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    1. Article L111-1Version en vigueur depuis le 26 août 2021Modifié par LOI n°2021-1109 du 24 août 2021 - art. 58L'éducation est la première priorité nationale. Le service public de l'éducation est conçu et organisé en fonction des élèves et des étudiants. Il contribue à l'égalité des chances et à lutter contre les inégalités sociales et territoriales en matière de réussite scolaire et éducative.
    1. Reviewer #1 (Public Review):

      Osteoarthritis (OA) is associated with painful, chronic inflammation that often leads to severe joint pain and joint stiffness for people over the age of 55. There is no effective therapeutic drug in the treatment of osteoarthritis. The authors found that mice without Cbfβ in their chondrocytes develop spontaneous OA. Authors uncovered that the deficiency of Cbfβ caused increased canonical Wnt signaling and inflammatory response, and decreased Hippo/YAP signaling and TGF-β signaling in articular cartilage. Authors showed that ACLT surgery-induced OA decreased Cbfβ and Yap expression and increased active β-catenin expression in articular cartilage, while local AAV-mediated Cbfβ overexpression promoted Yap expression, diminished active β-catenin expression in OA lesions. The authors demonstrated that AAV-mediated Cbfβ overexpression in knee joints of mice with OA showed the significant protective effect of Cbfβ on articular cartilage in the ACLT OA mouse model. The results from the study demonstrated Cbfβ maintains articular cartilage homeostasis through inhibiting Wnt/β-catenin signaling and increasing Hippo/Yap, and TGFβ signaling. Importantly, the authors proved that local Cbfβ overexpression could be an effective strategy for treatment of OA. The data shown in the study demonstrated that the findings are novel and very significant, and the authors' claims and conclusions are justified by their data. The paper is generally excellent with an interesting scientific premise and strong scientific rigor. The findings in this manuscript are novel, the manuscript is clearly written, and the findings will make a significant impact in the field.

    1. Reviewer #1 (Public Review):

      In this paper, Nikolaou et al. demonstrated that CYRI-B expression is upregulated in a mouse model of pancreatic ductal adenocarcinoma (PDAC). Interestingly, they found that, while CYRI-B KO promotes the early stages of tumour progression, it prevents the formation of metastasis at later stages. Focusing on the latter, the authors highlight a role for CYRI-B in controlling the membrane availability of the LPA receptor LPAR1, which is required to support PDAC cell chemotaxis towards serum or LPA.

      Strengths: the in vivo and imaging data are very solid, and convincingly support the authors' conclusions. The KPC model is well-established in PDAC research and is a very powerful tool to investigate disease onset and progression. The imaging approaches used are of a very high standard. Good data presentation with the use of super-plots.

      Weaknesses: the authors focused on chemotaxis, but did not present any evidence with regard to the role of CYRI-B in 3D cell invasion, which is a key process associated with cell invasion. The data presented clearly show a specific effect towards liver metastasis, while diaphragm and bowel metastasis were not affected by CYRI-B deletion. It would be beneficial to include a discussion about this, providing some potential explanation behind this observation.

      This work is of interest to cell biologists not only working in pancreatic cancer but also more broadly to researchers interested in vesicular trafficking, plasma membrane receptor dynamics and cell migration.

    1. Reviewer #1 (Public Review):

      Hoving and colleagues investigated the mechanisms of contact inhibition of locomotion (CIL) in Schwann cells using cell migration assays, in combination with siRNA as well as an ex-vivo model for collective cell migration of the peripheral nervous system. They found that N-cadherin is needed for proper cell repulsion during CIL. Schwann Cells depleted of N-cadherin failed CIL when encountering other Schwann cells depleted of N-cadherin, however they maintained CIL when encountering Schwann cells expressing N-cadherin. Depletion of alpha-catenin and to some degree p120 did not have the same effect as N-cadherin depletion. Further, they determined that the extracellular domain is needed for CIL as well as an interaction with Glypican-4. Glypicans often act as co-receptors for other signaling molecules, and so the authors further narrowed CIL's dependence to Slit signaling. N-cadherin was needed for proper Slit surface expression, again, dependent on the extracellular domain, and depletion of both Slit2 and 3 lead to a cell clumping and rounding phenotype. Finally, using an ex-vivo model of Schawnn cell migration they showed that rSlit lead to a similar cell rounding and clumping phenotype, ultimately leading to an inhibition of cell migration.

      Strengths

      This was a very methodical examination of what is needed for CIL in cultured Schwann cells. The data presented largely supports the findings and the linking of N-cad to glypican-4 to Slit signaling further illuminates this process helping to define the molecular players. The mechanistic insight goes further in that they demonstrate the Slit does not get to the cell surface without the expression of the extracellular domain of N-cad.

      Weaknesses

      The conclusions that can be drawn from this study remain a little narrow since only Schwann cells were used. This is not so much a weakness in that authors were indeed investigating the periphery nervous system regeneration but it does limit their findings. The experiments carried out in the ex-vivo system only touch on one aspect of their cell culture work, the mechanism of Slit. No other aspects of their cell culture system was tested ex-vivo which

    1. Reviewer #1(Public Review):

      In this manuscript the authors report an experiment to assess how training on a perceptual task may not only increase performance on that task but impact on the appearance of the trained stimuli. They compare discrimination performance, coherence thresholds, and estimation biases for random dot motion direction relative to horizontal rightward in three groups of observers before and after 3 days in which they either trained on a discrimination task, an estimation task, or did not train. The authors report significant increases in discrimination performance post training compared to not training. They also report increases in estimation biases when assessed as the average estimate (over a bimodal distribution that crosses 0) but not when assessed as the mode of the bimodal distribution. They conclude that training resulted in "increases in already-large estimation biases away from horizontal".

      The methods and results are strengthened by the combination of classical psychophysical techniques and sophisticated computational modelling. One weakness is the possibility is misleading summary statistics when dealing with bimodal distributions. Convincing evidence that observers perceived stimulus directions as further from horizontal (in the absolute sense) following training is not presented in the current manuscript. Irrespective, this work is likely to impact the field.

    1. Reviewer #1 (Public Review):

      Identifying individual BCR/Ab chain sequences that are members of the same clone is a long-standing problem in the analysis of BCR/Ab repertoire sequencing data. The authors propose a new method designed to be scalable for application to huge repertoire data sets without sacrificing accuracy. Their approach utilizes Hamming Distance between CDR3 sequences followed by clustering for a fast, high-precision approach to classifying pairs of sequences as related or not, and then refines the classification using mutation information from germline-encoded regions. They compare their method with other state-of-the-art methods using synthetic data.

      The authors address an important problem in an interesting, innovative, and rigorous way, using probabilistic representations of CDR3 differences, frequencies of shared and not-shared mutations, and the relationships between the two under hypotheses of related pairs and unrelated pairs, and from these develop an approach for determining thresholds for classification and lineage assignment. Benchmarking shows that the proposed method, the complete method including both steps, outperforms other methods.

      Strengths of the method include its theoretical underpinnings which are consistent with an immunologist's intuition about how related and unrelated sequences would compare with each other in terms of the metrics to use and how those metrics are related to each other.

      I have two high-level concerns:<br /> (1) It isn't clear how the real and synthetic data are being used to estimate parameters for the classifier and evaluate the classifier to avoid circularity. It seems like the approach is used to assign lineages in the data from [1], and then properties of this set of lineages are used to estimate parameters that are then used to refine the approach and generate synthetic data that is used to evaluate the approach. This may not be a problem with the approach but rather with its presentation, but it isn't entirely clear what data is being used and where for what purpose. An understanding of this is necessary in order to truly evaluate the method and results.<br /> (2) Regarding the data used for benchmarking - given the intertwined fashion by which the classification approach and synthetic data generation approach appear to have been developed, it is not surprising that the proposed approach outperforms the other methods when evaluated on the synthetic data presented here. It would be better to include in the benchmark the data used by the other methods to benchmark themselves or also generate synthetic data using their data generation procedures.

      An improved method for BCR/Ab sequence lineage assignment would be a methodologic advancement that would enable more rigorous analyses of BCR/Ab repertoires across many fields, including infectious disease, cancer, autoimmune disease, etc., and in turn, enable advancement in our understanding of humoral immune responses. The methods would have utility to a broad community of researchers.

    1. Reviewer #1 (Public Review):

      Summary:

      Doxorubincin has long been known to cause bone loss by increasing osteoclast and suppressing osteoblast activities. The study by Wang et al. reports a comprehensive investigation into the off-target effects of doxorubicin on bone tissues and potential mechanisms.. They used a tumor-free model with wild type mice and found that even a single dose of doxorubicin has a major influence by increasing leukopenia and DAMPs and inflammasomes in macrophages and neutrophils, and inflammation-related cell death (pyroptosis and NETosis). The gene knockout study shows that AIM2 and NLRP3 are the major contributors to bone loss. Overall, the study confirmed previous findings regarding the impact of doxorubicin on tissue inflammation and expands the research further into bone tissue. The presented data presented are consistent; however, a major question remains regarding whether doxorubicin drives inflammation and its related events. Most in vitro study showed that the effect of doxorubincin cannot be demonstrated without LPS priming. This observation raises the question of whether doxorubincin itself could activate the inflammasome and the related events. In vivo study, on the other hand, suggested that it doesn't require LPS. The inconsistency here was not explained further. Moreover, a tumor-free mouse model was used for the study; however, immune responses in tumor bearing models would likely be distinct from tumor-free ones. The justification for using tumor-free models is not well-established.

      Strengths:

      The paper includes a comprehensive study that shows the effects of doxorubincin on cytokine levels in serum, release of DAMPs and NETosis, and leukopenia using both in vivo and in vitro models. Bone marrow cells, macrophages and neutrophils were isolated from the bone marrow, and the levels of cytokines in serum were also determined.

      They employed multiple knockout models with deficiency in Aim 2, Nlirp3, and double deficiencies to dissect the functional involvement of these two inflammasomes.

      The experiments in general are well designed. The paper is also logically written, and figures were clearly labeled.

      Weaknesses:

      Most of the data presented are correlative, and there is not much effort to dissect the underlying molecular mechanism.

      It is not entirely clear why a tumor free model is chosen to study immune responses, as immune responses can differ significantly with or without tumor-bearing.

      Immune responses in isolated macrophages, neutrophils and bone marrow cells require priming with LPS, while such responses are not observed in vivo. There is no explanation for these differences.

      The band intensities on Western blots in Fig. 4 and Fig. 5 are not quantified, and the numbers of repeats are also not provided.

      Many abbreviations are used throughout the text, and some of the full names are not provided.

      Fig. 5B needs a label on X axis.

    1. Reviewer #1 (Public Review):

      In this manuscript by Buchanan and colleagues, the authors set out to determine if mutations associated with resistance to the Plasmodium apicoplast inhibitor azithromycin (AZ) had a measurable impact on the fitness of Plasmodium berghei and P. falciparum parasites as they traverse both the mosquito host and vertebrate liver.

      The Plasmodium endosymbiotic organelles - the mitochondrion and apicoplast - are attractive drug targets as they (1) possess essential functions across the multi-host multi-compartment life cycle of these parasites, and (2) are of bacterial origin and thus are vulnerable to inhibition both to extant antibiotics, and novel drugs with high parasite specificity.

      Historically however the high resistance propensity of drug targets encoded in the organellar genomes (most notably atovaquone and doxycycline) has precluded the use of these drugs in an endemic setting, limiting these potent compounds to use in prophylaxis for travelers from non-endemic countries. Several studies in the last decade now fairly definitively show that mutations conferring resistance to atovaquone in the mitochondrial gene cytochrome b are, in a mutation-dependent manner, totally or near-totally compromised in their ability to infect, grow, and escape the mosquito host, leading to a reexamination of the potential utility of this extraordinarily potent drug in endemic settings. Symmetries exist between the Plasmodium mitochondrion and apicoplast, which both appear to have highly fexpanded roles in the mosquito and liver relative to the blood stages. Thus, the authors set out to explore whether mutations in essential apicoplast genes were, in a similar manner to mutations in cytochrome B, associated with fitness effects in the mosquito and/or liver.

      Towards this, the authors selected for several AZ-resistant parasite populations, all of which acquired mutations in the apicoplast genome-encoded ribosomal protein Rpl4. Interestingly, the authors observed contrasting fitness effects caused by these mutations, both between mutants within Plasmodium species, and between species. In P. berghei, AZ mutants were compromised in their ability to form oocysts and sporozoites, and a large proportion of sporozoites lacked an intact apicoplast and displayed aberrant gliding behaviour. Similarly, in the liver, Rpl4 mutant P. berghei liver schizonts were smaller, had fewer nuclei, and appeared extremely limited in their ability to cause a patent infection - crucially in particular via mosquito bites. Surprisingly, a P. falciparum Rpl4 mutant (notably in a different position of the protein) had no impact on sporogony but appeared to have a strong impact on liver schizont development in a liver-humanized mouse model, suggesting that establishment of blood stage infection in a subsequent human host would be less likely for mutant parasites.

      This is a well-executed study, that presents novel and noteworthy findings. The impact of drug-resistance-conferring mutations in Plasmodium outside of the blood stage is woefully understudied, primarily due to significant challenges associated with studying Plasmodium, especially P. falciparum, in both the mosquito and liver which the authors navigate commendably. The results presented in this manuscript leverage state-of-the-art techniques and clearly support the authors' conclusion that AZ-conferring resistance mutations have a strong negative effect on the ability of Plasmodium parasites to both reinfect and cause symptomatic infection in a subsequent vertebrate host. This could indicate that apicoplast-targeted inhibitors are more attractive as co-drugs for malaria treatment than previously thought, due to the reduced probability of the spread of resistance, which has been a perennial issue in malaria therapeutic care.

    1. Reviewer #1 (Public Review):

      Mohseni and Elhaik's article offers a critical evaluation of Geometric Morphometrics (GM), a common tool in physical anthropology for studying morphological differences and making phylogenetic inferences. I read their article with great interest, although I am not a geneticist or an expert on PCA theory since the problem of morphology-based classification is at the core of paleoanthropology.

      The authors developed a Python package for processing superimposed landmark data with classifier and outlier detection methods, to evaluate the adequacy of the standard approach to shape analysis via modern GM. They call into question the accuracy, robustness, and reproducibility of GM, and demonstrate how PCA introduces statistical artefacts specific to the data, thus challenging its scientific rigor. The authors demonstrate the superiority of machine learning methods in classification and outlier detection tasks. The paper is well-written and provides strong evidence in support of the authors' argument. Thus, in my opinion, it constitutes a major contribution to the field of physical anthropology, as it provides a critical and necessary evaluation of what has become a basic tool for studying morphology, and of the assumptions allowing its application for phylogenetic inferences. Again, I am not an expert in these statistical methods, nor a geneticist, but the authors' contribution is of substantial relevance to our field (physical anthropology). The examples of NR fossils and HLD 6 are cases in point, in line with other notable examples of critical assessment of phylogenetic inferences made on the basis of PCA results of GM analysis. For example, see Lordkipanidze et al.'s (2014) GM analyses of the Dmanisi fossils, suggesting that the five crania represent a single regional variant of Homo erectus; and see Schwartz et al.'s (2014) comment on their findings, claiming that the dental, mandibular, and cranial morphology of these fossils suggest taxic diversity. Schwartz et al. (2014) ask, "Why did the GMA of 78 landmarks not capture the visually obvious differences between the Dmanisi crania and specimens commonly subsumed H. erectus? ... one wonders how phylogenetically reliable a method can be that does not reflect even easily visible gross morphological differences" (p. 360).

      As an alternative to the PCA step in GM, the authors tested eight leading supervised learning classifiers and outlier detection methods on three-dimensional datasets. The authors demonstrated inconsistency of PCA clustering with the taxonomy of the species investigated for the reconstruction of their phylogeny, by analyzing a database comprising landmarks of 6 known species that belong to the Old World monkeys tribe Papionini, using PCA for classification. The authors also demonstrated that high explained variance should not be used as an estimate of high accuracy (reliability). Then, the authors altered the dataset in several ways to simulate the characteristic nature of paleontological data.

      The authors excluded taxa from the database to study how PCA and alternative classifiers are affected by partial sampling, and the results presented in Figures 4 and 5, among others, are quite remarkable in showing the deviations from the benchmark data. These results expose the perils of applying PCA and GM for interpreting morphological data. Furthermore, they provide evidence showing that the alternative classifiers are superior to PCA, and that they are less susceptible to experimenter intervention. Similar results, i.e., inconsistencies in the PC plots, were obtained in examinations of the effect of removing specimens from the dataset and in the interesting test of removing landmarks to simulate partial morphological data, as is often the case with fossils. To test the combined effect of these data alterations, the authors combined removal of taxa, specific samples, and landmarks from the dataset. In this case, as well, the PCA results indicate deviation from the benchmark data. However, the ML classifiers could not remedy the situation. The authors discuss how these inconsistencies may lead to different interpretations of the data, and in turn, different phylogenetic conclusions. Lastly, the authors simulated the situation of a specimen of unknown taxonomy using outlier detection methods, demonstrating LOF's ability to identify a novelty in the morphospace.

      References<br /> Bookstein FL. 1991. Morphometric tools for landmark data: geometry and biology [Orange book]. Cambridge New York: Cambridge University Press.<br /> Cooke SB, and Terhune CE. 2015. Form, function, and geometric morphometrics. The Anatomical Records 298:5-28.<br /> Lordkipanidze D, et al. 2013. A complete skull from Dmanisi, Georgia, and the evolutionary biology of early Homo. Science 342: 326-331.<br /> Schwartz JH, Tattersall I, and Chi Z. 2014. Comment on "A complete skull from Dmanisi, Georgia, and the evolutionary biology of Early Homo". Science 344(6182): 360-a.

    1. Joint Public Review:

      Roget et al. build on their previous work developing a simple theoretical model to examine whether ageing can be under natural selection, challenging the mainstream view that ageing is merely a byproduct of other biological and evolutionary processes. The authors propose an agent-based model to evaluate the adaptive dynamics of a haploid asexual population with two independent traits: fertility timespan and mortality onset. Through computational simulations, their model demonstrates that ageing can give populations an evolutionary advantage. Notably, this observation arises from the model without invoking any explicit energy tradeoffs, commonly used to explain this relationship.

      Additionally, the theoretical model developed here indicates that mortality onset is generally selected to start before the loss of fertility, irrespective of the initial values in the population. The selected relationship between the fertility timespan and mortality onset depends on the strength of fertility and mortality effects, with larger effects resulting in the loss of fertility and mortality onset being closer together. By allowing for a trans-generational effect on ageing in the model, the authors show that this can be advantageous as well, lowering the risk of collapse in the population despite an apparent fitness disadvantage in individuals. Upon closer examination, the authors reveal that this unexpected outcome is a consequence of the trans-generational effect on ageing increasing the evolvability of the population (i.e., allowing a more effective exploration of the parameter landscape), reaching the optimum state faster.

      The simplicity of the proposed theoretical model represents both the major strength and weakness of this work. On one hand, with an original and rigorous methodology, the logic of their conclusions can be easily grasped and generalised, yielding surprising results. Using just a handful of parameters and relying on direct competition simulations, the model qualitatively recapitulates the negative correlation between lifespan and fertility without requiring energy tradeoffs. This alone makes this work an important milestone for the rapidly growing field of adaptive dynamics, opening many new avenues of research, both theoretically and empirically.

      On the other hand, the simplicity of the model also makes its relationship with living organisms difficult to gauge, leaving open questions about how much the model represents the reality of actual evolution in a natural context. In particular, a more explicit discussion on how the specifics of the model can impact the results and their interpretation is needed. For example, the lack of mechanistic details on the trans-generational effect on ageing makes the results difficult to interpret. Even if analytical results are obtained, most of the observations appear derived from simulations as they are currently presented. Also, the choice of parameters for the simulations shown in the paper and how it relates to our biological knowledge is not fully addressed by the authors. Finally, the conclusions of evolvability are insufficiently supported, as the authors do not show if the wider genotypic variability in populations with the ageing trans-generational effect is, in fact, selected.

    1. Reviewer #1 (Public Review):

      Summary:

      In this study, Faniyan and colleagues build on their recent finding that renal Glut2 knockout mice display normal fasting blood glucose levels despite massive glucosuria. Renal Glut2 knockout mice were found to exhibit increased endogenous glucose production along with decreased hepatic metabolites associated with glucose metabolism. Crh mRNA levels were higher in the hypothalamus while circulating ACTH and corticosterone was elevated in this model. While these mice were able to maintain normal fasting glucose levels, ablating afferent renal signals to the brain resulted in substantially lower blood glucose levels compared to wildtype mice. In addition, the higher CRH and higher corticosterone levels of the knockout mice were lost following this denervation. Finally, acute phase proteins were altered, plasma Gpx3 was lower, and major urinary protein MUP18 and its gene expression were higher in renal Glut2 knockout mice. Overall, the main conclusion that afferent signaling from the kidney is required for renal glut2 dependent increases in endogenous glucose production is well supported by these findings.

      Strengths:

      An important strength of the paper is the novelty of the identification of kidney to brain communication as being important for glucose homeostasis. Previous studies had focused on other functions of the kidney modulated by or modulating brain function. This work is likely to promote interest in CNS pathways that respond to afferent renal signals and the response of the HPA axis to glucosuria. Additional strengths of this paper stem from the use of incisive techniques. Specifically, the authors use isotope enabled measurement of endogenous glucose production by GC-MS/MS, capsaicin ablation of afferent renal nerves, and multifiber recording from the renal nerve. The authors also paid excellent attention to rigor in the design and performance of these studies. For example, they used appropriate surgical controls, confirmed denervation through renal pelvic CGRP measurement, and avoided the confounding effects of nerve regrowth over time. These factors strengthen confidence in their results. Finally, humans with glucose transporter mutations and those being treated with SGLT2 inhibitors show a compensatory increase in endogenous glucose production. Therefore, this study strengthens the case for using renal Glut2 knockout mice as a model for understanding the physiology of these patients.

      Weaknesses:

      A few weaknesses exist. Most concerns relate to the interpretation of this study's findings. The authors state that loss of glucose in urine is sensed as a biological threat based on the HPA axis activation seen in this mouse model. This interpretation is understandable but speculative. Importantly, whether stress hormones mediate the increase in endogenous glucose production in this model and in humans with altered glucose transporter function remains to be demonstrated conclusively. For example, the paper found several other circulating and local factors that could be causal. This model is also unable to shed light on how elevated stress hormones might interact with insulin resistance, which is known to increase endogenous glucose production. That issue is of substantial clinical relevance for patients with T2D and metabolic disease. Finally, while findings from the Glut2 knockout mice are of scientific interest, it should be noted that the Glut2 receptor is critical to the function of pancreatic islets and as such is not a good candidate for pharmacological targeting

    1. Reviewer #1 (Public Review):

      Summary:

      The study investigated how root cap cell corpse removal affects the ability of microbes to colonize Arabidopsis thaliana plants. The findings demonstrate how programmed cell death and its control in root cap cells affect the establishment of symbiotic relationships between plants and fungi. Key details on molecular mechanisms and transcription factors involved are also given. The study suggests reevaluating microbiome assembly from the root tip, thus challenging traditional ideas about this process. While the work presents a key foundation, more research along the root axis is recommended to gain a better understanding of the spatial and temporal aspects of microbiome recruitment.

    1. Reviewer #1 (Public Review):

      Summary:

      In this paper, the authors performed molecular dynamics (MD) simulations to investigate the molecular basis of the association of alpha-synuclein chains under molecular crowding and salt conditions. Aggregation of alpha-synuclein is linked to the pathogenesis of Parkinson's disease, and the liquid-liquid phase separation (LLPS) is considered to play an important role in the nucleation step of the alpha-synuclein aggregation. This paper re-tuned the Martini3 coarse-grained force field parameters, which allows long-timescale MD simulations of intrinsically disordered proteins with explicit solvent under diverse environmental perturbation. Their MD simulations showed that alpha-synuclein does not have a high LLPS-forming propensity, but the molecular crowding and salt addition tend to enhance the tendency of droplet formation and therefore modulate the alpha-synuclein aggregation. The MD simulation results also revealed important intra and inter-molecule conformational features of the alpha-synuclein chains in the formed droplets and the key interactions responsible for the stability of the droplets. These MD simulation data add biophysical insights into the molecular mechanism underlying the association of alpha-synuclein chains, which is important for understanding the pathogenesis of Parkinson's disease.

      Strengths:

      (1) The re-parameterized Martini 3 coarse-grained force field enables the large-scale MD simulations of the intrinsically disordered proteins with explicit solvent, which will be useful for a more realistic description of the molecular basis of LLPS.

      (2) This paper showed that molecular crowding and salt contribute to the modulation of the LLPS through different means. The molecular crowding minimally affects surface tension, but adding salt increases surface tension. It is also interesting to show that the aggregation pathway involves the disruption of the intra-chain interactions arising from C-terminal regions, which potentially facilitates the formation of inter-chain interactions.

      Weaknesses:

      (1) Although the authors emphasized the advantage of the Martini3 force field for its explicit description of solvent, the whole paper did not discuss the water's role in the aggregation and LLPS.

      (2) This paper discussed the effects of crowders and salt on the surface tension of the droplets. The calculation of the surface tension relies on the droplet shape. However, for the formed clusters in the MD simulations, the typical size is <10, which may be too small to rigorously define the droplet shape. As shown in previous work cited by this paper [Benayad et al., J. Chem. Theory Comput. 2021, 17, 525−537], the calculated surface tension becomes stable when the chain number is larger than 100.

      (3) In this work, the Martini 3 force field was modified by rescaling the LJ parameters \epsilon and \sigma with a common factor \lambda. It has not been very clearly described in the manuscript why these two different parameters can be rescaled by a common factor and why it is necessary to separately tune these two parameters, instead of just tuning the coefficient \epsilon as did in a previous work [Larsen et al., PLoS Comput Biol 16: e1007870].

      (4) Both the sizes and volume fractions of the crowders can affect the protein association. It will be interesting to perform MD simulations by adding crowders with various sizes and volume fractions. In addition, in this work, the crowders were modelled by fullerenes, which contribute to protein aggregation mainly by entropic means as discussed in the manuscript. It is not very clear how the crowder effect is sensitive to the chemical nature of the crowders (e.g., inert crowders with excluded volume effect or crowders with non-specific attractive interactions with proteins, etc) and therefore the force field parameters.

    1. Reviewer #1 (Public Review):

      Summary:

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

      Strengths:

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

      Weaknesses:

      It is essential to note that the manuscript has issues with experimental designs and lacks consistency in the presented data. Here is a list of the major concerns:

      (1) The authors culture the cells in the presence of fetal bovine serum (FBS) in the culture medium. Given that FBS contains a substantial amount of EVs, this raises a significant issue, as it becomes challenging to differentiate between EVs derived from FBS and those released by the cells. This concern extends to all transmission electron microscopy (TEM) images (Figure 1, 2P-S, S5, Figure 4 P-U) and the quantification of EV numbers in Figure 3. The authors need to use an FBS-free cell culture medium.

      (2) The data presented in Figure 2 is not convincingly supportive of the authors' conclusion. The authors argue that "...CD81 was present in the plasma membrane-derived limiting membrane (Figures 2B, D, F), while CD63 was only found inside the MV-lEVs (Fig. 2A, C, E)." However, in Figure 2G, there is an observable CD63 signal in the limiting membrane (overlapping with the green signals), and in Figure 2J, CD81 also exhibits overlap with MV-IEVs.

      (3) Following up on the previous concern, the authors argue that CD81 and CD63 are exclusively located on the limiting membrane and MV-IEVs, respectively (Figure 2-A-M). However, in lines 104-106, the authors conclude that "The simultaneous presence of CD63, CD81, TSG101, ALIX, and the autophagosome marker LC3B within the MV-lEVs..." This statement indicates that CD63 and CD81 co-localize to the MV-IEVs. The authors need to address this apparent discrepancy and provide an explanation.

      (4) The specificity of the antibodies used in Figure 2 should be validated through knockout or knockdown experiments. Several of the antibodies used in this figure detect multiple bands on western blots, raising doubts about their specificity. Verification through additional experimental approaches is essential to ensure the reliability and accuracy of all the immunostaining data in this manuscript.

      (5) In Figures 2P-R, the morphology of the MV-IEVs does not resemble those shown in Figures 1-A, H, and D, indicating a notable inconsistency in the data.

      (6) There are no loading controls provided for any of the western blot data. Additionally, for Figures 2-S4B, the authors should run the samples from lanes i-iii in a single gel.

      (7) In Figure 2-S4, is there co-localization observed between LC3RFP (LC3A?) with other MV-IFV markers? How about LC3B? Does LC3B co-localize with other MV-IFV markers?

      (8) The TEM images presented in Figure 2-S5, specifically F, G, H, and I, do not closely resemble the images in Figure 2-S5 K, L, M, N, and O. Despite this dissimilarity, the authors argue that these images depict the same structures. The authors should provide an explanation for this observed discrepancy to ensure clarity and consistency in the interpretation of the presented data.

      (9) For Figures 3C and 3-S1, the authors should include the images used for EV quantification. Considering the concern regarding potential contamination introduced by FBS (concern 1), it is advisable for the authors to employ an independent method to identify EVs, thereby confirming the reliability of the data presented in these figures.

      (10) Do the amphiectosomes released from other cell types as well as cells in mouse kidneys or liver contain LC3B positive and CD63 positive ILVs?

    1. Reviewer #1 (Public Review):

      Summary:

      This study aims to understand how cell fusion contributes to wound healing using a laser-induced injury in the notum epithelium of a developing fruit fly. The authors meticulously characterize the epithelial fusion events using a live imaging approach and report that syncytia arise by 'border breakdown' and 'cell shrinking'. The syncytial epithelial cells also appear to outcompete mononucleated cells and preferentially dissolve their tangential borders, which correlates with the accumulation of actin at the leading edge.

      Strengths:

      The strength of this study is the authors' live imaging approach to capture these dynamic fusion events that are a fundamental, yet poorly understood biological process.

      Weaknesses:

      A major weakness is that all the authors' conclusions are based on descriptive studies, in which the role of cell fusion is not directly tested. This is particularly important because other models of wound-induced polyploidization have demonstrated that another cytoskeletal protein, myosin, was upregulated and dependent on endoreplication, and not cell fusion. Therefore it remains unclear to what extent cell fusion, endoreplication, or both are required to outcompete mononucleated cells as well as pool actin as described in this study.

    1. Reviewer #1 (Public Review):

      Summary:

      This is an original manuscript submission by Tatekoshi et al entitled, "Human induced pluripotent stem cell-derived cardiomyocytes to study inflammation-induced diastolic dysfunction." Based on the premise that treated HIV individuals commonly have heart failure with preserved ejection fraction, yet robust animal models have not been established, the team developed iPS-CM models to study HFpEF with this angle in mind. The group established iPS-CMs using standard methods and studied TNFa and IFNy effects on calcium transients. They observed that both cytokines increased calcium transient decay and downstroke times, which could be reversed by mitoTempo treatment in the case of TNFa. To determine how mitochondrial dysfunction may impact the cytokine-induced calcium transient changes, the team measured OCR treatment changes. They observed that NAC and TNFa co-treated cells demonstrated reduced OCR. The team went on to test the effects of antiretroviral therapies including tenofovir, relategravir, elvitegravir, and darunavir at 3-10 uM levels in iPS-CMs. The team noted that ART treatments reversed the diastolic dysfunction associated with TNFa treatment suggesting that ART therapies may improve diastolic dysfunction that is associated with TNFa signaling directly in cardiomyocytes. Following up on this treatment effect, the team screened several other candidates across drug classes and identified that dapagliflozin (SGLT2i) reversed diastolic dysfunction induced by TNFa. Finally, the team collected human serum from patients with HIV+ patients from two hospitals - Northwestern with diastolic dysfunction by cMRI, and UCSF with normal diastolic function by echo. Both cohort serum samples did not change calcium transients in iPS-CMs. However, due to numerous and significant major methodological concerns, and the potential low impact of the study results, this manuscript is expected to be of very low impact to the field in its current form.

      Strengths:

      1) From a significance standpoint, understanding the mechanisms of HFpEF, particularly in conditions such as HIV would be very impactful.

      2) Collecting HIV patient serum and identifying a plasma factor that impacts cardiac function could be very significant if successful.

      Weaknesses:

      (1) I am not convinced how this study relates to HIV individual HFpEF, and the study design does not seem to be well thought out.

      (2) The connectivity of the study experiments is loose, and data analysis and conclusions are broadly overstated and misinterpreted.

      (3) For example the study lacks any measure of diastolic contractile function, and even if performed, the relevance of TNFa treatments to cells in vitro in these immature cell contexts would remain unclear. There is surprisingly no reported molecular analyses of potential mechanisms of the calcium transient changes. The study falls short in molecular detail and instead relies on drug treatments and responses that are hard to interpret with dosages that are not well justified and treatments that are numerous. Unclear what changes in calcium transients mean functionally without a comprehensive assessment of CM biomechanical contraction and relaxation measurements, and this would also require parallel molecular investigations of potential targets of any phenotypes observed.

      (4) Calcium transient data need to be better illustrated such as with representative peak tracings. The data overall is with too few samples, particularly given the inherent heterogeneity of iPS-CM studies. The iPS-CM system as a model for diastolic dysfunction remains unestablished.

      (5) There are unclear dose choices for the various ART drugs tested, as well as the other drugs tested such as SGLT2i. Besides the observation that SLC5A2 (SGLT2 target) is not established to be expressed in adult mammalian cardiomyocytes.

      (6) HIV plasma samples were not tested for cytokine levels, but this could be done to assess the validity of the final experiments. It is unclear what is being tested with these experiments.

      (7) The choice of serum controls from a second institution (UCSF) opens up concerns over batch effects unrelated to differences in diastolic dysfunction. However, there were no differences with the Northwestern samples. It is unclear why this data is included as it does not add to the impact of the study.

      (8) There are concerns about the quality of the iPS-CMs since there is no cell imaging or molecular analyses. Figure 5 Supplement 1 images are of low quality and low resolution to assess cell quality. Overall the iPS-CM QC data is extremely sparse

    1. Reviewer #1 (Public Review):

      Summary:

      The manuscript by Relovska and colleagues aims to decipher the importance of sterol homeostasis on male reproduction and, in particular, the impact of altered sterol homeostasis in sperm cells. To this end, they are generating a global line of Dhcr24 transgenic mice by mating Dhcr24fl/fl mice (overexpressing the construct in the Rosa269c gene locus) with EIla 100 CRE mice (expressing Cre recombinase in the early mouse embryo).

      The data provided are robust, using a range of approaches from sperm analysis (structure, function) to lipid analysis. Results show that overexpression of DHCR24 (TG) leads to altered sterol homeostasis in spermatozoa. Sperm from TG mice have abnormal mitochondria and sperm tails. TG spermatozoa have reduced efficiency in undergoing the acrosomal reaction. Furthermore, the data suggest that TG spermatozoa have an altered metabolism with increased oxygen consumption. These data highlight that desmosterol depletion and/or altered sterol homeostasis impact sperm morphology, number, motility, and metabolism, resulting in reduced male fertility.

      Strengths:

      The manuscript is clear and well-written, and the results are presented in high-quality figures.

      Weaknesses:

      The main concern is the clear analysis of the rodent model. Indeed, the use of this particular Cre leads to whole-body overexpression, the remaining question is whether the observed effects are directly mediated by the testicular impacts of Dhcr24 overexpression. Even if it is a testicular effect, we can't conclude where it comes from. Is it at the level of spermatogenesis, as the authors speculate? Furthermore, the authors mention that normally, Dhcr24c is mainly expressed in spermatogonia, so it's not clear why they focus only on spermatozoa, which in WT males do not normally express dhcr24 according to the authors. It is worth clarifying the testicular phenotype in more detail.

      The lower level of TG in aged mice could suggest an extinction of the transgene at least in sperm during aging, which could be difficult to reconcile with the observed phenotype. With this in mind, it would be interesting to define the penetrance of the phenotype during aging, to define variability between mice, and to clearly define potential correlations between sterol levels and fertility disorders, or altered sperm parameters.

    1. Reviewer #1 (Public Review):

      Summary:

      The goal of this study was to use in vitro cell populations to determine mechanisms that may be important for the propagation of epimutations induced by EDCs in vivo. To do this, authors exposed induced pluripotent stem cells (iPS), somatic cells (Sertoli, granulosa), and primordial germ cell like cells (PGCLCs) to BPS, and conducted epigenomic and transcriptomic analyses on outcomes. The importance of estrogen receptors, and the relationship of epigenomic results to genomic sites expressing EREs, were also determined in the different cell types. Results revealed differential effects of BPS in each cell population on each of these endpoints, and that epimutations were prevalent in enhancer regions with EREs with the exception of PGCLCs (which do not express ERs). The authors speculate that because epimutations also occurred in regions without EREs, especially in PGCLCs, other mechanisms may be in place. Finally, epimutations induced in iPSCs exposed to BPS that were subsequently differentiated into PGCLCs demonstrated that most epimutations were corrected.

      Strengths:

      A strength of this work is the use of different cell types representing somatic cells that would be the major recipient of EDC exposure; pluripotent cells representing preimplantation embryos; and PGCLCs that model the early germline in which epigenetic reprogramming takes place. Work differentiating the iPSCs from PGCLCs with or without BPS exposure at the iPSC level is also very informative as it suggests that most epimutations are corrected, at least in vitro. The paper is well-written and studies were technically well-executed and validated. Results are novel and likely to be of interest to those interested in transgenerational inheritance of environmentally-induced traits, as well as others more broadly interested in epigenetic mechanisms.

      Critique/Weaknesses:

      (1) A problem with in vitro work is that homogeneous cell lines/cultures are, by nature, absent from the rest of the microenvironment. The authors need to discuss this.

      (2) What are n's/replicates for each study? Were the same or different samples used to generate the data for RNA sequencing, methylation beadchip analysis, and EM-seq? This clarification is important because if the same cultures were used, this would allow comparisons and correlations within samples.

      (3) In Figure 1, it is interesting that the 50 uM BPS dose mainly resulted in hypermethylation whereas 100 uM appears to be mainly hypomethylation. (This is based on the subjective appearance of graphs). The authors should discuss and/or present these data more quantitatively. For example, what percentage of changes were hypo/hypermethylation for each treatment? How many DMRs did each dose induce? For the RNA-seq results, again, what were the number of up/down-regulated genes for each dose?

      (4) Also in Figure 1, were there DMRs or genes in common across the doses? How did DMRs relate to gene expression results? This would be informative in verifying or refuting expectations that greater methylation is often associated with decreased gene expression.

      (5) In Figure 2, was there an overlap in the hypo- and/or hyper-methylated DMCs? Please also add more description of the data in 2b to the legend including what the dot sizes/colors mean, etc. Some readers (including me) may not be familiar with this type of data presentation. Some of this comes up in Figure 4, so perhaps allude to this earlier on, or show these data earlier.

      (6) iPSCs were derived from male mice MEFs, and subsequently used to differentiate into PGCLCs. The only cell type from an XX female is the granulosa cells. This might be important, and should be mentioned and its potential significance discussed (briefly).

      (7) EREs are only one type of hormone response element. The authors make the point that other mechanisms of BPS action are independent of canonical endocrine signaling. Would authors please briefly speculate on the possibility that other endocrine pathways including those utilizing AREs or other HREs may play a role? In other words, it may not be endocrine signaling independent. The statement that the differences between PGCLCs and other cells are largely due to the absence of ERs is overly simplistic.

      (8) Interpretation of data from the GO analysis is similarly overly simplistic. The pathways identified and discussed (e.g. PI3K/AKT and ubiquitin-like protease pathways are involved in numerous functions, both endocrine and non-endocrine. Also, are the data shown in Figure 6a from all 4 cell types? I am confused by the heatmap in 6c, which genes were significantly affected by treatment in which cell types?

      (9) In Figure 7, what were the 138 genes? Any commonalities among them?

      (10) The Introduction is very long. The last paragraph, beginning line 105, is a long summary of results and interpretations that better fit in a Discussion section.

      (11) Provide some details on husbandry: e.g. were they bred on-site? What food was given, and how was water treated? These questions are to get at efforts to minimize exposure to other chemicals.

    1. Reviewer #1 (Public Review):

      Wang et al investigated the evolution, expression, and function of the X-linked miR-506 miRNA family. They showed that the miR-506 family underwent rapid evolution. They provided evidence that miR-506 appeared to have originated from the MER91C DNA transposons. Human MER91C transposon produced mature miRNAs when expressed in cultured cells. A series of mouse mutants lacking individual clusters, a combination of clusters, and the entire X-linked cluster (all 22 miRNAs) were generated and characterized. The mutant mice lacking four or more miRNA clusters showed reduced reproductive fitness (litter size reduction). They further showed that the sperm from these mutants were less competitive in polyandrous mating tests. RNA-seq revealed the impact of deletion of miR-506 on the testicular transcriptome. Bioinformatic analysis analyzed the relationship among miR-506 binding, transcriptomic changes, and target sequence conservation. The miR-506-deficient mice did not have apparent effect on sperm production, motility, and morphology. Lack of severe phenotypes is typical for miRNA mutants in other species as well. However, the miR-506-deficient males did exhibit reduced litter size, such an effect would have been quite significant in an evolutionary time scale. The number of mouse mutants and sequencing analysis represent a tour de force.

      Strengths:

      This study is a comprehensive investigation of the X-linked miR-506 miRNA family. It provides important insights into the evolution and function of the miR-506 family.

    1. Reviewer #1 (Public Review):

      The present study conducted by Berger et al. delves into the extent to which memory formation relies on available energy reserves. While aversive memory formation has been extensively studied in this context, the investigation into appetitive memory formation has been comparatively sparse. It has long been recognized that flies can only form appetitive memory under conditions of starvation. However, the authors of this study go beyond this understanding by revealing that not only the duration of starvation matters, but it also dictates the type of memory formed, whether short- or long-term memory. The authors illustrate that internal glycogen stores play a crucial role in this process, facilitated by insulin-like signaling in octopaminergic reward neurons, which integrates internal energy reserves into memory formation. Consequently, the authors propose that octopamine serves as a negative regulator of various forms of memory, shedding light on the enduring question of the octopaminergic neuronal system's involvement in appetitive memory formation, which has been overshadowed by the focus on the dopaminergic system in recent years. Additionally, the findings contribute to the ongoing debate concerning the role of insulin receptors, whether they function within neurons themselves or in glial cells. Moreover, the authors not only convincingly demonstrate that octopamine negatively regulates appetitive memory formation, but they also propose a mechanism whereby the insulin receptor in octopaminergic neurons senses the internal energy status and subsequently modulates the activity of these neurons. The experiments are meticulously designed, employing a variety of behavioral assays, genetic tools for manipulating neuronal activity, and state-of-the-art imaging techniques. The conclusions are well supported by the data and carefully performed controlled experiments, yielding high-quality data.

    1. Reviewer #2 (Public Review):

      Summary:

      This paper acknowledges that most development occurs in social contexts, with other social partners. The authors put forth two main frameworks of how development occurs within a social interaction with a caregiver. The first, is that although social interaction with mature partners is somewhat bi-directional, mature social partners exogenously influence infant behaviors and attention through "attentional scaffolding", and that in this case infant attention is reactive to caregiver behavior. The second framework posits that caregivers support and guide infant attention by contingently responding to reorientations in infant behavior, thus caregiver behaviors are reactive to infant behavior. The aim of this paper is to use moment-to-moment analysis techniques to understand the directionality of dyadic interaction.

      Strengths:

      The question driving this study is interesting and a genuine gap in the literature. Almost all development occurs in the presence of a mature social partner. While it is known that these interactions are critical for development, the directionality of how these interactions unfold in real-time is less known.

      The analyses are appropriate for the question at hand, capturing small moment-to-moment dynamics in both infant and child behavior, and their relationships with themselves and each other. Autocorrelations and cross-correlations are powerful tools that can uncover small but meaningful patterns in data that may not be uncovered with other more discretized analyses (i.e. regression).

      Weaknesses:

      While the authors improved their explanation of why they are using cross-correlations and the resting EEG patterns and what they mean, they did not address this specific piece of feedback: to explain their rationale for only focussing on fronto-temporal channels, rather than averaging channels across the whole brain.

    1. Reviewer #2 (Public Review):

      Summary:

      Through a set of experiments and model simulations, the authors tested whether the commonly assumed world model of gravity was a faithful replica of the physical world. They found that participants did not model gravity as single, fixed vector for gravity but instead as a distribution of possible vectors. Surprisingly, the width of this distribution was quite large (~20 degrees). While previous accounts had suggested that this uncertainty was due to perceptual noise or an inferred external perturbation, the authors suggest that this uncertainty simply arises from a noisy distribution of the representation of gravity's direction. A reinforcement learning model with an initial uniform distribution for gravity's direction ultimately converged to a precision on the same order as the human participants, which lends support to the authors' conclusion and suggests that this distribution is learned through experience. What's more, further simulations suggest that representing gravity with such a wide distribution may balance speed and accuracy, providing a potentially normative explanation for the world model with gravity as a distribution.

      Strengths:

      The authors present surprising findings in a relatively straight-forward in a now classic experimental task. They provide a normative explanation based on a resource-rational framework for why people may have a stochastic world model instead of a deterministic world model. While the stochastic world model could be the result of people mentally simulating an external perturbation, the authors include several control experiments to test this possibility.

      Weaknesses:

      The possibility of inferred external perturbations, as opposed to a stochastic world model, is difficult to rule out. This could stem from how people interpret task instructions and it will likely take many, clever studies, to fully reconcile these two alternative accounts.

    1. Reviewer #1 (Public Review):

      Summary:

      The study examines the role of release site clearance in synaptic transmission during repetitive activity under physiological conditions in two types of central synapses, calyx of Held and hippocampal CA1 synapses. After acute block of endocytosis by pharmacology, deeper synaptic depression or less facilitation was observed in two types of synapses. Acute block of CDC42 and actin polymerization, which possibly inhibits the activity of Intersectin, affected synaptic depression at the calyx synapse, but not at CA1 synapses. The data suggest an unexpected, fast role of the site clearance in counteracting synaptic depression.

      Strengths:

      The study uses acute block of the molecular targets with pharmacology together with precise electrophysiology. The experimental results are clear cut and convincing. The study also examines the physiological roles of the site clearance using action potential-evoked transmission at physiological Ca and physiological temperature at mature animals. This condition has not been examined.

      Weaknesses:

      Pharmacology may have some off-target effects, though acute manipulation should be appreciated and the authors have tried several reagents to verify the overall conclusions.

    1. Reviewer #2 (Public Review):

      Gillespie et al. introduced a novel neurofeedback (NF) procedure to train rats in enhancing their sharp-wave ripple (SWR) rate within a short duration, a key neural mechanism associated with memory consolidation. The training, embedded within a spatial memory task, spanned 20-30 days and utilized food rewards as positive reinforcement upon SWR detection. Rats were categorized into NF and control groups, with the NF group further divided into NF and delay trials for within-subject control. While single trial differences were elusive due to the variability of SWR occurrence, the study revealed that statistically rats in NF trials exhibited a notably higher SWR rate before receiving rewards compared to delay trials. This difference was even more pronounced when juxtaposed with rats not exposed to NF training (control group). The unique design of blending the NF phase with the memory dependent spatial task enabled the authors to analyze whether the NF training influence the task performance and replay content during SWRs across three different conditions (NF trials, delay trials and control group). Interestingly, despite the NF training, there was no significant improvement or decline in the performance of the spatial memory task, and the replay content remained consistent across all three conditions. Hence, the operant conditioning only amplified the SWR rate before reward in NF trials without altering the task performance and the replay content during SWR. Moreover, considering the post-reward period, the total SWR count was consistent across all conditions as well, meaning the NF training also do not affect the total SWR count. The study concludes with the hypothesis of a potential homeostatic mechanism governing the total SWR production in rats. This research significantly extends previous work by Ishikawa et al. (2014), offering insights into the NF training with external reward on the SWR rate/counts, replay content and task performance.

      Strengths:

      - Integration of NF task and spatial memory task in a single trial<br /> The integration of NF training within a spatial memory task poses significant challenges. Gillespie and colleagues overcame this by seamlessly blending the NF task and the spatial memory task into a single trial. Each trial involved a rat undergoing three steps: First, initiating a trial. Second, moving to either the NF port or the delay trial port, as indicated by an LED, and then maintaining a nosepoke at one of the center ports. During this step, the rat had to keep its nose (in the NF port) until a sharp-wave ripple (SWR) exceeding a set threshold was detected, which then triggered a reward, or until a variable time elapsed (in the delay port). Third, the rat would choose one of eight arms to explore before starting the next trial. This integration of the two tasks (step two as the NF task and step three as the spatial memory task) facilitated a direct analysis of the impact of NF training on behaviorally relevant replay content during SWRs and the performance in the spatial memory task.

      - Clear Group Separation<br /> A robust study design necessitates clear distinctions between experimental conditions to ensure that observed differences can be attributed to the variable under investigation. This study meticulously categorized rats into three distinct conditions: NF trials, delay trials (for within-subject control), and a control group (for across-subject control). Furthermore, for each trial, the times of interest (TOI) were separated into pre-reward and post-reward periods. This clear separation ensures that any observed differences in SWR rates and other outcomes can be confidently attributed to the effects of neurofeedback training during specific time periods, minimizing potential confounding factors.

      - Evidence of SWR rate modulation<br /> The study's results offer compelling evidence that rats can be trained to modulate their SWR rates during the pre-reward period. This is evident from the observation that rats in the NF trials consistently displayed a higher SWR rate before receiving rewards compared to those in delay trials or the control group (Fig. 2). Such findings not only validate the efficacy of the NF paradigm but also underscore the potential of operant conditioning in influencing neural mechanisms. The observation that rats were able to produce larger SWR events by modulating their occurrence rate, rather than merely waiting for these events, suggests a learned strategy to generate them more efficiently.

      - Evidence of SWR count homeostasis<br /> A notable finding from the study was the observation of a consistent total SWR count during both pre-reward and post-reward periods across all conditions, despite the evident increase in SWR rates during the pre-reward period in NF trials. This points to a potential homeostatic mechanism governing SWR production in rats. This balance suggests that while NF training can modulate the timing and rate of SWRs over a short duration, it doesn't influence the overall count of SWRs over a longer period. Such a mechanism might be essential in ensuring that the brain neither overcompensates nor depletes its capacity for SWRs, maintaining the overall neural balance and functionality. This discovery deepens our understanding of neural mechanisms and highlights potential avenues for future research into the regulatory processes governing neural activity.

      In this revision, the paper explores a neurofeedback technique in rats that modulates hippocampal sharp-wave ripple (SWR) rates, crucial for memory replay, without altering the content of those replays. The study demonstrates that neurofeedback can specifically increase SWR rates during a task's pre-reward phase. Revisions address concerns about movement's impact on SWR rates, clarify the statistical approach used, and modify the title for accuracy, now emphasizing the modulation of memory replay rates rather than suggesting alterations to memory content itself. I think all the concerns in the previous version have been addressed.

    1. Reviewer #1 (Public Review):

      The study investigates parafoveal processing during natural reading, combining eye-tracking and MEG techniques, building upon the RIFT paradigm previously introduced by Pan et al. (2021). Overall, the manuscript is well-written with a clear structure, and the data analysis and experimental results are presented in a lucid manner.

      The authors have addressed the issues I raised in the previous round of review to my satisfaction. However, I still have two concerns that require the authors' consideration.

      Firstly, the similarity between the RIFT analysis process in this study and traditional ERP analysis could lead readers to equate RIFT with components like N400, potentially influencing their interpretation of the results. Although the author's response has somewhat clarified my queries, I seek confirmation: does RIFT itself signify "visual attention" or the "allocation of attentional resources to the flickering target words" (line 208) in this study? While this may not be pivotal, as it primarily serves as an indicator to evaluate whether contextual congruity can indeed modulate the RIFT response rather than indicating early parafoveal semantic integration, I recommend that the authors explicitly address this point in the manuscript, maybe in the discussion section, to enhance reader comprehension of the article's rationale.

      Secondly, regarding the study's conclusions, there appears to be an overemphasis in stating that "semantic information ... can also be integrated with the sentence context ..." (line 21-22). As raised by Reviewer 2 (Major Point 1) and acknowledged by the authors in the limitations of the revised manuscript (lines 403-412), the RIFT effect observed likely stems from local congruency. Therefore, adjusting the conclusion to "integrated with previous context" may offer a more precise reflection of the findings.

    1. Reviewer #1 (Public Review):

      Zhu, et al present a genome-wide histone modification analysis comparing patients with schizophrenia (on or off antipsychotics) to non-psychiatric controls. The authors performed analyses across the dorsolateral prefrontal cortex and tested for enrichment of nearby genes and pathways. The authors performed analysis measuring the effect of age on the epigenomic landscape as well. This paper provides a unique resource around SCZ and its epigenetic correlates, and some potentially intriguing findings in the antipsychotic response dataset.

      Comments on revised version:

      The authors have adequately responded to my review comments.

    1. Reviewer #2 (Public Review):

      Sasaki et al. investigated methods to entrain vasomotion in awake wild-type mice across multiple regions of the brain using a horizontally oscillating visual pattern which induces an optokinetic response (HOKR) eye movement. They found that spontaneous vasomotion could be detected in individual vessels of their wild-type mice through either a thinned cranial window or intact skull preparation using a widefield macro-zoom microscope. They showed that low-resolution autofluorescence signals coming from the brain parenchyma could be used to capture vasomotion activity using a macro-zoom microscope or optical fibre, as this signal correlates well with the intensity profile of fluorescently-labelled single vessels. They show that vasomotion can also be entrained across the cortical surface using an oscillating visual stimulus with a range of parameters (with varying temporal frequencies, amplitudes, or spatial cycles), and that the amplitude spectrum of the detected vasomotion frequency increases with repeated training sessions. The authors include some control experiments to rule out fluorescence fluctuations being due to artifacts of eye movement or screen luminance and attempt to demonstrate some functional benefit of vasomotion entraining as HOKR performance improves after repeat training. These data add in an interesting way to the current knowledge base on vasomotion, as the authors demonstrate the ability to entrain vasomotion across multiple brain areas and show some functional significance to vasomotion with regards to information processing as HOKR task performance correlates well with vascular oscillation amplitudes.

    1. Reviewer #1 (Public Review):

      Summary:

      Most studies in sensory neuroscience investigate how individual sensory stimuli are represented in the brain (e.g., the motion or color of a single object). This study starts tackling the more difficult question of how the brain represents multiple stimuli simultaneously and how these representations help to segregate objects from cluttered scenes with overlapping objects.

      Strengths

      The authors first document the ability of humans to segregate two motion patterns based on differences in speed. Then they show that a monkey's performance is largely similar; thus establishing the monkey as a good model to study the underlying neural representations.

      Careful quantification of the neural responses in the middle temporal area during the simultaneous presentation of fast and slow speeds leads to the surprising finding that, at low average speeds, many neurons respond as if the slowest speed is not present, while they show averaged responses at high speeds. This unexpected complexity of the integration of multiple stimuli is key to the model developed in this paper.

      One experiment in which attention is drawn away from the receptive field supports the claim that this is not due to the involuntary capture of attention by fast speeds.

      A classifier using the neuronal response and trained to distinguish single-speed from bi-speed stimuli shows a similar overall performance and dependence on the mean speed as the monkey. This supports the claim that these neurons may indeed underlie the animal's decision process.

      The authors expand the well-established divisive normalization model to capture the responses to bi-speed stimuli. The incremental modeling (eq 9 and 10) clarifies which aspects of the tuning curves are captured by the parameters.

      Weaknesses

      While the comparison of the overall pattern of behavioral performance between monkeys and humans is important, some of the detailed comparisons are not well supported by the data. For instance, whether the monkey used the apparent coherence simply wasn't tested and a difference between 4 human subjects and a single monkey subject cannot be tested statistically in a meaningful manner. I recommend removing these observations from the manuscript and leaving it at "The difference between the monkey and human results may be due to species differences or individual variability" (and potentially add that there are differences in the task as well; the monkey received feedback on the correctness of their choice, while the humans did not.)

      A control experiment aims to show that the "fastest speed takes all" behavior is general by presenting two stimuli that move at fast/slow speeds in orthogonal directions. The claim that these responses also show the "fastest speed takes all" is not well supported by the data. In fact, for directions in which the slow speed leads to the largest response on its own, the population response to the bi-speed stimulus is the average of the response to the components. Only for the directions where the fast speed stimulus is the preferred direction is there a bias towards the faster speed (Figure 7A). The quantification of this effect in Figure 7B seems to suggest otherwise, but I suspect that this is driven by the larger amplitude of Rf in Figure 8, and the constraint that ws and wf are constant across directions. The interpretation of this experiment needs to be reconsidered.

    1. Reviewer #1 (Public Review):

      Summary:

      This study compares experimental data recorded from the PFC of monkeys to the activity of recurrent neural networks trained to perform the same `task' as the monkeys, namely, to predict the delivery of reward following the presentation of visual stimuli. The visual information varied along 3 dimensions, color, shape, and width. Shape was always relevant for reward prediction, width was always irrelevant, and color was irrelevant at the beginning of the trial but became relevant later on, once it could be assessed together with shape. The neural data showed systematic changes in the representations of these features and of the expected reward as the learning progressed, and the objective of this study was to try to understand what principles could underlie these changes. The simulations and theoretical calculations indicated that the changes in PFC activity (throughout learning and throughout a trial) can be understood as an attempt by the circuitry to use an efficient representational strategy, i.e., one that uses as few spikes as possible, given that the resulting representation should be accurate enough for task performance.

      Strengths:

      - The paper is concise and clearly written.

      - The paper shows that, in a neural circuit, the information that is decodable and the information that is task-relevant may relate in very different ways. Decodable information may be very relevant or very irrelevant. This fact is critical for interpreting the results of pure decoding studies, which often assume an equivalence. This take-home message is not emphasized by the authors, but I think is quite important.

      - The results provide insight as to how neural representations may be transformed as a task is learned, which often results in subtle changes in selectivity and overall activity levels whose impact or reason is not entirely clear just by looking at the data.

      Weaknesses:

      The match between the real PFC and the model networks is highly qualitative, and as noted by the authors, comparisons only make sense in terms of *changes* between early and late learning. The time scales, activity levels, and decoding accuracies involved are all different between the model and recording data. This is not to disregard what the authors have done, but simply to point out an important limitation.

    1. Reviewer #1 (Public Review):

      Summary:

      The authors bring together implanted radiofrequency coils, high-field MRI imaging, awake animal imaging, and sensory stimulation methods in a technological demonstration. The results are very detailed descriptions of the sensory systems under investigation.

      Strengths:

      - The maps are qualitatively excellent for rodent whole-brain imaging.<br /> - The design of the holder and the coil is pretty clever.

      Weaknesses:

      - Some unexpected regions appear on the whole brain maps, and the discussion of these regions is succinct.<br /> - The authors do not make the work and effort to train the animals and average the data from several hundred trials apparent enough. This is important for any reader who would like to consider implementing this technology.<br /> - The data is not available. This does not let the readers make their own assessment of the results.

    1. Reviewer #1 (Public Review):

      The authors perform RNA-seq on FACS-isolated neurons from adult worms at days 1 and 8 of adulthood to profile the gene expression changes that occur with cognitive decline. Supporting data are included indicating that by day 7 of adulthood, learning and memory are reduced, indicating that this time point or after represents cognitively aged worms. Neuronal identity genes are reduced in expression within cognitively aged worms, whereas genes involved in proteostasis, transcription/chromatin, and stress response are elevated. A number of specific examples are provided, representing markers of specific neuronal subtypes, and correlating expression changes to the erosion of particular functions (e.g. motor neurons, chemosensory neurons, aversive learning neurons, etc).

      To investigate whether the upregulation of genes in neurons with age is compensatory or deleterious, the authors reduced the expression of a set of three significantly upregulated genes and performed behavioral assays in young adults. In each case, reduction of expression improved memory, consistent with a model in which age-associated increases impair neuronal function. This claim would be bolstered by an experiment elevating the expression of these genes in young neurons, which should reduce the learning index if the hypothesis is correct.

      The authors then characterize learning and memory in wild-type, daf-2, and daf-2/daf-16 worms with age and find that daf-2 worms have an extended ability to learn for approximately 10 days longer than wild types. This was daf-16 dependent. Memory was extended in daf-2 as well, and strikingly, daf-2;daf-16 had no short-term memory even at day 1. Transcriptomic analysis of FACS-sorted neurons was performed on the three groups at day 8. The authors focus their analysis on daf-2 vs. daf-2;daf-16 and present evidence that daf-2 neurons express a stress-resistance gene program. One question that remains unanswered is how well the N2 and daf-2;daf-16 correlate overall, and are there differences? This may be informative as wild type and daf-2;daf-16 mutants are not phenotypically identical when it comes to memory, and there may be differences that can be detected despite the overlap in the PCA. This analysis could reveal the daf-16 targets involved in memory.

      The authors tested eight candidate genes that were more highly expressed in daf-2 neurons vs. daf-2;daf-16 and showed that reduction of 2 and 5 of these genes impaired learning and memory, respectively, in daf-2 worms. This finding implicates specific neuronal transcriptional targets of IIS in maintaining cognitive ability in daf-2 with age, which, importantly, are distinct from those in young wild type worms.

    1. Reviewer #1 (Public Review):

      Huber proposes a theory where the role of the medial temporal lobe (MTL) is memory, where properties of spatial cells in the MTL can be explained through memory function rather than spatial processing or navigation. Instantiating the theory through a computational model, the author shows that many empirical phenomena of spatial cells can be captured, and may be better accounted through a memory theory. It is an impressive computational account of MTL cells with a lot of theoretical reasoning and aims to tightly relate to various spatial cell data.

      In general, the paper is well written, but likely due to the complexity, there are various aspects of the paper that are difficult to understand. One point is that it is not entirely clear to me that it is a convincing demonstration of purely memory rather than navigation, but rather an account of the findings through the lens of memory. Below, I raise several big-picture theoretical questions. I also have some clarification questions about the model (where I also have some theoretical question marks - due to not achieving a full understanding).

      (1) Although the theory is based on memory, it also is based on spatially-selective cells. Not all cells in the hippocampus fulfill the criteria of place/HD/border/grid cells, and place a role in memory. E.g., Tonegawa, Buszaki labs' work does not focus on only those cells, and there are certainly a lot of non-pure spatial cells in monkeys (Martinez-Trujillo) and humans (iEEG). Does the author mainly focus on saying that "spatial cells" are memory, but do not account for non-spatial memory cells? This seems to be an incomplete account of memory - which is fine, but the way the model is set up suggests that *all* memory is, place (what/where), and non-spatial attributes ("grid") - but cells that don't fulfil these criteria in MTL (Diehl et al., 2017, Neuron; non-grid cells; Schaeffer et al., 2022, ICML; Luo et al., 2024, bioRxiv) certainly contribute to memory, and even navigation. This is also related to the question of whether these cell definitions matter at all (Luo et al., 2024).

      The authors note "However, this memory conjunction view of the MTL must be reconciled with the rodent electrophysiology finding that most cells in MTL appear to have receptive fields related to some aspect of spatial navigation (Boccara et al., 2010; Grieves & Jeffery, 2017). The paucity of non-spatial cells in MTL could be explained if grid cells have been mischaracterized as spatial." Is the author mainly talking about rodent work?

      (2) Related to the last point, how about non-grid multi-field mEC cells? In theory, these also should be the same; but the author only presents perfect-look grid cells. In empirical work, clearly, this is not the case, and many mEC cells are multi-field non-grid cells (Diehl et al., 2017). Does the model find these cells? Do they play a different role?

      As noted by the author "Because the non-spatial attributes are constant throughout the two-dimensional surface, this results in an array of discrete memory locations that are approximately hexagonal (as explained in the Model Methods, an "online" memory consolidation process employing pattern separation rapidly turns an approximately hexagonal array into one that is precisely hexagonal). "

      If they are indeed all precisely hexagonal, does that mean the model doesn't have non-grid spatial cells?

      (3) Theoretical reasons for why the model is put together this way, and why grid cells must be coding a non-spatial attribute: Is this account more data-driven (fits the data so formulated this way), or is it theoretical - there is a reason why place, border, grid cells are formulated to be like this. For example, is it an efficient way to code these variables? It can be both, like how the BVC model makes theoretical sense that you can use boundaries to determine a specific location (and so place cell), but also works (creates realistic place cells).

      But in this case, the purpose of grid cell coding a non-spatial attribute, and having some kind of system where it doesn't fire at all locations seems a little arbitrary. If it's not encoding a spatial attribute, it doesn't have to have a spatial field. For example, it could fire in the whole arena - which some cells do (and don't pass the criteria of spatial cells as they are not spatially "selective" to another location, related to above).

      (4) Why are grid cells given such a large role for encoding non-spatial attributes? If anything, shouldn't it be lateral EC or perirhinal cortex? Of course, they both could, but there is less reason to think this, at least for rodent mEC.

      (5) Clarification: why do place cells and grid cells differ in terms of stability in the model? Place cells are not stable initially but grid cells come out immediately. They seem directly connected so a bit unclear why; especially if place cell feedback leads to grid cell fields. There is an explanation in the text - based on grid cells coding the on-average memories, but these should be based on place cell inputs as well. So how is it that place fields are unstable then grid fields do not move at all? I wonder if a set of images or videos (gifs) showing the differences in spatial learning would be nice and clarify this point.

      (6) Other predictions. Clearly, the model makes many interesting (and quite specific!) predictions. But does it make some known simple predictions?<br /> • More place cells at rewarded (or more visited) locations. Some empirical researchers seem to think this is not as obvious as it seems (e.g., Duvellle et al., 2019; JoN; Nyberg et al., 2021, Neuron Review).<br /> • Grid cell field moves toward reward (Butler et al., 2019; Boccera et al., 2019).<br /> • Grid cells deform in trapezoid (Krupic et al., 2015) and change in environments like mazes (Derikman et al., 2014).

    1. Reviewer #1 (Public Review):

      Summary:

      This paper presents two experiments, both of which use a target detection paradigm to investigate the speed of statistical learning. The first experiment is a replication of Batterink, 2017, in which participants are presented with streams of uniform-length, trisyllabic nonsense words and asked to detect a target syllable. The results replicate previous findings, showing that learning (in the form of response time facilitation to later-occurring syllables within a nonsense word) occurs after a single exposure to a word. In the second experiment, participants are presented with streams of variable-length nonsense words (two trisyllabic words and two disyllabic words) and perform the same task. A similar facilitation effect was observed as in Experiment 1. The authors interpret these findings as evidence that target detection requires mechanisms different from segmentation. They present results of a computational model to simulate results from the target detection task and find that an "anticipation mechanism" can produce facilitation effects, without performing segmentation. The authors conclude that the mechanisms involved in the target detection task are different from those involved in the word segmentation task.

      Strengths:

      The paper presents multiple experiments that provide internal replication of a key experimental finding, in which response times are facilitated after a single exposure to an embedded pseudoword. Both experimental data and results from a computational model are presented, providing converging approaches for understanding and interpreting the main results. The data are analyzed very thoroughly using mixed effects models with multiple explanatory factors.

      Weaknesses:

      In my view, the main weaknesses of this study relate to the theoretical interpretation of the results.

      (1) The key conclusion from these findings is that the facilitation effect observed in the target detection paradigm is driven by a different mechanism (or mechanisms) than those involved in word segmentation. The argument here I think is somewhat unclear and weak, for several reasons:

      First, there appears to be some blurring in what exactly is meant by the term "segmentation" with some confusion between segmentation as a concept and segmentation as a paradigm.<br /> Conceptually, segmentation refers to the segmenting of continuous speech into words. However, this conceptual understanding of segmentation (as a theoretical mechanism) is not necessarily what is directly measured by "traditional" studies of statistical learning, which typically (at least in adults) involve exposure to a continuous speech stream followed by a forced-choice recognition task of words versus recombined foil items (part-words or nonwords). To take the example provided by the authors, a participant presented with the sequence GHIABCDEFABCGHI may endorse ABC as being more familiar than BCG, because ABC is presented more frequently together and the learned association between A and B is stronger than between C and G. However, endorsement of ABC over BCG does not necessarily mean that the participant has "segmented" ABC from the speech stream, just as faster reaction times in responding to syllable C versus A do not necessarily indicate successful segmentation. As the authors argue on page 7, "an encounter to a sequence in which two elements co-occur (say, AB) would theoretically allow the learner to use the predictive relationship during a subsequent encounter (that A predicts B)." By the same logic, encoding the relationship between A and B could also allow for the above-chance endorsement of items that contain AB over items containing a weaker relationship.

      Both recognition performance and facilitation through target detection reflect different outcomes of statistical learning. While they may reflect different aspects of the learning process and/or dissociable forms of memory, they may best be viewed as measures of statistical learning, rather than mechanisms in and of themselves.

      (2) The key manipulation between experiments 1 and 2 is the length of the words in the syllable sequences, with words either constant in length (experiment 1) or mixed in length (experiment 2). The authors show that similar facilitation levels are observed across this manipulation in the current experiments. By contrast, they argue that previous findings have found that performance is impaired for mixed-length conditions compared to fixed-length conditions. Thus, a central aspect of the theoretical interpretation of the results rests on prior evidence suggesting that statistical learning is impaired in mixed-length conditions. However, it is not clear how strong this prior evidence is. There is only one published paper cited by the authors - the paper by Hoch and colleagues - that supports this conclusion in adults (other mentioned studies are all in infants, which use very different measures of learning). Other papers not cited by the authors do suggest that statistical learning can occur to stimuli of mixed lengths (Thiessen et al., 2005, using infant-directed speech; Frank et al., 2010 in adults). I think this theoretical argument would be much stronger if the dissociation between recognition and facilitation through RTs as a function of word length variability was demonstrated within the same experiment and ideally within the same group of participants.

      (3) The authors argue for an "anticipation" mechanism in explaining the facilitation effect observed in the experiments. The term anticipation would generally be understood to imply some kind of active prediction process, related to generating the representation of an upcoming stimulus prior to its occurrence. However, the computational model proposed by the authors (page 24) does not encode anything related to anticipation per se. While it demonstrates facilitation based on prior occurrences of a stimulus, that facilitation does not necessarily depend on active anticipation of the stimulus. It is not clear that it is necessary to invoke the concept of anticipation to explain the results, or indeed that there is any evidence in the current study for anticipation, as opposed to just general facilitation due to associative learning.

      In addition, related to the model, given that only bigrams are stored in the model, could the authors clarify how the model is able to account for the additional facilitation at the 3rd position of a trigram compared to the 2nd position?

      (4) In the discussion of transitional probabilities (page 31), the authors suggest that "a single exposure does provide information about the transitions within the single exposure, and the probability of B given A can indeed be calculated from a single occurrence of AB." Although this may be technically true in that a calculation for a single exposure is possible from this formula, it is not consistent with the conceptual framework for calculating transitional probabilities, as first introduced by Saffran and colleagues. For example, Saffran et al. (1996, Science) describe that "over a corpus of speech there are measurable statistical regularities that distinguish recurring sound sequences that comprise words from the more accidental sound sequences that occur across word boundaries. Within a language, the transitional probability from one sound to the next will generally be highest when the two sounds follow one another within a word, whereas transitional probabilities spanning a word boundary will be relatively low." This makes it clear that the computation of transitional probabilities (i.e., Y | X) is conceptualized to reflect the frequency of XY / frequency of X, over a given language inventory, not just a single pair. Phrased another way, a single exposure to pair AB would not provide a reliable estimate of the raw frequencies with which A and AB occur across a given sample of language.

      (5) In experiment 2, the authors argue that there is robust facilitation for trisyllabic and disyllabic words alike. I am not sure about the strength of the evidence for this claim, as it appears that there are some conflicting results relevant to this conclusion. Notably, in the regression model for disyllabic words, the omnibus interaction between word presentation and syllable position did not reach significance (p= 0.089). At face value, this result indicates that there was no significant facilitation for disyllabic words. The additional pairwise comparisons are thus not justified given the lack of omnibus interaction. The finding that there is no significant interaction between word presentation, word position, and word length is taken to support the idea that there is no difference between the two types of words, but could also be due to a lack of power, especially given the p-value (p = 0.010).

      (6) The results plotted in Figure 2 seem to suggest that RTs to the first syllable of a trisyllabic item slow down with additional word presentations, while RTs to the final position speed up. If anything, in this figure, the magnitude of the effect seems to be greater for 1st syllable positions (e.g., the RT difference between presentation 1 and 4 for syllable position 1 seems to be numerically larger than for syllable position 3, Figure 2D). Thus, it was quite surprising to see in the results (p. 16) that RTs for syllable position 1 were not significantly different for presentation 1 vs. the later presentations (but that they were significant for positions 2 and 3 given the same comparison). Is this possibly a power issue? Would there be a significant slowdown to 1st syllables if results from both the exact replication and conceptual replication conditions were combined in the same analysis?

      (7) It is difficult to evaluate the description of the PARSER simulation on page 36. Perhaps this simulation should be introduced earlier in the methods and results rather than in the discussion only.

    1. Reviewer #1 (Public Review):

      Summary:

      The authors introduce two preparations for observing large-scale cortical activity in mice during behavior. Alongside, they present intriguing preliminary findings utilizing these methods. This paper is poised to be an invaluable resource for researchers engaged in extensive cortical recording in behaving mice.

      Strengths:

      Comprehensive methodological detailing:<br /> The paper excels in providing an exceptionally detailed description of the methods used. This meticulous documentation includes a step-by-step workflow, complemented by thorough workflow, protocols and list of materials in the supplementary materials.

      Minimal of movement artifacts:<br /> A notable strength of this study is the remarkably low movement artifacts, with specific strategies outlined to attain this outcome.

      Insightful preliminary data and analysis:<br /> The preliminary data unveiled in the study reveal interesting heterogeneity in the relationships between neural activity and detailed behavioral features, particularly notable in the lateral cortex. This aspect of the findings is intriguing and suggests avenues for further exploration.

      Weaknesses:

      Clarification about the extent of the method in title:<br /> The title of the paper, using the term "pan-cortical", may inadvertently suggest that both the top and lateral view preparations are utilized in the same set of mice, while the authors employ either the dorsal view (which offers limited access to the lateral ventral regions) or the lateral view (which restricts access to the opposite side of the cortex).

      Despite the authors not identifying qualitative effects, tilting the mouse's head could potentially influence behavioral outcomes in certain paradigms.

    1. Reviewer #1 (Public Review):

      Summary:

      For many years, there has been extensive electrophysiological research investigating the relationship between local field potential patterns and individual cell spike patterns in the hippocampus. In this study, using state-of-the-art imaging techniques, they examined spike synchrony of hippocampal cells during locomotion and immobility states. In contrast to conventional understanding of the hippocampus, the authors demonstrated that hippocampal place cells exhibit prominent synchronous spikes locked to theta oscillations.

      Strengths:

      The voltage imaging used in this study is a highly novel method that allows recording not only suprathreshold-level spikes but also subthreshold-level activity. With its high frame rate, it offers time resolution comparable to electrophysiological recordings. Moreover, it enables the visualization of actual cell locations, allowing for the examination of spatial properties (e.g., Figure 4G).

      Weaknesses:

      There is a notable deviation from several observations obtained through conventional electrophysiological recordings. Particularly, as mentioned below in detail, the considerable differences in baseline firing rates and no observations of ripple-triggered firing patterns raise some concerns about potential artifacts from imaging and analsyis, such as cell toxicity, abnormal excitability, and false detection of spikes. While these findings are intriguing if the validity of these methods is properly proven, accepting the current results as new insights is challenging.

    1. Reviewer #1 (Public Review):

      Summary:

      The manuscript examines the contribution of the dorsal and intermediate hippocampus to goal-directed navigation in a wide virtual environment where visual cues are provided by the scenery on the periphery of a wide arena. Among a choice of 2 reward zones located near the arena periphery, rats learn to navigate from the center of the arena to the reward zone associated with the highest reward. Navigation performance is largely assessed from the rats' body orientation when they leave the arena center and when they reach the periphery, as well as the angular mismatch between the reward zone and the site rats reach the periphery. Muscimol inactivation of the dorsal and intermediate hippocampus alters rat navigation to the reward zone, but the effect was more pronounced for the inactivation of the intermediate hippocampus, with some rat trajectories ending in the zone associated with the lowest reward. Based on these results, the authors suggest that the intermediate hippocampus is critical, especially for navigating to the highest reward zone.

      Strengths:

      _ The authors developed an effective approach to study goal-directed navigation in a virtual environment where visual cues are provided by the peripheral scenery.

      _ In general, the text is clearly written and the figures are well-designed and relatively straightforward to interpret, even without reading the legends.

      _ An intriguing result, which would deserve to be better investigated and/or discussed, was that rats tended to rotate always in the counterclockwise direction. Could this be because of a hardware bias making it easier to turn left, some aspect of the peripheral landscape, or a natural preference of rats to turn left that is observable (or reported) in a real environment?

      _ Another interesting observation, which would also deserve to be addressed in the discussion, is the fact that dHP/iHP inactivations produced to some extent consistent shifts in departing and peripheral crossing directions. This is visible from the distributions in Figures 6 and 7, which still show a peak under muscimol inactivation, but this peak is shifted to earlier angles than the correct ones. Such change is not straightforward to interpret, unlike the shortening of the mean vector length.

      Maybe rats under muscimol could navigate simply by using the association of reward zone with some visual cues in the peripheral scene, in brain areas other than the hippocampus, and therefore stopped their rotation as soon as they saw the cues, a bit before the correct angle. While with their hippocampus is intact, rats could estimate precisely the spatial relationship between the reward zone and visual cues.

      Weaknesses:

      _ I am not sure that the differential role of dHP and iHP for navigation to high/low reward locations is supported by the data. The current results could be compatible with iHP inactivation producing a stronger impairment on spatial orientation than dHP inactivation, generating more erratic trajectories that crossed by chance the second reward zone.

      To make the point that iHP inactivation affects the disambiguation of high and low reward locations, the authors should show that the fraction of trajectories aiming at the low reward zone is higher than expected by chance. Somehow we would expect to see a significant peak pointing toward the low reward zone in the distribution of Figures 6-7.

    1. Reviewer #1 (Public Review):

      Summary:

      This work describes a simple mechanical model of worm locomotion, using a series of rigid segments connected by damped torsional springs and immersed in a viscous fluid. It uses this model to simulate forward crawling movement, as well as omega turns.

      Strengths:

      The primary strength is in applying a biomechanical model to omega-turn behaviors. The biomechanics of nematode turning behaviors are relatively less well described and understood than forward crawling, and the increase in power during omega turns is one of the more novel results. The model itself may be a useful implementation to other researchers, particularly owing to its simplicity.

      Weaknesses:

      The strength of the model presented in this work relative to prior approaches is not well supported, and in general the paper would be improved with a better description of the broader context of existing modeling literature related to undulatory locomotion. This paper claims to improve on previous approaches to taking body shapes as inputs. However, the sole nematode model cited aims to do something different, and arguably more significant, which is to use experimentally derived parameters to model both the neural circuits that induce locomotion as well as the biomechanics and to subsequently compare the model to experimental data. Other modeling approaches do take experimental body kinematics as inputs and use them to produce force fields, however, they are not cited or discussed. Finally, the overall novelty of the approach is questionable. A functionally similar approach was developed in 2012 to describe worm locomotion in lattices (Majmudar, 2012, Roy. Soc. Int.), which is not discussed and would provide an interesting comparison and needed context.

      In some sense, because the model takes kinematics as an input and uses previously established techniques to model mechanics, it is unsurprising that it can reproduce experimentally observed kinematics, however, the forces calculated and the variation of parameters could be of interest, but other methods derived from kinematics could provide similar results. It is unclear what the predictive power of the model is.

      Relatedly, a justification of why the drag coefficients had to be changed by a factor of 100 should be explored. Plate conditions are difficult to replicate and the rheology of plates likely depends on several factors, but is for example, changes in hydration level likely to produce a 100-fold change in drag? or something more interesting/subtle within the model producing the discrepancy?

      Finally, the language used to distinguish different modeling approaches was often unclear. For example, it was unclear in what sense the model presented in Boyle, 2012 was a "kinetic model" and in many situations, it appeared that the term kinematic might have been more appropriate. Other phrases like "frictional forces caused by the tension of its muscles" were unclear at first glance, and might benefit from revision and more canonical usage of terms.

    1. Reviewer #2 (Public Review):

      Summary:

      Laham et al. investigate how the projection from adult born granule cells into CA2 affects the retrieval of social memories at various developmental points. They use chemogenetic manipulations and electrophysiological recordings to test how this projection affects hippocampal network properties during behavior. The study is of relevant interest for the neuroscience community and the results are important for our understanding of how social memories of different nature (remote or immediate) are encoded and supported by the hippocampal circuitry. The behavioral experiments after abGC projections to CA2 are compelling as they show clearly distinct behavioral readout. While the electrophysiological experiments are difficult to interpret without more single cell responses quantifications, they clearly show that more than one region in the hippocampus is involved in the formation of social memories.

    1. Reviewer #1 (Public Review):

      Summary:

      In this study, Diana et al. present a Monte Carlo-based method to perform spike inference from calcium imaging data. A particular strength of their approach is that they can estimate not only averages but also uncertainties of the modeled process. The authors then focus on the quantification of spike time uncertainties in simulated data and in data recorded with a high sampling rate in cerebellar slices with GCaMP8f.

      Strengths:

      - The authors provide a solid groundwork for sequential Monte Carlo-based spike inference, which extends previous work of Pnevmatikakis et al., Greenberg et al., and others.

      - The integration of two states (silence vs. burst firing) seems to improve the performance of the model.

      - The acquisition of a GCaMP8f dataset in the cerebellum is useful and helps make the point that high spike time inference precision is possible under certain conditions.

      Weaknesses:

      - The algorithm is designed to predict single spike times. Currently, it is not benchmarked against other algorithms in terms of single spike precision and spike time errors. A benchmarking with the most recent other SMC model and another good model focused on single spike outputs (e.g., MLSpike) would be useful to have.

      - Some of the analyses and benchmarks seem too cursory, and the reporting simply consists of a visual impression of results instead of proper analysis and quantification. For example, the authors write "The spike patterns obtained using our method are very similar across trials, showing that PGBAR can reliably detect single-trial action potential-evoked GCaMP8f fluorescence transients." This is a highly qualitative statement, just based on the (subjective) visual impression of a plot. Similarly, the authors write "we could reliably identify the two spikes in each trial", but this claim is not supported by quantification or a figure, as far as I can see. The authors write "but the trade-off between temporal accuracy, SNR and sampling frequency must be considered", but they don't discuss these trade-offs systematically.

      - It has been shown several times from experimental data that spike inference with single spike resolution does not work well (Huang et al. eLife, 2021; Rupprecht et al., Nature Neuroscience, 2021) in general. This limitation should be discussed with respect to the applicability of the proposed algorithm for standard population calcium imaging data.

      - Several analyses are based on artificial, simulated data with simplifying assumptions. Ever since Theis et al., Neuron, 2016, it has been known that artificially generated ground truth data should not be used as the primary means to evaluate spike inference algorithms. It would have been informative if the authors had used either the CASCADE dataset or their cerebellum dataset for more detailed analyses, in particular of single spike time precision.

      - In its current state, the sum of the current weaknesses makes the suggested method, while interesting for experts, rather unattractive for experimentalists who want to perform spike inference on their recorded calcium imaging data.

      Other comments:

      - One of the key features of the SMC model is the assumption of two states (bursting vs. non-bursting). However, while it seems clear that this approach is helpful, it is not clear where this idea comes from, from an observation of the data or another concept.

      - Another SMC algorithm (Greenberg et al., 2018) stated that the fitted parameters showed some degeneracy, resulting in ambiguous fitting parameters. It would be good to know if this problem was avoided by the authors.

    1. Reviewer #1 (Public Review):

      Short Assessment

      In this work the authors propose a new regulatory role for one the most abundant circRNAs, circHIPK3. They demonstrate that circHIPK3 interacts with an RNA binding protein (IGF2BP2), sequestering it away from its target mRNAs. This interaction is shown to regulates the expression of hundreds of genes that share a specific sequence motif (11-mer motif) in their untranslated regions (3'-UTR), identical to one present in circHIPK3 where IGF2BP2 binds. The study further focuses on the specific case of STAT3 gene, whose mRNA product is found to be downregulated upon circHIPK3 depletion. This suggests that circHIPK3 sequesters IGF2BP2, preventing it from binding to and destabilizing STAT3 mRNA. The study presents evidence supporting this mechanism and discusses its potential role in tumor cell progression. These findings contribute to the growing complexity of understanding cancer regulation and highlight the intricate interplay between circRNAs and protein-coding genes in tumorigenesis.

      Strengths:<br /> The authors show mechanistic insight into a proposed novel "sponging" function of circHIPK3 which is not mediated by sequestering miRNAs but rather a specific RNA binding protein (IGF2BP2). They address the stoichiometry of the molecules involved in the interaction, which is a critical aspect that is frequently overlooked in this type of studies. They provide both genome-wide analysis and a specific case (STAT3) which is relevant for cancer progression. Overall, the authors have significantly improved their manuscript in their revised version.

      Weaknesses:<br /> While the authors have performed northern blots to measure circRNA levels, an estimation of the circRNA overexpression efficiency, namely the circular-to-linear expression ratio, would be desired. The seemingly contradictory effects of circHIPK3 and STAT3 depletion in cancer progression, are now addressed by the authors in their revised manuscript, incorporating potential reasons that might explain such complexity.

      Major points about revised manuscript

      (1) In Supplementary Figure S5H, the membrane may have been trimmed too closely to the circRNA band, potentially resulting in the absence of the linear RNA band. Could the authors provide a full image of the membrane that includes the loading points? Having access to the complete image would allow for a more comprehensive evaluation of the results, including the presence or absence of expected linear and circular RNA bands.

    1. Reviewer #1 (Public Review):

      Summary:

      Authors were attempting to determine the extent that CIH altered swallowing motor function; specifically, the timing and probability of the activation of the larygneal and submental motor pools. The paper describes a variety of different motor patterns elicited by optogenetic activation of individual neuronal phenotypes within PiCo in a group of mice exposed to CIH. They show that there are a variety of motor patterns that emerge in CIH mice; this is apparently different than the more consistent motor patterns elicited by PiCo activation in normoxic mice (previously published)

      Strengths:

      The preparation is technically challenging and gives valuable information related to the role of PiCo in the pattern of motor activation involved in swallowing and its timing with phrenic activity. Genetic manipulations allow for the independent activation of the individual neuronal phenotypes of PiCo (glutamatergic, cholinergic) which is a strength.

      Weaknesses:

      (1) Comparisons made between experimental data acquired currently with those previously published are extremely problematic, with the potential confounding influence of changing environments, genetics and litter effects. For example, were the current mice tested at the same time as those exposed to normoxia? Are they littermates (or at least from the same colony) as those previously examined? If they were tested at the same time and age, then the authors should explicitly state this in the methods. The authors have provided no statistical analyses to determine whether there is an effect of CIH on the motor patterns. In short, how can they be sure that the phenomena they observe with respect to motor patterns is due to CIH?

      (2) The data are descriptive in nature, reporting only differences (diversity) of motor patterns in this cohort of animals exposed to CIH. There is limited mechanistic insight into how PiCo manipulation alters the pattern and probability of motor activation. Can they utilize Fos or marker of activation within the nTS or other regions to provide initial insight? Or in another nucleus that contributes as part of the circuit.

      (3) The differences between the genotypes (ChaTcre; Vglut2Cre; ChatCre:Vglut2FlpO) with regard to the probability of generating a swallow are not sufficiently discussed, in my view. If, as the authors state, it is "reasonable to suggest that CIH differentially affects" these populations, then what are some viable reasons? What are the known differences in these populations of neurons that could lead to variable responses? Do they project to different places?

      (4) The Results section is difficult to follow and interpret. It would be beneficial to have a couple of sentences after each sub-section stating what the data actually mean. As of now it reads like a statistical report of the data with little "basic" interpretation of the data.

      (5) I have a hard time understanding the functional significance of calculating and plotting the degree of correlation between shifting/delaying the following inspiratory burst and triggering a swallow.

    1. Reviewer #1 (Public Review):

      Summary:

      In this work, the authors examine the activity and function of D1 and D2 MSNs in dorsomedial striatum (DMS) during an interval timing task. In this task, animals must first nose poke into a cued port on the left or right; if not rewarded after 6 seconds, they must switch to the other port. Critically, this task thus requires animals to estimate if at least 6 seconds have passed after the first nose poke - this is the key aspect of the task focused on here. After verifying that animals reliably estimate the passage of 6 seconds by leaving on average after 9 seconds, the authors examine striatal activity during this interval. They report that D1-MSNs tend to decrease activity, while D2-MSNs increase activity, throughout this interval. They suggest that this activity follows a drift-diffusion model, in which activity increases (or decreases) to a threshold after which a decision (to leave) is made. The authors next report that optogenetically inhibiting D1 or D2 MSNs, or pharmacologically blocking D1 and D2 receptors, increased the average wait time of the animals to 10 seconds on average. This suggests that both D1 and D2 neurons contribute to the estimate of time, with a decrease in their activity corresponding to a decrease in the rate of 'drift' in their drift-diffusion model. Lastly, the authors examine MSN activity while pharmacologically inhibiting D1 or D2 receptors. The authors observe most recorded MSNs neurons decrease their activity over the interval, with the rate decreasing with D1/D2 receptor inhibition.

      Major strengths:

      The study employs a wide range of techniques - including animal behavioral training, electrophysiology, optogenetic manipulation, pharmacological manipulations, and computational modeling. The behavioral task used by the authors is quite interesting and a nice way to probe interval timing in rodents. The question posed by the authors - how striatal activity contributes to interval timing - is of importance to the field and has been the focus of many studies and labs; thus, this paper can meaningfully contribute to that conversation. The data within the paper is presented very clearly, and the authors have done a nice job presenting the data in a transparent manner (e.g., showing individual cells and animals). Overall, the manuscript is relatively easy to read and clear, with sufficient detail given in most places regarding the experimental paradigm or analyses used.

      Major weaknesses:

      I perceive two major weaknesses. The first is the impact or contextualization of their results in terms of the results of the field more broadly. More specifically, it was not clear to me how the authors are interpreting the striatal activity in the context of what others have observed during interval timing tasks. In other words - what was the hypothesis going into this experiment? Does observing increasing/decreasing activity in D2 versus D1 support one model of interval timing over another, or does it further support a more specific idea of how DMS contributes to interval timing? Or was the main question that we didn't know if D2 or D1 neurons had differential activity during interval timing?

      In the second, I felt that some of the conclusions suggested by the authors don't seem entirely supported by the data they present, or the data presented suggests a slightly more complicated story. Below I provide additional detail on some of these instances.

      Regarding the results presented in Figures 2 and 3:

      I am not sure the PC analysis adds much to the interpretation, and potentially unnecessarily complicates things. In particular, running PCA on a matrix of noisy data that is smoothed with a Gaussian will often return PCs similar to what is observed by the authors, with the first PC being a line up/down, the 2nd PC being a parabola that is up/down, etc. Thus, I'm not sure that there is much to be interpreted by the specific shape of the PCs here. I think an alternative analysis that might be both easier and more informative is to compute the slope of the activity of each neuron across the 6 seconds. This would allow the authors to quantify how many neurons increase or decrease their activity much like what is shown in Figure 2.

      Relatedly, it seems that the data shown in Figure 2D *doesn't* support the authors' main claim regarding D2/D1 MSNs increasing/decreasing their activity, as the trial-by-trial slope is near 0 for both cell types.

      Regarding the results in Figure 4:

      The authors suggest that their data is consistent with a drift-diffusion model. However, it is unclear how well the output from the model fits the activity from neurons the authors recorded. Relatedly, it is unclear how the parameters were chosen for the D1/D2 versions of this model. I think that an alternate approach that would answer these questions is to fit the model to each cell, and then examine the best-fit parameters, as well as the ability of the model to predict activity on trials held out from the fitting process. This would provide a more rigorous method to identify the best parameters and would directly quantify how well the model captures the data.

      Relatedly, looking at the raw data in Figure 2, it seems that many neurons either fire at the beginning or end of the interval, with more neurons firing at the end, and more firing at the beginning, for D2/D1 neurons respectively. Thus, it's not clear to me whether the drift-diffusion model is a good model of activity. Or, perhaps the model is supposed to be related to the aggregate activity of all D1/D2 neurons? (If so, this should be made more explicit. The comment about fitting the model directly to the data also still stands).

      Further, it's unclear to me how, or why, the authors changed the specific parameters they used to model the optogenetic manipulation. Were these parameters chosen because they fit the manipulation data? This I don't think is in itself an issue, but perhaps should be clearly stated, because otherwise it sounds a bit odd given the parameter changes are so specific. It is also not clear to me why the noise in the diffusion process would be expected to change with increased inhibition.

      Regarding the results in Figure 6:

      My comments regarding the interpretation of PCs in Figure 2 apply here as well. In addition, I am not sure that examining PC2 adds much here, given that the authors didn't examine such nonlinear changes earlier in the paper.

      A larger concern though that seems potentially at odds with the authors' interpretation is that there seems to be very little change in the firing pattern after D1 or D2 blockade. I see that in Figure 6F the authors suggest that many cells slope down (and thus, presumably, they are recoding more D1 cells), and that this change in slope is decreased, but this effect is not apparent in Figure 6C, and Figure 6B shows an example of a cell that seems to fire in the opposite direction (increase activity). I think it would help to show some (more) individual examples that demonstrate the summary effect shown by the authors, and perhaps the authors can comment on the robustness (or the variability) of this result.

      Also, it seems that if the authors want to claim that this manipulation lowers the drift rate. I think to make this claim, they could fit the DDM model and examine whether D is significantly lower.

      Regarding the results in Figure 7:

      I am overall a bit confused about what the authors are trying to claim here. In Figure 7, they present data suggesting that D1 or D2 blockade disrupts their ability to decode time in the interval of interest (0-6 seconds). However, in the final paragraph of the results, the authors seem to say that by using another technique, they didn't see any significant change in decoding accuracy after D1 or D2 blockade. What do the authors make of this?

      Impact:

      The task and data presented by the authors are very intriguing, and there are many groups interested in how striatal activity contributes to the neural perception of time. The authors perform a wide variety of experiments and analysis to examine how DMS activity influences time perception during an interval-timing task, allowing for insight into this process. However, the significance of the key finding - that D2/D1 activity increases/ decreases with time - remains somewhat ambiguous to me. This arises from a lack of clarity regarding the initial hypothesis and the implications of this finding for advancing our understanding of striatal functions.

    1. Reviewer #1 (Public Review):

      Summary:

      This is well-performed research with solid results and thorough control. The authors did a good job of finding the relationship between the 5-HT1A receptor and megakaryocytopoiesis, which demonstrated the potential of vilazodone in the management of thrombocytopenia. It emphasizes the regulatory mechanism of 5-HT1A receptor signaling on hematopoietic lineages, which could further advance the field of thrombocytopenia for therapeutic purposes.

      Strengths:

      This is a comprehensive and detailed research using multiple methods and model systems to determine the pharmacological effects and molecular mechanisms of vilazodone. The authors conducted in vitro experiments using HEL and Meg-01 cells and in vivo experiments using Zebrafish and Kunming-irradiated mice. The experiments and bioinformatics analysis have been performed with a high degree of technical proficiency. The authors demonstrated how vilazodone binds to 5-HTR1A and regulates the SRC/MAPK pathway, which is inhibited by particular 5-HTR1A inhibitors. The authors determined this to be the mechanistic underpinning for the effects of vilazodone in promoting megakaryocyte differentiation and thrombopoiesis.

      Weaknesses:

      (1) Which database are the drug test sets and training sets for the creation of drug screening models obtained from? What criteria are used to grade the results?<br /> (2) What is the base of each group in Figure 3b for the survival screening of zebrafish? The positivity rate of GFP-labeled platelets is too low, as indicated by the quantity of eGFP+ cells. What gating technique was used in Figure 3e?<br /> (3) In Figure 4C, the MPV values of each group of mice did not show significant downregulation or upregulation. Please explain the possible reasons.<br /> (4) The PPI diagram and the KEGG diagram in Figure 6 both provide a possible mechanism pathway for the anti-thrombocytopenia effect of vilazodone. How can the author analyze the differences in their results?<br /> (5) 5-HTR1A protein expression is measured only in the Meg-01 cells assay. Similar quantitation through western blot is not shown in other cell models.

    1. Reviewer #1 (Public Review):

      Summary:

      In this manuscript the authors have applied an asymmetric split mNeonGreen2 (mNG2) system to human iPSCs. By integrating a constitutively expressed long fragment of mNG2 at the AAVS1 locus, this allows other proteins to be tagged through the use of available ssODN donors. This removes the need to generate long AAV donors for tagging, thus greatly facilitating high-throughput tagging efforts. The authors then demonstrate the feasibility of the method by successfully tagging 9 markers expressed in iPSC at various, and one expressed upon endoderm differentiation. Several additional differentiation markers were also successfully tagged but not subsequently tested for expression/visibility. As one might expect for high-throughput tagging, a few proteins, while successfully tagged at the genomic level, failed to be visible. Finally, to demonstrate the utility of the tagged cells, the authors isolated clones with genes relevant to cytokinesis tagged, and together with an AI to enhance signal to noise ratios, monitored their localization over cell division.

      Strengths

      Reviewer Comment: Characterization of the mNG2 tagged parental iPSC line was well and carefully done including validation of a single integration, the presence of markers for continued pluripotency, selected off-target analysis and G-banding-based structural rearrangement detection.<br /> The ability to tag proteins with simple ssODNs in iPSC capable of multi-lineage differentiation will undoubtedly be useful for localization tracking and reporter line generation.<br /> Validation of clone genotypes was carefully performed and highlights the continued need for caution with regards to editing outcomes.

      Weaknesses

      Reviewer Comment: IF and flow cytometry figures lack quantification and information on replication. How consistent is the brightness and localization of the markers? How representative are the specific images? Stability is mentioned in the text but data on the stability of expression/brightness is not shown.

      Author Response: To address this comment, we have quantified the mean fluorescence intensity of the tagged cell populations in Fig. S3B-T. This data correlates well with the expected expression levels of each gene relative to the others (Fig. S3A), apart from CDH1 and RACGAP1, which are described in the discussion.

      Reviewer Reply: Great, thanks.

      Reviewer Comment: The localization of markers, while consistent with expectations, is not validated by a second technique such as antibody staining, and in many cases not even with Hoechst to show nuclear vs cytoplasmic.

      Author Response: We find that the localization of each protein is distinct and consistent with previous studies. To address this comment, we have added an overlay of the green fluorescence images with brightfield images to better show the location of the tagged protein relative to the nuclei and cytoplasm. We have also added references to other studies that showed the same localization patterns for these proteins in iPSCs and other relevant cell lines.

      Reviewer Reply: There was no question that the localization fit with expectations, however, this still doesn't show that in the same cell the tag is in the same spot. It would have been fairly simple to do for at least a handful of markers, image, fix and stain to demonstrate unequivocally the tag and protein are co-localized. Of course, this isn't damning by any means, it just would have been nice.

      Reviewer Comment: For the multi-germ layer differentiation validation, NCAM is also expressed by ectoderm, so isn't a good solo marker for mesoderm as it was used. Indeed, the kit used for the differentiation suggests Brachyury combined with either NCAM or CXCR4, not NCAM alone.

      Author Response: Since Brachyury is the most common mesodermal marker, we first tested differentiation using anti-Brachyury antibodies, but they did not work well for flow cytometry. We then switched to anti-NCAM antibodies. Since we used a kit for directed differentiation of iPSCs into the mesodermal lineage, NCAM staining should still report for successful differentiation. In the context of mixed differentiation experiments (embryoid body formation or teratoma assay), NCAM would not differentiate between ectoderm and mesoderm. The parental cells (201B7) have also been edited at the AAVS1 locus in multiple other studies, with no effect on their differentiation potential.

      Reviewer Reply: This is placing a lot of trust in the kit that it only makes what it says it makes. It could have been measured by options other than flow such as qPCR, Western blot, or imaging, but fine.

      Reviewer Comment: Only a single female parental line has been generated and characterized. It would have been useful to have several lines and both male and female to allow sex differences to be explored.

      Author Response: We agree that it would be interesting (and important) to study differences in protein localization between female and male cell types, and from different individuals with different genetic backgrounds. We see our tool as opening a door for cell biology to move away from randomly collected, transformed, differentiated cell types to more directed comparative studies of distinct normal cell types. Since few studies of cell biological processes have been done in normal cells, a first step is to understand how processes compare in an isogenic background, then future studies can reveal how they compare with other individuals and sexes. We hope that either our group or others will continue to build similar lines so that these studies can be done.

      Reviewer Reply: Fair enough.

      Reviewer Comment: The AI-based signal to noise enhancement needs more details and testing. Such models can introduce strong assumptions and thus artefacts into the resolved data. Was the model trained on all markers or were multiple models trained on a single marker each? For example, if trained to enhance a single marker (or co-localized group of markers), it could introduce artefacts where it forces signal localization to those areas even for others. What happens if you feed in images with scrambled pixel locations, does it still say the structures are where the training data says they should be? What about markers with different localization from the training set. If you feed those in, does it force them to the location expected by the training data or does it retain their differential true localization and simply enhance the signal?

      Author Response: The image restoration neural network was used as in Weigert et al., 2018. The model was trained independently for each marker. Each trained model was used only on the corresponding marker and with the same imaging conditions as the training images. From visual inspection, the fluorescent signal in the restored images was consistent with the signal in the raw images, both for interphase and mitotic cells. We found very few artefacts of the restoration (small bright or dark areas) that were discarded. We did not try to restore scrambled images or images of mismatched markers.

      Reviewer Reply: I understand. What I'm saying is that for the restoration technique to be useful you need to know that it won't introduce artefacts if you have an unexpected localization. Think of it this way, if you already know the localization, then there's no point measuring it. If you don't, or there's a possibility that it is somewhere unexpected, then you need to know with confidence that your algorithm will be able to accurately detect that unexpected localization. As such, it would be extremely important to validate that your restoration algorithm will not bias the results to the expected localization if the true localization is unexpected/not seen in the training dataset. It would have been extremely trivial to run this analysis and I do not feel this comment has been in any way adequately addressed.

    1. Reviewer #1 (Public Review):

      Summary:

      This study offers a comprehensive examination of the early postnatal development of the patch and matrix compartments within the striatum. These are segregated circuits within the striatum circuits with distinct embryonic origins and functional roles in mature brain physiology. Despite the recognized significance of these circuits, a comprehensive understanding of their postnatal maturation remains elusive.

      Strengths:

      The authors undertake a thorough investigation, characterizing the intrinsic properties of direct pathway spiny projection neurons (dSPNs) and indirect pathway spiny projection neurons (iSPNs) across both matrix and striosome compartments throughout development. The authors identify the regulatory role of M1 receptors in modulating spontaneous activity in SPNs, and demonstrate the impact of chemogenetic inhibition of MOR-positive neurons during development on GABAergic synapses in substantia nigra pars compacta (SNc) dopamine (DA) neurons. These findings significantly advance our understanding of striatal development and function.

      Weaknesses:

      Certain methodological considerations warrant attention. Notably, the reliance on TdTomato expression for the identification of striosomes raises concerns, particularly regarding the substantial difference in slice thickness between the immunohistochemistry (IHC) images (50um) shown in Figure 2 and those utilized for whole-cell recordings (300um).

      Enhanced clarification regarding the identification of cell patches is possible in the electrophysiology rig conditions. Using a widefield microscope rather than a confocal would strengthen the reliability of this methodology.

      In the Ca2+ imaging experiments of Figure 2, striosomes were defined as the regions of brighter GCaMP fluorescence. This presents a potential limitation because it presupposes higher activity levels within patch cells, which is what the experiment is designed to test. Based on this criteria, neurons of this region will necessarily have more activity than in others.

      There is also no information on how Ca2+ imaging traces were analyzed. In the examples provided, putative matrix neurons seem to exhibit different Ca2+ dynamics compared to striosome neurons. The plateau responses might reflect even higher activity than the transient signals observed in striosome neurons. It'll be important to know how the data was quantified. For example, calculations of F0 based on rolling functions tend to underestimate dF/F in traces like this. Calculations of the area under the curve can also provide valuable information in these cases.

      There is no description of the 8mM KCl treatment in the methods. Was this only used for the Ca2+ imaging experiments? The percentage of active cells in Figures 2C-D is similar to or lower than that described in Figure 2B, which is confusing. Were recordings always performed in 8mM KCl?

      Lastly, while the findings of Figure 6 suggest a deficit in striosomal inputs to SNc DA neurons, they do not conclusively demonstrate this point (DA neurons receive many sources of inhibition, and local interneurons in SNc are highly plastic). Given the availability of Opmr1-Cre mice and the utilization of multiple viruses in Figure 6 experiments, the inclusion of experiments employing ChR2 to directly assess striatal/striosome inputs would substantially strengthen this claim. This is the main claim stated in the manuscript title, so it is important to provide evidence of specific striatonigral deficits.

    1. Reviewer #1 (Public Review):

      Summary:

      The authors seek to elucidate the structural role of N-glycosylation at the N343 position of the SARS-CoV-2 Spike protein's Receptor Binding Domain (RBD) and its evolution across different variants of concern (VoCs). Specifically, they aim to understand the impact of this glycosylation on the RBD's stability and function, which could have implications for the virus's infectivity and, eventually, the effectiveness of vaccines.

      Strengths:

      The major strength of the study stems from the molecular-level picture emerging from the use of over 45 μs of cumulative molecular dynamics (MD) simulations, including both conventional and enhanced sampling schemes, which provide detailed insights into the structural role of N343 glycosylation. The combination of these simulations with experimental assays, such as electron-spray ionization mass spectrometry (ESI-MS) for affinity measurements, bolsters the reliability of the findings. At the same time, one potential weakness is the inherent limitation of the current computational models to fully capture the complexities of in vivo systems. While the authors acknowledge the difficulty in completely gauging the N343 glycosylation's impact on RBD folding due to the dynamic nature of glycan structures, their computational/experimental approach lends support to their claims.

      Weaknesses:

      One potential weakness is the inherent limitation of computational models to fully capture the complexities of in vivo systems. While the authors acknowledge the difficulty in completely gauging the N343 glycosylation's impact on RBD folding due to the dynamic nature of glycan structures, their multi-faceted approach lends solid support to their claims.

      Other Comments:

      The study shows that N343 glycosylation plays a structural role in stabilizing the RBD across various SARS-CoV-2 strains. The removal of this glycan led to conformational changes that could affect the virus's infectivity. The results correlate with a reported reduction in viral infectivity upon deletion of glycosylation sites, supporting the authors' conclusion that N343 glycosylation is functionally essential for viral infection.

      By providing molecular insights into the spike protein's architectural changes, the work could influence the design of more effective vaccines and therapeutic agents. The data and methods used could serve as a valuable resource for researchers looking into viral evolution, protein-glycan interactions, and the development of glycan-based interventions.

    1. Reviewer #1 (Public Review):

      Summary:

      This is an interesting and potentially important paper, which however has some deficiencies.

      Strengths:

      A significant amount of potentially useful data.

      Weaknesses:

      One issue is a confusion of thermal stability with solubility. While thermal stability of a protein is a thermodynamic parameter that can be described by the Gibbs-Helmholtz equation, which relates the free energy difference between the folded and unfolded states as a function of temperature, as well as the entropy of unfolding. What is actually measured in PISA is a change in protein solubility, which is an empirical parameter affected by a great many variables, including the presence and concentration of other ambient proteins and other molecules. One might possibly argue that in TPP, where one measures the melting temperature change ∆Tm, thermal stability plays a decisive or at least an important role, but no such assertion can be made in PISA analysis that measures the solubility shift.

      Another important issue is that the authors claim to have discovered for the first time a number of effects well described in prior literature, sometimes a decade ago. For instance, they marvel at the differences between the solubility changes observed in lysate versus intact cells, while this difference has been investigated in a number of prior studies. No reference to these studies is given during the relevant discussion.

      The validity of statistical analysis raises concern. In fact, no calculation of statistical power is provided. As only two replicates were used in most cases, the statistical power must have been pretty limited. Also, there seems to be an absence of the multiple-hypothesis correction.

      Also, the authors forgot that whatever results PISA produces, even at high statistical significance, represent just a prediction that needs to be validated by orthogonal means. In the absolute majority of cases such validation is missing.

      Finally, to be a community-useful resource the paper needs to provide the dataset with a user interface so that the users can data-mine on their own.

    1. Joint Public Review:

      Summary:

      Brauns et al. work to decipher the respective contribution of active versus passive contributions to cell shape changes during germ band elongation. Using a novel quantification tool of local tension, their results suggest that epithelial convergent extension results from internal forces.

      Strengths:

      The approach developed here, tension isogonal decomposition, is original and the authors made the demonstration that we can extract comprehensive data on tissue mechanics from this type of analysis.

      They present an elegant diagram that quantifies how active and passive forces interact to drive cell intercalations.

      The model qualitatively recapitulates the features of passive and active intercalation for a T1 event.

      Regions of high isogonal strains are consistent with the proximity of known active regions.

      They define a parameter (the LTC parameter) which encompasses the geometry of the tension triangles and allows the authors to define a criterium for T1s to occur.

      The data are clearly presented, going from cellular scale to tissue scale, and integrating modeling approach to complement the thoughtful description of tension patterns.

      Weaknesses:

      The modeling is interesting, with the integration of tension through tension triangulation around vertices and thus integrating force inference directly in the vertex model. However, the authors are not using it to test their hypothesis and support their analysis at the tissue level. Thus, although interesting, the analysis at the tissue level stays mainly descriptive.

      Major points:

      (1) The authors mention that from their analysis, they can predict what is the tension threshold required for intercalations in different conditions and predict that in Snail and Twist mutants the T1 tension threshold would be around √2. Since movies of these mutants are most probably available, it would be nice to confirm these predictions.

      (2) While the formalism is very elegant and convincing, and also convincingly allows making sense of the data presented in the paper, it is not all that clear whether the claims are compatible with previous experimental observations. In particular, it has been reported in different papers (including Collinet et al NCB 2015, Clement et al Curr Biol 2017) that affecting the initial Myosin polarity or the rate of T1s does not affect tissue-scale convergent extension. Analysis/discussion of the Tor phenotype (no extension with myosin anisotropy) and the Eve/Runt phenotype (extension without Myosin anisotropy), which seem in contradiction with an extension mostly driven by myosin anisotropy.

    1. Reviewer #1 (Public Review):

      The manuscript by Long et al. focused on SUL1, a gene encoding a sulfate transporter with signaling roles in yeast. The authors claim that the deletion of SUL1, rather than SUL2 (encoding a similar transporter), extended yeast replicative lifespan independent of sulfate transport. They also show that SUL1 loss-of-function mutants display decreased PKA activity, indicated by stress-protective carbohydrate accumulation, relevant transcription factor relocalization (measured during aging in single cells), and changes in gene expression. Finally, they show that loss of SUL1 increases autophagy, which is consistent with the longer lifespan of these cells. Overall, this is an interesting paper, but additional work should strengthen several conclusions, especially for the role of sulfate transport. Specific points include the following:

      - What prompted the authors to measure the RLS of sul1 mutants? Prior systematic surveys of RLS in the same strain background (which included the same sul1 deletion strain they used) did not report lifespan extension in sul1 cells (PMID: 26456335).

      - Cells carrying a mutant Sul1 (E427Q), which was reported to be disrupted in sulfate transport, did not have a longer lifespan (Figure 1), leading them to conclude that "lifespan extension by SUL1 deletion is not caused by decreased sulfate uptake". They would need to measure sulfate uptake in the mutants they test to draw that conclusion firmly.

      - Related to my previous point, another simple experiment would be to repeat the assays in Figure 1 with exogenous sulfur added to see if the lifespan extension is suppressed.

      - There needs to be more information in the text or the methods about how they did the enrichment analysis in Figure 2B. P-values are typically insufficient, and adjusted FDR values are reported from standard gene ontology platforms (e.g., PANTHER).

      - It is somewhat puzzling that relocalization of Msn2 was not seen in very old cells (past the 17th generation), but it was evident in younger cells. The authors could consider another possibility, that it was early and midlife experiences that made those cells live longer. Past that window, loss of Sul1 may have no impact on longevity. A conditional shutoff system to regulate SUL1 expression would be needed to test the above, albeit this is probably beyond the scope of this report.

      - The connections between glucose restriction, autophagy, and sul1 (Figure 4) could be further tested by measuring the RLS of sul1 cells in glucose-restricted cells. If RLS is further extended by glucose restriction, then whatever effects they see should be independent of glucose restriction.

      - They made and tested the double (sul1, msn2) mutants, but they should also test the sul1, msn4 combination since Msn4 functions similarly to Msn2.

    1. Reviewer #1 (Public Review):

      This is an interesting study investigating the mechanisms underlying membrane targeting of the NLRP3 inflammasome and reporting a key role for the palmitoylation-depalmitoylation cycle of cys130 in NRLP3. The authors identify ZDHHC3 and APT2 as the specific ZDHHC and APT/ABHD enzymes that are responsible for the s-acylation and de-acylation of NLRP3, respectively. They show that the levels of ZDHHC3 and APT2, both localized at the Golgi, control the level of palmitoylation of NLRP3. The S-acylation-mediated membrane targeting of NLRP3 cooperates with polybasic domain (PBD)-mediated PI4P-binding to target NLRP3 to the TGN under steady-state conditions and to the disassembled TGN induced by the NLRP3 activator nigericin.

      However, the study has several weaknesses in its current form as outlined below.

      (1) The novelty of the findings concerning cys130 palmitoylation in NLRP3 is unfortunately compromised by recent reports on the acylation of different cysteines in NLRP3 (PMID: 38092000), including palmitoylation of the very same cys130 in NLRP3 (Yu et al https://doi.org/10.1101/2023.11.07.566005), which was shown to be relevant for NLRP3 activation in cell and animal models. What remains novel and intriguing is the finding that NLRP3 activators induce an imbalance in the acylation-deacylation cycle by segregating NLRP3 in late Golgi/endosomes from de-acylating enzymes confined in the Golgi. The interesting hypothesis put forward by the authors is that the increased palmitoylation of cys130 would finally contribute to the activation of NLRP3. However, the authors should clarify the trafficking pathway of acylated-NLRP3. This pathway should, in principle, coincide with that of TGN46 which constitutively recycles from the TGN to the plasma membrane and is trapped in endosomes upon treatment with nigericin.

      (2) To affect the S-acylation, the authors used 16 hrs treatment with 2-bromopalmitate (2-BP). In Figure 1f, it is quite clear that NLRP3 in 2-BP treated cells completely redistributed in spots dispersed throughout the cells upon nigericin treatment. What is the Golgi like in those cells? In other words, does 2-BP alter/affect Golgi morphology? What about PI4P levels after 2-BP treatment? These are important missing pieces of data since both the localization of many proteins and the activity of one key PI4K in the Golgi (i.e. PI4KIIalpha) are regulated by palmitoylation.

      (3) The authors argue that the spots observed with NLRP-GFP result from non-specific effects mediated by the addition of the GFP tag to the NLRP3 protein. However, puncta are visible upon nigericin treatment, as a hallmark of endosomal activation. How do the authors reconcile these data? Along the same lines, the NLRP3-C130S mutant behaves similarly to wt NLRP3 upon 2-BP treatment (Figure 1h). Are those NLRP3-C130S puncta positive for endosomal markers? Are they still positive for TGN46? Are they positive for PI4P?

      (4) The authors expressed the minimal NLRP3 region to identify the domain required for NLRP3 Golgi localization. These experiments were performed in control cells. It might be informative to perform the same experiments upon nigericin treatment to investigate the ability of NLRP3 to recognize activating signals. It has been reported that PI4P increases on Golgi and endosomes upon NG treatment. Hence, all the differences between the domains may be lost or preserved. In parallel, also the timing of such recruitment upon nigericin treatment (early or late event) may be informative for the dynamics of the process and of the contribution of the single protein domains.

      (5) As noted above for the chemical inhibitors (1) the authors should check the impact of altering the balance between acyl transferase and de-acylases on the Golgi organization and PI4P levels. What is the effect of overexpressing PATs on Golgi functions?

    1. Reviewer #1 (Public Review):

      Summary<br /> In this work, Mouelhi et al investigated how the nucleus responds to long term confinement. They find that short-term confinement does not affect nuclear volume, whereas long-term confinement leads to a decrease in volume. The authors propose this decrease occurs after mitosis and relies on cPLA2 and myosin contractility.

      Strengths

      The ability to accurately control cell confinement allows authors to determine its effects on cellular function with high resolution. This provides a good addition to the existing collection of tools used for cellular micromanipulation. The results provided are relevant and timely and could help understand how cancer cells adapt to conditions of confinement.

      Weaknesses

      I have a few concerns which I believe should be addressed:

      (1) It is unclear whether the authors took into consideration the contribution of nuclear blebs for nuclear volume measurements. This would be particularly relevant in situations of very strong confinement. Blebs were previously shown to affect volume (Mistriotis et al., JCB 2019). One could argue that the decreased nuclear volume was due to the increased blebbing observed in very strong confinements.

      (2) From their experimental setup, it is unclear whether the reduced nuclear volume observed after confined cell division arises from a geometrical constraint or is due to an intrinsic nuclear feature. One could argue that cells exiting mitosis under confinement have clustered chromosomes and, therefore, will have decreased volume. This would imply that the nucleus is not "reset" but rather that a geometrical constraint is forcing nuclei to be smaller. One way to test this would be to follow individual cells under confinement, let them enter mitosis, and then release the confinement. If, under these conditions, the daughter nuclei are smaller, then it supports their model. If daughter nuclei recover to their initial value, then it´s simply due to a geometrical constraint that forces the clustering of chromosomes and the reassembly of the NE in a confined space.

      (3) The authors claim that the nucleus adapts to confinement based on evidence that the nucleus no longer shrinks in the second division following the first division. I would argue no further decrease is possible because the DNA is already compacted in the smallest possible volume. If indeed nuclei are in a new homeostatic state as the authors claim, then one would expect nuclei to remain smaller even after confinement is removed. This analysis is missing.

      (4) Also, if the authors want to claim that this is a mechanism used for cancer cells to adapt to confined situations as the title says, they need to show that normal, near-diploid cells do not behave in the same way. This analysis is missing.

      (5) Authors state that "Loss of nuclear blebs is clearly linked to mitosis, suggesting that nuclear volume and nuclear envelope tension are tightly coupled, and supports the hypothesis that mitosis is a key regulator of nuclear envelope tension". I have a few issues with the way this sentence is written. Firstly, one could say that all nuclear structures (and not only blebs) are lost during mitosis because the nucleus disassembles. Hence, the new homeostatic state could be determined by envelope reassembly after mitosis and not mitosis itself. Secondly, I don´t understand why the loss of nuclear blebs suggests that volume and tension are tightly coupled. Thirdly, how can mitosis be a key regulator of nuclear envelope tension when the nucleus is disassembled during the process? These require clarification.

      (6) The authors claim that, unlike previous studies (Lomakin et al), this work shows a "gradual nuclear adaptation". From their results, this is difficult to conclude simply because they do not analyse cPLA2 levels. This is solely based on indirect evidence obtained from cPLA2 inhibition. A gradual adaptation would mean that based on the level of confinement we would expect to have increasingly higher levels of cPLA2 (and therefore nuclear tension).

      (7) The authors should refrain from saying that the mechanism behind DNA repair is coupled to the nuclear adaptation they show. There are several points regarding this statement. Firstly, increased DNA damage could be due to nuclear ruptures imposed by confinement at 2h. In fact, the authors show leakage of NLS from the nucleus after confinement (Figure S3A). Secondly, the decrease in DNA damage at 24h could be because these nuclei did not rupture. How can they ensure that cells with low DNA damage at 24h had increased DNA damage at 2h? Finally, one needs to confirm if the nuclei they are analysing at 24h did undergo a round of cell division previously. From the evidence provided, the authors cannot conclude that DNA damage regulation is occurring in confined cells. Moreover, cell cycle arrest is a known effect of DNA damage. Cells with high damage at 2h most likely are arrested or will present with increased mitotic errors (which the authors exclude from their analyses).

    1. Reviewer #1 (Public Review):

      Summary:

      The authors aim to address a critical challenge in the field of bioinformatics: the accurate and efficient identification of protein binding sites from sequences. Their work seeks to overcome the limitations of current methods, which largely depend on multiple sequence alignments or experimental protein structures, by introducing GPSite, a multi-task network designed to predict binding residues of various molecules on proteins using ESMFold.

      Strengths:

      (1) Benchmarking. The authors provide a comprehensive benchmark against multiple methods, showcasing the performances of a large number of methods in various scenarios.

      (2) Accessibility and Ease of Use. GPSite is highlighted as a freely accessible tool with user-friendly features on their website, enhancing its potential for widespread adoption in the research community.

      Weaknesses:

      (1) Lack of significant insights. The paper reproduces results and analyses already presented in previous literature, without providing significant novel analysis or interpretation. However, they show a novel method with an original approach.

      The work is useful for the field, especially in disease mechanism elucidation and novel drug design. The availability of genome-scale binding residue annotations GPSite offers is a significant advancement.

    1. Reviewer #1 (Public Review):

      Summary:

      This manuscript examined the impact of prenatal alcohol exposure on genome-wide DNA methylation in the brain and liver, comparing ethanol-exposed mice to unexposed controls. They also investigated whether a high-methyl diet (HMD) could prevent the DNA methylation alterations caused by alcohol. Using bisulfite sequencing (n=4 per group), they identified 78 alcohol-associated differentially methylated regions (DMRs) in the brain and 759 DMRs in the liver, of which 85% and 84% were mitigated by the HMD group, respectively. The authors further validated 7 DMRs in humans using previously published data from a Canadian cohort of children with FASD.

      Overall, the findings from this study provide new insight into the impact of prenatal alcohol exposure, while also showing evidence for methyl-rich diets as an intervention to prevent the effects of alcohol on the epigenome. Some methodological concerns and confounders limit the robustness of these results, and should be addressed in future studies to further strengthen the conclusions of this study and its applicability to broader settings.

      Strengths:

      - The use of whole genome bisulfite sequencing allowed for the interrogation of the entire DNA methylome and DMR analysis, rather than a subset of CpGs.<br /> - The combination of data from animal models and humans allowed the authors to make stronger inferences regarding their findings<br /> - The authors investigated a potential mechanism (high methyl diet) to buffer against the effects of prenatal alcohol exposure, which increases the relevance and applicability of this research.

      Weaknesses:

      - The sample size was small for the epigenetic analyses, which limits the robustness of the findings.<br /> - The authors could not account for potential confounders in their analyses, including birthweight, alcohol levels, and sex. This is a particular problem for the high-methyl diet analyses, in which the alcohol-exposed mice consumed less alcohol than their non-diet counterparts.

    1. Joint Public Review:

      In this manuscript, Xue and colleagues investigate the fundamental aspects of cellular fate decisions and differentiation, focusing on the dynamic behaviour of gene regulatory networks. It explores the debate between static (noise-driven) and dynamic (signal-driven) perspectives within Waddington's epigenetic landscape, highlighting the essential role of gene regulatory networks in this process. The authors propose an integrated analysis of fate-decision modes and gene regulatory networks, using the Cross-Inhibition with Self-activation (CIS) network as a model. Through mathematical modelling, they differentiate two logic modes and their effect on cell fate decisions: requires both the presence of an activator and absence of a repressor (AA configuration) with one where transcription occurs as long the repressor is not the only species on the promoter (OO configuration).

      The authors establish a relationship between noise profiles, logic-motifs, and fate-decision modes, showing that defining any two of these properties allows the inference of the third. They also identify, under the signal-driven mode, two fundamental patterns of cell fate decisions: either prioritising progression or accuracy in the differentiation process. The authors apply this analysis to available high-throughput datasets of cell fate decisions in hematopoiesis and embryogenesis, proposing the underlying driving force in each case and utilising the observed noise patterns to nominate key regulators.

      The paper significantly advances our understanding of gene regulatory networks through a well-described computational study, where the authors rigorously evaluate assumptions in modelling. Particularly commendable is their introduction of the concept of combinatorial logic, exemplified by the double 'and' and double 'or' (AA/OO) logic motifs, which they successfully map to previously described cell fate decision processes. This theoretical and computational exploration sheds light on the dynamic landscape of epigenetic cell fate decisions, emphasising the role of combinatorial logic in coordinating noise and signal-driven processes. The thorough comparison of two model configurations underscores the importance of integration logic, contributing to a clearer understanding of gene regulatory network dynamics. Importantly, the results of the simulations are presented clearly, enhancing accessibility and intuitive understanding. The paper's strength also lies in its predictive power, as the authors use simulations to make insightful predictions about the regulatory organisation of stem cell differentiation systems. While the exploration is restricted to specific scenarios, these limitations serve to highlight areas for future research rather than detract from the paper's strengths.

      While the paper presents an intriguing framework for understanding gene regulatory networks and cell fate decisions, there are some weaknesses that warrant attention. Firstly, the framework would benefit from validation with more experimental data and application to diverse systems beyond those explored in the study, such as de-differentiation in adult tissues and regeneration processes. Additionally, while the authors successfully make predictions about the regulatory organisation of stem cell differentiation systems, there is a lack of discussion regarding how perturbations in the regulatory network could affect cell fate decisions. Furthermore, the paper could be strengthened by addressing the effects of mutations and other perturbations that may significantly influence cell fate decision-making processes, thus enhancing the robustness of the findings. Finally, there are instances where the clarity of the writing could be improved to enhance understanding and accessibility for readers.

    1. Reviewer #1 (Public Review):

      Induction of beta cell regeneration is a promising approach for the treatment of diabetes. In this study, Massoz et.al., identified calcineurin (CaN) as a new potential modulator of beta cell regeneration by using zebrafish as model. They also showed that calcineurin (CaN) works together with Notch signaling to promote the beta cell regeneration. Overall, the paper is well organized, and technically sound. However, some evidences seem weak to get the conclusion.

    1. Reviewer #1 (Public Review):

      Summary:

      The research study under review investigated the relationship between the gut and identified potential biomarkers derived from the nasopharyngeal and gut microbiota-based that could aid in predicting COVID-19 severity. The study reported significant changes in the richness and Shannon diversity index in nasopharyngeal microbiome associated with severe symptoms. The study showed a high abundance of Bacillota and Pesudomonadota in patients exhibiting severe symptomatology. Positive correlations were also found between Corynebacterium, Acinetobacter, Staphylococcus, and Veillonella, with the severity of SARS-CoV-2 infection.

      Strengths:

      The study successfully identified differences in the microbiome diversity that could indicate or predict disease severity. Furthermore, the authors demonstrated a link between individual nasopharyngeal organisms and the severity of SARS-CoV-2 infection. The density of the nasopharyngeal organism was shown to be a potential predictor of the severity of COVID-19.

      Weaknesses:

      The authors claimed an association between nasopharyngeal organisms and severity of SARS-CoV-2 infection but omitted essential data on the statistical significance of these associations between groups. The authors frequently referred to a p-value < 0.05 without presenting the actual p-values and percentages to show the significance of their results. The discussion is hard to understand (lacked clarity), as it contained an extensive literature review without discussing the study findings. A more focused discussion and results section on the main findings could have improved the overall readability of the paper. The role of potential confounders, such as HIV infection, and ethnicity which impacts the nasopharyngeal microbiome composition, was not included in the paper. Addressing the potential confounders would contribute to a more comprehensive understanding of the study's implications, specifically the role of the nasopharyngeal microbiome as a predictor of COVID-19 severity.

    1. Joint Public Review:

      This study investigates the role of Ikaros, a zinc finger family transcription factor related to Helios and Eos, in T-regulatory (Treg) cell functionality in mice. Through genome-wide association studies and chromatin accessibility studies, the authors find that Ikaros shares similar binding sites to Foxp3. Ikaros cooperates with Foxp3 to establish a major portion of the Treg epigenome and transcriptome. Ikaros-deficient Treg exhibits Th1-like gene expression with abnormal expression of IL-2, IFNg, TNFa, and factors involved in Wnt and Notch signalling. Further, two models of inflammatory/ autoimmune diseases - Inflammatory Bowel Disease (IBD) and organ transplantation - are employed to examine the functional role of Ikaros in Treg-mediated immune suppression. The authors provide a detailed analysis of the epigenome and transcriptome of Ikaros-deficient Treg cells.

      These studies establish Ikaros as a factor required in Treg for tolerance and the control of inflammatory immune responses. The data are of high quality. Overall, the study is well organized, and reports new data consolidating mechanistic aspects of Foxp3 mediated gene expression program in Treg cells.

      Strengths:

      The authors have performed biochemical studies focusing on mechanistic aspects of molecular functions of the Foxp3-mediated gene expression program and complemented these with functional experiments using two models of autoimmune diseases, thereby strengthening the study. The studies are comprehensive at both the cellular and molecular levels. The manuscript is well organized and presents a plethora of data regarding the transcriptomic landscape of these cells.

      Weakness:

      The findings of markedly increased percentages of activated conventional T cells (CD44hi), major increases in TFH cells, and elevated serum Ig levels indicate disrupted immune homeostasis even in the absence of overt autoimmune manifestations seen in histopathology. Thus, some of the observed genetic changes observed by the authors are likely Treg cell extrinsic. Further, clear conclusions from the genome-wide studies are lacking.

    1. Reviewer #1 (Public Review):

      The manuscript describes the development of a mouse model that co-expresses a fluorescent protein ZsGreen) marker in gene fusion with the FSHR gene.

      The authors are correct in that there is a lack of reliable antibodies against many of the GPCR family members. The approach is novel and interesting, with the potential to help understand the expression pattern of gonadotropin receptors. There has been a very long debate about the expression of gonadotropin receptors in other tissues other than gonads. While their expression of the FSHR in some of those tissues has been detected by a variety of methods, their physiological, or pathophysiological, function(s) remain elusive.

      The authors in this manuscript assume that the expression of ZsGren and the FSHR are equal. While this is correct genetically (transcription->translation) it does not go hand in hand with other posttranslational processes.

      (1) One of the shocking observations in this manuscript is the expression of FSHR in Leydig cells. Other observations are in the osteoblasts and endothelial cells as well as epithelial cells in different organs. The expression of ZsGreen in these tissues seems high and one shall start questioning if there are other mechanisms at play here.

      First, the turnover of fluorescent proteins is long, longer than 48h, which means that they accumulate at a different speed than the endogenous FSHR This means that ZsGreen will accumulate in time while the FSHR receptor might be degraded almost immediately. This correlated with mRNA expression (by the authors) but does not with the results of other studies in single-cell sequencing (see below).

      The expression of ZsGreen in Leydig cells seems much higher than in Sertoli cells, this is "disturbing" to put it mildly. This is visible in both the ZsGreen expression and the FISH assay (Figure 2 B-D).

      (2) The expression in WAT and BAT is also questionable as the expression of ZsGreen is high everywhere. That makes it difficult to believe that the images are truly informative. For example, the stainings of aorta show the ZsGreen expression where elastin and collagen fibres are - these are not "cells" and therefore are not expressing ZsGreen.

      (3) FISH expression (for FSHR) in WT mice is missing.

      Also, the tissue sections were stained with the IgG only (neg control) but in practice both the KI and the WT tissues should be stained with the primary and secondary antibodies. The only control that I could think of to truly get a sense of this would be a tagged receptor (N-terminal) that could then be analysed by immunohistochemistry.

      (4) The authors also claim:<br /> To functionally prove the presence of FSHR in osteoblasts/osteocytes, we also deleted FSHR in osteocytes using an inducible model. The conditional knockout of FSHR triggered a much more profound increase in bone mass and decrease in fat mass than blockade by FSHR antibodies (unpublished data).

      This would be a good control for all their images. I think it is necessary to make the large claim of extragonadal expression, as well as intragonadal such as Leydig cells.

      (5) Claiming that the under-developed Leydig cells in FSHR KO animals are due to a direct effect of the FSHR, and not via a cross-talk between Sertoli and Leydig cells, is too much of a claim. It might be speculated to some degree but as written at the moment it suggests this is "proven".

      (6) We also do not know if this FSHR expressed is a spliced form that would also result in the expression of ZsGreen but in a non-functional FSHR, or whether the FSHR is immediately degraded after expression. The insertion of the ZsGreen might have disturbed the epigenetics, transcription, or biosynthesis of the mRNA regulation.

      (7) The authors should go through single-cell data of WT mice to show the existence of the FSHR transcript(s).<br /> For example here:<br /> https://www.nature.com/articles/sdata2018192

    1. Reviewer #1 (Public Review):

      As a pathogen, S. aureus has evolved strategies to evade the host's immune system. It effectively remains 'under the radar' in the host until it reaches high population densities, at which point it triggers virulence mechanisms, enabling it to spread within the host. The agr quorum sensing system is central to this process, as it coordinates the pathogen's virulence in response to its cell density.

      In this study, Podkowik and colleagues suggest that cells activating agr signaling also benefit from protection against H2O2 stress, whereas inactivation of agr increases cell death. The underlying cause of this lack of protection is tied to an ATP deficit in the agr mutant, leading to increased glucose consumption and NADH production, ultimately resulting in a redox imbalance. In response to this imbalance, the agr mutant increases respiration, resulting in the endogenous production of ROS which synergizes with H2O2 to mediate killing of the agr mutant. Suppressing respiration in the agr mutant restored protection against H2O2 stress.

      Additionally, the authors establish that agr-dependent protection against oxidative stress is also linked to RNAIII activation, and the subsequent block of Rot translation. However, the specific protective genes regulated by Rot remain unidentified. Thus, according to the evidence provided, agr triggers intrinsic mechanisms that not only decrease harmful ROS production within the cell but also alleviate its detrimental effects.

      Interestingly, these protective mechanisms are long-lived, and guard the cells against external oxidative stressors such as H2O2, even after the agr system has been 'turned off' in the population.

    1. Reviewer #1 (Public Review):

      Summary:

      Del Rosario et al characterized the extent and cell types of sibling chimerism in marmosets. To do so, they took advantage of the thousands of SNPs that are transcribed in single-nucleus RNA-seq (snRNA-seq) data to identify the sibling genotype of origin for all sequenced cells across 4 tissues (blood, liver, kidney, and brain) from many marmosets. They found that chimerism is prevalent and widespread across tissues in marmosets, which has previously been shown. However, their snRNA-seq approach allowed them to identify precisely which cells were of sibling origin, and which were not. In doing so they definitively show that sibling chimerism across tissues is limited to cells of myeloid and lymphoid lineages. The authors then focus on a large sample of microglia sequenced across many brain regions to quantify: (1) variation in chimerism across brain regions in the same individual, and (2) the relative importance of genetic vs. environmental context on microglia function/identity.

      (1) Much like across different tissues in the same individual, they found that the proportion of chimeric microglia varies across brain regions collected from the same individuals (as well as differing from the proportion of sibling cells found in the blood of the same animals), suggesting that cells from different genetic backgrounds may differ in their recruitment and/or proliferation across regions and local tissue contexts, or that this may be linked to stochastic bottleneck effects during brain development.

      (2) Their (admittedly smaller sample size) analyses of host-sibling gene expression showed that the local environment dominates genotype.

      All told, this thoughtful and thorough manuscript accomplishes two important goals. First, it all but closes a previously open question on the extent and cell origins of sibling chimerism. Second, it sets the stage for using this unique model system to examine, in a natural context, how genetic variation in microglia may impact brain development, function, and disease.

      The conclusions of this paper are well supported by the data, and the authors exert appropriate care when extrapolating their results that come from smaller samples. However, there are a few concerns that should be addressed.

      The "modest correlation" mentioned in lines 170-172 does not take into account the uncertainty in estimates of each chimeric cell proportion (although the plot shows those estimates nicely). This is particularly important for the macrophages, which are far less abundant. Perhaps a more appropriate way to model this would be in a binomial framework (with a random effect for individuals of origin). Here, you could model the sibling identity of each macrophage as a function of the proportion of sibling-origin microglia and then directly estimate the percent variance explained.

      A similar (albeit more complicated because of the number of regions being compared) approach could be applied to more rigorously quantify the variation in chimerism across brain regions (L198-215; Figure 4). This would also help to answer the question of whether specific brain regions are more "amenable" to microglia chimerism than others.

      While the sample size is small, it would be exciting to see if any microglia eQTL are driven by sibling chimerism across the marmosets.

      L290-292: The authors should propose ways in which they could test the two different explanations proposed in this paragraph. For instance, a simulation-based modeling approach could potentially differentiate more stochastic bottleneck effects from recruitment-like effects.

      While intriguing, the gene expression comparison (Figure 5) is extremely underpowered. It would be helpful to clarify this and note the statistical thresholds used for identifying DEGs (the black points in the figure).

    1. Reviewer #1 (Public Review):

      Summary:<br /> The study presented by Atsumi et al. is about using smartphone-driven, community-sourced data to enhance biodiversity monitoring. The idea is to leverage the widespread use of smartphones to gather data from the community quickly, contributing to a more comprehensive understanding of biodiversity. The authors discuss the importance of ecosystem services linked to biodiversity and the threats posed by human activities. It emphasizes the need for comprehensive biodiversity data to implement the Kunming-Montreal Global Biodiversity Framework. The 'Biome' mobile app, launched in Japan, uses species identification algorithms and gamification to gather over 6 million observations since 2019. While community-sourced data may have biases, incorporating it into Species Distribution Models (SDMs) improves accuracy, especially for endangered species. The app covers urban-natural gradients uniformly, enhancing traditional survey data biased towards natural areas. Combining these sources provides valuable insights into species distributions for conservation, protected area designation, and ecosystem service assessment.

      Strengths:

      The use of a smartphone app ('Biome') for community-driven species occurrence data collection represents an innovative and inclusive approach to biodiversity monitoring, leveraging the widespread use of smartphones. The app has successfully accumulated a large volume of species occurrence data since its launch in 2019, showcasing its effectiveness in rapidly gathering information from diverse locations. Despite challenges with certain taxa, the study highlights high species identification accuracy, especially for birds, reptiles, mammals, and amphibians, making the 'Biome' app a reliable tool for species observation. The integration of community-sourced data into Species Distribution Models (SDMs) improves the accuracy of predicting species distributions. This has implications for conservation planning, including the designation of protected areas and assessment of ecosystem services. The rapid accumulation of data and advancements in machine learning methods open up opportunities for conducting time-series analyses, contributing to the understanding of ecosystem stability and interaction strength over time. The study emphasizes the collaborative nature of the platform, fostering collaboration among diverse stakeholders, including local communities, private companies, and government agencies. This inclusive approach is essential for effective biodiversity assessment and decision-making. The platform's engagement with various stakeholders, including local communities, supports biodiversity assessment, management planning, and informed decision-making. Additionally, the app's role in fostering nature-positive awareness in society is highlighted as a significant contribution to creating a sustainable society.

      Weaknesses:

      While the studies make significant contributions to biodiversity monitoring, they also have some weaknesses. Firstly, relying on smartphone-driven, community-sourced data may introduce spatial and taxonomic biases. The 'Biome' app, for example, showed lower accuracy for certain taxa like seed plants, molluscs, and fishes, potentially impacting the reliability of the gathered data. Furthermore, the effectiveness of Species Distribution Models (SDMs) relies on the assumption that biases in community-sourced data can be adequately accounted for. The unique distribution patterns of the 'Biome' data, covering urban-natural gradients uniformly, might not fully represent the diversity of certain ecosystems, potentially leading to inaccuracies in the models. Moreover, the divergence in data distribution patterns along environmental gradients between 'Biome' data and traditional survey data raises concerns. The app data shows a more uniform distribution across natural-urban gradients, while traditional data is biased towards natural areas. This discrepancy may impact the representation of certain ecosystems and influence the accuracy of Species Distribution Models (SDMs). While the integration of 'Biome' data into SDMs improves accuracy, the study notes that controlling the sampling efforts is crucial. Spatially-biased sampling efforts in community-sourced data need careful consideration, and efforts to control biases are essential for reliable predictions.

  2. Mar 2024
    1. Reviewer #1 (Public Review):

      Summary:

      The manuscript presents a short report investigating mismatch responses in the auditory cortex, following previous studies focused on the visual cortex. By correlating the mouse locomotion speed with acoustic feedback levels, the authors demonstrate excitatory responses in a subset of neurons to halts in expected acoustic feedback. They show a lack of responses to mismatch in the visual modality. A subset of neurons show enhanced mismatch responses when both auditory and visual modalities are coupled to the animal's locomotion.

      While the study is well-designed and addresses a timely question, several concerns exist regarding the quantification of animal behavior, potential alternative explanations for recorded signals, correlation between excitatory responses and animal velocity, discrepancies in reported values, and clarity regarding the identity of certain neurons.

      Strengths:

      (1) Well-designed study addressing a timely question in the field.

      (2) Successful transition from previous work focused on the visual cortex to the auditory cortex, demonstrating generic principles in mismatch responses.

      (3) The correlation between mouse locomotion speed and acoustic feedback levels provides evidence for a prediction signal in the auditory cortex.

      (4) Coupling of visual and auditory feedback shows putative multimodal integration in the auditory cortex.

      Weaknesses:

      (1) Lack of quantification of animal behavior upon mismatches, potentially leading to alternative interpretations of recorded signals.

      (2) Unclear correlation between excitatory responses and animal velocity during halts, particularly in closed-loop versus playback conditions.

      (3) Discrepancies in reported values in a few figure panels raise questions about data consistency and interpretation.

      (4) Ambiguity regarding the identity of the [AM+VM] MM neurons.

    1. Reviewer #1 (Public Review):

      Summary:

      The authors were trying to understand the relation between the development of large trunks and longirrostrine mandibles in bunodont proboscideans of Miocene, and how it reflects the variation in diet patterns.

      Strengths:

      The study is very well supported, written, and illustrated, with plenty Supplementary materials. The authors included all Asian bunodont proboscideans with long mandibles and I suggest that they should use the expression "bunodont proboscideans" instead of gomphotheres.

      Weaknesses:

      I believe that the only weakness is the lack of discussion comparing their results with the development of gigantism and long limbs in proboscideans from the same epoch.

      The authors reviewed the manuscript according to my suggestions and responded well to all my comments.

    1. Reviewer #1 (Public Review):

      Summary:

      This manuscript investigates the regulation of chlorophyll biosynthesis in rice embryos, focusing on the role of OsNF-YB7. The rigorous experimental approach, combining genetic, biochemical, and molecular analyses, provides a robust foundation for these findings. The research achieves its objectives, offering new insights into chlorophyll biosynthesis regulation, with the results convincingly supporting the authors' conclusions.

      Strengths:

      The major strengths include the detailed experimental design and the findings regarding OsNF-YB7's inhibitory role.

      Weaknesses:

      However, the manuscript's discussion on the practical implications for agriculture and the evolutionary analysis of regulatory mechanisms could be expanded.

    1. Reviewer #1 (Public Review):

      Summary:

      The authors describe that the endocytic pathway is crucial for ColI fibrillogenesis. ColI is endocytosed by fibroblasts, prior to exocytosis and formation of fibrils, which can include a mixture of endogenous/nascent ColI chains and exogenous ColI. ColI uptake and fibrillogenesis are regulated by circadian rhythm as described by the authors in 2020, thanks to the dependence of this pathway on circadian-clock-regulated protein VPS33B. Cells are capable of forming fibrils with recently endocytosed ColI when nascent chains are not available. Previously identified VPS33B is demonstrated not to have a role in endocytosis of ColI, but to play a role in fibril formation, which the authors demonstrate by showing the loss of fibril formation in VPS33B KO, and an excess of insoluble fibrils - along-side a decrease in soluble ColI secretion - in VPS33B overexpression conditions. A VPS33B binding protein VIPAS39 is also shown to be required for fibrillogenesis and to colocalise with ColI. The authors thus conclude that ColI is internalised into endosomal structures within the cell, and that ColI, VPS33B, and VIPA39 are co-trafficked to the site of fibrillogenesis, where along with ITGA11, which by mass spectrometric analysis is shown to be regulated by VPS33B levels, ColI fibrils are formed. Interestingly, in involved human skin sections from idiopathic pulmonary fibrosis (IPF) patients, ITGA11 and VPS33B expression is increased compared to healthy tissue, while in patient-derived fibroblasts, uptake of fluorescently-labelled ColI is also increased. This suggests that there may be a significant contribution of endocytosis-dependent fibrillogenesis in the formation of fibrotic and chronic wound-healing diseases in humans.

      Strengths:

      This is an interesting paper that contributes an exciting novel understanding of the formation of fibrotic disease, which despite its high occurrence, still has no robust therapeutic options. The precise mechanisms of fibrillogenesis are also not well understood, so a study devoted to this complex and key mechanism is well appreciated. The dependence of fibrillogenesis on VPS33B and VIPA39 is convincing and robust, while the distinction between soluble ColI secretion and insoluble fibrillar ColI is interesting and informative.

      Weaknesses:

      There are a number of limitations to this study in its current state. Inhibition of ColI uptake is performed using Dyngo4a, which although proposed as an inhibitor of Clathrin-dependent endocytosis is known to be quite un-specific. This may not be a problem however, as the endocytic mechanism for ColI also does not seem to be well defined in the literature, in fact, the principle mechanism described in the papers referred to by the authors is that of phagocytosis. It would be interesting to explore this important part of the mechanism further, especially in relation to the intracellular destination of ColI. The circadian regulation does not appear as robust as the authors' last paper, however, there could be a larger lag between endocytosis of ColI and realisation of fibrils. The authors state that the endocytic pathway is the mechanism of trafficking and that they show ColI, VPS33B, and VIPA39 are co-trafficked. However, the only link that is put forward to the endosomes is rather tenuously through VPS33B/VIPA39. There is no direct demonstration of ColI localisation to endosomes (ie. immunofluorescence), and this is overstated throughout the text. Demonstrating the intracellular trafficking and localisation of ColI, and its actual relationship to VPS33B and VIPA39, followed by ITGA11, would broaden the relevance of this paper significantly to incorporate the field of protein trafficking. Finally, the "self-formation" of ColI fibrils is discussed in relation to the literature and the concentration of fluorescently-tagged ColI, however as the key message of the paper is the fibrillogenesis from exocytosed colI, I do not feel like it is demonstrated to leave no doubt. Specific inhibition of intracellular trafficking steps, or following the progressive formation of ColI fibrils over time by immunofluorescence would demonstrate without any further doubt that ColI must be endocytosed first, to form fibrils as a secondary step, rather than externally-added ColI being incorporated directly to fibrils, independent of cellular uptake.

    1. Reviewer #1 (Public Review):

      Summary:

      In this manuscript, Herrmannova et al explore changes in translation upon individual depletion of three subunits of the eIF3 complex (d, e, and f) in mammalian cells. The authors provide a detailed analysis of regulated transcripts, followed by validation by RT-qPCR and/or Western blot of targets of interest, as well as GO and KKEG pathway analysis. The authors confirm prior observations that eIF3, despite being a general translation initiation factor, functions in mRNA-specific regulation, and that eIF3 is important for translation re-initiation. They show that the global effects of eIF3e and eIF3d depletion on translation and cell growth are concordant. Their results support and extend previous reports suggesting that both factors control the translation of 5'TOP mRNAs. Interestingly, they identify MAPK pathway components as a group of targets coordinately regulated by eIF3 d/e. The authors also discuss discrepancies with other reports analyzing eIF3e function.

      Strengths:

      Altogether, a solid analysis of eIF3 d/e/h-mediated translation regulation of specific transcripts. The data will be useful for scientists working in the Translation field.

      Weaknesses:

      The authors could have explored in more detail some of their novel observations, as well as their impact on cell behavior.

    1. Reviewer #1 (Public Review):

      The authors observed a decline in autophagy and proteasome activity in the context of Milton knockdown. Through proteomic analysis, they identified an increase in the protein levels of eIF2β, subsequently pinpointing a novel interaction within eIF subunits where eIF2β contributes to the reduction of eIF2α phosphorylation levels. Furthermore, they demonstrated that overexpression of eIF2β suppresses autophagy and leads to diminished motor function. It was also shown that in a heterozygous mutant background of eIF2β, Milton knockdown could be rescued. This work represents a novel and significant contribution to the field, revealing for the first time that the loss of mitochondria from axons can lead to impaired autophagy function via eIF2β, potentially influencing the acceleration of aging. To further support the authors' claims, several improvements are necessary, particularly in the methods of quantification and the points that should be demonstrated quantitatively. It is crucial to investigate the correlation between aging and the proteins eIF2β and eIF2α.

    1. Reviewer #1 (Public Review):

      Here the authors discuss mechanisms of ligand binding and conformational changes in GlnBP (a small E Coli periplasmic binding protein, which binds and carries L-glutamine to the inner membrane ATP-binding cassette (ABC) transporter). The authors have distinguished records in this area and have published seminal works. They include experimentalists and computational scientists. Accordingly, they provide comprehensive, high-quality, experimental and computational work.

      They observe that apo- and holo- GlnBP does not generate detectable exchange between open and (semi-) closed conformations on timescales between 100 ns and 10 ms. Especially, the ligand binding and conformational changes in GlnBP that they observe are highly correlated. Their analysis of the results indicates a dominant induced-fit mechanism, where the ligand binds GlnBP prior to conformational rearrangements. They then suggest that an approach resembling the one they undertook can be applied to other protein systems where the coupling mechanism of conformational changes and ligand binding.

      They argue that the intuitive model where ligand binding triggers a functionally relevant conformational change was challenged by structural experiments and MD simulations revealing the existence of unliganded closed or semi-closed states and their dynamic exchange with open unbound conformations, discuss alternative mechanisms that were proposed, their merits and difficulties, concluding that the findings were controversial, which, they suggest is due to insufficient availability of experimental evidence to distinguish them. As to further specific conclusions they draw from their results, they determine that a conformational selection mechanism is incompatible with their results, but induced fit is. They thus propose induced fit as the dominant pathway for GlnBP, further supported by the notion that the open conformation is much more likely to bind substrate than the closed one based on steric arguments.

      Considering the landscape of substrate-free states, in my view, the closed state is likely to be the most stable and, thus most highly populated. As the authors note and I agree that state can be sterically infeasible for a deep-pocketed substrate. As indeed they also underscore, there is likely to be a range of open states. If the populations of certain states are extremely low, they may not be detected by the experimental (or computational) methods. The free energy landscape of the protein can populate all possible states, with the populations determined by their relative energies. In principle, the protein can visit all states. Whether a particular state is observed depends on the time the protein spends in that state. The frequencies, or propensities, of the visits can determine the protein function. As to a specific order of events, in my view, there isn't any. It is a matter of probabilities which depend on the populations (energies) of the states. The open conformation that is likely to bind is the most favorable, permitting substrate access, followed by minor, induced fit conformational changes. However, a key factor is the ligand concentration. Ligand binding requires overcoming barriers to sustain the equilibrium of the unliganded ensemble, thus time. If the population of the state is low, and ligand concentration is high (often the case in in vitro experiments, and high drug dosage scenarios) binding is likely to take place across a range of available states.

      This is however a personal interpretation of the data. The paper here, which clearly embodies massive careful, and high-quality work, is extensive, making use of a range of experimental approaches, including isothermal titration calorimetry, single-molecule Förster resonance energy transfer, and surface-plasmon resonance spectroscopy. The problem the authors undertake is of fundamental importance.

    1. Reviewer #2 (Public Review):

      Summary:

      In this manuscript Nie et al investigate the effect of PARG KO and PARG inhibition (PARGi) on pADPR, DNA damage, cell viability and synthetic lethal interactions in HEK293A and Hela cells. Surprisingly, the authors report that PARG KO cells are sensitive to PARGi and show higher pADPR levels than PARG KO cells, which is abrogated upon deletion or inhibition of PARP1/PARP2. The authors explain the sensitivity of PARG KO to PARGi through incomplete PARG depletion and demonstrate complete loss of PARG activity when incomplete PARG KO cells are transfected with additional gRNAs in the presence of PARPi. Furthermore, the authors show that the sensitivity of PARG KO cells to PARGi is not caused by NAD depletion but by S-phase accumulation of pADPR on chromatin coming from unligated Okazaki fragments, which are recognized and bound by PARP1. Consistently, PARG KO or PARG inhibition show synthetic lethality with Pol beta, which is required for Okazaki fragment maturation. PARG expression levels in ovarian cancer cell lines correlate negatively with their sensitivity to PARGi.

    1. Reviewer #1 (Public Review):

      Summary:

      The paper measures the prevalence and mortality of stroke and its comorbidities across geographic regions in order to find differences in risks that may lead to more effective guidance for these subpopulations. It also does a genetic analysis to look for variants that may drive these phenotypic variations.

      Strengths:

      The data provided here will provide a foundation for a lot of future research into the causes of the observed correlations as well as whether the observed differences in comorbidities across regions have clinically relevant effects on risk management.

      Weaknesses:

      As with any cross-national analysis of rates, the data is vulnerable to differences in classification and reporting across jurisdictions. Furthermore, given the increased death rate from COVID-19 associated with many of these comorbid conditions and the long-term effects of COVID-19 infection on vascular health, it is expected that many of the correlations observed in this dataset will shift along with the shifting health of the underlying populations.

    1. Reviewer #1 (Public Review):

      Summary:

      The manuscript by Duilio M. Potenza et al. explores the role of Arginase II in cardiac aging, majorly using whole-body arg-ii knock-out mice. In this work, the authors have found that Arg-II exerts non-cell-autonomous effects on aging cardiomyocytes, fibroblasts, and endothelial cells mediated by IL-1b from aging macrophages. The authors have used arg II KO mice and an in vitro culture system to study the role of Arg II. The authors have also reported the cell-autonomous effect of Arg-II through mitochondrial ROS in fibroblasts that contribute to cardiac aging. These findings are sufficiently novel in cardiac aging and provide interesting insights. While the phenotypic data seems strong, the mechanistic details are unclear. How Arg II regulates the IL-1b and modulates cardiac aging is still being determined. The authors still need to determine whether Arg II in fibroblasts and endothelial contributes to cardiac fibrosis and cell death. This study also lacks a comprehensive understanding of the pathways modulated by Arg II to regulate cardiac aging.

      Strengths:

      This study provides interesting information on the role of Arg II in cardiac aging.

      The phenotypic data in the arg II KO mice is convincing, and the authors have assessed most of the aging-related changes.

      The data is supported by an in vitro cell culture system.

      Weaknesses:

      The manuscript needs more mechanistic details on how Arg II regulates IL-1b and modulates cardiac aging.

      The authors used whole-body KO mice, and the role of macrophages in cardiac aging is not studied in this model. A macrophage-specific arg II Ko would be a better model.

      Experiments need to validate the deficiency of Arg II in cardiomyocytes.

      The authors have never investigated the possibility of NO involvement in this mice model.

      A co-culture system would be appropriate to understand the non-cell-autonomous functions of macrophages.

      The Myocardial infarction data shown in the mice model may not be directly linked to cardiac aging.

    1. Reviewer #1 (Public Review):

      Summary:

      Semenova et al. have studied a large cross-sectional cohort of people living with HIV on suppressive ART, N=115, and performed high dimensional flow cytometry to then search for associations between immunological and clinical parameters and intact/total HIV DNA levels.

      A number of interesting data science/ML approaches were explored on the data and the project seems a serious undertaking. However, like many other studies that have looked for these kinds of associations, there was not a very strong signal. Of course, the goal of unsupervised learning is to find new hypotheses that aren't obvious to human eyes, but I felt in that context, there were (1) results slightly oversold, (2) some questions about methodology in terms mostly of reservoir levels, and (3) results were not sufficiently translated back into meaning in terms of clinical outcomes.

      Strengths:

      The study is evidently a large and impressive undertaking and combines many cutting-edge statistical techniques with a comprehensive experimental cohort of people living with HIV, notably inclusive of populations underrepresented in HIV science. A number of intriguing hypotheses are put forward that could be explored further. Sharing the data could create a useful repository for more specific analyses.

      Weaknesses:

      Despite the detailed experiments and methods, there was not a very strong signal for the variable(s) predicting HIV reservoir size. The Spearman coefficients are ~0.3, (somewhat weak, and acknowledged as such) and predictive models reach 70-80% prediction levels, though sometimes categorical variables are challenging to interpret.

      There are some questions about methodology, as well as some conclusions that are not completely supported by results, or at minimum not sufficiently contextualized in terms of clinical significance.

      On associations: the false discovery rate correction was set at 5%, but data appear underdetermined with fewer observations than variables (144vars > 115ppts), and it isn't always clear if/when variables are related (e.g inverses of one another, for instance, %CD4 and %CD8).

      The modeling of reservoir size was unusual, typically intact and defective HIV DNA are analyzed on a log10 scale (both for decays and predicting rebound). Also sometimes in this analysis levels are normalized (presumably to max/min?, e.g. S5), and given the large within-host variation of level we see in other works, it is not trivial to predict any downstream impact of normalization across population vs within-person.

      Also, the qualitative characterization of low/high reservoir is not standard and naturally will split by early/later ART if done as above/below median. Given the continuous nature of these data, it seems throughout that predicting above/below median is a little hard to translate into clinical meaning.

      Lastly, the work is comprehensive and appears solid, but the code was not shared to see how calculations were performed.

    1. Reviewer #1 (Public Review):

      Summary:

      The current study aims to quantify associations between the regular use of proton-pump inhibitors (PPI) - defined as using PPI most days of the week during the last 4 weeks at one cross-section in time - with several respiratory outcomes up to several years later in time. There are 6 respiratory outcomes included: risk of influenza, pneumonia, COVID-19, other respiratory tract infections, as well as COVID-19 severity and mortality).

      Strengths:

      Several sensitivity analyses were performed, including i) estimation of the e-value to assess how strong unmeasured confounders should be to explain observed effects, ii) comparison with another drug with a similar indication to potentially reduce (but not eliminate) confounding by indication.

      Weaknesses:

      (1) The main exposure of interest seems to be only measured at one time-point in time (at study enrollment) while patients are considered many years at risk afterwards without knowing their exposure status at the time of experiencing the outcome. As indicated by the authors, PPI are sometimes used for only short amounts of time. It seems biologically implausible that an infection was caused by using PPI for a few weeks many years ago.

      (2) Previous studies have shown that by focusing on prevalent users of drugs, one often induces several biases such as collider stratification bias, selection bias through depletion of susceptible, etc.

      (3) It seems Kaplan Meier curves are not adjusted for confounding through e.g. inverse probability weighting. As such the KM curves are currently not informative (or the authors need to make clearer that curves are actually adjusted for measured confounding).

      (4) Throughout the manuscript the authors seem to misuse the term multivariate (using one model with e.g. correlated error terms to assess multiple outcomes at once) when they seem to mean multivariable.

      (5) Given multiple outcomes are assessed there is a clear argument for accounting for multiple testing, which following the logic of the authors used in terms of claiming there is no association when results are not significant may change their conclusions. More high-level, the authors should avoid the pitfall of stating there is evidence of absence if there is only an absence of evidence in a better way (no statistically significant association doesn't mean no relationship exists).

      (6) While the authors claim that the quantitative bias analysis does show results are robust to unmeasured confounding, I would disagree with this. The e-values are around 2 and it is clearly not implausible that there are one or more unmeasured risk factors that together or alone would have such an effect size. Furthermore, if one would use the same (significance) criteria as used by the authors for determining whether an association exists, the required effect size for an unmeasured confounder to render effects 'statistically non-significant' would be even smaller.

      (7) Some patients are excluded due to the absence of follow-up, but it is unclear how that is determined. Is there potentially some selection bias underlying this where those who are less healthy stop participating in the UK biobank?

      (8) Given that the exposure is based on self-report how certain can we be that patients e.g. do know that their branded over-the-counter drugs are PPI (e.g. guardium tablets)? Some discussion around this potential issue is lacking.

      (9) Details about the deprivation index are needed in the main text as this is a UK-specific variable that will be unfamiliar to most readers.

      (10) It is unclear how variables were coded/incorporated from the main text. More details are required, e.g. was age included as a continuous variable and if so was non-linearity considered and how?

      (11) The authors state that Schoenfeld residuals were tested, but don't report the test statistics. Could they please provide these, e.g. it would already be informative if they report that all p-values are above a certain value.

      (12) The authors would ideally extend their discussion around unmeasured confounding, e.g. using the DAGs provided in https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7832226/, in particular (but not limited to) around severity and not just presence/absence of comorbidities.

      (13) The UK biobank is known to be highly selected for a range of genetic, behavioural, cardiovascular, demographic, and anthropometric traits. The potential problems this might create in terms of collider stratification bias - as highlighted here for example: https://www.nature.com/articles/s41467-020-19478-2 - should be discussed in greater detail and also appreciated more when providing conclusions.

    1. Reviewer #1 (Public Review):

      The manuscript, "A versatile high-throughput assay based on 3D ring-shaped cardiac tissues generated from human induced pluripotent stem cell-derived cardiomyocytes," developed a unique culture platform with PEG hydrogel that facilitates the in-situ measurement of contractile dynamics of the engineered cardiac rings. The authors optimized the tissue seeding conditions, demonstrated tissue morphology with expressions of cardiac and fibroblast markers, mathematically modeled the equation to derive contractile forces and other parameters based on imaging analysis, and concluded by testing several compounds with known cardiac responses.

      The authors answered my questions with appropriate experiments and explanation.

      (1) This paper presents an intriguing platform that creates miniature cardiac rings with merely thousands of cardiomyocytes per tissue in a 96-well plate format. The shape of the ring and the squeezing motion can recapitulate the contraction of the cardiac chamber to a certain degree. However, Thavandiran et al. (PNAS 2013) created a larger version of the cardiac ring and found that electrical propagation revealed spontaneous infinite loop-like cycles of activation propagation traversing the ring. This model was used to mimic a reentrant wave during arrhythmia. Therefore, there are concerns about whether a large number of cardiac tissues experience arrhythmia due to geometry-induced re-entry current and cannot be used as a healthy tissue model.

      In the new experiment, the authors demonstrated with voltage-sensitive dye that these miniaturized tissues do not experience any arrhythmia, potentially due to their small size.

      (2) The platform can produce 21 cardiac rings per well in 96-well plates, with the throughput being the highest among competing platforms. The resulting tissues exhibit good sarcomere striation due to the strain from the pillars. However, emerging questions pertain to culture longevity and reproducibility among tissues. According to Figure 1E, uneven ring formation around the pillar leads to tissue thinning and breakage. Only 50% survival is observed after 20 days of culture in the optimized seeding group. Are there any strategies to improve this survival rate? Additionally, do the cardiac rings detach from the glass slides and roll up, given the two compartments with cardiac and fibroblast-rich regions where fibroblasts maintain attachment to the glass slides? Moreover, the standard deviation of force measurement is a quarter of the value, which is suboptimal given the high replicate number. As the platform utilizes imaging analysis to derive contractile dynamics, calibration based on the angle and distance of the camera lens to individual tissues should be conducted to reduce error. On the other hand, how reproducible are the pillars? It is highly recommended to mechanically evaluate the consistency of the hydrogel-based pillars across different wells and within wells to understand the variance.

      The authors stated that the platform has been tested and improved with multiple cell lines to enhance tissue survival rates. The methodology of image capture and calculation of contractile dynamics were explained in detail to address concerns. Moreover, the reproducibility of the pillars was demonstrated by consistent results of Young's Modulus (AFM) from each pillar, showing low standard deviations.

      (3) Does the platform allow the observation of non-synchronized beating when testing with compounds? This can be extremely important as the intended applications of this platform are drug testing and cardiac disease modeling. The author should elaborate on the method in the manuscript and explain the obtained results in detail.

      Referring to Question #1, the platform does not present arrythmia potentially due to the small size of the tissue.

      (4) The results of drug testing are interesting. Isoperenoral is typically causing positive chronotropic and positive inotropic responses, where inotropic responses are difficult to obtain due to low tissue maturity. It is inconsistent with other reported results that cardiac rings do not exhibit increased beating frequency, but slightly increased forces only.

      The authors repeated the experiment with the same results and hypothesized that the results would be line-dependent, since the maturation of iPSC-CM is not consistent. The additional dose curves provided more information on the tissue behaviors against well-known compounds.

      Overall, the manuscript is well-written, and the designed platform presents unique advantages for high-throughput cardiac tissue culture. The paper has adequate data to demonstrate the proof-of-concept study of the platform. The throughput, consistency of the tissue, and the potential integration of high-throughput automation would be the highlights of this platform.

    1. Reviewer #1 (Public Review):

      Thermogenic adipocyte activity associate with cardiometabolic health in humans, but decline with age. Identifying the underlying mechanisms of this decline is therefore highly important.

      To address this task, Holman and co-authors present compelling data from their investigations of the effects of two major determinants of thermogenic activity: cold, which induce thermogenic de novo differentiation as well as conversion of dormant thermogenic inguinal adipocytes: and aging, which strongly reduce thermogenic activity. The authors study young and middle-aged mice at thermoneutrality and following cold exposure.

      Using linage tracing, the authors conclude that the older group produce less thermogenic adipocytes from progenitor differentiation. However, they found no differences between thermogenic differentiation capacity between the age groups when progenitors are isolated and differentiated in vitro. This finding is consistent with previous findings in humans, demonstrating that progenitor cells derived from dormant perirenal brown fat of humans differentiate into thermogenic adipocytes in vitro. Taken together, this underscores that age-related changes in the microenvironment rather than autonomous alterations in the ASPCs explain the age related decline in thermogenic capacity, This is an important finding in terms of identifying new approaches to switch dormant adipocytes into an active thermogenic phenotype.

      To gain insight into the age-related changes, the authors use single cell and single nuclei RNA sequencing mapping of their two age groups, comparing thermoneutral and cold conditions between the two groups. Interestingly, where the literature previously demonstrated that de novo lipogenesis (DNL) occurs in relation to thermogenic activation, the authors show that DNL in fact is activated in a white adipocyte cell type, whereas the beige thermogenic adipocytes form a separate cluster.

      Considering recent findings, that adipose tissue contains several subtypes of ASPCs and adipocytes, mapping the changes at single cell resolution following cold intervention provides an important contribution to the field, in particular as an older group with limited thermogenic adaptation is analyzed in parallel with a younger, more responsive group. This model also allowed for detection of microenvironment as a determining factor of thermogenic response.

      The use of only two time points (young and middle-aged) along the aging continuum limits the conclusions that can be made on aging as the only driver of the observed differences between the groups. Furthermore, as the authors also discuss, aging is a complex phenotype, and in this case the older mice were heavier and had larger fat depots, which should be taken into consideration when interpreting the data.

      In conclusion, this study provides an important resource for further studies, which should investigate how the findings can be translated into humans for reactivation of dormant thermogenic fat and a potential improvement of metabolic health.

    1. Reviewer #1 (Public Review):

      Metzger et al develop a rigorous method filling an important unmet need in protein evolution - analysis of protein genetic architecture and evolution using data from combinatorially complete 20^N variant libraries. Addressing this need has become increasingly valuable, as experimental methods for generating these datasets expand in scope and scale. Their method integrates two key features - (1) it reports the effects of mutations relative to the average across all variants, rather than a particular genotype, making it useful for examining global genetic architecture, and (2) it does this for all possible 20 states at each site, in contrast to the binary analyses in prior work. These features are not individually novel but integrating them into a single analysis framework is novel and will be valuable to the protein evolution community. Using a previously published dataset generated by two of the authors, they conclude that (1) changes in function are largely attributable to pairwise but not higher-order interactions, and (2) epistasis potentiates, rather than constrains, evolutionary paths. These findings are well-supported by the data. Overall, this work has important implications for predicting the relationship between genotype and phenotype, which is of considerable interest to protein biochemistry, evolutionary biology, and numerous other fields.

    1. Reviewer #1 (Public Review):

      The authors have generated a set of yeast S. cerevisiae strains containing different numbers of chromosomes.<br /> Elimination of telomerase activates homologous recombination (HR) to maintain telomeres in cells containing the original 16 chromosomes. However, elimination of telomerase leads to circularization of cells containing a single or two chromosomes. The authors examined whether the subtelomeric sequences X and Y' promote HR-mediated telomere maintenance using the strain SY12 carrying three chromosomes. They found that the subtelomeric sequences X and Y' are dispensable for cell proliferation and HR-mediated telomere maintenance in telomerase-minus SY12 cells. They conclude that subtelomeric X and Y' sequences do not play essential roles in both telomerase-proficient and telomerase-null cells and propose that these sequences represent remnants of genome evolution.

      Interestingly, telomerase-minus SY12 generates survivors that are different from well-established Type I or Type II survivors. The authors uncover atypical telomere formation which does not depend on the Rad52 homologous recombination pathway.

      Strengths:

      The authors examined whether the subtelomeric sequences X and Y' promote HR-mediated telomere maintenance using the strain SY12 carrying three chromosomes. They show that subtelomeres do not have essential roles in telomere maintenance and cell proliferation.

      Weaknesses:

      It is not fully addressed how atypical survivors are generated independently of Rad52-mediated homologous recombination.<br /> It remains possible that X and Y elements influence homologous recombination, type 1 and type 2 (type X), at telomeres. In particular, the presence of X and Y elements appears to be important for promoting type 1 recombination, although the authors conclude "Elimination of subtelomeric repeat sequences exerts little effect on telomere functions".

    1. Reviewer #1 (Public Review):

      Summary: In this article, Mirza et al developed a continuum active gel model of actomyosin cytoskeleton that account for nematic order and density variations in actomyosin. Using this model, they identify the requirements for the formation of dense nematic structures. In particular, they show that self-organization into nematic bundles requires both flow-induced alignment and active tension anisotropy in the system. By varying model parameters that control active tension and nematic alignment, the authors show that their model reproduces a rich variety of actomyosin structures, including tactoids, fibres, asters as well as crystalline networks. Additionally, discrete simulations are employed to calculate the activity parameters in the continuum model, providing a microscopic perspective on the conditions driving the formation of fibrillar patterns.

      Strengths: The strength of the work lies in its delineation of the parameter ranges that generate distinct types of nematic organization within actomyosin networks. The authors pinpoint the physical mechanisms behind the formation of fibrillar patterns, which may offer valuable insights into stress fiber assembly. Another strength of the work is connecting activity parameters in the continuum theory with microscopic simulations.

      Weaknesses: This paper is a very difficult read for nonspecialists, especially if you are not well-versed in continuum hydrodynamic theories. Efforts should be made to connect various elements of theory with biological mechanisms, which is mostly lacking in this paper. The comparison with experiments is predominantly qualitative. It is unclear if the theory is suited for in vitro or in vivo actomyosin systems. The justification for various model assumptions, especially concerning their applicability to actomyosin networks, requires a more thorough examination. The classification of different structures demands further justification. For example, the rationale behind categorizing structures as sarcomeric remains unclear when nematic order is perpendicular to the axis of the bands. Sarcomeres traditionally exhibit a specific ordering of actin filaments with alternating polarity patterns. Similarly, the criteria for distinguishing between contractile and extensile structures need clarification, as one would expect extensile structures to be under tension contrary to the authors' claim. Additionally, its unclear if the model's predictions for fiber dynamics align with observations in cells, as stress fibers exhibit a high degree of dynamism and tend to coalesce with neighboring fibers during their assembly phase. Finally, it seems that the microscopic model is unable to recapitulate the density patterns predicted by the continuum theory, raising questions about the suitability of the simulation model.

    1. Reviewer #1 (Public Review):

      Summary:

      In this manuscript, Liu et al. used scRNA-seq to characterize cell type-specific responses during allergic contact dermatitis (ACD) in a mouse model, specifically the hapten-induced DNFB model. Using the scRNA-seq data, they deconvolved the cell types responsible for the expression of major inflammatory cytokines such as IFNG (from CD4 and CD8 T cells), IL4/13 (from basophils), IL17A (from gd T cells), and IL1B from neutrophils and macrophages. They found the highest upregulation of a type 1 inflammatory response, centering around IFNG produced by CD4 and CD8 T cells. They further identified a subpopulation of dermal fibroblasts that upregulate CXCL9/10 during ACD and provided functional genetic evidence in their mouse model that disrupting IFNG signaling to fibroblasts decreases CD8 T cell infiltration and overall inflammation. They identify an increase in IFNG-expressing CD8 T cells in human patient samples of ACD vs. healthy control skin and co-localization of CD8 T cells with PDGFRA+ fibroblasts, which suggests this mechanism is relevant to human ACD. This mechanism is reminiscent of recent work (Xu et al., Nature 2022) showing that IFNG signaling in dermal fibroblasts upregulates CXCL9/10 to recruit CD8 T cells in a mouse model of vitiligo. Overall, this is a very well-presented, clear, and comprehensive manuscript. The conclusions of the study are mostly well supported by data, but some aspects of the work could be improved by additional clarification of the identity of the cell types shown to be involved, including the exact subpopulation discovered by scRNA-seq and the subtype of CD8 T cell involved. The study was limited by its use of one ACD model (DNFB), which prevents an assessment of how broadly relevant this axis is. The human sample validation is slightly circumstantial and limited by the multiplexing capacity of immunofluorescence markers.

      Strengths:

      Through deep characterization of the in vivo ACD model, the authors were able to determine which cell types were expressing the major cytokines involved in ACD inflammation, such as IFNG, IL4/13, IL17A, and IL1B. These analyses are well-presented and thoughtful, showing first that the response is IFNG-dominant, then focusing on deeper characterization of lymphocytes, myeloid cells, and fibroblasts, which are also validated and complemented by FACS experiments using canonical markers of these cell types as well as IF staining. Crosstalk analyses from the scRNA-seq data led the authors to focus on IFNG signaling fibroblasts, and in vitro experiments demonstrate that CXCL9 and CXCL10 are expressed by fibroblasts stimulated by IFNG. In vivo functional genetic evidence demonstrates an important role for IFNG signaling in fibroblasts, as KO of Ifngr1 using Pdgfra-Cre Ifngr1 fl/fl mice, showed a reduction in inflammation and CD8 T cell recruitment.

      Weaknesses:

      The use of one model limits an understanding of how broad this fibroblast-T cell axis is during ACD. However, the authors chose the most commonly employed model and cited additional work in a vitiligo model (another type 1 immune response). The identity of the involved fibroblasts and T cells in the mouse model is difficult to assess as scRNA-seq identified subpopulations of these cell types, but most work in the Pdgfra-Cre Ifngr1 fl/fl mice used broad markers for these cell types as opposed to matched subpopulation markers from their scRNA-seq data. Human patient samples of ACD were co-stained with two markers at a time, demonstrating the presence of CD8+IFNG+ T cells, PDGFRA+CXCL10+ fibroblasts, and co-localization of PDGFRA+ fibroblasts and CD8+ T cells. However, no IF staining demonstrates co-expression of all 4 markers at once; thus, the human validation of co-localization of CD8+IFNG+ T cells and PDGFRA+CXCL10+ fibroblasts is ultimately indirect, although not a huge leap of faith. Although n=3 samples of healthy control and ACD samples are used, there is no quantification of any results to demonstrate the robustness of differences.

    1. Reviewer #1 (Public Review):

      Summary:

      The authors report compound heterozygous deleterious variants in the kinase domains of the non-receptor tyrosine kinases (NRTK) TNK2/ACK1 in familial SLE. They suggest that ACK1 and BRK deficiencies are associated with human SLE and impair efferocytosis.

      Strengths:

      The identification of similar mutations in non-receptor tyrosine kinases (NRTKs) in two different families with familial SLE is a significant finding in human disease. Furthermore, the paper provides a detailed analysis of the molecular mechanisms behind the impairment of efferocytosis caused by mutations in ACK1 and BRK.

      Weaknesses:

      A critical point in this paper is whether the loss of function of ACK1 or BRK contributes to the onset of familial SLE. The authors emphasize that inhibitors of ACK1/BRK worsened IgG deposition in the kidneys in a pristane-induced SLE model, which contributes not to the onset but to the exacerbation of SLE, thus only partially supporting their claim.

    1. Reviewer #1 (Public Review):

      Summary:

      In this manuscript, the authors employed direct RNA sequencing with nanopores, enhanced by 5' end adaptor ligation, to comprehensively interrogate the human transcriptome at single-molecule and nucleotide resolution. They conclude that cellular stress induces prevalent 5' end RNA decay that is coupled to translation and ribosome occupancy. Contrary to the literature, they found that, unlike typical RNA decay models in normal conditions, stress-induced RNA decay is dependent on XRN1 but does not depend on the removal of the poly(A) tail. The findings presented are interesting but a substantial amount of work is needed to fully establish these paradigm-shifting findings.

      Strengths:

      These are paradigm-shifting observations using cutting-edge technologies.

      Weaknesses:

      The conclusions do not appear to be fully supported by the data presented.

    1. Reviewer #1 (Public Review):

      In this study Cacho-Navas et al. describes the role of ICAM-1 expressed on the apical membrane of bile canaliculi and its function to control the homeostasis of the bile canaliculi (BCs). This is a previously unrecognized function of this protein in hepatocytes. The same authors have previously shown that basolateral ICAM-1 plays a role in controlling lymphocyte adhesion to hepatocytes during inflammation and that this interaction is responsible on the loss of polarity of hepatocytes during the disease.<br /> In this new study they show that ICAM-1, is mainly localized in the apical domain of the BC and in association with EBP-50, comunicates with the subapical acto-myosin ring to regulate the size and morphology of the BC.<br /> In this study they used the well-known immortal cell line of liver cells (HepG2) in which they knocked-out ICAM-1 using CRISPR-Cas9 editing and hepatic organoid derived from WT and ICAM-1-KO mice. alternating knocking-out as well as rescue experiments they show that in the absence of apical ICAM-1, the BC dimension and shape are altered.<br /> The conclusions of the study are sufficiently supported by the data.

      Comments on revision:

      The authors have addressed most of the reviewer's comments in the re-submission, however the use of the organoids as a model to study bile canaliculi is still not convincing.<br /> The HA-4 staining and the space wehere CFDA is secreted does not overlap considering the nuclei position in the middle z-stack section. Also, the interdigitations between cells identified by EM do not form an enclosed space as we should expect for a bile canaliculi.<br /> I understand that other studies have used these organoids to show some hepatocytic functions but at the same time none has characterized before the formation of bile canaliculi as suggested in this study. Therefore a characterization showing the expression of specific markers (i.e mrp2, bsep) should be provided to support this claim.<br /> I would suggest the authors to carefully read the helpful review by Marsee et al., Cell Stem Cell 2021 that clearly and carefully address the classification and validation of liver organoids from experts in the field.

    1. Reviewer #1 (Public Review):

      Tu et al investigated how LFPs recorded simultaneously with rsfMRI explain the spatiotemporal patterns of functional connectivity in sedated and awake rats. They find that connectivity maps generated from gamma band LFPs (from either area) explain very well the spatial correlations observed in rsfMRI signals, but that the temporal variance in rsfMRI data is more poorly explained by the same LFP signals. The authors excluded the effects of sedation in this effect by investigating rats in the awake state (a remarkable feat in the MRI scanner), where the findings generally replicate. The authors also performed a series of tests to assess multiple factors (including noise, outliers, and nonlinearity of the data) in their analysis.

      This apparent paradox is then explained by a hypothetical model in which LFPs and neurovascular coupling are generated in some sense "in parallel" by different neuron types, some of which drive LFPs and are measured by ePhys, while others (nNOS, etc.) have an important role in neurovascular coupling but are less visible in Ephys data. Hence the discrepancy is explained by the spatial similarity of neural activity but the more "selective" LFPs picked up by Ephys account for the different temporal aspects observed.

      This is a deep, outstanding study that harnesses multidisciplinary approaches (fMRI and ephys) for observing brain activity. The results are strongly supported by the comprehensive analyses done by the authors, which ruled out many potential sources for the observed findings. The study's impact is expected to be very large.

      There are very few weaknesses in the work, but I'd point out that the 1-second temporal resolution may have masked significant temporal correlations between LFPs and spontaneous activity, for instance, as shown by Cabral et al Nature Communications 2023, and even in earlier QPP work from the Keilholz Lab. The synchronization of the LFPs may correlate more with one of these modes than the total signal. Perhaps a kind of "dynamic connectivity" analysis on the authors' data could test whether LFPs correlate better with the activity at specific intervals. However, this could purely be discussed and left for future work, in my opinion.

    1. Reviewer #1 (Public Review):

      Summary:

      In this paper, Song, Shi, and Lin use an existing deep learning-based sequence model to derive a score for each haplotype within a genomic region, and then perform association tests between these scores and phenotypes of interest. The authors then perform some downstream analyses (fine-mapping, various enrichment analyses, building polygenic scores) to ensure that these associations are meaningful. The authors find that their approach allows them to find additional associations, the associations have biologically interpretable enrichments in terms of tissues and pathways, and can slightly improve polygenic scores when combined with standard SNP-based PRS.

      Strengths:

      - I found the central idea of the paper to be conceptually straightforward and an appealing way to use the power of sequence models in an association testing framework.

      - The findings are largely biologically interpretable, and it seems like this could be a promising approach to boost power for some downstream applications.

      Weaknesses:

      - While not a weakness of the manuscript, the proposed method is computationally intensive.

    1. Reviewer #1 (Public Review):

      In this study, Hoops et al. showed that Netrin-1 and UNC5c can guide dopaminergic innervation from nucleus accumbens to cortex during adolescence in rodent models. They found that these dopamine axons project to the prefrontal cortex in a Netrin-1 dependent manner and knocking down Netrin-1 disrupted motor and learning behaviors in mice. Furthermore, the authors used hamsters, a seasonal model that is affected by the length of daylight, to demonstrate that the guidance of dopamine axons is mediated by the environmental factor such as daytime length and in sex dependent manner.

      Regarding the cell type specificity of Netrin-1 expression, the authors began by stating "this question is not the focus of the study and we consider it irrelevant to the main issue we are addressing, which is where in the forebrain regions we examined Netrin-1+ cells are present." This statement contradicts the exact issue regarding the specificity issue I raised. They then went on to show the RNAscope data for Netriin-1 in Figure 2, which showed Netrin-1 mRNA was actually expressed quite ubiquitously in anterior cingulate cortex, dorsopeduncular cortex, infralimbic cortex, prelimbic cortex, etc. In addition, contrary to the authors' statement that Netrin-1 is a "secreted protein", the confocal images in Figure 1 in the rebuttal letter actually show Netrin-1 present in "granule-like" organelles inside the cytoplasm of neurons. Finally, the authors presented Figure 7 to indicate the location where virus expressing Netrin-1 shRNA might be located. Again, the brain region targeted was quite focal and most likely did not cover all the Netrin-1+ brain regions in Figure 2. Collectively, these results raised more questions regarding the specificity of Netrin-1 expression in brain regions that are behaviorally relevant to this study.

      With respect to the effectiveness of Netrin-1 knockdown in the animals in this study, the authors cited data in HEK293 cells (Figure 5), which did not include any statistics, and previously published in vivo data in a separate, independent study (Figure 6). They do not provide any data regarding the effectiveness of Netrin-1 knockdown in THIS study.

      Similar concerns regarding UNC5C knockdown (points #6, #7, and #8) were not adequately addressed.

      In brief, while this study provides a potential role of Netrin-1-UNC5C in target innervation of dopaminergic neurons and its behavioral output in risk-taking, the data lack sufficient evidence to firmly establish the cause-effect relationship.

    1. Reviewer #1 (Public Review):

      In this paper, the authors developed an image analysis pipeline to automatically identify individual ‎‎neurons within a population of fluorescently tagged neurons. This application is optimized to deal with ‎‎multi-cell analysis and builds on a previous software version, developed by the same team, to resolve ‎‎individual neurons from whole-brain imaging stacks. Using advanced statistical approaches and ‎‎several heuristics tailored for C. elegans anatomy, the method successfully identifies individual ‎‎neurons with a fairly high accuracy. Thus, while specific to C. elegans, this method can ‎become ‎instrumental for a variety of research directions such as in-vivo single-cell gene expression ‎analysis ‎and calcium-based neural activity studies.‎

    1. Reviewer #1 (Public Review):

      In this manuscript, Yao et al. explored the transcriptomic characteristics of neural stem cells (NSCs) in the human hippocampus and their changes under different conditions using single-nucleus RNA sequencing (snRNA-seq). They generated single-nucleus transcriptomic profiles of human hippocampal cells from neonatal, adult, and aging individuals, as well as from stroke patients. They focused on the cell groups related to neurogenesis, such as neural stem cells and their progeny. They revealed genes enriched in different NSC states and performed trajectory analysis to trace the transitions among NSC states and towards astroglial and neuronal lineages in silico. They also examined how NSCs are affected by aging and injury using their datasets and found differences in NSC numbers and gene expression patterns across age groups and injury conditions. One major issue of the manuscript is questionable cell type identification. For example, more than 50% of the cells in the astroglial lineage clusters are NSCs, which is extremely high and inconsistent with classic histology studies.

      While the authors have made efforts to address previous critics, major concerns have not been adequately addressed, including a very limited sample size and patient information. In addition, some analytical approaches are still questionable and the authors acknowledge some issues they cannot address. Therefore, while the topic is interesting, some results are preliminary and some conclusions are not fully supported by the data presented.

    1. Reviewer #1 (Public Review):

      The authors show that concurrently presenting foreign words and their translations during sleep leads to the ability to semantically categorize the foreign words above chance. Specifically, this procedure was successful when stimuli were delivered during slow oscillation troughs as opposed to peaks, which has been the focus of many recent investigations into the learning & memory functions of sleep. Finally, further analyses showed that larger and more prototypical slow oscillation troughs led to better categorization performance, which offers hints to others on how to improve or predict the efficacy of this intervention.

      Comments on the revised version:

      I applaud the authors on a nice rebuttal. Many responses use solid arguments based on the existing literature, such as their response regarding the possibility that low-level acoustic characteristics explaining EEG differences between conditions. Their new analyses also clarify the paper. Additionally, I appreciate their labeling their more speculative claims as such. Below are my remaining thoughts:

      Major point:

      The largest remaining issue for me regards the term 'episodic'. Before I begin, I should say that I imagine the authors have thought considerably about this definition and may disagree with what I will say. That would be fine - it's their choice at this journal. My main point in writing this is to help them clarify their case further. R3 had a similar concern on the first round of review, and I imagine others holding the "traditional" view of episodic memory would be similarly skeptical. If the authors have a great rebuttal to these points, I imagine it will address others' concerns too.<br /> I believe I understand the authors' argument: I read the Henke (2010, Nature Reviews Neuroscience) piece years back with great interest and again now, and I've gone back to read their other papers cited in this manuscript. Again, I applaud the authors on producing a large collection of fascinating findings expanding knowledge of what can be accomplished via unconscious learning. That includes this paper! But I still disagree with the term 'episodic' for what is measured here. The authors state in the Methods section that they prompted participants to 'guess whether the presented pseudoword designates an animal, a tool, or a place'. IMHO, the main issue of using 'episodic' is the nature of the memory representation - 'guessing' does not ask participants anything about the source (the who-what-when-why-where) of the information (anything about an episode).<br /> Notably, it does seem to fit their own definition from Henke (2010). Rapid? I believe so - 4 trial-learning is fairly quick. Certainly, there are studies of supposed episodic memory that use a few rounds of learning the same stimuli (rather than single trial learning) and one can still get away with calling the nature of the memories 'episodic'. Flexible? I believe the authors mean that their task is flexible because participants learn a category exemplar during sleep (e.g., 'aryl'-'bird') but then only respond based on its category membership ('animal'?). If this is the case, I agree that the representations are flexible. Reliant on the 'episodic memory system' (lines 495-9)? Reasonably likely, given their prior findings (e.g., Züst et al., 2019). However, there is considerable data suggesting the hippocampus contributes to functions beyond episodic memory, including statistical learning (e.g., Schapiro et al., 2013, Current Biology), motor learning (e.g., Schendan et al., 2003, Neuron; Dohring et al., 2017, Cortex; Jacobacci et al., 2020, PNAS), attention (e.g., Aly & Turk-Browne, 2016, Cerebral Cortex), perception (e.g., Lee et al., 2012), and semantic memory (e.g., Cutler et al., 2019, Frontiers in Human Neuroscience). Therefore, given that the hippocampus contributes to other tasks too, saying the task is episodic in part because it likely relies on the hippocampus (the 'episodic memory system') is an incorrect reverse inference. But regardless of this concern, it seems true to me that the term fits 'episodic' according to Henke (2010).<br /> So, it seems I'm raising an issue with this entire way of defining memory. IMHO, the biggest issue is that there is no reason to assume the participant relies upon any source-related information in making their guess. There is room in the field for a new type of rapid, unconscious, flexible, hippocampal-dependent learning that does not need to align with the term, 'episodic', for it to be important and fascinating! The term, 'episodic', is convenient for a reason - namely, for labeling the behavioral output of what it measures, not the process that underlies it. The authors have continually made an excellent case for rapid, unconscious, flexible, hippocampal-dependent learning, and it would seem even more beneficial for the field for the authors to just call this its own thing.

      A related point:<br /> - I see that the authors do not use 'episodic' in prior papers with similar tasks (e.g., Züst et al., 2019), and I am curious if anything changed in their thinking or why they use the term now. They can ignore this if they'd like, but it would perhaps give useful context.

      Other points:<br /> IMHO, the issue of repeated tests is more legitimate than the authors suggest. They state in their response letter, "However, recent literature suggests that retrieval practice is only beneficial when corrective feedback is provided (Belardi et al., 2021; Metcalfe, 2017)." This is incorrect. While retrieval practice is often less effective without feedback, it can be effective without feedback if retrieval accuracy is high and if the experimenters later employ a long enough retention interval to witness long-term effects. This is clear in various papers (e.g., Roediger & Karpicke, 2006, Psychological Science; Karpicke & Roediger, 2008, Science) and there is a nice theoretical model explaining how these complex effects could arise (Halamish & Bjork, 2011, JEP:LMC; Kornell et al., 2011, JML). The authors do not heavily rely on this in their paper, but they could consider tempering their claims that it is 'unlikely' (line 509) that delayed retrieval was affected by the first retrieval.<br /> The authors claim that fast spindles are part of a speculative model underlying their learning effects (lines 605-6). However, they did not find any differential spindle effects in determining later performance, so they could consider keeping just points #1&2 or mentioning that spindles differ by condition but may not directly influence the learning effects here.

    1. Reviewer #1 (Public Review):

      This manuscript from Clayton and co-authors, entitled "Mechanism of dimer selectivity and binding cooperativity of BRAF inhibitors", aims to clarify the molecular mechanism of BRAF dimer selectivity. Indeed, first-generation BRAF inhibitors, targeting monomeric BRAFV600E, are ineffective in treating resistant dimeric BRAF isoforms. Here, the authors employed molecular dynamics simulations to study the conformational dynamics of monomeric and dimeric BRAF, in the presence and absence of inhibitors. Multi-microsecond MD simulations showed an inward shift of the αC helix in the BRAFV600E mutant dimer. This helped in identifying a hydrogen bond between the inhibitors and the BRAF residue Glu501 as critical for dimer compatibility. The stability of the aforementioned interaction seems to be important to distinguish between dimer-selective and equipotent inhibitors.

      The study is overall valuable and robust. The authors used the recently developed particle mesh Ewald constant pH molecular dynamics, a state-of-the-art method, to investigate the correct histidine protonation considering the dynamics of the protein. Then, multi-microsecond simulations showed differences in the flexibility of the αC helix and DFG motif. The dimerization restricts the αC position in the inward conformation, in agreement with the result that dimer-compatible inhibitors can stabilize the αC-in state. Noteworthy, the MD simulations were used to study the interactions between the inhibitors and the protein, suggesting a critical role for a hydrogen bond with Glu501. Finally, simulations of a mixed state of BRAF (one protomer bound to the inhibitor and the other apo) indicate that the ability to stabilize the inward αC state of the apo protomer could be at the basis of the positive cooperativity of PHI1.

      One potential weakness in the manuscript is the lack of reported uncertainties related to the analyzed quantities. Providing this information would significantly enhance the clarity regarding the reliability of the analyses and the confidence in the claims presented.

    1. Reviewer #1 (Public Review):

      Zhang et al. tackle the important topic of primate-specific structural features of the brain and the link with functional specialization. The authors explore and compare gyral peaks of the human and macaque cortex through non-invasive neuroimagery, using convincing techniques that have been previously validated elsewhere. They show that nearly 60% of the macaque peaks are shared with humans, and use a multi-modal parcellation scheme to describe the spatial distribution of shared and unique gyral peaks in both species.

      The claim is made that shared peaks are mainly located in lower-order cortical areas whereas unique peaks are located in higher-order regions, however, no systematic comparison is made. The authors then show that shared peaks are more consistently found across individuals than unique peaks, and show a positive but small and non-significant correlation between cross-individual counts of the shared peaks of the human and the macaque i.e. the authors show a non-significant trend for shared peaks that are more consistently found across humans to be those that are also more found across macaques.

      In order to identify if unique and shared peaks could be identified based on the structural features of the cortical regions containing them, the authors compared them with t-tests. A correction for multiple comparisons should be applied and t-values reported. Graph-theoretical measures were applied to functional connectivity datasets (resting-state fMRI) and compared between unique and shared peak regions for each species separately. Again the absence of multiple comparison correction and t-values make the results hard to interpret. The same comment applies to the analysis reporting that shared peaks are surrounded by a larger number of brain regions than unique peaks. Finally, the potentially extremely interesting results about differential human gene expression of shared and unique peaks regions are not systematically reported e.g. the 28 genes identified are not listed and the selection procedure of 7 genes is not fully reported.

      The paper is well written and the methods used for data processing are very compelling i.e. the peak cluster extraction pipeline and cross-species registration.

      Comments on revision:

      The authors have convincingly addressed all my previous concerns such that, as the revised paper stands now, the presented results provide solid support for the conclusions of the authors. The revised paper is now of interest for a large part of the neuroscience community and specifically for those interested in primate-specific structural features of the brain and the link with functional specialization.

    1. Reviewer #1 (Public Review):

      Summary:

      The authors re-analysed the data of a previous study in order to investigate the relation between asymmetries of subcortical brain structures and the hemispheric lateralization of alpha oscillations during visual spatial attention. The visual spatial attention task crossed the factors of target load and distractor salience, which made it possible to also test the specificity of the relation of subcortical asymmetries to lateralized alpha oscillations for specific attentional load conditions. Asymmetry of globus pallidus, caudate nucleus, and thalamus explained inter-individual differences in attentional alpha modulation in the left versus right hemisphere. Multivariate regression analysis revealed that the explanatory potential of these regions' asymmetries varies as a function of target load and distractor salience.

      In the revision of the article, the authors addressed my concerns.

      However, my concern with regard to the statistical analysis of the specificity of certain subcortical regions predicting HLM seems to be not fully addressed. The authors added an additional statistical analysis for "testing the null hypothesis that a given regressor does not impact all dependent variables". To my understanding, this is a somewhat unusual definition of a null hypothesis. Typically, the null hypothesis is the hypothesis of no effect, meaning here it should state that the effect is the same across predictors.

      In the new statistical analysis, the authors seem to take non-significant results (p>.05) as evidence for the specificity of subcortical regions in predicting HLM. The rationale of this statistical approach is difficult to follow and was somewhat unclear to me.

      A much simpler and more straight-forward approach would be to contrast beta-estimates per subcortical region between experimental conditions. For instance, if the beta estimates in the thalamus for the "low-load target, non-salient distractor" condition would be significantly larger than the beta estimates for the other conditions, this would speak to specificity.

    1. Reviewer #1 (Public Review):

      SUMMARY:

      Parkinson's disease (PD) and other synucleinopathies, including Parkinson's Disease Dementia (PDD), Dementia with Lewy Bodies (DLB), and Multiple System Atrophy (MSA), pose significant challenges for early diagnosis, as their clinical manifestations often emerge after substantial neurodegeneration has occurred. In this context, the Alpha-Synuclein Seeding Amplification Assay (SAA) has garnered considerable attention for its potential as a diagnostic tool, capable of detecting pathological forms of alpha-synuclein (αSyn) even before the onset of classical clinical symptoms and signs. The assay exploits αSyn's intrinsic property to convert healthy forms into pathological ones, subsequently amplifying these pathological forms for visualization. This study aims to investigate the efficacy of SAA in accurately identifying subtypes of synucleinopathies, including PD, PDD, DLB, and MSA. To achieve this, the results from the patient brain-derived αSyn SAA are compared with those obtained through conformational stability assays, immunolabeling, and electron microscopy. Study shows that brain-derived αSyn fibrils exhibit significant differences across various synucleinopathies in their conformation, biochemical profile and phosphorylation patterns. Importantly, the SAA method appears to fall short in capturing these distinctions.

      The study's findings are highly relevant given the rapidly advancing landscape of utilizing the SAA for the diagnosis and differentiation of various forms of PD and synucleinopathies using patient biofluids. It is somewhat surprising that the authors primarily characterize SAA as a research tool without delving into its potential as a biomarker detection assay, especially in the context of the field's excitement about its diagnostic applications. Additionally, a missed opportunity lies in not referencing a recent study that employed SAA successfully to diagnose PD and subtype the condition using a vast sample size. To further strengthen the results, the inclusion of healthy control brains in the biochemical and immunostaining/immunoblot experiments would provide more robust comparisons. Overall, the authors have conducted their experiments diligently, and their study offers valuable insights that align with the ongoing efforts to enhance early diagnosis and subtype differentiation in the domain of synucleinopathies.

      STRENGTH:

      The strengths of this research article are indeed notable and contribute to the credibility and significance of the study:

      Important Research Question: The study addresses a crucial question in the field of neurodegenerative diseases by evaluating the effectiveness of the αSyn SAA in diagnosing and differentiating synucleinopathies. This question is of significant clinical and scientific interest.<br /> Comprehensive Introduction: The article provides a thorough and well-structured introduction to the topic with an illustration, setting the stage for the research. It ensures that readers, including those unfamiliar with the subject matter, can grasp the context and significance of the study.<br /> Use of Patient Brain Tissue: The use of patient-derived brain tissue samples from various synucleinopathies, including PD, PDD, DLB, and MSA, enhances the clinical relevance and applicability of the findings.<br /> Replication and Statistical Significance: Conducting the experiments six times for each sample demonstrates the rigor of the study and the robustness of the results, and increases the confidence in the conclusions drawn.<br /> Clarity in Experimental Results and Discussion: The authors have presented the experimental results in a clear and understandable manner. I was personally impressed by images showing twisted and straight conformations of αSyn, as well as immunogold labeling for phosphorylation of αSyn, which aids in conveying the findings effectively to the readers. The results clearly show distinct differences in the characteristics of αSyn fibrils across different synucleinopathies. It also highlights the more aggressive seeding capacities and higher biochemical stability of αSyn in PDD and DLB patients, offering valuable insights into the pathophysiology of these conditions. The authors also clearly show that SAA fails in differentiating the disease types within the synucleinopathies.<br /> Clinical relevance: The study underscores the importance of considering complementary diagnostic methods alongside SAA for a more comprehensive understanding of synucleinopathy subtypes. The study might also play an important role in potential FDA approval of SAA as a diagnostic tool for synucleinopathies, especially for PD.<br /> These strengths collectively make the study a valuable contribution to the field of neurodegenerative diseases, shedding light on the limitations and potential applications of SAA in the diagnosis and differentiation of various synucleinopathies.

      WEAKNESS:

      While this study is overall robust, there are several aspects that could further enhance the quality and interpretation of the findings.

      Clinical Data on Patient Brain Samples: The inclusion of specific details such as post-mortem intervals and the age at disease onset for patient brain samples would be valuable. These factors could significantly affect the quality of the tissues and their relevance to the study. Moreover, given the large variation in disease duration between PD and PDD, it's important to consider disease duration as a potential confounding factor, especially when concluding that PDD patients have a more severe form of synucleinopathy compared to PD.<br /> Inclusion of Healthy Controls in Multiple Tests: Given the importance of healthy controls in scientific studies, especially those involving human brain samples, the authors could consider using healthy controls in more tests to strengthen the robustness of the findings. Expanding the use of healthy controls in biochemical profiling and phosphorylation profiles would provide a better basis for comparison and clarify the significance of results in a disease context.<br /> This will help the authors to elaborate on the interpretation of results, for example, in Figure 3, where the authors claim that PD brains show mostly monomeric αSyn forms (line 119 and 120, and also in 222 and 223). Whether it implies the absence of alpha-syn pathology in PD brains? If there are differences from healthy controls? What are these low molecular weight bands (<15kD) (line 125-126) and whether they are also present in healthy controls? Also, we do not have a perfect pS129-specific (anti-p𝛼Syn) antibody. They are known for non-specific labeling. Investigating the phosphorylation levels in healthy controls and comparing them to PD brains, especially considering the predominance of monomeric (healthy αSyn?) in PD brains, would help clarify the observed changes.<br /> Age of Healthy Controls: Providing information about the age at death for healthy controls is crucial, as age can impact the accumulation of αSyn. Also include if the brain samples were age-matched, or analyses were age-adjusted.<br /> Braak Staging Discrepancy: The study reports the same Braak staging for both PD and PDD, despite the significant difference in disease duration. Maybe other reviewers with clinical experience might have a better take on this. This observation merits discussion in the paper, allowing readers to better understand the implications of this finding.<br /> Citation of Relevant Studies: The paper should consider citing and discussing a recent celebrated study on PD biomarkers that used thousands of cerebrospinal fluid (CSF) samples from different PD patient cohorts to demonstrate the effectiveness of SAA as a biochemical assay for diagnosing PD and its subtypes (https://doi.org/10.1016/S1474-4422(23)00109-6).<br /> In summary, these suggestions aim to enhance the study's quality and the clarity of its findings, ultimately contributing to a more comprehensive understanding of synucleinopathies and the diagnostic potential of SAA.

    1. Reviewer #1 (Public Review):

      Summary:

      Songbirds provide a tractable system to examine neural mechanisms of sequence generation and variability. In past work, the projection from LMAN to RA (output of the anterior forebrain pathway) was shown to be critical for driving vocal variability during babbling, learning, and adulthood. LMAN is immediately adjacent to MMAN, which projects to HVC. MMAN is less well understood but, anatomically, appears to resemble LMAN in that it is the cortical output of a BG-thalamocortical loop. Because it projects to HVC, a major sequence generator for both syllable phonology and sequence, a strong prediction would be that MMAN drives sequence variability in the same way that LMAN drives phonological variability. This hypothesis predicts that MMAN lesions in a Bengalese finch would reduce sequence variability. Here, the authors test this hypothesis. They provide a surprising and important result that is well motivated and well analyzed: MMAN lesions increase sequence variability - this is exactly the opposite result from what would be predicted based on the functions of LMAN.

      Strengths:

      (1) A very important and surprising result shows that lesions of a frontal projection from MMAN to HVC, a sequence generator for birdsong, increase syntactical variability.

      (2) The choice of Bengalese finches, which have complex transition structures, to examine the mechanisms of sequence generation, enabled this important discovery.

      (3) The idea that frontal outputs of BG-cortical loops can generate vocal variability comes from lesions/inactivations of a parallel pathway from LMAN to RA. The difference between MMAN and LMAN functions is striking and important.

    1. Reviewer #1 (Public Review):

      Summary:

      This manuscript reports a series of experiments examining category learning and subsequent generalization of stimulus representations across spatial and nonspatial domains. In Experiment 1, participants were first trained to make category judgments about sequences of stimuli presented either in nonspatial auditory or visual modalities (with feature values drawn from a two-dimensional feature manifold, e.g., pitch vs timbre), or in a spatial modality (with feature values defined by positions in physical space, e.g., Cartesian x and y coordinates). A subsequent test phase assessed category judgments for 'rotated' exemplars of these stimuli: i.e., versions in which the transition vectors are rotated in the same feature space used during training (near transfer) or in a different feature space belonging to the same domain (far transfer). Findings demonstrate clearly that representations developed for the spatial domain allow for representational generalization, whereas this pattern is not observed for the nonspatial domains that are tested. Subsequent experiments demonstrate that if participants are first pre-trained to map nonspatial auditory/visual features to spatial locations, then rotational generalization is facilitated even for these nonspatial domains. It is argued that these findings are consistent with the idea that spatial representations form a generalized substrate for cognition: that space can act as a scaffold for learning abstract nonspatial concepts.

      Strengths:

      I enjoyed reading this manuscript, which is extremely well written and well presented. The writing is clear and concise throughout, and the figures do a great job of highlighting the key concepts. The issue of generalization is a core topic in neuroscience and psychology, relevant across a wide range of areas, and the findings will be of interest to researchers across areas in perception and cognitive science. It's also excellent to see that the hypotheses, methods and analyses were pre-registered.

      The experiments that have been run are ingenious and thoughtful; I particularly liked the use of stimulus structures that allow for disentangling of one-dimensional and two-dimensional response patterns. The studies are also well powered for detecting effects of interest. The model-based statistical analyses are thorough and appropriate throughout (and it's good to see model recovery analysis too). The findings themselves are clear-cut: I have little doubt about the robustness and replicability of these data.

      Weaknesses:

      In my original review I raised a concern related to a potential alternative interpretation of the findings: the idea that participants have substantial experience of representing space in terms of multiple, independent, and separable dimensions, whereas this may not be the case for the visual and auditory stimuli used here. As I noted in that prior review, on this view "the impact of spatial pre-training and (particularly) mapping is simply to highlight to participants that the auditory / visual stimuli comprise two separable (and independent) dimensions."

      In addressing this point, the authors note that performance in the visual/auditory "mapping" task in Experiments 2c and 3c suggests that most participants were paying attention to both dimensions of auditory and visual stimuli. I agree that seems to have been the case. But there is a difference between making use of information from both dimensions, and realizing that ***the two dimensions are separable and independent*** (which is what is required for rotational generalization in this task).

      As an analogy, suppose I have a task where participants have to map a pillow and a shuttlecock to category A, and a surfboard and a bicycle to category B. A participant could learn to do this just by memorizing the correct response for each item considered as a "whole thing". Or they could realize that the items contain component information, learning that "things with feathers" belong in category A, and "things that can carry people" go in category B. Performance may be the same in both cases, but the underlying process is quite different.

      The "attention to dimensions" account that I advanced in my previous review was referring to something more like the latter (feathers/vehicle) case: that spatial pre-training helps people to understand that items can be decomposed into separable pieces of information. Above-chance performance in the visual-auditory mapping task does not (necessarily) demonstrate this ability because it could reflect memorization of "whole" stimuli rather than reflecting decomposition into separable component parts. I agree that it does at least show that participants were paying attention to and making use of information from both dimensions when making their mapping decisions; it's just that they may not have *realized* that they were using information from two separable dimensions.

    1. Reviewer #1 (Public Review):

      Summary:

      This is an interesting report examining activity patterns in mouse ACC and in the OFC neurons projecting to ACC. In addition, the effects of inactivation are examined. In aggregate, the results provide new and interesting information about these two brain areas and they translate motivation into action - a function that it seems intuitively plausible that ACC might perform but, despite this intuition, there have been comparatively few direct tests of the idea and little is known of the specific mechanisms. The study is performed carefully and is written up clearly. There were just a few points where I wondered if a little more clarification might be helpful.

      Strengths:

      The combination of recording and inactivation/inhibition experiments and the combination of investigation of ACC neurons and of OFC regions projecting to ACC are very impressive.

      Weaknesses:

      These are all minor points of clarification.

      (1) An important conclusion (Figure 4) is that when mice are trained to run through no reward (N) cues in order to reach reward (R) cues, the OFC neurons projecting to ACC each respond to different specific events in a manner that ensures that collectively they tile the extended behavioural sequence. What I was less sure of was whether the ACC neurons do the same or not. Figure 3 suggests that on average ACC neurons maintain activity across N cues in order to get to R cues but I was not sure whether this was because all individual neurons did this or whether some had activity patterns like the OFC neurons projecting to ACC.

      (2) Figure 1 versus Figure 2: There does not seem to be a particular motivation for whether chemogenetic inactivation or optogenetic inhibition were used in different experiments. I think that this is not problematic but, if I am wrong and there were specific reasons for performing each experiment in a certain way, then further clarification as to why these decisions were made would be useful. If there is no particular reason, then simply explaining that this is the case might stop readers from seeking explanations.

      (3) P5, paragraph 2. The authors argue that OFC and anteriomedial (AM) thalamic inputs into ACC are especially important for mediating motivation through N cues in order to reach R cues. Is this based on a statistical comparison between the activity in OFC or AM inputs as opposed to the other inputs?

      (4) P3, paragraph 2. Some papers by Khalighinejad and colleagues (eg Neuron 2020, Current Biology, 2022) might be helpful here in as much as they assess ACC roles in determining action frequency, initiation, and speed and mediating the relationship between reward availability and action frequency and speed.

      (5) Paragraph 1 "This learning is of a more deliberate, informed nature than habitual learning, as they are sensitive to the current value of outcomes and can lead to a novel sequence of actions for a desired outcome1-3." Should "they" be "it"?

    1. Reviewer #1 (Public Review):

      Summary:

      Wang and colleagues present a study aimed at demonstrating the feasibility of repeated ultrasound localization microscopy (ULM) recording sessions on mice chronically implanted with a cranial window transparent to ultrasound. They provided quantitative information on their protocol, such as the required number of contrast-enhancing microbubbles (MBs) to get a clear image of the vasculature of a brain coronal section. Also, they quantified the co-registration quality over time-distant sessions and the vasodilator effect of isoflurane.

      Strengths:

      The study showed a remarkable performance in recording precisely the same brain coronal section over repeated imaging sessions. In addition, it sheds light on the vasodilator effect of isoflurane (an anesthetic whose effects are not fully understood) on the different brain vasculature compartments, although, as the authors stated, some insights in this aspect have already been published with other imaging techniques. The experimental setting and protocol are very well described.

      Weaknesses:

      While the title is fair with respect to the data shown, in the summary and the rest of the paper, the comparison between anesthetized and awake conditions is systematically stated, while more caution should be used.

      First, isoflurane is one of the (many) anesthetics commonly used in pre-clinical research, and its effect on the brain vasculature cannot be generalized to all the anesthetics. Indeed, other anesthesia approaches do not produce evident vasodilation; see ketamine + medetomidine mixtures. Second, the imaged awake state is head-fixed and body-constrained in mice. A condition that can generate substantial stress in the animals. In this study, there is no evaluation of the stress level of the mice. In addition, the awake imaging sessions were performed a few minutes after the mouse woke up from isoflurane induction, which is necessary to inject the MB bolus. It is known that the vasodilator effects of isoflurane last a long time after its withdrawal. This aspect would have influenced the results, eventually underestimating the difference with respect to the awake state.

      These limitations should be clearly described in the Discussion.

      Looking at Figure 2e, it takes more than 5' to reach the 5 Millions MB count useful for good imaging. However, the MB count per pixel drops to a few % at that time. This information tells me that (i) repeated measurements are feasible but with limited brain coverage since a single 'wake up' is needed to acquire a single brain section and (ii) this approach cannot fit the requirements of functional ULM that requires to merge the responses to multiple stimuli to get a complete functional image. Of course, a chronic i.v. catheter would fix the issue, but this configuration is not trivial to test in the experimental setup proposed by the authors, hindering the extension of the approach to fULM.

      Statistics are often poor or not properly described. The legend and the text referring to Figure 2 do not report any indication of the number of animals analyzed. I assume it is only one, which makes the findings strongly dependent on the imaging quality of THAT mouse in THAT experiment. Three mice have been displayed in Figure 3, as reported in the text, but it is not clear whether it is a mouse for each shown brain section. Figure 5 reports quantitative data on blood vessels in awake VS isoflurane states but: no indication about the number of tested mice is provided, nor the number of measured blood vessels per type and if statistics have been done on mice or with a multivariate method. Also, a T-test is inappropriate when the goal is to compare different brain regions and blood vessel types. Similar issues partially apply to Figure 6, too.

    1. Reviewer #2 (Public Review):

      The authors combine genetic tools, dye fills and connectome analysis techniques to generate a "first-of-its-kind", near complete, synaptic resolution map of the head bristle neurons of Drosophila. While some of the BMN anatomy was already known based on previous work by the authors and other researchers, this is the first time a near complete map has been created for the head BMNs at electron microscopy resolution.

      Strengths:

      (1) The authors cleverly use techniques that allow moving back and forth between periphery (head bristle location) and brain, as well as moving between light microscopy and electron microscopy data. This allows them to first characterize the pathways taken by different head BMNs to project to the brain and also characterize anatomical differences among individual neurons at the level of morphology and connectivity.<br /> (2) The work is very comprehensive and results in a near complete map of all head BMNs.<br /> (3) Authors also complement this anatomical characterization with a first-level functional analysis using optogenetic activation of BMNs that results in expected directed grooming behavior.

      Weaknesses:<br /> (1) While not strictly needed here, it could help provide context if authors revealed some of the important downstream pathways that could explain optogenetics behavioral phenotypes: This point was addressed by authors in the revisions and I agree a detailed description of downstream circuits is not needed at this point.<br /> (2) In contrast to the rigorous quantitative analysis of the anatomical data, the behavioral data is analyzed using much more subjective methods. While I do not think it is necessary to perform a rigorous analysis of behaviors in this anatomy focused manuscript, the conclusions based on behavioral analysis should be treated as speculative in the current form e.g. calling "nodding + backward motions" as an avoidance response is not justified as it currently stands. Strong optogenetic activation could lead to sudden postural changes that due to purely biomechanical constraints could lead to a couple of backward steps as seen in the example videos. Moreover since the quantification is manual, it is not clear what the analyst interprets as backward walking or nodding. Interpretation is also concerning because controls show backward walking (although in fewer instances based on subjective quantification): This point was addressed by the authors during revisions and I'm mostly satisfied with their response, where authors agree that the behavioral results are currently used to speculate about the role of BMNs in aversive behaviors. Still, the fact that controls show some "backward motions" is a bit concerning when talking about "significant differences" between control and test groups based on manual annotations and I would recommend future studies focusing on these behaviors to use more unbiased quantitative analysis wherever possible.

      Summary:

      The authors end up generating a near-complete map of head BMNs that will serve as a long-standing resource to the Drosophila research community. This will directly shape future experiments aimed at modeling or functionally analyzing the head grooming circuit to understand how somatotopy guides behaviors. I appreciate the authors taking the time to revise the manuscript and address reviewer concerns.

    1. Reviewer #1 (Public Review):

      Summary:

      The motivating questions are an accurate reflection of the current state of knowledge surrounding striatal pathway function. The comparisons of pathway function across striatal subregion, activation & inhibition, and task context are laudable and extremely important for advancing the subfield. Had these manipulations, to the largest extent possible been performed in single animals (e.g. activate dSPNs of DMS or DLS in the same mouse across the 3 tasks), this would have significantly strengthened the impact and conclusions that could be drawn by making this set of studies even more so internally consistent and directly comparable. While this is no longer possible, a conceptually related and fantastic contribution to the subfield (and likely beyond in terms of Opto manipulations of brain areas) would be to directly demonstrate that within their studies their DMS pathway manipulations do not impact nearby DLS activity (and vice versa). This is a significant and non-essential request. More feasibly and reasonably, it would be fantastic and strengthen the conclusions here to more fully detail their opsin expression patterns in DMS vs DLS groups and perhaps attempt to relate individual opsin profiles and fiberoptic targeting with behavioral outcomes across tests.

      Strengths:

      A comprehensive and paired comparison of inhibition and activation of striatal pathways across subregions and tasks is a very important and meaningful step towards reconciling contradictory results on striatal pathway function that are observed across labs (who typically focus on one subregion, one task setting, and often do not directly report comparisons of activation and inhibition).

      Weaknesses:

      Figure 1A - the example DMS vs DLS opsin expression and fiber targeting are not terribly convincing that the manipulations will be specific to each subregion (the example in Figure 2A is a little better but I have a similar concern still). The specificity of these manipulations is key to interpretation and conclusions and I strongly feel they should be strengthened here. The best evidence would be direct neural recordings (light in DMS, no effect in DLS, and vice versa), but this is a tall ask and not expected. The next best option, which is readily feasible, is to show not only fiberoptic targeting summaries (as in Figure 1A, Figure 2A) but also a summary of opsin spread for all animals (especially given the two examples appear to have significant spread across DMS and DLS). It would be of great benefit to the field to have these in the Allen Common Coordinate Framework. It would also be fine and useful to utilize the authors' current classical histological atlas alignment methods (e.g. Paxinos pdf). These histological summary figures would also benefit from being larger and more visible (perhaps as separate supplemental figures associated with the main figures).

      Related to the above, it is a concern that the classic view is supported or not because of individual variations in virus/fiber targeting to striatal subregions which likely have greater granularity than the traditional dorsal medial vs lateral (e.g. Hunnicutt et al 2016, Foster et al 2021, Hintiryan et al 2016). Although there may not be enough animals or variation in targeting in the present study to find meaningful relationships, it would strengthen the paper and be a great benefit to the field to know whether for key findings if the strength of behavioral effects correlated with anterior/posterior or medial/lateral or dorsal/ventral fiberoptic coordinates (or the volume of opsin expression profiles).

      Conceptually, a clear new idea or integrative interpretation of prior work (nor even the large body of results within this work) comes to the fore, save for the already appreciated fact that the classic view of opposing pathways is sometimes supported and sometimes not. Two tangible suggestions that I believe would facilitate the influence of this study - (1) can the authors more thoughtfully bridge the logical steps in their results sections and the prior context around them (some topic sentences jump right into results, e.g. line 195: "The inhibition experiment showed), and (2) in discussion, rather than emphasizing when/where the classic view is supported and not, more content on precisely why would be helpful. Some questions more specifically, if DMS/DLS pathway activation/inhibition is *mostly* oppositely appetitive/aversive, what does that mean in the context of spontaneous or reward-guided locomotion? Self-initiated pathway activation/inhibition is in part learned (with very intriguing differences across pathways in the expression across learning) - how should we think about striatal pathway function with regards to learning, spontaneous/innate behaviors, vs over-trained behaviors? When the classic view fails in the dorsal striatum - why? And is a complimentary "model" an actual alternative concept, a distinct mode of circuit function, or just a negative result on the classic view?

    1. Reviewer #1 (Public Review):

      Gazula and co-workers presented in this paper a software tool for 3D structural analysis of human brains, using slabs of fixed or fresh brains. This tool will be included in Freesurfer, a well-known neuroimaging processing software. It is possible to reconstruct a 3D surface from photographs of coronal sliced brains, optionally using a surface scan as model. A high-resolution segmentation of 11 brain regions is produced, independent of the thickness of the slices, interpolating information when needed. Using this method, the researcher can use the sliced brain to segment all regions, without the need of ex vivo MRI scanning.

      The software suite is freely available and includes 3 modules. The first accomplishes preprocessing steps, for correction of pixel sizes and perspective. The second module is a registration algorithm that registers a 3D surface scan obtained prior to sectioning (reference) to the multiple 2D slices. It is not mandatory to scan the surface, -a probabilistic atlas can also be used as reference- however the accuracy is lower. The third module uses machine learning to perform the segmentation of 11 brain structures in the 3D reconstructed volume. This module is robust, dealing with different illumination conditions, cameras, lens and camera settings. This algorithm ("Photo-SynthSeg") produces isotropic smooth reconstructions, even in high anisotropic datasets (when the in-plane resolution of the photograph is much higher than the thickness), interpolating the information between slices.

      To verify the accuracy and reliability of the toolbox, the authors reconstructed 3 datasets, using real and synthetic data. Real data of 21 postmortem confirmed Alzheimer's disease cases from the Massachusetts Alzheimer's Disease Research Center (MADRC)and 24 cases from the AD Research at the University of Washington(who were MRI scanned prior to processing)were employed for testing. These cases represent a challenging real-world scenario. Additionally, 500 subjects of the Human Connectome project were used for testing error as a continuous function of slice thickness. The segmentations were performed with the proposed deep-learning new algorithm ("Photo-SynthSeg") and compared against MRI segmentations performed to "SAMSEG" (an MRI segmentation algorithm, computing Dice scores for the segmentations. The methods are sound and statistically showed correlations above 0.8, which is good enough to allow volumetric analysis. The main strengths of the methods are the datasets used (real-world challenging and synthetic) and the statistical treatment, which showed that the pipeline is robust and can facilitate volumetric analysis derived from brain sections and conclude which factors can influence in the accuracy of the method (such as using or not 3D scan and using constant thickness).

      Although very robust and capable of handling several situations, the researcher has to keep in mind that processing has to follow some basic rules in order for this pipeline to work properly. For instance, fiducials and scales need to be included in the photograph, and the slabs should be photographed against a contrasting background. Also, only coronal slices can be used, which can be limiting for certain situations.

      The authors achieved their aims, and the statistical analysis confirms that the machine learning algorithm performs segmentations comparable to the state-of-the-art of automated MRI segmentations.<br /> Those methods will be particularly interesting to researchers who deal with post-mortem tissue analysis and do not have access to ex vivo MRI. Quantitative measurements of specific brain areas can be performed in different pathologies and even in the normal aging process. The method is highly reproducible, and cost-effective since allows the pipeline to be applied by any researcher with small pre-processing steps.

    1. Reviewer #1 (Public Review):

      The authors have developed and optimized a footprinting assay to monitor the recruitment of mRNAs to a reconstituted translation initiation system. This assay is named Recruitment-Sequencing (Rec-Seq) and enables the analysis of many purified mRNAs in the reconstituted system.

      This system possesses the ability to determine how competition occurs between mRNAs for the initiation machinery. This is the first approach using a reconstituted system that enables this important feature, and this is an important advance for the field.

      Using purified mRNAs in a fully reconstituted system together with the ability to monitor start site selection is an important advance. The method enables one to observe for the first time how competition between mRNAs is altered in response to the absence or presence of different initiation components or accessory proteins.

      Start site fidelity in purified reconstituted systems can be altered in different buffer conditions and by the concentration of various initiation factors involved in start site fidelity. Future experiments will reveal how these variables can regulate start site selection in this powerful system.

      Comments on revised version:

      The authors have addressed all of my original comments. This is an impressive manuscript.

    1. Reviewer #1 (Public Review):

      Summary:

      The CPC plays multiple essential roles in mitosis such as kinetochore-microtubule attachment regulation, kinetochore assembly, spindle assembly checkpoint activation, anaphase spindle stabilization, cytokinesis, and nuclear envelope formation, as it dynamically changes its mitotic localization: it is enriched at inner centromeres from prophase to metaphase but it is relocalized at the spindle midzone in anaphase. The business end of the CPC is Aurora B and its allosteric activation module IN-box, which is located at the C-terminus of INCENP. In most well-studied eukaryotic species, Aurora B activity is locally controlled by the localization module of the CPC, Survivin, Borealin and the N-terminal portion of INCENP. Survivin and Borealin, which bind the N-terminus of INCENP, recognize histone residues that are specifically phosphorylated in mitosis, while anaphase spindle midzone localization is supported by the direct microtubule-binding capacity of the SAH (single alpha helix) domain of INCENP and other microtubule-binding proteins that specifically interact with INCENP during anaphase, which are under the regulation of CDK activity. One of these examples includes the kinesin-like protein MKLP2 in vertebrates. Trypanosoma is an evolutionarily interesting species to study mitosis since its kinetochore and centromere proteins do not show any similarity to other major branches of eukaryotes, while orthologs of Aurora B and INCENP have been identified. Combining molecular genetics, imaging, biochemistry, cross-linking IP-MS (IP-CLMS), and structural modeling, this manuscript reveals that two orphan kinesin-like proteins KIN-A and KIN-B act as localization modules of the CPC in Trypanosoma brucei. The IP-CLMS, AlphaFold2 structural predictions, and domain deletion analysis support the idea that (1) KIN-A and KIN-B form a heterodimer via their coiled-coil domains, (2) Two alpha helices of INCENP interact with the coiled-coil of the KIN-A-KIN-B heterodimer, (3) conserved KIN-A C-terminal CD1 and CD2 interact with the heterodimeric KKT9-KKT11 complex, which is a submodule of the KKT7-KKT8 kinetochore complex composed of KKT7, KKT8, KKT9, KKT11, and KKT12 unique to Trypanosoma, (4) KIN-A and KIN-B coiled-coil domains and the KKT7-KKT8 complex are required for CPC localization at the centromere, (5) CD1 and CD2 domains of KIN-A support its centromere localization. The authors further introduced a KIN-A rigor mutant and knocked-down wild-type KIN-A to show that the ATPase activity of KIN-A seems dispensable for centromere targeting but critical for spindle midzone enrichment of the CPC. The imaging data of the KIN-A rigor mutant suggest that dynamic KIN-A-microtubule interaction is required for metaphase alignment of the kinetochores and proliferation. Overall, the study reveals novel pathways of CPC localization regulation via KIN-A and KIN-B by multiple complementary approaches.

      Strengths:

      The major conclusion is collectively supported by multiple approaches, combining CRISPR-mediated gene deletion and complementation/site specific genome engineering, epistasis analysis of cellular localization, AlphaFold2 structure prediction of protein complexes, IP-CLMS and biochemical reconstitution (the complex of KKT8, KKT9, KKT11 and KKT12)

      Weaknesses:

      Minor weakness. The authors imply that KIN-A, but not KIN-B, interacts with microtubules based on microtubule pelleting assay (Fig. S6), but the substantial insoluble fractions of 6HIS-KINA and 6HIS-KIN-B make it difficult to conclusively interpret the data. It is possible that these two proteins are not stable unless they form a heterodimer.

    1. Reviewer #1 (Public Review):

      Summary:

      This is a follow-up study to the authors' previous eLife report about the roles of an alpha-arrestin called protein thioredoxin interacting protein (Txnip) in cone photoreceptors and in the retinal pigment epithelium. The findings are important because they provide new information about the mechanism of glucose and lactate transport to cone photoreceptors and because they may become the basis for therapies for retinal degenerative diseases.

      Strengths:

      Overall, the study is carefully done and, although the analysis is fairly comprehensive with many different versions of the protein analyzed, it is clearly enough described to follow. Figure 4 greatly facilitated my ability to follow, understand and interpret the study. The authors have appropriately addressed a few concerns about statistical significance and the relationship between their findings and previous studies of the possible roles of Txnip on GLUT1 expression and localization on the surfaces of RPE cells.

    1. Reviewer #1 (Public Review):

      Summary:

      In this manuscript, Sang et al. proposed a pair of IR60b-expressing pharyngeal neurons in Drosophila use IR25a, IR76b, and IR60b channels to detect high Na+ and limit its consumption. Some of the key findings that support this thesis are: 1) animals that lacked any one of these channels - or with their IR60b-expressing neurons selectively silenced - showed much reduced rejection of high Na+, but restored rejection when these channels were reintroduced back in the IR60b neurons; 2) animals with TRPV artificially expressed in their IR60b neurons rejected capsaicin-laced food whereas WT did not; 3) IR60b-expressing neurons exhibited increased Ca2+ influx in response to high Na+ and such response went away when animals lacked any of the three channels.

      The experiments were thorough and well designed and further improved after revision. The results are compelling and support the main claim. The development and the use of the DrosoX two-choice assay put forward for a more quantitative and automatic/unbiased assessment for ingestion volume and preference.

    1. Joint Public Review:

      The present study focuses on the structure and function of human PURA, a regulator of gene transcription and mRNA transport and translation whose mutation causes the neurodevelopmental PURA syndrome, characterized by developmental delay, intellectual disability, hypotonia, epileptic seizures, a.o. deficits. The authors combined structural biology, molecular dynamics simulation, and various cell biological assays to study the effects of disease-causing PURA mutations on protein structure and function. The corresponding data reveal a highly dynamic PURA structure and show that disease-related mutations in PURA cause complex defects in folding, DNA-unwinding activity, RNA binding, dimerization, and partitioning into processing bodies. These findings provide first insights into how very diverse PURA mutations can cause penetrant molecular, cellular, and clinical defects. This will be of substantial interest to cell biologists, neurogeneticists, and neurologists alike.

      A particular strength of the present study is the structural characterization of human PURA, which is a challenging target for structural biology approaches. The molecular dynamics simulations are state-of-the-art, allowing a statistically meaningful assessment of the differences between wild-type and mutant proteins. The functional consequences of PURA mutations at the cellular level are fascinating, particularly the differential compartmentalization of wild-type and mutant PURA variants into certain subcellular condensates.

    1. Reviewer #4 (Public Review):

      Summary:

      Walker et al. investigated the function of TMEM127 on RET regulation and function that could contribute to the development of pheochromocytoma (PCC). The authors showed that deletion of TMEM127 causes RET protein accumulation on the cell surface and, thereby, increased its constitutive ligand-independent activity and downstream signaling. They also unveiled the mechanism of how TMEM127 regulates cell membrane dynamics, particularly focusing on clathrin distribution and its effects on cargo internalization.

      Strengths:

      They showed that the deletion of TMEM127 affected multiple classes of transmembrane proteins, including RTKs (RET, EGFR), cell adhesion molecules (N-Cadherin, Integrin Beta-3), and carrier proteins (Transferrin Receptor-1), suggesting a global effect on cell surface proteins. This, at least in part, may explain how TMEM127 mutations act as drivers in PCC as well as in other cancers, such as renal cell carcinoma, where RET is not highly expressed. Overall, these findings provide new insights into the understanding of pheochromocytoma pathogenesis and potentially other cancers.

      Weaknesses:

      The major weakness of this study is the lack of human PCC cell lines for evaluating the function of TMEM127. Currently, the cell line models for pheochromocytoma are unavailable, and manipulation of patient-derived organoids is challenging. To complement this weakness, they provided immunohistochemical data showing that RET protein is highly expressed in TMEM127-mutant PCC.

      Furthermore, some of the authors in this manuscript recently published a paper titled 'TMEM127 suppresses tumor development by promoting RET ubiquitination, positioning, and degradation' (Guo et al. Cell Reports 42, 113070, 2023, which is also cited in the current manuscript). In this manuscript, they showed that TMEM127 binds to RET and recruits the NEDD4 E3 ubiquitin ligase for RET ubiquitination and degradation via TMEM127. In general, the ubiquitination of proteins is highly specific to each molecule. In the current version of the manuscript, there is no description of the relevance between these two potentially different mechanisms (clathrin-mediated or ubiquitin-mediated) of accumulating RET and/or other proteins mentioned in two separate papers. I believe the authors should at least discuss this.

    1. Reviewer #2 (Public Review):

      The manuscript from deHaro-Arbona et al, entitled "Dynamic modes of Notch transcription hubs conferring memory and stochastic activation revealed by live imaging the co-activator Mastermind", uses single molecule microscopy imaging in live tissues to understand the dynamics and molecular determinants of transcription factor recruitment to the E(spl)-C locus in Drosophila salivary gland cells under Notch-ON and -OFF conditions. Previous studies have identified the major players that are involved in transcription regulation in the Notch pathway, as well as the importance of general transcriptional coregulators, such as CBP/P300 and the Mediator CDK module, but the detailed steps and dynamics involved in these processes are poorly defined. The authors present a wealth of single molecule data that provides significant insights into Notch pathway activation, including:

      (1) Activation complexes, containing CSL and Mam, have slower dynamics than the repressor complexes, containing CSL and Hairless.<br /> (2) Contribution of CSL, NICD, and Mam IDRs to recruitment.<br /> (3) CSL-Mam slow-diffusing complexes are recruited and form a hub of high protein concentrations around the target locus in Notch-ON conditions.<br /> (4) Mam recruitment is not dependent on transcription initiation or RNA production.<br /> (5) CBP/P300 or its associated HAT activity is not required for Mam recruitment<br /> (6) Mediator CDK module and CDK8 activity is required for Mam recruitment, and vice-versa, but not CSL recruitment.<br /> (7) Mam is not required for chromatin accessibility but is dependent on CSL and NICD.<br /> (8) CSL recruitment and increased chromatin accessibility persist after NICD removal and loss of Mam, which confers a memory state that enables rapid re-activation in response to subsequent Notch activation<br /> (9) Differences in the proportions of nuclei with both Pol II and with Mam enrichment, which results in transcription being probabilistic/stochastic. These data demonstrate that presence of Mam-complexes is not sufficient to drive all the steps required for transcription in every Notch-ON nucleus.<br /> (10) The switch from more stochastic to robust transcription initiation was elicited when ecdysone was added.

      Overall, the manuscript is well written, concise, and clear, and makes significant contributions to the Notch field, which are also important for a general understanding of transcription factor regulation and behavior in the nucleus. The authors have satisfactorily addressed all my criticisms of their initial submission and therefore congratulate the authors on an excellent paper.

    1. Reviewer #1 (Public Review):

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

      This paper seeks to address the challenging question of how quantitative trait variation and expression variation are related. scRNAseq represents an appealing approach to eQTL mapping as it is possible to simultaneously genotype individual cells and measure expression in the same cell. As eQTL mapping requires large sample sizes to identify statistical relationships, this approach is likely to dramatically increase the statistical power of such studies. However, there are several technical challenges associated with scRNAseq and the authors' study is focused on addressing those challenges. Although the authors present results suggesting the feasibility of the approach there are limitations in the conclusions that can be drawn in the current study owing to the lack of clarity in the presentation of the results. Ultimately, this study presents a proof of concept with limited novel biological insights that would nonetheless make a useful contribution to the literature if the following major points were addressed:

      (1) There is insufficient information provided about the nature of data. At a minimum, the following information should be provided to enable assessment of the study: What is the total library size, how many genes are identified per cell, how many UMIs are found per cell, what is the doublet rate, and how are doublets identified (e.g. on the basis of heterozygous calls at polymorphic loci?), how many times is each genotype observed, and how many polymorphic sites are identified per cell that are the basis of genotype inferences?

      (2) The prior study analyzed 18 different conditions, whereas this study only assays expression in a single condition. However, the power of the authors' approach is that its efficiency enables testing eQTLs in multiple conditions. The study would be greatly strengthened through analysis of at least one more condition, and ideally several more conditions. The previous fitness study would be a useful guide for choosing additional conditions as identifying those conditions that result in the greatest contrasts in fitness QTL would be best suited to testing the generalizations that can be drawn from the study.

      (3) Alternatively, the authors could demonstrate the power of their approach by applying it to a cross between two other yeast strains. As the cross between BY and RM has been exhaustively studied, applying this approach to a different cross would increase the likelihood of making novel biological discoveries.

      (4) Figure 1 is misleading as A presents the original study from 2022 without important details such as how genotypes were identified. It is unclear what the barcode is in this study and how it is used in the analysis. Is the barcode for each lineage transcribed so that it is identified in the scRNAseq data? Or, does the barcode in B refer to the cell index barcode? A clearer presentation and explanation of terms are needed to understand the method.

      (5) The rationale for the analysis reported in Figure 2B is unclear. The fitness data are from the previous study and the goal is to estimate the heritability using the genotyping data from the scRNAseq data. What is the explanation for why the data don't agree for only one condition, i.e. 37C? And, what are we to understand from the overall result?

      (6) Figure 3 presents an analysis of variance partitioning as a Venn diagram. This summarized result is very hard to understand in the absence of any examples of what the underlying raw data look like. For example, what does trait variation look like if only genotype explains the variance or if only gene expression explains the variance? The presented highly summarized data is not intuitive and its presentation is poor - the result that is currently provided would be easier to read in a table format, but the reader needs more information to be able to interpret and understand the result.

      (7) I am concerned about the conclusions that can be drawn about expression heritability. The authors claim that expression heritability is correlated with expression levels. It seems likely that this reflects differing statistical power. How can this possibility be excluded?

      (8) Conversely, the authors claim that the genes with the lowest heritability are genes involved in the cell cycle. However, uniquely in scRNAseq, cell cycle regulated genes appear to have the highest variance in the data as they are only expressed in a subset of cells. Without incorporating this fact one would erroneously conclude that the variation is not heritable. To test the heritability of cell cycle regulation genes the authors should partition the cells into each cell cycle stage based on expression.

      (9) I do not understand Figure S5 and how eQTL sites are assigned to these specific classes given that the authors say that causative variation cannot be resolved because of linkage disequilibrium.

      (10) The paragraph starting at line 305 is very confusing. In particular, the authors state that they identify a hotspot of regulation at the mating type locus. It is not obvious why this would be the case. Moreover, they claim that they find evidence for both MATa and MATalpha gene expression. Information is not provided about how segregants were isolated, but assuming that the authors did not dissect 25,000 tetrads to obtain 100,000 segregants I would infer that random spore using SGA was used. In that case, all cells should be MATa. The authors should clarify and explain this observation.

      (11) Ultimately, it is not clear what new biological findings the authors have made. There are no novel findings with respect to causative variation underlying eQTLs and I would encourage the authors to make clearer statements in their abstract, introduction, and conclusion about the key discoveries. E.g. What are the "new associations between phenotypic and transcriptomic variations" mentioned in the abstract?

      The following minor points should be addressed:

      (1) The segregants should be referred to as F2 segregants as they are derived from an F1 cross.

      (2) The connections to eQTLs in other organisms should be addressed in the introduction and conclusion. For example, in humans, there has been little evidence for trans eQTLs in contrast to what has been found in yeast.

      (3) The authors state that an advantage of scRNAseq over bulk is that it captures rare cell populations (line 79), but this advantage is not exploited in this study.

      (4) The authors use ~5% of the lineages from the original study. There is no rationale for why this is an appropriate sample size. Is there an argument for using more cells in eQTL mapping or conversely could the authors ask if fewer cells would provide similar conclusions by downsampling?

      (5) I do not agree that the use of UMIs overcomes the challenges of low sequencing depth. UMIs mitigate the possible technical artifacts due to massive PCR amplification.

      (6) There is an inadequate reference to prior work on scRNAseq in yeast that established the methods used by the authors and eQTL mapping in human cells using scRNAseq.

      (7) The use of empty quotes in Figure 4A is confusing and an alternative presentation method should be used.

      (8) The authors speculate about the use of predicted fitness instead of observed fitness, but this is something they could explicitly address in their current study.

    1. Reviewer #1 (Public Review):

      Summary:

      This is an experimentally soundly designed work and a very well-written manuscript. There is a very clear logic that drives the reader from one experiment to the next, the experimental design is clearly explained throughout and the relevance of the acquired data is well analyzed and supports the claims made by the authors. The authors made an evident effort to combine imaging, genetic, and molecular data to describe previously unknown early embryonic movement patterns and to identify regulatory mechanisms that control several aspects of it.

      Strengths:

      The authors develop a new method to analyze, quantitatively, the onset of movement during the latter embryonic stages of Drosophila development. This setup allows for a high throughput analysis of general movement dynamics based on the capture of variations of light intensity reflected by the embryo. This setup is capable of imaging several embryos simultaneously and provides a detailed measure of movement over time, which proves to be very useful for further discoveries in the manuscript. This setup already provides a thorough and quantifiable description of a process that is little known and identifies two different phases during late embryonic movements: a myogenic phase and a neurogenic phase, which they elegantly prove is dependent on neuronal activity by knocking down action potentials across the nervous system.

      However, in this system, movement is detected as a whole, and no further description of the type of movement is provided beyond frequency and amplitude; it would be interesting to know from the authors if a more precise description of the movements that take place at this stage can be achieved with this method (e.g. motion patterns across the A-P body axis).

      Importantly, this highly quantitative experimental setup is an excellent system for performing screenings of motion regulators during late embryonic development, and its use could be extended to search for different modulators of the process, beyond miRNAs (genetic mutants, drugs, etc.).

      Using their newly established motion detection pipeline, the authors identify miR-2b-1 as required for proper larval and embryonic motion, and identify an overall reduction in the quantity of both myogenic and neurogenic movements, as well as an increased frequency in neurogenic movement "pulses".

      Focusing on the neurogenic movement phenotype the authors use in situ probes and perform RT-PCR on FACS-sorted CNS cells to unambiguously detect miR-2b-1 expression in the embryonic nervous system. The neurogenic motion defects observed in miR-2b-1 mutant embryos and early larvae can be completely rescued by the expression of ectopic miR-2b-1 specifically in the nervous system, providing solid evidence of the requirement and sufficiency of miR-2b-1 expressed in the nervous system to regulate these phases of movement.

      To explore the mechanism through which miR-2b-1 impacts embryonic movement, the authors use a state-of-the-art bioinformatic approach to identify potential targets of miR-2b-1, and find that the expression levels of an uncharacterized gene, CG3638, are indeed regulated by miR-2b-1. Furthermore, they prove that by knocking down the expression of CG3638 in a miR-2b-1 mutant background, the neurogenic embryonic movement defects are rescued, pointing that the repression of CG3638 by miR-2b-1 is necessary for correct motion patterns in wild-type embryos. Therefore, this paper provides the first functional characterization of CG3638, and names this gene Janus.

      Finally, the authors aim to discriminate which elements of the embryonic motor system miR-2b-1/Janus are required. Using directed overexpression of miR-2b-1 and Janus knockdown in the motor neurons and the chordotonal (sensory) organs, they prove that the miR-2b-1/Janus regulatory axis is specifically required in the sensory organs to promote normal embryonic and larval movement.

      Weaknesses:

      The authors do not describe properly how the miRNA screening was performed and just claim that only miR-2b-1 mutants presented a defective motion phenotype in early L1. How many miRNAs were tested, and how candidates were selected is never explicitly mentioned in the text or the Methods section.

      The initial screening to identify miRNAs involved in motion behaviors is performed in early larval movement. The logic presented by the authors is clear - it is assumed that early larval movement cannot proceed normally in the absence of previous embryonic motion - and ultimately helped them identify a miRNA required for modulation of embryonic movement. However, it is possible that certain miRNAs play a role in the modulation of embryonic movement while being dispensable for early L1 behaviors. Such regulators might have been missed with the current screening setup.

      Although similar changes to those described for the neurogenic phase of embryonic movement are described for the myogenic phase in miR-2b-1 mutants (reduction in motion amplitude), this phenotype goes unexplored. This is not a big issue, as the authors convincingly demonstrate later that miR-2b-1 is specifically required in the nervous system for proper embryonic and larval movement, and the effects of miR-2b-1 on myogenic movement might as well be the focus of future work. However, it will be interesting to discuss here the implications of a reduced myogenic movement phase, especially as miR-2b-1 is specifically involved in regulating the activity of the chordotonal system - which precisely detects early myogenic movements.

      FACS-sorting of neuronal cells followed by RT-PCR convincingly detects the presence of miR-2b-1 in the embryonic CNS. However, control of non-neuronal cells would be required to explore whether miR-2b-1 is not only present but enriched in the nervous system compared to other tissues. This is also the case in the miR-2b-1 and Janus expression analysis in the chordotonal organs: a control sample from the motor neurons would help discriminate whether miR-2b-1/Janus regulatory axis is specifically enriched in chordotonal organs or whether both genes are expressed throughout the CNS but operate under a different regulation or requirements for the movement phenotypes.

    1. Reviewer #1 (Public Review):

      Summary:

      This manuscript employs yolk sac visceral endoderm cells as a novel model for studying endosomal fusion, observing two distinct fusion behaviors: quick homotypic fusion between late endosomes, and slower heterotypic fusion between late endosomes and lysosomes. The mathematical modeling suggests that vesicle size critically influences the mode of fusion. Further investigations reveal that actin filaments are dynamically associated with late endosomal membranes, and are oriented in the x-y plane and along the apical-basal axis. Actin and Arf2/3 were shown to appear at the rear end of the endosomes along the moving direction suggesting polymerization of actin may provide force for the movement of endosomes. Additionally, the authors found that actin dynamics regulate homotypic and heterotypic fusion events in a different manner. The authors also provide evidence to suggest that Cofilin-dependent actin dynamics are involved in late endosome fusion.

      Strengths:

      The unique feature of this study is that the authors use yolk sac visceral endoderm cells to study endosomal fusion. Yolk sac visceral endoderm cells have huge endocytic vesicles, endosomes, and lysosomes, offering an excellent system to explore endosomal fusion dynamics and the assembly of cellular factors on membranes. The manuscript provides a valuable and convincing observation of the modes of endosomal fusion and the roles of actin dynamics in this process, and the conclusions of the study are justified by the data.

      Weaknesses:

      While the study offers compelling observations, it falls short of delivering clear mechanistic insights. Key questions remain unaddressed, such as the functional significance of actin filaments that extend apically in positioning late endosomes, the ways in which actin dynamics influence fusion events, and the functional implications of the slower bridge fusion process.

    1. Reviewer #1 (Public Review):

      This work provides new mechanistic insights into the competitive inhibition in the mammalian P2X7 receptors using structural and functional approaches. The authors solved the structure of panda (pd) P2X7 in the presence of the classical competitive antagonists PPNDS and PPADS. They find that both the drugs bind to the orthosteric site employed by the physiological agonist ATP. However, owing to the presence of a single phosphate group, they prevent movements in the flipper domain required for channel opening. The authors performed structure based mutational analysis together with electrophysiological characterization to understand the subtype specific binding of these drugs. It is known from previous studies that P2X1 and P2X3 are more sensitive to these drugs as compared to P2X7, hence, the residues adjacent to the ATP binding site in pdP2X7 were mutated to those present in P2X1. They observed that mutations of Q143, I214 and Q248 into lysine (hP2X1) increased the P2X7 sensitivity to PPNDS, whereas in P2X1, mutations of these lysines to alanine reduced sensitivity to PPNDS, suggesting that these key residues contribute to the subunit specific sensitivity to these drugs. Similar experiments were done in hP2X3 to demonstrate its higher sensitivity to PPNDS. This preprint provides a useful framework for developing subtype specific drugs for the family of P2X receptor channels, an area that is currently relatively unexplored.<br /> The conclusions of the paper are well supported.

      The revised manuscript is well written and presents its data with more clarity.

    1. Reviewer #1 (Public Review):

      Summary:

      The manuscript describes the crystal structures of Streptococcus pneumoniae NOXs. Crystals were obtained for the wild-type and mutant dehydrogenase domain, as well as for the full-length protein comprising the membrane domain. The manuscript further carefully studies the enzyme's kinetics and substrate-specificity properties. Streptococcus pneumoniae NOX is a non-regulated enzyme, and therefore, its structure should provide a view of the NOX active conformation. The structural and biochemical data are discussed on this ground.

      Strengths:

      This is very solid work. The protein chemistry and biochemical analysis are well executed and carefully described. Similarly, the crystallography must be appreciated given the difficulty of obtaining good enzyme preparations and the flexibility of the protein. Even if solved at medium resolution, the crystal structure of the full-length protein conveys relevant information. The manuscript nicely shows that the domain rotations are unlikely to be the main mechanistic element of NOX regulation. It rather appears that the NADPH-binding conformation is pivotal to enzyme activation. The paper extensively refers to the previous literature and analyses the structures comprehensively with a comparison to previously reported structures of eukaryotic and prokaryotic NOXs.

    1. Reviewer #1 (Public Review):

      Using a pharmacological and knock-down approach, the authors could demonstrate that ROCK activity is required for the normal development of the larval skeleton. The presence of ROCK in the pluteus stage depends on the activity of VEGF that is responsible for the formation of the tubular syncytial sheath of the calcifying primary mesenchyme cells in which the skeleton forms. The importance of ROCK in skeleton formation was confirmed in cell culture experiments, demonstrating that ROCK inhibition leads to decreased elongation and abnormal branching of spicules. µCT analyses underline this finding demonstrating that the inhibition of ROCK mainly affects elongation of spicules while growth in girth is little affected. F-actin labeling experiments could demonstrate that ROCK inhibition interferes with the organization of the actomyosin network in the early phase of skeleton formation, while f-actin organization in the tips of the elongating spicule is unaffected by the pharmacological inhibition of ROCK. Finally, ROCK inhibition strongly affects the expression of major regulatory and calcification-related genes in the calcifying cells. Based on these findings the authors propose a model for the regulatory interaction between the skeletogenic GRN, ROCK and the f-actin system relevant for skeletogenesis.

      Comments on revised version:

      In their manuscript Hijaze et al. adequately addressed the majority of my previous concerns in a satisfactory manner. In particular, they validated their morpholino knock-down experiments by explaining how they determined the optimal concentrations and provided an immunohistological evidence for the reduction in ROCK protein abundance. The authors also added new antibody stainings providing evidence that ROCK and F-actin do not interact directly but likely through other kinases that modulate f-actin, and that the localization of f-actin at the spicule tips remains unaffected by the knock-down. In addition, the authors revised their discussion to not overstate their observations, and by focusing on the potential mechanisms by which ROCK may affect biomineralization (i.e. mechano sensing and exocytosis of vesicles). Here I would like to add, that f-actin mediated exocytosis does not necessarily target mineral baring vesicles but may also promote the exocytosis of matrix proteins that are essential for the normal formation of the spicules and that are an integral component of other biominerals, as well. I strongly encourage the authors to continue on this exciting research, including the development of methods to analyze the molecular mechanisms that control vesicular trafficking in mineralizing systems.

    1. Reviewer #1 (Public Review):

      Summary:

      The authors want to understand fundamental steps in ligand binding to muscle nicotinic receptors using computational methods. Overall, although the work provides new information and support for existing models of ligand activation of this receptor type, some limitations in the methods and approach mean that the findings are not as conclusive as hoped.

      Strengths:

      The strengths include the number of ligands tried, and the comparison to the existing mature analysis of receptor function from the senior author's lab.

      Weaknesses:

      The weakness are the brevity of the simulations, the concomitant lack of scope of the simulations, the lack of depth in the analysis and the incomplete relation to other relevant work. The free energy methods use seem to lack accuracy - they are only correct for 2 out of 4 ligands.

    1. Reviewer #1 (Public Review):

      Summary:

      The work by Joseph et al "Impact of the clinically approved BTK inhibitors on the conformation of full-length BTK and analysis of the development of BTK resistance mutations in chronic lymphocytic leukemia" seeks to comparatively analyze the effect of a range of covalent and noncovalent clinical BTK inhibitors upon BTK conformation. The novel aspect of this manuscript is that it seeks to evaluate the differential resistance mutations that arise distinctly from each of the inhibitors.

      Strengths:

      This is an exciting study that builds upon the fundamental notion of ensemble behavior in solutions for enzymes such as BTK. The HDX-MS and NMR experiments are adequately and comprehensively presented.

      Weaknesses:

      While I commend the novelty of the study, the absence of important controls greatly tempers my enthusiasm for this work. As stated in the abstract, there are no broad takeaways for how resistance mutation bias operated from this study, although the mechanism of action of 2 common resistance mutations is useful. How these 2 resistance mutations connect to ensemble behavior, is not obvious. This is partly because BTK does not populate just binary "open"/"closed" conformations, but there are likely multiple intermediate conformations. Each inhibitor appears to preferentially "select" conformations by the authors' own assessment (line 236) and this carries implications for the emergence of resistance mutations. The most important control that would help is to use ADP or nonhydrolyzable and ATP as a baseline to establish the "inactive" and "active" conformations. All of the HDX-MS and NMR studies use protein that has no nucleotide present. A major question that remains is whether each of the inhibitors preferentially favors/blocks ADP or ATP binding. This then means it is not equivalent to correlate functional kinase assay conditions with either HDX-MS or NMR experiments.

    1. Reviewer #1 (Public Review):

      The Calcium Homeostasis Modulators (CALHM) are a family of large pore channels, of which the physiological role of CALHM1 and 3 is well understood, in particular their key role in taste sensation via the release of the neurotransmitter ATP. The activation mechanism of CALHM1 involves membrane depolarization and a decrease in extracellular Ca concentration, allowing the passage of large cellular metabolites. However, the activation mechanism and physiological roles of other family members are much less well understood. Many structures of homomeric CALHM proteins have been determined, revealing distinct oligomeric assemblies despite a common transmembrane domain topology. CALHM1 and 3 have been shown functionally to form heteromeric assemblies with properties distinct from those of homomeric CALHM1. However, the structural basis of heteromeric CALHM1 and 3 remains unexplored.

      In this paper, Drozdzyk et al. present an important study on the structures of heteromeric channels composed of CALHM2 and CALHM4, extending the structural understanding of the CALHM family beyond homomeric channels. The study relies primarily on cryo-EM. Despite the inherent challenges of structural determination due to the similar structural features of CALHM2 and CALHM4, the authors innovatively use synthetic nanobodies to distinguish between the subunits. Their results show a broad distribution of different heteromeric assemblies, with CALHM4 conformation similar to its homomeric form and CALHM2 conformation influenced by its proximity to CALHM4, and provide detailed insights into the interaction between CALHM2 and CALHM4.

      The manuscript is well-structured and presents clear results that support the conclusions drawn. The discovery of heteromeric CALHM channels, although currently limited to an overexpressed system, represents a significant advance in the field of large-pore channels and will certainly encourage further investigation into the physiological relevance and roles of heteromeric CALHM channels. The manuscript would benefit from further insight into the functional properties of these heteromeric channels. However, this is not a weakness as the identification of precise activation stimuli for CALHM2 and 4 is beyond the scope of this work.

      A challenge noted is the wide distribution of heteromeric assemblies in the 3D classification, resulting in insufficient particles for high-resolution structure determination of each assembly. The authors choose to combine particles from assemblies with 2-4 copies of CALHM4, which reveals the interface between CALHM2 and 4 but may compromise the quality of structural details. I recommend an alternative data processing strategy. First, refine particles with 2-4 CALHM4 subunits with symmetry imposed. This is followed by symmetry expansion, signal subtraction of two adjacent subunits, and subsequent classification and refinement of the subtracted particles. This approach, while not guaranteed, can potentially provide a clearer definition of CALHM2 and CALHM4 interfaces and show whether CALHM2 subunits adopt different conformations based on their proximity to CALHM4 subunits.

    1. Reviewer #1 (Public Review):

      Summary:

      This paper shows that E. coli exhibits a chemotactic response to potassium by measuring both the motor response (using a bead assay) and the intracellular signaling response (CheY phosporylation level via FRET) to step changes in potassium concentration. They find increase in potassium concentration induces a considerable attractant response, with amplitude comparable to aspartate, and cells can quickly adapt (and generally over-adapt). The authors propose that the mechanism for potassium response is through modifying intracellular pH; they find both that potassium modifies pH and other pH modifiers induce similar attractant responses. It is also shown, using Tar- and Tsr-only mutants, that these two chemoreceptors respond to potassium differently. Tsr has a standard attractant response, while Tar has a biphasic response (repellent-like then attractant-like). Finally, the authors use computer simulations to study the swimming response of cells to a periodic potassium signal secreted from a biofilm and find a phase delay that depends on the period of oscillation.

      Strengths:

      The finding that E. coli can sense and adapt to potassium signals and the connection to intracellular pH is quite interesting and this work should stimulate future experimental and theoretical studies regarding the microscopic mechanisms governing this response. The evidence (from both the bead assay and FRET) that potassium induces an attractant response is convincing, as is the proposed mechanism involving modification of intracellular pH. The updated manuscript controls for the impact of pH on the fluorescent protein brightness that can bias the measured FRET signal. After correction the response amplitude and sharpness (hill coefficient) are comparable to conventional chemoattractants (e.g. aspartate), indicating the general mechanisms underlying the response may be similar. The authors suggest that the biphasic response of Tar mutants may be due to pH influencing the activity of other enzymes (CheA, CheR or CheB), which will be an interesting direction for future study.

      Weaknesses:

      The measured response may be biased by adaptation, especially for weak potassium signals. For other attractant stimuli, the response typically shows a low plateau before it recovers (adapts). In the case of potassium, the FRET signal does not have an obvious plateau following the stimuli of small potassium concentrations, perhaps due to the faster adaptation compared to other chemoattractants. It is possible cells have already partially adapted when the response reaches its minimum, so the measured response may be a slight underestimate of the true response. Mutants without adaptation enzymes appear to be sensitive to potassium only at much larger concentrations, where the pH significantly disrupts the FRET signal; more accurate measurements would require development of new mutants and/or measurement techniques.

    1. Reviewer #1 (Public Review):

      Summary:

      This paper provides a straightforward mechanism of how mycobacterial cAMP level is increased under stressful conditions and shows that the increase is important for the survival of the bacterium in animal hosts. The cAMP level is increased by decreasing the expression of an enzyme that degrades cAMP.

      Strengths:

      The paper shows that under different stresses the response regulator PhoP represses a phosphodiesterase (PDE) that degrades cAMP specifically. Identification of<br /> PhoP as a regulator of cAMP is significant progress in understanding Mtb pathogenesis, as increase in cAMP apparently increases bacterial survival upon infection. On the practical side, reduction of cAMP by increasing PDE can be a means to attenuate the growth of the bacilli. The results have wider implications since PhoP is implicated in controlling diverse mycobacterial stress responses and many bacterial pathogens modulate host cell cAMP level. The results here are straightforward, internally consistent, and of both theoretical and applied interests. The results also open considerable future work, especially how increases in cAMP level help to increase survival of the pathogen.

      Weaknesses:

      It is not clear whether PhoP-PDE Rv0805 is the only pathway to regulate cAMP level under stress.

    1. Reviewer #1 (Public Review):

      Summary:

      This work describes the mechanism of protein disaggregation by the ClpL AAA+ protein of Listeria monocytogenes. Using several model subtrate proteins the authors first show that ClpL possesses a robust disaggregase activity that does not further require the endogenous DnaK chaperone in vitro. In addition, they found that ClpL is more thermostable than the endogenous L. monocytogenes DnaK and has the capacity to unfold tightly folded protein domains. The mechanistic basis for the robust disaggregase activity of ClpL was also dissected in vitro and in some cases, supported by in vivo data performed in chaperone-deficient E. coli strains. The data presented show that the two AAA domains, the pore-2 site and the N-terminal domain (NTD) of ClpL are critical for its disaggregase activity. Remarkably, grafting the NTD of ClpL to ClpB converted ClpB into an autonomous disaggregase, highlighting the importance of such a domain in the DnaK-independent disaggregation of proteins. The role of the ClpL NTD domain was further dissected, identifying key residues and positions necessary for aggregates recognition and disaggregation. Finally, using sets of SEC and negative staining EM experiments combined with conditional covalent linkages and disaggregation assays the authors found that ClpL shows significant structural plasticity, forming dynamic hexameric and heptameric active single rings that can further form higher assembly states via their middle domains.

      Strengths:

      The manuscript is well written and the experimental work well executed. It contains a robust and complete set of in vitro data that push further our knowledge of such important disaggregases. It shows the importance of the atypical ClpL N-terminal domain in the disaggregation process as well as the structural malleability of such AAA+ proteins. More generally, this work expands our knowledge of heat resistance in bacterial pathogens.

      Weaknesses:

      There is no specific weakness in this work, although it would have helped to have a drawing model showing how ClpL performs protein disaggregation based on their new findings. The function of the higher assembly states of ClpL remains unresolved and will need further extensive research. Similarly, it will be interesting in the future to see whether the sole function of the plasmid encoded ClpL is to cope with general protein aggregates under heat stress.

    1. Joint Public Review:

      Summary:

      Previously, this group showed that Tgfbr1 regulates the reorganization of the epiblast and primitive streak into the chordo-neural hinge and tailbud during the trunk-to-tail transition. Gdf11 signaling plays a crucial role in orchestrating the transition from trunk to tail tissues in vertebrate embryos, including the reallocation of axial progenitors into the tailbud and Tgfbr1 plays a key role in mediating its signaling activity. Progenitors that contribute to the extension of the neural tube and paraxial mesoderm into the tail are located in this region. In this work, the authors show that Tgfbr1 also regulates the reorganization of the posterior primitive streak/base of allantois and the endoderm as well.

      By analyzing the morphological phenotypes and marker gene expression in Tgfbr1 mutant mouse embryos, they show that it regulates the merger of somatic and splanchnic layers of the lateral plate mesoderm, the posterior streak derivative. They also present evidence suggesting that Tgfbr1 acts upstream of Isl1 (key effector of Gdf11 signaling for controlling differentiation of lateral mesoderm progenitors) and regulates the remodelling of the major blood vessels, the lateral plate mesoderm and endoderm associated with the trunk-to-tail transition. Through a detailed phenotypic analysis, the authors observed that, similarly to Isl1 mutants, the lack of Tgfbr1 in mouse embryos hinders the activation of hindlimb and external genitalia maker genes and results in a failure of lateral plate mesoderm layers to converge during tail development. As a result, they interpret that ventral lateral mesoderm, which generates the peri cloacal mesenchyme and genital tuberculum, fails to specify.

      They also show defects in the morphogenesis of the dorsal aorta at the trunk/tail juncture, resulting in an aberrant embryonic/extraembryonic vascular connection. Endoderm reorganization defects following abnormal morphogenesis of the gut tube in the Tgfbr1 mutants cause failure of tailgut formation and cloacal enlargement. Thus, Tgfbr1 activity regulates the morphogenesis of the trunk/tail junction and the morphogenetic switch in all germ layers required for continuing post-anal tail development. Taken together with the previous studies, this work places Gdf11/8 - Tgfbr1 signaling at the pivot of trunk-to-tail transition and the authors speculate that critical signaling through Tgfbr1 occurs in the posterior-most part of the caudal epiblast, close to the allantois.

      Strengths:

      The data shown is solid with excellent embryology/developmental biology. This work demonstrates meticulous execution and is presented in a comprehensive and coherent manner. Although not completely novel, the results/conclusions add to the known function of Gdf11 signaling during the trunk-to-tail transition.

      Weaknesses:

      The authors rely on the expression of a small number of key regulatory genes to interpret the developmental defects. The alternative possibilities remain to be ruled out thoroughly. The manuscript is also quite descriptive and would benefit from more focused highlighting of the novelty regarding the absence of Tgfbr1 in the mouse embryo. They should also strengthen some of their conclusions with more details in the results.

    1. Reviewer #1 (Public Review):

      Summary:<br /> In the present study, Rincon-Torroella et al. developed ME3BP-7, a microencapsulated formulation of 3BP, as an agent to target MCT1 overexpressing PDACs. They provided evidence showing the specific killing of PDAC cells with MCT1 overexpressing in vitro, along with demonstrating the safety and anti-tumor efficacy of ME3BP-7 in PDAC orthotopic mouse models.

      Strengths:<br /> * Developed a novel agent.<br /> * Well-designed experiments and an organized presentation of data that support the conclusions drawn.

      Weaknesses:<br /> There are some minor issues that could enhance the clarity and completeness of the study:

      (1) Statistical results should be visually presented in Figure 4 and Figure S1.

      (2) Given the tumor heterogeneity and the identification of focal high expression of MCT1 in Figure 7 and Figure S5B, it is suggested that the authors include the results of immunohistochemical (IHC) analysis of MCT1 expression in both control and ME3BP-7 treated tumor tissues. This addition may offer insight into whether the remaining tumors are composed of PDAC cells with negative MCT1 expression, while the cells with relatively high levels of MCT1 expression were eliminated by ME3BP-7 treatment.

      (3.)The authors are encouraged to discuss the future directions for improving the efficacy of this study. For example, exploring the combination of ME3BP-7 with a glutaminase-1 inhibitor (PMID 37891897) could be a valuable avenue for further research.

    1. Reviewer #1 (Public Review):

      Summary:

      This work presents an in-depth characterization of the factors that influence the structural dynamics of the Clostridium botulinum guanidine-IV riboswitch (riboG). Using a single-molecule FRET, the authors demonstrate that riboG undergoes ligand and Mg2+ dependent conformational changes consistent with the dynamic formation of a kissing loop (KL) in the aptamer domain. Formation of the KL is attenuated by Mg2+ and Gua+ ligand at physiological concentrations as well as the length of the RNA. Interestingly, the KL is most stable in the context of just the aptamer domain compared to longer RNAs capable of forming the terminator stem. To attenuate transcription, binding of Gua+ and formation of the KL must occur rapidly after transcription of the aptamer domain but before transcription of the rest of the terminator stem.

      Strengths:

      (1) Single-molecule FRET microscopy is well suited to unveil the conformational dynamics of KL formation and the authors provide a wealth of data to examine the effect of the ligand and ions on riboswitch dynamics. The addition of complementary transcriptional readthrough assays provides further support for the author's proposed model of how the riboswitch dynamics contribute to function.

      (2) The single-molecule data strongly support that the effect of Gua+ ligand and Mg2+ influence the RNA structure differently for varying lengths of the RNA. The authors also demonstrate that this is specific for Mg2+ as Na+ and K+ ions have little effect.

      (3) The PLOR method utilized is clever and well adapted for both dual labeling of RNAs and examining RNA at various lengths to mimic co-transcriptional folding. Using PLOR, they demonstrate that a change in the structural dynamics and ligand binding can occur after the extension of the RNA transcript by a single nucleotide. Such a tight window of regulation has intriguing implications for kinetically controlled riboswitches.

      Weaknesses:

      (1) The authors use only one mutant to confirm that their FRET signal indicates the formation of the KL. Importantly, this mutation does not involve the nucleotides that are part of the KL interaction. It would be more convincing if the authors used mutations in both strands of the KL and performed compensatory mutations that restore base pairing. Experiments like this would solidify the structural interpretation of the work, particularly in the context of the full-length riboG RNA or in the co-transcriptional mimic experiments, which appear to have more conformational heterogeneity.

      (2) The existence of the pre-folded state (intermediate FRET ~0.5) is not well supported in their data and could be explained by an acquisition artifact. The dwell times are very short often only a single frame indicating that there could be a very fast transition (< 0.1s) from low to high FRET that averages to a FRET efficiency of 0.5. To firmly demonstrate that this intermediate FRET state is metastable and not an artifact, the authors need to perform measurements with a faster frame rate and demonstrate that the state is still present.

      (3) The PLOR method employs a non-biologically relevant polymerase (T7 RNAP) to mimic transcription elongation and folding near the elongation complex. T7 RNAP has a shorter exit channel than bacterial RNAPs and therefore, folding in the exit channel may be different between different RNAPs. Additionally, the nascent RNA may interact with bacterial RNAP differently. For these reasons, it is not clear how well the dynamics observed in the T7 ECs recapitulate riboswitch folding dynamics in bacterial ECs where they would occur in nature.

    1. Reviewer #1 (Public Review):

      Summary:

      In this manuscript, Zeng and Staley provide a valuable analysis of the molecular requirements for the export of a reporter mRNA that contains a lariat structure at its 5' end in the budding yeast S. cerevisiae. The authors provide evidence that this is regulated by the main mRNA export machinery (Yra1, Mex67, Nab2, Npl3, Tom1, and Mlp1). Of note, Mlp1 has been mainly implicated in the nuclear retention of unspliced pre-mRNA (i.e. quality control), and relatively little has been done to investigate its role in mRNA export in budding yeast.

      Strengths:

      There is relatively little information in the current literature about the nuclear export of splicing intermediates. This paper provides one of the first analyses of this process and dissects the molecular components that promote this form of RNA export. Overall, the strength of the data presented in the manuscript is solid. The paper is well written and the message is clear and of general interest to the mRNA community.

      Weaknesses:

      There are three problems with the paper, although these are not major and likely would not affect the final model as most aspects of the molecular details are confirmed by multiple complementary assays.

      (1) The brG reporter produces both unspliced pre-mRNA and a lariat-containing intermediate RNA. Based on the primer extension assay the authors claim that only 33% of the final product is in pre-mRNA form and that this "is insufficient to account for the magnitude of the cytoplasmic signal from the brG reporter (83%)". Nevertheless, it is possible that primer extension is incomplete or that the lariat-containing RNA is inaccessible for smFISH. The authors could easily perform a dual smFISH experiment (similar to Adivarahan et l., Molecular Cell 2018) where exon 1 is labelled with probes of one color, and the region that overlaps the lariat-containing intermediate is labelled with probes of a second color. If the authors are correct, then one-third of the smFISH foci should have both labels and the rest would have only the second label. This would also confirm that the latter (i.e. the lariat-containing RNAs) are exported to the cytoplasm. Using this approach, the authors could then show that MLP1-depletion (or depletion of any of the other factors) affect(s) one pool of RNAs (i.e. those that are lariat-containing) but not the other (i.e. pre-mRNA). Including these experiments would make the evidence for their model more convincing.

      (2) In some cases, the number of smFISH foci appears to change drastically depending on the genetic background. This could either be due to the stochastic nature of mRNA expression between cells or reflect real differences between the genetic backgrounds that could alter the interpretation of the other observations.

      (3) The authors state in the discussion that "the general mRNA export pathway transports discarded lariat intermediates into the cytoplasm". Although this appears to be the case for the reporters that are investigated in this paper, I don't think that the authors should make such a broad sweeping claim. It may be that some discarded lariat intermediates are exported to the cytoplasm while others are targeted for nuclear retention and/or decay.

    1. Reviewer #1 (Public Review):

      Summary:

      The major result in the manuscript is the observation of the higher order structures in a cryoET reconstruction that could be used for understanding the assembly of toroid structures. The cross-linking ability of ZapD dimers result in bending of FtsZ filaments to a constant curvature. Many such short filaments are stitched together to form a toroid like structure. The geometry of assembly of filaments - whether they form straight bundles or toroid like structures - depends on the relative concentrations of FtsZ and ZapD.

      Strengths:

      In addition to a clear picture of the FtsZ assembly into ring-like structures, the authors have carried out basic biochemistry and biophysical techniques to assay the GTPase activity, the kinetics of assembly, and the ZapD to FtsZ ratio.

      Weaknesses:

      The discussion does not provide an overall perspective that correlates the cryoET structural organisation of filaments with the biophysical data.

      The crosslinking nature of ZapD is already established in the field. The work carried out is important to understand the ring assembly of FtsZ. However, the availability of the cryoET observations can be further analysed in detail to derive many measurements that will help validate the model, and obtain new insights.

    1. Reviewer #1 (Public Review):

      Summary:

      Hippo pathway activity is required for pancreas morphogenesis, but its role in endocrine pancreas function remains elusive. The author aims to study the function of the TEAD1 gene in b-cells.

      Strengths:

      The authors generated TEAD1 conditional knockout animals by crossing the TEAD1f/f mice with three Cre strains (RIP-Cre, Ins1-Cre, and MIP-CreERT). In all of them, the KO animals showed progressive loss of insulin secretion with normal beta cell mass. Further characterization of the animals indicated glucose-induced insulin secretion defect and increased beta cell proliferation rate. RNA-Seq and ChIP-Seq experiments identified Pdx1, MafA, and Glut2, etc. as direct targets of TEAD1, which might be responsible for the insulin secretion defect in the animals. Of interest, the authors also uncovered the cell cycle-related gene p16 as a direct target of TEAD1. Reduction of p16 is likely to drive the beta cell proliferation in the TEAD1 knockout model. Thus, they proposed that TEAD1 is a regulator of the proliferative quiescence process in beta cells. Overall, the evidence provided by the authors is highly relevant and supports their conclusion.

      Weaknesses:

      (1) The authors don't explicitly mention that some results appeared in a previous publication (https://doi.org/10.1093/nar/gkac1063) from them.

      (2) The authors begin their story by introducing TEAD1 as part of the Hippo pathway. They showed Taz expression data in Figure 1. Did they do any experiments to detect Taz in their TEAD1 model? Did the authors detect any expression changes in CTGF following TEAD1 knockout? I could not see this changed. The phenotype characterization data presented here contrasts with what has been shown in TAZ b-cell knockout mice (https://doi.org/10.1101/2022.05.31.494216). Based on the data presented here, Hippo is not involved, which should at least be discussed in length.

      (3) Figure 1B - TAZ staining looks different in the three-month age group.

      (4) TEAD ChIP-seq data doesn't look very convincing to me. It's hard to tell whether those highlighted regions in Figures 3A and 5J were signals or background noise. Although the authors also performed ChIP-qPCR in MIN6, it's unclear whether these binding events occur in vivo. The analysis of ChIP-seq dataset is limited as well. How many peaks called? What proportion of differentially expressed genes are bound by TEAD1? Was TEAD1 also detectable at NGN3 and NEUROD1 gene regions? If acquiring enough cells is not possible, the authors could try CUT&RUN or CUT&Tag to improve the data quality.

      (5) The authors should perform RNA-seq or gene expression studies in MIP-CreERT to confirm, which could help narrow down the actual targets of TEAD1 as well.

      (6) Figure 6 - the experiment lacks a control: Ezh2 beta cell KO. In addition to p16, Ezh2, and PRC2 have other targets in beta-cells, the authors could not rule out the contribution of those to the phenotype, so the implication of this experiment is vague.

    1. Reviewer #1 (Public Review):

      Summary:

      Ewing sarcoma is an aggressive pediatric cancer driven by the EWS-FLI oncogene. Ewing sarcoma cells are addicted to this chimeric transcription factor, which represents a strong therapeutic vulnerability. Unfortunately, targeting EWS-FLI has proven to be very difficult, and a better understanding of how this chimeric transcription factor works is critical to achieving this goal. Towards this perspective, the group had previously identified a DBD-𝛼4 helix (DBD) in FLI that appears to be necessary to mediate EWS-FLI transcriptomic activity. Here, the authors used multi-omic approaches, including CUT&tag, RNAseq, and MicroC to investigate the impact of this DBD domain. Importantly, these experiments were performed in the A673 Ewing sarcoma model where endogenous EWS-FLI was silenced, and EWS-FLI-DBD proficient or deficient isoforms were re-expressed (isogenic context). They found that the DBD domain is key to mediating EWS-FLI cis activity (at msat) and to generating the formation of specific TADs. Furthermore, cells expressing DBD-deficient EWS-FLI display very poor colony-forming capacity, highlighting that targeting this domain may lead to therapeutic perspectives.

      Strengths:

      The group has strong expertise in Ewing sarcoma genetics and epigenetics and also in using and analyzing this model (Theisen et al., 2019; Boone et al., 2021; Showpnil et al., 2022).

      They aim at better understanding how EWS-FLI mediated its oncogenic activity, which is critical to eventually identifying novel therapies against this aggressive cancer.

      They use the most recent state-of-the-art omics methods to investigate transcriptome, epigenetics, and genome conformation methods. In particular, Micro-C enables achieving up to 1kb resolved 3D chromatin structures, making it possible to investigate a large number of TADs and sub-TADs structures where EWS-FLI1 mediates its oncogenic activity.

      They performed all their experiments in an Ewing sarcoma genetic background (A673 cells) which circumvents bias from previously reported approaches when working in non-orthologous cell models using similar approaches.

      Weaknesses:

      The main weakness comes from the poor reproducibility of Micro-C data. Indeed, it appears that the distances/clustering observed between replicates are typically similar or even larger than between biological conditions. For instance, in Figure 1B, I do not see any clustering when considering DBD1, DBD2, DBD+1, DBD+2.

      Lanes 80-83: "KD replicates clustered together with DBD replicate 1 on both axes and with DBD replicate 2 on the y-axis. DBD+ replicates, on the other hand, clustered away from both KD and DBD replicates. These observations suggest that the global chromatin structure of DBD replicates is more similar to KD than DBD+ replicates."

      When replacing DBD replicate 1 with DBD replicate 2, their statement would not be true anymore.

      Additional replicates to clarify this aspect seem absolutely necessary since those data are paving the way for the entire manuscript.

      Similarly:<br /> - In Figure 1C, how would the result look when comparing DBD2/KD2/DBD+2? Same when comparing DBD 1 with KD1 and DBD+1. Would the difference go in the same direction?<br /> - Figure 1D-E. How would these plots look like when comparing each replicate to each other's? How much difference would be observed when comparing, for instance, DBD1/DBD2 ? or DBD1/DBD+1?<br /> - Figure 2: again, how would these analyses look like when performing the analysis with only DBD1/DBD+1/KD1 or DBD2/DBD+2/KD?

      Another major question is the stability of EWS-FLI DBD vs EWS-FLI DBD+ proteins. Indeed, it seems that they have more FLAG (i.e., EWS-FLI) peaks in the DBD+ condition compared to the DBD condition (Figure 2B). In the WB, FLAG intensities seem also higher (2/3 replicates) in DBD+ condition compared to the DBD condition (Figure S1B).

      Would it be possible that DBD+ is just more expressed or more stable than DBD? The higher stability of the re-expressed DBD+ could also partially explain their results independently of the 3D conformational change. In other words, can they exclude that DBD+ and DBD binding are not related to their respective protein stability or their global re-expression levels?

      Surprisingly, WB FLI bands in DBD+ conditions are systematically (3/3 replicates) fainter than in DBD conditions (Figure S1B). How do the authors explain these opposite results between FLI and FALG in the WB?

    1. Reviewer #1 (Public Review):

      Summary:

      In this manuscript, the authors provide strong evidence that the cell surface E3 ubiquitin ligases RNF43 and ZNRF3, which are well known for their role in regulating cell surface levels of WNT receptors encoded by FZD genes, also target EGFR for degradation. This is a newly identified function for these ubiquitin ligases beyond their role in regulating WNT signaling. Loss of RNF43/ZNRF3 expression leads to elevated EGFR levels and signaling, suggesting a potential new axis to drive tumorigenesis, whereas overexpression of RNF43 or ZNRF3 decreases EGFR levels and signaling. Furthermore, RNF43 and ZNRF3 directly interact with EGFR through their extracellular domains.

      Strengths:

      The data showing that RNF43 and ZNRF3 interact with EGFR and regulate its levels and activity are thorough and convincing, and the conclusions are largely supported.

      Weaknesses:

      While the data support that EGFR is a target for RNF43/ZNRF3, some of the authors' interpretations of the data on EGFR's role relative to WNT's roles downstream of RNF43/ZNRF3 are overstated. The authors, perhaps not intentionally, promote the effect of RNF43/ZNRF3 on EGFR while minimizing their role in WNT signaling. This is the case in most of the biological assays (cell and organoid growth and mouse tumor models). For example, the conclusion of "no substantial activation of Wnt signaling" (page 14) in the prostate cancer model is currently not supported by the data and requires further examination. In fact, examination of the data presented here indicates effects on WNT/b-catenin signaling, consistent with previous studies.<br /> Cancers in which RNF43 or ZNRF3 are deleted are often considered to be "WNT addicted", and inhibition of WNT signaling generally potently inhibits tumor growth. In particular, treatment of WNT-addicted tumors with Porcupine inhibitors leads to tumor regression. The authors should test to what extent PORCN inhibition affects tumor (and APC-min intestinal organoid) growth. If the biological effects of RNF43/ZNRF3 loss are mediated primarily or predominantly through EGFR, then PORCN inhibition should not affect tumor or organoid growth.

    1. Reviewer #1 (Public Review):

      Wang and colleagues conducted a study to determine the neurotransmitter identity of all neurons in C. elegans hermaphrodites and males. They used CRISPR technology to introduce fluorescent gene expression reporters into the genomic loci of NT pathway genes. This approach is expected to better reflect in vivo gene expression compared to other methods like promoter- or fosmid-based transgenes, or available scRNA datasets. The study presents several noteworthy findings, including sexual dimorphisms, patterns of NT co-transmission, neuronal classes that likely use NTs without direct synthesis, and potential identification of unconventional NTs (e.g. betaine releasing neurons). The data is well-described and critically discussed, including a comparison with alternative methods. Although many of the observations and proposals have been previously discussed by the Hobert lab, the current study is particularly valuable due to its comprehensiveness. This NT atlas is the most complete and comprehensive of any nervous system that I am aware of, making it an extremely useful tool for the community.

    1. Reviewer #1 (Public Review):

      Summary:

      Dong et al here have studied the impact of the small Ras-like GTPase Rab10 on the exocytosis of dense core vesicles (DVC), which are important mediators of neuropeptide signaling in the brain. They use optical imaging to show that lentiviral depletion of Rab10 in mouse hippocampal neurons in culture independent of the established defects in neurite outgrowth hamper DCV exocytosis. They further demonstrate that such defects are paralleled by changes in ER morphology and defective ER-based calcium buffering as well as reduced ribosomal protein expression in Rab10-depleted neurons. Re-expression of Rab10 or supplementation of exogenous L-leucine to restore defective neuronal protein synthesis rescues impaired DCV secretion. Based on these results they propose that Rab10 regulates DCV release by maintaining ER calcium homeostasis and neuronal protein synthesis.

      Strengths:

      This work provides interesting and potentially important new insights into the connection between ER function and the regulated secretion of neuropeptides via DCVs. The authors combine advanced optical imaging with light and electron microscopy, biochemistry, and proteomics approaches to thoroughly assess the effects of Rab10 knockdown at the cellular level in primary neurons. The proteomic dataset provided may be valuable in facilitating future studies regarding Rab10 function. This work will thus be of interest to neuroscientists and cell biologists.

      Weaknesses:

      While the main conclusions of this study are comparably well supported by the data, I see three major weaknesses:

      (1) For some of the data the statistical basis for analysis remains unclear. I.e. is the statistical assessment based on N= number of experiments or n = number of synapses, images, fields of view etc.? As the latter cannot be considered independent biological replicates, they should not form the basis of statistical testing.

      (2) As it stands the paper reports on three partially independent phenotypic observations, the causal interrelationship of which remains unclear. Based on prior studies (e.g. Mercan et al 2013 Mol Cell Biol; Graves et al JBC 1997) it is conceivable that defective ER-based calcium signaling and the observed reduction in protein synthesis are causally related. For example, ER calcium release is known to promote pS6K1 phosphorylation, a major upstream regulator of protein synthesis and ribosome biogenesis. Conversely, L-leucine supplementation is known to trigger calcium release from ER stores via IP3Rs. Given the reported impact of Rab10 on axonal transport of autophagosomes and, possibly, lysosomes via JIP3/4 or other mediators (see e.g. Cason and Holzbaur JCB 2023) and the fact that mTORC1, the alleged target of leucine supplementation, is located on lysosomes, which in turn form membrane contacts with the ER, it seems worth analyzing whether the various phenotypes observed are linked at the level of mTORC1 signaling.

      (3) The claimed lack of effect of Rab10 depletion on SV exocytosis is solely based on very strong train stimulation with 200 Aps, a condition not very well suited to analyze defects in SV fusion. The conclusion that Rab10 loss does not impact SV fusion thus seems premature.

    1. Reviewer #1 (Public Review):

      Summary:

      Zhang et al., investigated the relationship between monocular and binocular responses of V1 superficial-layer neurons using two-photon calcium imaging. They found a strong relationship in their data: neurons that exhibited a greater preference for one eye or the other (high ocular dominance) were more likely to be suppressed under binocular stimulation, whereas neurons that are more equivalently driven by each other (low ocular dominance) were more likely to be enhanced by binocular stimulation. This result chiefly demonstrates the relationship between ocular dominance and binocular responses in V1, corroborating what has been shown previously using electrophysiological techniques with now much finer spatial resolution. The binocular responses were well-fitted by a model that institutes divisive normalization between the eyes that accounts for both the suppression and enhancement phenomena observed in the subpopulation of binocular neurons. In so doing, the authors reify the importance of incorporating ocular dominance in computational models of binocular combination.

      The conclusions of this paper are well supported by the data. The authors deftly contextualize these important findings in the literature while also acknowledging the limitations of the methodology employed. Future work would do well to combine the spatial power of 2P imaging with the temporal power of electrophysiology to assess ocular dominance-dependent binocular combination across the V1 laminar microcircuit.

      Strengths:

      The two-photon imaging technique used to resolve the activity of individual neurons within intact brain tissue grants a host of advantages. Foremost, two-photon imaging confers considerably high spatial resolution. As a result, the authors were able to sample and analyze the activity from thousands of verified superficial-layer V1 neurons. The animal model used, awake macaques, is also highly relevant for the study of binocular combination. Macaques, like humans, are binocular animals, meaning they have forward-facing eyes that confer overlapping visual fields. Importantly, macaque V1 is organized into cortical columns that process specific visual features from the separate eyes just like in humans. In combination with a powerful imaging technique, this allowed the authors to evaluate the monocular and binocular response profiles of V1 neurons that are situated within neighboring ocular dominance columns, a novel feat. To this aim, the approach was well-executed and should instill confidence in the notion that V1 neurons combine monocular information in a manner that is dependent on the strength of their ocular dominance.

      Weaknesses:

      This study suffers no major weaknesses. The authors address the limitations of the methodology and have calibrated the interpretations accordingly.

    1. Reviewer #1 (Public Review):

      Summary:

      This study examines lipid profiles in cancer patients treated with the neurotoxic chemotherapy paclitaxel. Multiple methods, including machine learning as well as more conventional statistical modelling, were used to classify lipid patterns before and after paclitaxel treatment and in conjunction with neuropathy status. Lipid profiles before and after paclitaxel therapy were analysed from 31 patients. The study aimed to characterize from the lipid profile if plasma samples were collected pre-paclitaxel or post-paclitaxel and their relevance to neuropathy status. Sphingolipids including sphinganine-1-phosphate (SA1P) differed between patients with and without neuropathy. To examine the potential role of SA1P, it was applied to murine primary sensory neuron cultures, and produced calcium transients in a proportion of neurons. This response was abolished by the application of a TRPV1 antagonist. The number of neurons responding to SA1P was partially reduced by the sphingosine 1-phosphate receptor (S1PR1) modulator fingolimod.

      Strengths:

      The strengths of this study include the use of multiple methods to classify lipid patterns and the attempt to validate findings from the clinical cohort in a preclinical model using primary sensory neurons.

      Weaknesses:

      There are a number of weaknesses in the study. The small sample size is a significant limitation of the study. Out of 31 patients, only 17 patients were reported to develop neuropathy, with significant neuropathy (grade 2/3) in only 5 patients. The authors acknowledge this limitation in the results and discussion sections of the manuscript, but it limits the interpretation of the results. Also acknowledged is the limited method used to assess neuropathy.

      Potentially due to this small number of patients with neuropathy, the machine learning algorithms could not distinguish between samples with and without neuropathy. Only selected univariate analyses identified differences in lipid profiles potentially related to neuropathy.

      Three sphingolipid mediators including SA1P differed between patients with and without neuropathy at the end of treatment. These sphingolipids were elevated at the end of treatment in the cohort with neuropathy, relative to those without neuropathy. However, across all samples from pre to post-paclitaxel treatment, there was a significant reduction in SA1P levels. It is unclear from the data presented what the underlying mechanism for this result would be. If elevated SA1P is associated with neuropathy development, it would be expected to increase in those who develop neuropathy from pre to post-treatment time points.

      Primary sensory neuron cultures were used to examine the effects of SA1P application. SA1P application produced calcium transients in a small proportion of sensory neurons. It is not clear how this experimental model assists in validating the role of SA1P in neuropathy development as there is no assessment of sensory neuron damage or other hallmarks of peripheral neuropathy. These results demonstrate that some sensory neurons respond to SA1P and that this activity is linked to TRPV1 receptors. However, further studies will be required to determine if this is mechanistically related to neuropathy.

      Impact:

      Taken in total, the data presented do not provide sufficient evidence to support the contention that SA1P has an important role in paclitaxel-induced peripheral neuropathy. Further, the results do not provide evidence to support the use of S1PR1 receptor antagonists as a therapeutic strategy. It is important to be careful with language use in the discussion, as the significance of the present results is overstated.

      However, based on the results of previous studies, it is likely that sphingolipid metabolism plays a role in chemotherapy-induced peripheral neuropathy. Based on this existing evidence, the S1PR1 receptor antagonist fingolimod has already been examined in experimental models and clinical trials. Further work is needed to examine the links between lipid mediators and neuropathy development and identify additional strategies for intervention.

    1. Reviewer #2 (Public Review):

      Summary:

      Previous work has shown subjects can use a form of short-term sensory memory, known as 'iconic memory', to accurately remember stimuli over short periods of time (several hundred milliseconds). Working memory maintains representations for longer periods of time but is strictly limited in its capacity. While it has long been assumed that sensory information acts as the input to working memory, a process model of this transfer has been missing. To address this, Tomic and Bays present the Dynamic Neural Resource (DyNR) model. The DyNR model captures the dynamics of processing sensory stimuli, transferring that representation into working memory, the diffusion of representations within working memory, and the selection of a memory for report.

      The DyNR model captures many of the effects observed in behavior. Most importantly, psychophysical experiments found the greater the delay between stimulus presentation and the selection of an item from working memory, the greater the error. This effect also depended on working memory load. In the model, these effects are captured by the exponential decay of sensory representations (i.e., iconic memory) following the offset of the stimulus. Once the selection cue is presented, residual information in iconic memory can be integrated into working memory, improving the strength of the representation and reducing error. This selection process takes time, and is slower for larger memory loads, explaining the observation that memory seems to decay instantly. The authors compare the DyNR model to several variants, demonstrating the importance of persistence of sensory information in iconic memory, normalization of representations with increasing memory load, and diffusion of memories over time.

      Strengths:

      The manuscript provides a convincing argument for the interaction of iconic memory and working memory, as captured by the DyNR model. The analyses are cutting-edge and the results are well captured by the DyNR model. In particular, a strength of the manuscript is the comparison of the DyNR model to several alternative variants.

      The results provide a process model for interactions between iconic memory and working memory. This will be of interest to neuroscientists and psychologists studying working memory. I see this work as providing a foundation for understanding behavior in continuous working memory tasks, from either a mechanistic, neuroscience, perspective or as a high-water mark for comparison to other psychological process models.

      Finally, the manuscript is well written. The DyNR model is nicely described and an intuition for the dynamics of the model are clearly shown in Figures 2 and 4.

      Weaknesses:

      The manuscript appropriately acknowledges and addresses several minor weaknesses that are due to the limited ability of the approach to disambiguate underlying neural mechanisms. Nevertheless, the manuscript adds significant value to the literature on working memory.

    1. Reviewer #1 (Public Review):

      Summary:

      This study assumes but also demonstrates that auditory rhythm processing is produced by internal oscillating systems and evaluates the properties of internal oscillators across individuals. The authors designed an experiment and performed analyses that address individuals' preferred rate and flexibility, with a special focus on how much past rhythms influence subsequent trials. They find evidence for such historical dependence and show that we adapt less well to new rhythms as we age. While I have some doubts about the entrainment-based interpretation of the results, this work offers a useful contribution to our understanding of individual differences in rhythm processing regardless.

      Strengths:

      The inclusion of two tasks -- a tapping and a listening task -- complement each other methodologically. By analysing both the production and tracking of rhythms, the authors emphasize the importance of the characteristics of the receiver, the external world, and their interplay. The relationship between the two tasks and components within tasks are explored using a range of analyses. The visual presentation of the results is very clear. The age-related changes in flexibility are useful and compelling.

      The paper includes a discussion of the study assumptions, and it contextualizes itself more explicitly as taking entrainment frameworks as a starting point. As such, even if the entrainment of oscillators cannot be decisively shown, it is now clear that this is nevertheless adopted as a useful theoretical lens.

      Weaknesses:

      The newly included analyses that justify an entrainment or oscillator-based interpretation of the result could be presented in a clearer manner so that readers can parse their validity better. For example, in line with an entrainment interpretation, the regression lines in Figure 2B show accuracy increases as the IOI moves towards the preferred rate -- but then beyond the preferred rate, accuracy appears to increase further still. Furthermore, the additional analyses on harmonic relationships could be enriched with justification and explanation of each of its steps.

    1. Reviewer #1 (Public Review):

      Summary:

      The paper presents a nice study investigating the impairments of biological motion perception in individuals with ADHD in comparison with neurotypical controls. Motivated by the idea that there is a relationship between biological motion perception and social capabilities, the authors investigated the impairments of local and global (holistic) biological motion perception, the diagnosis status, and several additional behavioral variables that are affected in ADHS (IQ, social responsiveness, and attention / impulsivity). As well local as global biological motion perception is impaired in ADHD individuals. In addition, the study demonstrates a significant correlation between local biological motion perception skills and the social responsiveness score in the ADHD group, but not in controls. A path analysis in the ADHD group suggests that general performance in<br /> biological motion perception is influenced mainly by global biological motion perception performance and attentional and perceptual reasoning skills.

      Strengths:

      It is true that there exists not much work on biological motion perception and ADHD. Therefore, the presented study contributes an interesting new result to the biological motion literature, and adds potentially also new behavioral markers for this clinical group. The design of the study is straightforward and technically sound, and the drawn conclusions are supported by the presented results.

      Weaknesses:

      Some of the claims about the relationship between genetic factors and ADHD and the components of biological motion processing have to remain speculative at this point because genetic influences were not explicitly tested in this paper. Specifically, the hypothesis that the perception of human social interaction is critically based on a local mechanism for the detection of asymmetry in foot trajectories of walkers (this is what 'BL-local' really measures), or on the detection of live agents in cluttered scenes seems not very plausible.

      Based on my last comments, now the discussion has been changed in a way that tries to justify the speculative claims by citing a lot of other speculative papers, which does not really address the problem. For example, the fact that chicks walk towards biological motion stimuli is interesting. To derive that this verifies a fundamental mechanism in human biological motion processing is extremely questionable, given that birds do not even have a cortex. Taking the argumentation of the authors serious, one would have to assume that the 'Local BM' mechanism is probably located in the mesencephalon in humans, and then would have to interact in some way with social perception differences of ADHD children. To me all this seems to make very strong (over-)claims. I suggest providing a much more modest interpretation of the interesting experimental result, based on what has been really experimentally shown by the authors and closely related other data, rather than providing lots of far-reaching speculations.

      In the same direction, in my view, go claims like 'local BM is an intrinsic trait' (L. 448) , which is not only imprecise (maybe better 'mechanisms of processing of local BM cues') but also rather questionable. Likely, this' local processing of BM' is a lower level mechanisms, located probably in early and mid-levels of the visual cortex, with a possible influence of lower structures. It seems not really plausible that this is related to a classical trait variables in the sense of psychology, like personality, as seems to be suggested here. Also here I suggest a much more moderate and less speculative interpretation of the results.

    1. Reviewer #1 (Public Review):

      This paper presents a cognitive model of out-of-distribution generalisation, where the representational basis is grid-cell codes. In particular, the authors consider the tasks of analogies, addition, and multiplication, and the out-of-distribution tests are shifting or scaling the input domain. The authors utilise grid cell codes, which are multi-scale as well as translationally invariant due to their periodicity. To allow for domain adaptation, the authors use DPP-A which is, in this context, a mechanism of adapting to input scale changes. The authors present simulations results demonstrating this model can perform out-of-distribution generalisation to input translations and re-scaling, whereas other models fail.

      This paper makes the point it sets out to - that there are some underlying representational bases, like grid cells, that when combined with a domain adaptation mechanism, like DPP-A, can facilitate out-of-generalisation. I don't have any issues with the technical details.

      The paper nicely demonstrates how neural codes can be transformed into a common representational space so that analogies, and presumably other useful tasks/computations, can be performed.

    1. Reviewer #1 (Public Review):

      It is known that aberrant habit formation is a characteristic of obsessive-compulsive disorder (OCD). Habits can be defined according to the following features (Balleine and Dezfouli, 2019): rapid execution, invariant response topography, action 'chunking' and resistance to devaluation.

      The extent to which OCD behavior is derived from enhanced habit formation relative to deficits in goal-directed behavior is a topic of debate in the current literature. This study examined habit-learning specifically (cf. deficits in goal-directed behavior) by regularly presenting, via smartphone, sequential learning tasks to patients with OCD and healthy controls. Participants engaged in the tasks every day over the course of a month. Automaticity, including the extent to which individual actions in the sequence become part of a unified 'chunk', was an important outcome variable. Following the 30 days of training, in-laboratory tasks were then administered to examine 1) if performing the learned sequences themselves had become rewarding 2) differences in goal-directed vs. habitual behavior.

      Several hypotheses were tested, including:<br /> Patients would have impaired procedural learning vs. healthy volunteers (this was not supported, possibly because there were fewer demands on memory in the task used here)<br /> Once the task had been learned, patients would display automaticity faster (unexpectedly, patients were slower to display automaticity)<br /> Habits would form faster under a continuous (vs. variable) reinforcement schedule

      Exploratory analyses were also conducted: an interesting finding was that OCD patients with higher self-reported symptoms voluntarily completed more sessions with the habit-training app and reported a reduction in symptoms.

      Strengths

      This paper is well situated theoretically within the habit learning/OCD literature.<br /> Daily training in a motor-learning task, delivered via smartphone, was innovative, ecologically valid and more likely to assay habitual behaviors specifically. Daily training is also more similar to studies with non-humans, making a better link with that literature. The use of a sequential-learning task (cf. tasks that require a single response) is also more ecologically valid.<br /> The in-laboratory tests (after the 1 month of training) allowed the researchers to test if the OCD group preferred familiar, but more difficult, sequences over newer, simpler sequences.

      Weaknesses

      The authors were not able to test one criterion of habits, namely resistance to devaluation, due to the nature of the task.<br /> The sample size was relatively small. Some potentially interesting individual differences within the OCD group could have been examined more thoroughly with a bigger sample (e.g., preference for familiar sequences). A larger sample may have allowed the statistical testing of any effects due to medication status.

      The authors achieved their aims in that two groups of participants (patients with OCD and controls) engaged with the task over the course of 30 days. The repeated nature of the task meant that 'overtraining' was almost certainly established, and automaticity was demonstrated. This allowed the authors to test their hypotheses about habit learning. The results are supportive of the author's conclusions.

      This article is likely to be impactful -- the delivery of a task across 30 days to a patient group is innovative and represents a new approach for the study of habit learning that is superior to an in-laboratory approach.

      An interesting aspect of this manuscript is that it prompts a comparison with previous studies of goal-directed/habitual responding in OCD that used devaluation protocols, and which may have had their effects due to deficits in goal-directed behavior and not enhanced habit learning per se.

    1. "Il résulte donc de ce qui précède, qu’en l’absence d’obstacle juridique, l’organe délibératif de l’EPLE est parfaitement libre d’adopter le principe d’une répartition de l’année scolaire en deux semestres, au lieu de trois trimestres. Une fois cette résolution arrêtée, il conviendra également de modifier en conséquence le règlement intérieur de l’établissement."

    1. Reviewer #1 (Public Review):

      Summary:

      In this study, participants completed two different tasks. A perceptual choice task in which they compared the sizes of pairs of items and a value-different task in which they identified the higher value option among pairs of items with the two tasks involving the same stimuli. Based on previous fMRI research, the authors sought to determine whether the superior frontal sulcus (SFS) is involved in both perceptual and value-based decisions or just one or the other. Initial fMRI analyses were devised to isolate brain regions that were activated for both types of choices and also regions that were unique to each. Transcranial magnetic stimulation was applied to the SFS in between fMRI sessions and it was found to lead to a significant decrease in accuracy and RT on the perceptual choice task but only a decrease in RT on the value-different task. Hierarchical drift-diffusion modelling of the data indicated that the TMS had led to a lowering of decision boundaries in the perceptual task and a lower of non-decision times on the value-based task. Additional analyses show that SFS covaries with model-derived estimates of cumulative evidence and that this relationship is weakened by TMS.

      Strengths:

      The paper has many strengths including the rigorous multi-pronged approach of causal manipulation, fMRI and computational modelling which offers a fresh perspective on the neural drivers of decision making. Some additional strengths include the careful paradigm design which ensured that the two types of tasks were matched for their perceptual content while orthogonalizing trial-to-trial variations in choice difficulty. The paper also lays out a number of specific hypotheses at the outset regarding the behavioural outcomes that are tied to decision model parameters and are well justified.

      Weaknesses:

      Unless I have missed it, the SFS does not actually appear in the list of brain areas significantly activated by the perceptual and value tasks in Supplementary Tables 1 and 2. Its presence or absence from the list of significant activations is not mentioned by the authors when outlining these results in the main text. What are we to make of the fact that it is not showing significant activation in these initial analyses?

      The value difference task also requires identification of the stimuli, and therefore perceptual decision-making. In light of this, the initial fMRI analyses do not seem terribly informative for the present purposes as areas that are activated for both types of tasks could conceivably be specifically supporting perceptual decision-making only. I would have thought brain areas that are playing a particular role in evidence accumulation would be best identified based on whether their BOLD response scaled with evidence strength in each condition which would make it more likely that areas particular to each type of choice can be identified. The rationale for the authors' approach could be better justified.

      TMS led to reductions in RT in the value-difference as well as the perceptual choice task. DDM modelling indicated that in the case of the value task, the effect was attributable to reduced non-decision time which the authors attribute to task learning. The reasoning here is a little unclear. If task learning is the cause, then why are similar non-decision time effects not observed in the perceptual choice task? Given that the value-task actually requires perceptual decision-making, is it not possible that SFS disruption impacted the speed with which the items could be identified, hence delaying the onset of the value-comparison choice?

      The sample size is relatively small. The authors state that 20 subjects is 'in the acceptable range' but it is not clear what is meant by this.

    1. Reviewer #1 (Public Review):

      Summary and strength:

      The authors undertook to assemble and annotate the genome sequence of the Malabar grouper fish, with the aim of providing molecular resources for fundamental and applied research. Even though this is more mainstream, the task is still daunting and labor-intensive. Currently, high-quality and fully annotated genome sequences are of strategic importance in modern biology. The authors make use of the resource to address the endocrine control of an ecologically and developmentally relevant life cycle transition, metamorphosis. As opposed to amphibian and flat fish where body plan changes, fish metamorphosis is anatomically more subtle and much less known, although it is clear that thyroid hormone (TH) signaling is a key player. The authors thus provide a repertoire of TH-relevant gene expression changes during development and across metamorphosis and correlate developmental stages with changes in gene expression. Overall, this work has a strong potential to meet its target.

      Weaknesses:

      The manuscript needs proper editing and is not complete. Some wordings lack precision and make it difficult to follow (e.g. line 98 "we assembled a chromosome-scale genome of ..." should read instead "we assembled a chromsome-scla genome sequence of ...". Also, panel Figure 2E is missing.

      The shortcomings of the manuscripts are not limited to the writing style, and important technical and technological information is missing or not clear enough, thereby preventing a proper evaluation of the resolution of the genomic resources provided:

      - Several RNASeq libraries from different tissues have been built to help annotate the genome and identify transcribed regions. This is fine. But all along the manuscript, gene expression changes are summarized into a single panel where it is not clear at all which tissue this comes from (whole embryo or a specific tissue ?), or whether it is a cumulative expression level computed across several tissues (and how it was computed) etc. This is essential information needed for data interpretation.

      - The bioinformatic processing, especially of the assemble and annotation, is very poorly described. This is also a sensitive topic, as illustrated by the numerous "assemblathon" and "annotathon" initiatives to evaluate tools and workflows. Importantly, providing configuration files and in-depth description of workflows and parameter settings is highly recommended. This can be made available through data store services and documents even benefit from DOIs. This provides others with more information to evaluate the resolution of this work. No doubt that it is well done,<br /> but especially in the field of genome assembly and annotation, high resolution is VERY cost and time-intensive. Not surprisingly, most projects are conditioned by trade-offs between cost, time, and labor. The authors should provide others with the information needed to evaluate this.

      - Quantifications of T3 and T4 levels look fairly low and not so convincing. The work would clearly benefit from a discussion about why the signal is so low and what are the current technological limitations of these quantifications. This would really help (general) readers.

      - Differential analysis highlights up to ~ 15,000 differentially expressed genes (DEG), out of a predicted 26k genes. This corresponds to more than half of all genes. ANOVA-based differential analysis relies on the simple fact that only a minority of genes are DEG. Having >50% DEG is well beyond the validity of the method. This should be addressed, or at least discussed.

    1. Reviewer #1 (Public Review):

      Summary:

      In their manuscript, "Nicotine enhances the stemness and tumorigenicity in intestinal stem cells via Hippo-YAP/TAZ and Notch signal pathway", authors Isotani et al claimed that this study identifies a NIC-triggered pathway regulating the stemness and tumorigenicity of ISCs and suggest the use of DBZ as a potential therapeutic strategy for treating intestinal tumors. However, the presented data do not support the primary claims.

      Weaknesses:

      My main reservation is that the quality of the results presented in the manuscript may not fully substantiate their conclusions. For instance, in Figure 2 A and B, it is challenging to discern a healthy organoid. This is significant, as the entirety of Figure 2 and several panels in Figures 3 - 5 are based on these organoid assays. Additionally, there seems to be a discrepancy in the quality of results from the western blot, as the lanes of actin do not align with other proteins (Figure 6B).

    1. Reviewer #1 (Public Review):

      Summary:

      Cognitive and brain development during the first two years of life is vast and determinant for later development. However, longitudinal infant studies are complicated and restricted to occidental high-income countries. This study uses fNIRS to investigate the developmental trajectories of functional connectivity networks in infants from a rural community in Gambia. In addition to resting-state data collected from 5 to 24 months, the authors collected growing measures from birth until 24 months and administrated an executive functioning task at 3 or 5 years old.

      The results show left and right frontal-middle and right frontal-posterior negative connections at 5 months that increase with age (i.e., become less negative). Interestingly, contrary to previous findings in high-income countries, there was a decrease in frontal interhemispheric connectivity. Restricted growth during the first months of life was associated with stronger frontal interhemispheric connectivity and weaker right frontal-posterior connectivity at 24 months. Additionally, the study describes that some connectivity patterns related to better cognitive flexibility at pre-school age.

      Strengths:

      - The authors analyze data from 204 infants from a rural area of Gambia, already a big sample for most infant studies. The study might encourage more research on different underrepresented infant populations (i.e., infants not living in occidental high-income countries).

      - The study shows that fNIRS is a feasible instrument to investigate cognitive development when access to fMRI is not possible or outside a lab setting.

      - The fNIRS data preprocessing and analysis are well-planned, implemented, and carefully described. For example, the authors report how the choices in the parameters for the motion artifacts detection algorithm affect data rejection and show how connectivity stability varies with the length of the data segment to justify the threshold of at least 250 seconds free of artifacts for inclusion.

      - The authors use proper statistical methods for analysis, considering the complexity of the dataset.

      Weaknesses:

      - No co-registration of the optodes is implemented. The authors checked for correct placement by looking at pictures taken during the testing session. However, head shape and size differences might affect the results, especially considering that the study involves infants from 5 months to 24 months and that the same fNIRS array was used at all ages.

      - The authors regress the global signal to remove systemic physiological noise. While the authors also report the changes in connectivity without global signal regression, there are some critical differences. In particular, the apparent decrease in frontal inter-hemispheric connections is not present when global signal regression is omitted, even though it is present for deoxy-Hb. The authors use connectivity results obtained after applying global signal regression for further analysis. The choice of regressing the global signal is questionable since it has been shown to introduce anti-correlations in fMRI data (Murphy et al., 2009), and fNIRS in young infants does not seem to be highly affected by physiological noise (Emberson et al., 2016). Systemic physiological noise might change at different ages, which makes its remotion critical to investigate functional network development. However, global signal regression might also affect the data differently. The study would have benefited from having short separation channels to measure the systemic psychological component in the data.

      - I believe the authors bypass a fundamental point in their framing. When discussing the results, the authors compare the developmental trajectories of the infants tested in a rural area of Gambia with the trajectories reported in previous studies on infants growing in occidental high-income countries (likely in urban contexts) and attribute the differences to adverse effects (i.e., nutritional deficits). Differences in developmental trajectories might also derive from other environmental and cultural differences that do not necessarily lead to poor cognitive development.

      - While the study provides a solid description of the functional connectivity changes in the first two years of life at the group level, the evidence regarding the links between adverse situations, developmental trajectories, and later cognitive capacities is weaker. The authors find that early restricted growth predicts specific connectivity patterns at 24 months and that certain connectivity patterns at specific ages predict cognitive flexibility. However, the link between development trajectories (individual changes in connectivity) with growth and later cognitive capacities is missing. To address this question adequately, the study should have compared infants with different growing profiles or those who suffered or did not from undernutrition. However, as the authors discussed, they lacked statistical power.

    1. Reviewer #1 (Public Review):

      The authors sought to test whether anterior insular cortex neurons increase or decrease firing during fear behavior and freezing, bi-directionally control fear via separate, anatomically defined outputs. Using a fairly simple behavior where mice were exposed to tone-shock pairings, they found roughly equal populations that do indeed either increase or decrease firing during freezing. Next, they sought to test whether these distinct populations may also have distinct outputs. Using retrograde tracers they found that the anterior insular cortex contains non-overlapping neurons which project to the mediodorsal thalamus or amygdala. Mediodorsal thalamus-projecting neurons tended to cluster in deep cortical layers while amygdala-projecting neurons were primarily in more superficial layers. Stimulation of insula-thalamus projection decreased freezing behavior, and stimulation of insula-amygdala projections increased fear behavior. Given that the neurons that increased firing were located in deep layers, that thalamus projections occurred in deep layers, and that stimulation of insula-thalamus neurons decreased freezing, the authors concluded that the increased firing neurons may be thalamus projections. Similarly, given that decreased-firing neurons tended to occur in more superficial layers, that insula-amygdala projections were primarily superficial, and that insula-amygdala stimulation increased freezing behavior, authors concluded that the decreased firing cells may be amygdala projections. The study has several strengths though also some caveats.

      Strengths:

      The potential link between physiological activity, anatomy, and behavior is well laid out and is an interesting question. The activity contrast between the units that increase/decrease firing during freezing is clear.

      It is nice to see the recording of extracellular spiking activity, which provides a clear measure of neural output, whereas similar studies often use bulk calcium imaging, a signal that rarely matches real neural activity even when anatomy suggests it might (see London et al 2018 J Neuro - there are increased/decreased spiking striatal populations, but both D1 and D2 striatal neurons increase bulk calcium).

      Weaknesses:

      The link between spiking, anatomy, and behavior requires assumptions/inferences: the anatomically/genetically defined neurons which had distinct outputs and opposite behavioral effects can only be assumed the increased/decreased spiking neurons, based on the rough area of the cortical layer they were recorded.

      The behavior would require more control to fully support claims about the associative nature of the fear response (see Trott et al 2022 eLife) - freezing, in this case, could just as well be nonassociative. In a similar vein, fixed intertrial intervals, though common practice in the fear literature, pose a problem for neurophysiological studies. The first is that animals learn the timing of events, and the second is that neural activity is dynamic and changes over time. Thus it is very difficult to determine whether changes in neural activity are due to learning about the tone-shock contingency, timing of the task, simply occur because of time and independently of external events, or some combination of the above.

    1. Reviewer #1 (Public Review):

      Summary:

      Cheong et al. use a synapse-resolution wiring map of the fruit fly nerve cord to comprehensively investigate circuitry between descending neurons (DNs) from the brain and motor neurons (MNs) that enact different behaviours. These neurons were painstakingly identified, categorised, and linked to existing genetic driver lines; this allows the investigation of circuitry to be informed by the extensive literature on how flights walk, fly, and escape from looming stimuli. New motifs and hypotheses of circuit function were presented. This work will be a lasting resource for those studying nerve cord function.

      Strengths:

      The authors present an impressive amount of work in reconstructing and categorising the neurons in the DN to MN pathways. There is always a strong link between the circuitry identified and what is known in the literature, making this an excellent resource for those interested in connectomics analysis or experimental circuits neuroscience. Because of this, there are many testable hypotheses presented with clear predictions, which I expect will result in many follow-up publications. Most MNs were mapped to the individual muscles that they innervate by linking this connectome to pre-existing light microscopy datasets. When combined with past fly brain connectome datasets (Hemibrain, FAFB) or future ones, there is now a tantalising possibility of following neural pathways from sensory inputs to motor neurons and muscle.

      Weaknesses:

      As with all connectome datasets, the sample size is low, limiting statistical analyses. Readers should keep this in mind, but note that this is the current state-of-the-art. Some figures are weakened by relying too much on depictions of wiring diagrams as evidence of circuit function, similarity between neuropils, etc. without additional quantitative justification.

    1. Reviewer #1 (Public Review):

      Summary:

      This paper reports the first results on the effects of a novel waveform for weak transcranial magnetic stimulation, which they refer to as "perturbation" (kTMP). The waveform is sinusoidal at kHz frequency with subthreshold intensities of 2V/m, instead of the suprathreshold pulses used in conventional TMS (~100V/m). The effect reported here concerns motor-evoked potentials (MEPs) elicited on the hand with single-pulse TMS. These MEPs are considered a marker of "corotico-spinal excitability. The manuscripts report that kTMP at 3.5kHz enhances MEPs with a medium effect size, and reports independent replications of this fining on 3 separate cohorts of subjects (N=16, 15, 16). This result is important for the field of non-invasive brain stimulation. The evidence in support of this claim is compelling.

      Strengths:

      • This is a novel modality for non-invasive brain stimulation.

      • Knowing the history in this field, is likely to lead to a large number of follow-up studies in basic and clinical research.

      • The modality cases practically no sensation which makes it perfectly suitable for control conditions. Indeed, the study itself used a persuasive double-blinding procedure.

      • The replication of the main result in two subsequent experiments is very compelling.

      • The effect size of Cohen's d=0.5 is very promising.

      • It is nice the E-fields were actually measured on a phantom, not just modeled.

      Weakness:

      • The within-subject design may have carry-over effects, although a 2-day gap is probably enough for washout.

      • It would have been nice to assess washout by comparing the per-conditions between days. Particularly problematic are the paired-pulse effects that are done within sessions in experiments 2 and 3 which could have carried over to the main metric of interest, which was the single pulse MEP.

      • Statistical analysis combining Experiments 1, 2, and 3 is a little muddled.

      • Related, the biorxiv version history of this work as experiments 1-3 came together to point to diverging results, and changing analysis methods. Specifically, an earlier version of the work claims that modulated kHz sinusoids are more effective than un-modulated sinusoids, yet the current version says that no differences were detected - which seems consistent with the data presented in this version. However, it does raise concerns about analytic methods, which seem to have shifted over time.

      • While sensation has been documented nicely, it does not seem like blinding has been directly assessed, by asking participants at the end which group they thought to be in.

    1. Reviewer #1 (Public Review):

      Summary:

      The work by Zeng et al. comprehensively explored the differences in the effects of leaf and soil microbes on the seed germination, seedling survival, and seedling growth of an invasive forb, Ageratina Adenophora, and found evidence of stronger effects of leaf microbes on Ageratina compared with soil microbes, which were negative for seed germination and seedling survival but positive for seedling growth. By further DNA sequencing and fungal strain cultivation, the authors were able to identify some of the key microbial guilds that may facilitate such negative and positive feedback.

      Strengths:

      (1) The theoretic framework is well-established.

      (2) Relating the direction of plant-microbe feedback to certain microbial guilds is always hard, but the authors have done a great job of identifying and interpreting such relationships.

      Weaknesses:

      (1) In the G0 and G21 inoculation experiments, allelopathic effects from leaf litters had not been accounted for, while these two experiments happened to be the ones where negative feedback was detected.

      (2) The authors did not compare the fungal strains accumulated in dead seedlings to those accumulated in live seedlings to prove that the live seedlings indeed accumulated lower abundances of the strains that were identified to increase seedling mortality.

      (3) The data of seed germination and seedling mortality could have been analyzed in the same manner as that of seedling growth, which makes the whole result section more coherent. I don't understand why the authors had not calculated the response index (RI) for germination/mortality rate and conducted analyses on the correlation between these RIs with microbial compositions.

      (4) The language of the manuscript could be improved to increase clarity.

    1. Reviewer #1 (Public Review):

      Summary:

      This finding shows a connection between cancer-associated beta-catenin mutations and extracellular vesicle secretion. A link between the beta-catenin mutation and expression of trafficking and exocytosis machinery. They used a multidisciplinary approach to explore expression levels of relevant proteins and single-particle imaging to directly explore the release of extracellular vesicles. These results suggest a role of extracellular vesicles in immune evasion in liver cancer with the role needing to be further explored in other forms of cancer. I find this work to be compelling and of strong significance.

      Strengths:

      This paper uses multidisciplinary methods to demonstrate the compelling role of beta-catenin mutations in suppressing EV secretion in tumors. The results and imaging are extremely convincing and compelling.

      Weaknesses:

      There is no major weakness in this work. There are only things that left me more intrigued about this work. While the role of Rab27 was strongly examined, the hits of the VAMP proteins were not explored in detail. I was wondering if the decrease in the presence of VAMPS directly suggests the final step of membrane fusion in the exocytosis of EVs is what is being impaired. Or if it is other trafficking steps along the EV secretion pathway.

    1. Reviewer #1 (Public Review):

      Summary:

      The present study's main aim is to investigate the mechanism of how VirR controls the magnitude of MEV release in Mtb. The authors used various techniques, including genetics, transcriptomics, proteomics, and ultrastructural and biochemical methods. Several observations were made to link VirR-mediated vesiculogenesis with PG metabolism, lipid metabolism, and cell wall permeability. Finally, the authors presented evidence of a direct physical interaction of VirR with the LCP proteins involved in linking PG with AG, providing clues that VirR might act as a scaffold for LCP proteins and remodel the cell wall of Mtb. Since the Mtb cell wall provides a formidable anatomical barrier for the entry of antibiotics, targeting VirR might weaken the permeability of the pathogen along with the stimulation of the immune system due to enhanced vesiculogenesis. Therefore, VirR could be an excellent drug target. Overall, the study is an essential area of TB biology.

      Strengths:

      The authors have done a commendable job of comprehensively examining the phenotypes associated with the VirR mutant using various techniques. Application of Cryo-EM technology confirmed increased thickness and altered arrangement of CM-L1 layer. The authors also confirmed that increased vesicle release in the mutant was not due to cell lysis, which contrasts with studies in other bacterial species.

      Another strength of the manuscript is that biochemical experiments show altered permeability and PG turnover in the mutant, which fits with later experiments where authors provide evidence of a direct physical interaction of VirR with LCP proteins.

      Transcriptomics and proteomics data were helpful in making connections with lipid metabolism, which the authors confirmed by analyzing the lipids and metabolites of the mutant.

      Lastly, using three approaches, the authors confirm that VirR interacts with LCP proteins in Mtb via the LytR_C terminal domain.

      Altogether, the work is comprehensive, experiments are designed well, and conclusions are made based on the data generated after verification using multiple complementary approaches.

      Weaknesses:

      The major weakness is that the mechanism of VirR-mediated EV release remains enigmatic. Most of the findings are observational and only associate enhanced vesiculogenesis observed in the VirR mutant with cell wall permeability and PG metabolism. The authors suggest that EV release occurs during cell division when PG is most fragile. However, this has yet to be tested in the manuscript - the AFM of the VirR mutant, which produces thicker PG with more pore density, displays enhanced vesiculogenesis. No evidence was presented to show that the PG of the mutant is fragile, and there are differences in cell division to explain increased vesiculogenesis. These observations, counterintuitive to the authors' hypothesis, need detailed experimental verification.

      Transcriptomic data only adds a little substantial. Transcriptomic data do not correlate with the proteomics data. It remains unclear how VirR deregulates transcription. TLCs of lipids are not quantitative. For example, the TLC image of PDIM is poor; quantitative estimation needs metabolic labeling of lipids with radioactive precursors. Further, change in PDIMs is likely to affect other lipids (SL-1, PAT/DAT) that share a common precursor (propionyl- CoA).

      The connection of cholesterol with cell wall permeability is tenuous. Cholesterol will serve as a carbon source and contribute to the biosynthesis of methyl-branched lipids such as PDIM, SL-1, and PAD/DAT. Carbon sources also affect other aspects of physiology (redox, respiration, ATP), which can directly affect permeability and import/export of drugs. Authors should investigate whether restoration of the normal level of permeability and EV release is not due to the maintenance of cell wall lipid balance upon cholesterol exposure of the VirR mutant.

      Finally, protein interaction data is based on experiments done once without statistical analysis. If the interaction between VirR and LCP protein is expected on the mycobacterial membrane, how the SPLIT_GFP system expressed in the cytoplasm is physiologically relevant. No explanation was provided as to why VirR interacts with the truncated version of LCP proteins and not with the full-length proteins.

    1. Reviewer #1 (Public Review):

      In this work, the authors provide a comprehensive description of transcriptional regulation in Pseudomonas syringae by investigating the binding characteristics of various transcription factors. They uncover the hierarchical network structure of the transcriptome by identifying top-, middle-, and bottom-level transcription factors that govern the flow of information in the network. Additionally, they assess the functional variability and conservation of transcription factors across different strains of P. syringae by studying DNA-binding characteristics. These findings notably expand our current knowledge of the P. syringae transcriptome.

      The findings associated with crosstalk between transcription factors and pathways, and the diversity of transcription factor functions across strains provide valuable insights into the transcriptional regulatory network of P. syringae. However, these results are at times underwhelming as their significance is unclear. This study would benefit from a discussion of the implications of transcription factor crosstalk on the functioning of the organism as a whole. Additionally, the implications of variability in transcription factor functions on the phenotype of the strains studied would further this analysis.

      Overall, this manuscript serves as a key resource for researchers studying the transcriptional regulatory network of P. syringae.

    1. Reviewer #1 (Public Review):

      Summary:

      The article written by Kazdaghli et al. proposes a modification of imputation methods, to better account and exploit the variability of the data. The aim is to reduce the variability of the imputation results.<br /> The authors propose two methods, one that still includes some imputation variability, but accounts for the distribution of the data points to improve the imputation. The other one proposes a determinantal sampling, that presents no variation in the imputation data, but it seems to be, that they measure the variation in the classification task, instead. As these methods grow easily in computation requirements and time, they also propose an algorithm to run these methods in quantum processors.

      Strengths:

      The sampling method for imputing missing values that account for the variability of the data seems to be accurate.

      Weaknesses:

      The authors state "Ultimately, the quality and reliability of imputations can be measured by the performance of a downstream predictor, which is usually the AUC (area under the receiver operating curve) for a classification task." but there is no citation of other scientists doing this. I think the authors could have evaluated the imputations directly, as they mention in the introduction, I understand that the final goal in the task is to have a better classification. In a real situation, they would have data that would be used for training the algorithm, and then new data that needs to be imputed and classified. Is there any difference between imputing all the data together and training the algorithm, versus doing the imputation, training a classifier, then imputing new data (for the testing set), and then testing the classification?<br /> I wonder if there could be some spurious interaction between the imputation and the classification methods, that could bias the data in the sense of having a better classification, but not imputing the real values; in particular when the deterministic DPP is used.

    1. Joint Public Review:

      This papers performs fine-mapping of the silkworm mutants bd and its fertile allelic version, bdf, narrowing down the causal intervals to a small interval of a handful of genes. In this region, the gene orthologous to mamo is impaired by a large indel, and its function is later confirmed using expression profiling, RNAi, and CRISPR KO. All these experiments are convincingly showing that mamo is necessary for the suppression of melanic pigmentation in the silkworm larval integument.

      The authors also use in silico and in vitro assays to probe the potential effector genes that mamo may regulate.

      The genotype-to-phenotype workflow, combining forward (mapping) and reverse genetics (RNAi and CRISPR loss-of-function assays) linking mamo to pigmentation are extremely convincing.

      Comments on latest version:

      This second revision took into account all the reviewers' comments. The authors added an interesting analysis of nucleotide diversity at the Bm-mamo locus, using available sequence data from 51 wild silkworms and 171 domesticated silkworms.<br /> The last paragraph added to the discussion, starting with "It has often been believed that changes in CREs are caused by random mutations", is speculative. There is currently no evidence that the mutation rate is biased at the Bm-mamo locus.

    1. Reviewer #1 (Public Review):

      Summary:<br /> In their manuscript, Schmidlin, Apodaca, et al try to answer fundamental questions about the evolution of new phenotypes and the trade-offs associated with this process. As a model, they use yeast resistance to two drugs, fluconazole and radicicol. They use barcoded libraries of isogenic yeasts to evolve thousands of strains in 12 different environments. They then measure the fitness of evolved strains in all environments and use these measurements to examine patterns in fitness trade-offs. They identify only six major clusters corresponding to different trade-off profiles, suggesting the vast genotypic landscape of evolved mutants translates to a highly constrained phenotypic space. They sequence over a hundred evolved strains and find that mutations in the same gene can result in different phenotypic profiles.

      Overall, the authors deploy innovative methods to scale up experimental evolution experiments, and in many aspects of their approach tried to minimize experimental variation.

      Weaknesses:<br /> (1) One of the objectives of the authors is to characterize the extent of phenotypic diversity in terms of resistance trade-offs between fluconazole and radicicol. To minimize noise in the measurement of relative fitness, the authors only included strains with at least 500 barcode counts across all time points in all 12 experimental conditions, resulting in a set of 774 lineages passing this threshold. This corresponds to a very small fraction of the starting set of ~21 000 lineages that were combined after experimental evolution for fitness measurements. As the authors briefly remark, this will bias their datasets for lineages with high fitness in all 12 environments, as all these strains must be fit enough to maintain a high abundance. One of the main observations of the authors is phenotypic space is constrained to a few clusters of roughly similar relative fitness patterns, giving hope that such clusters could be enumerated and considered to design antimicrobial treatment strategies. However, by excluding all lineages that fit in only one or a few environments, they conceal much of the diversity that might exist in terms of trade-offs and set up an inclusion threshold that might present only a small fraction of phenotypic space with characteristics consistent with generalist resistance mechanisms or broadly increased fitness. This has important implications regarding the general conclusions of the authors regarding the evolution of trade-offs.

      (2) Most large-scale pooled competition assays using barcodes are usually stopped after ~25 to avoid noise due to the emergence of secondary mutations. The authors measure fitness across ~40 generations, which is almost the same number of generations as in the evolution experiment. This raises the possibility of secondary mutations biasing abundance values, which would not have been detected by the whole genome sequencing as it was performed before the competition assay.

      (3) The approach used by the authors to identify and visualize clusters of phenotypes among lineages does not seem to consider the uncertainty in the measurement of their relative fitness. As can be seen from Figure S4, the inter-replicate difference in measured fitness can often be quite large. From these graphs, it is also possible to see that some of the fitness measurements do not correlate linearly (ex.: Med Flu, Hi Rad Low Flu), meaning that taking the average of both replicates might not be the best approach. Because the clustering approach used does not seem to take this variability into account, it becomes difficult to evaluate the strength of the clustering, especially because the UMAP projection does not include any representation of uncertainty around the position of lineages. This might paint a misleading picture where clusters appear well separate and well defined but are in fact much fuzzier, which would impact the conclusion that the phenotypic space is constricted.

      (4) The authors make the decision to use UMAP and a gaussian mixed model to cluster and represent the different fitness landscapes of their lineages of interest. Their approach has many caveats. First, compared to PCA, the axis does not provide any information about the actual dissimilarities between clusters. Using PCA would have allowed a better understanding of the amount of variance explained by components that separate clusters, as well as more interpretable components. Second, the advantages of dimensional reduction are not clear. In the competition experiment, 11/12 conditions (all but the no drug, no DMSO conditions) can be mapped to only three dimensions: concentration of fluconazole, concentration of radicicol, and relative fitness. Each lineage would have its own fitness landscape as defined by the plane formed by relative fitness values in this space, which can then be examined and compared between lineages. Third, the choice of 7 clusters as the cutoff for the multiple Gaussian model is not well explained. Based on Figure S6A, BIC starts leveling off at 6 clusters, not 7, and going to 8 clusters would provide the same reduction as going from 6 to 7. This choice also appears arbitrary in Figure S6B, where BIC levels off at 9 clusters when only highly abundant lineages are considered. This directly contradicts the statement in the main text that clusters are robust to noise, as more a stringent inclusion threshold appears to increase and not decrease the optimal number of clusters. Additional criteria to BIC could have been used to help choose the optimal number of clusters or even if mixed Gaussian modeling is appropriate for this dataset.

      (5) Large-scale barcode sequencing assays can often be noisy and are generally validated using growth curves or competition assays. Having these types of results would help support the accuracy of the main assay in the manuscript and thus better support the claims of the authors.

    1. Reviewer #1 (Public Review):

      Summary:

      The authors investigated how the presence of interspecific introgressions in the genome affects the recombination landscape. This research was intended to inform about genetic phenomena influencing the evolution of introgressed regions, although it should be noted that the research itself is based on examining only one generation, which limits the possibility of drawing far-reaching evolutionary conclusions. In this work, yeast hybrids with large (from several to several dozen percent of the chromosome length) introgressions from another yeast species were crossed. Then, the products of meiosis were isolated and sequenced, and on this basis, the genome-wide distribution of both crossovers (COs) and noncrossovers (NCOs) was examined. Carrying out the analysis at different levels of resolution, it was found that in the regions of introduction, there is a very significant reduction in the frequency of COs and a simultaneous increase in the frequency of NCOs. Moreover, it was confirmed that introgressions significantly limit the local shuffling of genetic information, and NCOs are only able to slightly contribute to the shuffling, thus they do not compensate for the loss of CO recombination.

      Strengths:

      - Previously, experiments examining the impact of SNP polymorphism on meiotic recombination were conducted either on the scale of single hotspots or the entire hybrid genome, but the impact of large introgressed regions from another species was not examined. Therefore, the strength of this work is its interesting research setup, which allows for providing data from a different perspective.

      - Good quality genome-wide data on the distribution of CO and NCO were obtained, which could be related to local changes in the level of polymorphism.

      Weaknesses:

      - The research is based on examining only one generation, which limits the possibility of drawing far-reaching evolutionary conclusions. Moreover, meiosis is stimulated in hybrids in which introgressions occur in a heterozygous state, which is a very unlikely situation in nature. Therefore, I see the main value of the work in providing information on the CO/NCO decision in regions with high sequence diversification, but not in the context of evolution.

      - The work requires greater care in preparing informative figures and, more importantly, re-analysis of some of the data (see comments below).

      More specific comments:

      - The authors themselves admit that the detection of NCO, due to the short size of conversion tracts, depends on the density of SNPs in a given region. Consequently, more NCOs will be detected in introgressed regions with a high density of polymorphisms compared to the rest of the genome. To investigate what impact this has on the analysis, the authors should demonstrate that the efficiency of detecting NCOs in introgressed regions is not significantly higher than the efficiency of detecting NCOs in the rest of the genome. If it turns out that this impact is significant, analyses should be presented proving that it does not entirely explain the increase in the frequency of NCOs in introgressed regions.

      - CO and NCO analyses performed separately for individual regions rarely show statistical significance (Figures 3 and 4). I think that the authors, after dividing the introgressed regions into non-overlapping windows of 100 bp (I suggest also trying 200 bp, 500 bp, and 1kb windows), should combine the data for all regions and perform correlations to SNP density in each window for the whole set of data. Such an analysis has a greater chance of demonstrating statistically significant relationships. This could replace the analysis presented in Figure 3 (which can be moved to Supplement). Moreover, the analysis should also take into account indels.

      - In Arabidopsis, it has been shown that crossover is stimulated in heterozygous regions that are adjacent to homozygous regions on the same chromosome (http://dx.doi.org/10.7554/eLife.03708.001, https://doi.org/10.1038/s41467- 022-35722-3). This effect applies only to class I crossovers, and is reversed for class II crossovers (https://doi.org/10.15252/embj.2020104858, https://doi.org/10.1038/s41467-023-42511-z). This research system is very similar to the system used by the authors, although it likely differs in the level of DNA sequence divergence. The authors could discuss their work in this context.

    1. Reviewer #1 (Public Review):

      Summary:<br /> The authors of this study developed a software application, which aims to identify images as either "friendly" or "unfriendly" for readers with deuteranopia, the most common color-vision deficiency. Using previously published algorithms that recolor images to approximate how they would appear to a deuteranope (someone with deuteranopia), authors first manually assessed a set of images from biology-oriented research articles published in eLife between 2012 and 2022. The researchers identified 636 out of 4964 images as difficult to interpret ("unfriendly") for deuteranopes. They claim that there was a decrease in "unfriendly" images over time and that articles from cell-oriented research fields were most likely to contain "unfriendly" images.<br /> The researchers used the manually classified images to develop, train, and validate an automated screening tool. They also created a user-friendly web application of the tool, where users can upload images and be informed about the status of each image as "friendly" or "unfriendly" for deuteranopes.

      Strengths:<br /> The authors have identified an important accessibility issue in the scientific literature: the use of color combinations that make figures difficult to interpret for people with color-vision deficiency. The metrics proposed and evaluated in the study are a valuable theoretical contribution. The automated screening tool they provide is well-documented, open source, and relatively easy to install and use. It has the potential to provide a useful service to the scientists who want to make their figures more accessible. The data are open and freely accessible, well documented, and a valuable resource for further research. The manuscript is well written, logically structured, and easy to follow.

      Weaknesses:<br /> (1) The authors themselves acknowledge the limitations that arise from the way they defined what constitutes an "unfriendly" image. There is a missed chance here to have engaged deuteranopes as stakeholders earlier in the experimental design. This would have allowed to determine to what extent spatial separation and labelling of problematic color combinations responds to their needs and whether setting the bar at a simulated severity of 80% is inclusive enough. A slightly lowered barrier is still a barrier to accessibility.

      (2) The use of images from a single journal strongly limits the generalizability of the empirical findings as well as of the automated screening tool itself. Machine-learning algorithms are highly configurable but also notorious for their lack of transparency and for being easily biased by the training data set. A quick and unsystematic test of the web application shows that the classifier works well for electron microscopy images but fails at recognizing red-green scatter plots and even the classical diagnostic images for color-vision deficiency (Ishihara test images) as "unfriendly". A future iteration of the tool should be trained on a wider variety of images from different journals.

      (3) Focusing the statistical analyses on individual images rather than articles (e.g. in figures 1 and 2) leads to pseudoreplication. Multiple images from the same article should not be treated as statistically independent measures, because they are produced by the same authors. A simple alternative is to instead use articles as the unit of analysis and score an article as "unfriendly" when it contains at least one "unfriendly" image. In addition, collapsing the counts of "unfriendly" images to proportions loses important information about the sample size. For example, the current analysis presented in Fig. 1 gives undue weight to the three images from 2012, two of which came from the same article. If we perform a logistic regression on articles coded as "friendly" and "unfriendly" (rather than the reported linear regression on the proportion of "unfriendly" images), there is still evidence for a decrease in the frequency of "unfriendly" eLife articles over time. Another issue concerns the large number of articles (>40%) that are classified as belonging to two subdisciplines, which further compounds the image pseudoreplication. Two alternatives are to either group articles with two subdisciplines into a "multidisciplinary" group or recode them to include both disciplines in the category name.

      (4.)The low frequency of "unfriendly" images in the data (under 15%) calls for a different performance measure than the AUROC used by the authors. In such imbalanced classification cases the recommended performance measure is precision-recall area under the curve (PR AUC: https://doi.org/10.1371%2Fjournal.pone.0118432) that gives more weight to the classification of the rare class ("unfriendly" images).

    1. Reviewer #1 (Public Review):

      In this study, the authors use prospective sorting and microarray analyses, extended by single-cell RNA sequencing, in the neural stem cell niche of the subventricular zone (SVZ) to identify and refine a series of states along the continuum from quiescent neural stem cells to mature progeny. Of note, changes in the levels and subgroups of RNA splicing regulators are detailed across this continuum. Using in vitro proliferation and differentiation assays, coupled with in vivo engraftment of some prospectively sorted subsets, the authors argue that a stage they define as immature neuroblasts (iNBs) retain proliferative and multilineage differentiation capacity that is not seen in the mature neuroblast population, and is unexpected based on prior models for lineage progression in this system. This iNB stage is accompanied by a change in RNA splicing regulator expression, which is of interest due to the emerging roles for RNA processing and preferential translation within this niche.

      The central tension driving the discussion between authors and reviewers, in my view, is what is required to define cells as a "molecularly distinct population" in a lineage. Is it transcript expression, in vitro potential, or more? The authors argue that sorted immature neuroblasts are a defined, separate step in the neurogenic lineage. An alternative possibility is that this population is simply cycling transit-amplifying progenitors that have initiated a transcriptional program associated with neuroblast fates - that these cells are an intermediate point on a continuum between stem cells, transit-amplifying progeny, and commitment to a neuronal (or other) fate. Despite some additions in response to initial reviews, it is still the case that much of the data presented would be equally or more effective in supporting the second model. For example, the differentially spliced gene sets in Figure S4, which are put forward by the authors to support a different molecular identity for immature neuroblasts, show that the terms enriched for immature neuroblasts are largely also found in transit amplifying progenitors (generation of neurons, neurogenesis, cell projection organization, neuron development) and/or mature neuroblasts (cell projection organization, generation of neurons), suggesting that "immature neuroblasts" are transiting between these two states and that one of their most relevant features is that they are still cycling.

      These data complement several additional sc-RNAseq studies of this stem cell niche, and use a different, but similar, sorting strategy to isolate and profile subpopulations of stem/progenitor cells and neuroblast progeny. The claim that immature neuroblasts retain multipotency - the ability to generate glia and neurons - is surprising and somewhat controversial given that this has largely not been reported before under homeostatic conditions. Some factors to consider when interpreting these data are that the "immature neuroblast" populations are studied in some experiments using a transcriptional signature and a functional assay, namely the timing of reappearance of these cells after use of agents that kill rapidly dividing cells (in this case, radiation), leading to reconstitution of the lineage by previously quiescent stem cells. In a separate set of experiments, a tamoxifen-inducible labeling system is used in combination with cell-surface markers to prospectively isolate and study the differentiation potential of neuroblast populations that are assumed to be equivalent to those found in transcriptional experiments. It would be of interest in the future to confirm that the exact sorted populations (using CD24/EGFR/DCX-CreERT2::CAG) have the same transcriptional profile as those studied in earlier experiments within the paper and to confirm the purity of the sorted populations. Finally, while use is made of engraftment of sorted populations to study the differentiation and lineage potential of these immature neuroblasts, a remaining question is the relative abundance of each lineage (neurons/astrocytes/oligodendrocytes) produced by the engrafted cells - is production of glia rare, or common? Could this be due to factors such as alteration of lineage potential due to culture conditions, a disconnect between transcript expression and protein expression, or an incompletely purified starter population?

      Overall, this manuscript presents an intriguing possible refinement of models for SVZ neurogenesis, and highlights the role of RNA splicing at specific stages in the lineage. It will be of interest to see if additional groups confirm these findings and whether multiplexed immunostaining, highly multiplexed flow cytometry, or other approaches focused at the proteomic level extend these findings, particularly given recent data in the developing brain that suggest transcript and protein levels are relatively poorly correlated in stem/progenitor populations.

    1. Reviewer #1 (Public Review):

      In this manuscript, authors have performed extensive imaging analysis of six human histone H1 variants, their enrichment and localization, their differential dynamics during interphase and mitosis, and their association with lamina-associated domains (LADs) or nucleolus-associated domains. The manuscript is well-written with high-quality confocal and super-resolution images. Various interesting observations are made on distribution patterns of H1 variants. H1.2, H1.3, and H1.5 are shown to be universally enriched at the nuclear periphery whereas H1.4 and H1X are found to be distributed throughout the nucleus. Interestingly, H1X was the only H1 variant found to be abundant in nucleoli. Depletion of H1 variants has been shown to affect chromatin structure in a variant-specific manner, with H1.2 knock-down resulting in global chromatin decompaction. Overall, the study presents several interesting insights on H1 variants conducted in a large number of cell lines.

    1. Reviewer #1 (Public Review):

      Summary:

      The authors combined high-speed video tracking of the limbs of freely moving mice with in vivo electrophysiology to demonstrate how striatal neurons encode single-limb gait. They also examine encoding other well-known aspects of locomotion, such as movement velocity and the initiation/termination of movement. The authors show that striatal neurons exhibit firing phase-locked with mouse gait at the single limb but also multi-limb level. Moreover, they describe gait deficits induced by severe unilateral dopamine neuron degeneration, and associate these deficits with a relative strengthening of gait-modulation in the firing of D2-expressing MSNs. Although the source and function of this gait-modulation remain unclear, this manuscript uncovers an important physiological correlate of striatal activity with gait, which may have implications for gait deficits in Parkinson's Disease.

      Strengths:

      While some previous work has looked at the encoding of gait variables in the striatum and other basal ganglia nuclei, this paper uses more careful quantification of gait with video tracking, comparing healthy and 6-OHDA-treated mice in the open field. The authors have collected a relatively large dataset of optically-identified striatal recordings to shed light on similarities and differences in the encoding of gait by striatal direct and indirect pathway neurons

      Weaknesses:

      There are some caveats to the interpretation of the analyses presented here, including how to compare encoding of gait variables when animals have markedly different behaviors (eg comparing sham and unilaterally 6-OHDA treated mice). The authors now address this caveat in the Discussion.

      In an effort to causally link striatal firing to gait, the authors have added data from N=4 mice in which D2-expressing MSNs are optogenetically activated, and measured the resulting changes in gait parameters. As the authors note, this experiment does not directly get at the question of whether gait modulation of firing in the striatum contributes to the kinematics of gait (an experiment in which they altered the pattern of firing, to reduce modulation, would likely be needed). Given that this experiment has very low N and there are no included controls (eg mice expressing a control construct with optical stimulation), I do not think this data should be included in the manuscript. I think commenting in the Discussion that causal experiments will be needed in the future is adequate.

      Many of the examples, as well as the average firing rates shown, are higher than typical for MSNs as reported in the literature. This is true even of the optically identified units that are shown in Figure 4. This may reflect the inclusion of neurons with interneuron-type properties (the authors report that there were some optically identified units with interneuron properties), the inclusion of some multi-unit activity in some recordings, or differences in recording/spike sorting techniques.

    1. Reviewer #1 (Public Review):

      Summary:

      In their article, the authors delve into the therapeutic potential of a newly identified liver-specific lncRNA, FincoR, regulated by the Farnesoid X Receptor (FXR) and induced by the agonist tropifexor, in treating nonalcoholic steatohepatitis (NASH). They demonstrate that FincoR significantly enhances tropifexor's effectiveness in reducing liver fibrosis and inflammation in NASH, presenting it as a promising therapeutic target. The manuscript revisions broaden the study to include both mouse and human data, showing elevated FincoR levels in various mouse models of liver disease and identifying a similar lncRNA in humans, potentially indicating a conserved therapeutic mechanism. This research offers valuable insights into FincoR's role in NASH and suggests further exploration into its functions and mechanisms in liver disease treatment.

      Strengths:

      This study enhances our understanding of FincoR, a liver-specific lncRNA, and its therapeutic potential in treating NASH through a multifaceted research approach. The revised manuscript further strengthens this contribution by incorporating additional experiments and human relevance, summarized as follows: 1) The use of GRO-seq and RNA-seq technologies has provided an in-depth and unbiased view of the transcriptional alterations driven by the FXR agonist tropifexor, especially emphasizing FincoR's pivotal role. 2) The research expands on the original findings by including diverse mouse models of NAFLD/NASH and cholestatic liver injury. These models demonstrate significant increases in hepatic FincoR levels across various conditions, such as diets high in fat and fructose, chemical induction of liver cholestasis with ANIT, and surgical induction via bile duct ligation. This broadened scope underscores FincoR's involvement in liver disease mechanisms beyond the initial models of FXR knockout (KO) and FincoR liver-specific knockdown (FincoR-LKD). 3) Incorporation of tropifexor, an FDA-approved FXR agonist, alongside these experimental models bridges experimental findings to potential therapeutic applications for NASH patients. 2) The manuscript revision includes promising data on the sequence similarity between mouse FincoR and a human locus, identifying a partially conserved human lncRNA (XR_007061585.1) with elevated levels in NAFLD and PBC patients. This addition enhances the study's relevance to human health. 3) The study's design, with the inclusion of both negative and positive controls and now enriched with a wider array of mouse models and human data, ensures that the observed therapeutic effects can be confidently attributed to FincoR's modulation by tropifexor.

      Weaknesses:

      The authors acknowledge that certain questions remain unanswered within the scope of this study on FincoR, due to feasibility and technical challenges. While it's important to note that such limitations are rooted in the practical and technical complexities, these unresolved issues might limit the study's immediate impact. The decision to focus on the discovery and initial characterization of FincoR, is strategically but not scientifically justified.

    1. Reviewer #3 (Public Review):

      Summary:

      In their paper Li et al. investigate the transcriptome of satellite cells obtained from different muscle types including hindlimb, diaphragm and extraocular muscles (EOM) from wild type and G93A transgenic mice (end stage ALS) in order to identify potential factors involved in the maintenance of the neuromuscular junction. The underlying hypothesis being that since EOMs are largely spared from this debilitating disease, they may secrete NMJ-protective factors. The results of their transcriptome analysis identified several axon guidance molecules including the chemokine Cxcl12, which are particularly enriched in EOM-derived satellite cells. Transduction of hindlimb-derived satellite cells with AAV encoding Cxcl12 reverted hindlimb-derived myotubes from the G93A mice into myotubes sharing phenotypic characteristics similar to those of EOM-derived satellite cells. Additionally, the authors were able to demonstrate that EOM-derived satellite cell myotube cultures are capable of enhancing axon extensions and innervation in co-culture experiments.

      Strengths:

      The strength of the paper is that the authors successfully isolated and purified different populations of satellite cells, compared their transcriptomes, identified specific factors release by EOM-derived satellite cells, overexpressed one of these factors (the chemokine Cxcl12) by AAV-mediated transduction of hindlimb-derived satellite cells. The transduced cells were then able to support axon guidance and NMJ integrity. They also show that administration of Na butyrate to mice decreased NMJ denervation and satellite cell-depletion of hind limbs. Furthermore, addition of Na Butyrate to hindlimb derived satellite cell myotube cultures increased Cxcl12 expression. These are impressive results providing important insights for the development of therapeutic targets to slow the loss on neuromuscular function characterizing ALS.

      Weaknesses:

      Several important aspects have not been addressed by the authors, these include the following points which weaken the conclusions and interpretation of the results.<br /> (a) Na Butyrate was shown to extend the survival of G93A mice by Zhang et al. Na butyrate has a variety of biological effects. For example, anti-inflammatory effects, inhibits mitochondrial oxidative stress, positively influences mitochondrial function, is a class I / II HDAC inhibitor etc. What is the mechanism underlying its beneficial effects both in the context of mouse muscle function in the ALS G93A mice and in the in vitro myotube assay? Cytokine quantification as well as histone acetylation/methylation can be assessed experimentally and this is an important point that has not been appropriately investigated.<br /> (b) In the context of satellite cell characterization, on line 151-152 the authors state that soleus muscles were excluded from further studies since they have a higher content of slow twitch fibers and are more similar to diaphragm. This justification is not valid in the context of ALS as well as many other muscle disorders. Indeed, soleus and diaphragm muscles contain a high proportion of slow twitch fibers (up to 80% and 50% respectively) but soleus muscles are more spared than diaphragm muscles. What makes soleus muscles (and EOMs) more resistant to ALS NMJ injury? Satellite cells from soleus muscles need to be characterized in detail as well.<br /> Furthermore, EOMs are complex muscles, containing many types of fibers and expressing different myosin heavy chain isoforms and muscle proteins. The fact that in mouse both the globular layer and orbital layers of EOMs express slow myosin heavy chain isoform as well as myosin heavy chain 2X, 2A and 2B (Zhou et al., 2010 IOVIS 51:6355-6363) also indicates that the sparing is not directly linked to the fast or slow twitch nature of the muscle fiber. This needs to be considered.<br /> (c) In the context of myotube formation from cultured satellite cells on line 178-179 the authors stained the myotubes for myosin heavy chain. Because of the diversity of myosin heavy chain isoforms and different muscle origin of the satellite cells investigated, the isoform of myosin heavy chain expressed by the myotubes needs to be tested and described. It is not sufficient to state anti-MYH.<br /> (d) The original RNAseq results have not been deposited and while it is true that the authors have analyzed the results and described them in Figures 6 and 7 and relative supplements, the original data needs to be shown both as an xls list as a Volcano plots (q value versus log2 fold change). This will facilitate the independent interpretation of the results by the readers as some transcripts may not be listed. As presented it is rather difficult to identify which transcripts aside Cxcl12 are commonly upregulated. Can the data be presented in a more visual way?<br /> (e) There is no section describing the statistical analysis methods used. In many figures more than 2 groups are compared so the authors need to use an ANOVA followed by a post hoc test.

      The authors have achieved their aim in showing that satellite cells derived from EOMs have a distinct transcriptome and that this may be the basis of their sparing in ALS. Furthermore, this work may help develop future therapeutic interventions for patients with ALS.

    1. Reviewer #1 (Public Review):

      Summary:

      In this manuscript, the authors set up a pipeline to predict insect repellents that are pleasant and safe for humans. This is done by daisy-chaining a new classification model based on predicting repellents with a published model on predicting human perception. Models use a feature-engineered selection of chemical features to make their predictions. The predicted molecules are then validated against a proxy humanoid (heated brick) and its safety is tested by molecular assays of human cells. The humanistic approach to modeling these authors have taken (which considers cosmetic/aesthetic appeal and safety) is novel and a necessary step for consumer usage. However, the importance of pleasantness over effectiveness is still up for debate (DEET is unpleasant but still used often) and the generalization of safety tests is unknown and assumed. The effectiveness of the prediction models is also still warranted. They pass the authors' own behavioral tests, but their contribution to the field is unknown as both models (new and published) have not been rigorously benchmarked to previous models. Moreover, the author's breadth of literature in this field is sparse, ignoring directly related studies.

      Strengths:

      Humanistic approach to modeling considers pleasantness and safety. Chaining models can help limit the candidate odorants from the vastness of odor space.

      Weaknesses:

      The current models need to be bench-marked against leading models predicting similar outcomes. Similarly, many of these papers need to be addressed and discussed in the introduction. The authors might even consider their data sources for model training to increase performance and lexical categorization for interoperability. For instance, the Dravnikes data lexicon, currently used in the human perception lexicon, has been highly criticized for its overlapping and hard-to-interpret descriptive terms ("FRAGRANT", "AROMATIC").

      Human Perception

      Khan, R. M., Luk, C. H., Flinker, A., Aggarwal, A., Lapid, H., Haddad, R., & Sobel, N. (2007). Predicting odor pleasantness from odorant structure: pleasantness as a reflection of the physical world. Journal of Neuroscience, 27(37), 10015-10023.

      Keller, A., Gerkin, R. C., Guan, Y., Dhurandhar, A., Turu, G., Szalai, B., ... & Meyer, P. (2017). Predicting human olfactory perception from chemical features of odor molecules. Science, 355(6327), 820-826.

      Gutiérrez, E. D., Dhurandhar, A., Keller, A., Meyer, P., & Cecchi, G. A. (2018). Predicting natural language descriptions of mono-molecular odorants. Nature communications, 9(1), 4979.

      Lee, B. K., Mayhew, E. J., Sanchez-Lengeling, B., Wei, J. N., Qian, W. W., Little, K. A., ... & Wiltschko, A. B. (2023). A principal odor map unifies diverse tasks in olfactory perception. Science, 381(6661), 999-1006.<br /> Related cleaned data: https://github.com/BioMachineLearning/openpom

      Insect Repellents:

      Wright, R. H. (1956). Physical basis of insect repellency. Nature, 178(4534), 638-638.

      Katritzky, A. R., Wang, Z., Slavov, S., Tsikolia, M., Dobchev, D., Akhmedov, N. G., ... & Linthicum, K. J. (2008). Synthesis and bioassay of improved mosquito repellents predicted from chemical structure. Proceedings of the National Academy of Sciences, 105(21), 7359-7364.

      Bernier, U. R., & Tsikolia, M. (2011). Development of Novel Repellents Using Structure− Activity Modeling of Compounds in the USDA Archival Database. In Recent Developments in Invertebrate Repellents (pp. 21-46). American Chemical Society.

      Wei, J. N., Vlot, M., Sanchez-Lengeling, B., Lee, B. K., Berning, L., Vos, M. W., ... & Dechering, K. J. (2022). A deep learning and digital archaeology approach for mosquito repellent discovery. bioRxiv, 2022-09.

      The current study assumes that insect repellents repel via their odor valence to the insect, but this is not accurate. Insect repellents also mask the body odor of humans making them hard to locate. The authors need to consult the literature to understand the localization and landing mechanisms of insects to their hosts. Here, they will understand that heat alone is not the attractant as their behavioral assay would have you believe. I suggest the authors test other behaviour assays to show more convincing evidence of effectiveness. See the following studies:

      De Obaldia, M. E., Morita, T., Dedmon, L. C., Boehmler, D. J., Jiang, C. S., Zeledon, E. V., ... & Vosshall, L. B. (2022). Differential mosquito attraction to humans is associated with skin-derived carboxylic acid levels. Cell, 185(22), 4099-4116.

      McBride, C. S., Baier, F., Omondi, A. B., Spitzer, S. A., Lutomiah, J., Sang, R., ... & Vosshall, L. B. (2014). Evolution of mosquito preference for humans linked to an odorant receptor. Nature, 515(7526), 222-227.

      Wei, J. N., Vlot, M., Sanchez-Lengeling, B., Lee, B. K., Berning, L., Vos, M. W., ... & Dechering, K. J. (2022). A deep learning and digital archaeology approach for mosquito repellent discovery. bioRxiv, 2022-09.

    1. Reviewer #1 (Public Review):

      Summary:

      In this report, the authors investigated the effects of reproductive secretions on sperm function in mice. The authors attempt to weave together an interesting mechanism whereby a testosterone-dependent shift in metabolic flux patterns in the seminal vesicle epithelium supports fatty acid synthesis, which they suggest is an essential component of seminal plasma that modulates sperm function by supporting linear motility patterns.

      Strengths:

      The topic is interesting and of general interest to the field. The study employs an impressive array of approaches to explore the relationship between mouse endocrine physiology and sperm function mediated by seminal components from various glandular secretions of the male reproductive tract.

      Weaknesses:

      Unfortunately, support for the proposed mechanism is not convincingly supported by the data, and the experimental design and methodology need more rigor and details, and the presence of numerous (uncontrolled) confounding variables in almost every experimental group significantly reduce confidence in the overall conclusions of the study.

      The methodological detail as described is insufficient to support replication of the work. Many of the statistical analyses are not appropriate for the apparent designs (e.g. t-tests without corrections for multiple comparisons). This is important because the notion that different seminal secretions will affect sperm function would likely have a different conclusion if the correct controls were selected for post hoc comparison. In addition, the HTF condition was not adjusted to match the protein concentrations of the secretion-containing media, likely resulting in viscosity differences as a major confounding factor on sperm motility patterns.

      There is ambiguity in many of the measurements due to the lack of normalization (e.g. all Seahorse Analyzer measurements are unnormalized, making cell mass and uniformity a major confounder in these measurements). This would be less of a concern if basal respiration rates were consistently similar across conditions and there were sufficient independent samples, but this was not the case in most of the experiments.

      The observation that oleic acid is physiologically relevant to sperm function is not strongly supported. The cellular uptake of 10-100uM labeled oleic acid is presumably due to the detergent effects of the oleic acid, and the authors only show functional data for nM concentrations of exogenous oleic acid. In addition, the effect sizes in the supporting data were not large enough to provide a high degree of confidence given the small sample sizes and ambiguity of the design regarding the number of biological and technical replicates in the extracellular flux analysis experiments.

      Overall, the most confident conclusion of the study was that testosterone affects the distribution of metabolic fluxes in a cultured human seminal vesicle epithelial cell line, although the physiological relevance of this observation is not clear.

      In the introduction, the authors suggest that their analyses "reveal the pathways by which seminal vesicles synthesize seminal plasma, ensure sperm fertility, and provide new therapeutic and preventive strategies for male infertility." These conclusions need stronger or more complete data to support them.

    1. Reviewer #1 (Public Review):

      The manuscript investigates the role of membrane contact sites (MCSs) and sphingolipid metabolism in regulating vacuolar morphology in the yeast Saccharomyces cerevisiae. The authors show that tricalbin (1-3) deletion leads to vacuolar fragmentation and the accumulation of the sphingolipid phytosphingosine (PHS). They propose that PHS triggers vacuole division through MCSs and the nuclear-vacuolar junction (NVJ). The study presents some solid data and proposes potential mechanisms underlying vacuolar fragmentation driven by this pathway. Although the manuscript is clear in what the data indicates and what is more hypothetical, the story would benefit from providing more conclusive evidence to support these hypothesis. Overall, the study provides valuable insights into the connection between MCSs, lipid metabolism, and vacuole dynamics.

    1. Reviewer #1 (Public Review):

      The authors investigate the alpha chain t cell receptor landscape in conventional vs regulatory CD4 T cells. Overall I think it is a very well thought out and executed study with interesting conclusions. Findings are valuable and are supported by convincing evidence. This work will be of interest for immunologists studying T cells.

      Strengths:

      - One of a kind evidence and dataset.

      - State of the art analyses using well accepted in the literature tools.

      - Interesting conclusions on the breadth of immune response to challenges across different types of challenges (tumor, viral and parasitic).

    1. Reviewer #1 (Public Review):

      In this manuscript, Shimonty and colleagues study the effects of FNDC5/irisin deletion on osteocytes in a sex-specific manner using models of lactation induced bone loss and bone loss due to low calcium diet (LCD). Consistent with the previous findings of Kim et al. (2018), the authors report 'protective' effects of irisin deficiency in lactating female FNDC5-null mice due to reduced osteocytic osteolysis. Interestingly, FNDC5 null mice show distinct changes when placed on LCD, with mutant females showing some protection from hyperparathyroidism-induced bone loss, while mutant males (which have more cortical bone at baseline) show increased LCD-induced bone loss. Furthermore, new insights into irisin's role in osteocytes regarding cellular energetic metabolism were provided by sex and gene-dependent transcriptomic datasets. Strengths of the well-written manuscript include clear description of sex-dependent effects, strong transcriptomic datasets, and focus on cortical bone changes using microCT, histomorphometry, BSEM, and serum analysis. Despite these strengths, important weaknesses are noted (below) which could be addressed to improve the impact of the work for a broad audience.

      Major comments:

      (1) Overall, the magnitude of the effect size due to FNDC5 deficiency in both male and female mice is rather modest at the level of bone mass. Looking at the data from a qualitative perspective, it is clear that knockout females still lose bone during lactation and on the low calcium diet (LCD). It is difficult to assess the physiologic consequence of the modest quantitative 'protection' seen in FNDC5 mutants since the mutants still show clear and robust effects of lactation and LCD on all parameters measured. Similarly, the magnitude of the 'increased' cortical bone loss in FNDC5 mutant males is also modest, and perhaps could be related to the fact that these mice are starting with slightly more cortical bone. Since the authors do not provide a convincing molecular explanation for why FNDC5 deficiency causes these somewhat subtle changes, I would like to offer a suggestion for the authors to consider (below, point #2) which might de-emphasize the focus of the manuscript on FNDC5. If the authors chose not to follow this suggestion, the manuscript could be strengthened by addressing the consequences of the modest changes observed in WT versus FNDC5 KO mice. I understand that the effects of FNDC5 are more obvious at the level of osteocyte morphology, and it is reasonable to emphasize these findings here.

      (2) The bone RNA-seq findings reported in Figures 4-6 are quite interesting. Although Youlten et al previously reported that the osteocyte transcriptome is sex-dependent, the work here certainly advances that notion to a considerable degree, and likely will be of high interest to investigators studying skeletal biology and sexual dimorphism in general. To this end, one direction for the authors to consider might be to refocus their manuscript towards sexually-dimorphic gene expression patterns in osteocytes and the different effects of LCD on male versus female mice. This would allow the authors to better emphasize these major findings, and then to use FNDC5 deficiency as an illustrative example of how sexually-dimorphic osteocytic gene expression patterns might be affected by deletion of an osteocyte-acting endocrine factor. Ideally, the authors would confirm RNA-seq data comparing male versus female mice in osteocytes using in situ hybridization or immunostaining. Of course, this point is only a suggestion for the authors to consider.

      (3) It would be appreciated if the authors could provide additional serum parameters (if possible) to clarify incomplete data in both lactation and low-calcium diet models: RANKL/OPG ratio, Ctx, PTHrP, and 1,25-dihydroxyvitamin D levels. I understand that this may not be possible due to lack of available material.

    1. Reviewer #1 (Public Review):

      The authors present a number of deep learning models to analyse the dynamics of epithelia. In this way they want to overcome the time-consuming manual analysis of such data and also remove a potential operator bias. Specifically, they set up models for identifying cell division events and cell division orientation. They apply these tools to the epithelium of the developing Drosophila pupal wing. They confirm a linear decrease of the division density with time and identify a burst of cell division after healing of a wound that they had induced earlier. These division events happen a characteristic time after and a characteristic distance away from the wound. These characteristic quantities depend on the size of the wound.

      Strengths:

      The methods developed in this work achieve the goals set by the authors and are a very helpful addition to the toolbox of developmental biologists. They could potentially be used on various developing epithelia. The evidence for the impact of wounds on cell division is compelling.

      The methods presented in this work should prove to be very helpful for quantifying cell proliferation in epithelial tissues.

    1. Reviewer #1 (Public Review):

      This is a clear account of some interesting work. The experiments and analyses seem well done and the data are useful. It is nice to see that VSDI results square well with those from prior extracellular recordings.

      The authors have done a good job responding to the main points of my previous review. One important question remains, as stated in that review:

      "My reading is that this is primarily a study of surround suppression with results that follow pretty directly from what we already know from that literature, and although they engage with some of the literature they do not directly mention surround suppression in the text. Their major effect - what they repeatedly describe as a "paradoxical" result in which the responses initially show a stronger response to matched targets and backgrounds and then reverse - seems to pretty clearly match the expected outcome of a stimulus that initially evokes additional excitation due to increased center contrast followed by slightly delayed surround suppression tuned to the same peak orientation. Their dynamics result seems entirely consistent with previous work, e.g. Henry at al 2020, particularly their Fig. 3 https://elifesciences.org/articles/54264, so it seems like a major oversight to not engage with that work at all, and to explain what exactly is new here."

      Their rebuttal of my first review is not convincing -- I still believe that surround influences are important and perhaps predominant in determining the outcome of the experiments. This is particularly clear for the "paradoxical" dynamics that they observe, which seem exactly to reflect the behavior of the surround.

      The authors' arguments to the contrary are based on three main points. First, their stimuli cover the center and surround, unlike those of many previous experiments, so they argue that this somehow diminishes the impact of the surround. But the argument is not accompanied by data showing the effects of center stimuli alone or surround stimuli alone. Second, their model -- a normalization model -- does not need surround influences to account for the masking effect. Third, they cite human psychophysical masking results from their collaborators (Sebastian et al 2017), but do not cite an equally convincing demonstration that surround contrast creates potent orientation selective masking when presented alone (Petrov et al 2005, https://doi.org/10.1523/JNEUROSCI.2871-05.2005).

      At the end of the day, these issues will be resolved by further experiments, not argumentation. The paper stands as an excellent contribution, but it might be wise for the authors to be less doctrinaire in their interpretations.

    1. Reviewer #1 (Public Review):

      Summary:

      In this work, the authors utilize recurrent neural networks (RNNs) to explore the question of when and how neural dynamics and the network's output are related from a geometrical point of view. The authors found that RNNs operate between two extremes: an 'aligned' regime in which the weights and the largest PCs are strongly correlated and an 'oblique' regime where the output weights and the largest PCs are poorly correlated. Large output weights led to oblique dynamics, and small output weights to aligned dynamics. This feature impacts whether networks are robust to perturbation along output directions. Results were linked to experimental data by showing that these different regimes can be identified in neural recordings from several experiments.

      Strengths:

      A diverse set of relevant tasks.

      A well-chosen similarity measure.

      Exploration of various hyperparameter settings.

      Weaknesses:

      One of the major connections found BCI data with neural variance aligned to the outputs. Maybe I was confused about something, but doesn't this have to be the case based on the design of the experiment? The outputs of the BCI are chosen to align with the largest principal components of the data.

      Proposed experiments may have already been done (new neural activity patterns emerge with long-term learning, Oby et al. 2019). My understanding of these results is that activity moved to be aligned as the manifold changed, but more analyses could be done to more fully understand the relationship between those experiments and this work.

      Analysis of networks was thorough, but connections to neural data were weak. I am thoroughly convinced of the reported effect of large or small output weights in networks. I also think this framing could aid in future studies of interactions between brain regions.

      This is an interesting framing to consider the relationship between upstream activity and downstream outputs. As more labs record from several brain regions simultaneously, this work will provide an important theoretical framework for thinking about the relative geometries of neural representations between brain regions.

      It will be interesting to compare the relationship between geometries of representations and neural dynamics across connected different brain areas that are closer to the periphery vs. more central.

      It is exciting to think about the versatility of the oblique regime for shared representations and network dynamics across different computations.

      The versatility of the oblique regime could lead to differences between subjects in neural data.

    1. Reviewer #1 (Public Review):

      Summary:

      The pioneering work of Eve Marder on central pattern generators in the stomatogastric ganglion (STG) has made a strong case for redundancy as a biological mechanism for ensuring functional robustness, where multiple configurations of biophysical parameters are equivalent in terms of their ability to generate desired patterns of periodic circuit activity. In parallel, normative theories of synaptic plasticity have argued for functional equivalences between learning objectives and corresponding plasticity rules in implementing simple unsupervised learning (see Brito & Gerstner 2016, although similar arguments have been made long before e.g. in Aapo Hyvarinen's ICA book). This manuscript argues that similar notions of redundancy need to be taken into account in the study of synaptic plasticity rules in the brain, more specifically in the context of data-driven approaches to extract the "true" synaptic plasticity rule operating in a neural circuit from neural activity recordings. Concretely, the modeling approach takes a set of empirical measurements of the evolution of neural activity and trains a flexibly parametrized model to match that in statistical terms. Instead of being predefined by the experimenter, the features that determine this match are themselves extracted from data using a generative adversarial network framework (GAN). They show that the flexible models manage to reproduce the neural activity to a reasonable degree (though not perfectly), but lead to very different synaptic trajectories.

      Strengths:

      The idea of learning rule redundancy is a good one, and the use of GANs for the learning rule estimation is a good complement to other data-driven approaches to extract synaptic plasticity ruled from neural data.

      Weaknesses:

      (1) Numerics provide only partial support to the statements describing the results.

      (2) Even if believing the results, I don't necessarily agree with the interpretation. First: unlike the Marder example where there is complementary evidence to argue that the parameter variations actually reflect across animal biophysical variations, here the statements are really about uncertainty that the experimenter has about what is going on in a circuit observed through a certain measurement lens. Second, while taking into account this uncertainty when using the outcomes of this analysis for subsequent scientific goals is certainly sensible, the biggest punchline for me is that simply observing neural activity in a simple and very restricted context does not provide enough information about the underlying learning mechanism, especially when the hypothesis space is very large (as is the case for the MLP). So it seems more useful to use this framework to think about how to enrich the experimental design/ learning paradigms/ or the measurements themselves to make the set of hypotheses more discriminable (in the spirit of the work by Jacob Portes et al, 2022 for instance). Conversely, one should perhaps think about other ways in which to use other forms of experimental data to reasonably constrain the hypothesis space in the first place.

    1. Reviewer #2 (Public Review):

      Summary:

      This study from Bamgbose et al. identifies a new and important interaction between H4K20me and Parp1 that regulates inducible genes during development and heat stress. The authors present convincing experiments that form a mostly complete manuscript that significantly contributes to our understanding of how Parp1 associates with target genes to regulate their expression.

      Strengths:

      The authors present 3 compelling experiments to support the interaction between Parp1 and H4K20me, including:

      (1) PR-Set7 mutants remove all K4K20me and phenocopy Parp mutant developmental arrest and defective heat shock protein induction.

      (2) PR-Set7 mutants have dramatically reduced Parp1 association with chromatin and reduced poly-ADP ribosylation.

      (3) Parp1 directly binds H4K20me in vitro.

      Weaknesses:

      (1) The RNAseq analysis of Parp1/PR-Set7 mutants is reasonable, but there is a caveat to the author's conclusion (Line 251): "our results indicate H4K20me1 may be required for PARP-1 binding to preferentially repress metabolic genes and activate genes involved in neuron development at co-enriched genes." An alternative possibility is that many of the gene expression changes are indirect consequences of altered development induced by Parp1 or PR-Set7 mutants. For example, Parp1 could activate a transcription factor that represses metabolic genes. The authors counter this model by stating that Parp1 directly binds to "repressed" metabolic genes. While this argument supports their model, it does not rule out the competing indirect transcription factor model. Therefore, they should still mention the competing model as a possibility.

      (2) The section on inducibility of heat shock genes is interesting but missing an important control that might significantly alter the author's conclusions. Hsp23 and Hsp83 (group B genes) are transcribed without heat shock, which likely explains why they have H4K20me without heat shock. The authors made the reasonable hypothesis that this H4K20me would recruit Parp-1 upon heat shock (line 270). However, they observed a decrease of H4K20me upon heat shock, which led them to conclude that "H4K20me may not be necessary for Parp1 binding/activation" (line 275). However, their RNA expression data (Fig4A) argues that both Parp1 and H40K20me are important for activation. An alternative possibility is that group B genes indeed recruit Parp1 (through H4K20me) upon heat shock, but then Parp1 promotes H3/H4 dissociation from group B genes. If Parp1 depletes H4, it will also deplete H4K20me1. To address this possibility, the authors should also do a ChIP for total H4 and plot both the raw signal of H4K20me1 and total H4 as well as the ratio of these signals. The authors could also note that Group A genes may similarly recruit Parp1 and deplete H3/H4 but with different kinetics than Group B genes because their basal state lacks H4K20me/Parp1. To test this possibility, the authors could measure Parp association, H4K20methylation, and H4 depletion at more time points after heat shock at both classes of genes.

    1. Reviewer #1 (Public Review):

      Summary:

      Knudstrup et al. use two-photon calcium imaging to measure neural responses in the mouse primary visual cortex (V1) in response to image sequences. The authors presented mice with many repetitions of the same four-image sequence (ABCD) for four days. Then on the fifth day, they presented unexpected stimulus orderings where one stimulus was either omitted (ABBD) or substituted (ACBD). After analyzing trial-averaged responses of neurons pooled across multiple mice, they observed that stimulus omission (ABBD) caused a small, but significant, strengthening of neural responses but observed no significant change in the response to stimulus substitution (ACBD). Next, they performed population analyses of this dataset. They showed that there were changes in the correlation structure of activity and that many features of sequence ordering could be reliably decoded. This second set of analyses is interesting and exhibited larger effect sizes than the first results about predictive coding. However, concerns about the design of the experiment temper my enthusiasm.

      Strengths:

      (1) The topic of predictive coding in the visual cortex is exciting, and this task builds on previous important work by the senior author (Gavornik and Bear 2014) where unexpectedly shuffling sequence order caused changes in LFPs recorded from the visual cortex.

      (2) Deconvolved calcium responses were used appropriately here to look at the timing of the neural responses.

      (3) Neural decoding results showing that the context of the stimuli could be reliably decoded from trial-averaged responses were interesting. However I have concerns about how the data was formatted for performing these analyses.

      Weaknesses:

      (1) All analyses were performed on trial-averaged neural responses that were pooled across mice. Owing to differences between subjects in behavior, experimental preparation quality, and biological variability, it seems important to perform at least some analyses on individual analyses to assess how behavioral training might differently affect each animal.

      (2) The correlation analyses presented in Figure 3 (labeled the second Figure 2 in the text) should be conducted on a single-animal basis. Studying population codes constructed by pooling across mice, particularly when there is no behavioral readout to assess whether learning has had similar effects on all animals, appears inappropriate to me. If the results in Figure 3 hold up on single animals, I think that is definitely an interesting result.

      (3) On Day 0 and Day 5, the reordered stimuli are presented in trial blocks where each image sequence is shown 100 times. Why wasn't the trial ordering randomized as was done in previous studies (e.g. Gavornik and Bear 2014)? Given this lack of reordering, did neurons show reduced predictive responses because the unexpected sequence was shown so many times in quick succession? This might change the results seen in Figure 2, as well as the decoder results where there is a neural encoding of sequence order (Figure 4). It would be interesting if the Figure 4 decoder stopped working when the higher-order block structure of the task was disrupted.

      (4) A primary advantage of using two-photon calcium imaging over other techniques like extracellular electrophysiology is that the same neurons can be tracked over many days. This is a standard approach that can be accomplished by using many software packages-including Suite2P (Pachitariu et al. 2017), which is what the authors already used for the rest of their data preprocessing. The authors of this paper did not appear to do this. Instead, it appears that different neurons were imaged on Day 0 (baseline) and Day 5 (test). This is a significant weakness of the current dataset.

    1. Reviewer #1 (Public Review):

      Summary:

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

      Strengths:

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

      Weaknesses:

      The main problem is that they do not show that Hyaluronan is necessary for SVZ astrogenesis and or migration to MCAO lesions. Such loss of function studies have been carried out by studies they cite (e.g. Girard et al., 2014 and Benner et al., 2013). Similar approaches seem to be necessary in this work.

      Major points:

      (1) How good of a marker for newborn astrocytes is Thbs4? Did you co-label with B cell markers like EGFr? Is the Thbs4 gene expressed in B cells? Do scRNAseq papers show it is expressed in B cells? Are they B1 or B2 cells?

      (2) It is curious that there was no increase in Type C cells after MCAO - do the authors propose a direct NSC-astrocyte differentiation?

      (3) The paper would be strengthened with orthogonal views of z projections to show co-localization.

      (4) It is not clear why the dorsal SVZ is analysed and focused on in Figure 4. This region emanates from the developmental pallium (cerebral cortex anlagen). It generates some excitatory neurons early postnatally and is thought to have differential signalling such as Wnt (Raineteau group).

      (5) Several of the images show the lesion and penumbra as being quite close to the SVZ. Did any of the lesions contact the SVZ? If so, I would strongly recommend excluding them from the analysis as such contact is known to hyperactivate the SVZ.

      (6) The authors switch to a rat in vitro analysis towards the end of the study. This needs to be better justified. How similar are the molecules involved between mouse and rat?

      (7) Similar comment for overexpression of naked mole rat HA.

    1. Reviewer #1 (Public Review):

      The present study examines whether one can identify kinematic signatures of different motor strategies in both humans and non-human primates (NHP). The Critical Stability Task (CST) requires a participant to control a cursor with complex dynamics based on hand motion. The manuscript includes datasets on performance of NHPs collected from a previous study, as well as new data on humans performing the same task. Further human experiments and optimal control models highlight how different strategies lead to different patterns of hand motion. Finally, classifiers were developed to predict which strategy individuals were using on a given trial.

      There are several strengths to this manuscript. I think the CST task provides a very useful behavioural task to explore the neural basis of voluntary control. While reaching is an important basic motor skill and commonly studied, there is much to learn by looking at other motor actions to address many fundamental issues on the neural basis of voluntary control.

      I also think the comparison between human and NHP performance is important as there is a common concern that NHPs can be overtrained in performing motor tasks leading to differences in their performance as compared to humans. The present study highlights that there are clear similarities in motor strategies of humans and NHPs. While the results are promising, I would suggest that the actual use of these paradigms and techniques likely need some improvement/refinement. Notably, the threshold or technique to identify which strategy an individual is using on a given trial needs to be more stringent given the substantial overlap in hand kinematics between different strategies.

      The most important goal of this study is to set up future studies to examine how changes in motor strategies impact neural processing. The revised manuscript has improved the technique for identifying which strategy appears to be performed by the individual. A pivotal assumption is that one can identify control strategies from differences in behaviour. As I'm sure the authors know, this inversion of the control problem is not trivial and so success requires that there are only a few 'reasonable' strategies to solve the control problem, and that these strategies lead to distinct patterns of behavior. Many of the concerns raised by myself and the other reviewers relate to this challenge. The revised manuscript now uses a more strict criteria which is good improvement.

      One of the values of this paper is to start to develop the tools and approaches to address neural basis of control. The strength of the present manuscript is that it includes modelling, explicit strategy instructions in humans, and then analysis of free-form performance in humans and non-human primates. Given the novelty of this question and approach, there likely are many ways that the techniques and approaches could be improved, but I think they've done a great start. Their approach is quite clever and provides an important blueprint for future studies.

      One weakness at this point is that there is still substantial overlap in behavoural performance predicted between strategies, as some human participants given an explicit strategy were almost equally categorized as reflecting the other strategy. I'm glad to see the addition of the model performance on perturbation trials as this additional figure clearly highlights much greater separation in performance than when observing natural behavior. While it is not reasonable to expand beyond this for the present manuscript, I think it is essential for this group to develop the perturbation paradigm (and potentially other approaches) that can better isolate behavioral signatures of different control strategies. I think future work will be strengthened by having multiple experimental angles to interpret the neural activity.

    1. Reviewer #1 (Public Review):

      Summary:<br /> In this manuscript, Fister et. al. investigate how amputational and burn wounds affect sensory axonal damage and regeneration in a zebrafish model system. The authors discovered that burn injury results in increased peripheral axon damage and impaired regeneration. Convincing experiments show altered axonal morphology and increased Ca2+ fluxes as a result of burn damage. Further experimental proof supports that early removal of the burnt tissue by amputation rescues axonal damage. Burn damage was also shown to markedly increase keratinocyte migration and increase localized ROS production as measured by the dye Pfbsf. These responses could be inhibited by Arp 2/3 inhibition and isotonic treatment.

      Strengths:<br /> The authors use state-of-the-art methods to study and compare transection and burn-induced tissue damage. Multiple experimental approaches (morphology, Ca2+ fluxing, cell membrane labeling) confirm axonal damage and impaired regeneration time. Furthermore, the results are also accompanied by functional response tests of touch sensitivity. This is the first study to extend the role of tissue-damage-related osmotic exposure beyond wound closure and leukocyte migration to a novel layer of pathology: axonal damage and regeneration.

      Weaknesses:<br /> The conclusions of the paper claiming a link between burn-induced epithelial cell migration, spatial redox signaling, and sensory axon regeneration are mainly based on correlative observations. Arp 2/3 inhibition impairs cell migration but has no significant effect on axon regeneration and restoration of touch sensitivity.

      Pharmacological or genetic approaches should be used to prove the role of ROS production by directly targeting the known H2O2 source in the system: DUOX.

      While the authors provide clear and compelling proof that osmotic responses lie at the heart of the burn-induced axonal damage responses, they did not consider the option of further exploring any biology related to osmotic cell swelling. Could osmotic ATP release maybe play a role through excitotoxicity? Could cPLA2 activation-dependent eicosanoid production relate to the process? Pharmacological tests using purinergic receptor inhibition or blockage of eicosanoid production could answer these questions.

      The authors provide elegant experiments showing that early removal of the burnt tissue can rescue damage-induced axonal damage, which could also be interpreted in an osmotic manner: tail fin transections could close faster than burn wounds, allowing for lower hypotonic exposure time. Axonal damage and slow regeneration in tail fin burn wounds could be a direct consequence of extended exposure time to hypotonic water.

    1. Reviewer #1 (Public Review):

      This manuscript proposes a new bioinformatics approach identifying several hundreds of previously unknown inhibitory immunoreceptors. When expressed in immune cells (such as neutrophils, monocytes, CD8+, CD4+, and T-cells), such receptors inhibit the functional activity of these cells. Blocking inhibitory receptors represents a promising therapeutic strategy for cancer treatment.

      As such, this is a high-quality and important bioinformatics study. One general concern is the absence of direct experimental validation of the results. In addition to the fact that the authors bioinformatically identified 51 known receptors, providing such experimental evaluation (of at least one, or better few identified receptors) would, in my opinion, significantly strengthen the presented evidence.

      I will now briefly summarize the results and give my comments.

      First, using sequence comparison analysis, the authors identify a large set of putative receptors based on the presence of immunoreceptor tyrosine-based inhibitory motifs (ITIMs), or immunoreceptor tyrosine-based switch motifs (ITSMs). They further filter the identified set of receptors for the presence of the ITIMs or ITSMs in an intracellular domain of the protein. Second, using AlphaFold structure modeling, the authors select only receptors containing ITIMs/ITSMs in structurally disordered regions. Third, the evaluation of gene expression profiles of known and putative receptors in several immune cell types was performed. Fourth, the authors classified putative receptors into functional categories, such as negative feedback receptors, threshold receptors, threshold disinhibition, and threshold-negative feedback. The latter classification was based on the available data from Nat Rev Immunol 2020. Fifth, using publicly available single-cell RNA sequencing data of tumor-infiltrating CD4+ and CD8+ cells from nearly twenty types of cancer, the authors demonstrate that a significant fraction of putative receptors are indeed expressed in these datasets.

      In summary, in my opinion, this is an interesting, important, high-quality bioinformatics work. The manuscript is clearly written and all technical details are carefully explained.

      One comment/suggestion regarding the methodology of evaluating gene expression profiles of putative receptors: perhaps it might be important to look at clusters of genes that are co-expressed with putative inhibitory receptors.

    1. Reviewer #1 (Public Review):

      Summary:

      In the paper entitled "PI3K/HSCB axis facilitates FOG1 nuclear translocation to promote erythropoiesis and megakaryopoiesis", the authors sought to determine the role of HSCB, a known regulator of iron-sulfur cluster transfer, in the generation of erythrocytes and megakaryocytes. They utilized a human primary cell model of hematopoietic differentiation to identify a novel mechanism whereby HSCB is necessary for the activation of erythroid and megakaryocytic gene expression through regulation of the nuclear localization of FOG-1, an essential transcription co-regulator of the GATA transcription factors. Their work establishes this novel regulatory axis as a mechanism by which cytokine signaling through EPO-R and MPL drives the lineage-specification of hematopoietic progenitors to erythrocytes and megakaryocytes, respectively.

      Impact:

      The major impact of this work is in a greater understanding of how cytokine signaling through EPO/TPO functions to promote lineage specification of hematopoietic stem/progenitor cells. While the major kinase cascades downstream of the EPO/TPO receptors have been elucidated, how those cascades affect gene expression to promote a specific differentiation program is poorly understood. For this work, we now understand that nuclear localization of FOG is a critical regulatory node by which EPO/TPO signaling is required to launch FOG-dependent gene expression. However, these cytokine receptors have many overlapping and redundant targets, so it still remains to be elucidated how signaling through the different receptors promotes divergent gene expression programs. Perhaps similar regulatory mechanisms exist for other lineage-specifying transcription factors.

      Strengths:

      The authors use two different cellular models of erythroid differentiation (K562 and human primary CD34+ cells) to elucidate the multi-factorial mechanism controlling FOG-1 nuclear localization. The studies are well-controlled and rigorously establish their mechanism through complementary approaches. The differentiation effects are established through cell surface marker expression, protein expression, and gene expression analyses. Novel protein interactions discovered by proteomics analyses were validated through bi-directional co-IP experiments in multiple experimental systems. Protein cellular localization findings are supported by both immunofluorescence and cell fractionation immunoblot analyses. The robustness of their experimental findings gives great confidence in the likelihood that the methods and findings can be reproduced in future work based on their conclusions.

      Weaknesses:

      The one unexplained step in this intricately described mechanism is how HSCB functions to promote TACC3 degradation. It appears that the proteasome is involved since MG-132 reverses the effect of HSCB deficiency, but no other details are provided. Does HSCB target TACC3 for ubiquitination somehow? Future studies will be required to understand this portion of the mechanism.

      One weakness of the study design is that no in vivo experiments are conducted. The authors comment that the HSCB mouse phenotype is too dramatic to permit studies of erythropoiesis in vivo; however, a conditional approach could have been pursued.

      It should also be noted that a previous study had already shown that TACC3 regulates the nuclear localization of FOG-1, so this portion of the mechanism is not entirely novel. However, the role of HSCB and the proteasomal degradation of TACC3 is entirely novel to my knowledge.

    1. Reviewer #1 (Public Review):

      Summary:

      The authors developed a new viral 'gene drive' based on an alternate CRISPR Cas system: UNCas12f1. They show in HSV-1 that the gene drive virus can transmit as hypothesized and is superior to Cas9 in terms of evolutionary robustness.

      Strengths:

      No doubt this is an impressive technological achievement and UNCas12f1 does appear superior to Cas9 in terms of taking longer to develop resistance. This is a strong body of work and Fig 3B is the crux of the paper for me showing that resistance does take longer in terms of % of viruses that are wildtype versus UNCas12f1 gene drive. I applaud the authors and I think this is a nice technological contribution.

      Weaknesses:

      I will focus on major conceptual issues.

      (1) Mechanism. It is not really that clear to me why the UNCas12f1 has a higher barrier to the evolution of resistance. Is this simply a temporal delay or is there something intrinsic about UNCas12f1 that does not allow resistance to arise? There is a some discussion about this but it is speculative and I could not understand why resistance would not develop.

      (2) Evolution. Fig 3B is the crux of the paper for me showing that resistance does take longer in terms of % of viruses that are wildtype versus UNCas12f1 gene drive. The authors did a nice job, however, I think they need to temper the claims somewhat as longer studies (other studies typically go out to >40 days) might show resistance arising. Also, I think absolute viral titers need to be shown in addition to percentage of viruses.

      (3) Therapeutic Utility. Is this proposed as a therapeutic strategy? If so, how would it work? Could it lower overall total viral burden (i.e., wt + gene drive)? Another issue that I think needs to be specifically addressed is the issue of MOI as typically HSV-1 is thought to be (i.e. shown to be) a low MOI infection in vivo and in patients, whereas this strategy appears to rely on high MOI. Overall, to me, this is probably the major weakness: i.e., whether this strategy has therapeutic potential.

      (4) Title. I don't think the subordinate clause of the title "virus that 'infect' viruses" is quite correct. This needs to be be reworded. This strategy converts the viral population from wild type to a gene drive virus but "infect" does not seem accurate.

    1. Reviewer #2 (Public Review):

      Summary:

      Here the authors examine how increased temperature affects pollen production and fertility of Arabidopsis thaliana plants grown at selected temperature conditions ranging from 16C to 30C. They show that pollen production and fertility decline with increasing temperature. To identify the cause of reduced pollen and fertility, they resort to living cell imaging of male meiotic cells to identify that duration of meiosis increases with an increase in temperature. They also show that pollen sterility is associated with the increased presence of micronuclei likely originating from heat stress-induced impaired meiotic chromosome segregation. They correlate abnormal meiosis to weakened centromere caused by meiosis-specific defective loading of the centromere-specific histone H3 variant (CenH3) to the meiotic centromeres. Similar is the case with kinetochore-associated spindle assembly checkpoint(SAC) protein BMF1. Intriguingly, they observe a reverse trend of strong CENH3 presence in the somatic cells of the tapetum in contrast to reduced loading of CENH3 in male meiocytes with increasing temperature. In contrast to CENH3 and BMF1, the SAC protein BMF3 persists for longer periods than the WT control, based on which authors conclude that the heat stress prolongs the duration of SAC at metaphase I, which in turn extends the time of chromosome biorientation during meiosis I. This study provides insights onto the processes that affect plant reproduction with increasing temperatures which may be relevant to develop climate-resilient cultivars.

      Strengths:

      This study shows that the centromere function is affected under heat stress in meiotic cells by modulating the dynamics of the centromere specific histone H3 (CENH3) that in turn compromises the assembly of kinetochore complex proteins. This they have demonstrated by the way of live cell imaging of male meiocytes by tracking the loading dynamics and resident time of fluorescently tagged centromere/kinetochore proteins and spindle assembly checkpoint proteins.

      Weaknesses:

      Though the results presented here are interesting and solid, the current study lacks a deeper mechanistic understanding of what causes the defective loading of CenH3 to the centromeres, and why the SAC protein BMF3 persists only at meiotic centromeres to prolong the spindle assembly checkpoint, which will be interesting to delve further to completely understand the process.

      Here the authors monitor one representative centromere protein CENH3, one kinetochore-associated SAC protein BMF1, and the SAC protein BMF3 to conclude that heat stress impairs centromere/kinetochore function and prolongs SAC with increased temperatures. Centromere and its associated protein complex the kinetochores and the SAC contains a multitude of proteins, some of which are well characterized in Arabidopsis thaliana. Hence the authors could have used additional such tagged proteins to further strengthen their claim.

    1. Reviewer #1 (Public Review):

      The authors aim to develop an easy-to-use image analysis tool for the mother machine that is used for single-cell time-lapse imaging. Compared with related software, they tried to make this software more user-friendly for non-experts with a design of "What You Put Is What You Get". This software is implemented as a plugin of Napari, which is an emerging microscopy image analysis platform. The users can interactively adjust the parameters in the pipeline with good visualization and interaction interface.

      Strengths:

      - Updated platform with great 2D/3D visualization and annotation support.<br /> - Integrated one-stop pipeline for mather machine image processing.<br /> - Interactive user-friendly interface.<br /> - The users can have a visualization of intermediate results and adjust the parameters.

      Weaknesses:

      - Based on the presentation of the manuscript, it is not clear that the goals are fully achieved.<br /> - Although there is great potential, there is little evidence that this tool has been adopted by other labs.<br /> - the diversity of datasets used in this study is limited.<br /> - Some paragraphs in the Discussion section are like blogs with general recommendations. Although the suggestions look pretty useful, it is not the focus of this manuscript. It might be more appropriate to put it in the GitHub repo or a documentation page. The discussion should still focus on the software, such as features, software maintenance, software development roadmap, and community adoption.

      A discussion of the likely impact of the work on the field, and the utility of the methods and data to the community.<br /> - The impact of this work depends on the adoption of the software MM3. Napari is a promising platform with an expanding community. With good software user experience and long-term support, there is a good chance that this tool could be widely adopted in the mother machine image analysis community.<br /> - The data analysis in this manuscript is used as a demo of MM3 features, rather than scientific research.

    1. Reviewer #1 (Public Review):

      Zhang et al. investigate the hypothesis that tRNA methyl transferase 1 (TRMT1) is cleaved by NSP5 (nonstructural protein 5 or MPro), the SARS-CoV-2 main protease, during SARS-CoV-2 infection. They provide solid evidence that TRMT1 is a substrate of Nsp5, revealing an Nsp5 target consensus sequence and evidence of TRMT1 cleavage in cells. Their conclusions are exceptionally strong given the co-submission by D'Oliveira et al showing cleavage of TRMT1 in vitro by Nsp5. The detection of the N-terminal TRMT1 fragment by western blot is not robust; however, the authors provide corroborating assays and detailed densitometry methods, providing confidence to this reviewer that a TRMT1 fragment is produced under some conditions. Separately, the authors convincingly demonstrate widespread downregulation of RNA modifications during CoV-2 infection, including a requirement for TRMT1 in efficient viral replication. This finding is congruent with the authors' previous work defining the impact of TRMT1 and m2,2g on global translation, which is most likely necessary to support infection and virion production. Based on the data provided here, TRMT1 cleavage may be an act by CoV-2 to self-limit replication, as expression of a non-cleavable TRMT1 (versus wild type TRMT1) supports enhanced viral RNA expression at certain MOIs. The authors propose a few fascinating ideas to why this may be so in "Ideas and Speculation." Theoretically, TRMT1 cleavage should inactivate the modification activity of TRMT1, which the authors thoroughly and elegantly investigate with rigorous biochemical assays. However, only a minority of TRMT1 undergoes cleavage during infection at low MOIs and thus whether TRMT1 cleavage serves an important functional role during CoV-2 replication will be an important topic for future work. The authors fairly assess their work in this regard. In summary, this study demonstrates an important finding that the tRNA modification landscape is altered during CoV-2 infection, and that TRMT1 is an important host factor supporting CoV-2 replication. Their data pushes forward the idea that control of tRNA expression and functionality is an important and understudied area of host-pathogen interaction.

    1. Reviewer #1 (Public Review):

      The study by Vengayil et al. presented a role for Ubp3 for mediating inorganic phosphate (Pi) compartmentalization in cytosol and mitochondria, which regulates metabolic flux between cytosolic glycolysis and mitochondrial processes. Although the exact function of increased Pi in mitochondria is not investigated, findings have valuable implications for understanding the metabolic interplay between glycolysis and respiration under glucose-rich conditions. They showed that UBP3 KO cells regulated decreased glycolytic flux by reducing the key Pi-dependent-glycolytic enzyme abundances, consequently increasing Pi compartmentalization to mitochondria. Increased mitochondria Pi increases oxygen consumption and mitochondrial membrane potential, indicative of increased oxidative phosphorylation. In conclusion, the authors reported that the Pi utilization by cytosolic glycolytic enzymes is a key process for mitochondrial repression under glucose conditions.

      Comments on revised version:

      This reviewer appreciates the author's responses addressing some of the concerns.

      However, the concern of reproducibility and experimental methods applied to the study is still valid, particularly considering that many conclusions were drawn from western blot analysis. The authors used separate gel loading controls for western blot analysis, which is not a valid method. Considering loading and other errors/discrepancies during the transfer phase of the assay, the direct control should be analyzing the membrane after transfer or using an internal control antibody on the same membrane. None of the western blots are indicated with marker sizes, and it isn't very clear how many repeats there are and whether those repeats are biological or technical repeats.

      Concern regarding citing the Ouyang et al. paper is still valid. This paper is an essential implication in phosphate metabolism and is directly related to some of the findings associated with mitochondrial function, along with conflicting results, which should be discussed in the discussion section. As a reviewer, I do not request citing any paper from the authors in general; however, considering some of the conflicting results here, citing and discussing paper from Ouyang et al. will improve the interoperation/value of their findings.

      Considering these factors, the presented results do not fully support the findings.

    1. Reviewer #1 (Public Review):

      This study explores whether the extreme polygenicity of common traits (the fact that variation in such traits is explained by a very large number of genetic variants) could be explained in part by competition among genes for limiting molecular resources involved in gene regulation, which would cause the expression of most genes to be correlated. While the hypothesis is interesting, I still have some concerns about the analysis and interpretation.

      As the authors say in their rebuttal, assuming extreme resource limitation, i.e., going from equation 2 to 5 essentially assumes assuming that 1/(gtot [G] ) <<1 and that terms that are order [ 1/(gtot [G] ) ] can neglected. However, then the authors derive so-called resource competition terms that are order (1/m) where m is the number of genes, so that gtot is proportional to m. My main criticism (which I am not sure was addressed) is thus: can we reliably derive small order (1/m) effects while neglecting order [ 1/(gtot [G] ) ] terms, when both are presumably similar in order of magnitude? Is this mathematically sound?

      I do not think the supplement that the authors have added actually gets to this. For example, section 7.1 just gives the textbook derivation of Michelis-Menten kinetics, and does not address my earlier criticism that the terms neglected in going from eq. 16 to eq. 17 (or from eq. 2 to 3) may be similar in magnitude to the terms being derived and interpreted in eqs. 6 and 7.<br /> Similarly, it is unclear from section 7.2 how the authors are doing the simulations. Are these true Michelis-Menten simulations involving equation 2? If yes, then what is the value of [G] and [P_0] in the simulations? If these are not true Michelis-Menten simulations, but instead something that already uses equation 5, then this still does not address my earlier criticism.

    1. Reviewer #2 (Public Review):

      Summary:

      Two early Cambrian taxa of linguliform brachiopods are assigned to the family Eoobolidae. The taxa exhibit a columnar shell structure and the phylogenetic implications of this shell structure in relation to other early Cambrian families is outlined.

      Strengths:

      Interesting idea regarding the evolution of shell structure.

      Weaknesses:

      The early record of shell structures of linguliform brachiopods is incomplete and partly contradictory. The authors maintain silence regarding contradictory information throughout the article to an extend that information is cited wrongly. The article is written under the assumption that all eoobolids have a columnar shell structure. Thus, the previously claimed columnar structure of Eoobolus incipiens which has been re-illustrated in the paper is not convincing and could be interpreted in other ways.

      The article still needs a proper results section. The Discussion is mainly a review of published data. Other potential results are hidden in this "discussion". Large sections of the paper appear irrelevant and can be deleted.

      A critical revision of the family Eoobolidae and Lingulellotretidae including a revision of the type species of Eoobolus and Lingulellotreta is needed first.

      The potential evolutionary patterns that are presented towards the end (summarized in Fig 6) are interesting but rather unconvincing. The stated numbers of shell laminae, whose origin has now been clarified in a still rather short Methods section, represent a mix of data and are not comparable. Achieved numbers of laminae based on literature data include laminae from the secondary and tertiary shell layer, a distinction between the two would be crucial for the proposed claims.<br /> The obtained evolutionary patterns as presented in Fig. 6 must, after the second revision and clarification of the methods used, be regarded as misleading and reflects a limited understanding of shell growths in linguliform brachiopods (despite the extensive review of the literature).

    1. Reviewer #1 (Public Review):

      Summary:

      This study by Lee et al. is a direct follow-up on their previous study that described an evolutionary conservancy among placental mammals of two motifs (a transmembrane motif and a juxtamembrane palmitoylation site) in CD4, an antigen co-receptor, and showed their relevance for T-cell antigen signaling. In this study, they describe the contribution of these two motifs to the CD4-mediated antigen signaling in the absence of CD4-LCK binding. Their approach was the comparison of antigen-induced proximal TCR signaling and distal IL-2 production in 58-/- T-cell hybridoma expressing exogenous truncated version of CD4 (without the interaction with LCK), called T1 and T1 version with the mutations in either or both of the conserved motifs. They show that the T1 CD4 can support signaling to extend similar to WT CD4, but the mutation of the conserved motifs substantially reduced the signaling. The authors conclude that the role of these motifs is independent of the LCK-binding.

      Strengths:

      The authors convincingly show that CD4 is capable of contributing to TCR signaling in a manner independent of LCK, but dependent on the two studied motifs in CD4.

      Weaknesses:

      (1) Experiments in primary T cells are required to estimate the relative contribution of LCK-dependent and LCK-independent mechanisms of CD4 signaling.

      (2) The mechanistic explanation (beyond the independence of LCK binding) of the role of these motifs is unclear at the moment.

    1. Reviewer #1 (Public Review):

      The authors have addressed most of the concerns I had about the original version in this revised version.

    1. Reviewer #1 (Public Review):

      Caprylic acid (CAP), i.e., octanoic acid, is a saturated fatty acid. CAP is commonly used as a food contact surface sanitizer. In mammals, caprylic acid is related to hunger sensation (i.e., food consumption). serine hydroxymethyl transferase (SHMT) has been previously known as a potential herbicidal target. The present study involves a huge amount of work. The results are useful and contribute well to the literature. The data does support the conclusion. It does not seem that SHMT is the only target of CAP though (CAP may act on other proteins as well). A major deficiency of this manuscript is that there are many unclear, inaccurate, or unconcise descriptions.

    1. Reviewer #1 (Public Review):

      Summary:

      This study used a unique acute HIV-1 infection cohort, RV217, to study the evolution of transmitted founder viral Envelope sequences under nascent immune pressure. The striking feature of the RV217 cohort is the ability to detect viremia in the first week of infection, which can be followed at discrete Fiebig stages over long time intervals. This study evaluated Env sequences at 1 week, 4 weeks, and 24 weeks to provide a picture of viral and immunological co-evolution from Fiebig Stage I (1 week), Fiebig Stages IV (4 weeks), and Fiebig Stage VI (>24 weeks). This study design enabled lineage tracing of viral variants from a single transmitted founder (T/F) over the Fiebig Stages I, IV, and VI under nascent immune pressure generated in response to the T/F virus and its subsequent mutants.

      Strengths:

      As expected, there were temporal differences in the appearance of virus quasispecies among the individuals, which were located predominantly in solvent-exposed residues of Env, which is consistent with prior literature. Interestingly, two waves of antibody reactivity were observed for variants with mutations in the V2 region that harbors V2i and V2p epitopes correlated with protection in the RV144 clinical trial. Two waves of antibody response, detected by SPR, were observed, with the first wave being predominated by antibodies specific for the T/F07 V2 epitope associated with H173 located on the C -strand in the V2 region. The second wave was dominated by antibodies specific for an H to Y mutation at 173 that emerged due to antibody-mediated pressure to the original H173 virus. This is a remarkable finding in three ways.

      First, the mutation is in the C β-strand, an unlikely paratope contact residue, as this region of the V2 loop is shielded by glycans in Env trimer structures with full glycan representation (see PDB:5t3x). The structure used for modeling in the current study was an earlier structure, PDB:4TVP, that had many truncated glycans. This does not detract from the finding that the H173Y mutation likely causes a conformational shift from a more rigid helical/coil conformation to a more dynamic conformation with a β-stranded and -sheet core preference as indicated by the literature and by the MD simulations carried out by the authors. This observation suggests that using V2 scaffolds with both the H173 and H173Y variants will increase the breadth of potentially protective antibody responses to HIV-1, as indicated in reference 42, cited by the authors. Interestingly, the H173Y mutation abrogates reactivity to mAb CH58 and attenuates reactivity to mAb CH59 isolated from RV144 volunteers. These mAbs recognize conformationally distinct V2 epitopes, adding further credence to the conclusion that the H173Y mutation results in a conformational switch of the V2 region.

      Second, the H173Y mutation affects the conformation of V2 epitopes recognized by mAbs that do not neutralize T/F HIV-1 while mediating potent ADCC. The ADCC data in the manuscript provides strong support for this hypothesis and augers for further exploration of the V2 epitopes as HIV-1 vaccine targets.

      Third, it is striking that immunogens based on the H173Y mutation successfully recapitulated the observed human antibody responses in wild-type Balb/c mice. The investigators used their extensive knowledge of V2 structures and scaffold-immunogens to create the library of constructs used for this study. In this case, the ΔDSV mutation increased the breadth and magnitude of the murine antibody responses.

    1. Reviewer #2 (Public Review):

      Summary:

      Liao and colleagues generated tagged SMAD1 and SMAD5 mouse models and identified genome occupancy of these two factors in the uterus of these mice using the CUT&RUN assay. The authors used integrative bioinformatic approaches to identify putative SMAD1/5 direct downstream target genes and to catalog the SMAD1/5 and PGR genome co-localization pattern. The role of SMAD1/5 on stromal decidualization was assayed in vitro on primary human endometrial stromal cells. The new mouse models offer opportunities to further dissect SMAD1 and SMAD5 functions without the limitation from SMAD antibodies, which is significant. The CUT&RUN data further support the usefulness of these mouse models for this purpose.

      Strengths:

      The strength of this study is the novelty of new mouse models and the valuable cistromic data derived from these mice. This revised manuscript provides lots of food for thought inside and outside of the field of reproductive biology.

      Weaknesses:

      Causal effects of SMAD1/5 on the genome occupancy of other major uterine transcription factors were discussed but not experimentally examined in the present manuscript, which is understandable.

    1. Reviewer #1 (Public Review):

      Summary:

      Wang and colleagues presented an investigation of pig-origin bacteria Bacillus velezensis HBXN2020, for its released genome sequence, in vivo safety issue, probiotic effects in vitro, and protection against Salmonella infection in a murine model. Various techniques and assays are performed.

      Strengths:

      An extensive study on the probiotic properties of the Bacillus velezensis strain HBXN2020.

      Weaknesses:

      - The main results are all descriptive, without new insight advancing the field or a mechanistic understanding of the observed protection.

      - Most of the results and analysis parts are separated without a link or any story-telling to deliver a concise message.

      - For the Salmonella Typhimurium-induced mouse model of colitis, it is not clear how an oral infection of C57BL/6 would lead to colitis. Streptomycin is always pretreated (https://link.springer.com/protocol/10.1007/978-1-0716-1971-1_17).

    1. Reviewer #1 (Public Review):

      Summary:

      In this paper, Hackwell and colleagues performed technically impressive, long-term, GCaMP fiber photometry recordings from Kiss1 neurons in the arcuate nucleus of mice during multiple reproductive states. The data show an immediate suppression of activity of arc Kiss1 neuronal activity during pregnancy that is maintained during lactation. In the absence of any apparent change in suckling stimulus or milk production, mice lacking prolactin receptors in arcuate Kiss1 neurons regained Kiss1 episodic activity and estrous cyclicity faster than control mice, demonstrating that direct prolactin action on Kiss1 neurons is at least partially responsible for suppressing fertility in this species. The effect of loss of prolactin receptors from CamK2a expressing neurons was even greater, indicating either that prolactin sensitivity in Kiss1 neurons of the RP3V contributes to lactational infertility or that other prolactin-sensitive neurons are involved. These data demonstrate the important role of prolactin in suppressing Kiss1 neuron activity and thereby fertility during the lactational period in the mouse.

      Strengths:

      This is the first study to monitor the activity of the GnRH pulse-generating system across different reproductive states in the same animal. Another strength in the study design is that it isolated the effects of prolactin by maintaining normal lactation and suckling (assessed indirectly using pup growth curves). The study also offers insight into the phenomenon of postpartum ovulation in mice. The results showed a brief reactivation of arcuate Kiss1 activity immediately prior to parturition, attributed to falling progesterone levels at the end of pregnancy. This hypothesis will be of interest to the field and is likely to inspire testing in future studies. With the exceptions mentioned below, the conclusions of the paper are well supported by the data, and the aims of the study were achieved. This paper is likely to raise the standard for technical expectations in the field and spark new interest in the direct impact of prolactin on Kiss1 neurons during lactation in other species.

      Weaknesses:

      A weakness in the approach is the use of genetic models that do not offer complete deletion of the prolactin receptor from targeted neuronal populations. A substantial proportion of Kiss1 neurons in both models retain the receptor. As a result, it is not clear whether the partial maintenance of cyclicity during lactation in the genetic models is due to incomplete deletion or to the involvement of other factors. This weakness should be more fully discussed in the text. In addition, results showing no impact of progesterone on LH secretion during lactation are surprising, given the effectiveness of progesterone-containing birth control in lactating women. The progesterone-related experiments were not well justified or discussed in the text. While the authors assert their findings may reflect an important role for prolactin in lactational infertility in other mammalian species, that remains to be seen. Hyperprolactinemia is known to suppress GnRH release, but its importance in the suppression of cyclicity during lactation is controversial. Indeed, in several species, the stimulus of suckling is considered to be the main driver of lactational fertility suppression. Data from rats shows that exogenous prolactin was unable to suppress LH release in dams deprived of their pups shortly after birth; both suckling and prolactin were necessary to suppress a post ovariectomy rise in LH levels. The duration of amenorrhea does not correlate with average prolactin levels in humans, and suckling but not prolactin was required to suppress the postpartum rise in LH in the rhesus monkey. The authors should discuss more thoroughly whether the protocol of this or other studies might result in discordant results and whether mice are likely to be an outlier in their mechanism of cycle suppression.

    1. Reviewer #1 (Public Review):

      Summary:

      The study investigates the role of cylicin-1 (CYLC1) in sperm acrosome-nucleus connections and its clinical relevance to male infertility. Using mouse models, the researchers demonstrate that cylicin-1 is specifically expressed in the post acrosomal sheath-like region in spermatids and plays a crucial role in mediating acrosome-nucleus connections. Loss of CYLC1 results in severe male subfertility, characterized by acrosome detachment and aberrant head morphology in sperm. Further analysis of a large cohort of infertile men reveals CYLC1 variants in patients with sperm head deformities. The study provides valuable insights into the role of CYLC1 in male fertility and proposes CYLC1 variants as potential risk factors for human male infertility, emphasizing the importance of mouse models in understanding the pathogenicity of such variants.

      Strengths:

      This article demonstrates notable strengths in various aspects. Firstly, the clarity and excellent writing style contribute to the accessibility of the content. Secondly, the employed techniques are not only relevant but also complementary, enhancing the robustness of the study. The precision in their experimental design and the meticulous interpretation of results reflect the scientific rigor maintained throughout the study. Furthermore, the decision to create a second mouse model with the exact CYLC1 mutation found in humans adds significant qualitative value to the research. This approach not only validates the clinical relevance of the identified variant but also strengthens the translational impact of the findings.

      Weaknesses:

      There are no obvious weaknesses. While a few minor refinements, as suggested in the recommendations to authors, could enhance the overall support for the data and the authors' messages, these suggested improvements in no way diminish the robustness of the already presented data.

    1. Reviewer #1 (Public Review):

      Summary:

      In this study, Nishi et al. claim that the ratio of long-term hematopoietic stem cell (LT-HSC) versus short-term HSC (ST-HSC) determines the lineage output of HSCs and reduced ratio of ST-HSC in aged mice causes myeloid-biased hematopoiesis. The authors used Hoxb5 reporter mice to isolate LT-HSC and ST-HSC and performed molecular analyses and transplantation assays to support their arguments. How the hematopoietic system becomes myeloid-biased upon aging is an important question with many implications in the disease context as well. However, their study is descriptive with remaining questions.

      Weaknesses:

      (1) The authors may need conceptual re-framing of their main argument because whether the ST-HSCs used in this study are functionally indeed short-term "HSCs" is questionable. The data presented in this study and their immunophenotypic definition of ST-HSCs (Lineage negative/Sca-1+/c-Kit+/Flk2-/CD34-/CD150+/Hoxb5-) suggest that authors may find hematopoietic stem cell-like lymphoid progenitors as previously shown for megakaryocyte lineage (Haas et al., Cell stem cell. 2015) or, as the authors briefly mentioned in the discussion, Hoxb5- HSCs could be lymphoid-biased HSCs. The authors disputed the idea that Hoxb5- HSCs as lymphoid-biased HSCs based on their previous 4 weeks post-transplantation data (Chen et al., 2016). However, they overlooked the possibility of myeloid reprogramming of lymphoid-biased population during regenerative conditions (Pietras et al., Cell stem cell., 2015). In other words, early post-transplant ST-HSCs (Hoxb5- HSCs) can be seen as lacking the phenotypic lymphoid-biased HSCs. Thinking of their ST-HSCs as hematopoietic stem cell-like lymphoid progenitors or lymphoid-biased HSCs makes more sense conceptually as well. ST-HSCs come from LT-HSCs and further differentiate into lineage-biased multipotent progenitor (MPP) populations including myeloid-biased MPP2 and MPP3. Based on the authors' claim, LT-HSCs (Hoxb5- HSCs) have no lineage bias even in aged mice. Then these LT-HSCs make ST-HSCs, which produce mostly memory T cells. These memory T cell-producing ST-HSCs then produce MPPs including myeloid-biased MPP2 and MPP3. This differentiation trajectory is hard to accept. If we think Hoxb5- HSCs (ST-HSCs by authors) as a sub-population of immunophenotypic HSCs with lymphoid lineage bias or hematopoietic stem cell-like lymphoid progenitors, the differentiation trajectory has no flaw.

      (2) Authors' experimental designs have some caveats to support their claims. Authors claimed that aged LT-HSCs have no myeloid-biased clone expansion using transplantation assays. In these experiments, authors used 10 HSCs and young mice as recipients. Given the huge expansion of old HSC by number and known heterogeneity in immunophenotypically defined HSC populations, it is questionable how 10 out of so many old HSCs can faithfully represent the old HSC population. The Hoxb5+ old HSC primary and secondary recipient mice data (Figure 2C and D) support this concern. In addition, they only used young recipients. Considering the importance of the inflammatory aged niche in the myeloid-biased lineage output, transplanting young vs old LT-HSCs into aged mice will complete the whole picture.

      (3) The authors' molecular data analyses need more rigor with unbiased approaches. They claimed that neither aged LT-HSCs nor aged ST-HSCs exhibited myeloid or lymphoid gene set enrichment but aged bulk HSCs, which are just a sum of LT-HSCs and ST-HSCs by their gating scheme (Figure 4A), showed the "tendency" of enrichment of myeloid-related genes based on the selected gene set (Figure 4D). Although the proportion of ST-HSCs is reduced in bulk HSCs upon aging, since ST-HSCs do not exhibit lymphoid gene set enrichment based on their data, it is hard to understand how aged bulk HSCs have more myeloid gene set enrichment compared to young bulk HSCs. This bulk HSC data rather suggests that there could be a trend toward certain lineage bias (although not significant) in aged LT-HSCs or ST-HSCs. The authors need to verify the molecular lineage priming of LT-HSCs and ST-HSCs using another comprehensive dataset.

      (4) Some data are too weak to fully support their claims. The authors claimed that age-associated extramedullary changes are the main driver of myeloid-biased hematopoiesis based on no major differences in progenitor populations upon transplantation of 10 young HSCs into young or old recipient mice (Figure 7F) and relatively low donor-derived cells in thymus and spleen in aged recipient mice (Figure 7G-J). However, they used selected mice to calculate the progenitor populations in recipient mice (8 out of 17 from young recipients denoted by * and 8 out of 10 from aged recipients denoted by * in Figure 7C). In addition, they calculated the progenitor populations as frequency in c-kit positive cells. Given that they transplanted 10 LT-HSCs into "sub-lethally" irradiated mice and 8.7 Gy irradiation can have different effects on bone marrow clearance in young vs old mice, it is not clear whether this data is reliable enough to support their claims. The same concern applies to the data Figure 7G-J. Authors need to provide alternative data to support their claims.

    1. Reviewer #1 (Public Review):

      Summary:

      Given knowledge of the amino acid sequence and of some version of the 3D structure of two monomers that are expected to form a complex, the authors investigate whether it is possible to accurately predict which residues will be in contact in the 3D structure of the expected complex. To this effect, they train a deep learning model which takes as inputs the geometric structures of the individual monomers, per-residue features (PSSMs) extracted from MSAs for each monomer, and rich representations of the amino acid sequences computed with the pre-trained protein language models ESM-1b, MSA Transformer, and ESM-IF. Predicting inter-protein contacts in complexes is an important problem. Multimer variants of AlphaFold, such as AlphaFold-Multimer, are the current state of the art for full protein complex structure prediction, and if the three-dimensional structure of a complex can be accurately predicted then the inter-protein contacts can also be accurately determined. By contrast, the method presented here seeks state-of-the-art performance among models that have been trained end-to-end for inter-protein contact prediction.

      Strengths:

      The paper is carefully written and the method is very well detailed. The model works both for homodimers and heterodimers. The ablation studies convincingly demonstrate that the chosen model architecture is appropriate for the task. Various comparisons suggest that PLMGraph-Inter performs substantially better, given the same input, than DeepHomo, GLINTER, CDPred, DeepHomo2, and DRN-1D2D_Inter.<br /> The authors control for some degree of redundancy between their training and test sets, both using sequence and structural similarity criteria. This is more careful than can be said of most works in the field of PPI prediction.<br /> As a byproduct of the analysis, a potentially useful heuristic criterion for acceptable contact prediction quality is found by the authors: namely, to have at least 50% precision in the prediction of the top 50 contacts.

      Weaknesses:

      The authors check for performance drops when the test set is restricted to pairs of interacting proteins such that the chain pair is not similar *as a pair* (in sequence or structure) to a pair present in the training set. A more challenging test would be to restrict the test set to pairs of interacting proteins such that *none* of the chains are separately similar to monomers present in the training set. In the case of structural similarity (TM-scores), this would amount to replacing the two "min"s with "max"s in Eq. (4). In the case of sequence similarity, one would simply require that no monomer in the test set is in any MMSeqs2 cluster observed in the training set. This may be an important check to make, because a protein may interact with several partners, and/or may use the same sites for several distinct interactions, contributing to residual data leakage in the test set.

      The training set of AFM with v2 weights has a global cutoff of 30 April 2018, while that of PLMGraph-Inter has a cutoff of March 7 2022. So there may be structures in the test set for PLMGraph-Inter that are not in the training set of AFM with v2 weights (released between May 2018 and March 2022). The "Benchmark 2" dataset from the AFM paper may have a few additional structures not in the training or test set for PLMGraph-Inter. I realize there may be only few structures that are in neither training set, but still think that showing the comparison between PLMGraph-Inter and AFM there would be important, even if no statistically significant conclusions can be drawn.

      Finally, the inclusion of AFM confidence scores is very good. A user would likely trust AFM predictions when the confidence score is high, but look for alternative predictions when it is low. The authors' analysis (Figure 6, panels c and d) seems to suggest that, in the case of heterodimers, when AFM has low confidence, PLMGraph-Inter improves precision by (only) about 3% on average. By comparison, the reported gains in the "DockQ-failed" and "precision-failed" bins are based on knowledge of the ground truth final structure, and thus are not actionable in a real use-case.

    1. Reviewer #1 (Public Review):

      Summary:

      In this paper, Li and colleagues overcome solubility problems to determine the structure of FtsEX bound to EnvC from E. coli.

      Strengths:

      The structural work is well done and the work is consistent with previous work on the structure of this complex from P. aerugionsa.

      Weaknesses:

      The model does not take into account all information that the authors obtained as well as known in vivo data.

      The work lacks a clear comparison to the Pseudomonas structure highlighting new information that was obtained so that it is readily available to the reader.

      The authors set out to obtain the structure of FtsEX-EnvC complex from E. coli. Previously, they were unable to do so but were able to determine the structure of the complex from P. aeruginosa. Here they persisted in attacking the E. coli complex since more is known about its involvement in cell division and there is a wealth of mutants in E. coli. The structural work is well done and recapitulates the results this lab obtained with this complex from P. aeruginosa. It would be helpful to compare more directly the results obtained here with the E. coli complex with the previously reported P. aeruginosa complex - are they largely the same or has some insight been obtained from the work that was not present in the previous complex from P. aeruginosa. This is particularly the case in discussing the symmetrical FtsX dimer binding to the asymmetrical EnvC, since this is emphasized in the paper. However, Figures 3C & D of this paper appear similar to Figures 2D & E of the P. aeruginosa structure. Presumably, the additional information obtained and presented in Figure 4 is due to the higher resolution, but this needs to be highlighted and discussed to make it clear to a general audience.

      The main issue is the model (Figure 6). In the model ATP is shown to bind to FtsEX before EnvC, however, in Figure 1c it is shown that ADP is sufficient to promote binding of FtsEX to EnvC.

      The work here is all done in vitro, however, information from in vivo needs to be considered. In vivo results reveal that the ATP-binding mutant FtsE(D162N)X promotes the recruitment of EnvC (Proc Natl Acad Sci U S A 2011 108:E1052-60). Thus, even FtsEX in vivo can bind EnvC without ATP (not sure if this mutant can bind ADP).

      Perhaps the FtsE protein from E. coli has to have bound nucleotides to maintain its 3D structure.

    1. Reviewer #1 (Public Review):

      Summary:

      The authors present a new application of the high-content image-based morphological profiling Cell Painting (CP) to single cell type classification in mixed heterogeneous induced pluripotent stem cell-derived mixed neural cultures. Machine learning models were trained to classify single cell types according to either "engineered" features derived from the image or from the raw CP multiplexed image. The authors systematically evaluated experimental (e.g., cell density, cell types, fluorescent channels) and computational (e.g., different models, different cell regions) parameters and convincingly demonstrated that focusing on the nucleus and its surroundings contains sufficient information for robust and accurate cell type classification. Models that were trained on mono-cultures (i.e., containing a single cell type) could generalize for cell type prediction in mixed co-cultures, and describe intermediate states of the maturation process of iPSC-derived neural progenitors to differentiation neurons.

      Strengths:

      Automatically identifying single-cell types in heterogeneous mixed-cell populations holds great promise to characterize mixed-cell populations and to discover new rules of spatial organization and cell-cell communication. Although the current manuscript focuses on the application of quality control of iPSC cultures, the same approach can be extended to a wealth of other applications including an in-depth study of the spatial context. The simple and high-content assay democratizes use and enables adoption by other labs.

      The manuscript is supported by comprehensive experimental and computational validations that raise the bar beyond the current state of the art in the field of high-content phenotyping and make this manuscript especially compelling. These include (i) Explicitly assessing replication biases (batch effects); (ii) Direct comparison of feature-based (a la cell profiling) versus deep-learning-based classification (which is not trivial/obvious for the application of cell profiling); (iii) Systematic assessment of the contribution of each fluorescent channel; (iv) Evaluation of cell-density dependency; (v) Explicit examination of mistakes in classification; (vi) Evaluating the performance of different spatial contexts around the cell/nucleus; (vii) Generalization of models trained on cultures containing a single cell type (mono-cultures) to mixed co-cultures; (viii) Application to multiple classification tasks.

      I especially liked the generalization of classification from mono- to co-cultures (Figure 4C), and quantitatively following the gradual transition from NPC to Neurons (Figure 5H).

      The manuscript is well-written and easy to follow.

      Weaknesses:

      I am not certain how useful/important the specific application demonstrated in this study is (quality control of iPSC cultures), this could be better explained in the manuscript. Another issue that I feel should be discussed more explicitly is how far can this application go - how sensitively can the combination of cell painting and machine learning discriminate between cell types that are more subtly morphologically different from one another?

      Regarding evaluations, the use of accuracy, which is a measure that can be biased by class imbalance, is not the most appropriate measurement in my opinion. The confusion matrices are a great help, but I would recommend using a measurement that is less sensitive for class imbalance for cell-type classification performance evaluations. Another issue is that the performance evaluation is calculated on a subset of the full cell population - after exclusion/filtering. Could there be a bias toward specific cell types in the exclusion criteria? How would it affect our ability to measure the cell type composition of the population?

      I am not entirely convinced by the arguments regarding the superiority of the nucleocentric vs. the nuclear representations. Could it be that this improvement is due to not being sensitive/ influenced by nucleus segmentation errors?

      GRADCAM shows cherry-picked examples and is not very convincing.

      There are many missing details in the figure panels, figure legend, and text that would help the reader to better appreciate some of the technical details, see details in the section on recommendations for the authors.

    1. Reviewer #1 (Public Review):

      Summary:

      SUMO proteins are processed and then conjugated to other proteins via a C-terminal di-glycine motif. In contrast, the N-terminus of some SUMO proteins (SUMO2/3) contains lysine residues that are important for the formation of SUMO chains. Using NMR studies, the N-terminus of SUMO was previously reported to be flexible (Bayer et al., 1998). The authors are investigating the role of the flexible (referred to as intrinsically disordered) N-terminus of several SUMO proteins. They report their findings and modeling data that this intrinsically disordered N-terminus of SUMO1 (and the C. elegans Smo1) regulates the interaction of SUMO with SUMO interacting motifs (SIMs).

      Strengths:

      Among the strongest experimental data suggesting that the N-terminus plays an inhibitory function are their observations that<br /> (1) SUMO1∆N19 binds more efficiently to SIM-containing Usp25, Tdp2, and RanBp2,<br /> (2) SUMO1∆N19 shows improved sumoylation of Usp25,<br /> (3) changing negatively-charged residues, ED11,12KK in the SUMO1 N-terminus increased the interaction and sumoylation with/of USP25.

      The paper is very well-organized, clearly written, and the experimental data are of high quality. There is good evidence that the N-terminus of SUMO1 plays a role in regulating its binding and conjugation to SIM-containing proteins. Therefore, the authors are presenting a new twist in the ever-evolving saga of SUMO, SIMs, and sumoylation.

      Weaknesses:

      Much has been learned about SUMO through structure-function analyses and this study is another excellent example. I would like to suggest that the authors take some extra time to place their findings into the context of previous SUMO structure-function analyses. Furthermore, it would be fitting to place their finding of a potential role of N-terminally truncated Smo1 into the context of the many prior findings that have been made with regard to the C. elegans SUMO field. Finally, regarding their data modeling/simulation, there are questions regarding the data comparisons and whether manipulations of the N-terminus also have an effect on the 70/80 region of the core.

    1. Reviewer #1 (Public Review):

      Summary:

      In this manuscript, Ngo et al. report a peculiar effect where a single base mismatch (CC) can enhance the mechanical stability of a nucleosome. In previous studies, the same group used a similar state-of-the-art fluorescence-force assay to study the unwrapping dynamics of 601-DNA from the nucleosome and observed that force-induced unwrapping happens more slowly for DNA that is more bendable because of changes in sequence or chemical modification. This manuscript appears to be a sequel to this line of projects, where the effect of CC is tested. The authors confirmed that CC is the most flexible mismatch using the FRET-based cyclization assay and found that unwrapping becomes slower when CC is introduced at three different positions in the 601 sequence. The CC mismatch only affects the local unwrapping dynamics of the outer turn of nucleosomal DNA.

      Strengths:

      These results are in good agreement with the previously established correlation between DNA bendability and nucleosome mechanical stability by the same group. This well-executed, technically sound, and well-written experimental study contains novel nucleosome unwrapping data specific to the CC mismatch and 601 sequence, the cyclizability of DNA containing all base pair mismatches, and the unwrapping of 601-DNA from xenophus and yeast histones. Overall, this work will be received with great interest by the biophysics community and is definitely worth attention.

      Weaknesses:

      The scope and impact of this study are somewhat limited due to the lack of sequence variation. Whether the conclusion from this study can be generalized to other sequences and other bendability-enhancing mismatches needs further investigation.

      Major questions:

      (1) As pointed out by the authors, the FRET signal is not sensitive to nucleosome position; therefore, the increasing unwrapping force in the presence of CC can be interpreted as the repositioning of the nucleosome upon perturbation. It is then also possible that CC-containing DNA is not positioned exactly the same as normal DNA from the start upon nucleosome assembly, leading to different unwrapping trajectories. What is the experimental evidence that supports identical positioning of the nucleosomes before the first stretch?

      (2) The authors chose a constant stretching rate in this study. Can the authors provide a more detailed explanation or rationale for why this rate was chosen? At this rate, the authors found hysteresis, which indicates that stretching is faster than quasi-static. But it must have been slow and weak enough to allow for reversible unwrapping and wrapping of a CC-containing DNA stretch longer than one helical turn. Otherwise, such a strong effect of CC at a single location would not be seen. I am also curious about the biological relevance of the magnitude of the force. Can such force arise during nucleosome assembly in vivo?

      (3) In this study, the CC mismatch is the only change made to the 601 sequence. For readers to truly appreciate its unique effect on unwrapping dynamics as a base pair defect, it would be nice to include the baseline effects of other minor changes to the sequence. For example, how robust is the unwrapping force or dynamics against a single-bp change (e.g., AT to GC) at the three chosen positions?

      (4) The last section introduces yeast histones. Based on the theme of the paper, I was expecting to see how the effect of CC is or is not preserved with a different histone source. Instead, the experiment only focuses on differences in the unwrapping dynamics. Although the data presented are important, it is not clear how they fit or support the narrative of the paper without the effect of CC.

      (5) It is stated that tRNA was excluded in experiments with yeast-expressed nucleosomes. What is the reason for excluding it for yeast nucleosomes? Did the authors rule out the possibility that tRNA causes the measured difference between the two nucleosome types?

    1. Reviewer #1 (Public Review):

      Summary:<br /> TMC7 knockout mice were generated by the authors and the phenotype was analyzed. They found that Tmc7 is localized to Golgi and is needed for acrosome biogenesis.

      Strengths:<br /> The phenotype of infertility is clear, and the results of TMC7 localization and the failed acrosome formation are highly reliable. In this respect, they made a significant discovery regarding spermatogenesis.

      Weaknesses:<br /> There are also some concerns, which are mainly related to the molecular function of TMC7 and Figure 5. It is understandable that TMC7 exhibits some channel activity in the Golgi and somehow affects luminal pH or Ca2+, leading to the failure of acrosome formation. On the other hand, since they are conducting the pH and calcium imaging from the cytoplasm, I do not think that the effect of TMC7 channel function in Golgi is detectable with their methods. Rather, it is more likely that they are detecting apoptotic cells that have no longer normal ion homeostasis. Another concern is that n is only 3 for these imaging experiments.

    1. Reviewer #1 (Public Review):

      Among the many challenges in the cilia field, is the question of how multicellular organisms assemble a variety of structurally and functionally specialized cilia, including cilia of different lengths. This study addresses the important question of how ciliary length differences are established in vertebrates. Specifically, the authors analyzed the role of intraflagellar transport (IFT) in ciliary length regulation in zebrafish, exploiting the transparency of the embryos. Zebrafish possess functionally specialized motile and non-motile cilia in a variety of tissues. Expression of GFP-tagged IFT88, a component of the IFT-B subcomplex, in a corresponding mutant, allowed the authors to image IFT in five distinct types of cilia. They note that IFT moves faster in longer cilia. Tagging and imaging of the IFT-A protein IFT43 further support this observation. IFT speed was largely unaffected in knock-out and morphants targeting the BBSome, various kinesin-2 motors, and the posttranslational modifications of tubulin polyglycylation and polyglutamylation. Using high-resolution STED imaging, the authors observe that IFT signals (likely, corresponding to IFT trains) are smaller in the shorter spinal cord cilia compared to the long cristae cilia. Based on these observations, the authors test the hypothesis that larger IFT trains recruit more motors or coordinate the motors better, resulting in faster trains, and causing cilia to be longer. This is further tested using partial knock-down of IFT88-GFP, which resulted in shorter crista cilia, reduced IFT particle number, size, and velocity. Some parts of the manuscript show "negative" data (e.g., ciliary length and IFT are not affected by the loss of BBS4) but these add beautifully to the overall story and allow for additional conclusions such as the minor role of ttll3 and ccp knockouts on ciliary length in this model. This is an excellent study, which documents IFT in a vertebrate species and explores its regulation. The data are of high quality and support most of the conclusions.

      (1) The main hypothesis/conclusion is summarized in the abstract: "Our study presents an intriguing model of cilia length regulation via controlling IFT speed through the modulation of the size of the IFT complex." The data clearly document the remarkable correlation between IFT velocity and ciliary length in the different cells/tissues/organs analyzed. The experimental test of this idea, i.e., the knock-down of GFP-IFT88, further supports the conclusion but needs to be interpreted more carefully. While IFT particle size and train velocity were reduced in the IFT88 morphants, the number of IFT particles is even more decreased. Thus, the contributions of the reduction in train size and velocity to ciliary length are, in my opinion, not unambiguous. Also, the concept that larger trains move faster, likely because they dock more motors and/or better coordinating kinesin-2 and that faster IFT causes cilia to be loner, is to my knowledge, not further supported by observations in other systems (see below).

      (2) I think the manuscript would be strengthened if the IFT frequency would also be analyzed in the five types of cilia. This could be done based on the existing kymographs from the spinning disk videos. As mentioned above, transport frequency in addition to train size and velocity is an important part of estimating the total number of IFT particles, which bind the actual cargoes, entering/moving in cilia.

      (3) Here, the variation in IFT velocity in cilia of different lengths within one species is documented - the results document a remarkable correlation between IFT velocity and ciliary length. These data need to be compared to observations from the literature. For example, the velocity of IFT in the quite long (~ 100 um) olfactory cilia of mice is similar to that observed in the rather short cilia of fibroblasts (~0.6 um/s). In Chlamydomonas, IFT velocity is not different in long flagella mutants compared to controls. Probably data are also available for C. elegans or other systems. Discussing these data would provide a broader perspective on the applicability of the model outside of zebrafish.

    1. Reviewer #1 (Public Review):

      Summary:

      The study "Effect of alpha-tubulin acetylation on the doublet microtubule structure" by S. Yang et al employs a multi-disciplinary approach, including cryo-electron microscopy (cryo-EM), molecular dynamics, and mass spectrometry, to investigate the impact of α-tubulin acetylation at the lysine 40 residue (αK40) on the structure and stability of doublet microtubules in cilia. The work reveals that αK40 acetylation exerts a small-scale, but significant, effect by influencing the lateral rotational angle of the microtubules, thereby affecting their stability. Additionally, the study provided an explanation of the relationship between αK40 acetylation and phosphorylation within cilia, despite that the details still remain elusive. Overall, these findings contribute to our understanding of how post-translational modifications can influence the structure, composition, stability, and functional properties of important cellular components like cilia.

      Strengths:

      (1) Multi-Disciplinary Approach: The study employs a robust combination of cryo-electron microscopy (cryo-EM), molecular dynamics, and mass spectrometry, providing a comprehensive analysis of the subject matter.<br /> (2) Significant Findings: The paper successfully demonstrates the impact of αK40 acetylation on the lateral rotational angles between protofilaments (inter-PF angles) of doublet microtubules in cilia, thereby affecting their stability. This adds valuable insights into the role of post-translational modifications in cellular components.<br /> (3) Exploration of Acetylation-Phosphorylation Relationship: The study also delves into the relationship between αK40 acetylation and phosphorylation within cilia, contributing to a broader understanding of post-translational modifications.<br /> (4) High-quality data: The authors are cryo-EM experts in the field and the data quality presented in the manuscript is excellent.<br /> (5) Depth of analysis: The authors analyzed the effects of αK40 acetylation in excellent depth which significantly improved our understanding of this system.

      Weaknesses:

      I have no major concerns about this paper.