reveal a limitation of such a stimulus-computable model: that it is (so far) strictly bottom-up.

      The manuscript rightly chooses to focus a lot of the work on speech, fitting the MCD model to predict behavioral responses to speech. The range of findings from AV speech experiments that the MCD can account for is very convincing. Given the provided context that speech is "often claimed to be processed via dedicated mechanisms in the brain," a statement claiming a "first end-to-end account of multisensory perception," and findings that the MCD model can account for speech behaviors, it seems the reader is meant to infer that energetic correlation detection is a complete account of speech perception. I think this conclusion misses some facets of AV speech perception, such as integration of higher-order, non-redundant/correlated speech features (Campbell, 2008) and also the existence of top-down and predictive processing that aren't (yet!) explained by MCD. For example, one important benefit of AV speech is interactions on linguistic processes - how complementary sensitivity to articulatory features in the auditory and visual systems (Summerfield, 1987) allow constraint of linguistic processes (Peelle & Sommers, 2015; Tye-Murray et al., 2007).

      References

      Campbell, R. (2008). The processing of audio-visual speech: empirical and neural bases. Philosophical Transactions of the Royal Society B: Biological Sciences, 363(1493), 1001-1010. https://doi.org/10.1098/rstb.2007.2155<br /> Nidiffer, A. R., Diederich, A., Ramachandran, R., & Wallace, M. T. (2018). Multisensory perception reflects individual differences in processing temporal correlations. Scientific Reports 2018 8:1, 8(1), 1-15. https://doi.org/10.1038/s41598-018-32673-y<br /> Parise, C. V, & Ernst, M. O. (2025). Multisensory integration operates on correlated input from unimodal transient channels. ELife, 12. https://doi.org/10.7554/ELIFE.90841<br /> Peelle, J. E., & Sommers, M. S. (2015). Prediction and constraint in audiovisual speech perception. Cortex, 68, 169-181. https://doi.org/10.1016/j.cortex.2015.03.006<br /> Summerfield, Q. (1987). Some preliminaries to a comprehensive account of audio-visual speech perception. In B. Dodd & R. Campbell (Eds.), Hearing by Eye: The Psychology of Lip-Reading (pp. 3-51). Lawrence Erlbaum Associates.<br /> Tye-Murray, N., Sommers, M., & Spehar, B. (2007). Auditory and Visual Lexical Neighborhoods in Audiovisual Speech Perception: Trends in Amplification, 11(4), 233-241. https://doi.org/10.1177/1084713807307409

    2. Reviewer #2 (Public review):

      Summary:

      Building on previous models of multisensory integration (including their earlier correlation-detection framework used for non-spatial signals), the author introduces a population-level Multisensory Correlation Detector (MCD) that processes raw auditory and visual data. Crucially, it does not rely on abstracted parameters, as is common in normative Bayesian models," but rather works directly on the stimulus itself (i.e., individual pixels and audio samples). By systematically testing the model against a range of experiments spanning human, monkey, and rat data, the authors show that their MCD population approach robustly predicts perception and behavior across species with a relatively small (0-4) number of free parameters.

      Strengths:

      (1) Unlike prior Bayesian models that used simplified or parameterized inputs, the model here is explicitly computable from full natural stimuli. This resolves a key gap in understanding how the brain might extract "time offsets" or "disparities" from continuously changing audio-visual streams.

      (2) The same population MCD architecture captures a remarkable range of multisensory phenomena, from classical illusions (McGurk, ventriloquism) and synchrony judgments, to attentional/gaze behavior driven by audio-visual salience. This generality strongly supports the idea that a single low-level computation (correlation detection) can underlie many distinct multisensory effects.

      (3) By tuning model parameters to different temporal rhythms (e.g., faster in rodents, slower in humans), the MCD explains cross-species perceptual data without reconfiguring the underlying architecture.

      Weaknesses:

      (1) The authors show how a correlation-based model can account for the various multisensory integration effects observed in previous studies. However, a comparison of how the two accounts differ would shed light on the correlation model being an implementation of the Bayesian computations (different levels in Marr's hierarchy) or making testable predictions that can distinguish between the two frameworks. For example, how uncertainty in the cue combined estimate is also the harmonic mean of the unimodal uncertainties is a prediction from the Bayesian model. So, how the MCD framework predicts this reduced uncertainty could be one potential difference (or similarity) to the Bayesian model.

      2) The authors show a good match for cue combination involving 2 cues. While Bayesian accounts provide a direction extension to more cues (also seen empirically, for eg, in Hecht et al. 2008), discussion on how the MCD model extends to more cues would benefit the readers.

      Likely Impact and Usefulness:

      The work offers a compelling unification of multiple multisensory tasks- temporal order judgments, illusions, Bayesian causal inference, and overt visual attention - under a single, fully stimulus-driven framework. Its success with natural stimuli should interest computational neuroscientists, systems neuroscientists, and machine learning scientists. This paper thus makes an important contribution to the field by moving beyond minimalistic lab stimuli, illustrating how raw audio and video can be integrated using elementary correlation analyses.

    1. Reviewer #1 (Public review):

      Summary:

      Identifying drugs that target specific disease phenotypes remains a persistent challenge. Many current methods are only applicable to well-characterized small molecules, such as those with known structures. In contrast, methods based on transcriptional responses offer broader applicability because they do not require prior information about small molecules. Additionally, they can be rapidly applied to new small molecules. One of the most promising strategies involves the use of "drug response signatures"-specific sets of genes whose differential expression can serve as markers for the response to a small molecule. By comparing drug response signatures with expression profiles characteristic of a disease, it is possible to identify drugs that modulate the disease profile, indicating a potential therapeutic connection.

      This study aims to prioritize potential drug candidates and to forecast novel drug combinations that may be effective in treating triple-negative breast cancer (TNBC). Large consortia, such as the LINCS-L1000 project, offer transcriptional signatures across various time points after exposing numerous cell lines to hundreds of compounds at different concentrations. While this data is highly valuable, its direct applicability to pathophysiological contexts is constrained by the challenges in extracting consistent drug response profiles from these extensive datasets. The authors use their method to create drug response profiles for three different TNBC cell lines from LINCS.<br /> To create a more precise, cancer-specific disease profile, the authors highlight the use of single-cell RNA sequencing (scRNA-seq) data. They focus on TNBC epithelial cells collected from 26 diseased individuals compared to epithelial cells collected from 10 healthy volunteers. The authors are further leveraging drug response data to develop inhibitor combinations.

      Strengths:

      The authors of this study contribute to an ongoing effort to develop automated, robust approaches that leverage gene expression similarities across various cell lines and different treatment regimen, aiming to predict drug response signatures more accurately. There remains a gap in computational methods for inferring drug responses at the cell subpopulation level, which the authors are trying to address.

      Weaknesses:

      The major deficiencies in this revised manuscript are a lack of benchmarking against established methods, clarification of method limitations, and experimental validation.

      (1) The manuscript still lacks a direct comparison between the retriever tool and well-established methods. How does it perform compared to metaLINCS? Evaluating its performance relative to existing approaches is essential to demonstrate its added value and robustness.<br /> (2) The study remains limited by the absence of experimental validation. Are there supporting data from biological models or clinical trials? Figure 5F is important as this is the validation of the identified compounds in three cell lines. In the previous review, it was noted that the identified drugs had only a modest effect on cell viability. Furthermore, the efficacy of QL-XII-47 and GSK-690693 was found to be cell-line specific-showing activity against BT20 (the cell line used for LINCS transcriptional signature generation) but not against CAL120 and DU4475, which were not included in the signature derivation process. This raises concerns about the tool's ability to predict effective drugs. Additionally, the combination may have an effect because the drugs were tested at high concentrations. How does this effect compare in non-TNBC or normal immortalized breast cell lines? Finally, the DU4475 data were not reproducible, and the experiment must be repeated to ensure reliable comparisons.<br /> (3) A previous review requested a discussion on the limitations of the retriever tool, but the authors instead focused on the well-documented constraints of the LINCS dataset. Clearly defining limitations of the retriever will be critical for evaluating its potential applications and reliability.<br /> (4) Description of the database that the authors used should be corrected. Two examples are below:<br /> "The LINCS-L1000 project published transcriptional profiles of several cell lines." Exploring LINCS metadata will help to introduce the reader to this impressive catalog.<br /> "The portal then returns a ranked list of compounds that are likely to have an inverse effect on disease-associated gene expression levels". When selecting small molecules for use in LINCS-L1000 platform, no link was established between the compounds and disease-associated gene expression levels.<br /> (5) Fig. 3 presents data on differentially expressed genes. However, without indicating whether these genes are up- or downregulated, it is difficult to assess their relevance to TNBC phenotypes and cancer burden.<br /> Additionally, presenting the new Biological Process Gene Ontology analysis in a format similar to Fig. 3C would be beneficial. The statement that these processes are closely related to cancer deregulation is somewhat vague. Instead, the findings may be discussed in relation to each enriched pathway, specifically in the context of TNBC biology and available treatments.

    2. Reviewer #2 (Public review):

      Summary:

      In their study, Osorio and colleagues present 'retriever,' an innovative computational tool designed to extract disease-specific transcriptional drug response profiles from the LINCS-L1000 project. This tool has been effectively applied to TNBC, leveraging single-cell RNA sequencing data to predict drug combinations that may effectively target the disease. The public review highlights the significant integration of extensive pharmacological data with high-resolution transcriptomic information, which enhances the potential for personalized therapeutic applications.

      Strengths:

      A key finding of the study is the prediction and validation of the drug combination QL-XII-47 and GSK-690693 for the treatment of TNBC. The methodology employed is robust, with a clear pathway from data analysis to experimental confirmation.

      Comments on revisions:

      I commend the authors for their thorough and thoughtful revisions, which have significantly strengthened the manuscript. The expanded discussion on the limitations of the LINCS-L1000 dataset and the inherent challenges of imputation techniques provides critical context for interpreting the tool's predictive accuracy. The addition of clinical implications, including strategies for integrating retriever into clinical trial design and its broader applicability to other diseases, enhances the translational relevance of the work. Addressing drug resistance mechanisms in the context of combination therapy further underscores the biological rationale for the approach.

      The transparency regarding computational requirements and ethical considerations-particularly data privacy, bias mitigation, and model validation-demonstrates a responsible and forward-thinking approach to computational biology. These additions not only improve the manuscript's rigor but also set a precedent for ethical practices in personalized medicine research.

      With these revisions, the authors have effectively addressed prior concerns and elevated the impact of their work. The manuscript now presents a compelling case for the retriever as a valuable tool in precision oncology.

    1. Reviewer #2 (Public review):

      In this study, the authors aim to investigate habituation, the phenomenon of increasing reduction in activity following repeated stimuli, in the context of its information theoretic advantage. To this end, they consider a highly simplified three-species reaction network where habituation is encoded by a slow memory variable that suppresses the receptor and therefore the readout activity. Using analytical and numerical methods, they show that in their model the information gain, the difference between the mutual information between the signal and readout after and before habituation, is maximal for intermediate habituation strength. Furthermore, they demonstrate that the Pareto front corresponding to an optimization strategy that maximizes the mutual information between signal and readout in the steady-state and minimizes dissipation in the system also exhibits similar intermediate habituation strength. Finally, they briefly compare predictions of their model to whole-brain recordings of zebrafish larvae under visual stimulation.

      The author's simplified model serves as a good starting point for understanding habituation in different biological contexts as the model is simple enough to allow for some analytic understanding but at the same time exhibits most basic properties of habituation in sensory systems. Furthermore, the author's finding of maximal information gain for intermediate habituation strength via an optimization principle is, in general, interesting. However, the following points remain unclear:

      (1) How general is their finding that the optimal Pareto front coincides with the region of maximal information gain? For instance, what happens if the signal H_st (H_max) isn't very strong? Does it matter that in this case, H_st only has a minor influence on delta Q_R? In the binary switching case, what happens if H_max is rather different from H_st (and not just 20% off)? Or in a case where the adapted value corresponds to the average of H_max and H_min?

      (2) The comparison to experimental data isn't very convincing. For instance, is PCA performed simultaneously on both the experimental data set and on the model or separately? What are the units of the PCs in Fig. 6(b,c)? Given that the model parameters are chosen so that the activity decrease in the model is similar to the one in the data (i.e., that they show similar habituation in terms of the readout), isn't it expected that the dynamics in the PC1/2 space look very similar?

    2. Reviewer #3 (Public review):

      The authors use a generic model framework to study the emergence of habituation and its functional role from information-theoretic and energetic perspectives. Their model features a receptor, readout molecules, and a storage unit, and as such, can be applied to a wide range of biological systems. Through theoretical studies, the authors find that habituation (reduction in average activity) upon exposure to repeated stimuli should occur at intermediate degrees to achieve maximal information gain. Parameter regimes that enable these properties also result in low dissipation, suggesting that intermediate habituation is advantageous both energetically and for the purpose of retaining information about the environment.

      A major strength of the work is the generality of the studied model. The presence of three units (receptor, readout, storage) operating at different time scales and executing negative feedback can be found in many domains of biology, with representative examples well discussed by the authors (e.g. Figure 1b). A key takeaway demonstrated by the authors that has wide relevance is that large information gain and large habituation cannot be attained simultaneously. When energetic considerations are accounted for, large information gain and intermediate habituation appear to be the favorable combination.

      Comments on the revision:

      The authors have adequately addressed the points I raised during the initial review. The text has been clarified at multiple instances, and the treatment of energy expenditure is now more rigorous. The manuscript is much improved both in terms of readability and scientific content.

    1. Reviewer #1 (Public review):

      This is a simple and potentially valuable approach to reduce Cre leak in amplified systems designed to improve CreER use across alleles. The revised work is improved with a direct comparison to the Benedito iSure-Cre line, providing some practical guidance for investigators. The authors do not address the issue of Cre toxicity or mosaic efficiency with low Tamoxifen use.

      The major improvement in my mind is the inclusion of Supp Fig 7 where the authors compare their loxCre to iSureCre. The discussion is somewhat improved, but still fails to discuss significant issues such as Cre toxicity in detail. As noted by most reviewers, without a biological question, the paper is entirely a technical description of a couple of new tools. Whether and to what extent journals such as eLife should publish every new technical innovation without rigorous functional comparison to prior tools is an important question raised by this study. There is already a plethora of available techniques, most of which look better on paper than they function in mice.

      However, I do feel that these tools will be of potential use to the field.

    2. Reviewer #2 (Public review):

      This work presents new genetic tools for enhanced Cre-mediated gene deletion and genetic lineage tracing. The authors optimise and generate mouse models that convert temporally controlled CreER or DreER activity to constitutive Cre expression, coupled with the expression of tdT reporter for the visualizing and tracing of gene-deleted cells. This was achieved by inserting a stop cassette into the coding region of Cre, splitting it into N- and C-terminal segments. Removal of the stop cassette by Cre-lox or Dre-rox recombination results in the generation of modified Cre that is shown to exhibit similar activity to native Cre. The authors further demonstrate efficient gene knockout in cells marked by the reporter using these tools, including intersectional genetic targeting of pericentral hepatocytes.

      The new models offer several important advantages. They enable tightly controlled and highly effective genetic deletion of even alleles that are difficult to recombine. By coupling Cre expression to reporter expression, these models reliably report Cre-expressing i.e. gene-targeted cells and circumvent false positives that can complicate analyses in genetic mutants relying on separate reporter alleles. Moreover, the combinatorial use of Dre/Cre permits intersectional genetic targeting, allowing for more precise fate mapping.

      The study and the new models have also limitations. The demonstration of efficient deletion of multiple floxed alleles in a mosaic fashion, a scenario where the lines would demonstrate their full potential compared to already existing models, has not been tested in the current study. Mosaic genetics is increasingly recognized as a key methodology for assessing cell-autonomous gene functions. The challenge lies in performing such experiments, as low doses of tamoxifen needed for inducing mosaic gene deletion may not be sufficient to efficiently recombine multiple alleles in individual cells while at the same time accurately reporting gene deletion. In addition, as discussed by the authors, a limitation of this line is the constitutive expression of Cre, which is associated with toxicity in some cases.

      Comments on revisions: I have no further comments.

    3. Reviewer #3 (Public review):

      Shi et al describe a new set of tools to facilitate Cre or Dre-recombinase-mediated recombination in mice. The strategies are not completely novel but have been pursued previously by the lab, which is world-leading in this field, and by others. The authors report a new version of the iSuRe-Cre approach, which was originally developed by Rui Benedito's group in Spain. Shi et al describe that their approach shows reduced leakiness compared to the iSuRe-Cre line. Furthermore, a new R26-roxCre-tdT mouse line was established after extensive testing, which enables efficient expression of the Cre recombinase after activation of the Dre recombinase. The authors carefully evaluated efficiency and leakiness of the new line and demonstrated the applicability by marking peri-central hepatocytes in an intersectional genetics approach. The paper represents the result of enormous, carefully executed efforts. Although I would have preferred to see a study which uses the wonderful new tools to address a major biological question, carefully conducted technical studies have an enormous value for the scientific community, clearly justifying publication.

      The new mouse lines generated in this study will enhance the precision of genetic manipulation in distinct cell types and greatly facilitate future work in numerous laboratories. The authors expertly eradicated weaknesses from initial submissions. Remaining open questions regarding potential toxicity of expressing multiple recombinases and fluorescence reports were convincingly answered.

    1. Reviewer #1 (Public review):

      Summary:

      This noteworthy paper examines the role of planar cell polarity and Wnt signalling in body axis formation of the hydrozoan Clytia. In contrast to the freshwater polyp Hydra or the sea anemone Nematostella, Clytia represents a cnidarian model system with a complete life cycle (planula larva-polyp-medusa). In this species, classical experiments have demonstrated that a global polarity is established from the oral end of the embryos (Freeman, 1981). Prior research has demonstrated that Wnt3 plays a role in the formation of the oral organiser in Clytia and other cnidarians, acting in an autocatalytic feedback-loop with β-catenin. However, the question of whether and to what extent an oral-aboral gradient of Wnt activity is established remained unanswered. This gradient is thought to control both tissue differentiation and tissue polarity. The planar cell polarity (PCP) pathway has been linked to this polarity, although it is generally considered to be β-catenin independent.

      Comments on major strengths and weaknesses:

      Beautiful and solid experiments to clarify the role of canonical Wnt signalling and PCP core factors in coordinating planar cell polarity of Clytia. The authors have conducted a series of sophisticated experiments utilising morpholinos, mRNA microinjections and immunofluorescent visualisation of PCP. The objective of these experiments was to address the function of Wnt3, β-catenin and PCP core proteins in the coordination of the global polarity of Clytia embryos. The authors conclude that PCP plays a role in regulating polarity along the oral-aboral axis of embryos and larvae. This offers a conceivable explanation for how polarity information is established and distributed globally during Clytia embryogenesis, with implications for our understanding of axis formation in cnidarians and the evolution of Wnt signalling in general. - While the experiments are well-designed and executed, there are some criticisms, questions or suggestions that should be addressed.

      (i) Wnt3 cue and global PCP. PCP has been described in detail in a previous paper on Clytia (Momose et al, 2012): its orientation along the oral-aboral body axis (ciliary basal body positioning studies), and its function in directional polarity during gastrulation (Stbm-, Fz1-, and Dsh-MO experiments). I wonder if this part could be shortened. What is new, however, are the knockdown and Wnt3-mRNA rescue experiments, which provide a deeper insight into the link between Wnt3 function in the blastopore organiser as a source or cue for axis formation. These experiments demonstrate that the Wnt3 knockdown induces defects equivalent to PCP factor knockdown, but can be rescued by Wnt3-mRNA injection, even at a distance of 200 µm away from the Wnt-positive area. The experimental set-up of these new molecular experiments follows in important aspects those of Freeman's experiments of 1981 (who in turn was motivated to re-examine Teissier's work of 1931/1933 ...). Freeman did not use the term "global polarity" but the concept of an axis-inducing source and a long-range tissue polarity can be traced back to both researchers.

      (ii) PCP propagation and β-catenin. The central but unanswered question in this study focuses on the interaction between Wnt3 and PCP and the propagation of PCP. Wnt3 has been described in cnidarians but also in vertebrates and insects as a canonical Wnt interacting with β-catenin in an autocatalytic loop. The surprising result of this study is that the action of Wnt3 on PCP orientation is not inhibited in the presence of a dominant-negative form of CheTCF (dnTCF) ruling out a potential function of β-catenin in PCP. This was supported by studies with constitutively active β-catenin (CA-β-cat) mRNA which was unable to restore PCP coordination nor elongation of Wnt3-depleted embryos but did restore β-catenin-dependent gastrulation. Based on these data, the authors conclude that Wnt3 has two independent roles: Wnt/β-catenin activation and initial PCP orientation (two step model for PCP formation). However, the molecular basis for the interaction of Wnt3 with the PCP machinery and how the specificity of Wnt3 for both pathways is regulated at the level of Wnt-receiving cells (Fz-Dsh) remains unresolved. - Also, with respect to PCP propagation, there is no answer with respect to the underlying mechanisms. The authors found that PCP components are expressed in the mid-blastula stage, but without any further indication of how the signal might be propagated, e.g., by a wavefront of local cell alignment. Here, it is necessary to address the underlying possible cellular interactions more explicitly.

      (iii) The proposed two step model for PCP formation has important evolutionary implications in that it excludes the current alternate model according to which a long-range Wnt3-gradient orients PCP ("Wnt/β-catenin-first"). Nevertheless, the initial PCP orientation by Wnt3 - as proposed in the two-step-model - is not explained at all on the molecular level. Another possible, but less well discussed and studied option for linking Wnt3 with PCP action could be a role of other Wnt pathways. The authors present compelling evidence that Wnt3 is the most highly expressed Wnt in Clytia at all stages of development. The authors convincingly show that Wnt3 is the most highly expressed Wnt in Clytia at all stages of development (Fig. S1). However, Wnt7 is also more highly expressed, which makes it a candidate for signal transduction from canonical Wnts to PCP Wnts. An involvement of Wnt7 in PCP regulation has been described in vertebrates (http://dx.doi.org/10.1016/j.celrep.2013.12.026). This would challenge the entire discussion and speculation on the evolutionary implications according to which PCP Wnt signaling comes first (PCP-first scenario") and canonical Wnt signaling later in metazoan evolution.

      (iv) The discussion, including Figure 6, is strongly biased towards the traditional evolutionary scenario postulating a choanzoan-sponge ancestry of metazoans. Chromosome-linkage data of pre-metazoans and metazoans (Schulz et al., 2023; https://doi.org/10 (1038/s41586-023-05936-6) now indicate a radically different scenario according to which ctenophores represent the ancestral form and are sister to sponges, cnidarians and bilaterians (the Ctenophora-sister hypothesis). This also has implications for the evolution of Wnt signalling, as discussed in the recent Nature Genetics Review by Holzem et al. (2024) (https://doi.org/10.1038/s41576-024-00699-w). Furthermore, it calls into question the hypothesis of a filter-feeding multicellular gastrula-like ancestor as proposed by Haeckel (Maegele et al., 2023). These papers have not yet been referenced, but they would provide a more robust discussion.

      General appraisal:

      The authors have carefully addressed all important points raised in this review. Aims and results support their conclusions.

      Impact of the work, utility of methods and data:

      As stated above, there will be a major impact on our understanding of the role of Wnt signaling in gradient formation and particularly the role of non canonical wnt signaling. As mentioned above, this will have a major impact on our understanding of the role of Wnt signalling in gradient formation, particularly the role of non-canonical Wnt signalling. - It will also be important to better understand the role of Wnt-Frizzled interactions in these basal organisms, as cnidarians have a smaller repertoire of Frizzled receptors compared to the relatively complete repertoire of Wnt subfamilies. This may imply that Wnt 3 is active in both canonical and PCP.

      Additional context:

      With regard to the question of the evolution of the body plan and Wnt signalling, it would be helpful and important for readers unfamiliar with cnidarians to know that the Hydrozoa/Medusozoa, to which Clytia belongs, are an "evolutionary derived group" within the Cnidaria, as opposed to the Anthozoa (e.g. sea anemone Nematostella). Hydrozoans possess planula larvae that are devoid of a mouth and any form of feeding mechanism, relying instead on the yolk of a fertilised egg for sustenance. The substantial divergence between the Anthozoa and Medusozoa was accompanied by significant gene reductions within the Medusozoa, which likely exerts an influence on the evolution of Wnt signalling in this group as well. This should not detract from the value of the work, but may help to put it in perspective.