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.

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]

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.

for - Medium article - cogress - Part 1 - progress trap - James Gien Wong - definition - cogress - to - Medium article cogress - Part 2 - progress trap - James Gien Wong - https://hyp.is/t8FhpDGAEfC4J7f0NEFujg/medium.com/@gien_SRG/human-cogress-part-2-d6fd075a55c7 - to - Stop Reset Go hypothesis annotations - progress trap - Ronald Wright - https://jonudell.info/h/facet/?max=100&expanded=true&user=stopresetgo&exactTagSearch=true&any=ronald+wright - General - https://jonudell.info/h/facet/?max=100&expanded=true&user=stopresetgo&exactTagSearch=true&any=progress+trap

for - definition - cogress - Medium article - Cogress - Part 2 - Stop Reset Go - Deep Humanity - James Gien Wong - from - Medium article - Cogress - Part 1 - Stop Reset Go - Deep Humanity - James Gien Wong - https://hyp.is/_Nyg2DF_EfCBeu_iuDroYg/medium.com/@gien_SRG/human-cogress-part-1-5159a575e1c4 - to - Stop Reset Go hypothesis annotations - progress trap - Ronald Wright - https://jonudell.info/h/facet/?max=100&expanded=true&user=stopresetgo&exactTagSearch=true&any=ronald+wright - General - https://jonudell.info/h/facet/?max=100&expanded=true&user=stopresetgo&exactTagSearch=true&any=progress+trap

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.

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.

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.

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?

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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?

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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).'

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

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.

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?

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.

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.

Reviewer #1 (Public review):

Summary:

Compelling and clearly described work that combines two elegant cell fate reporter strains with mathematical modelling to describe the kinetics of CD4+ TRM in mice. The aim is to investigate the cell dynamics underlying maintenance of CD4+TRM.

The main conclusions are that 1) CD4+ TRM are not intrinsically long-lived 2) even clonal half lives are short: 1 month for TRM in skin, even shorter (12 days) for TRM in lamina propria 3) TRM are maintained by self-renewal and circulating precursors.

Strengths:

(1) Very clearly and succinctly written. Though in some places too succinctly! See suggestions below for areas I think could benefit from more detail.

(2) Powerful combination of mouse strains and modelling to address questions that are hard to answer with other approaches.

(3) The modelling of different modes of recruitment (quiescent, neutral, division linked) is extremely interesting and often neglected (for simpler neutral recruitment).

Comments on revised version: This reviewer is satisfied with the author responses and the changes made in the manuscript.

Reviewer #1 (Public review):

Summary:

Wang et al. identify Hamlet, a PR-containing transcription factor, as a master regulator of reproductive development in Drosophila. Specifically, the fusion between the gonad and genital disc that is necessary for development of a continuous testes and seminal vesicle tissue essential for fertility. To do so, the authors generate novel Hamlet null mutants by CRISPR/Cas9 gene editing and characterize the morphological, physiological, and gene expression changes of the mutants using immunofluorescence, RNA-seq, cut-tag, and in-situ analysis. Thus, Hamlet is discovered to regulate a unique expression program, which includes Wnt2 and Tl, that is necessary for testis development and fertility.

Strengths:

This is a rigorous and comprehensive study that identifies the Hamlet dependent gene expression program mediating reproductive development in Drosophila. The Hamlet transcription targets are further characterized by Gal4/UAS-RNAi confirming their role in reproductive development. Finally, the study points to a role for Wnt2 and Tl as well as other Hamlet transcriptionally regulated genes in epithelial tissue fusion.

Weaknesses:

None noted.

Reviewer #1 (Public review):

Summary:

In this manuscript the Treisman and colleagues address the question of how protein phosphatase 1 (PP1) regulatory subunits (or PP1-interacting protein (PIPs)) confer specificity on the PP1 catalytic subunit which by itself possesses little substrate specificity. In prior work the authors showed that the PIP Phactrs confers specificity by remodelling a hydrophobic groove immediately adjacent to the PP1 catalytic site through residues within the RVxF- ø ø -R-W string of Phactrs. Specifically, the residues proximal and including the 'W' of the RVxF- ø ø -R-W string remodel the hydrophobic groove. Other residues the of the RVxF- ø ø -R-W string (i.e. the RVxF- ø ø -R) are not involved in this remodelling.

The authors suggest that the RVxF- ø ø -R-W string is a conserved feature of many PIPs including PNUTS, Neurabin/spinophilin and R15A. However from a sequence and structural perspective only the RVxF- ø ø -R- is conserved. The W is not conserved in most and in the R15A structure (PDB:7NZM) the Trp side chain points away from the hydrophobic channel - this could be a questionable interpretation due to model building into the low resolution cryo-EM map (4 A).

In this paper the authors convincingly show that Neurabin confers substrate specificity through interactions of its PDZ domain with the PDZ domain-binding motif (PBM) of 4E-BP. They show the PBM motif is required for Neurabin to increase PP1 activity towards 4E-BP and a synthetic peptide modelled on 4E-BP and also a synthetic peptide based on IRSp53 with a PBM added. The PBM of 4E-BP1 confers high affinity binding to the Neurabin PDZ domain. A crystal structure of a PP1-4E-BP1 fusion with Neurabin shows that the PBM of 4E-BP interacts with the PDZ domain of Neurabin. No interactions of 4E-BP and the catalytic site of PP1 are observed. Cell biology work showed that Neurabin-PP1 regulates the TOR signalling pathway by dephosphorylating 4E-BPs.

Strengths:

This work demonstrates convincingly using a variety of cell biology, proteomics, biophysics and structural biology that the PP1 interacting protein Neurabin confers specificity on PP1 through an interaction of its PDZ domain with a PDZ-binding motif of 4E-BP1 proteins. Remodelling of the hydrophobic groove of the PP1 catalytic subunit is not involved in Neurabin-dependent substrate specificity, in contrast to how Phactrs confers specificity on PP1. The active site of the Neurabin/PP1 complex does not recognise residues in the vicinity of the phospho-residue, thus allowing for multiple phospho-sites on 4E-BP to be dephosphorylated by Neurabin/PP1. This contrasts with substrate specificity conferred by the Phactrs PIP that confers specificity of Phactrs/PP1 towards its substrates in a sequence-specific context by remodelling the hydrophobic groove immediately adjacent to the catalytic. The structural and biochemical insights are used to explore the role of Neurabin/PP1 in dephosphorylation 4E-BPs in vivo, showing that Neurabin/PP1 regulates the TOR signalling pathway, specifically mTORC1-dependent translational control.

Weaknesses:

The only weakness is the suggestion that a conserved RVxF- ø ø -R-W string exists in PIPs. The 'W' is not conserved in sequence and 3-dimensions in most of the PIPs discussed in this manuscript. The lack of conservation of the W would be consistent with the finding based on multiple PP1-PIP structures that apart from Phactrs, no other PIP appears to remodel the PP1 hydrophobic channel.

Comments on revisions:

The authors have addressed my comments.

One aspect of the manuscript and response to reviewers is misleading regarding the statement: 'Like many PIPs, they interact with PP1 using the previously defined "RVxF", "ΦΦ", and "R" motifs (Choy et al, 2014).' This statement, and similar in the authors' response, implies that Choy et al discovered the "RVxF" and "ΦΦ" motifs. The Choy et al, 2014 paper reports the discovery of the "R" motif. The "RVxF" and "ΦΦ" motifs were discovered and reported in earlier papers not cited in the authors' manuscript. Perhaps the authors can correct this.

Reviewer #1 (Public review):

Summary:

This article presents an analysis that challenges established abundance-occupancy relationships (AORs) by utilizing the largest known bird observation database. The analysis yields contentious outcomes, raising the question of whether these findings could potentially refute AORs.

Strengths:

The study employed an extensive aggregation of datasets to date to scrutinize the abundance-occupancy relationships (AORs).

Weaknesses:

The authors should thoroughly address the correlation between checklist data and global range data, ensuring that the foundational assumptions and potential confounding factors are explicitly examined and articulated within the study's context.

In the revision, the authors have refined their findings to birds and provided additional clarifications and discussion. However, the primary concerns raised by reviewers remain inadequately addressed. My main concern continues to be whether testing AOR at a global scale is meaningful given the numerous confounding factors involved. With the current data and analytical approach, these confounders appear inseparable. The study would be significantly strengthened if the authors identified specific conditions under which AORs are valid.

Reviewer #1 (Public review):

Summary:

This paper investigates the physical mechanisms underlying cell intercalation, which then enables collective cell flows in confluent epithelia. The authors show that T1 transitions (the topological transitions responsible for cell intercalation) correspond to the unbinding of groups of hexatic topological defects. Defect unbinding, and hence cell intercalation and collective cell flows, are possible when active stresses in the tissue are extensile. This result helps to rationalize the observation that many epithelial cell layers have been found to exhibit extensile active nematic behavior.

Strengths:

The authors obtain their results based on a combination of active hexanematic hydrodynamics and a multiphase field (MPF) model for epithelial layers, whose connection is a strength of the paper. With the hydrodynamic approach, the authors find the active flow fields produced around hexatic topological defects, which can drive defect unbinding. Using the MPF simulations, the authors show that T1 transitions tend to localize close to hexatic topological defects.

Weaknesses:

Citations are sometimes not comprehensive. Cases of contractile behavior found in collective cell flows, which would seemingly contradict some of the authors' conclusions, are not discussed.

I encourage the authors to address the comments and questions below.

(1) In Equation 1, what do the authors mean by the cluster's size \ell? How is this quantity defined? The calculations in the Methods suggest that \ell indicates the distance between the p-atic defects and the center of the T1 cell cluster, but this is not clearly defined.

(2) The multiphase field model was developed and reviewed already, before the Loewe et al. 2020 paper that the authors cite. Earlier papers include Camley et al. PNAS 2014, Palmieri et al. Sci. Rep. 2015, Mueller et al. PRL 2019, and Peyret et al. Biophys. J. 2019, as reviewed in Alert and Trepat. Annu. Rev. Condens. Matter Phys. 2020.

(3) At what time lag is the mean-squared displacement in Figure 3f calculated? How does the choice of a lag time affect these data and the resulting conclusions?

(4) The authors argue that their results provide an explanation for the extensile behavior of cell layers. However, there are also examples of contractile behavior, such as in Duclos et al., Nat. Phys., 2017 and in Pérez-González et al., Nat. Phys., 2019. In both cases, collective cell flows were observed, which in principle require cell intercalations. How would these observations be rationalized with the theory proposed in this paper? Can these experiments and the theory be reconciled?

Reviewer #1 (Public review):

This manuscript uses a well-validated behavioural estimation task to investigate the degree to which optimistic belief updating was attenuated during the 2020 global pandemic. Online participants estimated how likely different negative life events were to happen to them in the future and were given statistics about these events. Belief updating (measured as the degree to which estimations changed after viewing the statistics) was less optimistically biased during the pandemic (compared to outside of it). This resulted from reduced updating from "good news" (better than expected information). Computational models were used to try to unpack how statistics were integrated and used to revise beliefs. Two families of models were compared - an RL set of models where "estimation errors" (analogous to prediction errors in classic RL models) predict belief change and a Bayesian set of models where an implied likelihood ratio was calculated (derived from participants estimations of their own risk and estimation of the base rate risk) and used to predict belief change. The authors found evidence that the former set of models accounted for updating better outside of the pandemic, but the latter accounted for updating during the pandemic. In addition, the RL model provides evidence that learning was asymmetrically positively biased outside of the pandemic but symmetric during it (as a result of reduced learning rates from good news estimation errors).

Strengths

Understanding whether biases in learning are fixed modes of information processing or flexible and adapt in response to environmental shocks (like a global pandemic or economic recession) is an important area of research relevant to a wide range of fields, including cognitive psychology, behavioural economics, and computational psychiatry. The study uses a well-validated task, and the authors conduct a power analysis to show that the sample sizes are appropriate. Furthermore, the authors test that their results hold in both a between-group analysis (the focus of the main paper) and a within-group analysis (mainly in the supplemental).

The finding that optimistic biases are reduced in response to acute stress, perceived threat, and depression has been shown before using this task both in the lab (social stress manipulation), in the real world (firefighters on duty), and clinical groups (patients with depression). However, the work does extend these findings here in important ways:

(1) Examining the effect of a new real-world adverse event (the pandemic).<br /> (2) The reduction in optimistic updating here arises due to reduced updating from positive information (previously, in the case of environmental threat, this reduction mainly arose from increased sensitivity to negative information).<br /> (3) Leveraging new RL-inspired computational approaches, demonstrating that the bias - and its attenuation - can be captured using trial-by-trial computational modelling with separate learning rates for positive and negative estimation errors.

The authors now take great care to caveat that the findings cannot directly attribute the observed lack of optimistically biased belief updating during lockdown to psychological causes such as heightened anxiety and stress.

The authors have added model recovery results. Whilst there are some cases within a family (RL or Bayesian) of models where they can be confused (e.g., Bayesian model 10-the winning model during the pandemic-sometimes gets confused with Bayesian model 9), there is no confusion between families of models (RL models don't get confused with Bayesian models and vice versa), which is reassuring.

Weaknesses

The authors now conduct model recovery (SI Figure 5) and show how the behaviour of the two best-fitting models (Rational Bayesian model and optimistically biased RL-like model) approximates the actual data observed by showing them alongside each other (Figure 1b). It seems from Figure 1b that the 2 models predict similar behaviour for bad news but diverge for good news, with the optimistically biased RL-like model predicting greater updates than the rational Bayesian model. However, it is difficult to tell from the figure (partly because of the y-axis scale) how much of a divergence this is and how distinctive a pattern relative to the other models. I think the interpretation could be improved further by a clearer sense of the behavioural signatures of each model, enabling them to be reliably teased apart from one another in the model recovery.

Reviewer #1 (Public review):

This study presents valuable findings on the GABA and BOLD changes induced by continuous theta burst stimulation (cTBS) and on the relationships between ATL GABA level and performance in a semantic task. However, I'm afraid that the current results are incomplete to support some primary claims of the paper, for example, the purported inverted-U-shaped relationship between GABA levels in the ATL and semantic task performance. The influence of practice effects also complicates the interpretation of the results. Additional concerns include potential double dipping in the analysis depicted in Figure 3A and the use of inconsistent behavioral measures (IE and accuracy) across various analyses.

The authors have made two beneficial revisions in this round: (1) acknowledging the insufficient data points supporting the inverted U-shaped curve; (2) attempting to control for practice effects. However, I believe unresolved issues remain:

(1) The authors have not addressed my specific concern about Figure 4D - the analysis attempts to fit an inverted U-shaped curve to the data without distinguishing between data points influenced by practice effects and those unaffected, rendering its reliability questionable.

(2) The authors appear to have misunderstood my question regarding Figure 3A. This issue is unrelated to practice effects. My point was that even if we randomly generated pre- and post-test data points and grouped/analyzed them according to the authors' methodology, we would still likely reproduce the pattern in Figure 3A due to the double dipping problem. Thus, this statistical analysis and its conclusions currently lack methodological validity.

(3) Regarding the inconsistency in behavioral measures, the authors' explanation fails to remove my concerns. If the authors argue that accuracy is the most appropriate behavioral dependent variable for this study, why did they employ inverse efficiency in some of their analyses? My understanding is that a study should either consistently use the single most suitable measure or report multiple measures while providing adequate discussion of inconsistent results.

Reviewer #1 (Public review):

Summary:

The results offer compelling evidence that L5-L5 tLTD depends on presynaptic NMDARs, a concept that has previously been somewhat controversial.

It documents the novel finding that presynaptic NMDARs facilitate tLTD through their metabotropic signaling mechanism.

Strengths:

The experimental design is clever and clean.

The approach of comparing the results in cell pairs where NMDA is deleted either presynaptically or postsynaptically is technically insightful and yields decisive data.

The MK801 experiments are also compelling.

Weaknesses:

No major weaknesses were noted by this reviewer.

Reviewer #1 (Public review):

Summary:

The authors describe a role of sumoylation at K81 in p66Shc which affects endothelial dysfunction. This explores a new mechanism for understanding the role of PTMs in cellular processes.

Strengths:

The experiments are well planned and the results are well represented.<br /> Vascular tonality experiments were carried out nicely, given the amount of time and effort one needs to put in to get clean results from these experiments.

Weaknesses:

(1) The production of ROS has been measured in a very superficial way.<br /> The term "ROS" confers a plethora of chemical species which exerts different physiological effects on different cells and situations.<br /> Mitochondria through one of the source , but not the only source of ROS production. Only measuring ROS with mitosox do not reflect the cellular condition of ROS in a specific condition. I would suggest authors consider doing IF of oxidative stress specific markers , carbonyl group and also, maybe, Amplex red for determining average oxidative stress and ros production in the cells.<br /> (2) 8-OHG signal seems very confusing in Figure 7E. 8-ohg is supposed to be mainly in the nucleus and to some extent in mitochondria. The signal is very diffused in the images. I would suggest a higher magnification and better resolution images for 8-ohg. Also, the VWF signal is pretty weak whereas it should be strong given the staining is in aorta. Authors should redo the experiments.<br /> (3) PCA analysis is quite not clear. Why is there a convergence among the plots? Authors should explain. Also, I would suggest that the authors do the analysis done in Figure 8B again with R based packages. IPA, though being user-friendly, mostly does not yield meaningful results and the statistics carried out is not accurate. Authors should redo the analysis in R or Python whichever is suitable for them.<br /> (4) The MS analysis part seems pretty vague in methods. Please rewrite.

Reviewer #1 (Public review):

Summary:

This manuscript assesses the utility of spatial image correlation spectroscopy (ICS) for measuring physiological responses to DNA damage. ICS is a long-established (~1993) method, similar to fluorescence correlation spectroscopy, for deriving information about the fluorophore density that underlies the intensity distributions of images.

The revisions to the current manuscript have improved the understanding of the strengths and limitations of the spatial ICS method. In particular, since the measurements are obtaining complementary information to traditional focus counting, one does not expect a simple linear relationship between the quantities obtained by ICS and by immunostaining. The explanations are satisfactory to me and, I expect, to the interested reader.

Additionally, I am satisfied with the code availability now that it is placed on Github.

Reviewer #2 (Public review):

Summary:

This study aims to explore the ferroptosis-related immune landscape of TNBC through the integration of single-cell and bulk RNA sequencing data, followed by the development of a risk prediction model for prognosis and drug response. The authors identified key subpopulations of immune cells within the TME, particularly focusing on T cells and macrophages. Using machine learning algorithms, the authors constructed a ferroptosis-related gene risk score that accurately predicts survival and the potential response to specific drugs in TNBC patients.

Strengths:

The study identifies distinct subpopulations of T cells and macrophages with differential expression of ferroptosis-related genes. The clustering of these subpopulations and their correlation with patient prognosis is highly insightful, especially the identification of the TREM2+ and FOLR2+ macrophage subtypes, which are linked to either favorable or poor prognoses. The risk model thus holds potential not only for prognosis but also for guiding treatment selection in personalized oncology.

Reviewer #1 (Public review):

Summary:

In this revised report, Yamanaka and colleagues investigate a proposed mechanism by which testosterone modulates seminal plasma metabolites in mice. Based on limited evidence in previous versions of the report, the authors softened the claim that oleic acid derived from seminal vesicle epithelium strongly affects linear progressive motility in isolated cauda epididymal sperm in vitro. Though the report still contains somewhat ambiguous references to the strength of the relationship between fatty acids and sperm motility.

Strengths:

Often, reported epidydimal sperm from mice have lower percent progressive motility compared with sperm retrieved from the uterus or by comparison with human ejaculated sperm. The findings in this report may improve in vitro conditions to overcome this problem, as well as add important physiological context to the role of reproductive tract glandular secretions in modulating sperm behaviors. The strongest observations are related to the sensitivity of seminal vesicle epithelial cells to testosterone. The revisions include the addition of methodological detail, modified language to reflect the nuance of some of the measurements, as well as re-performed experiments with more appropriate control groups. The findings are likely to be of general interest to the field by providing context for follow-on studies regarding the relationship between fatty acid beta oxidation and sperm motility pattern.

Weaknesses:

The connection between media fatty acids and sperm motility pattern remains inconclusive.

Reviewer #1 (Public review):

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

I think the potentially most significant contribution of this database is the manual data curation to fix errors present in the PDB entries, by cross-referencing with the literature. The authors also seem to describe, whenever possible, the procedures they took to correct the annotations.

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

"(1) the species of the antibody in 7WRL was incorrectly labeled as "SARS coronavirus B012" in both PDB and SabDab" → I have verified the mistake and fix, and that SAbDab does not fix is, just uses the pdb annotation.<br /> "(2) 1NSN, the resolution should be 2.9 , but it was incorrectly labeled as 2.8" → I have verified the mistake and fix, and that saabdab does not fix it, just uses the PDB annotation.<br /> "(3) mislabeling of antibody chains as other proteins (e.g. in 3KS0, the light chain of B2B4 antibody was misnamed as heme domain of flavocytochrome b2)" → SAbDab fixes this as well in this case.<br /> "(4) misidentification of heavy chains as light chains (e.g. both two chains of antibody were labeled as light chain in 5EBW)" → SAbDab fixes this as well in this case.

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

I think the annotation of interface residues is a very useful addition to structural datasets.

I am, however, not convinced of the utility of *change* in SASA as a useful metric for identifying interacting residues, beyond what is already identified via pairwise distances between the antibody and antigen residues. If we had access to the unbound conformation of most antibodies and antigens, then we could analyze the differences in structural conformations upon binding, which can be in part quantified by change in SASA. However, as only bound structures are usually available, one is usually force to approximate a protein's unbound structure by computationally removing its binding partner - as it seems to me the authors of this work are doing.

Some obvious limitations of AACDB in its current form include:

AACDB only contains entries with protein-based antigens of at most 50 amino-acids in length. This excludes non-protein-based antigens, such as carbohydrate- and nucleotide-based, as well as short peptide antigens.<br /> AACDB does not include annotations of binding affinity, which are present in SAbDab and have been proven useful both for characterizing drivers of antibody-antigen interactions (cite https://www.sciencedirect.com/science/article/pii/S0969212624004362?via%3Dihub) and for benchmarking antigen-specific antibody-design algorithms (cite https://www.biorxiv.org/content/10.1101/2023.12.10.570461v1))

Reviewer #1 (Public review):

In their manuscript, Papadopoli et al explore the role of ETFDH in transformation. They note that ETFDH protein levels are decreased in cancer, and that deletion of ETFDH in cancer cell lines results in increased tumorigenesis, elevated OXPHOS and glycolysis, and a reduction in lipid and amino acid oxidation. The authors attribute these effects to increased amino acid levels stimulating mTORC1 signaling and driving alterations in BCL6 and EIF4EBP1. They conclude that ETFDH1 is epigenetically silenced in a proportion of neoplasms, suggesting a tumor-suppressive function. Overall, the authors logically present clear data and perform appropriate experiments to support their hypotheses. I only have a few minor points related to the semantics of a few of the author's statements.

Minor Points

Authors state, "we identified ETF dehydrogenase (ETFDH) as one of the most dispensable metabolic genes in neoplasia." Surely there are thousands of genes that are dispensable for neoplasia. Perhaps the authors can revise this sentence and similar sentiments in the text.

Authors state, " These findings show that ETFDH loss elevates glutamine utilization in the CAC to support mitochondrial metabolism." While elevated glutamine to CAC flux is consistent with the statement that increased glutamine, the authors have not measured the effect of restoring glutamine utilization to baseline on mitochondrial metabolism. Thus, the causality implied by the authors can only be inferred based on the data presented. Indeed, the increased glutamine consumption may be linked to the increase in ROS, as glutamate efflux via system xCT is a major determinant of glutamine catabolism in vitro.

Authors state that the mechanism described is an example of "retrograde signaling". However, the mechanism seems to be related to a reduction in BCAA catabolism, suggesting that the observed effects may be a consequence of altered metabolic flux rather than a direct signaling pathway. The data presented do not delineate whether the observed effects stem from disrupted mitochondrial communication or from shifts in nutrient availability and metabolic regulation.

The authors should discuss which amino acids that are ETFDH substrates might affect mTORC1 activity, or consider whether other ETFDH substrates might also affect mTORC1 in their discussion. Along these lines, the authors might consider discussing why amino acids that are not ETFDH substrates are increased upon ETFDH loss.

Reviewer #1 (Public review):

To elucidate the mechanisms and evolution of animal biomineralization, Voigt et al. focused on the sponge phylum - the earliest branching extant metazoan lineages exhibiting biomineralized structures - with a particular emphasis on deciphering the molecular underpinnings of spicule formation. This study centered on calcareous sponges, specifically Sycon ciliatum, as characterized in previous work by Voigt et al. In S. ciliatum, two morphologically distinct spicule types are produced by a set of two different types of cells that secrete extracellular matrix proteins, onto which calcium carbonate is subsequently deposited. Comparative transcriptomic analysis between a region with active spicule formation and other body regions identified 829 candidate genes involved in this process. Among these, the authors focused on the calcarine gene family, which is analogous to the Galaxins, the matrix proteins known to participate in coral calcification. The authors performed three-dimensional structure prediction using AlphaFold, examined mRNA expression of Calcarin genes in spicule-forming cell types via in situ hybridization, conducted proteomic analysis of matrix proteins isolated from purified spicules, and carried out chromosome arrangement analysis of the Calcarin genes.

Based on these analyses, it was revealed that the combination of Calcarin genes expressed during spicule formation differs between the founder cells-responsible for producing diactines and triactines-and the thickener cells that differentiate from them, underscoring the necessity for precise regulation of Calcarin gene expression in proper biomineralization. Furthermore, the observation that 4 Calcarin genes are arranged in tandem arrays on the chromosome suggests that two rounds of gene duplication followed by neofunctionalization have contributed to the intricate formation of S. ciliatum spicules. Additionally, similar subtle spatiotemporal expression patterns and tandem chromosomal arrangements of Galaxins during coral calcification indicate parallel evolution of biomineralization genes between S. ciliatum and aragonitic corals.

Strengths:

(1) An integrative research approach, encompassing transcriptomic, genomic, and proteomic analyses as well as detailed FISH.

(2) High-quality FISH images of Calcarin genes, along with a concise summary clearly illustrating their expression patterns, is appreciated.

(3) It was suggested that thickener cells originate from founder cells. To the best of my knowledge, this is the first study to demonstrate trans-differentiation of sponge cells based on the cell-type-specific gene expression, as determined by in situ hybridization.

(4) The comparison between Calcarins of Calcite sponge and Galaxins of aragonitic corals from various perspective-including protein tertiary structure predictions, gene expression profiling during calcification, and chromosomal sequence analysis to reveal significant similarities between them.

(5) The conclusions of this paper are generally well supported by the data; however, some FISH images require clearer indication or explanation.

(6) Figure S2 (B, C, D): The fluorescent signals in these images are difficult to discern. If the authors choose to present signals at such low magnification, enhancing the fluorescence signals would improve clarity. Additionally, incorporating Figure S2A as an inset within Figure S2E may be sufficient to convey the necessary information about signal localization.

(7) Figure S3A: The claim that Cal2-expressing spherical cells are closely associated with the choanoderm at the distal end of the radial tube is difficult to follow. Are these Cal2-expressing spherical cells interspersed among choanoderm cells, or are they positioned along the basal surface of the choanoderm? Clarifying their precise localization and indicating it in the image would strengthen the interpretation.

(8) To further highlight the similarities between S.ciliatum and aragonitic corals in the molecular mechanisms of calcification, consider including a supplementary figure providing a concise depiction of the coral calcification process. This would offer valuable context for readers.

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors investigated factors required for neural progenitors to exit the cell cycle before the adult stage. They first show that Kr is turned on in pupal stage MBNBs, and depletion of Kr from pupal stage NBs leads to retention of MBNBs into the adult stage. Then they demonstrate that these retained NBs maintain the expression of Imp, and co-depletion of Imp abolishes the extended neurogenesis. Further, they show that co-depletion of kr-h1 significantly reduces the retained MBNBs caused by loss of kr, suggesting antagonistic genetic interactions between these two. In addition, they demonstrate that over-expressing Kr-h1 leads to the striking phenotype of tumor-like neuroblast overgrowth in adult brains.

Strengths:

(1) The authors leveraged well-controlled, powerful genetic tools (including temporal control of RNAi knockdown using the Gal80ts system), and provided strong evidence that Kr expression in pupal stage MBNBs is required to repress Imp and promote the end of neurogenesis. Similarly, the experimental result of co-depleting Kr-h1 and Kr, and the striking phenotype upon Kr-h1 mis-expression, support the antagonistic roles played by Kr-h1 and Kr in this process.

(2) The sample sizes, quantification methods, and p-values are well documented for all experiments. In most parts, the data presented strongly support their conclusions.

(3) Identification of two transcription factors with opposite roles in controlling cell cycle exit, and their possible interactions with the Imp/Syp axis, is highly significant for the study on how the proliferation of neural progenitors is regulated and limited before the adult stage.

Weaknesses:

(1) The nature of the KrIf-1 allele is not clear. It is mentioned that this allele leads to misexpression of Kr in various tissues. However, it is not clear if Kr is mis-expressed or lost in MBNBs in the KrIf-1 mutant. If Kr is mis-expressed in MBNBs in the KrIf-1 mutant, then it would be difficult to explain why both loss of Kr and mis-expression of Kr in MBNBs lead to the same NB retention phenotype. The authors should examine Kr expression in MBNBs in the KrIf-1 mutant.

(2) Some parts of the regulations and interactions between Kr, Kr-h1, Imp, Syp, and E93 are not well-defined. For example, the data suggest that Kr is turned on in the pupal stage MBNBs, and is required to end neurogenesis through repressing Imp and Kr-h1. To further support this conclusion, the authors can examine if Kr-h1 expression is up-regulated in kr-RNAi. The authors suggested that Kr-h1 may act upstream or in parallel to Imp/Syp, but also suggested that Kr-h1 may repress E93. The expression of Imp, Syp, and E93 can be examined in brains with Kr-h1 mis-expression to determine where Kr-h1 acts. If Imp expression is elevated when Kr-h1 is mis-expressed, then Kr-h1 may act upstream of Imp. If Imp/Syp expression does not change, then Kr-h1 may act on the E93 level.

Reviewer #1 (Public review):

Summary:

The manuscript by Garcia et al. describes how the expression of a respiratory chain alternative oxidase (AOX) from the tunicate Ciona intestinalis, capable of transferring electrons directly from reduced coenzyme Q (CoQ) to oxygen, is able to induce an increase in the mass of Drosophila melanogaster larvae and an accelerated development, especially when the larvae are kept at low temperatures. In order to explain this phenomenon, the paper addresses the modifications in the activity and levels of the 'canonical' electron transfer system (ETS), i.e., complexes I-IV and of the ATP synthase. In addition, the abundance of different metabolites as well as the NAD+/NADH ratios are measured, finding significant differences between the larvae.

Strengths:

The observations of differences in growth, body mass and food intake in the wt D. melanogaster larvae vs. those expressing the AOX transgene are solid. The evidence that mild uncoupling of the ETS might accelerate development of the fly larvae is convincing.

Weaknesses:

Some of the observations, especially those concerning the origin of the metabolic remodelling in AOX-expressing larvae, are left unexplained, and the argumentation is somewhat speculative. What the authors mean by "reconfiguration" of the mitochondrial electron transfer system is not clear. If this implies that there is an actual change in ETS function and/or structural organisation in the presence of AOX, this conclusion is not supported by the experimental data. In addition, the influence of AOX activity in the mitochondrial ETS system is tested in vitro in the presence of saturating concentrations of substrates. The real degree to which AOX activity is actually influencing ETS activity in vivo remains unknown.

Reviewer #1 (Public review):

Summary:

The authors have used gene deletion approaches in zebrafish to investigate the function of genes of the hox clusters in pectoral fin "positioning" (but perhaps more accurately pectoral fin "formation").

Strengths:

The authors have employed a robust and extensive genetic approach to tackle an important and unresolved question.

The results are largely presented in a very clear way.

Weaknesses:

The Abstract suggests that no genetic evidence exists in model organisms for a role of Hox genes in limb positioning. There are, however, several examples in mouse and other models (both classical genetic and other) providing evidence for a role of Hox genes in limb position, which is elaborated on in the Introduction.

It would perhaps be more accurate to state that several lines of evidence in a range of model organisms (including the mouse) support a role for Hox genes in limb positioning. The author's work is not weakened by a more inclusive introduction that cites the current literature more comprehensively.

It would be helpful for the authors to make a clear distinction between "positioning" of the limb/fin and whether a limb/fin "forms" at all, independent of the relative position of this event along the body axis.

Discussion of why the zebrafish is sensitive to Hoxb loss with reference to the fin, but mouse Hoxb mutants do make a limb?

Is this down to exclusive expression of Hoxbs in the zebrafish pectoral fin forming region rather than a specific functional role of the protein? This is important as it has implications for the interpretation of results throughout the paper and could explain some apparently conflicting results.

Why is Hoxba more potent than Hoxbb? Is this because Hoxba has Hox4/5 present, while Hoxbb has only Hoxb5? Hoxba locus has retained many more Hox genes in cluster than hoxbb; therefore, one might expect to see greater redundancy in this locus).

Deletion of either Hoxa or Hoxd in the background of the Hoxba mutant does have some effect. Is this a reflection of protein function or expression dynamics of Hoxa/Hoxd genes?

Can we really be confident that there is a "transformation of pectoral fin progenitor cells into cardiac cells"?

The failure to repress Nkx2.5 in the posterior (pelvic fin) domain is clear, but have these cells actually acquired cardiac identity? They would be expected to express Tbx5a (or b) as cardiac precursors, but this domain does not broaden. There is no apparent expansion of the heart (field)/domain or progenitors beyond the 16 somite stage. The claimed "migration" of heart precursors in the mutant is not clear. The heart/cardiac domain that does form in the mutant is not clearly expanded in the mutant. The domain of cmlc2 looks abnormal in the mutant, but I am not convinced it is "enlarged" as claimed by the authors. The authors have not convincingly shown that "the cells that should form the pectoral fin instead differentiate into cardiac cells."

The only clear conclusion is the loss of pectoral fin-forming cells rather than these fin-forming cells being "transformed" into a new identity. It would be interesting to know what has happened to the cells of the pectoral fin-forming region in these double mutants.

It is not clear what the authors mean by a "converse" relationship between forelimb/pectoral fin and heart formation. The embryological relationship between these two populations is distinct in amniotes.

The authors show convincing data that RA cannot induce Tbx5a in the absence of Hob clusters, but I am not convinced by the interpretation of this result. The results shown would still be consistent with RA acting directly upstream of tbx5a, but merely that RA acts in concert with hox genes to activate tbx5a. In the absence of one or the other, Tbx5a would not be expressed. It is not necessary that RA and hoxbs act exclusively in a linear manner (i.e., RA regulates hoxb that in turn regulates tbx5a).

The authors have carried out a functional test for the function of hoxb6 and hoxb8 in the hemizygous hoxb mutant background. What is lacking is any expression analysis to demonstrate whether Hoxb6b or Hoxb8b are even expressed in the appropriate pectoral fin territory to be able to contribute to pectoral fin development, either in this assay or in normal pectoral fin development.

(The term "compensate" used in this section is confusing/misleading.)

The authors' confounding results described in Figures 6-7 are consistent with the challenges faced in other model organisms in trying to explore the function of genes in the hox cluster and the known redundancy that exists across paralogous groups and across individual clusters.

Given the experimental challenges in deciphering the actual functions of individual or groups of hox genes, a discussion of the normal expression pattern of individual and groups of hox genes (and how this may change in different mutant backgrounds) could be helpful to make conclusions about likely normal function of these genes and compensation/redundancy in different mutant scenarios.

Reviewer #1 (Public review):

The manuscript by Ivan et al aimed to identify epitopes on the Abeta peptide for a large set of anti-Abeta antibodies, including clinically relevant antibodies. The experimental work was well done and required a major experimental effort, including peptide mutational scanning, affinity determinations, molecular dynamics simulations, IP-MS, WB, and IHC. Therefore, it is of clear interest to the field. The first part of the work is mainly based on an assay in which peptides (15-18-mers) based on the human Abeta sequence, including some containing known PTMs, are immobilized, thus preventing aggregation. Although some results are in agreement with previous experimental structural data (e.g. for 3D6), and some responses to disease-associated mutations were different when compared to wild-type sequences (e.g. in the case of Aducanumab) - which may have implications for personalized treatment - I have concerns about the lack of consideration of the contribution of conformation (as in small oligomers and large aggregates) in antibody recognition patterns. The second part of the study used full-length Abeta in monomeric or aggregated forms to further investigate the differential epitope interaction between Aducanumab, donanemab, and lecanemab (Figures 5-7). Interestingly, these results confirmed the expected preference of these antibodies for aggregated Abeta, thus reinforcing my concerns about the conclusions drawn from the results obtained using shorter and immobilized forms of Abeta. Overall, I understand that the work is of interest to the field and should be published without the need for additional experimental data. However, I recommend a thorough revision of the structure of the manuscript in order to make it more focused on the results with the highest impact (second part).

Reviewer #1 (Public review):

Summary:

The manuscript "Targeted Protein Degradation by KLHDC2 Ligands Identified by High Throughput Screening" by Zhou, H. et al. describes the development of a high-throughput FP-based screen and the identification of a KLHDC2 ligand from a small molecule library. A counter screen and other filtering criteria led to the identification of lead compounds that contained a tetrahydroquinoline scaffold. Commercially available analogs (52 compounds) that shared this scaffold were characterized by a KLHDC2 competitive binding assay. Optimized compounds were obtained that demonstrated improved potency and increased binding affinity by SPR. Docking of a lead candidate (compound 6) suggested it bound at a distal lipophilic site within the SelK binding pocket of KLHDC2. Based on this model, the authors then synthesized PROTACs that linked the KLHDC2 binder to a BRD4-binding molecule, JQ1. These PROTAC candidates possessed different linker configurations, and PROTAC 8 was able to cause BRD4 degradation in cells, with a half-maximal degradation concentration (DC50) of 80 nM. The authors demonstrate the identification and characterization of small-molecule KLHDC2 ligands that can be used to generate PROTACs that result in BRD4 degradation in cells.

Strengths:

The study by Zhou, H. et al. expands the E3 ligase toolkit by targeting KLHDC2 to identify ligands for PROTAC development, which has predominantly relied on VHL and CRBN. This was accomplished using a described FP-based high-throughput screening strategy (high Z' values in 1536 well format). Both target-specific and counter-specific assays were performed, along with subsequent stringent follow-up assays designed to address non-specific binding/specificity concerns. Label-free direct binding validations by SPR were used to determine binding affinity/kinetics. A strength of the study is the characterization of the interaction between candidate compounds and KLHDC2 versus related KEAP1.

Structural insight into the potential mode of binding was inferred by computational docking studies of the newly discovered KLHDC2 ligands. This was performed to identify where the identified scaffolds could be modified by linker incorporation for the design of PROTACs. The computational predictions were evaluated by linking a solvent-exposed site on the KLHDC2 ligand to JQ1. Three linkers were tested, and two compounds were found to result in BRD4 degradation in cells by HiBiT degradation assay and western blot. These findings demonstrate the feasibility of these compounds for the design of PROTAC-based degraders.

The authors present compelling KLHDC2 binding data for their lead compounds and demonstrate degradation of a target using a PROTAC strategy. Accordingly, the screening approach and compounds identified are likely to be of interest to the field and are likely to be generalizable to other PROTAC targets of interest.

Weaknesses:

The specificity of compounds for KLHDC2 was assessed by using a counter screen against KEAP1 and in vitro binding assays. However, off-target effects might occur in a cellular context, which weren't fully explored in the study. Notably, the authors do not demonstrate that the degradation induced by their PROTACs in cells is KLHDC2-dependent. A requirement for KLHDC2-mediated degradation could be evaluated, for example, by using knockout/knockdown of KLHDC2, or other means, to demonstrate specificity. Addressing specificity is deemed important to evaluate the proposed PROTAC mechanism of action in a cellular context that results in the degradation of BRD4. Specificity is important when considering the utility of these new compounds for PROTAC design.

Additional rationale behind the selection of linkers used to generate candidate PROTACs would be informative and would benefit from additional discussion and/or citation. The reasons for the lack of activity, such as for compound 9, were not fully explored or discussed, such as whether complex assembly is potentially affected by linker choice. Perhaps related to this point, the authors note that a trifluoromethoxy group increased the binding affinity of compound 6. However, the subsequent docking analysis revealed this moiety to be solvent-exposed. The relationship between this site of functionalization, linker selection, and the resulting binding affinity or effect on DC50 was not clear and/or could be developed further.

Minor issues related to the presentation of the manuscript include sections that would benefit from either additional citation and/or description, such as the KI-696 inhibitor used and the BRD4 HiBiT degradation assay that was used to assess PROTAC potency. Figure captions should be reviewed to ensure that the number of independent experiments is indicated, and what data points and error bars represent, as these are not indicated in several figures. BRD4 levels were quantified in 4E; however, error/reproducibility (n) is not indicated.

Reviewer #1 (Public review):

The study aims to determine the role of Slit-Robo signaling in the development and patterning of cardiac innervation, a key process in heart development. Despite the well-studied roles of Slit axon guidance molecules in the development of the central nervous system, their roles in the peripheral nervous system are less clear. Thus, the present study addresses an important question. The study uses genetic knockout models to investigate how Slit2, Slit3, Robo1, and Robo2 contribute to cardiac innervation.

Using constitutive and cell type-specific knockout mouse models, they show that the loss of endothelial-derived Slit2 reduces cardiac innervation. Additionally, Robo1 knockout, but not Robo2 knockout, recapitulated the Slit2 knockout effect on cardiac innervation, leading to the conclusion that Slit2-Robo1 signaling drives sympathetic innervation in the heart. Finally, the authors also show a reduction in isoproterenol-stimulated heart rate but not basal heart rate in the absence of endothelial Slit2.

The conclusions of this paper are mostly well supported by the data, but some should be modified to account for the study's limitations and discussed in the context of previous literature.

(1) It is well established that Slit ligands undergo proteolytic cleavage, generating N- and C-terminal fragments with distinct biological functions. Full-length Slit proteins and their fragments differ in cell association, with the N-terminal fragment typically remaining membrane-bound, while the C-terminal fragment is more diffusible. This distinction is crucial when evaluating the role of Slit proteins secreted by different cell types in the heart. However, this study does not examine or discuss the specific contributions of different Slit2 fragments, limiting its mechanistic insight into how Slit2 regulates cardiac innervation.

(2) The endothelial-specific deletion of Slit2 leads to its loss in endothelial cells across various organs and tissues in the developing embryo. Therefore, the phenotypes observed in the heart may be influenced by defects in other parts of the embryo, such as the CNS or sympathetic ganglia, and this possibility cannot be ruled out.

Reviewer #1 (Public review):

Summary:

Lysosomal damage is commonly found in many diseases including normal aging and age-related disease. However, the transcriptional programs activated by lysosomal damage have not been thoroughly characterized. This study aimed to investigate lysosome damage-induced major transcriptional responses and the underlying signaling basis. The authors have convincingly shown that lysosomal damage activates a ubiquitination-dependent signaling axis involving TAB, TAK1, and IKK, which culminates in the activation of NF-kB and subsequent transcriptional upregulation of pro-inflammatory genes and pro-survival genes. Overall, the major aims of this study were successfully achieved.

Strengths:

This study is well-conceived and strictly executed, leading to clear and well-supported conclusions. Through unbiased transcriptomics and proteomics screens, the authors identified NF-kB as a major transcriptional program activated upon lysosome damage. TAK1 activation by lysosome damage-induced ubiquitination was found to be essential for NF-kB activation and MAP kinase signaling. The transcriptional and proteomic changes were shown to be largely driven by TAK1 signaling. Finally, the TAK1-IKK signaling was shown to provide resistance to apoptosis during lysosomal damage response. The main signaling axis of this pathway was convincingly demonstrated.

Weaknesses:

One weakness was the claim of K63-linked ubiquitination in lysosomal damage-induced NF-kB activation. While it was clear that K63 ubiquitin chains were present on damaged lysosomes, no evidence was shown in the current study to demonstrate the specific requirement of K63 ubiquitin chains in the signaling axis being studied. Clarifying the roles of K63-linked versus other types of ubiquitin chains in lysosomal damage-induced NF-kB activation may improve the mechanistic insights and overall impact of this study.

Another weakness was that the main conclusions of this study were all dependent on an artificial lysosomal damage agent. It will be beneficial to confirm key findings in other contexts involving lysosomal damage.

Reviewer #1 (Public review):

Summary:

This work by Govorunova et al. identified three naturally blue-shifted channelrhodopsins (ChRs) from ancyromonads, namely AnsACR, FtACR, and NlCCR. The phylogenetic analysis places the ancyromonad ChRs in a distinct branch, highlighting their unique evolutionary origin and potential for novel applications in optogenetics. Further characterization revealed the spectral sensitivity, ionic selectivity, and kinetics of the newly discovered AnsACR, FtACR, and NlCCR. This study also offers valuable insights into the molecular mechanism underlying the function of these ChRs, including the roles of specific residues in the retinal-binding pocket. Finally, this study validated the functionality of these ChRs in both mouse brain slices (for AnsACR and FtACR) and in vivo in Caenorhabditis elegans (for AnsACR), demonstrating the versatility of these tools across different experimental systems.

In summary, this work provides a potentially valuable addition to the optogenetic toolkit by identifying and characterizing novel blue-shifted ChRs with unique properties.

Strengths:

This study provides a thorough characterization of the biophysical properties of the ChRs and demonstrates the versatility of these tools in different ex vivo and in vivo experimental systems. The mutagenesis experiments also revealed the roles of key residues in the photoactive site that can affect the spectral and kinetic properties of the channel.

Weaknesses:

While the novel ChRs identified in this work are spectrally blue-shifted, there still seems to be some spectral overlap with other optogenetic tools. The authors should provide more evidence to support the claim that they can be used for multiplex optogenetics and help potential end-users assess if they can be used together with other commonly applied ChRs. Additionally, further engineering or combination with other tools may be required to achieve truly orthogonal control in multiplexed experiments.

In the C. elegans experiments, partial recovery of pharyngeal pumping was observed after prolonged illumination, indicating potential adaptation. This suggests that the effectiveness of these ChRs may be limited by cellular adaptation mechanisms, which could be a drawback in long-term experiments. A thorough discussion of this challenge in the application of optogenetics tools would prove very valuable to the readership.

Reviewer #1 (Public review):

Summary:

The authors assess the role of map3k1 in adult Planaria through whole body RNAi for various periods of time. The authors' prior work has shown that neoblasts (stem cells that can regenerate the entire body) for various tissues are intermingled in the body. Neoblasts divide to produce progenitors that migrate within a "target zone" to the "differentiated target tissues" where they differentiate into a specific cell type. Here the authors show that map3k1-i animals have ectopic eyes that form along the "normal" migration path of eye progenitors (Fig. 1), ectopic neurons and glands along the AP axis (Fig. 2) and pharynx in ectopic anterior positions (Fig. 3). The rest of the study show that positional information is largely unaffected by loss of map3k1 (Fig. 4,5). However, loss of map3k1 leads to premature differentiated of progenitors along their normal migratory route (Fig. 6). They also show that an ill-defined "long-term" whole body depletion of map3k1 results in mis-specified organs and teratomas.

Strengths:

(1) The study has appropriate controls, sample sizes and statistics.<br /> (2) The work appears to be high-quality.<br /> (3) The conclusions are supported by the data.<br /> (4) Planaria is a good system to analyze the function of map3k1, which exists in mammals but not in other invertebrates.

Weaknesses:

(1) The paper is largely descriptive with no mechanistic insights.<br /> (2) Given the severe phenotypes of long-term depletion of map3k1, it is important that this exact timepoint is provided in the methods, figures, figure legends and results.<br /> (3) Fig. 1C, the ectopic eyes are difficult to see, please add arrows.<br /> (4) line 217 - why does the n=2/12 animals not match the values in Fig. 3B, which is 11/12 and 12/12. The numbers don't add up. Please correct/explain.<br /> (5) Figure panels do not match what is written in the results section. There is no Fig. 6E. Please correct.

Reviewer #1 (Public review):

Summary:

This manuscript investigated the mechanism underlying boundary formation necessary for proper separation of vestibular sensory end organs. In both chick and mouse embryos, it was shown that a population of cells abutting the sensory (marked by high Sox2 expression) /nonsensory cell populations (marked by Lmx1a expression) undergo apical expansion, elongation, alignment and basal constriction to separate the lateral crista (LC) from the utricle. Using Lmx1a mouse mutant, organ cultures, pharmacological and viral-mediated Rock inhibition, it was demonstrated that the Lmx1a transcription factor and Rock-mediated actomyosin contractility is required for boundary formation and LC-utricle separation.

Strengths:

Overall, the morphometric analyses were done rigorously and revealed novel boundary cell behaviors. The requirement of Lmx1a and Rock activity in boundary formation was convincingly demonstrated.

Weaknesses:

However, the precise roles of Lmx1a and Rock in regulating cell behaviors during boundary formation were not clearly fleshed out. For example, phenotypic analysis of Lmx1a was rather cursory; it is unclear how Lmx1a, expressed in half of the boundary domain, control boundary cell behaviors and prevent cell mixing between Lmx1a+ and Lmx1a- compartments? Well-established mechanisms and molecules for boundary formation were not investigated (e.g. differential adhesion via cadherins, cell repulsion via ephrin-Eph signaling). Moreover, within the boundary domain, it is unclear whether apical multicellular rosettes and basal constrictions are drivers of boundary formation, as boundary can still form when these cell behaviors were inhibited. Involvement of other cell behaviors, such as radial cell intercalation and oriented cell division, also warrant consideration. With these lingering questions, the mechanistic advance of the present study is somewhat incremental.

Reviewer #1 (Public review):

Summary:

In this manuscript, Taujale et al describe an interdisciplinary approach to mine the human channelome and further discover orthologues across diverse organisms, culminating in delineating co-conserved patterns in an example ion channel: CALHM. Overall, this paper comes in two sections, one where 419 human ion channels and 48,000+ channels from diverse organisms are found through a multidisciplinary data mining approach, and a second where this data is used to find co-conserved sequences, whose functional significance is validated via experiments on CALHM1 and CALHM6. Overall, this is an intriguing data-first approach to better understand even understudied ion channels like CALHM6. However, more needs to be done to pull this story together into a single, coherent narrative.

Strengths:

This manuscript takes advantage of modern-day LLM tools to better mine the literature for ion channel sequences in humans and other species with orthologous ion channel sequences. They explore the 'dark channome' of understudied ion channels to better reveal the information evolution has to tell us about our own proteins, and illustrate the information this provides access to in experimental studies in the final section of the paper. Finally, they provide a wealth of information in the supplementary tables (in the form of Excel spreadsheets) for others to explore. Overall, this is a creative approach to a wide-reaching problem that can be applied to other families of proteins.

Weaknesses:

Overall, while a considerable amount of work has been done for this manuscript, the presentation, both in terms of writing and figures, leaves much to be desired. One can imagine a story that clearly describes the need for a better-curated sequence database of ion channels, and clearly describes how existing resources fall short, but here this is not very clearly illustrated.

One question that arises with the part of the manuscript that discusses the identification and classification of ion channels is whether they plan to make these sequences available to the wider public. For the 419 human sequences, making a small database to share this result so that these sequences can be easily searched and downloaded would be desirable. There are a variety of acceptable formats for this: GitHub/figshare/zenodo/university website that allows a wider community to access their hard work. The authors have included enough information in the supplementary tables that this could be done by a motivated reader, but providing such a resource would greatly expand the impact of this paper. The same question can be asked of the 48,000+ ion channels from diverse organisms. For these, one is even worried that these are not properly sequenced genes? What checks have been done to confirm this? Uniport contains a good deal of unreviewed sequences, especially from single-celled organisms. Potentially, this is covered in the sentence in the Methods: "Finally, the results obtained from both the full-length and pore domains were retained as true orthologous relationships to remove extraneous hits." But this process could be discussed in more detail, clearly illustrating that the risk of gene duplicates and fragments in this final set of ion channel orthologues has been avoided. Related to this, does this analysis include or exclude isoforms?

Another aspect of the identification and classification of ion channel genes that could be improved is the figures for this section. One is relatively used to seeing trees as shown in Figures 3 and 4, which show relationships between genes as distances or evolutionary relationships. The decision to show the families of ion channels in Figure 1 as pie charts within a UMAP embedding is intriguing but somewhat non-intuitive and difficult to understand. Illustrating these results with a standard tree-like visualization of the relationship of these channels to each other would be preferred.

One aspect of the pie-chart/UMAP visualization that works well is the highlighting of the 'dark' ion channels according to the status as designated by IDG, which highlights a strength of this whole paper. However, throughout the paper, this could be emphasized more as the key advantage of this approach and how this or similar approaches could be used for other families of proteins. Specifically, in the initial statement describing 'light' vs 'dark channels', the importance of this distinction and the historical preference in science to study that which has already been studied can be discussed more, even including references to other studies that take this kind of approach. An example of a relevant reference here is to the Structural Genomics Consortium and its goals to achieve structures of proteins for which functions may not be well-characterized. Furthermore, this initial statement mentioning 'light channels' was initially confusing -- does this mean light-sensing channels? As one reads on this is clearly not the case, but for such an important central focus of this paper, these kinds of misunderstandings do not serve the authors well. Clarifying these motivations throughout the entire paper would strengthen it considerably.

Additionally, since the authors have generated this UMAP visualization, it would be interesting to understand how the human vs orthologue gene sets compare in this space. Furthermore, Figure 1, for just the human analysis, should say more clearly that this is an analysis of the human gene set and include more of the information in the text: 419 human ion channel sequences, 75 sequences previously unidentified, 4 major groups and 55 families, 62 outliers, etc. Clearer visualizations of these categories and numbers within the UMAP (and newly included tree) visualization would help guide the reader to better understand these results.

One of the most peculiar aspects of this paper is that it feels like two papers, one about better documenting the ion channel genes across species, and another with well-executed experiments on CALHM channels. One suggestion for how to link these two sections together better is to show that previous methods to analyze conserved residues in CALHM were significantly lacking. What results would that give? Why was this not enough? Were there just not enough identified CALHM orthologues to give strong signals in conservation analysis?

Some of the analysis pipeline is unclear. Specifically, the RAG analysis seems critical, but it is unclear how this works - is it on top of the GPT framework and recursively inquires about the answer to prompts? Some example prompts would be useful to understand this. Furthermore, the existence of 76 auxiliary non-pore containing 'ion channel' genes in this analysis is a little confusing, as it seems a part of the pipeline is looking for pore-lining residues. Furthermore, how many of these are picked up in the larger orthologues search? Are these harder to perform checks on to ensure that they are indeed ion channel genes? A further discussion of the choice to include these auxiliary sequences would be relevant. This could just be further discussion of the literature that has decided to do this in the past.

Overall, this manuscript is a valuable contribution to the field, but it requires a few main things to make it truly useful. Namely, how has this approach really improved the ability to identify conserved residues over a less-involved approach? A better description of their methods and results is required in the first section of the paper, as well as some cosmetic improvements.

Reviewer #1 (Public review):

Summary:

This useful work extends a prior study from the authors to observe distance changes within the CNBD domains of a full-length CNG channel based on changes in single photon lifetimes due to tmFRET between a metal at an introduced chelator site and a fluorescent non-canonical amino acid at another site. The data are excellent and convincingly support the authors' conclusions. The methodology is of general use for other proteins. The authors also show that coupling of the CNBDs to the rest of the channel stabilizes the CNBDs in their active state, relative to an isolated CNBD construct.

Strengths:

The manuscript is very well written and clear.

Reviewer #1 (Public review):

The authors investigate how the viscoelasticity of the fingertip skin can affect the firing of mechanoreceptive afferents and they find a clear effect of recent physical skin state (memory), which is different between afferents. The manuscript is extremely well-written and well-presented. It uses a large dataset of low threshold mechanoreceptive afferents in the fingertip, where it is particularly noteworthy that the SA-2s have been thoroughly analyzed and play an important role here. They point out in the introduction the importance of the non-linear dynamics of the event when an external stimulus contacts the skin, to the point at which this information is picked up by receptors. Although clearly correlated, these are different processes, and it has been very well-explained throughout. I have some comments and ideas that the authors could think about that could further improve their already very interesting paper. Overall, the authors have more than achieved their aims, where their results very much support the conclusions and provoke many further questions. This impact of the previous dynamics of skin affecting current state can be explored further in so many ways and may help us in understanding skin aging and the effects of anatomical changes of the skin better.

Comments on revised submission:

The authors have taken all my considerations into account and provided excellent responses to them. They have modified their paper accordingly, which improves its clarity even more. Very interesting work and I have no further comments.

Reviewer #1 (Public review):

Summary:

The authors demonstrate that two human preproprotein human mutations in the BMP4 gene cause a defect in proprotein cleavage and BMP4 mature ligand formation, leading to hypomorphic phenotypes in mouse knock-in alleles and in Xenopus embryo assays.

Strengths:

They provide compelling biochemical and in vivo analyses supporting their conclusions, showing the reduced processing of the proprotein and concomitant reduced mature BMP4 ligand protein from impressively mouse embryonic lysates. They perform excellent analysis of the embryo and post-natal phenotypes demonstrating the hypomorphic nature of these alleles. Interesting phenotypic differences between the S91C and E93G mutants are shown with excellent hypotheses for the differences. Their results support that BMP4 heterodimers act predominantly throughout embryogenesis whereas BMP4 homodimers play essential roles at later developmental stages.

Weaknesses:

In the revision the authors have appropriately addressed the previous minor weaknesses.

Reviewer #1 (Public review):

This study investigates the role of microtubules (MT) in regulating insulin secretion from pancreatic islet beta cells. This is of great importance considering that controlled secretion of insulin is essential to prevent diabetes. Previously, it has been shown that KIF5B plays an essential role in insulin secretion by transporting insulin granules to the plasma membrane. High glucose activates KIF5B to increase insulin secretion resulting in cellular uptake of glucose. In order to prevent hypoglycemia, insulin secretion needs to be tightly controlled. Notably, it is known that KIF5B plays a role in MT sliding. This is important, as the authors described previously that beta cells establish a peripheral sub-membrane MT array, which is critical for withdrawal of excessive insulin granules from the secretion sites. At high glucose, the sub-membrane MT array is destabilized to allow for robust insulin secretion. Here the authors aim to answer the question how the peripheral array is formed. Based on the previously published data the authors hypothesize that KIF5B organizes the sub-membrane MT array via microtubule sliding.

General comment:<br /> This manuscript provides data that indicate that KIF5B, like in many other cells, mediates MT sliding in beta cells to establish a non-radial sub-membrane MT array. This study is based mainly on in vitro assays and one cell line. To demonstrate the importance of KIF5B in vivo/under physiological conditions, the MT pattern and directionality in beta cells within whole isolated pancreatic islets from KIF5B KO mice was analyzed in comparison to their WT littermates. While the presented effects appear often rather small, it is important to note that small changes in MT configuration can have strong effects. However, the authors provide no link to insulin secretion and glucose uptake. Finally, it remains unclear whether a KIF5B-dependent mechanism regulating microtubule sliding plays a major role in controlling insulin secretion.

Specific comments:<br /> (1) It is difficult to appreciate that there is a "peripheral sub-membrane microtubule array" as it is not well defined in the manuscript. This reviewer assumes that this is in the respective field clear. Yet, while it is appreciated that there is an increased amount of MTs close to the cytoplasmic membrane, the densities appear very variable along the membrane. Please provide a clear description in the Introduction what is meant with "peripheral sub-membrane microtubule array".<br /> (2) The authors described a "consistent presence of a significant peripheral array in the C57BL/6J control mice, while the KO counterparts exhibited a partial loss of this peripheral bundle. Specifically, the measured tubulin intensity at the cell periphery was significantly reduced in the KO mice compared to their wild-type counterparts". In vitro "control cells had convoluted non-radial MTs with a prominent sub-membrane array, typical for β cells (Fig. 2A), KIF5B-depleted cells featured extra-dense MTs in the cell center and sparse receding MTs at the periphery (Fig. 2B,C)". Please comment/discuss why in vivo there are no "extra-dense MTs in the cell center".<br /> (3) Authors should include in the Discussion a paragraph discussing the fact that small changes in MT configuration can have strong effects.

Reviewer #1 (Public review):

Summary:

This short report shows that the transcription factor gene mirror is specifically expressed in the posterior region of the butterfly wing imaginal disk, and uses CRISPR mosaic knock-outs to show it is necessary to specify the morphological features (scales, veins, and surface) of this area.

Strengths:

The data and figures support the conclusions. The article is swiftly written and makes an interesting evolutionary comparison to the function of this gene in Drosophila. Based on the data presented, it can now be established that mirror likely has a similar selector function for posterior-wing identity in a plethora of insects.

Comments on revisions:

The revision is satisfactory. I agree with the authors that this article provides interesting insights on the evolution of insect wings. Of note, butterfly and fly wing imaginal disks differ in their mode of development: while fly wing disks grow as epithelial sacs that evaginate during metamorphosis, butterfly wing disks develop as relatively flat epithelial sheets that expand and differentiate progressively. This makes the similar role of mirror all the more interesting.

The revised text appropriately discuss how selector genes like mirror regionalize the wing during larval and pupal development. This article makes a reasonable use of CRISPR mosaic knock outs and uses contralateral controls to show the nature of the phenotypic transformations.

Reviewer #1 (Public review):

The study addresses how faces and bodies are integrated in two STS face areas revealed by fMRI in the primate brain. It is building upon recordings and analysis of the responses of large populations of neurons to three sets of images, that vary face and body positions. These sets allowed the author to thoroughly investigate invariance to position on the screen (MC HC), to pose (P1 P2), to rotation (0 45 90 135 180 225 270 315), to inversion, to possible and impossible postures (all vs straight), to presentation of head and body together or in isolation. By analyzing neuronal responses, they find that different neurons showed preferences for body orientation, or head orientation or for the interaction between the two. By using a linear support vector machine classifier, they show that the neuronal population can decode head-body angle presented across orientations, in the anterior aSTS patch (but not middle mSTS patch), except for mirror orientation. On the contrary, mSTS neurons show less invariance for head-body angle and more specialization for head or body orientation.

Strengths:

These results expand prior work on the role of Anterior STS fundus face area in face-body integration and its invariance to mirror symmetry, with a rigorous set of stimuli revealing the workings of these neuronal populations in processing individuals as a whole, in an important series of carefully designed conditions.

It also raises questions for future investigations comparing humans and monkeys expertise with upright and inverted configurations, in light of monkey-specific hanging upside-down behavior. Further, using two types of body postures (sitting, standing), they show a correlation in head-body angle between postures, suggesting that monkey orientation might be more meaningful to these neurons than precise posture.

Reviewer #1 (Public review):

Summary:

Kv2 subfamily potassium channels contribute to delayed rectifier currents in virtually all mammalian neurons and are encoded by two distinct types of subunits: Kv2 alpha subunits that have the capacity to form homomeric channels (Kv2.1 and Kv2.2), and KvS or silent subunits (Kv5,6,8.9) that can assemble with Kv2.1 or Kv2.2 to form heteromeric channels with novel biophysical properties. Many neurons express both types of subunits and therefore have the capacity to make both homomeric Kv2 channels and heteromeric Kv2/KvS channels. Determining the contributions of each of these channel types to native potassium currents has been very difficult because the differences in biophysical properties are modest and there are no Kv2/KvS-specific pharmacological tools. The authors set out to design a strategy to separate Kv2 and Kv2/KvS currents in native neurons based on their observation that Kv2/KvS channels have little sensitivity to the Kv2 pore blocker RY785 but are blocked by the Kv2 VSD blocker GxTx. They clearly demonstrate that Kv2/KvS currents can be differentiated from Kv2 currents in native neurons using a two-step strategy to first selectively block Kv2 with RY785, and then block both with GxTx. The manuscript is beautifully written; takes a very complex problem and strategy and breaks it down so both channel experts and the broad neuroscience community can understand it.

Strengths:

The compounds the authors use are highly selective and unlikely to have significant confounding cross-reactivity to other channel types. The authors provide strong evidence that all Kv2/KvS channels are resistant to RY785. This is a strength of the strategy - it can likely identify Kv2/KvS channels containing any of the 10 mammalian KvS subunits and thus be used as a general reagent on all types of neurons. The limitation then of course is that it can't differentiate the subtypes, but at this stage, the field really just needs to know how much Kv2/KvS channels contribute to native currents and this strategy provides a sound way to do so.

Weaknesses:

The authors are very clear about the limitations of their strategy, the most important of which is that they can't differentiate different subunit combinations of Kv2/KvS heteromers. This study is meant to be a start to understanding the roles of Kv2/KvS channels in vivo. As such, this is a minor weakness, far outweighed by the potential of the strategy to move the field through a roadblock that has existed since its inception.

The study accomplishes exactly what it set out to do: provide a means to determine the relative contributions of homomeric Kv2 and heteromeric Kv2/KvS channels to native delayed rectifier K+ currents in neurons. It also does a fabulous job laying out the case for why this is important to do.

Comments on revisions:

I liked this manuscript the first time and thought it was a great attempt to address a difficult problem, made more difficult by confusing background literature and conventions. The authors have kept all the strong points I liked from the first round and made it even stronger with their thoughtful and substantive responses to reviews. My first review was strongly supportive, and my initial short assessment/public review was written with the assumption that they would be public and the paper would be published essentially in its original form. All those points still apply so I am going to leave the initial reviews as is. The paper is a pleasure to read and a nice contribution to the field.

Reviewer #2 (Public review):

Summary:

I found this an interesting manuscript describing a study investigating the role of MC4R signalling on kisspeptin neurons. The initial question is a good one. Infertility associated with MC4 mutations in humans has typically been ascribed to the consequent obesity and impaired metabolic regulation. Whether there is a direct role for MC4 in regulating the HPG axis has not been thoroughly examined. Here, the researchers have put together an elegant combination of targeted loss of function and gain of function in vivo experiments, specifically targeting MC4 expression in kisspeptin neurons. This excellent experimental design should provide compelling evidence for whether melanocortin signalling has a direct role in arcuate kisspeptin neurons to support normal reproductive function. There were definite effects on reproductive function (irregular estrous cycle, reduced magnitude of LH surge induced by exogenous estradiol). However, the magnitude of these responses and the overall effect on fertility were relatively minor. The mice lacking MC4R in kisspeptin neurons remained fertile despite these irregularities. The second part of the manuscript describes a series of electrophysiological studies evaluating the pharmacological effects of melanocortin signalling in kisspeptin cells in ex-vivo brain slides. These studies characterised interesting differential actions of melanocortins in two different populations of kisspeptin neurons. Collectively, I think the study provides novel insights into how direct actions of melanocortin signalling, via the MC4 receptor in kisspeptin neurons, contribute to the metabolic regulation of the reproductive system. Importantly, however, it is clear that other mechanisms are also at play.

Strengths:

The loss of function/gain of function experiments provide a conceptually simple but hugely informative experimental design. This is the key strength of the current paper - especially the knock-in study that showed improved reproductive function even in the presence of ongoing obesity. This is a very convincing result that documents that reproductive deficits in MC4R knockout animals (and humans with deleterious variants of the MC4R gene) can be ascribed to impaired signalling in the hypothalamic kisspeptin neurons and not necessarily simply caused as a consequence of obesity. As concluded by the authors: "reproductive impairments observed in MC4R deficient mice, which replicate many of the conditions described in humans, are largely mediated by the direct action of melanocortins via MC4R on Kiss1 neurons and not to their obese phenotype." This is important, as it might change the way such fertility problems are treated.

Limitation:

The mechanistic studies evaluating melanocortin signalling in kisppetin neurons were all completed in ovariectomized animals (with and without exogenous hormones). This reductionist approach allowed a focus on the direct actions of estradiol to regulate responses but missed an opportunity to evaluate how cyclical changes in hormones might impact the system. Such cyclical changes are fundamental to how these neurons function in vivo and may dynamically alter the way they respond to hormones and neuropeptides. However, the inclusion of gonad-intact animals would have significantly increased the complexity of experiments and can reasonably be considered outside of the scope of the present study.

Reviewer #1 (Public review):

Summary:

The authors track the motion of multiple consortia of Multicellular Magnetotactic Bacteria moving through an artificial network of pores and report a discovery of a simple strategy for such consortia to move fast through the network: an optimum drift speed is attained for consortia that swim a distance comparable to the pore size in the time it takes to align the with an external magnetic field. The authors rationalize their observations using dimensional analysis and numerical simulations. Finally, they argue that the proposed strategy could generalize to other species by demonstrating the positive correlation between the swimming speed and alignment time based on theoretical analysis and parameters derived from literature.

Strengths:

The underlying dimensional analysis and model convincingly rationalize the experimental observation of an optimal drift velocity: the optimum balances the competition between the trapping in pores at large magnetic fields and random pore exploration for weak magnetic fields.

Weaknesses:

The convex pore geometry studied here creates convex traps for cells, which I expect enhances their trapping. Natural environments may create a much smaller concentration of such traps. In this case, whether a non-monotonic dependence of the drift velocity on the Scattering number would persist is unclear.

Comments on revisions:

Thank you very much for addressing my comments. I think the revisions have improved the paper.

Reviewer #1 (Public review):

Summary:

This manuscript reports the investigation of PriC activity during DNA replication initiation in Escherichia coli. It is reported that PriC is necessary for growth and control of DNA replication initiation under diverse conditions where helicase loading is perturbed at the chromosome origin oriC. A model is proposed where PriC loads helicase onto ssDNA at the open complex formed by DnaA at oriC. Reconstituted helicase loading assays in vitro are consistent with the model.

Strengths:

The complementary combination of genetics in vivo and reconstituted assays in vitro provide solid evidence to support the role of PriC at a replication origin.

The manuscript is well written and has a logical narrative.

The data provide new insight to how bacteria might load helicase at the replication origin when the wild-type DnaA-dependent loading pathway is perturbed.

Weakness:

It has not yet been established whether PriC localises at oriC in vivo under the conditions tested.

Reviewer #1 (Public review):

Summary:

This paper focuses on understanding how covalent inhibitors of peroxisome proliferator-activated receptor-gamma (PPARg) show improved inverse agonist activities. This work is important because PPARg plays essential roles in metabolic regulation, insulin sensitization, and adipogenesis. Like other nuclear receptors, PPARg, is a ligand-responsive transcriptional regulator. Its important role, coupled with its ligand-sensitive transcriptional activities, makes it an attractive therapeutic target for diabetes, inflammation, fibrosis, and cancer. Traditional non-covalent ligands like thiazolininediones (TZDs) show clinical benefit in metabolic diseases, but utility is limited by off-target effects and transient receptor engagement. In previous studies, the authors characterized and developed covalent PPARg inhibitors with improved inverse agonist activities. They also showed that these molecules engage unique PPARg ligand binding domain (LBD) conformations whereby the c-terminal helix 12 penetrates into the orthosteric binding pocket to stabilize a repressive state. In the nuclear receptor superclass of proteins, helix 12 is an allosteric switch that governs pharmacologic responses, and this new conformation was highly novel. In this study, the authors did a more thorough analysis of how two covalent inhibitors, SR33065 and SR36708 influence the structural dynamics of PPARg LBD.

Strengths:

(1) The authors employed a compelling integrated biochemical and biophysical approach.

(2) The cobinding studies are unique for the field of nuclear receptor structural biology, and I'm not aware of any similar structural mechanism described for this class of proteins.

(3) Overall, the results support their conclusions.

(4) The results open up exciting possibilities for the development of new ligands that exploit the potential bidirectional relationship between the covalent versus non-covalent ligands studied here.

Weaknesses:

(1) The major weakness in this work is that it is hard to appreciate what these shifting allosteric ensembles actually look like on the protein structure. Additional graphical representations would really help convey the exciting results of this study.

Reviewer #1 (Public review):

Summary:

This manuscript by Harris and Gallistel investigates how the rate of learning and strength of conditioned behavior post learning depend on the various temporal parameters of Pavlovian conditioning. They replicate results from Gibbon and Balsam (1981) in rats to show that the rate of learning is proportional to the ratio between the cycle duration and the cue duration. They further show that the strength of conditioned behavior post learning is proportional to the cue duration, and not the above ratio. The overall findings here are interesting, provide context to many conflicting recent results on this topic, and are supported by reasonably strong evidence. Nevertheless, there are some major weaknesses in the evidence presented for some of the stronger claims in the manuscript.

Strengths:

This manuscript has many strengths including a rigorous experimental design, several different approaches to data analysis, careful consideration of prior literature, and a thorough introduction and discussion. The central claim-that animals track the rates of events in their environment, and that the ratio of two rates determine the rate of learning-is supported with solid evidence.

Weaknesses:

Despite the above major strengths, some key aspects of the paper need major improvement. These are listed below.

(1) A key claim made here is that the same relationship (including the same parameter) describes data from pigeons by Gibbon and Balsam (1981) and the rats in this study. I think the evidence for this claim is weak as presented here. First, the exact measure used for identifying trials to criterion makes a big difference in Fig 3. As best as I understand, the authors do not make any claims about which of these approaches is the "best" way. Second, the measure used for identifying trials to criterion in Fig 1 appears different from any of the criteria used in Fig 3. If so, to make the claim that the quantitative relationship is one and the same in both datasets, the authors need to use the same measure of learning rate on both datasets and show that the resultant plots are statistically indistinguishable. Currently, the authors simply plot the dots from the current dataset on the plot in Fig 1 and ask the readers to notice the visual similarity. This is not at all enough to claim that both relationships are the same. In addition to the dependence of the numbers on the exact measure of learning rate used, the plots are in log-log axis. Slight visual changes can mean a big difference in actual numbers. For instance, between Fig 3 B and C, the highest information group moves up only "slightly" on the y-axis but the difference is a factor of 5. The authors need to perform much more rigorous quantification to make the strong claim that the quantitative relationships obtained here and in Gibbon and Balsam 1981 are identical.

(2) Another interesting claim here is that the rates of responding during ITI and the cue are proportional to the corresponding reward rates with the same proportionality constant. This too requires more quantification and conceptual explanation. For quantification, it would be more convincing to calculate the regression slope for the ITI data and the cue data separately and then show that the corresponding slopes are not statistically distinguishable from each other. Conceptually, I am confused why the data used to the test the ITI proportionality come from the last 5 sessions. Specifically, if the model is that animals produce response rates during the ITI (a period with no possible rewards) based on the overall rate of rewards in the context, wouldn't it be better to test this before the cue learning has occurred? Before cue learning, the animals would presumably only have attributed rewards in the context to the context and thus, produce overall response rates in proportion to the contextual reward rate. After cue learning, the animals could technically know that the rate of rewards during ITI is zero. Why wouldn't it be better to test the plotted relationship for ITI before cue learning has occurred? Further, based on Fig 1, it seems that the overall ITI response rate reduces considerably with cue learning. What is the expected ITI response rate prior to learning based on the authors' conceptual model? Why does this rate differ pre and post cue learning? Finally, if the authors' conceptual framework predicts that ITI response rate after cue learning should be proportional to contextual reward rate, why should the cue response rate be proportional to cue reward rate instead of cue reward rate plus contextual reward rate?

(3) I think there was a major conceptual disconnect between the gradual nature of learning shown in Figs 7 and 8 and the information theoretic model proposed by the authors. To the extent that I understand the model, the animals should simply learn the association once the evidence crosses a threshold (nDKL > threshold) and then produce behavior in proportion to the expected reward rate. If so, why should there be a gradual component of learning as shown in these figures? In terms of the proportional response rule to rate of rewards, why is it changing as animals go from 10% to 90% of peak response? I think the manuscript would be much strengthened if these results are explained within the authors' conceptual framework. If these results are not anticipated by the authors' conceptual framework, please do explicitly state this in the manuscript.

(4) I find the idea stated in the Conclusion section that any model considering probability of reinforcement cannot be correct because it doesn't have temporal units to be weak. I think the authors might mean that existing models based on probability do not work and not that no possible model can work. For any point process, the standard mathematical treatment of continuous time is to compute the expected count of events as p*dt where p is the probability of occurrence of the event in that time bin and dt is an infinitesimal time bin. There is obviously a one-to-one mapping between probability of an event in a point process and its rate. Existing models use an arbitrary time bin/trial and thus, I get the authors' argument in the discussion. However, I think their conclusion is overstated.

(5) The discussion states that the mutual information defined in equation 1 does not change during partial reinforcement. I am confused by this. The mean delay between reinforcements increases in inverse proportion to the probability of reinforcement, but doesn't the mean delay between cue and next reinforcement increase by more than this amount (next reinforcement is greater than or equal to the cue-to-cue interval away from the cue for many trials)? Why is this ratio invariant to partial reinforcement?

Comments on revisions:

Update following revision

(1) This point is discussed in more detail in the attached file, but there are some important details regarding the identification of the learned trial that require more clarification. For instance, isn't the original criterion by Gibbon et al. (1977) the first "sequence of three out of four trials in a row with at least one response"? The authors' provided code for the Wilcoxon signed rank test and nDkl thresholds looks for a permanent exceeding of the threshold. So, I am not yet convinced that the approaches used here and in prior papers are directly comparable. Also, there's still no regression line fitted to their data (Fig 3's black line is from Fig 1, according to the legends). Accordingly, I think the claim in the second paragraph of the Discussion that the old data and their data are explained by a model with "essentially the same parameter value" is not yet convincing without actually reporting the parameters of the regression. Related to this, the regression for their data based on my analysis appears to have a slope closer to -0.6, which does not support strict timescale invariance. I think that this point should be discussed as a caveat in the manuscript.

(2) The authors report in the response that the basis for the apparent gradual/multiple step-like increases after initial learning remains unclear within their framework. This would be important to point out in the actual manuscript. Further, the responses indicating the fact that there are some phenomena that are not captured by the current model would be important to state in the manuscript itself.

(3) There are several mismatches between results shown in figures and those produced by the authors' code, or other supplementary files. As one example, rat 3 results in Fig 11 and Supplementary Materials don't match and neither version is reproduced by the authors' code. There are more concerns like this, which are detailed in the attached review file.

Reviewer #1 (Public review):

Summary:

Systemic and partial Tcf7l2 repression is effective in protecting cancer mice from cachexia-induced death. Hence, this is a promising treatment strategy for cancer patients suffering from cachexia.

Strengths:

The method is well-designed and clearly explained.

Weaknesses:

(1) Abbreviations should be mentioned in full terms for the first time.

(2) Relatively old or even very old references in the Introduction and Discussion.

(3) The result section contains discussion with references, as well.

(4) The number of mice in individual groups is relatively small (3 mice in some groups).

Reviewer #1 (Public review):

This is a very elegant and convincing study. Using systematic screening of actin tail formation in two bacterial strains and employing a panel of CRISPR-CAS ko cell lines, the authors identify a novel dynamin-related GTPase GVIN, which forms an oligomeric coat around an intracellular Burkholderia strain. The bacterial O-antigen LPS layer is required for the formation of the GVIN coat, which disturbs the polar localization of the bacterial actin-polymerizing BimA protein.

I am not an expert in infection studies, but the experiments appear to be of high quality, the figures are well prepared, and clean and statistically significant results are provided. I have no criticism of the presented approaches.

The identification of a novel GBP1-independent pathway targeting intracellular bacteria is not only of fundamental importance for the immunity field but also of high interest to researchers in other areas, for example, evolutionary or structural biologists.

Reviewer #1 (Public review):

Summary:

This manuscript describes a novel magnetic steering technique to target human adipose derived mesenchymal stem cells (hAMSC) or induce pluripotent stem cells to the TM (iPSC-TM). The authors show delivery of the stem cells lowered IOP, increased ouflow facility, and increased TM cellularity.

Strengths:

The technique is novel and shows promise as a novel therapeutic to lower in IOP in glaucoma. hAMSC are able to lower IOP below baseline as well as increase outflow facility above baseline with no tumorigenicity. These data will have a positive impact on the field and will guide further research using hAMSC in glaucoma models.

Weaknesses:

The transgenic mouse model of glaucoma the authors used did not show ocular hypertensive phenotypes as previously reported; therefore, the Tg-MYOCY437H model should be used with caution in the future. However, the results presented here clearly show magnetically steered cell therapy as a viable treatment strategy to lower intraocular pressure even from baseline. Future studies are needed to demonstrate the effects in ocular hypertensive eyes.

Örsted hat das derzeit weltweit größte Windenergieprojekt Harnz-ZV unterbrochen. Die Pause stellt die Realisierung des britischen Ziels, Bis 2030 50 Gigawatt Strom durch Auf Schrahr, Windenergie Windenergie zu produzieren in Frage. Der Windpark Hornzzi vor hätte alleine oder soll alleine 2400 Megawatt Strom produzieren. Für die Unterbrechung wurden vor allem kostengründelverantwortlich gemacht. In den USA wurden von der Trump-Administration mehrere Windenergieprojekte aus Tanz gestorpt.

https://www.connaissancedesenergies.org/afp/eolien-offshore-le-geant-orsted-met-en-pause-lexpansion-du-plus-grand-parc-du-monde-250507?utm_source=newsletter&utm_medium=fil-info-energies&utm_campaign=/newsletter/cde-aujourdhui-7-mai-2025&sstc=u36579nl166 571

Reviewer #1 (Public review):

The authors attempted to replicate previous work showing that counterconditioning leads to more persistent reduction of threat responses, relative to extinction. They also aimed to examine the neural mechanisms underlying counterconditioning and extinction. They achieved both of these aims, and were able to provide some additional information, such as how counterconditioning impacts memory consolidation. Having a better understanding of which neural networks are engaged during counterconditioning may provide novel pharmacological targets to aid in therapies for traumatic memories. It will be interesting to follow up by examining the impact of varying amounts of time between acquisition and counterconditioning phases, to enhance replicability to real world therapeutic settings.

Major strengths

• This paper is very well written and attempts to comprehensively assess multiple aspects counterconditioning and extinction processes. For instance, the addition of memory retrieval tests is not core to the primary hypotheses, but provides additional mechanistic information on how episodic memory is impacted by counterconditioning. This methodical approach is commonly seen in animal literature, but less so in human studies.

• The Group x Cs-type x Phase repeated measure statistical tests with 'differentials' as outcome variables are quite complex, however the authors have generally done a good job of teasing out significant F test findings with post hoc tests and presenting the data well visually. It is reassuring that there is convergence between self-report data on arousal and valence and the pupil dilation response. Skin conductance is a notoriously challenging modality, so it is not too concerning that this was placed in the supplementary materials. Neural responses also occurred in logical regions with regards to reward learning.

• Strong methodology with regards to neuroimaging analysis, and physiological measures.

• The authors are very clear on documenting where there were discrepancies from their pre-registration and providing valid rationales for why.

Major Weaknesses

• The statistics showing that counterconditioning prevents differential spontaneous recovery are the weakest p values of the paper (and using one tailed tests, although this is valid due to directions being pre-hypothesised). This may be due to relatively small number of participants and some variability in responses.

Reviewer #1 (Public review):

Summary:

Audio et al. present an interesting study examining cerebral blood volume (CBV) across cortical areas and layers in non-human primates (NHPs) using high-resolution MRI. While with contrast agents are frequently employed to improve fMRI sensitivity in NHP research, its application for characterizing baseline CBV distribution is less common. This study quantifies large-vessel distribution as well as regional and laminar CBV variations, comparing them with other metrics.

Strengths:

(1) Noninvasive mapping of relative cerebral blood volume is novel for non-human primates.<br /> (2) A key finding was the observation of variations in CBV across regions; primary sensory cortices had high CBV, whereas other higher areas had low CBV.<br /> (3) The measured relative CBV values correlated with previously reported neuronal and receptor densities, potentially providing valuable physiological insights.

Weaknesses:

(1) A weakness of this manuscript is that the quantification of CBV with postprocessing approaches to remove susceptibility effects from pial and penetrating vessels is not fully validated, especially on a laminar scale.<br /> (2) High-resolution MRI with a critical sampling frequency estimated from previous studies (Weber 2008, Zheng 1991) was performed to separate penetrating vessels. However, this approach depends on multiple factors, including spatial resolution, contrast agent dosage, and data processing methods. This raises concerns about the generalizability of these findings to other experimental setups or populations.<br /> (3) Baseline R2* is sensitive to baseline R2, vascular volume, iron content, and susceptibility gradients. Additionally, it is sensitive to imaging parameters; higher spatial resolution tends to result in lower R2* values (closer to the R2 value). Although baseline R2* correlates with several physiological parameters, drawing direct physiological inferences from it remains challenging.<br /> (4) CBV-weighted deltaR2*, which depends on both CBV and contrast agent dose, correlates with various metrics (cytoarchitectural parcellation, myelin/receptor density, cortical thickness, CO, cell-type specificity, etc.). While such correlations may be useful for exploratory analyses, all comparisons depend on measurement accuracy. A fundamental question remains whether CBV-weighted ΔR2* can provide reliable and biologically meaningful insights into these metrics, particularly in diseased or abnormal brain states.

Reviewer #1 (Public review):

Summary:

This study explores how heterozygosity for specific neurodevelopmental disorder-associated Trio variants affects mouse behavior, brain structure, and synaptic function, revealing distinct impacts on motor, social, and cognitive behaviors linked to clinical phenotypes. Findings demonstrate that Trio variants yield unique changes in synaptic plasticity and glutamate release, highlighting Trio's critical role in presynaptic function and the importance of examining variant heterozygosity in vivo.

Strengths:

This study generated multiple mouse lines to model each Trio variant, reflecting point mutations observed in human patients with developmental disorders. The authors employed various approaches to evaluate the resulting behavioral, neuronal morphology, synaptic function, and proteomic phenotypes.