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    1. Reviewer #1 (Public Review): 

      Alexej Schatz and York Winter wrote "LabNet," a C++ tool to control Raspberry Pi (raspi) GPIO (General Purpose Input-Output) and other hardware using a network messaging protocol using protobuf. The authors were primarily concerned with performance, specifically low execution latencies, as well as extensibility to a variety of hardware. LabNet's network architecture is asymmetrical and treats one or many raspis as servers that can receive control signals from one or more clients. Servers operate as (approximately) stateless "agents" that execute instructions received in message boxes using a single thread or pool of threads. The authors describe several examples of basic functionality like time to write and read GPIO state to characterize the performance of the system, the code for which is available in a linked GitHub repository. 

      The described performance of LabNet is impressive, with near- or sub-millisecond latency across the several tests when conducted over a LAN TCP/IP connection. The demonstrated ability to interact with the server from three programming languages (C++, C#, and Python) also would be quite useful for a tool that intends to be as general-purpose as this one. The design decisions that led to the use of protobuf and SObjectizer seem sound and supportive of the primary performance goal. 

      As far as I'm concerned, the authors accomplished their goals and give a convincing demonstration in their performance tests. 

      The authors compare LabNet to: 

      - Whisker ( https://web.archive.org/web/20200222133946/http://egret.psychol.cam.ac.uk/whisker/index.shtml ), an aging proprietary experimental package typically sold along with purpose-built hardware; <br /> - pyControl and Bpod, both of which are open-source software frameworks for performing behavioral experiments using a specific combination of microcontrollers and an ecosystem of extension parts; <br /> - Autopilot, a software framework for performing behavioral experiments on the raspberry pi as well as modular development of hardware controllers and other common experimental components. 

      Each of these packages has a different enough scope and accompanying differences in design priorities that I think are worth noting to give context to the niche LabNet fills. For example, pyControl and Autopilot emphasize ease of use, pyControl and Bpod are built around state machines for controlling experiments, etc. All have some facility for designing and performing experiments themselves and are thus a bit "higher level" than LabNet, which is intended more as a GPIO and hardware control system specifically. I think LabNet is more aptly compared to something like pigpio (https://web.archive.org/web/20220130033233/https://abyz.me.uk/rpi/pigpio/) which is also a low-level GPIO control library with network control capabilities. In that respect LabNet fills at least two needs that aren't well-served by existing tools: first, it provides a means to extend the server with additional commands that can be exposed to multiple programming languages. Second, that lets users control additional hardware and implement custom logic aside from simple on/off commands (for example, the ability to output sound) - this would be particularly useful as a way of controlling HATs and other devices. LabNet's agent-based concurrency architecture also seems like it will allow the number of simultaneously controlled devices to scale well. LabNet's network-first design positions it well for behavioral experiments that are often better served by a swarm of networked computers rather than a single controlling computer. 

      The largest point of improvement that I expect will unfold over this project's development lifecycle will be its documentation. LabNet has no documentation to speak of, outside a brief description of the build process for a relatively voluminous body of code (~27k lines) with relatively few comments. There is no established norm as to what stage in a scientific software package's development a paper should be written, so I take the lack of documentation at this stage as just a sign that this project is young. The primary barrier for the broader landscape of scientific software is less that of availability of technically proficient packages, but the ease with which they can be adopted and used by people outside the development team. The ability of downstream researchers to use and extend the library to suit their needs will depend on future documentation. For example, at the moment the Python adapter to the client and server is present in the examples folder but relatively un-annotated, so it might be challenging to adapt to differing needs at the moment (https://github.com/WinterLab-Berlin/LabNet/blob/34e71c6827d2feef9b65d037ee5f2e8ca227db39/examples/python/perf_test/LabNetClient_pb2.py and https://github.com/WinterLab-Berlin/LabNet/blob/34e71c6827d2feef9b65d037ee5f2e8ca227db39/examples/python/perf_test/LabNetServer_pb2.py ). Documentation for projects like this that aim to serve as the basis from which to build experimental infrastructure can be quite challenging, as it often needs to spread beyond the package itself to more general concerns like how to use Raspberry Pis, how to set them up to be available over a network, and so on, so I look forward to seeing the authors meet that challenge. 

      I would like to thank the authors for their work and thank them for bringing us a fast way to control experimental hardware over the network.

    2. Reviewer #2 (Public Review): 

      The manuscript introduces LabNet as a network-based platform for the control of hardware in Neuroscience. The authors recognize and attempt to address two fundamental problems in constructing systems neuroscience experiments: on one hand the importance of precise timing measurements of behavior; on the other hand, the need for flexibility in experimental design. These two goals are often at great odds with each other. Precise timing is more easily achieved when using fewer, dedicated homogeneous devices such as embedded microcontrollers. Flexibility can be found in the diversity of devices and programming languages available for commercial personal computers, but this often comes at the cost of a non-real-time operating system, where timing can be much harder to predict accurately. There is also a limitation on the number of devices which can be simultaneously controlled by a single processor, which can be an impediment for high-throughput behavior studies where the ability to run dozens of experiments in parallel is desirable. 

      LabNet proposes to address this tension by focusing on the design of a pure hardware control and instrumentation layer implemented on top of the Raspberry Pi family of microprocessors. The idea is to keep coordination of experimental hardware in a central computer, but keep time-critical components at the edge, each node running the same control software in a Raspberry Pi to provide precise timing guarantees. Flexibility would be provided by the ability to connect an arbitrary number of nodes to the central computer using a unified message-passing protocol by which the computer can receive events and send commands to each node. 

      The authors propose the use of the C++ programming language and the actor-model as a unifying framework for implementing individual nodes and present a series of benchmarks comparing their system against other established hardware control platforms. 

      The idea of keeping time-critical components at the edge, and the use of network communication protocols, and in particular message-passing systems such as the actor-model, to scale up experimental control is reasonable. These principles have undoubtedly been very successful in enabling the creation of massively distributed systems such as web applications connecting millions of devices to each other every second. 

      The Design section then introduces the actor model, the C++ library SObjectivizer used to implement it, and the binary message protocol used for transmission of data across nodes. As currently written, however, this section seems overly technical and hard to grasp for readers who might be interested in experimental neuroscience, but who lack the expertise to understand all mentioned functional constructs and required expertise in the C++ language. Several concepts are mentioned only in passing and without introductory references for the non-expert reader. The level of detail also seems to distract from conveying a more meaningful understanding of the remaining trade-offs involved between network communication, latency, synchronization, and bandwidth. 

      The essence of the actor-model could probably be captured more succinctly, and more time spent discussing some of these critical decisions underlying LabNet's design principles. For example, although each Raspberry Pi device runs a LabNet server, the current implementation allows only one client connection per node. This might be surprising for some readers as it excludes a large number of possible network topologies, and the reason presented for the design decision as currently detailed is hard to understand without further clarification. 

      The main method for evaluating the performance of LabNet is a series of performance tests in the Raspberry Pi comparing clients written in C++, C# and Python, followed by a series of benchmarks comparing LabNet against other established hardware control platforms. While these are undoubtfully useful, especially the latter, the use of benchmarking methods as described in the paper should be carefully revisited, as there are a number of possible confounding factors. 

      For example, in the performance tests comparing clients written in C++, C# and Python, the Python implementation is running synchronously and directly on top of the low-level interface with system sockets, while the C++ and C# versions use complex, concurrent frameworks designed for resilience and scalability. This difference alone could easily skew the Python results in the simplistic benchmarks presented in the paper, which can leave the reader skeptical about all the comparisons with Python in Figure 3. Similarly, the complex nature of available frameworks also raises questions about the comparison between C# and C++. I don't think it is fair to say that Figure 3 is really comparing languages, as much as specific frameworks. In general, comparing the performance of languages themselves for any task, especially compiled languages, is a very difficult topic that I would generally avoid, especially when targeting a more general, non-technical audience. 

      The second set of benchmarks comparing LabNet to other established hardware control platforms is much more interesting, but it doesn't currently seem to allow a fair assessment of the different systems. Specifically, from the authors' description of the benchmarking procedure, it doesn't seem like the same task was used to generate the different latency numbers presented, and the values seem to have been mostly extracted from each of the platform's published results. This unfortunately reduces the value of the benchmarks in the sense that it is unclear what conditions are really being compared. For example, while the numbers for pyControl and Bpod seem to be reporting the activation of simple digital input and output lines, the latency presented for Autopilot uses as reference the start of a sound waveform on a stereo headphone jack. Audio DSP requires specialized hardware in the Pi which is likely to intrinsically introduce higher latency versus simply toggling a digital line, so it is not clear whether these scenarios are really comparable. Similarly, the numbers for Whisker and Bpod being presented without any variance make it hard to interpret the results. 

      One of the stated aims of LabNet was to provide a system where implementing new functionality extensions would be as simple as possible. This is another aspect of experimental neuroscience that is under active discussion and where more contributions are very much needed. Surprisingly, this topic receives very little attention in the paper itself. It is not clear whether the actor model is by itself supposed to make the implementation of new functionality easier, but if this is the case, this is not obvious from the way the design and evaluation sections are currently written, especially given the choice of language being C++. 

      One of the reasons behind the choice of Python for other hardware platforms such as pyControl and Autopilot is the growing familiarity and prevalence of Python within the neuroscience research community, which might assist researchers in implementing new functionality. Other open-hardware projects in neuroscience allowing for community extensions in C++ such as Open-Ephys have informally expressed the difficulty of the C++ language as a point of friction. I feel that the aim of "ease of extensibility" should merit much more discussion in any future revision of the paper. 

      Indeed, they only mention in passing that user extensibility is in the conclusion where it is stated that it is not currently possible to modify LabNet without directly modifying and recompiling the entire code base. A software plug-in system is suggested, and indeed this would be extremely beneficial in achieving the second stated aim. 

      Finally, a set of example experimental applications would have been extremely useful to ground the design of LabNet in practical terms, in addition to the example listings. Even in diagrammatic form, describing how specific experiments have been powered by LabNet would give readers a better sense of the kind of designs that might be currently more appropriate for this platform. For example, video is being increasingly used in behavioral experiments, and Raspberry Pi drivers are available for several camera models, but this important aspect is not mentioned at all in the discussion, so readers interested in video would not know from reading this paper whether LabNet would be appropriate for their goals. 

      As the manuscript currently stands, I don't feel the authors have achieved their second stated aim, and I am unfortunately not fully convinced that the experimental results are adequate to demonstrate the achievement of the first aim. I fully agree, however, that a robust, high-performance and flexible hardware layer for combining neuroscience instruments is desperately needed, and so I do expect that a more thorough treatment of the methods developed in LabNet will in the future have a very positive impact on the field.

    1. Reviewer #1 (Public Review): 

      In this manuscript, the authors test their previously proposed model (also presented in Figure 1A) that ImuB interacts with the DnaN DNA polymerase III β clamp to recruit DnaE2. The previously identified mutasome components ImuA', ImuB, and DnaE2 and essential for DNA-damage induced mutagenesis. Although the exact function of ImuA' and ImuB is unknown, ImuB has long before been proposed to interact with DnaN via an interaction domain within ImuB that has already been identified. Since the experiments herein test and validate a well-establish model, the results are somewhat expected. However, all models should be tested experimentally, making this an important confirmation. The manuscript nicely makes use of both in vivo and in vitro approaches and the data is convincing for the most part, although the inability of the fusion proteins used in this study to complement the knockout strains during exposure to the DNA damaging agent MMC does raise an important limitation of the tools used herein. The major concern is the limited new biological insight gained from the study.

    2. Reviewer #2 (Public Review): 

      The imuABC genes from Caulobacter crescentus were first described almost 20 years ago, yet very little biochemical analysis regarding their mechanism of action has been published. It is now appreciated that these imuABC genes are present in a large number of clinically important bacterial pathogens, underscoring their potential importance to adaptation and antibiotic resistance. The imuA gene in M. tuberculosis is somewhat diverged from imuA in C. crescentus (the first described ImuABC), and is thus named imuA' to reflect this. The goal of the work described in this report was to gain further insights into the function of the M. tuberculosis ImuA', ImuB, and ImuC proteins in DNA damage-induced mutagenesis. The authors used fluorescent fusions of the M. tuberculosis imuA'BC gene products to demonstrate that ImuA', ImuB, and ImuC each colocalized with the beta sliding clamp protein in live cells. The beta clamp helps to organize replication and repair proteins on the DNA at replication forks; thus their colocalization implies functional complexes. These in vivo results were correlated with biochemical results in which they used size exclusion chromatography to measure interactions between the purified beta sliding clamp protein and the ImuB protein or ImuA'-ImuB protein complex. These biochemical results support earlier published results describing these interactions using yeast-two-hybrid (Warner et al., 2010, PNAS). Importantly, the authors also demonstrate that both a mutant ImuB containing a disruption to its beta clamp binding motif, as well as griselimycin (GSR), an antimicrobial that binds the beta clamp at the same site required for its interaction with the clamp binding motif, both impaired ImuB-beta clamp interactions in vitro, and resulted in the loss of their colocalization in vivo. A potentially powerful conclusion of this work is that adjuvant therapies such as GSR may inhibit mutagenesis, limiting M. tuberculosis drug resistance, facilitating the ability of traditional antimicrobial therapies to treat the infection. While the work has several strengths, there are also some shortcomings. 


      1) The authors describe fluorescent fusions of the M. tuberculosis ImuA', ImuB, and ImuC proteins that support ImuABC function in UV-induced mutagenesis. Using these fusions, they demonstrate colocalization of the different Imu proteins and the beta clamp in live cells following treatment with MMC. 

      2) This is the first report of purified ImuA' and ImuB, which allowed the authors to biochemically test for their interactions with each other, as well as interaction of ImuB with the beta clamp, both of which were previously reported based on results of yeast-two-hybrid experiments (Warner et al., 2010, PNAS). This, together with the live cell imaging work, goes a long way towards testing and refining the model for ImuA'BC 'mutasome' function first proposed in 2010 (Warner et al., PNAS). 

      3) The authors provide in vitro results suggesting GSR, in addition to its known role in blocking replication, may also inhibit mutagenesis in M. tuberculosis. This is an exciting possibility, and provides support for the view that anti-evolution therapies may be possible. 


      1) Colocalization studies were performed using ImuA' and ImuB fluorescent fusions that failed to complement MMS sensitivity. Given their in vivo inactivity of these fusions, what does their colocalization with the beta clamp actually mean? Since these same fusions supported UV mutagenesis, it seems UV may be a superior means of analyzing colocalization of these fluorescently tagged proteins. 

      2) The methods used to analyze the colocalization is not explained for non-experts. A more complete description of how colocalization was established along the z-axis is needed. Likewise, a more thorough discussion of exactly what the foci consist of is needed. 

      3) ImuC could not be purified for in vitro analysis. This is unfortunate since it is thought to harbor the polymerase activity involved in mutagenesis. While not the fault of the authors, this significantly limits the scope of the in vitro work regarding ImuA'BC function in mutagenesis. 

      4) The in vitro analysis of ImuA'-ImuB, ImuB-beta clamp, and ImuA'-ImuB-beta clamp interactions lack quantitative descriptions, and a complete analysis of the possible interactions among these proteins was not explored, limiting our understanding of their possible function in mutagenesis.

    3. Reviewer #3 (Public Review): 

      In this study, Gessner et al., characterize the localization dynamics of the mycobacterial mutasome complex, comprising ImuA', ImuB and DnaE2, in order to understand the molecular composition and mechanism of action of this complex in living cells. For this, they construct semi-functional fluorescent fusion constructs of ImuB (as well as ImuA' and DnaE2) in M. smegmatis, a non-pathogenic model system used to study several pathways present in M. tuberculosis. They find that ImuB localizes with the beta-clamp upon damage exposure. They further show that the clamp binding motif in ImuB is essential for its localization as well as in vitro beta-clamp interaction. Finally, they treat cells with the beta-clamp targeting antibiotic griselimycin and find that it abrogates ImuB interaction with the clamp. They suggest that ImuB localization (or its disruption) can serve as a reliable proxy to screen for mutasome-inhibiting antibacterial drugs. 


      The in vivo dynamics of the ImuA'-ImuB-DnaE2 complex is not well-studied, when compared with its E. coli counterpart UmuDC-RecA. In this direction, the authors generate a set of tools that can be utilized to understand the molecular mechanism of action of this complex in detail. The distinct localization dynamics of ImuA' and ImuB is of particular interest, given the several unresolved questions associated with ImuA' function in induced mutagenesis. 


      The strength of the manuscript lies in the imaging data, which is rich with information with regards to the dynamics of localizations over time for clamp, ImuB and DnaE2, under damage. In the current form, the authors do not utlize this to provide insights into any "real-time" dynamics of the mutasome. The results with regards to the clamp interaction are well-done, but largely confirmatory. Importantly, it remains unclear whether the localization read-out is reliably indicative of mutasome function, given the discrepancy in performance of the constructs in UV vs MMC-induced damage. The authors primarily rely on MMC for all their in vivo read-outs and the tags do not perform as wild type in this damage condition. This needs to be resolved.

    1. Reviewer #1 (Public Review):

      Streptococcus pyogenes expresses coiled-coil M proteins that interact with host proteins to promote virulence. One example is M protein association with antimicrobial peptide LL-37. However, since the coiled-coil M proteins lack canonical protein-protein interaction sites, it remained unclear how these interactions occurred. In this manuscript, the authors solved a crystal structure of a complex of LL-37 with the M protein M87. The M87 coiled coil unfurled to exposed its hydrophobic core to interact with LL-37. The authors then aim to show that this mechanism contributed to LL-37 resistance of S. pyogenes. These studies have provided important new information regarding the mechanism of interaction between coiled coil proteins and the alpha helical LL-37. Overall, this is an interesting and convincing manuscript. The only major concern relates to the conclusions made based on the E to R substitutions when alanine substitutions yielded no effect on the interactions. The interpretation would be that the electrostatic interaction with the Lysine in LL-37 is not important for the association of M87 with LL-37. The E85R mutant could be causing repulsion effects that are dominant negative. These and associated caveats raise concern that some data may be over-interpreted or requires further analysis.

    2. Reviewer #2 (Public Review):

      This is an excellent paper on an original and exciting discovery from the Ghosh group. The paper is also nicely and concisely written and in a style that will be accessible to a wide audience.

      In short, the paper shows that the dimeric coiled coil of a bacterial surface (M) protein can open up to trap a human antimicrobial peptide by forming a new 2:1 heteromeric 3-helix bundle. This is a really neat discovery, which has clear implications for understanding how M proteins protect bacteria against attack by the hosts, and it could also inspire protein designers and synthetic biologists to design mimetic systems along these lines.

    1. Reviewer #1 (Public Review): 

      In this article Farrell et al. leverage existing datasets which measure frailty longitudinally in mice and humans to model 'robustness' (the ability to resist damage) and 'resilience' (the ability to recover from damage), their dynamics across age, and their relative contributions to overall frailty and mortality. The concept of separating damage/robustness from recovery/resilience is valid and has many important applications including better assessment and prediction of effective intervention strategies. I also appreciate the authors' sophisticated attempts to effectively model longitudinal data, which is a challenge in the field. The use of human and mouse data is another strength of the study, and it is quite interesting to see overlapping trends between the two species. 

      While I find the rationale sound and appreciate the approach taken at a high level, there are a few key considerations of the specific data used which are lacking. The authors conceptualize resilience based on studies which primarily use short time scales and dynamic objective measures (ex. complete blood cell counts in Pyrkov et al.) often in conjunction with an acute stress stimulus. For example, they heavily cite Ukraintseva et al. who define resilience as "the ability to quickly and completely recover after deviation from normal physiological state or damage caused by a stressor or an adverse health event." 

      Given these definitions, the human data used seem to fit within this framework, but we should carefully consider the mouse data. The mouse frailty index is a very useful tool for efficiently measuring the organismal state in large cohorts. A tradeoff for quickly measuring a broad range of health domains is that the individual measurements are low resolution (categorical) and involve inherent subjectivity (which may be considered part of the measurement error). Some transitions in individual components are due to random measurement error and I believe this is especially likely with decreases (or 'resilience' transitions). 

      The reason I think the resilience transitions are subject to high measurement error is that I am skeptical as to whether many of the deficits in the mouse index are reversible under normal physiologic conditions. For example, it is exceptionally unlikely for a palpable/visible tumor to resolve in an aged mouse over the time scales studied here, thus any reversal that was observed is very likely due to random measurement error. Other components which I have doubts about reversibility are alopecia, loss of fur color, loss of whiskers, tumors, kyphosis, hearing loss, cataracts, corneal capacity, vision loss, rectal prolapse, genital prolapse. 

      In summary, I applaud the authors' efforts in generating complex models to better understand longitudinal aging data. This is an important area that needs further development. I appreciate their conceptualization of resilience and robustness and think this framework has an important place in aging research. I also appreciate their cross-species approach. However, the authors may have over-conceptualized and made some assumptions about the mouse data which may not be valid. It will be important to assess the results with careful consideration of the time scales of the underlying biology and the resolution and measurement error inherent to these tools.

    2. Reviewer #2 (Public Review): 

      This study uses repeated measurements of the frailty index (FI), composed of multiple binary parameters. It is posited that newly detected changes in the number of these parameters represent damage and that the parameters that have previously been detected but are not detected currently represent damage repair. Statistical treatment then follows, deriving resilience and robustness and their changes over time. This is an interesting idea. Strengths of the study include analyses across species (mice and humans), including multiple datasets in mice. 

      What are the elements of FI that increase at each period of life, and what are those that decrease? For example, humped phenotype or alopecia are more likely to appear in old mice and are essentially irreversible, whereas weight loss due to infection may be more common in young mice and is reversible. Therefore, the choice of health deficits would affect the model and, for example, may artificially lead to a decreased value of what the authors call damage repair. 

      More generally, information on the frailty index lacks sufficient details. I doubt that this method has sufficient accuracy to draw conclusions from as little as 32 female mice (21 + 11 animals in datasets 1 and 2) and 63 males (13 + 6 + 44 animals in datasets 1, 2 and 3). Also, only 25 enalapril-treated mice of each sec were analyzed, and only 17 exercised mice (11 females and 6 males). The number of human participants is large, but the total follow-up period is not shown, and the subjects were assessed based on 23 parameters only. 

      A key assumption in this work is that increased FI is equivalent to the rise in damage. However, the relationship between changes in FI and damage is unknown. One can imagine a situation when damage increases, but protection also increases. In this case, fitness may increase, decrease or remain unchanged. What is the basis for calling an increased number of health deficits damage? Is there a more reliable method to measure damage that could support the authors' claims?

    3. Reviewer #3 (Public Review): 

      In this work, the authors aimed at investigating two related components of aging-related processes of health deficits accumulation in mice and humans: the processes of damage (representing the robustness of an organism) and repair (corresponding to resilience), and at determining how different interventions (the angiotensin-converting enzyme inhibitor enalapril and voluntary exercise) in mice and a representative measure of socio-economic status (household wealth) in humans affect the rates of damage and repair. Two key elements in this study allowed the authors to achieve their goals: 1) the use of relevant data containing repeated measurements of health deficits from which they were able to compute the cumulative indices of health deficits in mice and humans and which are also necessary to evaluate the processes of damage and repair; 2) the methodological approach that allowed them to formulate the concepts of damage and repair, model them and estimate from the available data. This methodological framework coupled with the data resulted in important findings about the contribution of the age-related decline in robustness and resilience in health deficits accumulation with age and the differential impact of interventions on the processes of damage and repair. This provides important insights into these key components of the process of aging and this research should be of interest to both lab researchers who plan experimental studies with laboratory animals to study potential mechanisms and interventions affecting health deficits accumulation as well as researchers working with human longitudinal studies who can apply this approach to further investigate the impact of different factors on robustness and resilience and their contribution to the overall health deterioration, onset of diseases and, eventually, death. 

      The key strength of this work is a rigorous analytic approach that includes joint modeling of longitudinal measurements of health deficits and mortality (in mice). This approach avoids biased inference which would be observed if longitudinal data were analyzed alone, ignoring attrition due to mortality. Another strength is a comprehensive analysis of both laboratory animal data that allows exploring the impact of different interventions on the processes of damage and repair and human data that allows investigating disparities in these processes in individuals with different socioeconomic backgrounds (represented by household wealth). 

      One weakness (which is commonplace for human studies) is self-reported data on health deficits in humans which makes it difficult to compare with lab data where deficits are assessed objectively by lab researchers. The subjective nature of health deficits measurements complicates the interpretation of findings, especially about repairs of deficits. In addition, it is not clear whether the availability/absence of caregivers at different exams/interviews factors into the answers on difficulty/not difficulty with specific activities constituting health deficits and, respectively, into their change over time reflected in damage/repair estimates.

    1. Reviewer #1 (Public Review): 

      Accumulating evidence indicates RNA N6-adenosine methylation as an important post-transcriptional RNA modification in the regulation of gene expression, organ development, and disease development. However, the role of m6A mRNA methylation in cardiomyocyte proliferation and heart regeneration in normal development and in heart injury is not known. The authors first identified increased m6A mRNA levels during heart regeneration following injury to the neonatal heart, in association with selectively up-regulated expression of Mettl3, the methyltransferase catalyzing RNA N6-adenosine methylation, a finding suggesting a potential role of m6A in heart regeneration. Using cardiomyocyte cell lines, primary cardiomyocytes, and neonatal heart regeneration models, the authors next showed that down-regulation of Mettl3 markedly increased cardiomyocyte proliferation and heart regeneration in association with expected down-regulation of m6A mRNA levels in cardiomyocytes. This effect was selective in the injured neonatal heart. Together, the data indicate an important role of Mettl3 in the regulation of cardiomyocyte proliferation and heart regeneration. The quality of the data is high and the conclusions are convincing. 

      Next, the authors assessed the role of Mettl3 in heart regeneration and tissue repair at non-regenerative stages in postnatal mouse models and even in adult mice. Down-regulation of Mettl3 expression improved heart regeneration and tissue repair, in association with cardiomyocyte proliferation and improved cardiac functions. The role of Mettl3 was also assessed using Mettl3 overexpression and largely opposite effects relative to the effects of down-regulation of Mettl3 expression were detected. The authors attributed the effect of down-regulated Mettl3 expression to its impact on the regulation of Fgf16 expression. This is supported by the finding that Mettl3 down-regulation was associated with decreased Fgf16 mRNA m6A methylation and increased Fgf16 mRNA levels. Finally, the authors assessed the role of Fgf16 in heart regeneration by introducing the expression of wild type vs mutant Fgf16, with the latter having m6A consensus sequence deleted. The mutant Fgf16 increased heart regeneration in neonatal heart injury models, with increased cardiomyocyte proliferation and improved cardiac function. Overall, the authors have identified a novel mechanism for regulating cardiomyocyte proliferation and heart regeneration during heart injury. There is an impressive amount of rigorous data. It is a significant contribution to bring all these mechanisms together in the context of cardiomyocyte proliferation and heart regeneration. However, in some cases, the claims are probably overstated based on the data shown. Some of the findings are inconsistent with the interpretations. There are places where additional evidence is required in order to justify the claims.

    2. Reviewer #2 (Public Review): 

      The manuscript identified m6A RNA methylation (via increased m6A writer, Mettl3 expression) as a critical regulator of cardiomyocyte proliferation during the initial regenerative window that was proposed earlier in the mouse heart. Although these processes are developmentally induced, the results of the manuscript show Mettl3-dependent m6A RNA modifications as a negative regulator of cardiomyocyte proliferation and cardiac regeneration. The authors have comprehensively profiled Mettl3 expression and Mettl3-dependent m6A regulation during cardiac regeneration using a variety of in vivo models (both neonatal heart development and post-MI injury) as well as using in vitro primary cardiomyocytes to identify Fgf16 as a key downstream mRNA transcript for m6A RNA modification by Mettl3 to further show that m6A-dependent cytoplasmic decay of Fgf16 mRNA in a Ythdf2-dependent pathway as the key underlying mechanism regulating cardiac regeneration in these models. Overall, a well-thought-out study that reports exciting new data that shows suppression of a developmentally induced phenomenon as a therapeutic option for inducing cardiac regeneration. 

      Strengths of the manuscript:

      The manuscript investigates an important topic relevant to cardiac regeneration, which carries great clinical significance given that the cardiomyocyte turnover, as well as the processes for replacement of cardiomyocyte loss following MI injury, are limited. Therefore, any major discoveries to enhance the regenerative ability of the heart is critical to treating cardiac disease and this manuscript underscores a previously unrecognized mechanism (m6A modification) as a critical regulator of cardiomyocyte proliferation and cardiac regeneration. 

      The manuscript's major finding is the identification of developmental induction for Mettl3/m6A within the cardiac regenerative window (p1-p7). Another critical finding is that modulation of Mettl3 proves to be a negative regulator of cardiomyocyte proliferation and cardiac regeneration. 

      In the therapeutic setting, using post-MI mouse models, the manuscript further shows that targeting Mettl3 can enhance cardiac regeneration by specific effects on cardiomyocyte proliferation than on non-cardiomyocytes. 

      At the molecular level, the authors have carefully looked at Mettl3/m6a pathways in cardiomyocytes and non-cardiomyocyte proportions of the heart to show that Mettl3 is a critical mediator of cardiomyocyte hypertrophy as well as their proliferation in non-cardiomyocytes thus strengthening their conclusion that m6A process is being regulated in cardiomyocytes thus having a direct impact on cardiac regeneration. 

      Furthermore, the manuscript characterizes the downstream mRNA targets of Mettl3-mediated m6A modifications and found that Fgf16 mRNA is a critical target for m6A modification by Mettl3, which in turn leads to m6A-dependent degradation for Fgf16 via YTHDF2 pathway. 

      Taken together, the manuscript provides critical evidence demonstrating Mettl3-dependent m6A pathways regulating cardiomyocyte proliferation and heart regeneration. 

      Weaknesses of the manuscript:

      It would be more appreciated if the manuscript can provide more insights and rationale by discussing why a developmentally induced Mettl3/m6A phenomenon (i.e. induced during normal developmental stages as well as post apical resection injury) would turn out to be a negative regulator of cardiac regeneration, than having a positive impact on cardiac regeneration.

    1. Reviewer #1 (Public Review): 

      The authors characterized the expression of DDR2 in the developing craniofacial skeleton. The authors showed that Ddr2-deficient mice exhibited defects in craniofacial bones including impaired calvarial growth and frontal suture formation, cranial base hypoplasia due to aberrant chondrogenesis, and delayed ossification at growth plate synchondroses. The histological studies are well done. However, the studies as shown in this manuscript do not provide cellular and molecular mechanisms beyond what is already known, particularly beyond what the authors have already published in a similar study in Bone Research (Mohamed et al., 2022 Feb 9;10(1):11). With the same Cre lines and analytic approaches, the authors already showed in the Bone Research paper that Ddr2 in the Gli1+ cells is required for chondrocyte proliferation and polarity in growth plate development and osteoblast differentiation. Cartilage development and bone formation occur in both long bones and craniofacial skeleton, the authors showed similar functions of Ddr2 in similar skeletal tissues, although the location is different. One new point in this manuscript might be: the authors indicated that loss of Ddr2 led to ectopic chondrocyte hypertrophic (Fig. 7I). But what the data actually showed was delayed chondrocyte hypertrophy and abnormal location of the delayed hypertrophic chondrocytes, which could be well caused by abnormal chondrocyte polarity. This interesting defect was superficially described with no mechanistic investigation at cellular or molecular level.

    2. Reviewer #2 (Public Review): 

      DDR2 is a collagen-binding receptor that is required for proper skull development. Ddr2 loss-of-function in humans is associated with the developmental disease spondylo-meta-epiphyseal dysplasia (SMED). Here, the authors aim to elucidate the role of DDR2 in skull development. In this work, the role of DDR2 in skull and face development is studied in mice, which exhibit SMED-like symptoms in the absence of Ddr2. Histological studies showed that Ddr2 knockout disrupts organization and proper differentiation within progenitor-rich regions of the skull from which bone growth occurs. Histology and lineage tracing studies revealed that DDR-expressing cells in/around these zones 1) generally also express the proliferation regulator Gli1, and 2) eventually contribute to osteogenic and chondrogenic lineages. Cell-type specific knockout studies were used to show that DDR2 has a development-specific role: knockout of Ddr2 in Gli+ cells re-capitulated the developmental abnormalities observed in global Ddr2 knockout mice; knockout in chondrocytes partially recapitulated developmental abnormalities, and osteoblast-specific knockout mice were indistinguishable from their wild-type littermates. This work also catalogues the locations of Ddr2 positive cells and their lineages at various stages of development. Additionally, the anatomical effects of loss of DDR2 function on skull and face development are thoroughly described in global and cell-type specific knockouts. 

      This work is a vital and stimulating contribution to the scientific literature. The authors' claims and conclusions are well supported by the evidence they present. 

      The scientific approach is sound and the conclusions important. However, a limitation of the work's discussion is a lack of attention paid to the specific biophysical mechanism that DDR2 is playing during development. The discussion of the positioning of the golgi is nice, but a lack of golgi polarity is likely a downstream effect of processes occurring within the cell adhesion and mechanotransduction machinery. Perhaps, like integrins, DDR2 is a mechanosensor that the cell needs to properly sense local collagen orientation, polarize, and secrete properly-organized COL2. It would be beneficial to put up some guideposts that will facilitate engagement from the molecular biophysics/mechanobiology community.

    3. Reviewer #3 (Public Review): 

      From this work, the authors investigated a number of parameters in order to profoundly understand and demonstrate the vital role of ongoing interaction between components of extracellular matrix and particular stem cells to induce normal Craniofacial development. Thus, there was a focus on the genetic manipulation (knockout) impact of molecules behind the above-mentioned interaction, and on determining how such modification would be reflected on skull bone morphogenesis. 

      Strengths and Weaknesses:

      • Using different animals' backgrounds in the same experiment might impact work outcomes.

      • Better to have (ethical approval) at the beginning of the material and methods in separate paragraphs.

      • It is great that the authors precisely explain all the measurements.

      • Supplementary file to have details of used antibodies might be required.

      • All methods have been written in academic and clear ways.

      • It is nice that there is a conclusion sentence by end of the results paragraph, which made it easy for readers to fully remember and understand.

      • It is possible to see a reduction in proliferative chondrocyte, with no change in apoptosis rate?

      • Results are supposed to be compatible.

      • Very nice and representative images from the immunofluorescence protocol.

      • Using different techniques to confirm observations is clearly manifested in methods and results.

      It is clear that the author has used different methods and techniques in order to meet his work's objectives. Importantly, there was more than one procedure to confirm observations that are related to one or more than one aim. 

      Although determining to what extent the outcomes of this work could be applied to community need might require a subspecialist physician's opinion, it seems that observations of the present study are likely to require a series of further investigations in order to take it to the level of human users. Notably, identification of molecules and pathways behind skull development abnormalities would open a door to early diagnosis reasons for such deformities, thus mitigating future abnormalities either by developing new prevention methods or discovering unique medications.

    1. Reviewer #1 (Public Review):

      The MS by Mehta et al, reports the first full-length structure of a polytopic membrane adenylyl cyclase from M. tuberculosis (Rv1625c/Cya). The authors used detergent-purified full-length Cya mixed with a stabilizing nanobody and determined the structure using cryo-EM yielding a 3D reconstruction at 3.8 Å resolution. Full-length Cya (443aa) consists of an N-terminal domain followed by 6 transmembrane (TM) helices, and a helical domain (HD) that connects to the catalytic domain. The full-length protein was active as a dimer and not affected by the presence of the nanobody. In contrast, a soluble form consisting of only HD-cat was not active. The structure of the full-length Cya shows a complex with three molecules of the nanobody, two bound symmetrically to the cytosolic domains and a third one bound asymmetrically to the extracellular surface. The density map covered residues 41-428 of the full-length Cya. The overall topology of each monomer in the Cya dimer corresponded to approximately half of the only known structure of the full-length mammalian membrane-bound AC9, previously solved by the same group. The overall conservation among the bacterial and mammalian cyclases is remarkable. The structure unmasked novel pockets (some conserved with AC9), allowing the authors to suggest a function for the TM bundle as an organizer of the HD for efficient communication to the catalytic domain, and more speculative, a role for the TM, via one of the identified pockets, to act as a putative receptor for yet unidentified ligands able to transduce information via the HD towards enzyme activation.

      The overall conclusions of this manuscript are well supported by the data, and although some are speculative, they provide a predictive framework for future studies aiming at biochemical validation.

    2. Reviewer #2 (Public Review):

      Mehta et al report a cryo-EM structure of membrane-bound adenylyl cyclase from Mycobacterium tuberculosis. The structure is a dimer, each subunit consisting of a 6TM membrane domain, a helical domain, and a cytoplasmic adenyl cyclase domain. The overall shape of the dimer is similar to the monomeric (12 TM) mammalian adenylyl cyclase AC9, although there is a swap in the position of the helices that link the TM domain to the helical domain.

      An intriguing question is what role the TM domains play in membrane-bound adenylyl cyclases. The authors speculate that they may provide binding sites for ligands and describe 4 different pockets (2 extracellular, 2 cytoplasmic). One extracellular pocket contains the density of a metal ion. Mutation of this pocket reduces the activity of the cytoplasmic catalytic domain and induces conformational changes in the whole cytoplasmic region. This provides some evidence for changes in the TM domain being propagated to control cytoplasmic catalytic activity.

      In summary, the work is an important structure of bacterial membrane-bound adenylyl cyclase. The description of potential binding pockets is interesting in the step towards understanding the role of the membrane domain although as yet the lack of a clear physiological ligand makes this role still an open question.

    1. Reviewer #1 (Public Review):

      In this online randomized clinical trial, Sudharsanan et al. used the nudge design/approach to examine the association between framing the risk of the Covid vaccine and the willingness of the participants in getting the vaccine. This is a timely and important study.

      The results indicate that adding a descriptive risk label by the numerical side effect and providing a comparison to mortality due to motor vehicle accidents increased willingness to get the Covid vaccine by 3% and 2.4%, respectively, and independently.

      The study is designed properly, and the manuscript is well written. The findings are important from a policy perspective. Using nudges is an easy and inexpensive way to pursue people toward better decision makings in public health.

      Authors should explain in more detail the methods used to minimize bias. Second, probably the same nudge does not produce homogenous results among different population cohorts. One thing that can strengthen future work is to conduct cluster analysis to examine different nudging mechanisms among different subpopulations.

    2. Reviewer #2 (Public Review):

      With participants recruited from the United States and the United Kingdom, Sudharsanan et al. conducted an online randomized controlled trial, which examined three framing strategies of the side effects of a hypothetical COVID-19 vaccine: adding a qualitative risk label next to the numerical risk, adding a comparison group (along with which comparison group is most effective), and for those with comparison groups, framing the comparison in relative terms. Two outcomes were considered in the study: the self-reported willingness to take the vaccine and the perceived safety of the vaccine. Analysis showed that adding a qualitative label and a comparison to motor-vehicle mortality significantly increased the propensity for vaccination, whereas adding a comparison to COVID-19 mortality or framing the comparison in relative terms did not have a significant effect on the willingness of vaccination. These findings add useful evidence to the literature on the behavioral incentives of vaccination uptake.

      The study is well designed and implemented, with the data being carefully collected and analyzed. However, as indicated by the authors, some evidence is nonsignificant or counterintuitive, suggesting a retrospective need to increase the sample size for more convincing and granular results.

      Self-reported willingness to get vaccinated somehow depends upon the regional situation of the pandemic, which is partially reflected by the incidence and mortality among the local population. This factor of potential importance was not accounted for in the study, especially given that participants are recruited from two different countries with different governmental mandates.

    3. Reviewer #3 (Public Review):

      The authors were trying to examine if different framing strategies and types of presentation affect an individual's willingness to take vaccines. The paper has several strengths that should be noted. First, they have used a large randomized control trial composed of 8988 participants of adults ages 18 and older conducted in the United States and the United Kingdom. They have found a significant increase in vaccine willingness (about 3%) when a low-risk descriptive risk label is attached to numerical side effects, parallelly when a comparison group is added vaccine willingness also increases by 2.4%. They found these effects to be additive and increase further when combined together (to about 6.1%). Furthermore, they analyzed whether framing strategies varied with respect to age, sex, and country and they found no significant differences. The authors have addressed their aims quite competently and the results also point out that framing strategies are linked to vaccine hesitancy. Overall, this paper does not have too many weaknesses. One area of concern was the loss of 3000 participants for their study which would have been really great for a secondary sub-analysis. Although in the limitation the authors mentioned that the racial composition of the participants varied from the national estimates, it should also be noted that the age composition of participants was also a lot younger than the general population. To sum it up, the authors did a fantastic job of incorporating a novel idea that can be repeated in other countries as well.

    1. Reviewer #1 (Public Review):

      This paper asks how hawkmoths stabilize their head during externally-imposed body rolls. It finds that when body rolls are fast or there is little light, moths use their antennae to stabilize their head. The authors use convincing manipulations to show the necessity of the antennae for these behaviors and for stable free flight. This finding expands beyond similar studies in dipteran flies, showing that mechanical sensing of rotation can be performed by Johnston's organ in the antennae in moths, while similar functions are performed by the halteres in dipteran flies.

    2. Reviewer #2 (Public Review):

      This manuscript from Chaterjee and colleagues examines head-stabilization reflexes in the hawkmoth. Using light level manipulations and surgical manipulations of the antennae, they show that hawkmoths combine visual and mechanosensory feedback in order to stabilize the head over a wide range of temporal frequencies. Similar to other systems studied, such as flight stablization and antennal positioning, they find that visual feedback makes a stronger contribution at low frequencies while mechanosensory feedback contributes at higher frequencies. Finally they show that loss of head movement during flight contributes to flight instability, suggesting that an inability to stabilize the head might contribute to the effects of antennal manipulation on flight stability. Overall this is a nicely done study with clear findings that support a general principle of multisensory integration for feedback control. One way in which the manuscript could be strengthened is by explicitly modeling the feedback controller shown in Figure 5, and quantitatively comparing results obtained from this model to experimental results. In addition, it might be helpful to further quantify flight trajectories in head-stabilized moths.

    3. Reviewer #3 (Public Review):

      Payel Chatterjee et al. investigated how compensatory head movements in the nocturnal hawkmoth Daphnis nerii are controlled. This was done by subjecting tethered moths to open-loop body rotations under different light conditions, while simultaneously measuring their ability to main head angle in the presence or absence (achieved by antennal ablation) of antennal mechanosensory feedback. They find that head stabilization is mediated primarily by visual feedback during roll movements at lower frequencies, while antennal mechanosensory feedback is required when roll occurs at higher frequencies or under dark conditions. The findings add to our understanding of how non-dipteran insects (that lack halteres) stabilize their heads. Compensatory head-movements are essential for stabilizing the visual field on the retina, reducing motion blur and supporting visual self-motion estimation. These are all important parameters to control flight and allow for fast manoeuvres in air. The conclusions of the paper are well supported by the data.

    1. Reviewer #1 (Public Review):

      Wosniack et al. perform the analysis of larval trajectories from behavioral experiments and build a phenomenological model and efficiently combine the two to dissect behavioral strategies that Drosophila larvae use during foraging. The paper touches upon several factors that influence foraging: from food quality and distribution to genetic polymorphism and finally the contribution of sensory cues. While the first two are well explored and characterized in the paper, the contribution of different sensory modalities is less investigated. They study how homogeneous food substrates or food distributed in patches influence foraging strategies. They find a modular organization of behavioral strategies that is dependent of food characteristics: food quality modulates crawling speed, turning and pausing while increases in the time spent inside the patches are the result of biasing turning towards the patch center when the larvae are at the food-no food interface. Furthermore, using anosmic animals they determine that olfaction is differentially involved in the foraging decisions depending on the type of food substrates that the larvae are exploring. Finally, they perform this analysis in rover and sitter larvae to determine the effect of the foraging gene polymorphism on these behaviors and show that its expression (where sitter larvae are slower, turn less and pause more compared to rover larvae) is dependent on the food distribution. They propose that larvae adapt the extent of their exploration to the quality of food. This detailed analysis of elements that constitute behavioral strategies sets the basis for identifying genes involved in foraging and the neural substrates of the different behavioral modules and ultimately understanding the neural circuit mechanisms involved.

      The paper efficiently combines analysis of larval trajectories from experiments with computational modeling and identifies the behavioral elements that contribute to foraging. The authors show that olfaction has an important role when foraging on yeast substrates but not on sugar-rich substrates using anosmic larvae. They propose that taste could contribute more on sugar and apple juice substates however they do not test this hypothesis. Did the authors try or consider testing the Gr43a mutant on these substrates? Determining to which extent taste contributes to the different strategies completes the picture of how sensory cues contribute to foraging decisions that the authors started to address by tackling the contribution of olfaction to foraging on the different substrates. Also on patchy substrates, is the border completely smooth or could the larvae also sense the border as a rough edge? Could other modalities be involved?

      In Figure 3C the crawling speed is lower in yeast and apple juice experiments both inside and outside of patches (and in both rovers and sitters) compared to sucrose experiments. Do the authors have an explanation for this? Also, as they note, surprisingly the turn bias persisted when the larvae exited the patches. Are these two related? Do larvae turn more frequently?

      The authors describe and discuss handedness in larval turning. While this in itself is an interesting characterisation, it does not appear to be thoroughly addressed in the context of its influence on foraging behavior. The authors conclude that the presence of patches induces turning bias that overrides handedness. It would be interesting to determine whether there are differences in turn size and/or reorientation frequency depending if the larvae are turning on the preferred side versus the non-preferred side.

      During different types of taxes, the larvae modulate crawling speed, duration, turn rate, size and direction to avoid unfavourable conditions and approach unfavourable conditions. This is true across different types of sensory gradients. Some of these strategies are also described in this paper. The authors make a link between behaviour on patch-no patch interface and taxis behaviour. It would be interesting to further develop the comparison between the behavioural elements described here and those in navigational strategies in sensory gradients. The commonalities and possible modular organisation of both could point to an existence of neural circuits for the different behavioural modules that are recruited differentially dependent on the sensory context, motivation state, or a combination of both (and based on different types of sensory information).

    2. Reviewer #2 (Public Review):

      The study by Wosniack et al. investigates the impact of polymorphism on effective foraging behavior in patchy environments. The paper combines behavioral tracking data and phenomenological modelling to effectively describe and understand the navigational strategies underlying patch foraging in Drosophila larvae.

      A major strength of the work is the use and integration of the model that accompanies the experimental findings and is refined with evidence from experiments throughout the paper. A key result is that the genetic differences between rover/sitter larvae only manifest in patchy environments and are effectively hidden when larvae are exposed to homogeneous environments.

      This is a well-written and clear manuscript that effectively uses relatively simple techniques of behavioral tracking to quantify larval navigation (and patch residency, albeit this is not connected explicitly to optimal foraging in the text.)

      One aspect where the work could be strengthened is by highlighting where the differences in patch residence times between the model and data might arise. Especially in the anosmic animals, this removes one sensory aspect and one might feasibly expect the trajectory models to match better with the measured data. If this is not the case, it would be helpful for the readers to discuss the differences more explicitly.

      The modeling approach taken here is generalizable to a number of model- and non-model species which can be tracked, e.g., nematodes, where similar models have been used to describe navigation albeit not in the context of sparse patches.

    3. Reviewer #3 (Public Review):

      The authors of the paper study foraging strategy in crawling Drosophila larvae. They utilize single-larva tracking in isotropic and patchy food nutrition environments, detailed quantitative analysis of the animals' behavioral states and transitions, and a random-walk-style Monte Carlo simulation setting. They investigate how specific components of behavior are modulated for the animal to locate suitable food resources.


      * The main results of the paper, laying out how crawling speed, turn/pause rates, and turn direction bias work together cause larvae to find the food they need are interesting, nicely presented, and important for ultimately understanding how foraging really works in detail, here at the behavioral level, and somewhere down the road at the circuit and/or molecular levels too.<br /> * Comparing rovers and sitters throughout the experimental parts of the paper was a really nice idea, with interesting results, and it is well motivated in the introduction.<br /> * The handedness of individuals is a nice finding as well, I think the first time this has been published for larval Drosophila.<br /> * Simulations that use empirical results as probability distributions make for a nice environment for testing ideas about larva behavior.<br /> * Creating the patchy food environments was a great idea, as it puts the larva behavior in a more realistic setting, but still controlled enough to be analyzed clearly.


      * For an animal that tends to have a very high variance in its behavior, the number of larvae used in each experiment seems pretty low to me. As a result, some of the secondary claims are perhaps not as well supported when they rely on "not significant" statistical test results.<br /> * The introduction is generally good, but could perhaps better motivate why fly larva foraging should be of interest to a more general audience.<br /> * The execution of the simulations seems reasonable, but perhaps don't add a lot to this particular paper, especially given how much of the manuscript they take up.

      Overall, the primary results of the paper do achieve the stated goals and set the stage nicely for further studies into the underlying mechanisms of foraging in larvae.

      For those studying foraging, especially in flies/larvae but probably other animals as well, this should be an important paper that highlights the utility of individual animal tracking with high resolution, analyzing specific components of behavior, and creating simulation environments as playgrounds for investigating the impact of those components.

    1. Reviewer #1 (Public Review):

      The synthesis and metabolism of sphingolipid (SL) are involved in wide range of biological processes. In the present study, the authors investigate the role of SPTLC1, one of the essential subunits of serine palmitoyl transferase complex, in both physiological and pathophysiological angiogenesis, via using inducible endothelial-specific SPTLC1 knockout mice. They found SPTLC1 deficiency in ECs inhibited retinal angiogenesis along with reducing several SL metabolites in plasma, red blood cells, and peripheral organs. In addition, the authors found SPTLC1 EC-KO mice are resistant to APAP-induced liver injury. Overall, the in vivo findings in the present study are of potential interest and the authors have given clear evidence that endothelial SPTLC1 is critical to retinal angiogenesis. However, the underlying mechanisms are completely lacking in the present study. Most of the evidence provided is circumstantial, associative, and indirect. To be specific,

      1. The authors found endothelial SPTLC1 is important to both angiogenesis and the plasma lipid profile. However, the authors did not present the data to demonstrate the relationship between them. The in vivo findings about the phenotype and the plasma lipid profile might be true and unrelated. It would be important to know whether supplementing the reduced lipid induced by SPTLC1 KO could rescue the angiogenesis related phenotype in mice, or, whether the alternative way to inhibit the SL synthesis could mimic the phenotype of KO mice.

      2. A major issue is that the present study did not reveal is a real downstream target. It is possible that VEGF signaling might be impaired by SPTLC1 knockout as discussed by the authors. However, the authors did not demonstrate this point with data. Including both in vivo and in vitro data to evaluate the effects of SPTLC1 deficiency on VEGF signaling might further strengthen the hypothesis. Besides, with in vitro experiments, the authors might further find the critical metabolite(s) involved in VEGF signaling and angiogenesis.

    2. Reviewer #2 (Public Review):

      Andrew Kuo et al. investigated the role of endothelial de novo sphingolipids (SL) synthesis using endothelial cell specific SPTLC1 knockout (ECKO) mice. They showed that these mice exhibited low concentration of various SL species in not only ECs but also RBC, circulation, and other non-EC tissues. They also showed that ECKO mice exhibited impaired angiogenesis in normal and oxygen-induced retinopathy models, consistent with the decrease of endothelial proliferation and tip cell formation. They finally revealed that these mice were resistant to acetaminophen-induced acute liver injury in early phase. The experiments were well-designed, and the results were clear and convincing. The authors concluded that endothelial cells were the major source of SL in circulation and various organs (liver and lung) other than retina (and probably brain). The weakness of the current version of the manuscript is that the authors did not elucidate the mechanisms underlying the observed phenomena.<br /> 1) The authors showed impaired angiogenesis in ECKO mice using neonatal retina model. Based on the fact that this phenotype was similar to that in endothelial VEGFR2 deficient mice, they suggested that VEGF responsiveness is altered in ECKO mice. Although this hypothesis is plausible, the authors would need to prove it by evaluating VEGFR signaling (VEGFR phosphorylation, Akt activation etc.) in ECKO mice.<br /> 2) The acetaminophen-induced liver injury was reduced in ECKO mice in early phase. However, it is still unclear whether SL production itself affects liver injury. The authors discussed the possibility that gene deficiency increases unconsumed serine resulting in GSH increase, but it is essentially independent to SL. If possible, it would be good if the authors could investigate the effect of SL administration on the liver injury progression.<br /> 3) This paper showed the impaired cell proliferation in Sptlc1 KO EC mice, and discussed it. Authors described that this phenotype was similar to that of Nos3 KO mice, but its inconsistency with Sptlc2 ECKO adult mice was only justified by a word "isoform-selective function". Authors could quantify eNOS expressions in Sptlc1 KO mice, compared results and then discuss this matter.

    1. Public Review:

      This paper reports on the development of a mechanistic model of bone remodeling that accounts for key regulatory factors of the remodeling process which control bone cell numbers. The model is used to simulate osteoporosis and a variety of combined drug treatments. A number of drug treatments were implemented in a pseudo pharmacokinetic fashion. The model was first calibrated on a large number of experimental data sets. Subsequently the model was tested/validated using complementary experimental data sets. Simulation results show that the model is able to predict a significant number of experimental data sets. In a further step, a variety of different combined drug therapies were tested in order to identify an optimum combination. The authors concluded that this computational modeling framework has great potential for future use in order to optimize combined dosing regimen.

      The paper is very well written and the methods, and results are clearly described. Also, the authors provided all the source codes for their simulation results to be reproducible. The mathematical model was well described and the accompanying figure helped identifying action of different regulatory mechanisms and drug actions.

      Some weaknesses of the paper are the following:

      1) Formulation of the equation for BMD: was simply assumed to be the product of bone density and mineral content. Particularly, the latter function is formulated in a very phenomenological way. There are more rigorous, materials science based, ways to formulate bone mineralization.

      2) Pharmacokinetic (PK) formulations of drugs: the representation of drug concentration for the different drugs is a simplification. Generally, PK models need to be used to provide values of drug concentrations in the bone compartment which interact with the remodeling process. The bisphosphonate PK model might be more complex due to the absorption of the drug into the bone matrix and dissolution of the drug during bone resorption.

      3) The discussion section was rather unconventional, as no links/comparisons with existing literature were made. However, given that the essential computational modeling results are on combined drug treatments that have not been tested experimentally nor with other computational simulations, this is ok.

      The authors demonstrated that use of a mechanistic bone remodeling model combined with different drug actions allows to explore optimal treatment regimens including combined drug therapies for osteoporosis. The results clearly showed that some drug combinations lead to significantly higher BMD gains than others.

    1. Reviewer #1 (Public Review):

      The authors comprehensively assess the measurement properties (reliability, criterion/predictive validity) of behavioral and neural measures of fear acquisition/extinction, with a focus on longitudinal reliability and consequences of analytic and processing choices via a multiverse approach. In a longitudinal design (6-mo interval), the authors collected fear acquisition and extinction measures (SCR, ratings, fMRI) at two time points in a relatively larger sample for this type of work. Most notably, test-retest reliability, which is identified as a key component in individual-difference and clinical translation work, was generally low, whereas internal consistency was generally high. Group-level (averaged) reliability and cross-phase prediction (i.e., criterion validity) were generally good. Most measurement indices varied as a function of modality, processing, or statistical decisions. This work is framed within a larger discussion of the role of measurement properties in individual difference work and clinical translation and will serve as an important building block towards improvement in both these areas.

      The conclusions of this work are largely supported by the data and methodological approach, and this is a good benchmark for the field. However, some aspects could be clearer or streamlined, and some analytic choices are relative weaknesses.


      The overall approach is excellent and represents the vanguard of open science practices (preregistration, all materials freely available, documentation of analysis deviations, multiverse analyses, etc.). Relatedly, this comprehensive approach reveals how different analytic choices/researcher degrees of freedom can have sometimes drastic effects on fundamental measurement properties. I think this underlines what I view as the key contribution of this manuscript: empirically highlighting the need for the fear conditioning field to pay more attention to measurement properties.

      Going beyond standard associative measures of reliability (ICCs) is an important contribution of this work, as they allow the authors to comment on nuances of individual-difference reliability that are not possible with the coarser ICCs. In turn, this facilitates researchers in making more informed decisions regarding the design of fear conditioning tasks to assess individual differences.

      The fMRI results are a particular strength, as fMRI continues to be a common fear conditioning index, yet its measurement properties within these studies are critically understudied. The choice to use standard ICCs in conjunction with similarity approaches is particularly fruitful here, as in conjunction with overlap metrics we now have a much better appraisal of the different components of reliability in fMRI data - and potential explanations for differences between behavioral and fMRI reliabilities.


      The authors structure their effort around the premise that reliability is essential in conducting solid individual-differences science, which I agree with wholeheartedly. However, I think the authors rely on relatively arbitrary cut-offs for classifying reliability as good/poor/etc to an extent that is not warranted, particularly in the context of the Discussion, and it takes away from the impact of this effort. As the authors point out, these categorical cut-offs are more guidelines than strict rules, yet the manuscript is structured around the premise that individual-level reliability is problematically poor. Many cut-off recommendations are based on psychometric work on trait self-report measures that usually assume fewer determinants/sources of error than would be seen in neuroscience experiments, which in turn allows for larger ceilings for effect sizes and reliability. The current manuscript does not address this issue and what meaningful (as opposed to good) fear conditioning reliability is when moving away from the categorical cut-offs. In other words, is it possible that the authors actually observed "good" reliability in the context of fear conditioning work, and that this reliability is lower than other types of paradigms is just inherent to the construct being studied?

      The internal consistency (cross-sectional reliability) calculation used is not well-justified, and potentially needs additional parameters. It is not clear why the authors deviate from the internal consistency calculation described in Parson, Kruijt, and Fox et al., 2019, especially given that these procedures are used for other metrics elsewhere in the manuscript.

      In fMRI analyses, the authors use an ROI approach based on prior studies of fear acquisition and extinction. The majority of the most consistently identified regions (as seen in meta-analyses, Fullana et al., 2016, 2018) are analyzed. However, it is not clear why other regions are omitted, particularly given meta-analytic evidence. Striatal regions and the thalamus are the most notable omissions. Further, a weakness is that functional ROIs in this study were based on peak coordinates from a handful of prior studies, instead of meta-analytically identified coordinates. As such, I do not think the authors present the strongest foundation for making conclusions about the reliability of fear conditioning fMRI Data.

    2. Reviewer #2 (Public Review):

      The manuscript describes a large set of statistical analyses on fear conditioning data from 107 participants (N=71 at two time points six months apart). The analyses comprise approaches to determine the reliability and predictability of conditioned fear responses: skin conductance, ratings, and fMRI data.

      The approach is thorough, with a range of analysis approaches, including within- and between-subjects similarity, the individual-level overlap of fMRI results, intraclass correlation coefficients, and cross-sectional reliability. It is important to determine these values so that researchers can discard incorrect assumptions, such as the belief that threat responses at baseline can be predictive of treatment responses in patient populations.

      The poor reliability identified by several of these approaches is likely to be of great importance to this large, translational field. A positive result was good reliability at the group level for fear learning, but not extinction.

    1. Reviewer #1 (Public Review):

      By performing a genome-wide association study that exploits a previously developed probabilistic model to discriminate between 'true' severe malaria and invasive bacterial infection, Gilchrist et al. identify a variant in the gene BIRC6 that increases the risk of invasive bacterial disease across a diverse range of bacterial pathogens in Kenyan children. Although discovered in a fairly modest sample size, the association at rs183868412 replicates in an independent sample with a fairly large effect size (OR=2.77, 95% CI 1.49-5.12). It, therefore, seems likely that the association is a true positive and that the variant has a fairly large effect on risk of invasive bacterial infection.

      The risk variant, rs183868412:T, is present at frequencies of only around 1-3% in African populations (Table 5), and is absent from non-African samples. This makes investigation of potential mechanisms by which the variant exerts its influence on invasive bacterial disease difficult. Leveraging a study of 100 European ancestry and 100 African ancestry samples, the authors find evidence that rs183868412:T affects the splicing of BIRC6 in stimulated monocytes and that this may explain the association signal they found. However, due to the fact that rs183868412:T is at relatively low frequency in Africans and not present in European samples, it is hard to be completely confident in this analysis since it must hinge on only a handful of carriers of the rs183868412:T allele.

      The paper gives an example of how a probabilistic model for phenotypic classification can be used via weighting to increase power to discover genetic variants with effects on a specific (sub)phenotype. Further investigation of the discovered variant may be useful for understanding the biology of general susceptibility to invasive bacterial disease.

    2. Reviewer #2 (Public Review):

      This is a thoughtful paper that infers likely bacteremia cases probabilistically amp amongst putative severe malaria cases and then uses them in a GWAS. Based on this approach it identifies a biologically interesting risk allele for bacteremia, which is found at low frequencies in African population which seems to increase risk uniformly amongst a variety of age groups and bacterial diseases.

    3. Reviewer #3 (Public Review):

      This manuscript describes host genetic data of several cohorts of Kenyan children with culture proven bacteremia, severe malaria, and controls, and the association with bacteraemia. We know that many children with severe malaria actually have a bacterial co-infection. Because it is difficult to get the numbers needed for such GWAS studies, the authors plus up their numbers by lumping together bacteremia and severe malaria cases - the latter in a weighted manner for the continuum of malaria and bacteraemia. In the next step they validate their findings in a new cohort of 434 bacteraemia cases and present functional studies in monocytes. The methods used are interesting and the data are valid. Findings are important. I am not an expert in statistics, so I cannot judge the statistical methods in detail, but they seemed to be valid.

      I have a few major points.

      1. Overview of cohorts - overview. A graphical overview of cohort could be helpful for the reader- including groups, comparisons, and time periods of collection.<br /> 2. Overview of cohorts - phenotypes. The datasets used have been published previously with clinical phenotypes in more detail. Would it be possible to include a supplementary table providing these clinical phenotypes per group? In how many patients in the severe malaria group cultures were performed?<br /> 3. The potential impact of the prevalence of Pf HRP2 gene deletions on the analysis is probably limited because the cohort was collected in the period 1995-2008; this should be mentioned.<br /> 4. BIRC6 is identified as risk factor for invasive bacterial infection. BIRC6 (or BRUCE) is rightfully discussed by the authors in detail. BIRC6/BRUCE indeed is a ubiquitin conjugating E2 enzyme and a well-established anti-apoptosis regulator. Interestingly, we identified UBE2U to be associated with outcome in invasive pneumococcal disease (Lees et al Nature Comm 2019). The author may well find a link here.<br /> 5. The discussion could a presented a bit more balanced. 2/3 is now used to discuss the potential role of BIRC 6- this could be condensed while limitations of the study should also be discussed.

    1. Reviewer #1 (Public Review):

      The authors examined using various optical live-imaging techniques the beat properties and coordination of motile cilia across the whole surface of the zebrafish nose embryo. As far as I know, this is a level of detail that had never been explored. This is an important "model organism" to understand vertebrates, and motile cilia and their carpets are a fascinating system, with aspects that one expects to be conserved across species.

      The experimental data is really impressive to me. I think there is in fact a wealth of data, and the analysis of it here is just one part of what can be extracted. The theory and the specific question are also posed clearly, and are a strength. I do wonder why the experiments did not include visualization of the fluid flow, on the same fish - this is actually a much easier experiment I think, and would have given an important other spatial map to relate to the cilia dynamics.

      The theoretical analysis backing the data allows the authors to discuss on what length scale cilia are strictly synchronized to each other, versus the (longer) scale over which there is an element of coordinated dynamics. This is an important conceptual point that is discussed here very clearly.

      I think the authors have made a clear point on the degree of coherence in the in-vivo system, and on the consequences on longer range coordination and fluid transport.

      Motile cilia are one of the most fascinating and conserved structures across eukaryotes, and motile cilia carpets deliver critical and poorly understood physiological fluid transport. Here the authors present a new level of detail in-vivo zebrafish embryo nose data on the coordination of cilia, and discuss it in the context of what is known from other systems and from basic physical models. I think this paper will have strong impact on the specific field of motile cilia, and is generally an important result in development. Both physicists and biologists will use this and build on it.

      I do wonder about the title: "Local synchronization of cilia and tissue-scale cilia alignment are sufficient for global metachronal waves". This is indeed what they find, but this title will not be very clear to many biologists. I would recommend naming the species. In fact I think a better title would focus on having discovered that there is a certain behavior in zebrafish... it sounds less "general" but represents the work better.

    2. Reviewer #2 (Public Review):

      The strength of this work is the quality and quantitative nature of the experimental data. Despite the complexity of the zebrafish olfactory pit architecture, the analysis resolution achieved is remarkable. The data reveals for the first time that cilia beating is heterogeneous and patchy, yet still becomes ordered across the entire tissue.

      The main weakness is the disconnect between the theoretical contribution and the experiments. There is some overinterpretation here - the authors should revise their conclusions accordingly. The main output of the theory is a comparison between pumping direction, rates and efficiencies for different wave and lattice parameters. The theory does not yet explain how global metachronism arises from local synchrony, what sets the wave direction etc... but only the implications of this once it does.

      A key (and unexpected) finding is the asymmetry between the left and right nose in terms of metachronal wave direction (detectable due to the quality and rigor of the analysis). The manuscript leaves open how this can arise, since the model does not help explain why the wave direction can differ, despite cilia being oriented similarly.

      I think the present title does not reflect the key findings presented in the paper and should be changed - see specific recommendations below. This is fundamentally a detailed study and mapping of cilia coordination in the zebrafish nose - the organism name should really appear in the title, there is no need to over-emphasize the implications for metachronal coordination in general. I suspect these conclusions may be specific to this organism, and so the overall message may not apply to other ciliated tissues.

    1. Reviewer #1 (Public Review):

      Staphylococcus epidermidis is a commensal that colonizes corneocytes of humans and other mammals. Colonization is crucial for many aspects of health including the development of our immune system and protection against invading pathogens causing skin and soft tissue infections including Staphylococcus aureus and Streptococcus pyogenes. However, certain strains of S. epidermidis, especially those that colonize hospitalized patients, have the ability to bind implantable foreign bodies (e.g. hip/knee/shoulder implants, catheters) and subsequently form biofilms. Biofilms are difficult to treat with antimicrobial agents, and, in many cases, the only method of treatment is to remove the device causing significant morbidity.

      Embp is a very long protein encoded by most strains of S. epidermidis that extends from the cell surface. It has been previously documented that Embp binds to the serum binding protein fibronectin, a glycoprotein that coats implanted foreign devices. Data from this manuscript documents that Embp does not bind to Fibronectin in its soluble form. However, once Fibronectin binds to a surface such as foreign bodies ( called the fibrillated form) other epitopes are exposed to which Embp binds. There are three major strengths of this manuscript. First, using elegant genetic techniques as well as advanced atomic force spectroscopy experiments, the investigators found that Embp binds only to fibrillated Fibronectin and not soluble Fibronectin. Second, they found that the large number of repeats associated with Embp function is similar to velcro where the strength of each interaction (between F- or FG- repeat and Fibronectin) is small but the additive nature of the interaction is significant. Lastly, they found using flow experiments that Embp functions in an environment with high shear stress similar to blood. This is a very exciting result and has a significant impact on the field, especially since other adhesins presumably facilitated adherence to Fibronectin in a low shear environment.

      In summation, the results suggest that new biomaterials can be designed to inhibit the formation of fibrillated fibrinogen. Indeed, studies in this manuscript document that Fibronectin primarily is found as a globular form when attached to compounds such as poly ethyl acrylate.

    2. Reviewer #2 (Public Review):

      The objective of this study was to use atomic force microscopy to analyse the strength of the binding interaction between the fibronectin binding repeats of Embp and the fibrillary form of the host protein and to study bacterial adhesions under shear-stress.


      -Using a surrogate host to study interactions between truncates of Embp with variable numbers of Fn binding repeats<br /> -Being able to attach globular Fn to a surface so that it either remains in the globular state or unfolds to reveal the buried binding domain<br /> -Studying bacterial attachment to immobilized Fn under flow conditions mimicking the blood stream<br /> -Applying atomic force microscopy to study the strength of binding between a single cell expressing Embp or a single molecule of Embp attached to the cantilever and surface-immobilized Fn allowing measurement of the strength of binding of individual and multiple Fn binding repeats


      -The application of AFM is not discussed in the context of the extensive studies by the Dufrene group who have published many papers on the binding of staphylococcal adhesins to immobilized ligands.<br /> -The AFM analysis lacks detail, please add more to enable replication<br /> -The Dufrene group showed that some interactions are promoted by shear stress under flow conditions (for example SdrG and ClfA binding to fibrinogen). In these cases the force need to separate molecules is very strong and equivalent to that needed to break a covalent bond. It is not clear how the Embp-Fn bond responds to shear stress<br /> -There is no discussion or comparison of Fn binding by the well characterized FnBPs of S.aureus (used as a control in Figure 1) which bind to the type I repeats in the Fn N-terminus<br /> The authors have achieved their objectives and have advanced the state of knowledge about the interaction in an incremental fashion. It does not provide any major new insights. They have confirmed that Embp only binds the fibrillar form of Fn and they have shown that the strength of the interaction is proportional to the number of binding repeats in Embp truncates. This study confirms the value of force microscopy to studying bacterial adhesin-ligand interactions

    1. Reviewer #1 (Public Review):

      The exact role of Gdown1 has remained something of an enigma. At the biochemical level, Gdown1 can be strongly inhibitory to both preinitiation complex assembly and transcript elongation. However, there are now many examples, including those shown here, where the loss of Gdown1 can have very little effect. Other work has shown that Gdown1 is typically cytoplasmic, focusing attention on the mechanisms that keep Gdown1 out of the nucleus. Here the authors conclusively demonstrate that Gdown1 has two independent protein domains that drive nuclear export and a separate domain that normally anchors Gdown1 on the cytoplasmic face of the nucleus. When these localization domains are mutated and Gdown1 has routine nuclear access transcription is inhibited, apparently in part because of reduced Pol II levels. This in turn compromises cell viability. Cell viability can also be reduced by exposure to arsenite. In that case, Gdown1 gains nuclear access and reduces the negative effect, presumably because temporary transcriptional shutdown is protective in that case.

    2. Reviewer #2 (Public Review):

      Zhu et al. studied GDOWN1, a known RNA Polymerase II transcriptional regulator, and specifically investigated regulation of its localization. Using fluorescent microscopy and pull-down assays, the authors determined the multi-valent nature by which GDOWN1 constitutively remains in the cytoplasm. They found three non-redundant regulator domains within GDOWN1 that ensure GDOWN1 remains in the cytoplasm and only localizes into the nucleus upon stress. Specifically, the authors found two independent domains that directly bind to exportin 1, which ensures the proper export out of the nucleus. Further, they find a third domain that directly interacts with the Nuclear Pore Complex. These three domains regulate nuclear import/export. Additionally, they demonstrate that GDOWN1 inhibits transcriptional activity upon nuclear localization. Their data suggests GDOWN1 is a transcriptional regulator that regulates transcription during cell-stress.

      Overall, this is a solid paper, the data supports the authors' claims, and only a few points require clarification.

      1. Throughout the paper, the authors use a BiFC assay to monitor direct interactions between GDOWN1 and other transcription factors in the cell. While this assay works well for their experiments, we are unsure why GDOWN1 appears to interact with every protein found in the cytoplasm. This is particularly concerning when we look at GDOWN1 interacting with itself (Figure 1D), as GDOWN1 is not known to self-oligomerize. The authors should provide a negative control that GDOWN1 does not non-specifically interact with any cytoplasm-localized protein.<br /> 2. Additionally, every GDOWN1 truncation tested was able to interact with NELF-E. We are unsure why each truncation tested (given that they tested multiple non-overlapping GDOWN1 regions) can interact with NELF-E. Do the authors believe that NELF-E directly interacts with every tested GDOWN1 construct? We believe that demonstration of BiFC specificity is critical for the conclusions drawn in the manuscript.<br /> 3. The authors note that the NES1 site is not as strong as the NES2 site at regulating exportin 1-dependent nuclear export. However, they suggest this is because mutating the NES2 site is more likely to disrupt the CAS site nearby. We ask the authors to expand on this concept. Do they have direct evidence that NES2 disrupts CAS activity (such as regulating its association with the nuclear pore complex)?<br /> 4. The authors show the critical role of the NES1, NES2, and CAS sites for the localization and function of GDOWN1. Have the authors checked post-translational modification databases to check if any of the identified sites could be post-translationally modified and thereby regulated? Elucidation of the mechanism by which GDOWN1 localization is regulated is of broad interest to the transcription community.

    1. Reviewer #1 (Public Review):

      This is an important paper that will be of broad interest. As the authors point out there has been a lot of work in the genetics of complex traits in the last decade-plus, but very few animal studies have actually validated candidate causative variants in a rigorous manner. The human and mouse literature is especially troublesome, so it will be up fly people to do the heavy lifting, although they have not done much yet. Lots of validation efforts exist, but they tend to be correlative and prone to various artifacts. Here the authors attempt to create fly strains that differ at only a single nucleotide, and show that this change has a measurable impact on fecundity (but not some other traits people have posited this nucleotide could impact). The work is rather trailblazing in this regard.

      Overall the RNAi knockdowns only add a little, and the work's real contribution is going to be the gene replacement experiment, and the DGRP crossing experiment. I thought there was a little more work that could be done to make the DGRP crossing experiment more valuable. I think it important to include heterozygous genotypes (is the variant recessive or additive?) and to quantify the contribution of the manipulated variant to total variation. Similarly, I thought the supplement could have described the replacement strategy a little better. It is unclear what the background is in which the replacements are measured? Is it isogenic (that is stated but never shown) ... what is the evidence? Are there other parts of the genome segregating ... can one totally exclude segregating variation as an explanation for the results. I guess this is perhaps an omission and not an error, but a little more validation of the backgrounds is required. Finally, I would have liked to have seen some speculation as to how this particular SNP mechanistically impacts female fecundity.

      I hope these concerns are mere speed-bumps for a paper that is otherwise strong. In some cases what was done just has to be explained a little better. In other cases a small amount of sequencing or a few more crosses would greatly strengthen the paper.

    2. Reviewer #2 (Public Review):

      The authors aim to link a SNP to variation in a complex phenotype. This paper builds on considerable prior research on Eip75B and uses three distinct functional genetic methods to measure the phenotypic effects of allelic variation. The work is thorough and, when combined with prior work identifying variation in this locus, represents an unusually complete look at the link between a specific genetic variant and its pleiotropic phenotypic effects.

      The major strength of this work is the depth to which variation at this specific locus is investigated. Conducting three separate experiments, each with independent line-level replication within them, is admirable. Together, these data provide a convincing demonstration that Eip75B has effects on egg production. The authors clearly demonstrate that a single SNP can have effects on a complex phenotype, albeit not the primary phenotype Eip75B had previously been associated with (lifespan).<br /> This finding is important because it provides validation that GWAS and evolution experiments are capable of locating single nucleotide variants with demonstrable effects on complex traits.

      A limitation of this work is that the authors do not provide much discussion of the implications of their findings. By necessity, their experiments focus primarily on one of many SNPs in a gene where prior work has detected many different candidate SNPs associated with phenotypic variation in complex traits. It would be good to learn whether future work should systematically functionally characterize all variants, whether this is a worthwhile pursuit for complex traits, and what should be done about those traits that are (nearly) omnigenic. I suspect the findings of this paper will be influential and it could be fruitful for the authors to provide additional perspective for readers seeking to build on it.

    1. Reviewer #1 (Public Review):

      Ma et al take a novel approach to an important problem of host cell susceptibility to HIV. They tackle an understudied area of glycan effects on HIV infection using a new method they developed called CyTOF-lec. This method allows single cell detection of infected cells when using a reporter virus for infection. Importantly, the authors go to considerable trouble to use biologically relevant systems, including a transmitted virus and tonsil, endometrial and peripheral T cells. They find that cells expressing higher levels of fucose and sialic acid are more likely to be infected with to HIV than those with low levels. The studies presented here suggest, although didn't fully resolve, that sialic acid itself may be important for infection in CD4, CCR5 positive cells, although they can't really rule out that sialic acid is simply a biomarker for other cell features, such as activation state and entry receptor levels, which are known to impact susceptibility to HIV. Nonetheless, the findings point to glycans as a biomarker and potential determinant for HIV cells susceptibility and open the door to new avenues for studies of the interplay between cell surface glycans and viral infections.

    2. Reviewer #2 (Public Review):

      In this manuscript by Ma et al., the authors develop a mass cytometry that includes 5 heavy metal conjugated lectins. After some validation of this panel, the authors use the panel to analyze human PBMCs, tonsils and endometrial CD4 T cells before and after infection with an HIV virus with HSA reporter tag. They found that HIV infection was associated with higher levels of staining with 4 out of 5 lectins (sialic acid and fucose binders). Using the PP-SLIDE algorithm they previously developed, and they predicted that HIV preferentially infected higher cells with higher lectin binding and led to an increase in staining after infection. To validate this hypothesis, sorting of high vs. low lectin staining cells was performed to show that cells with higher lectin staining also had higher rates of HIV infection. They also used sialidase to reduce sialic acid levels and showed that it reduced HIV infection in PBMCs from two different donors. In addition to the development, validation and demonstration of mass cytometry lectin staining, the finding that glycosylation can influence HIV infectivity is novel and could open up new avenues for investigation. I think this work will be generally useful to the mass cytometry and HIV communities.

    1. Reviewer #1 (Public Review):

      Lurie et al. investigate the effects of stimulation applied to the lateral temporal lobe on the ipsilateral hippocampus with respect to endogenous hippocampal theta phase. They find that the magnitude of evoked hippocampal potentials correlated with hippocampal theta phase at the time of stimulation. The experiments are novel and could be valuable for showing how to use cortical stimulation to modulate the hippocampus. However, I did not find the paper to be suitable in its current form because of the high variance in hipp-cortical stimulation latency between subjects, which is not properly measured. This and other concerns make the findings challenging to interpret.

      1 - Problems with the analysis of stimulation latency<br /> The data in this paper show a variable latency in signal propagation from stimulation sites to hippocampal recording electrodes. In an attempt to measure this latency, the authors examine the theta phase offset between each pair of stimulation and recording electrodes (Figure 9). They interpret their results as showing a consistent 90-degree phase offset. However, their data do not support this interpretation because in fact their measurements show a bimodal distribution of phase differences with peaks at 0 and 180 degrees. It is not valid to interpret the circular mean of a bimodal distribution because the result is not well defined. Further, individual electrodes do not show a mean difference of 90 degrees.

      Because the results do not reliably support the claim of a consistent 90 phase difference between the hippocampus and cortex, it is a substantial problem for the paper, given the importance of hippocampal-cortical timing in their interpretation. In particular, the authors should reconsider how they frame their results in relation to the Siegle and Wilson work and others.

      2 - Problems with the figures<br /> Some figures in the paper were hard to interpret and I felt it would benefit readers for many to be combined. The results from Figures 3 through 7 would be helpful to see side by side, as they show various investigations of the same data. In Figure 4, it would be helpful to see both plots from (a) on the same axis, as is in (b). I did not find that the accuracy estimation paper in Figure 2 was important to include in the main paper. It would be better suited for the supplement, in my view, unless I am missing something.

    2. Reviewer #2 (Public Review):

      Lurie and colleagues attempted to assess whether electrical inputs to the human hippocampus are affected by theta phase. Building off a long history of rodent and computational research showing that hippocampal theta phase affects a multitude of hippocampal processes, including evoked excitatory potentials, the authors examined this effect in a group of human epilepsy patients. Each patient had recording wires in the hippocampus and stimulating electrodes in the lateral temporal cortex. Subjects were given stimulating pulses at random and then the evoked hippocampal potentials were compared according to which theta phase stimulation occurred on. After accounting for conduction delays between the lateral temporal stim site and the hippocampal recording site, they found that evoked potentials delivered on the failing phase of hippocampal theta had larger evoked responses for both an early (~70-110ms) and late (~120-200ms) components. Similar protocols were tried for evoked potentials in both the amygdala and orbitofrontal cortex, and only the only theta phase-dependent effect was found for early components in amygdalar evoked potentials. The current work is consistent with a large body of rodent research and also adds interesting new wrinkles to how we should consider oscillatory linked neural interactions.

      The data set is large (8 subjects with hundreds of stimulation events each) and is well analyzed. The approach to theta phase estimation is well thought out and consistent with past efforts. The combination of oscillatory synchrony offset based phase lag estimation with stimulation provides a new window in which we should conceptualize neural interactions. While conduction delays are obviously well known, most rodent experiments studying evoked activity rely on internal hippocampal stimulation, so no phase lag. In contrast, most studies of area-to-area theta based communication rely upon hippocampal theta phase and fail to consider any possible conduction delays, which could significantly alter the impact of phase-locked activity in distant areas. This is an important point that neural network simulations need to carefully consider and the current publication provides a blueprint for how to conceptualize (and quantify) these effects.

    3. Reviewer #3 (Public Review):

      The authors are interested in understanding how increased input to the hippocampus from entorhinal cortex at particular phases of the theta rhythm alters hippocampal function. Rodent studies have shown memory enhancement from phase specific stimulation of the hippocampus. The authors examine hippocampal responses to intracranial electrical stimulation of the lateral temporal lobe in patients with medically refractory epilepsy. Specifically, they examine the amplitude of the resulting evoked potentials relative to stimulation at different phases of the hippocampal LFP between 3-8 Hz. They are not stimulating entorhinal cortex, but the electrodes they stimulate were determined based on their ability to evoke hippocampal responses in the LFP. They hypothesize that this in-network stimulation should modulate hippocampal responses. The authors find that hippocampal responses are indeed enhanced, but in an opposite pattern to what they expected. Responses are larger when hippocampus stimulation occurs at the peak of the LFP, which they suggest is likely due to a ~90 degree phase difference between the lateral temporal input region and the hippocampus. The effects are small but significant and would be bolstered by 1) controlling for the timing at which stimulation was delivered, 2) showing data for every subject, 3) controlling for re-referencing scheme, and 4) showing results in a richer frequency and phase context. With adequate controls and supportive data, these results will provide an important contribution to understanding how stimulation in concert with the brain's natural rhythms can be used to more effectively modulate brain activity, which is an important step towards understanding how such precise modulation of brain activity can be used to alter memory, perception, and behavior.

    1. Reviewer #1 (Public Review):

      The manuscript entitled "Spatial modeling reveals nuclear 1 phosphorylation and 2 subcellular shuttling of YAP upon drug-induced liver injury" by Wehling et al. sought to provide increased resolution for the dynamic regulation of the HIPPO/Yap signaling pathway in hepatocytes. The authors employed a multi-tier approach utilizing computational/machine learning as well as classical biochemical and molecular biology techniques both in vitro and in vivo. The authors demonstrate the nuclear phosphorylation of YAP as a critical regulator of subcellular localization. Furthermore, the authors provide a mechanism by which drug-induced liver injury activates a cascade of ROS/Akt to culminate on YAP phosphorylation to direct its activation/inactivation in vivo. This work extends our current understanding of the HIPPO/Yap pathway and provides novel signaling pathways that converge on Yap activation during pathophysiological conditions. The claims are well-supported by a variety of experimental conditions.

    2. Reviewer #2 (Public Review):

      The study used two different model versions to distinguish two competing hypotheses concerning the mechanisms of YAP and TAZ phosphorylation and their regularization of nuclear localization. In the first (canonical) model, YAP and TAZ are phosphorylated in the cytosol, whereas in the second (alternative) model YAP and TAZ are phosphorylated in the nucleus. By comparing the model predictions to spatially resolved data, the authors could convincingly reject the first model and show that the second model explains the observed data better. The authors conclude that YAP and TAZ cytoplasmic-nuclear shuttling is regulated by phosphorylation in the nucleus and this conclusion is well supported by their data. Albeit not modeled, the authors also show this phosphorylation depends on ROS and AKT signaling.

      Strengths: The study is a very nice example of systems biology and how modelling can be used to make different assumptions explicit to test different hypotheses. A key strength is the mathematical modelling with partial differential equations and the use of spatially resolved data. Spatial features of the data were compared to the models and used to distinguish two different, competing hypotheses. The results reveal a new mechanism of YAP and TAZ phosphorylation in the nucleus. Uncertain parameters in the model were estimated and predictions arising from the model were validated using multiple experimental techniques, increasing the confidence of the findings. In vivo significance was validated in a mouse model.

      Weaknesses: As is to be expected, the parameters in the model were not identifiable leading to large variability in the estimates. However, this is typical for these types of systems biology models. Another potential weakness is that different diffusion rates were assumed for the canonical and alternative models, and the significance of having different diffusion rates for two different model versions remains unclear. To implement the different hypotheses, two extreme model versions were analyzed (the alternative model had no phosphorylation in the cytosol, only the unphosphorylated form of the YAP/TAZ is shuttled into the nucleus and only the phosphorylated YAP/TAZ is exported). The reality is most likely less discrete and somewhere in between with some phosphorylation/dephosphorylation occurring in both compartments and nuclear shuttling occurring for both the unphosphorylated and phosphorylated forms. The time-dependency of the model simulations was not analyzed, and the nature of the observed biphasic time-dependent APAP response remains elusive. It would be interesting to see how the model can explain the time course of the APAP stimulation experiment.

    3. Reviewer #3 (Public Review):

      In this manuscript, Wehling and colleagues discovered an important role of nuclear phosphorylation in the Hippo pathway that regulates the cellular localization of YAP and TAZ in the nucleus and cytoplasm. Using PDE modeling and imaging assays, they showed that the difference in shuttling dynamics between YAP and TAZ can be accounted for by a single parameter determining the nuclear phosphorylation capability. The authors further demonstrated that in a drug-induced liver injury model, YAP shuttling is also regulated by nuclear phosphorylation, which may be mediated by the induction of reactive oxygen species and subsequently AKT. The work has added the cell nucleus as a critical compartment in the regulation of the Hippo pathway, which challenged and extended the conventional model. The conclusions are mostly well supported by the experimental evidence, although some improvements can be made in data presentation.

      Overall, the work is of general interest to the field of developmental and regenerative biology. It will benefit a broader audience if the role of nuclear phosphorylation can be further linked to tissue damage and the regeneration process in future studies.

    1. Reviewer #1 (Public Review):

      Recent studies in rodents suggest the existence of conditioned responses (CRs) to fearful stimuli beyond the classically measured freezing behavior. Here, Trott and colleagues acknowledge that if valid, these results represent an important theoretical issue for prior research using conditioning paradigms. To assess the validity of these prior conclusions, the authors first replicated these behavioral results using similar experimental conditions. Next, they conducted a series of important controls to assess whether these alternative CRs (i.e., darting, flight) are indeed the result of associative processes. In contrast to this model, the authors found that darting and flight where largely the result of nonassociative processes. In general, the experimental design and their accompanying results largely support the authors conclusions. Precisely, they support a model in which what authors refer to as Peak Activity Ratio (PAR) responses are the result from nonassociative processes akin to potentiated startle (or sensitization). As such, in my view, the paper is already quite compelling. It is an excellent study that offers an important contribution to the field. Still, the addition of direct statistical comparisons of data presented in Fig. 2 and S2 and a few clarification statements should aid in the interpretation of the data and the study itself.

    2. Reviewer #2 (Public Review):

      Trott et al examine associative and nonassociative influences on freezing and flight-like responses (locomotion/darting) during and after Pavlovian fear conditioning in mice. Initial experiments use a paradigm popularized recently by Fadok et al 2017 - where a serial conditioned stimulus (SCS) precedes a shock US (tone->noise->shock). They replicate the main result: freezing develops primarily to the tone and activity bursts/darting develop to noise. Control groups demonstrate that noise-elicited flight does not depend on embedding in a compound SCS or pairing noise with shock in the training phase. Velocity measurements during early shock-free tests indicate that vigorous movements occur mostly at noise onset and are bigger when the noise is novel. Since the tone is not necessary for noise-elicited flight, all subsequent experiments focus on reactions to noise after treatments in the same context to probe the contribution of associative vs non-associative processes directly. In brief, they find the strongest flight-like reactions when the noise is novel at test and there is a history of shock in the chamber. Habituating the noise or pairing it with shock both blunt subsequent flight reactions to noise onset. Interestingly, less vigorous movements later in the noise cue were potentiated by noise shock pairings (relative to shock-only or unpaired controls), suggesting that a component of the response is associative. Other data suggest that noise behaves as a weak US (disrupts ongoing freezing, repeated presentations support low levels of freezing & darting). The authors conclude that apparent conditioned flight responses are primarily due to non-associative pseudoconditioning/dishabituation of alpha responses to a sudden stimulus change - akin to fear-potentiated startle (FPS). This is an important addition to a recent literature that has generally assumed that active responses after conditioning are associative. The velocity data are especially clear and support the FPS analogy. The conclusions regarding the SCS-flight paradigm are justified by the current data but are partially inconsistent with findings recently published by another lab (Totty et al 2021). It is also unclear if the non-associative processes identified in the present studies apply to conditioned darting studies using a pure tone.

      Specific questions/concerns:

      1. The section relating SCS-flight reactions to alpha responses and fear potentiated startle (FPS) is interesting and potentially important. However, parts of this narrative are unclear. First, FPS has a strong associative component but the flight reaction studied here apparently does not. Second, pairing a tone with shock increases startle reactions preceded by a tone. Here, pairing noise with shock suppresses alpha reactions. Is there evidence that pairing startle-eliciting noises with shock reduces typical startle reactions? Is the issue here that SCS-flight studies are designed poorly to demonstrate the phenomenon (pairing the whole SCS compound with shock vs pairing just the tone with shock then testing noise reactions after a tone)? Lastly, an important experiment by Totty et al (Fig 5) is not discussed. They show that SCS presentations fail to elicit flight reactions in a threatening context (previously paired with unsignaled shocks) unless they were paired with shock in an earlier phase (different context). This seems inconsistent with the FPS interpretation of SCS-flight, since the threatening context should have increased alpha reactions to the novel noise. Along with other control experiments, it also suggests that associative processes related to SCS-shock pairings make a strong contribution to flight. Perhaps there is something unique about compound stimuli paired with shock that cannot be addressed with the simple noise-shock control experiments reported here? This should be discussed in the manuscript.

      2. Sex differences have been reported for darting behavior in a Pavlovian paradigm using a single tone CS, but have not been observed in studies using a tone-noise SCS. The combined analyses here (lines 424-429) also finds no sex differences for mice conditioned with a single noise CS. However, the original reports identified only a subset of females that showed prominent darting and stronger shock reactions. Is there any evidence for this Darter vs. Non-darter classification in your dataset? Either way it would be helpful to add graphs illustrating the sex difference analysis that include data points for individuals, at least in supplemental.

      3. Lines 432-439: This concluding paragraph is a missed opportunity for a more nuanced discussion of "active vs. passive" defense and perhaps different categories of "flight". The papers cited do not suggest that rats freeze because no other response is available (thought the Blanchards may have said this elsewhere). All the studies investigate CRs in situations where both freezing and locomotor movements are possible. Although it is true that freezing is not the absence of a response, it is the absence of movement. The distinction between movement/ambulation and immobility in threatening situations is important for describing brain circuits of defense and necessary to explain transitions to flight, escape, active avoidance, and even "choosing locations to freeze" by moving down threat gradients. Similar passive vs active terminology goes back at least to Konorski (1967), though "stationary" may be more appropriate than "passive" (Sigmundi, 1987). Related:<br /> -Line 66: "but not activity bursts", Line 77: "Gruene et al suggest that freezing and darting were competing CRs to the same level of threat". Please clarify the Predatory Imminence Theory views on this. If conditioned rats move to the safest spot to freeze (de Oca et al, 2007), is this not an activity burst? Does the velocity of the movement matter? How do these movements relate to the startle-like responses seen at CS onset vs. the more sustained activity reported here for paired groups? de Oca 2007 describes conditioned flight to a familiar enclosure and freezing as compatible post-encounter responses to the same threat, but flight and freezing cannot occur at the same time and must be competitive.

      4. The notion that noise is a (weak) US requires further discussion. Specifically, how do you define a US? And are these properties necessary for the argument that apparent conditioned flight/darting reactions are non-associative startle-like reactions? Freezing goes up when rats experience noise alone trials, but this does not appear to be a result of context conditioning (no BL freezing on day 2 of training). Further, there appears to be no summation once the context is paired with shock (freezing during habituated noise; Exp 4). Noise-elicited freezing appears to sensitize in phase 1 but at the same time darting responses habituate. This pattern is unlike what one might expect for even a very weak shock. One reason this seems important: the paper begins by explaining the challenge posed by the SCS-flight paradigm and the conditioned darting paradigm. However, the studies presented here focus on noise-elicited behavior and imply that similar phenomena occur in the conditioned darting paradigm. The conditioned darting studies all use a pure tone that may not be characterized as a US. Tone-elicited behavior isn't discussed much in the manuscript, but tone-elicited darts in Experiment 2 (pseudoconditioning control) appear lower than those elicited after tone-shock pairings in Experiment 1. So it remains unclear if conditioned darting results from non-associative processes, especially if the tone does not act as a weak US.

      5. Baseline data for Darts is missing throughout and should be added to all trial-by-trial graphs. This is important since all phases occurred in same chambers and baseline fear levels could drive darting before stimuli are presented.

      6. Line 133: "noise was never paired with shock". This is an important point -- but the white noise stimulus contains the tone frequency, and this was paired with shock in the previous phase.

    3. Reviewer #3 (Public Review):

      In this manuscript, Trott et al report a series of elegant experiments designed to parse associative and non-associative influences on escape-like locomotor responses elicited by conditioned stimuli. The work is timely, and the experiments are well designed to assess the contribution of novelty, surprise, and learning to behaviors such as darting and flight. The results paint a complicated picture, the complexities of which need better exploration in the discussion.

      The authors' do indeed demonstrate that increased locomotion in response to a white noise can be driven by non-associative factors. This seems particularly true in the 1st and 2nd experiments, in which a novel/surprising serial compound stimulus or a white noise alone triggered a switch from freezing to locomotion more effectively than any associative factor. The picture becomes more complex with experiment 3, however, which compared paired and unpaired groups, as well as a variety of other controls. Contrary to what one might predict on the basis of the first two experiments, the paired group showed less freezing and more darts than the unpaired group (a straightforward interpretation of experiments 1 & 2 would suggest that paired and unpaired should be equal across all measures). Further, the pattern of locomotion driven by non-associative novelty (shock-only group) is very similar to the pattern observed in the unpaired group - a strong spike at the onset of the white noise (alpha response) that returns to near baseline levels by the end of the stimulus. In contrast, the paired group showed a more enduring, multi-peak pattern of elevated locomotion that filled the white noise. The 4th experiment revealed that a locomotor pattern dominated by alpha responses (ie similar to unpaired and shock-only groups in experiment 2) is indeed attenuated by habituation, and thus can be considered non-associative. However, habituation does not seem to have a strong effect on the white noise-filling pattern of locomotion observed in the paired group. Indeed, the descriptions of experiments 3 & 4 in the results section do acknowledge that paired groups produce a particular pattern of locomotor activity/darting that differs from non-associative groups. The discussion, however, does not address this point. Though the paper provides good evidence for the role of non-associative factors in flight-like behavior, it does not totally refute a role for associative factors, as well. Associative factors may not be necessary to produce darting and increased locomotion, but in certain cases they seem sufficient to do so.

    1. Reviewer #1 (Public Review):

      The authors presented updated results for a clinical trial described in a previous publication (Zhang J et al 2017). With the updated results, the authors were able to further support the validity of their evolution-based model proposed before. These datasets also allow the authors to fit individual-level evolution models and examine critical parameters in their models.

      The concept of adaptive therapy is critical and has previously attracted broad attention in the field. The earlier work (Zhang J et al 2017) showed promising results in improving prognosis in prostate cancer patients. In this paper, the follow-up data for this clinical trial clearly confirms its previous findings that adaptive therapy was able to improve TTP and OS.<br /> The authors also went on to infer an evolution model of treatment sensitive and resistant cells for each individual patient. With a small number of parameters, the authors can fit most patients' longitudinal data tightly. The authors found some parameters are important to determine the outcome of adaptive therapy. These results are interesting and could have clinical implications, but some model assumptions are strong (like assuming a shared competition coefficient across patients) and some claims need more explicit analysis.<br /> One particularly interesting result from the modeling analysis is that failure of adaptive therapy is caused by overtreatment. However, the readers need to keep in mind that this conclusion is under the simple model described in the paper. More complicated clone composition, interaction and evolution paths will affect this conclusion.

    2. Reviewer #2 (Public Review):

      In this study, Zhang et al. expand on their previous work on using mathematical modelling to guide the timing and dosing of arbiterone treatment in castrate-resistant prostate cancer. The study presents the results of a follow-up pilot trial with 33 patients and adapts an updated mathematical model to fit longitudinal patient data. While the sample size is limited, the implications of the study outcome are broad and compelling. The manuscript can be strengthened by showing that there are no statistically significant differences between the two treatment groups in terms of additional clinical features, such as prior therapies.

    1. Reviewer #1 (Public Review):

      This work addresses a so far much overlooked aspect in plant signaling systems being the physiological reality of how components primarily identified by genetic means work together to achieve a functional physiological system. The genetically well-defined system of brassinosteroid signaling in Arabidopsis roots is employed as a convenient model system.<br /> For this accurate determination of the number of proteins involved, the kinetics of activation and ODE-based modelling are used by the authors.

      The results focus on one aspect of root brassinosteroid signaling, expansion of root cells as they enter into the extension zone of the root. Several parameters such as wall acidification and cell wall swelling are used as early output determinants of the BR signaling system.

      The authors show convincingly that BR signaling can be used to identify hitherto missing components of the system such as a potassium channel protein presumed to be used in rectifying the effects of the ion flow across the membrane.<br /> It would benefit from a clear perspective concerning the time frame of the events measured and a simplification by removing certain data sets from the main story.

    2. Reviewer #2 (Public Review):

      The manuscript entitled "Computational modeling and quantitative cell physiology reveal central parameters for the brassinosteroid-regulated cell growth of the Arabidopsis root" by Ruth Grobeholz et al. presents a hybrid computational and experimental study on the fast response to brassinosteroids of epidermal cells in the elongation and in the meristematic zones of Arabidopsis primary root. The study focuses on the regulation of ionic transport through the plasma membrane that is elicited upon BL induction. This is supported by experimental data on ion fluxes and pH dependence with BRI1 receptor in Arabidopsis roots, analyzing WT and bri1-301 mutant. The combination of modeling and experimental data reveal a new component of the fast BR response. This new component is a cation channel, CNGC10, which the authors show, through the analysis of a CNGC10 loss-of-function mutant to be required in epidermal cells of the elongation zone to change the apoplastic pH upon BL application. In addition, the experimental results show a gradient of the proton pumping activity, through AHA2, along the root, with highest activity in the elongation zone. Finally, the study analyses the role of BIR3 on this fast response.<br /> The study is very interesting because it addresses a part of BR pathway that has been little investigated, which is that of the fast-response. The manuscript continues the proposal made by one of the leading authors of this manuscript on the existence of this fast response (Caesar et al, 2011) by extending it to epidermal cells in Arabidopsis primary root and by finding a novel factor involved, the cation channel CNGC10. The finding of AHA2 gradient is also promising, albeit, as discussed below, partially inconclusive in my opinion. The manuscript is also very appealing because of the many different techniques that have been used, both computational and experimental.

    3. Reviewer #3 (Public Review):

      The authors create a computational model that aims to understand how the regulation of proton pumps by brassinosteroid receptor complexes translates into membrane potential changes and cell wall pH. They build the model using known facts from the brassinosteroid literature as well as published cell compartment volumes and membrane densities of some signaling components. To obtain further parameters for their model, the authors quantify the densities of BIR3 and AHA2 and found that the density of the proton pump increases along the differentiation gradient in the root, and that the ratio of AHA to BRI1 dramatically increases in the elongation zone.

      Their model focuses on the rapid responses to the external application of brassinosteroid BL, and as such it could predict the dose response of apoplastic pH to BL. The model was further broadened to involve the gradients of protein concentrations along the root developmental axis.

      The model predicted a significantly larger membrane hyperpolarization in response to BL than the one observed experimentally, indicating a missing component that would depolarize the PM, such as a cation channel. This prediction led the authors to identification of a missing component in the BRI1-BIR3-AHA module, the CNGC10 cation channel.

      The combination of quantitative cell physiology and mathematical modelling for the complex regulation of ion fluxes in the root cells is probably the only way how to understand the non-linear relationships and emergent properties that occur in growing root cells. This manuscript is an initial attempt to understand these phenomena in Arabidopsis roots, but as such it is, in my opinion, overly simplified, particularly when it comes to the involvement of ion channels that regulate and respond to membrane potential. The paper gives a somewhat unfinished impression, and most importantly, it lacks experimental validation of some of the crucial conclusions. Here I summarize the main points which I find problematic or weakly supported by the data:

      1) The central component of the model is the fast activation of AHA by BRI1, a rapid, non-transcriptional response. More experimental support is needed to establish that, in the root, AHAs are activated rapidly and not by the transcriptional pathway. Minami et al., 2019 showed that AHA activation in the hypocotyls requires tens of minutes and is likely mediated by the accumulation of SAUR proteins. In other words, the activation is not a rapid BRI1-mediated phosphorylation. The model, however, uses the findings from Minami et al 2019 as the support for the immediate activation of AHAs by phosphorylation (at the line 143).

      2) Further, one of the crucial outputs that is used to compare experimental a modelled data - the apoplastic pH - seems very noisy in the provided figures. This is particularly apparent in the time-course response of apoplastic pH to 10nM BL application. Figure 4B should show that there is a rapid acidification that is maintained, however the figure shows rather a noisy behavior (in particular when we consider that the errors represent SEM) and, moreover, the figure 4B does not fit the results from 4A. Similar noisy results are shown in the figure 6A and B and the model does not seem to fit the experimental data in the meristematic cells. In the case of these figures, the conclusions in the text do not seem to fit with the data presented in the figures.

      3) Further, the cngc10 mutant pH responses are not very convincing: the cells of the meristematic zone of the control line do not respond to BL (Appendix Fig3) while in figure 7C the meristematic zone of control does respond to BL. However, I think other physiological phenotypes of the mutant lines should be tested that would determine whether CNGC10 is involved in the response of roots to brassinosteroids. What is the expression of CNGC10 - is it expressed in the same cells as BRI1 and AHA2? What are the densities of CNGC10 molecules along the root developmental gradient? Such questions should be clarified to substantiate the conclusion that this channel is a major player in the regulation of membrane potential.

      4) Why the predictions of the model regarding the BIR3 involvement were not tested experimentally? This could again show that the model predicts the cellular behavior correctly. It would be particularly interesting to test the model predictions along the longitudinal root axis, where the ratio of signaling components is changing.

    1. Reviewer #1 (Public Review):

      This paper is potentially interesting to many researchers who are looking for ways to enhance the CTL response to tumor immunity in combination with checkpoint antibodies.


      1. The authors engineered a non-replicating pseudotyped influenza virus to deliver the well-known cancer testis antigen, NY-ESO-1 (NY-ESO-1 S-FLU). One problem in using virus vectors for vaccination is the immune reaction to viral protein. They clearly showed that Switching HA coat of virus in S-FLU construct would easily overcome the pre-existing anti FLU immunity. This finding is new and interesting.

      2. The authors also showed that intranasal or intramuscular immunization of NY-ESO-1 S-FLU virus in mice elicited a strong NY-ESO-1 specific CD8+T cell response in lungs and spleen that resulted in the regression of NY-ESO-1 expressing lung tumor and subcutaneous tumor respectively. In addition, they demonstrated that NY-ESO-1 S-FLU synergistically works with anti-PD1, which is also interesting.


      1. Mechanisms by which NY-ESO-1 S-FLU works better than other types of viral vectors are unclear.

      2. To most readers who do not have much information on cancer vaccine, it is unclear whether the anti-tumor effect of NY-ESO-1 S-FLU was high or modest compared with those reported in the similar studies using other types of vaccines (e.g., peptide vaccine, mRNA vaccine,----).

    2. Reviewer #2 (Public Review):

      Virus-based tumor vaccines can induce a robust T cell response capable of limiting tumor progression. The authors sought to investigate whether the influenza virus can be used to induce a protective tumor antigen-specific T cell response. They generated a replication deficient influenza virus expressing a testis cancer antigen and found that infection of mice with this virus can reduce lung or subcutaneous tumor burden depending on the route of immunization. Furthermore, the authors show that PD1 blockade can further augment the tumor protection elicited by this approach.

      One limitation of this study is that authors do not show that their approach induces long-lived anti-tumor immunity. While the authors show that their approach can induce a memory T cell response in the lungs, they do not perform any experiments in which mice are challenged at a memory time point. Rather their prophylactic tumor model challenges the mice two days after booster infection. Furthermore, their therapeutic model relies on repeat infection 14 days apart which is also not sufficient to generate a mature memory T cell response. Therefore, it is difficult to conclude from this study whether their approach will induce long-lasting immunity.

      An additional limitation of their approach is that most humans have been infected by influenza or received influenza vaccines many times in their lives. Therefore, most individuals would be expected to have some influenza-specific antibodies that can bind to conserved elements of the hemagglutinin protein. To circumvent this they showed that Switching HA coat of virus in S-FLU construct could overcome the pre-existing anti FLU immunity.

    1. Reviewer #2 (Public Review):

      Dalton and colleagues present an interesting and timely manuscript on diffusion weighted imaging analysis of human hippocampal connectivity. The focus is on connectivity differences along the hippocampal long axis, which in principle would provide important insights into the neuroanatomical underpinnings of functional long axis differences in the human brain. In keeping with current models of long-axis organisation, connectivity profiles show both discrete areas of higher connectivity in long axis portions, as well as an anterior-to-posterior gradient of increasing connectivity. Endpoint density mapping provided a finer grained analysis, by allowing visualisation of the spatial distribution of hippocampal endpoint density associated with each cortical area. This is particularly interesting in terms of the medial-lateral distribution with hippocampal head, body and tail. Specific areas map to precise hippocampal loci, and some hippocampal loci receive inputs from multiple cortical areas.

      This work is well-motivated, well-written and interesting. The authors have capitalised on existing data from the Human Connectome Project. I particularly like the way the authors try to link their findings to human histological data, and to previous NHP tracing results.

      I do, however, have some concerns about the interpretation of the results.

      There are some important surprises in the results, particularly the relatively strong connectivity between hippocampus and early visual areas (including V1) and low connectivity with areas highly relevant from functional perspectives, such as the medial prefrontal cortex (rank order by strength of connectivity 7th and 78th of all cortical structures, respectively). This raises a concern that the fibre tracking method may be joining hippocampal connections with other tracts. In particular, given the anatomical proximity of the lateral geniculate nucleus to the body and tail of the hippocampus, the reported V1 connectivity potentially reflects a fusion of tracked fibres with the optic radiation. In visualizing the putative posterior hippocampus-to-V1 projection (Figure 4B, turquoise), the tract does indeed resemble the optic radiation topography. Although care was taken to minimise the hippocampus mask 'spilling' into adjacent white matter, this was done with focus on the hippocampal inferior margin, whereas the different components of the optic radiation lie lateral and superior to the hippocampus.

      A second concern pertains to the location of endpoint densities within the hippocampus from the cortical mantle. These are almost entirely in CA1/subiculum/presubiculum. It is, however, puzzling why, in Supp Figure 2, the hippocampal endpoints for entorhinal projections is really quite similar to what is observed for other cortical projections (e.g., those from area TF). One would expect more endpoint density in the superior portions of the hippocampal cross section in head and body, in keeping with DG/CA3 termination. I note that streamlines were permitted to move within the hippocampus, but the highest density of endpoints is still around the margins.

      On a related point, the use of "medial" and "lateral" hippocampus can be confusing. In the head, CA2/3 is medial to CA1, but so are subicular subareas, just that the latter are inferior.

    2. Reviewer #1 (Public Review):

      This manuscript presents novel results which suggest that networks of cortical regions show different patterns of structural connectivity with hippocampal subregions. The results build on prior work, but also provide a spatially precise characterization of whole-brain structural connectivity patterns along the anterior-posterior hippocampal gradient. The paper is well-motivated and well-written. The authors discuss their findings in the context of previous investigations in non-human primates, and draw a number of parallels between these bodies of evidence. However, there were also some interesting differences between the connectivity patterns uncovered in resting-state fMRI and those identified using the present approach. It would also be helpful to highlight the key methodological advances or differences compared to prior work to contextualize the present findings.

      1. The connectivity patterns along the anterior-posterior hippocampal axis broadly follow an anterior-posterior cortical bias, such that posterior regions, e.g. the visual cortex, are preferentially connected to the hippocampal tail, and anterior regions, e.g. the temporal pole, are preferentially connected to the hippocampal head. The authors focus on the twenty regions with the highest connectivity profiles, which appears to capture the majority of all connections. However, some of the present structural connectivity patterns differ in interesting ways from previously described cortical networks reported in resting-state fMRI studies. Most notably, the medial PFC and orbitofrontal regions combined account for less than 1% of all connections in the present investigation (Table S1 & S2). This is an interesting contrast to functional investigations which tend to find that these regions cluster with the aHPC (e.g., Adnan et al. 2016 Brain Struct Func; Barnett et al. 2021 PLoS Biol; Robinson et al. 2016 NeuroImage). In contrast, the present DWI results suggesting preferential pHPC-medial parietal connectivity dovetail with those observed in fMRI studies. It seems important to discuss why these differences may arise: whether this is a differentiation between structural and functional networks, or whether this is due to a difference in methods.

      2. While the analytic pipeline is described in sufficient detail in the Methods, it is somewhat unclear to a non-DWI expert what the major methodological advance is over prior approaches. The authors refer to a tailored processing pipeline and 'an advance in the ability to map the anatomical connectivity (p. 5), but it's not immediately clear what these entail. It would be useful to highlight the key methodological differences or advances in the Introduction to help with the interpretation of the similarities and differences with previous connectivity findings.

      3. Related to the point above, it was a bit unclear to me how the present connections map onto canonical white matter tracts. In Fig., 4A, the tracts are shown for a single participant, but it would be helpful to map or quantify know how many of the connections for a given hippocampal subregion are associated with a given tract to provide a link to prior work or clarify the approach. A fairly large body of prior research on hippocampal white matter connectivity has focused on the fornix, but it's a little difficult to align these prior findings with the connectivity density results in the current paper.

      4. Finally, on a more speculative note: based on the endpoint density maps, there seems to be a lot of overlap between the EDMs associated with different cortical regions (which makes sense given the subregion results). Does this effectively mean that the same endpoints may be equally connected with multiple different cortical regions? Part of the answer can be found in Fig. 3D showing the combined EDM for three different regions, but how spatially unique is each endpoint? This is likely not a feasible question to address analytically but it might be helpful to provide some more context for what these maps represent and how they might relate to differences across individuals.

    1. Reviewer #1 (Public Review):

      The authors showed that longer reverberation time prolongs inhibitory receptive fields in cortex and suggest that this helps producing sound representations that are more stable to reverberation effects. The claims is qualitatively well supported by two controls based on probe responses to the same type of white noise in two different reverberation contexts and based on receptive fields measured at different time points after the switch between two reverberation conditions. The latter gives stronger results and thus constitutes a more convincing control that the longer decay of inhibition is not an artefact of stimulus statistics. The limits of the study include the use of anesthesia and the fact that cortex shows a very broad range of dereverberation effects, actually much broader than predicted by a simple model. This result confirms that reverberation produces cortical adaptation as suggested before, and suggests as a mechanistic hypothesis that rapid plasticity of inhibition underlies this adaptation. However the paper does not address whether this adaptation occurs in cortex or in subcortical structures. The fact that an effect is observed under anesthesia suggests a subcortical origin.

    2. Reviewer #2 (Public Review):

      Ivanov et al. examined how auditory representations may become invariant to reverberation. They illustrate the spectrotemporal smearing caused by reverberation and explain how dereverberation may be achieved through neural tuning properties that adapt to reverberation times. In particular, inhibitory responses are expected to be more delayed for longer reverberation times. Consistently, inhibition should occur earlier for higher frequencies where reverberation times are naturally shorter. In the manuscript, these two dependent relationships were derived not directly from acoustic signals but from estimated relationships between reverberant and anechoic signal representations after introducing some basic nonlinearity of the auditory periphery. They found consistent patterns in the tuning properties of auditory cortical neurons recorded from anesthetized ferrets. The authors conclude that auditory cortical neurons adapt to reverberation by adjusting the delay of neural inhibition in a frequency-specific manner and consistent with the goal of dereverberation.

      Strengths:<br /> This main conclusion is supported by the data. The dynamic nature of the observed changes in neural tuning properties are demonstrated mainly for naturalistic sounds presented in persistent virtual auditory spaces. The use of naturalistic sounds supports the generalization of their findings to real live scenarios. In addition, three control investigations were conducted to backup their conclusions: they investigated the build-up of the adaptation effect in a paradigm switching the reverberation time after every 8 seconds; they analyzed to which degree the observed changes in tuning properties may result from differences in the stimulus sets and unknown non-linearities; and, most convincingly, they demonstrated after-effects on anechoic probes.

      Weaknesses:<br /> 1) The strength of neural adaptation appears overestimated in the main body of the text. The effect sizes obtained in control conditions with physically identical stimuli (anechoic probes, Fig. 3-Supp. 3B; build-up after switching, Fig. 3-Supp. 4B-C) are considerably smaller than the ones obtained for the main analyses with physically different stimuli. In fact, the effect sizes for the control conditions are similar to those attributed to the physical differences alone (Fig. 3-Supp. 2B).<br /> 2) All but one analysis depends on so-called cochleagrams that very roughly approximate the spectrotemporal transfer characteristics of the auditory periphery. Basically, logarithmic power values of a time-frequency transformation with a linear frequency scale are grouped into logarithmically spaced frequency bins. This choice of auditory signal representation appears suboptimal in various contexts:<br /> On the one hand, for the predictions generated from the proposed "normative model" (linear convolution kernels linking anechoic with reverberant cochleagrams), the non-linearity introduced by the cochleagrams are not necessary. The same predictions can be derived from purely acoustical analyses of the binaural room impulse responses (BRIRs). Perfect dereverberation of a binaural acoustic signal is achieved by deconvolution with the BRIR (first impulse of the BRIR may be removed before deconvolution in order to maintain the direct path).<br /> On the other hand, the estimation of neural tuning properties (denoted as spectro-temporal receptive fields, STRFs) assumes a linear relationship between the cochleagram and the firing rates of cortical neurons. However, there are well-described nonlinearities and adaptation mechanisms taking place even up to the level of the auditory nerve. Not accounting for those effects likely impedes the STRF fits and makes all subsequent analyses less reliable. I trust the small but consistent effect observed for the anechoic probes (Fig. 3-Supp. 3B) the most because it does not rely on STRF fits.<br /> Finally, the simplistic nature of the cochleagram is not able to partial out the contribution of peripheral adaptation from the adaptation observed at cortical sites.

    3. Reviewer #3 (Public Review):

      The paper by Ivanov et. al. examines how the auditory system adapts in reverberant acoustic conditions. Using a linear dereverberation framework, the study tests whether the tuning properties of neurons change in a similar manner to what is predicted by a linear dereverberation filter. The study shows that dereverberation is achieved by an extension of the inhibitory regions of receptive fields in a frequency-dependent manner. Notably, this result is complemented by showing a change in the cortical responses to probe sounds presented in the context of different reverberant conditions. Together, the similarity of the computational predictions and experimental findings supports an adaptive cortical mechanism that can reduce the effect of reverberation and in turn, support noise robust auditory perception.

    1. Reviewer #1 (Public Review): 

      This study provides relatively convincing in vivo phenotype data in mice related to vertical sleeve gastrectomy (VSG) and provides some potential mechanistic insight. This study can potentially provide some therapeutic intervention strategies on combining VSG and immunotherapy in treating breast cancer. On the other hand, this paper also has some weaknesses especially related to the detailed molecular mechanism and characterization as described below: 

      1. The major weakness lies on the detailed characterization on which inflammatory response factors that may mediate the phenotype of HFS VSG mice when compared to WM Sham mice. The data presented currently is mainly limited to RNA-Seq data, which lacks detailed characterization. 

      2. The other significant weakness also is related to the descriptive nature on characterizing the effect of immune features in Fig.4 for these mice. What is the potential mechanism on regulating T cell signaling or Cytolysis in HFS VSG mice vs WM sham mice? This at least needs some preliminary exploration and characterization.

    2. Reviewer #2 (Public Review): 

      This is a study based on the clinical observation that bariatric surgery in patients appears to be beneficial to reduce breast cancer risk. In mice with diet-induced obesity, followed by vertical sleeve gastrectomy (VSG) or dietary weight loss, tumor graft growth and response to immune checkpoint blockade were investigated. Bariatric surgery was found to be not as effective as dietary interventions in suppressing tumor growth despite achieving a similar extent of weight and adiposity loss. Leptin-mediated signaling was ruled out as a potential mechanism that could account for that difference. Notably, tumors in mice that received VSG displayed elevated inflammation and expression of the immune checkpoint ligand, PD-L1. In addition, mice that received VSG had reduced tumor-infiltrating T lymphocytes and cytolysis suggesting an ineffective anti-tumor microenvironment. Anti-PD-L1 immunotherapy suppressed tumor progression after VSG but not in control obese mice. Genomic analysis of adipose tissue after bariatric surgery from both patients and mouse models revealed a conserved gene expression signature.

    3. Reviewer #3 (Public Review): 

      In this manuscript, the authors have investigated how weight loss by bariatric surgery or weight-matched dietary intervention impairs breast cancer growth. They have shown that post-bariatric surgery, the tumors show augmented inflammation and an immune checkpoint; PD-L1 expression, which suppresses the anti-tumor immune responses. In addition, anti-PD-L1 therapy in these mice has shown to be more effective at slowing tumor growth. The authors report interesting observations, and the findings are well supported by the data, however, the use of only one syngeneic model tampers the reviewer's enthusiasm. Overall, the study is clinically important and helps in stratifying obese breast cancer patients that may respond to anti-PD-L1 immune checkpoint inhibition.

    1. Reviewer #1 (Public Review):

      In this manuscript, authors found Halo tag become resistant to lysosome degradation upon ligand binding, using this unique property, they developed a highly sensitive assay to monitor the autophagy flux. Measuring autophagy flux is one of the most important assays for studying autophagy, there are a few widely used assays to monitor the autophagy flux, such as p62 degradation, and LC3 processing, however, each of them has its own limitation, which is well known in the field. In this regard, this assay provides a simple, straight forward and sensitive assay for measuring autophagy flux, which I personally think is very likely it will be widely used by the autophagy community. This is a well-controlled, rigorous study and the manuscript is clearly written.

    2. Reviewer #2 (Public Review):

      Yim et al have utilised the HaloTag system to generate tools and assays to measure autophagy flux. The assays are highly accessible and straight forward to conduct. The study does not have any major weaknesses, with all key conclusions strongly supported by clear data. A major strength of the study is the robustness of the assay and its ease of use across SDS-PAGE and imaging techniques that I expect will help with its uptake by the research community. The assay utilises the HaLo tag and its inherent stability within lysosomes once pulsed with a HaLo ligand. This enables analysis of autophagy flux over a set period of time. The approach is highly complementary to the recently published study by Rudinskiy et al (2022) MBoC, but also includes additional tools to measure different types of selective autophagy and bulk autophagy. The inclusion of limitations of their approach within the discussion will be very useful for researchers planning to use the assay in their work. Overall, this is an excellent study that has generated very valuable tools for the study of autophagy.

    3. Reviewer #3 (Public Review):

      Monitoring autophagy induction and flux in mammalian cells is challenging and depends largely on the mammalian ATG8 proteins (LC3 and GABARAP), typically tagged at the N-terminus with a small tag (HA, flag, myc) or a range of fluorescent tags. When autophagy is induced these ATG8 proteins get captured into autophagosomes and delivered to lysosomes for degradation. Monitoring flux by western blots relies on a molecular weight shift caused by lipidation, and quantification of loss of signal from degradation (analysis of initiation), or accumulation by the addition of inhibition of lysosomal inhibitors (analyses of flux). Fluorescent tags provide similar results but the measurements rely on counting degradation sensitive or resistant fluorescent signals. Image-based analysis is more challenging than western blot but both require significant optimization. In this manuscript these existing assays are modified by the use of a probe (Halo tag) again appended to the N-terminus of ATG8s which becomes resistant to lysosomal degradation after binding a ligand (TMR). The ligand can be pulsed-in to allow detection of acute induction of autophagy eliminating the background from basal accumulation. Generation of the Halo-TMR is then monitored by western blot or using an in gel-fluorescent assay. The authors present data which shows the adaptability of the system for imaging analysis, and for both quantitative analysis using western blot and imaging of selective autophagy or bulk, non-selective autophagy. The authors have developed a robust, useful alternative to existing assay and present the results in a careful, well described brief manuscript. These modifications are important for the field and for those who require quantitative results. The drawbacks are similar to existing assays and will usually require the generation of stable cell lines because over-expressed ATG8s can aggregate and confound the measurements.

    1. Reviewer #1 (Public Review):

      Engert et al. provides a nearly complete synaptic level description of the anatomy of gustatory receptor neurons (GRNs), reveals their connectivity, and shows that they segregate based on taste modality. They find that pre and post-synaptic sites are distributed along the axons of all GRNs, as well as that each GRN receives synaptic inputs from other GRNs. Using hierarchical clustering Engert et al. defined six GRN classes based on their distinct morphology and connectivity. Next, they matched the anatomy of the six classes to the anatomy of known GRN classes that detect different taste modalities. They reveal that GRNs of each class are highly connected with each other, as well as weaker connected between different classes. Therefore, the authors performed calcium and voltage imaging to elucidate the role of the low-level connectivity of different GRN classes with each other. Interestingly, no crosstalk between the different classes could be detected.

      The data are appropriately controlled and analyzed and support the conclusions drawn in this paper.

      Strength<br /> This manuscript provides the first nearly complete synaptic level description of gustatory receptor neurons. Given that the authors reconstructed GRNs in the FAFB electron microscopy dataset manually or using a combination of automated segmentation and manual tracing using Catmaid, the synapse numbers presented here are all verified and trustworthy.

      The authors went to great lengths to test every possible synaptic connection between different GRN classes using calcium and voltage imaging. Unfortunately, no mechanism for the alteration of the transmission of gustatory signals between different GRN classes was uncovered yet, however, it shows that it is extremely important to test the functionality of synapses seen in EM datasets.

      Weaknesses<br /> Given that manual tracing in Catmaid is very time-consuming it is difficult to complete the entire population of GRNs and be competitive with similar work being performed using other EM platforms with automated segmentation. Therefore, the authors could not determine how large the actual GRN population is and whether there might be additional unknown classes.

    2. Reviewer #2 (Public Review):

      This paper utilizes the whole brain EM volume of the adult Drosophila to elucidate the synaptic connectivity patterns of the gustatory sensory neurons in the brain. The reconstruction is focused on the gustatory sensory axons of the labial nerve, which houses sensory axons from the labellum, maxillary palp and the eye, as well as motor axons that innervate the proboscis. The labellar gustatory axons terminate in specific regions of the SEZ termed the anterior central sensory center (ACSC). 87 projections were reconstructed on the right side (representing 83-96% of the total estimated); 57 projections were done for the left side (54-63% total), attributed to registration error in the volume. Morphological and synaptic clustering led to the identification of 6 different regions/zones in the ACSC on which the axons terminate. Critically, and this is the really great part of this work, the authors were able to correlate the EM data with light microscopic data in terms of the identity of neurons reconstructed, thus enabling the use of published functional data already available in terms of different taste modalities. This revealed that extensive synaptic connections are found between neurons of the same modality. The functional analysis further showed that activation of neurons of one modality does not affect those of others.

    1. Reviewer #1 (Public Review):

      This study investigated potential links between sleep structure elements and Alzheimer's disease (AD) development in healthy individuals in the late midlife to capture early signs of cognitive decline. Full polysomnography sleep recording and EEG analysis showed that slow waves are classified into two types (slow and fast switchers) and spindles are preferentially coupled only to slow switcher slow waves. The authors revealed that among sleep parameters including sleep duration and SW density, only this spindle-slow switcher SW coupling showed a negative correlation with Aβ burden in mPFC. Furthermore, the follow-up memory test revealed that uncoupling of spindle and slow switcher SWs is predictive of a memory worsening over 2 years. Therefore, this study successfully identified an early marker of Aβ deposit in mPFC and cognitive decline, which may help earlier diagnosis of AD.

    2. Reviewer #2 (Public Review):

      Chylinski et al. investigate sleep EEG properties in a cohort of older individuals, to test how sleep microarchitecture is linked to amyloid burden and memory changes over time, which is important for understanding the evolution of neurodegenerative disease. They report that the temporal coupling of spindles to a specific slow wave type, which they term 'slow switchers', is correlated with A-beta and predictive of subsequent memory decline years later. Strengths of the study are the extensive sleep phenotyping, relatively large cohort, and the acquisition of a follow-up cognitive timepoint two years later. The effect sizes are small, which may be expected due to the nature of this scientific question. The analyses are interesting, but some additional analyses and reporting would be beneficial in the methods and results, particularly the analyses focused on differentiating SW types.

      Main issues:

      The EEG signal processing and analysis methods need additional details. A coincidence of slow wave peaks and spindles is defined as 'co-occurrence' - within what time window do the two events have to co-occur to be considered coincident?

      In Fig. 1, the analysis does not control for the fact that slow switcher SWs will have a longer time period before the peak than spindles. Fig. 1b's result that more spindles occur in the same phase period could be partially explained by the fact that this phase simply takes a longer period of time for slow switcher SWs (i.e. greater chance of having a spindle if it takes 5x as much time to get from phase -1 to 0, as suggested in Fig. 1c). A control analysis is needed to account for this.

      The green shading in Fig. 1c seems to suggest some phase-coupling for fast switchers too, so it would be appropriate to add a statistic for the statement "no such preferred coupling was detected for fast switcher SWs".

      The precise implementation of the main statistical tests is a bit unclear in the Methods. When stated "slow wave spindle coupling" is an independent variable, what precisely is in the variable? Is it the phase of the coupling? Is it the proportion of SWs with a spindle for one individual?

      Given the small effect size reported for slow switcher SWs, it seems a potential reason for not finding the same result in fast switcher SWs is that there are ~4 times fewer fast switcher SWs. Even if fast switcher SWs had the same size as the underlying effect, is this sample size sufficient to detect it? Is it possible that the difference in the slow wave types reflects the different number of events in each group? Since the analysis does not directly test for a difference between fast and slow (but rather detects a significant effect with slow SWs, and fails to detect it with a smaller number of fast SWs, which does not specifically test for a difference between the two), it seems there is still additional evidence needed if aiming to draw conclusions about these fast and slow SWs being different.

    3. Reviewer #3 (Public Review):

      Strengths:<br /> - EEG analyses are novel, extensive, and carefully done.<br /> - Inclusion of baseline amyloid PET is a strength.<br /> - There is great interest in the transition from normal cognition to cognitive impairment in the earliest stages of disease, and therefore this study population is quite relevant.

      Weaknesses:<br /> - The abstract isn't clear regarding the number of participants supporting the principal conclusions. The conclusion RE amyloid was based on the stated n=100, while the one concerning cognitive decline was based only on a subset of n=66.<br /> - In the statistical methods, the authors' stated primary analyses were 1) coupling of spindles to slow switching slow waves and 2) coupling of spindles to fast switching slow waves, neither of which has anything specific to do with cognition or dementia. They adjusted these two analyses for 2 comparisons with a threshold of p=0.025. The remainder of the analyses are considered by the authors to be exploratory and therefore not to require adjustment for multiple comparisons. However, in the abstract, the stated goal of the study is to investigate "whether 22 the coupling of spindles and slow waves are associated with early amyloid-beta (Aβ) brain burden, a hallmark of AD neuropathology, and cognitive change over 2 years". This doesn't align with the stated primary analyses in the statistical methods. Moreover, it suggests at a minimum 2 primary outcomes (amyloid burden and cognitive change), and 2 predictors (spindle-slow-switch phase, and spindle-fast-switch phase) for 4 primary analyses that need to be corrected for, resulting in a p-value threshold of 0.05/4 = 0.0125. Neither of the study's primary conclusions (1. that earlier occurrence of spindles on slow-depolarization slow waves is associated with higher prefrontal Ab burden p=0.014 and 2. that earlier occurrence of spindles on slow-depolarization slow waves is associated with greater longitudinal memory decline p=0.032) meets this cutoff. This is even if we disregard the many other comparisons that were made (in the study, there are at least 3 outcomes of interest - baseline cognition, baseline amyloid, and change in cognition) and many EEG predictors examined. Indeed, if we consider all the analyses performed in this study (3 outcomes as above [amyloid, baseline cognition, change in cognition] x 7-8 different EEG measures = 24 comparisons) the 2 significant results at p<0.05 are not all that much more than would be expected by chance.<br /> - It is not 100% clear how the authors selected specifically phase angles between spindles and slow waves (rather than, for instance, percent coincidence, or dispersion of phase angle as a measure of the "tightness" of coupling) as their primary predictors. If these were looked at they would require even more extensive adjustment for multiple comparisons.<br /> - The authors conclude that their findings suggest that "altered coupling of sleep microstructure elements, key to its mnesic function, contributes to poorer brain and cognitive trajectories in ageing." In their discussion, they do acknowledge that this sort of causal inference is not possible based on the non-interventional nature of this study. Indeed, it is certainly plausible that differences in the phase relationship between spindles and slow waves, rather than being contributors to cognitive decline, may instead be markers of early AD-related brain changes, not picked up on by amyloid PET (e.g. amyloid oligomers, or non-amyloid processes) that are the proximate cause of 2-year cognitive decline.

    1. Reviewer #1 (Public Review):

      The authors shed light on the role that non-CSC exerts in promoting cancer progression, revealing that non-CSC secreted fibromodulin is crucial in mediating angiogenesis in glioma via integrin-dependent Notch signaling. The data volume is sufficient and the argumentation is rigorous enough to support the conclusions. The results are important for gaining insight into the less concerned non-CSC component in promoting cancer, and would potentially enrich the treatment strategy for glioma.

    2. Reviewer #2 (Public Review):

      Tumors such as glioblastoma contain several types of cells: cancerous and reactive non-cancerous cells, and among cancerous cells, cancer cells with tumorigenic properties so-called "stem" and pseudo-differentiated cancer cells.

      Strengths: a multidisciplinary international cooperation gathering complementary expertises. An impressive quantity of experiments and presented data (28 supplementary figures with multiple panels!). First description of Fibromodulin as a secreted factor acting in a paracrine manner to activate an Integrin-dependent Notch signaling in endothelial cells. A detailed analysis of the molecular signaling triggered by integrin activation. Most of the results support this claim.

      Weaknesses: Several formulations in the introduction are controversial. Several results should be more clearly explained and the precise methods used are difficult to find since they are dispersed between the text, the "methods section" and often lacking in the legend of the figure. More precisely the following points should be addressed:

      1- The formulation "non-cancer stem cells" is confusing since these are cancer cells but without the functional characteristics of cancer stem cells and within the tumor exist non-cancer cells co-opted to the tumor, such cells being called "microenvironment" even if they are bona fide part of the tumor.<br /> 2- Lines 91 to 95 are particularly controversial and even erroneous since CD133- GSC have been reported by several authors and nestin is not a selective marker of CSC. This is most-likely due to referencing reviews of a single group that promoted this dichotomy that do not correspond to most of the reported results. Furthermore it is well known that GSC proliferation or DGC reprogrammation to GSC are favored by hypoxia, illustrated in vivo by the failure of anti-VEGF treatment to increase life expectancy.<br /> 3- As soon as 2012-2013 (thus before the referenced Suva et al 2014 paper), the group of Thierry Virolle demonstrated that stem cell-like properties of GSC fuel glioblastoma development by providing the different cell types that comprise the tumor. Reference to their work is surprisingly missing. Of note, after describing that the miR-302-367 cluster is strongly induced during stemness suppression, they showed that stable miR-302-367 cluster expression is sufficient to suppress the stemness signature, self-renewal, and cell infiltration within a host brain tissue, through inhibition of the CXCR4 pathway involving the SHH-GLI-NANOG network. Micro-RNA profiling studies to search for regulators of stem cell plasticity, allowed them to identified miR-18a* as a potential candidate and its expression correlated with the stemness state. MiR-18a* expression promotes clonal proliferation in vitro and tumorigenicity in vivo. Turchi L, Debruyne DN, Almairac F, Virolle V, Fareh M, Neirijnck Y, Burel-Vandenbos F, Paquis P, Junier MP, Van Obberghen-Schilling E, Chneiweiss H, Virolle T. Tumorigenic potential of miR-18A* in glioma initiating cells requires NOTCH-1 signaling. Stem Cells. 2013 Jul;31(7):1252-65. doi: 10.1002/stem.1373. PMID: 23533157 Fareh M, Turchi L, Virolle V, Debruyne D, Almairac F, de-la-Forest Divonne S, Paquis P, Preynat-Seauve O, Krause KH, Chneiweiss H, Virolle T. The miR 302-367 cluster drastically affects self-renewal and infiltration properties of glioma-initiating cells through CXCR4 repression and consequent disruption of the SHH-GLI-NANOG network. Cell Death Differ. 2012 Feb;19(2):232-44. doi: 10.1038/cdd.2011.89.<br /> 4- A main question arises from the use of multiple cellular models, some highly valuables such as MGG4, MGG6 and MGG8, that correspond to patient-derived cell line maintained in culture conditions known to preserve the phenotype and genotype encountered in real patient tumors, and other cell lines (LN229, U251, U87) known to be highly unrepresentative since grown for a long time in serum conditions. However, after the first set of experiments, only MGG8 is used in the rest of the paper, with no validation on MGG4 and MGG6 and one should wonder why.<br /> 5- Results presented in Fig2C and 2D are really strange and do not support the claim that "results indicate that FMOD secreted by DGCs is essential for the growth of tumors initiated by GSCs. FMOD induces angiogenesis of host-derived and tumor-derived endothelial cells" First, one should wonder about the subcutaneous model used since such xenograft do not raise a glioblastoma-like tumor but a mesenchymal-like highly undifferentiated tumor. Second, considering the development of the graft at day 5 and the growth curve of GSC alone, one should wonder why inhibiting expression of FMOD in DGC triggers a necrosis of the initial tumor and not a slower growth parallel to the one of GSC alone.<br /> 6- Line 445: "Cellular hierarchy is well established in GBM." This is an old view mimicking normal differentiation. Since GSC can pseudo-differentiate into DGC and DGC can be reprogrammed into GSC, no hierarchy exists, only cells with different properties and functions in tumor growth. GSC is not the origin of glioblastoma but the ultimate state of aggressiveness.<br /> 7- Lines 452-53 "GSCs are known to promote the establishment of a highly vascularized microenvironment by being in close physical contact with endothelial cells (Calabrese et al., 2007)." This in only partially true since many soluble factors have been described to support the dialog between endothelial cells and GSC: secreted proteins such as VEGF, HDGF, GDF15, and multiple types of microRNA.

    3. Reviewer #3 (Public Review):

      This article proposed a hypothesis that non-cancer stem cells secreted factor-FMOD could impressively promote angiogenesis to induce tumor growth in vivo. The finding uncovered a potential interesting and important protein therapeutic target from non-cancer stem cells but not from the glioma stem-like cells. Authors utilized diverse in vitro and in vivo methods to elucidate their hypothesis. The logic is smooth and clear and the results are solid. This article showed us that it might be worth also looking at non-cancer stem cells more in tumor growth.

    1. Joint Public review:

      Endonuclease G (EndoG) is best known for its role in caspase-independent degradation of nuclear DNA during apoptosis, when the protein is released from mitochondria upon oxidative stress. This manuscript reveals a role of EndoG in generating a 9 bp deletion that is commonly found in the mitochondrial genome. The authors combine bioinformatics and experimental analysis to identify mitochondrial genome sequences that have the potential to form G4 tetraplexes. They focus the further analysis on the intergenic region of cytochrome c oxidase II/tRNALys that contains the most common mitochondrial deletion, a 9 bp deletion involving a repeated DNA sequence. Furthermore, using BG4, a presumptive anti-G4 DNA antibody, the authors use immunofluorescence and ChIP to provide evidence for the occurrence of G4 tetraplexes in the mitochondrial genome. Using purified Endo G and mitochondrial extracts, the authors show preferential binding of EndoG to mitochondrial genome sequences with the potential to form G4DNA and induction of DNA breaks at such sites using mutant oligonucleotide substrates that are predicted to break the G4 potential as controls. Moreover, the authors reconstitute in vitro the reaction to generate the 9 bp deletion using oligonucleotides and mitochondrial extracts, leading to the model that EndoG-mediated cleavage at G4 tetraplex forming sequences in the mitochondrial genome generates breaks that are repaired by MMEJ generating rearrangements including the common 9 bp deletion. Finally, the authors show that under oxidative stress induced by menadione, EndoG is released into the mitochondrial matrix, possibly making it more available to act on the mitochondrial genome. The manuscript spans a significant number of experiments ranging from bioinformatic analysis, protein biochemistry, cell genetics, and immunofluorescence. The data generally support the conclusions with some significant exceptions. The authors often inaccurately describe the results mixing in their interpretation, and the manuscript is difficult to read. Major problems are the specificity of the BG4 antibody, the resolution of the microscopy, and the lack of a critical control of separate incubation of the substrates for the reconstitution experiment. Conceptually, it is unclear what the physiological roles of EndoG release into the mitochondrial matrix and the generation of mitochondrial genome arrangements might be.

    1. Reviewer #1 (Public Review):

      The authors of this manuscript analyzed the lung morphology of five osteichthyan vertebrate species (four that do not belong to amniotes or teleost fishes plus a salamander) and based on their phylogenetic relationship, they concluded that the lung first arose as an unpaired organ and later became bilaterally paired in the amniote lineage and secondarily lost in the teleost fish lineage. I found the authors' anatomical investigation of morphology enabled by sample collection covering the long-missing osteichthyan lineages appealing and sufficient to draw the conclusion.

    2. Reviewer #2 (Public Review):

      This is an interesting paper that gives novel insights into the evolution of lungs. The study is straightforward. It is well written and the data are clear. The authors have analysed lung development in a number of species of bony fish, which have been chosen as they occupy an informative position on the evolutionary tree. They use synchrotron X-ray microtomography and histology to probe lung development. They conclude that the primitive state of vertebrate lungs was as unpaired structures but with the evolution of land-dwelling vertebrates, truly paired lungs emerged. These are a defining feature of the tetrapods and are crucial for life on land. The authors present a scenario for how they believe lungs evolved which is believable and insightful.

    3. Reviewer #3 (Public Review):

      This paper presents significant new data on the anatomy and developmental biology and evolution of lungs in key basal, key osteichthyan fishes, and a primitive tetrapod. The paper provides a good background to the problem, but one main point that should be considered is missing (see below). The 3D CT data combined with thin-section images substantiate their findings well. The work holds important implications for understanding how paired lungs first evolved in fishes and tetrapods, which has been a major evolutionary conundrum up to now. The methods and data presented are sound, and the illustrations are clear and relevant to the development of the intellectual arguments presented in the discussion.

      There is a controversial old description of paired lungs in the placoderm (basal gnathostome) Bothriolepis (Denison 1941; Arsenault et al 2004; Goujet 2011, Janvier et a.l 2007) which is completely contrary to the main conclusions of this paper. One cannot simply ignore it - the authors must confront the data and discuss it, or else there is standing evidence that the basal condition for all gnathostomes is a paired lung.

    1. Reviewer #1 (Public Review):

      The study by Giulieri and colleagues focuses on the detection of genetic loci that experience selection in S. aureus during the transition from colonization to infection. The authors assembled a large collection of S. aureus genomes from prior studies and systematically analyzed them for genetic variation and signatures of genome degradation. They found significant convergent evolution in genes linked to antibiotic response and pathogenesis. The result is a high-resolution picture of S. aureus adaptation during the transition from colonization to infection.

      The major strength of the paper is the large scale of the analysis and the inclusion of additional variants besides SNPs, which are frequently ignored because they can be hard to reliably detect and study. One additional strength is the use of "multilayered" annotation (i.e. including intergenic variants) to increase signals of convergent evolution. One weakness of the study is a lack of a classification of the variants detected in convergent loci. For example, which genes do the authors think are acquiring gain-of-function versus loss-of-function mutations? One other weakness is a lack of functional studies exploring some of the more novel signals detected (such as hypothetical proteins with "no data on S. aureus").

      In general, the results support the conclusions drawn by the authors, the likely impact of the work is quite high, and overall it is a useful example of how to perform systematic detection of pathogen loci under selection in vivo during infection.

    2. Reviewer #2 (Public Review):

      This work provides a comprehensive within-host evolution analysis of all publicly available Staphylococcus aureus genome. The authors combined variant and chromosome structural variants detecting, internal variant annotation, gene and operon enrichment analysis, mutation co-occurence analysis and network analysis of adaptation signatures, to compile a comprehensive catalogue of bacterial genetic variation arising during host infection. This strategy enabled the detection of convergent adaptation patterns at an unprecedented resolution. Through study, they found evidence of a distinctive evolutionary pattern within the infecting populations compared to colonising bacteria. In addition to reported agr-mediated adaptation, they identified non-canonical genome-wide significant loci including sucA-sucB and stp1. The prevalence of adaptive changes increased with infection extent, emphasising the clinical significance of these signatures. These findings provide a high-resolution picture of the molecular changes when S. aureus transitions from colonisation to severe infection and may inform the correlation of infection outcomes with adaptation signatures.

    1. Reviewer #1 (Public Review):

      In this manuscript, Dard et al. investigate hippocampal dynamics over the course of early postnatal development. They find evidence for an abrupt developmental transition in this neural activity at the end of the first postnatal week in rodents and postulate that it is related to the emergence of internal representations. This work is interesting as it explores the developmental expression of neural activity patterns and contributes to the understanding of how cognitive functions could emerge from the immature brain. Additional methodological and statistical analysis is necessary to support the suggested conclusions of this work.

      Strengths:<br /> The authors employ in vivo imaging of the hippocampus in developing mice and merge these techniques with cell labeling to try to establish clues as to the mechanisms of the neural activity patterns they observe. This work provides information in a relatively understudied field and could help to provide developmental timelines and cellular mechanisms for the maturation of neural networks.

      Weaknesses:<br /> 1) The experiments involve an invasive neurosurgical procedure used to perform hippocampal imaging, which removes the ipsilateral overlying somatosensory cortex, and it is not possible to evaluate from the data provided that this surgery does not disrupt network function, especially given the focus on movement-related activity patterns.<br /> 2) State-dependent parameters are not adequately described, controlled, and examined quantitatively to ensure that data from similar behavioral states is being used for analysis across ages. Network activity from wakefulness, REM/active sleep and NREM/quiet sleep should not be presumed to be indistinguishable.<br /> 3) Currently employed statistics are not rigorous, unified, or sensitive, and do not support all of the authors' claims. Data shown suggest potentially significant changes that have not been identified due to suboptimal statistical approach and/or underpowering.<br /> 4) The authors use an artificial neural network approach to infer cell classification (pyramidal cell vs. interneuron). From the data provided, it is not possible to adequately evaluate whether these 'inferred' interneurons represent the same population as conventionally labeled interneurons.<br /> 5) Functional GABAergic activity is not assessed across development (only at P9-10), limiting mechanistic conclusions that can be drawn.<br /> 6) The present analyses are almost exclusively focused on movement-related epochs, substantially limiting conclusions that can be drawn as to what neural dynamics are actually occurring during epochs that the authors propose comprise internal representations.

      Overall:<br /> The authors aimed to demonstrate a shift in hippocampal neural activity from primarily responding to external stimuli (i.e. body movements) to manifesting internal network dynamics. They identify local GABAergic innervation as a likely candidate mechanism for this shift. While interesting, the current analytic methods used are insufficient to fully support the authors' claims.

    2. Reviewer #2 (Public Review):

      The study by Dard et al aims to uncover the post-natal emergence of mature network dynamics in the hippocampus, with a particular focus on how pyramidal cells and interneurons change their response to spontaneous limb movement. Several previous studies have investigated this topic using electrophysiology, but this study is the first to utilize 2-photon calcium imaging, enabling the recording of hundreds of individual neurons, and discrimination between pyramidal cell and interneuron activity. The aims of the study are of broad interest to all neuroscientists studying development (including neurodevelopmental disorders) and the basic science of network dynamics.

      The main conclusions of the study are that (1) in early life, most pyramidal cell activity occurs in bursts synchronized to spontaneous movement, (2) by P12, pyramidal cell activity is largely desynchronized from spontaneous movement, and indeed movement triggers an inhibition in the pyramidal network (approximately 2-4sec following movement), (3) unlike pyramidal cells, interneuron activity remains positively modulated by movement, throughout the period P1-P12, (4) the changes in pyramidal cell activity are achieved by means of increases in perisomatic inhibition, between P8 and P10.

      It should be noted that conclusion (1) and to some extent conclusion (2) have already been reported, by previous studies using electrophysiology (as clearly acknowledged by the authors).

      A principal strength of this manuscript is the extremely high quality of the data that the authors are able to use in support of (1) and (2), with very large numbers of neurons being analyzed to clearly delineate the relationship between neural activity and movement. The finding that pyramidal cells become inhibited following movement is novel, I believe. Furthermore, this study offers the first description of the development of interneuron activity, in this experimental context.

      The main weakness of the manuscript is that the authors cannot provide direct functional evidence for the conclusion (4). As shown by the analysis in support of conclusion (3), interneuron activity with respect to movement does not actually change during the developmental period being studied, making it prima facie unlikely that this is the cause of changes in pyramidal network responses to movement. To overcome this, the study describes the activity of GABA-ergic axon terminals in the pyramidal cell layer at P9-10, but it appears that due to technical problems this was not possible in younger animals. It, therefore, remains unknown if the functional inhibitory inputs to pyramidal cells are changing over the ages studied. The study does describe increases in the protein synaptotagmin-2, in the pyramidal cell layer, between P3 and P11, but in my opinion, this molecular evidence for increases in perisomatic inhibition does not match the (very high) standards of neuronal function/activity reported elsewhere in the manuscript.

    3. Reviewer #3 (Public Review):

      Dard and colleagues use both in vivo calcium imaging and computational modelling to explore the relationship between the early movement of CA1 hippocampal activity in neonatal mice.

      The manuscript represents a significant technical advance in that the authors have pioneered the use of multiphoton imaging to record activity in the hippocampus of awake neonates. Overall the presentation of the data is convincing although I would recommend a number of tweaks to the figures and the inclusion of some raw data to better direct and inform non-expert readers. I also believe that the assessment of long-range inputs using pseudo-rabies virus should be present in the main body of the manuscript as opposed to supplemental material.

      The computational modelling supports their idea but does not exclude other possibilities. Further, it is not clear to what extent the strengthening of local excitatory input onto the interneurons - the dominant route of recurrent input in the hippocampus, is important; something that the authors acknowledge in the discussion.

      Overall, I believe the paper adds to our knowledge of the timeline of development and further identified the postnatal day (P)9-P10 window as important in emergent cortical processing. The fact that this is linked to an increase in GABAergic innervation has implications for our understanding of both normal and dysfunctional brain development.

    1. Reviewer #1 (Public Review):

      Sadeh and Clopath analyze two mouse datasets from the Allen Brain Atlas and show that sensory representations can have apparent representational drift that is entirely due to behavioral modulation. The analysis serves as a caution against over-interpreting shifts in the neural code. The analysis of data is coupled with careful modeling work that shows that the behavioral state reliably shifts sensory representations independently of stimulus modulation (rather than acting as a gain factor), and further show that it is reproducibly shifted when the behavioral state is adequately controlled for. The methods presented point towards a more careful consideration and measurement of behavioral states during sensory recordings, and a re-analysis of previous findings. The findings held up for both standard drifting grating stimuli as well as natural movies.

      The fact that neurons may have different tuning depending on the behavioral state of the animal raises obvious questions about readout. The authors show that neurons with strong behavioral shifts should simply be ignored and that this can be achieved if the downstream decoder weights inputs with more stimulus information. While questions remain about why behavior shifts representations and how that could be more effectively utilized by downstream circuits, the results presented clearly show that sensory representations might not always be simply drifting over time, and will spark some careful analysis of past and future experimental results.

    2. Reviewer #2 (Public Review):

      Studies from recent years have shown that neuronal responses to the same stimuli or behavior can gradually change with time - a phenomenon known as representational drift. Other recent studies have shown that changes in behavior can also modulate neuronal responses to a given sensory stimulus. In this manuscript, Sadeh and Clopath analyzed publicly available data from the Allen Institute to examine the relationship between animal behavioral variability and changes in neuronal representations. The paper is timely and certainly has the potential to be of interest to neuroscientists working in different fields. However, there are currently several important issues with the analysis of the data and their interpretations that the authors should address. We believe that after these concerns are addressed, this study will be an important contribution to the field.

      1. The manuscript raises a potential problem: while previous work suggested that the passage of time leads to gradual changes in neuronal responses, the causality structure is different: i.e., the passage of time leads to gradual changes in behavior, which in turn lead to gradual changes in neuronal responses. The authors conclude that "variable behavioral signal might be misinterpreted as representational drift". While this may be true, in its current form, the paper lacks critical analyses that would support such a claim. It is possible that both factors - time and behavior - have a unique contribution to changes in neuronal responses, or that only time elicits changes in neuronal responses (and behavior is just correlated with time). Thus, the authors should demonstrate that these changes cannot be explained solely by the passage of time and elucidate the unique contributions of behavior (and elapsed time) to changes in representations.

      2. There are also several issues with the analysis of the data and the presentation of the results. The most concerning of which is that the data shows a non-linear (and non-monotonic) relationship between behavioral changes and representational similarity. In many of the presented cases, the data points fall into two or more discrete clusters. This can lead to the false impression that there is a monotonic relationship between the two variables, even though there is no (or even opposite) relationship within each cluster. This is a crucial point since the clusters of data points most likely represent different blocks that were separated in time (or separation between within-block and across-block comparisons).

      3. The authors also suggest that using measures of coding stability such as 'population-vector correlations' may be problematic for quantifying representational drift because it could be influenced by changes in the neuronal activity rates, which may be unrelated to the stimulus. We agree that it is important to carefully dissociate between the effects of behavior on changes in neuronal activity that are stimulus-dependent or independent, but we feel that the criticism raised by the authors ignores the findings of multiple previous papers, which (1) did not purely attribute the observed changes to the sensory component, and (2) did dissociate between stimulus-dependent changes (in the cells' tuning) and off-context/stimulus-independent changes (in the cells' activity rates).

      4. Another important issue relates to the interchangeable use of the terms 'representational drift' and 'representational similarity'. Representational similarity is a measure to identify changes in representations, and drift is one such change. This may confuse the reader and lead to the misconception that all changes in neuronal responses are representational drift.

    3. Reviewer #3 (Public Review):

      Although it is increasingly realized that cortical neural representations are inherently unstable, the meaning of such "drift" can be difficult or impossible to interpret without knowing how the representations are being read out and used by the nervous system (i.e. how it contributes to what the experimental animal is actually doing now or in the future). Previous studies of representational drift have either ignored or explicitly rejected the contribution of what the animal is doing, mostly due to a lack of high-dimensional behavioural data. Here the authors use perhaps the most extensive open-source and rigorous neural data available to take a more detailed look at how behaviour affects cortical neural representations as they change over repeated presentations of the same visual stimuli.

      The authors apply a variety of analyses to the same two datasets, all of which convincingly point to behavioural measures having a large impact on changing neural representations. They also pit models against each other to address how behavioural and stimulus signals combine to influence representations, whether independently or through behaviour influencing the gain of stimuli. One analysis uses subsets of neurons to decode the stimulus, and the independent model correctly predicts the subset to use for better decoding. However, one caveat may be that the nervous system does not need to decode the stimulus from the cortex independently of behaviour; if necessary, this could be done elsewhere in the nervous system with a parallel stream of visual information.

      Overall the authors' claims are well-supported and this study should lead to a re-assessment of the concept of "representational drift". Nonetheless, a weakness of all analyses presented here is that they are all based on data in head-fixed mice that were passively viewing visual stimuli, such that it is unclear what relevance the behaviour has. Furthermore, the behavioural measurements available in the open-source dataset (pupil movements and running speed) are still a very low dimensional representation of what the mice were actually doing (e.g. detailed kinematics of all body movements and autonomic outputs). Thus, although the authors here as well as other large-scale neural recording studies in the past decade or so make it clear that relatively basic measures of behaviour can dramatically affect cortical representations of the outside world, the extent to which any cortical coding might be considered purely sensory remains an important question. Moreover, it is possible that lower-dimensional signals are overly represented in visual areas, and that in other areas of the cortex (e.g. somatosensory for proprioception), the line between behaviour parameters and sensory processing is blurred.

    1. Reviewer #1 (Public Review):

      Huang et al. sought to study the cellular origin of Tuft cells and the molecular mechanisms that govern their specification in severe lung injury. First the authors show ectopic emergence of Tuft cells in airways and distal parenchyma following different injuries. The authors also used lineage tracing models and uncovered that p63-expressing cells and to some extent Scgb1a1-lineaged labeled cells contribute to tuft cells after injury. Further, the authors modulated multiple pathways and claim that Notch inhibition blocks tuft cells whereas Wnt inhibition enhances Tuft cell development in basal cell cultures. Finally, the authors used Trpm5 and Pou2f3 knock-out models to claim that tuft cells are indispensable for alveolar regeneration.

      In summary, the findings described in this manuscript are somewhat preliminary. The claim that the cellular origin of Tuft cells in influenza infection was not determined is incorrect. Current data from pathway modulation is preliminary and this requires genetic modulation to support their claims.

      Major comments:

      1. The abstract sounds incomplete and does not cover all key aspects of this manuscript. Currently, it is mainly focusing on the cellular origin of Tuft cells and the role of Wnt and notch signaling. However, it completely omits the findings from Trpm5 and Pou2f3 knock-out mice. In fact, the title of the manuscript highlights the indispensable nature of tuft cells in alveolar regeneration.

      2. In lines 93-94, the authors state that "It is also unknown what cells generate these tuft cells.....". This statement is incorrect. Rane et al., 2019 used the same p63-creER mouse line and demonstrated that all tuft cells that ectopically emerge following H1N1 infection originate from p63+ lineage labeled basal cells. Therefore, this claim is not new.

      3. Lines 152-153 state that "21.0% +/- 2.0 % tuft cells within EBCs are labeled with tdT when examined at 30 dpi...". It is not clear what the authors meant here ("within EBC's")? And also, the same sentence states that "......suggesting that club cell-derived EBCs generate a portion of tuft cells....". In this experiment, the authors used club cell lineage tracing mouse lines. So, how do the authors know that the club cell lineage-derived tuft cells came through intermediate EBC population? Current data do not show evidence for this claim. Is it possible that club cells can directly generate tuft cells?

      4. Based on the data from Fig-3A, the authors claim that treatment with C59 significantly enhances tuft cell development in ALI cultures. Porcupine is known to facilitate Wnt secretion. So, which cells are producing Wnt in these cultures? It is important to determine which cells are producing Wnt and also which Wnt? Further, based on DBZ treatments, it appears that active Notch signaling is necessary to induce Tuft cell fate in basal cells. Where are Notch ligands expressed in these tissues? Is Notch active only in a small subset of basal cells (and hence generate rate tuft cells)? This is one of the key findings in this manuscript. Therefore, it is important to determine the expression pattern of Wnt and Notch pathway components.

      5. How do the authors explain different phenotypes observed in Trpm5 knockout and Pou2f3 mutants? Is it possible that Trpm5 knockout mice have a subset of tuft cells and that they might be something to do with the phenotypic discrepancy between two mutant models?

      6. One of the key findings in this manuscript is that Wnt and Notch signaling play a role in Tuft cell specification. All current experiments are based on pharmacological modulation. These need to be substantiated using genetic gain loss of function models.

    2. Reviewer #2 (Public Review):

      In this manuscript, the authors describe the ectopic differentiation of tuft cells that were derived from lineage-tagged p63+ cells post influenza virus infection. These tuft cells do not appear to proliferate or give rise to other lineages. They then claim that Wnt inhibitors increase the number of tuft cells while inhibiting Notch signalling decreases the number of tuft cells within Krt5+ pods after infection in vitro and in vivo. The authors further show that genetic deletion of Trpm5 in p63+ cells post-infection results in an increase in AT2 and AT1 cells in p63 lineage-tagged cells compared to control. Lastly, they demonstrate that depletion of tuft cells caused by genetic deletion of Pou2f3 in p63+ cells has no effect on the expansion or resolution of Krt5+ pods after infection, implying that tuft cells play no functional role in this process.

      Overall, in vivo and in vitro phenotypes of tuft cells and alveolar cells are clear, but the lack of detailed cellular characterization and molecular mechanisms underlying the cellular events limits the value of this study.

      1. Origin of tuft cells: Although the authors showed the emergence of ectopic tuft cells derived from labelled p63+ cells after infection, it cannot be ruled out that pre-existing p63+Krt5- intrapulmonary progenitors, as previously reported, can also contribute to tuft cell expansion (Rane et al. 2019; by labelling p63+ cells prior to infection, they showed that the majority of ectopic tuft cells are derived from p63+ cells after viral infection). It would be more informative if the authors show the differentiation of tuft cells derived from p63+Krt5+ cells by tracing Krt5+ cells after infection, which will tell us whether ectopic tuft cells are differentiated from ectopic basal cells within Krt5+ pods induced by virus infection.

      2. Mechanisms of tuft cell differentiation: The authors tried to determine which signalling pathways regulate the differentiation of tuft cells from p63+ cells following infection. Although Wnt/Notch inhibitors affected the number of tuft cells derived from p63+ labelled cells, it remains unclear whether these signals directly modulate differentiation fate. The authors claimed that Wnt inhibition promotes tuft cell differentiation from ectopic basal cells. However, in Fig 3B, Wnt inhibition appears to trigger the expansion of p63+Krt5+ pod cells, resulting in increased tuft cell differentiation rather than directly enhancing tuft cell differentiation. Further, in Fig 3D, Notch inhibition appears to reduce p63+Krt5+ pod cells, resulting in decreased tuft cell differentiation. Importantly, a previous study has reported that Notch signalling is critical for Krt5+ pod expansion following influenza infection (Vaughan et al. 2015; Xi et al. 2017). Notch inhibition reduced Krt5+ pod expansion and induced their differentiation into Sftpc+ AT2 cells. In order to address the direct effect of Wnt/Notch signalling in the differentiation process of tuft cells from EBCs, the authors should provide a more detailed characterization of cellular composition (Krt5+ basal cells, club cells, ciliated cells, AT2 and AT1 cells, etc.) and activity (proliferation) within the pods with/without inhibitors/activators.

      3. Impact of Trpm5 deletion in p63+ cells: It is interesting that Trpm5 deletion promotes the expansion of AT2 and AT1 cells derived from labelled p63+ cells following infection. It would be informative to check whether Trpm5 regulates Hif1a and/or Notch activity which has been reported to induce AT2 differentiation from ectopic basal cells (Xi et al. 2017). Although the authors stated that there was no discernible reduction in the size of Krt5+ pods in mutant mice, it would be interesting to investigate the relationship between AT2/AT1 cell retaining pods and the severity of injury (e.g. large Krt5+ pods retain more/less AT2/AT1 cells compared to small pods. What about other cell types, such as club and goblet cells, in Trpm5 mutant pods? Again, it cannot be ruled out that pre-existing p63+Krt5- intrapulmonary progenitor cells can directly convert into AT2/AT1 cells upon Trpm5 deletion rather than p63+Krt5+ cells induced by infection.

      4. Ectopic tuft cells in COVID-19 lungs: The previous study by the authors' group revealed the presence of ectopic tuft cells in COVID-19 patient samples (Melms et al. 2021). There appears to be no additional information in this manuscript.

      5. Quantification information and method: Overall, the quantification method should be clarified throughout the manuscript. Further, in the method section, the authors stated that the production of various airway epithelial cell types was counted and quantified on at least 5 "random" fields of view. However, virus infection causes spatially heterogeneous injury, resulting in a difficult to measure "blind test". The authors should address how they dealt with this issue.

    3. Reviewer #3 (Public Review):

      In this manuscript Huang et al. study how the lung regenerates after severe injury due to viral infection. They focus on how tuft cells may affect regeneration of the lung by ectopic basal cells and come to the conclusion that they are not required. The manuscript is intriguing but also very puzzling. The authors claim they are specifically targeting ectopic basal progenitor cells and show that they can regenerate the alveolar epithelium in the lung following severe injury. However, it is not clear that the p63-CreERT2 line the authors are using only labels ectopic basal cells. The question is what is a basal cell? Is an ectopic basal progenitor cell only defined by Trp63 expression?

      The accompanying manuscript by Barr et al. uses a Krt5-CreERT2 line to target ectopic basal cells and using that tool the authors do not see a signification contribution of ectopic basal cells towards alveolar epithelial regeneration. As such the claim that ectopic basal cell progenitors drive alveolar epithelial regeneration is not well-founded.

      The title itself is also not very informative and is a bit misleading. That being said I think the manuscript is still very interesting and can likely easily be improved through a better validation of which cells the p63-CreERT2 tool is targeting.

      I, therefore, suggest the following experiments.

      1) Please analyze which cells p63-CreERT2 labels immediately after PR8 and tamoxifen treatment. Are all the tdTomato labeled cells also Krt5 and p63 positive or are some alveolar epithelial cells or other airway cell types also labeled?

      2) Please also show if p63-CreERT2 labels any cells in the adult lung parenchyma in the absence of injury after tamoxifen treatment.

      3) Please analyze if p63-CreERT2 labels any cells with tdTomato in the absence of injury or after PR8 infection but without tamoxifen treatment.

      4) Please analyze when after PR8 infection do the first p63-CreERT2 labeled tdTomato positive alveolar epithelial cells appear.

      5) A clonal analysis of p63-CreERT2 labeled cells using a confetti reporter might also help interpret the origin of p63-CreERT2 labeled cells.

      6) Lastly could the authors compare the single-cell RNAseq transcription profile of p63-CREERT2 labeled cells immediately after PR8 and tamoxifen treatment and also at 60dpi. A pseudotime analysis and trajectory interference analysis could help elucidate the identity of p63-CreERT2 labeled cells that are actually not ectopic basal progenitor cells.

    1. Reviewer #1 (Public Review):

      The manuscript by Tatli et al. entitled "Nanoscale resolution of microbial fiber degradation in action" characterizes how the cellulosome-producing, anaerobic bacterium Clostridium thermocellum responds to the presence of crystalline cellulose substrate and its subsequent degradation in real-time. Using state-of-the-art cryo-electron structural methods (i.e., microscopy and tomography) in combination with biochemistry, molecular biology, imaging, and microbial genetics and physiology the authors assess the location, density, enzyme composition of the cellulosomal complexes on the bacterial surface, its interactions with the crystalline cellulose substrate, and the corresponding changes in these properties that result from decomposition the substrate over time.

      Specifically, using cryo-electron-based methods and imaging the authors showed extracellular cellulosomal densities at resolutions not seen previously and were able to measure distances between the bacterial S-layer and the cellulosome layer as well of the thickness of the latter. Taking advantage of cryo-electron tomography methods and data processing, the authors present nano-scale images of cellulosome-crystalline cellulose interactions, where the cellulosomal machinery is seen to envelop the substrate and disrupt the well-order compact, packing of cellulose microfibrils. They also present the cryo-EM structure of Cel48S, the most abundant cellulosomal glycoside hydrolase, which had a similar fold to the catalytic module previously determined by X-ray crystallography but also the topology of the linker region tethering the catalytic module to its type-I dockerin module that had not been previously observed. Expression of Cel48S in C. thermocellum was dramatically increased upon exposure to the crystalline cellulose substrate for the first 10-15 hr after which there was a subsequent decrease to basal levels between hours 15-20, which was associated with substrate availability and increased presence of degradation bioproducts. Finally, the authors used cryo-electron microscopy to assess single-cell cellulosome distribution across the bacterial population and its substrate dependency. Rather than a distribution of cellulosome densities on the cell surface across the microbial population, two predominant phenotypes were observed - a high-density phenotype and a low-density phenotype that shifted from a 1:5 to 5:1 ratio upon exposure to cellulose. The authors associate these latter observations with division-of-labour and bet-hedging evolutionary strategy whereby a population invested significant energy to produce the cellulosomal machinery and are thus primed for a substrate-rich environment while the low-density population ensures continued cell growth and nimble response to changing environmental conditions.


      The manuscript is well-written and represents an influential body of work that will have broad appeal, including the environmental microbe, carbohydrate/biomass degradation, microbial and biopolymer engineering communities. The experiments are well-designed comprising ingenious use of microbial genetics, various substrates, and recombinant protein constructs with the C. thermocellum system to address observations in cyro-electron, biochemical and microbiology studies. The experimental analysis and interpretation are first-rate. The methods are appropriate, diverse yet truly complementary, and state-of-the-art.

      While much effort has been dedicated to the structural characterization of the cellulosome, it has largely involved a dissection approach involving recombinant proteins. As such, there remains a significant gap in knowledge of the in vivo cellulosome structure and its interaction with crystalline cellulose. Furthermore, little is currently known as to how cellulosome-producing bacteria respond to changing environments, including C. thermocellum, which serves as the model cellulosomal bacterium. The data provides unprecedented in situ views of the cellulosomal machinery on the bacterial cell surface, its interaction of cellulose, and the disruption of the latter's structural organization. The quantitative nature of the work, particularly those associated with revealing the dynamic yet quite specific phenotypic heterogeneity of cellulosome-producing C. thermocellum (a high-density and a low-density population), is innovative in its approach and novel. These findings present intrigues and previously unforeseen insights into the response of C. thermocellum to the cellulose substrate. The authors have done an excellent job of linking previous observations to one made here using them to establish a foundation from which they formulate their conclusions.


      There are no major weaknesses that significantly detract from the novelty and impact of the study. Not so much a weakness as much as simply unfortunate is the lack of the type-I dockerin module in the cryo-EM structure of Cel48S. The authors correctly note the apparent inherent flexibility of the N-terminal region of the dockerin module and the low calcium concentrations used, which may contribute to its absence in the structure.

    2. Reviewer #2 (Public Review):

      The manuscript by Itzhak Mizrahi is an original study using state-of-the-art integrative structural biology at multiple scales, using cryo-EM, imaging, electron tomography, microbiology and genetics to capture anaerobic bacterial cellulosomes from C. thermocellum in action. The study depicts the presence of cellulosomes at the bacterial extracellular surface, the impact of cellulosomal action on micro-crystalline cellulose, and identifies the presence of large globular enzymes in interaction with the substrate. Major findings are the multi-scale description of a 65 nm thick "belt" of cellulosomal particles around a single bacterial cell when displaying a high density of cellulosomes, down to the identification of specific components, such as the catalytic enzyme Cel48S, within this region when interacting and degrading cellulose micro-fibrils. These single-cell data are put in perspective with physiological growth properties of native and genetically modified C. thermocellum, showing that the bacterial population is heterogeneous with respect to the presence of cellulosomal complexes. Two types of populations, one displaying high density and a second with a much lower density of cellulosomes, co-exist in a ratio that depends on the available monosaccharides in solution, leading the authors to speculate that a division-of-labor strategy takes place in the C. thermocellum population.

      The conclusions drawn are clearly justified by the presented data, interpretations and even speculations are designed as such by the authors and are plausible in view of the obtained results. The strength of the paper is the clever application of methods that allow spanning scales by several orders of magnitude, and that allow connecting single-cell data to physiological observations in the bulk of cultured cells.

    1. Reviewer #1 (Public Review):

      Overall a very interesting paper. There have been calls for discovery of herbicides that are multi-site inhibitors as a predicted way to delay resistance evolution to those herbicides. Fungicides are known that are multi-site inhibitors and these are known to have lower risk for resistance evolution. The authors provide evidence that their novel inhibitors of lysine synthesis inhibit both the first enzyme (previously shown) and the second enzyme in the lysine synthesis pathway. Inhibition of the first enzyme was shown to be due to inhibition at an allosteric site, while the same compound is shown in this paper to be a competitive inhibitor of the second enzyme. This two-site inhibition explains the relatively higher in vivo activity of the compound compared to its in vitro activity on the first enzyme alone. The authors show that this two-site inhibitor of lysine synthesis has biological activity to reduce growth of the global weed Lolium rigidum. The modeling work to show the specific amino acids in the target binding site that are predicted to interact with the compound is really interesting and gives insights into how target site resistance could eventually evolve to this herbicide.

    2. Reviewer #2 (Public Review):

      The paper contains a continued investigation of previously described dihydrodipicolinate synthase (DHDPS) inhibitor (MBDTA-2) to determine if the MBDTA-2 could be activated in planta to a more potent inhibitor. The hypothesis of potential demethylation of the methoxy was solid and the authors clearly showed that the hydroxy analog has lower affinity for inhibition of both forms of Arabidopsis DHDPS. The authors show diligence looking at MBDTA-2 inhibition of other plant enzymes to explain the previously described in vivo data. Dose response curves for MBDTA-2 inhibition of the second enzyme in the lysine biosynthesis pathway, dihydrodipicolinate reductase (DHDPR), show that MBDTA-2 provides about 10-fold greater inhibition for both forms of Arabidopsis DHDPR than it did for DHDPS with IC50 values for inhibition of DHDPR in the single micromolar concentration. This rate of inhibition suggests more relevance for translation to whole plant growth inhibition.

      It is unfortunate that the co-crystallization attempts with DHDPR and MBDTA-2 were not successful as the physical interaction would be very useful for additional analog synthesis and structure-activity relationship (SAR) evaluation. Use of binding models is common in rational drug design so it is understandable for the authors to pursue a binding model for MBDTA-2. It is difficult to assess the utility of the docking model for SAR development without a better understanding of how many docking conformation predictions the software provided and/or a measure of the docking score. The increase in IC50 values under saturated substrate and co-factor concentrations does help support that MBDTA-2 is a competitive inhibitor with respect to either the substrate and/or the co-factor. The measure of the apparent Km values for the substrate and co-factor with MBDTA-2 at the sub-saturated IC50 values (6.92 and 8.58 micromolar) would help better understand the potential interaction between MBDTA-2 and the substrate and co-factor at the binding site.

      The translation of enzyme inhibition to whole organism inhibition is a common barrier in ration drug design. The use of the model dicot plant Arabidopsis (previous publication) and, the agronomically important monocot weed, Lolium rigidum to assess potential translation of inhibition to whole plant activity is key to understanding the potential of lysine biosynthesis inhibition to be a herbicidal target site. The authors utilized a unique method to assess the growth inhibition of Lolium with multiple applications directly to the Lolium seed. Interpretation of the whole plant data for such an application would be clearer with the inclusion of the application rate and whole plant data for the positive control, chlorosulfuron PESTANAL.

      Novel herbicidal target site are desperately needed and this paper has identified new opportunities to investigate. A key discussion point in this paper is that a dual-target enzyme inhibitor as a commercial herbicide would be beneficial, especially for the potential prevention of target-site based resistance in weeds. As the authors state, this has been addressed through the use of mixtures of herbicide active ingredient with different modes of action (MoA) targeting the same weed. One of the largest challenges in developing novel MoA herbicides, other than identifying novel herbicidal MoA, is the translation of in vitro activity to whole plant control in field applications for a single-target herbicide. It would be interesting to get the authors' perspectives on opportunities to utilize the binding data for MBDTA-2 on DHDPS and the docking model data for MBDTA-2 on DHDPR to identify new analogs that could have increased affinity for both enzymes with the goal to increase the whole plant activity.

    3. Reviewer #3 (Public Review):

      The authors provide further information on the mode action of a lysine synthesis inhibitor (MBTA-2) that is a potential herbicide. The authors determine that MBDTA-2 is not a proherbicide for DHDPS, but do not show that it is not a proherbicide for DHDPR. This should be done, especially since the findings support a very unusual conclusion: inhibition of consecutive enzymes of the lysine synthesis pathway by the same compound through binding an allosteric site for one enzyme and as a competitive inhibitor of the other. Having two molecular targets in the same pathway could hinder evolution of target site-base herbicide resistance. The bioassay of activity of MBDTA-2 on Lolium rigidum was done in such a way that it is difficult to determine if the activity is sufficient to be considered a herbicide.

    1. Reviewer #1 (Public Review):

      In this work, the authors determine the structure of ATAD1, a AAA protein responsible for removal of mistargeted tail anchored (TA) proteins from the mitochondria. In prior work, this group determined the structure of the yeast ortholog Msp1 and found that aromatic residues in key pore-loops were important for engaging substrates. In the current manuscript, the cryo-EM structure of ATAD1 reveals large similarities with the yeast ortholog but elaborates some details about interactions between subunits and unresolved regions from the prior work. Most important is the presence of an extended helix (a11) nestled between the subunits that was not visible in the prior cryo-EM Msp1 structure but was present in a published crystal structure from another group (Wohlever, et al. 2017).

      Based on similar structural motifs in related AAA proteins, the authors hypothesize that a11 is important for oligomerization and ATAD1 activity. Indeed SEC and activity assays suggest that ADAT1∆a11 assembles poorly, has reduced ATP hydrolysis activity, and fails to bind peptide substrates as readily. Using a novel in vivo mislocalization assay, the authors also show that there are defects with the function of this variant consistent with reduced activity and a failure to form oligomers.

      Overall this work extends our understanding of a family of AAA proteins responsible for extracting TA proteins mistargeted to the mitochondria.

    2. Reviewer #2 (Public Review):

      The AAA+ proteins ATAD1/Msp1 extract mislocalized TA-proteins from the outer membrane of mitochondria allowing for substrate retargeting to the ER. Msp1/ATAD1 belong to the meiotic clade of AAA+ proteins also including katanin and spastin that sever microtubules. The authors previously determined the cryo EM structure of C. thermophilum Msp1, now they report on the structure of the human homolog ATAD1. ATAD1 hexamers were determined two distinct (open vs closed) structures. The main difference between these structures is the position of the seam (M6) subunit, which contacts the clockwise subunit in the closed state. This structure represents an intermediate state in the ATPase and threading cycle of the AAA+ protein.

      The authors additionally report on unique structural features that may enable ATAD1 fulfilling its specific function in membrane extraction of TA-proteins. They show that ATAD1 harbors a particularly long C-terminal a-helix 11. This extension was not visible in the former Ct Msp1 structure. Notably, other meiotic family members harbor a shorter a11 but an additional a12. ATAD1 a11 contacts the counterclockwise subunit in the hexameric AAA ring, implicating a role in hexamer stabilization.

      Finally, the authors established a new, microscopic assay to study ATAD1/Msp1 activity in vivo. This assay is based on the direct visualization of mistargeting of the TA-protein GFP-Gos28. By co-expressing the fluorescent reporter and ATAD1 mutants in ATAD1+/+ and ATAD1-/- cells, the authors can differentiate between dominant loss-of-function mutants (toxic in ATAD1+/+ cells) and recessive mutants. This assay proves to be very useful for analysis of ATAD1 activity and allowed documenting oligomerization defects of ATAD1 a11 deletions, which was confirmed in vitro by analysis of respective purified proteins.

    3. Reviewer #3 (Public Review):

      AAA protein are involved in a variety of cellular activity. They all share the same structural fold and still they are all incredibly specialised. This study works towards the direction of understanding the unique specialisation of the AAA protein ATAD1. While the general mechanism of substrate threading by AAA proteins is by now fairly well-elucidated, it remains to describe and understand the finer structural protein details that make each specific AAA perform unfolding (threading) of certain substrate rather than others. Additionally, regulation and stabilisation of each AAA is also finely regulated by specific subdomain.

      This work is definitively strong in addressing these two points for ATAD1.<br /> The structural data are solid and the analysis of the pore loops residues and the role of a11 overall convincing.<br /> The cell fluorescence microscopy assay is a very good tool for checking in the cell the hypothesis risen by analysing of the structure. However, the assay is currently only based on the localisation of the Gos28 substrate, which leaves open the possibility that ATAD1 a11 mutants will have a different phenotype on different substrates.<br /> Overall the work is a solid follow up of the work on Msp1 and advances slowly but soundly the knowledge on ATAD1 and its mechanism in the rescue of mislocalised TA proteins.

    1. Reviewer #1 (Public Review):

      In this manuscript John Lovell and colleagues introduce GENESPACE. GENESPACE is a computational tool that filters (gene sequence based) ortholog annotations by considering the location in the genome to restrict orthologous relationships to syntenic regions. The syntenic regions can be selected according to the context of the study, for example to in- or exclude homeologous regions. In addition, GENESPACE uses its ortholog annotation for the definition of syntenic regions across the focal genomes so that broad-scale chromosomal events can be visualized in an evolutionary context. The manuscript then continues to show the application of GENESPACE in three different scenarios. The first analysis makes use of the broad-scale synteny annotation of GENESPACE to analyze the origin of vertebrate sex chromosomes. The second analysis explores synteny in grass genomes, and evaluates the possibility to find PAV in these genomes given three previously defined QTL regions where a single parental allele induced the phenotypic variation. The third application deals with the assignment of paralogs within grass genomes introduced by the ancient Rho WGD. Using GENESPACE's feature to ignore the first (best) hits (orthologs), it is possible to assign WGD-induced paralogs. GENESPACE seems to be highly useful in practice, and I do not know any other tool that would perform a similar task. I would envision the broad application of GENESPACE as it is agnostic to the species or species group as long as chromosome-level assemblies are available.

    2. Reviewer #2 (Public Review):

      The new tool GENESPACE implements a pipeline in R that combines two existing tools, OrthoFinder and MCScanX. OrthoFinder is a popular tool for finding certain groups of homologous genes within the sets of protein sequences of multiple species. It thereby constructs gene trees as well as a species tree in order to distinguish orthologs from paralogs and produces 'orthogroups'. OrthoFinder does not use the positions of the genes in the genome. The older MCScanX finds syntenic regions between multiple genomes. The GENESPACE pipeline calls OrthoFinder and MCScanX to identify orthogroups, using synteny to prevent that gene pairs are in an orthogroup that are not syntenically matched.

      The R package is relatively easy to install and run, the provided example runs through smoothly and it is straightforward to apply it to another annotated set of related genomes. The riparian plots give a good overview over large scale rearrangements and look neat although they are generated automatically. The 'pangenome' table of orthologous genes provide copy number differences and can be used to start any downstream analysis for orthologous sets of genes, such as a search for positive selection or accelerated evolution.

      The paper discusses several application cases of GENESPACE that are likely of great interest to the respective genomics communities. Unfortunately, though, it is not going into details when describing the algorithm. The method description that was given is not always clear.

      The plausibility and a better performance than OrthoFinder and MCScanX on their respective tasks is shown on polyploid and relatively closely related cotton genomes. However, a more comprehensive benchmark, in particular on data where the synteny is less pronounced was not done. It is therefore not clear up to what degree of synteny GENESPACE is better than OrthoFinder at inferring orthogroups.

    1. Reviewer #1 (Public Review):

      Sadhukhan and Nandi study theoretically the variation of cell shapes in an epithelial layer. Specifically, they consider the aspect ratio of the cell surface area and the surface area distribution. The authors use an effective equilibrium theory, where they restrict themselves to a regime, where the cell areas make a negligible contribution to the monolayer energy, which only depends on the cell perimeters. The energy is governed by the target perimeter P_0 and the perimeter elastic constant \lambda_P. The authors compute the distributions for the aspect ratio and the area. Each distribution depends on a single parameter, respectively called \alpha and \mu. A priori, neither of the two distributions is universal, but if the average aspect ratio shows a certain relation to \alpha, then the distribution of the scaled aspect ratio is universal. The deviation between the dependence of the mean aspect ratio on \alpha required for universality and the actual relation are small, such that shape fluctuations are nearly universal. The authors' derivation puts earlier experimental findings on a solid ground and very importantly shows that the distribution is NOT a consequence of jamming.

      The authors also find that the relation between the standard deviation and the mean aspect ratio is universal. They check their analytical results by simulations of epithelia in terms of a cellular Potts model and a vertex model for which they explicitly verify their assumptions. Due to the success of these approaches had in the past to describe salient features of epithelial tissues, these comparisons strongly support the relevance of the authors' calculations for real epithelia.

      The results obtained by the authors clarify the origin of the very intriguing near universality of aspect ratio fluctuations found in epithelial monolayers and should be of interest to all researchers with an interest in tissue properties. Unfortunately, the presentation is not at par with the quality of the results the authors obtain. Most importantly they often make use of jargon that is hard to understand for readers without a formal training in physics.

    1. Reviewer #1 (Public Review):

      Dotov et al. took joint drumming as a model of human collective dynamics. They tested interpersonal synchronization across progressively larger groups composed of 1, 2, 4 and 8 individuals. They conducted several analyses, generally showing that the stability of group coordination increases with group numerosity. They also propose a model that nicely mirrors some of the results.

      The manuscript is very clear and very well written. The introduction covers a lot of relevant literature, including animal models that are very relevant in this field but often ignored by human studies. The methods cover a wide range of distinct analyses, including modelling, giving a comprehensive overview of the data. There are a few small technical differences across the experiments conducted with small vs. large groups, but I think this is to some extent unavoidable (yet, future studies might attempt to improve this). Furthermore, the currently adopted model accounts well for behaviors where all individuals produce a similar output and therefore are "equally important". However, it might be interesting to test to what extent this can be generalized to situations where each individual produces a distinct sound (as in a small orchestra) and therefore might selectively adapt to (more clearly) distinguishable individuals. Similarly, it would be interesting to test to what extent the current results (and model) can be generalized to interactions that more strongly rely on predictive behavior (as there is not much to predict here given that all participants have to drum at a stable, non-changing tempo).

      An important implication of this study is that some well-known behaviors typically studied in dyadic interaction might be less prominent when group numerosity increases. I am specifically referring to "speeding up" (also termed "joint rushing") and "tap-by-tap error correction" (Wolf et al., 2019 and Konvalinka et al., 2010, also cited in the manuscript, are two recent examples). I am not sure whether this depends on how the data is analyzed (e.g. averaging the behavior of multiple drummers), yet this might be an important take-home message.

      I am confident that this study will have a significant impact on the field, bringing more researchers close to the study of large groups, and generally bridging the gap between human and animal studies of collective behavior.

    2. Reviewer #2 (Public Review):

      In this manuscript Dotov et al. study how individuals in a group adjust their rhythms and maintain synchrony while drumming. The authors recognize correctly that most investigation of rhythm interaction examines pairs (dyads) rather than larger groups despite the ubiquity of group situations and interactions in human as well as non-human animals. Their study is both empirical, using human drummers, and modeling, evaluating how well variations of the Kuramoto coupled-oscillator describe timing of grouped drummers. Based on temporal analyses of drumming in groups of different sizes, it is concluded that this coupled oscillator model provides a 'good fit' to the data and that each individual in a group responds to the collective stimulus generated by all neighbors, the 'mean field'.

      I have concerns about 1) the overall analysis and testing in the study and about 2) specific aspects of the model and how it relates to human cognition. Because the study is largely empirical, it would be most critical for the authors to propose two - or more - alternative hypotheses for achieving and maintaining synchrony in a group. Ideally, these alternatives would have different predictions, which could be tested by appropriate analyses of drummer timing. For example, in non-human animals, where the problem of rhythm interaction in groups has been examined more thoroughly than in humans, many acoustic species organize their timing by attending largely to a few nearby neighbors and ignoring the rest. Such 'selective attention' is known to occur in species where dyads (and triads) keep time with a Kuramoto oscillator, but the overall timing of the group does not arise from individual responses to the mean field. Can this alternative be evaluated in the drumming data ?<br /> Would this alternative fit the drumming data as well as, or better than , the mean field, 'wisdom of the crowd' model ?

      A second concern arises from relying on a hybrid, continuous - pulsed version of the Kuramoto coupled oscillator. If the human drummers in the test could only hear but not see their neighbors, this hybrid model would seem appropriate: Each drummer only receives sensory input at the exact moment when a neighbor's drumstick strikes the drum. But the drummers see as well as hear their neighbors, and they may be receiving a considerable amount of information on their neighbors' rhythms throughout the drum cycle. Can this potential problem be addressed? In general, more attention should be paid to the cognitive aspects of the experiment: What exactly do the individual drummers perceive, and how might they perceive the 'mean field' ?

    3. Reviewer #3 (Public Review):

      The contribution provides approaches to understanding group behaviour using drumming as a case of collective dynamics. The experimental design is interestingly complemented with the novel application of several methods established in different disciplines. The key strengths of the contribution seem to be concentrated in 1) the combination of theoretical and methodological elements brought from the application of methods from neurosciences and psychology and 2) the methodological diversity and creative debate brought to the study of musical performance, including here the object of study, which looks at group drumming as a cultural trait in many societies.

      Even though the experimental design and object of study do not represent an original approach, the proposed procedures and the analytical approaches shed light on elements poorly addressed in music studies. The performers' relationships, feedbacks, differences between solo and ensemble performance and interpersonal organization convey novel ideas to the field and most probably new insights to the methodological part.<br /> It must be mentioned that the authors accepted the challenge of leaving the nauseatic no-frills dyadic tests and tapping experiments in the direction of more culturally comprehensive (and complex) setups. This represents a very important strength of the paper and greatly improves the communication with performers and music studies, which have been affected by the poor impact of predictable non-musical experimental tasks (that can easily generate statistical significant measurements). More specifically, the originality of the experiment-analysis approach provided a novel framework to observe how the axis from individual to collective unfolds in interaction patterns. In special, the emergence of mutual prediction in large groups is quite interesting, although similar results might be found elsewhere.

      On another side, important issues regarding the literature review, experimental design and assumptions should be addressed.<br /> I miss an important part of the literature that reports similar experiments under the thematic framework of musical expressivity/expression, groove, microtiming and timing studies. From the participatory discrepancies proposed in 1980's Keil (1987) to the work of Benadon et al (2018), Guy Madison, colleagues and others, this literature presents formidable studies that could help understand how timing and interactions are structured and conceptualized in the music studies and by musicians and experts. (I declare that I have no recent collaborations with the authors I mentioned throughout the text and that I don't feel comfortable suggesting my own contributions to the field). This is important because there are important ontological concerns in applying methods from sciences to cultural performances. One ontological issue that different cultural phenomena differ from, for example, animal behaviour. For example, the authors consider timing and synchrony in a way that does not comply with cultural concepts: p.4 "Here we consider a musical task in which timing consistency and synchrony is crucial". A large part of the literature mentioned above and evidence found in ethnographic literature indicate that the ability to modulate timing and synchrony-asynchrony elements are part of explicit cultural processes of meaning formation (see, for example, Lucas, Glaura and Clayton, Martin and Leante, Laura (2011) 'Inter-group entrainment in Afro-Brazilian Congado ritual.', Empirical musicology review., 6 (2). pp. 75-102.). Without these idiosyncrasies, what you listen to can't be considered a musical task in context and lacks basic expressivity elements that represent musical meaning on different levels (see, for example, the Swanwick's work about layers/levels of musical discourse formation). Such plain ideas about the ontology of musical activities (e.g. that musical practice is oriented by precision or synchrony) generate superficial constructs such as precision priority, dance synchrony, imaginary internal oscillators, strict predictive motor planning that are not present in cultural reports, excepting some cultures of classical European music based on notation and shaped by industrial models. The lack of proper cultural framing of the drumming task might also have induced the authors to instruct the participants to minimize "temporal variability" (musical timing) and maintain the rate of the stimulus (musical tempo), even though these limiting tasks mostly take part of musical training in some societies (examples of social drumming in non-western societies barely represent isochronous tempo or timing in any linguistic or conceptual way). The authors should examine how this instruction impacts the validity of results that describe the variability since it was affected by imposed conditions and might have limited the observed behaviour. The reporting of the results in the graphs must also allow the diagnosis of the effect of timing in such small time frame windows of action.

    1. Reviewer #1 (Public Review):

      It is well established that the energy expenditure and metabolic rate of metazoan organisms scale inversely to body mass, based on the measurement of oxygen consumption and caloric intake. However, the underlying regulatory mechanisms for this observation are poorly defined. To investigate whether metabolic scaling is associated with reduced levels of transcription of metabolic genes in larger animals, the authors reviewed existing transcriptional datasets from liver tissues of five animals (mice, rats, monkeys, humans and cattle) with a 30,000-fold range in average adult body weights. They identified a number of metabolic genes in different pathways of central carbon metabolism whose expression inversely scaled with body size, a majority of which required oxygen, NAD/H or ATP/ADP. Metabolic flux studies on intact liver sections, as well as in live animals also revealed decreased liver metabolic fluxes in rats compared to mice. Interestingly, these differences were not observed in primary hepatocyte cultures, indicating that metabolic scaling is primarily regulated by cell-extrinsic factors and tissue context. These are interesting findings and highlight the importance of measuring metabolic processes in vivo. The measurement of cellular metabolic fluxes in different contexts (cultured, ex vivo tissue sections and live animals) is a major strength of this study. The lack of direct evidence that enzyme levels correlate with mRNA, and the absence of both transcriptional and enzyme activity measurements in cultured cells are potential weaknesses.

    2. Reviewer #2 (Public Review):

      Akingbesote et al. aim to determine the molecular basis of metabolic scaling - the phenomenon that metabolic rates scale inversely with (0.75) body mass. More specifically, they test the hypothesis that expression of genes involved in the regulation of oxygen consumption and substrate metabolism as well as respective fluxes provide a molecular basis for metabolic scaling across five species: mice, rats, monkeys, humans, and cattle. To this end, Akingbesote et al. use publicly available transcriptomics data and identify genes that show decreasing (normalized) expression with increasing mass of organisms. This descriptive analysis is followed by discussing a few relevant examples and (KEGG) pathway enrichment analysis. The authors then used their published PINTA approach with data from their experiments with mice and rats to provide estimates of selected cytosolic and mitochondrial fluxes in vitro, ex vivo, and in vivo; these estimates are then employed in determining if metabolic fluxes scale. The conclusion drawn from these analyses is that estimates of selected fluxes do not differ in vitro between plated hepatocytes of mice and rats, but that differences can be detected using metabolic flux analysis in vivo. As a result, in vivo flux profiling is more relevant to assessing metabolic scaling.

      The conclusions are only in part supported by the data and clarifications are needed both with respect to the analysis of transcriptomics data as well as flux estimates:

      1. In looking for scaling in gene expression, the authors rely on the assumption that mRNA expression correlates well with protein abundance (citing Schwanhäusser et al., 2011); however, transcripts explain about 40% of variance in protein abundance (this observation holds across multiple species). Hence, the identified patterns based on the transcript data may have little implications for protein abundance or flux.<br /> 2. While the procedure used to identify transcripts whose expression scale is clearly described, focusing the enrichment on KEGG pathways can only identify metabolic genes that scale. It would be informative and instructive to investigate if and to what extent genes involved in non-metabolic processes, that affect metabolic rates, also scale.<br /> 3. The result on flux ratios and absolute fluxes, based on the equations in Table S1, rely on certain assumptions (e.g. metabolic and isotopic steady state, among the others listed in PINTA); the current presentation does not ensure that all assumptions of PINTA are met in the present setting, so the estimates may be biased, leading to alternative explanations for the observed differences in vivo or the lack thereof in vitro.<br /> 4. The findings regarding the flux estimates seem to be fully determined by observed differences in gluconeogenesis (as demonstrated in Fig. 4). Usage of more involved approaches for metabolic flux analysis may provide wider-reaching conclusions beyond selected fluxes that appear fully coupled.

    3. Reviewer #3 (Public Review):

      This manuscript addresses a fundamental aspect of mammalian biology referred to as scaling, in which metabolic processes calibrate to the size of the organism. Longstanding observations related to scaling have been established based on rates of oxygen consumption. This manuscript extends these observations to gene expression and metabolic fluxes in order to discover the metabolic pathways that scale with body mass. The analyses are focused on the liver, which is the metabolic hub of the organism. Gene expression levels gleaned from available databases for organisms of varied sizes are analyzed and queried for scaling based on body mass. This analysis reveals that scaling is mainly a characteristic of metabolic genes. These data inform metabolic flux studies in cultured cells, liver slices and whole organisms. These studies demonstrate that scaling of metabolic fluxes occurs, but not out of the context of the whole organism or intact liver (in the form of liver slices). Scaling of metabolic fluxes is not observed in cultured hepatocytes. Overall, this is an interesting line of inquiry. The data are largely correlative in nature but add important texture to traditional characterization of oxygen consumption rates. The application of flux studies is a particular strength because these reflect the true metabolic processes. Enthusiasm was tempered by certain claims that extend beyond data (e.g., the title that suggests that metabolic scaling applies to tissues other than the liver, which was studied), as well as low numbers of biological replicates in some experiments, studies conducted in a single-gender and a writing style that includes excessive technical jargon.

    1. Reviewer #1 (Public Review):

      This manuscript reports on a very extensive molecular and cellular study of the effect of splicing factor Srsf10 on spermatogenesis in male mice. Using Srsf10 knockout mice, the investigators determined that loss of Srsf10 specifically inhibited spermatogonia differentiation (into spermatocytes) and entrance into meiosis (essential for fertility). The deletion of Srsf10, a factor already well characterized and known to be involved in alternative splicing of mRNA (i.e, post-transcriptional), was responsible for male infertility. It had been shown previously that Srsf10 controls alternative splicing by binding to exons as well as to splicing factors during mitosis. It is of interest that spermatogonia are produced, suggesting that loss of Srsf10 with its effects on alternative splicing may not affect early steps in spermatogenesis. The extensive analysis of alternative splicing was carried out in mouse testes and accounts primarily for the novelty of the research. This manuscript should be of interest to molecular, developmental, and reproductive biologists.

    2. Reviewer #2 (Public Review):

      SRSF10, also known as SRp38, is an atypical serine/arginine-rich splicing factor that regulates the generation of isoforms of messenger RNAs (mRNAs) from common precursor pre-mRNAs, so that cells can express protein variants in need. It has been shown that SRSF10 regulates alternative splicing via binding of exons and constitutive splicing factors in response to cellular stimuli during mitotic cell cycle progression. Liu et al. now utilize genetically modified mouse model that lacks SRSF10 specifically in male germ cells to show that SRSF10 is required for spermatogenesis at a very early stage and thus male fertility by regulating alternative splicing of hundreds of genes.

      Spermatogenesis encompasses a series of consecutive events to produce male gametes, sperm, including mitosis, meiosis and post-meiotic cellular morphogenesis. Although alternative splicing has been known as an important step to regulate gene expression in spermatogenic cells, the underlying molecular mechanisms remain to be fully understood. Using a genetic approach, the authors created mice that carry alleles of Vasa-Cre and floxed Srsf10 (Srsf10Flox/Flox:Vasa-Cre). Since Vasa (known as Mvh in mouse, the mouse Vasa homolog) is specifically expressed in germ cells, its promoter would drive the expression of Cre recombinase specifically in germ cells and remove floxed DNA fragment, generating mutant Srsf10 gene (missing exon 3 in this case) in germ cells. To find out whether removal of functional Srsf10 gene would affect male germline development (females are not mentioned in this manuscript), the authors analyzed the fertility of male mice, testis development and spermatogenic cells using various cell biological approaches. Their results showed that Srsf10 mutants suffered severe defects in spermatogenesis and produced no spermatogenic cells beyond meiotic stage, leading to male infertility. The authors further showed, by analyzing more detailed spermatogenic steps, that mutant mice retained only earliest stage spermatogonia at decreased levels, comparing to the control counterparts that still express SRSF10.

      Spermatogonial stem cells (SSCs) are the founder cells of spermatogenesis, which contain heterogenous cell populations probably due to the progressive proliferation and differentiation of self-renewing SSCs. To find out whether defected spermatogenesis of Srsf10 mutants was caused by defects in SSCs. The authors applied known marker proteins of SSCs to analyze their sub-populations in more details, using immunofluorescent staining, cell sorting and single cell RNA sequencing. The results showed that the proliferating population of SSCs expressing PLZF (PLZF+) were decreased in number, whereas the earlier stage of un-differentiated SSCs expressing GFRa1 were less affected. Consequently, meiotic spermatocytes were severely disrupted. Analysis of cell proliferation using nascent DNA labeling (Edu) supported the notion that deletion of SRSF10 impeded mitotic cell cycle of PLZF+ differentiating progenitors. This is further supported by the single cell RNA sequencing analyses. Characterization of cellular transcriptome at single cell level can not only identify changes of gene expression but also be used to classify cell types according to their similar expression patterns of typical marker genes. Bioinformatics analyses of single cell RNAseq data indeed showed that Srsf10 mutants contained a DSSC3 cell group that was not presented in the controls. In addition, they also showed that the ratios of USSC1 and USSC2 groups, two undifferentiated SSC sub-populations, are altered in mutants, comparing to the controls, supporting their cellular analyses in SSC sub-populations.

      To further determine how SRSF10 affected gene expression in spermatogenic cells, especially for SSCs, the authors conducted both bulk RNA sequencing and Isoseq experiments using sorted SSCs (THY1+KIT-). They found that expression of hundreds of genes was differentially affected in Srsf10 mutant SSCs, especially for genes involved in cell cycle regulation, cellular iron ion homeostasis and spermatogenesis. The authors went on, using Isoseq data, to show that isoforms of many transcripts (mRNAs) were altered in SSCs lacking SRSF10, mainly due to exon skipping and altered first exon splicing events. Consistently, these affected genes are mostly involved in mitotic cell cycle progression and stress responses.

      Overall, the authors presented convincing evidence on the defects of spermatogenesis and male sterility due to Srsf10 mutation. The RNA sequencing results support the role of SRSF10 in regulating alternative splicing of cell cycle regulators and post-translational modifiers that may impede mitotic cell cycle progression and stress responses. The RNA sequencing data also provided a rich source to further study the molecular mechanisms that underlie the SRSF10-mediated alternative splicing involved in the regulation of mouse SSCs.

      However, some caveats can be seen in the manuscript that may undermine the significance of the study. For example, the authors concluded that the main causative event leading to male infertility in Srsf10 mutants is due to the defected expansion of differentiating progenitors, the progenies of un-differentiated SSCs, but not the formation of SSCs in neonatal mice. This should be further tested. Since the deletion of Srsf10 gene mediated by Mvh-Cre starts in embryonic stage of pro-spermatogonia, experiments designated to the proliferation status and population changes of pro-spermatogonia and un-differentiated SSCs should be carried out. In fact, single cell analyses and comparison of GFRa1+ cells suggested that SSCs may be altered at the beginning as well. Consequently, defects in the initiation of meiosis, as the authors concluded, may not be a causal but consequential effect due to the defective proliferation of progenitors. The manuscript also contains some in-consistency in the description of SSC sub-populations at various stages, in data presentation and interpretation, lack of sufficient introduction of research rationale, materials and methods used, as well as discussions of possibilities the current results indicate. These issues should be amenable using their current mouse models and experimental approaches. It will also be of interest to see if spermatogenic cells are maintained in adult or even aged mice in the absence of SRSF10.

    3. Reviewer #3 (Public Review):

      The maintenance of spermatogonia stem cells is essential for fertility and a model for stem cell homeostasis. In this study the authors investigate the role of the alternative splicing factor SRSF10 in spermatogenesis, following on the discovery that germ cell-specific knock out of SRSF10 in mice caused a loss of spermatogonia and fertility in males.

      This study begins by crossing SRSF10 floxed mice to those expressing Cre in the male germline. This resulted in infertile male mice with significant defects in spermatogonia differentiation. To investigate the molecular defects associated with these developmental defects, the authors carried out transcriptomic analysis in full testes at several different developmental time points. RNA-Seq of this bulk tissue revealed differential gene expression in the SRSF10-depleted testes that is consistent with reduced expansion and proliferation of spermatogonia. Subsequent single-cell RNA-Seq also revealed a gene expression profile consistent with loss of spermatogonia, while cell cycle analysis demonstrated a reduction in cell division and increase in apoptosis in the SRSF10-depleted cells. Finally analysis of alternative splicing in SRSF10-depleted cells identified several hundred impacted splicing changes, consistent with previous studies implicating SRSF10 as a splicing regulatory protein. Notably, many of the confirmed changes in splicing occurred in genes with known activities in spermatogonia development.

      Together these studies provide useful physiologic and descriptive data on the impact of SRSF10 in mouse fertility. Future studies will be needed to determine which of the gene expression changes observed in the SRSF10-depleted cells drive the differentiation defects and which are a consequence of stalled development and proliferation. Moreover, the molecular mechanism by which SRSF10 impacts key splicing or gene expression events, and how many of these are direct targets of SRSF10 remains unexplored.

    1. Reviewer #1 (Public Review):

      Lee and Chen investigate the representation of between-movie boundaries in the brain, with a particular focus on the spontaneous boundaries that occur as people shift between movie recalls. Are these sorts of recall boundaries represented the same as those that occur (a) between the visual presentation of different movies (between-movies boundaries at encoding) and/or (b) between events within a single movie (within-movie boundaries at encoding)-or are these recall boundaries different? The main findings were that between-movie boundaries were quite similar regardless of the task phase (encoding/retrieval), but dissimilar from within-movie boundaries.

      This paper has many strengths, including the interesting research question and sophisticated analytic approach. The authors have done an excellent job presenting many important controls and, despite its complexity, the work is presented in a way that is clear and enjoyable to engage with. While a relatively brief paper and simple story, as the authors note in the discussion, there are many possible interpretations or underlying mechanisms that could be giving rise to this phenomenon-which is quite exciting. So, the paper may be a source of more questions than answers! I see this as a great feature. I think this work will inspire many new investigations into boundary representation and event segmentation.

      I do have a few suggestions and questions for the authors to address the current weaknesses, as outlined below:

      1. I am generally interested in better understanding how differences in the sensory experience (most notably, presentation of visual input in the movies versus its absence during the between-movie boundaries) across timepoints could be playing a role in these results. If I understand correctly, the between-movie boundaries will always contain a (mostly) blank screen (with simple white text at encoding) along and silence, for both encoding and recall phases. In contrast, the no-boundary periods as well as the within-movie boundaries would always contain visual input (movies). There are a few reasons why this is concerning to me. First, the boundary periods are relatively much more homogenous in terms of input/experience, and so it intuitively makes sense to me that the neural pattern would also therefore be quite similar across different boundary periods (even across phases). The primary comparisons as shown in Fig 2 are comparing these homogenous boundary experiences with highly variable within-movie experiences (either due to ongoing recall/speech, or movie viewing). It seems to follow that for this reason alone one should expect the boundary patterns to be more similar to one another, and I am not sure whether that is the sort of boundary processing that is of interest here. Second, as is evident in Figure 3A, the "middle" (no-boundary movie) patterns are a much more heterogeneous bunch, with some pairs showing positive and others showing negative correlations with one another-potentially reflecting variability in the input. Given this, it of course has to be the case that the average correlation of within-movie patterns is low (near zero; Figure 3C) but it also may follow that the boundary patterns are negatively correlated with the event offset (within-movie boundary) patterns. I appreciated the analysis related to the audio controls, but am not sure the authors were able to account for the visual differences.

      2. I was not sure I fully understood the offset vs. onset yoking analysis-both how it was performed, and how the conclusions followed from the results. First, I was a bit confused about how the difference in delay duration between movies at encoding (6s) versus at recall (9.3s on average, but variable; see also comment #5) would play into this and whether those are meaningful time points to display on the Figure 3D charts that might help the reader interpret those findings. Second, the authors state that this analysis shows the boundary patterns were driven by offset (more than onset) responses, but I was not sure what aspect of the results led to that conclusion. Can the authors say more about the evidence supporting this conclusion? It looks to me like there are strong correlations that emerge both after offset and onset (i.e., just above and to the right of the origin there are numerous time points with positive [red] correlations). Perhaps it is because the red positive correlations start earlier, prior to the recall itself, when yoked to the onset, but I am not sure why this means it is related to offset and not some preparation for the onset of recall (see also comment #5). Also, is it interesting or meaningful that the patterns seem more compressed at recall than at encoding (i.e., the outlined red areas are skinnier than they are tall)?

      3. I am not sure the reason for masking Figure 2B and C with the a>0 and c>0 maps. First of all, it seems as though in RSA the actual correlation being positive or negative is not terribly meaningful and can depend on preprocessing decisions, etc. In addition to that potential issue though I'm also just generally interested in more understanding the logic behind this decision. Can the authors explain that and include it in the main paper? Were there any regions that showed for example a<br /> 4. I am inferring (though could be incorrect) that some of the pattern similarity analyses would be directly comparing (i.e., correlating) patterns derived from the same scanning run. Can the authors confirm if this is the case? If so, it would be important to consider in the paper how temporal autocorrelation within scanning run may be impacting the results (for example, how does temporal distance between the different events vary (or not) across the different comparisons?). Ideally, the authors would be able to demonstrate that the same pattern of results can be found when limiting to cross-run comparisons only. Relatedly, it would be important to know whether the across-phase comparisons (e.g., in that there are more regions that show significant recall-recall similarity vs. encoding-recall in Figure 2B/C) might also be impacted by differences in whether the patterns were derived from the same run (whereas half of the comparisons could be same-run for recall-recall, none of the comparisons would be from the same run in encoding-recall, and so the overall correlation may be higher for recall-recall... or encoding-encoding).

      5. The definition of the time periods of interest were a bit confusing to me. For example in the main analysis, the duration seemed arbitrary at 15 seconds, and I believe it always began at the offset of the preceding movie (shifted by 4.5s for hemodynamic lag). To clarify, at encoding, this means that it would always include the beginning of the "next" movie, but never the end of the preceding movie, is that correct? So the boundary between movie A and movie B (looking at Fig 1) would include some activation associated with the beginning of movie B viewing-is that correct? It seems a bit strange to me given the goals and framing of the study that movie B would be included here, given I thought most of the "action" would be happening with the movie A memory in this case. It seems as though this definition may also produce systematic differences between encoding and recall: for one, the delays between recalls are variable and longer (9.3s on average with an SD of 16.8s) than the fixed 6s title screen, so the contents going into the neural patterns at recall would be different (contain less recall time and more blank-screen time); but also during recall, it seems as though the participant would be bringing to mind memories of the upcoming movie B they are about to recall, while there is no way for participants to anticipate anything specific about the upcoming movie during encoding. Can the authors clarify these points in the paper?

    2. Reviewer #2 (Public Review):

      This experiment by Lee and Chen sought to examine internally-guided boundaries between events during recall, specifically recall of audiovisual short movies viewed a few minutes prior. They performed a set of well thought out pattern similarity analyses to determine if activity in the Default Mode Network (DMN) and more specifically Poster Medial Cortex (PMC) were related across encoding and retrieval of separate movies. Briefly, the authors found characteristic univariate activation patterns in the brain's 'Default Mode Network' (DMN) during event transitions, but extend prior work to show that these activations are present during internally-guided event transitions. Furthermore, fascinatingly, the authors report increases in pattern similarity at event offsets that persist across encoding and recall, and which were not present during the middle of events. This is taken as evidence of a general cognitive state that exists at event transitions, and exists beyond the level of a single event.

      In our view, the authors' results support their interpretations. Not only are there internally generated boundaries that mark shifts between broader contexts, but these boundaries appear to be distinct from those that are found within continuous narratives. This is a very interesting dataset, cleverly analyzed, which points to interesting new directions for the fields of event cognition and event memory. Namely, the distinction between within-event and between-event boundaries adds new depth to the discussion of event boundaries more broadly, and the notion of a general common cognitive state during event transitions is a thought-provoking result that will certainly influence our group's thinking on this topic, and likely many others.

      We truthfully do not have substantive criticisms of this manuscript. We think the study is well done, the analyses seem properly conducted, and the manuscript is generally written well and clearly. There are a few minor requests for clarification that we will note in our separate recommendation to the authors, but the only critique bordering on a 'major' concern is the very short 'Discussion' portion of the manuscript. While we recognize that this is a short-format submission, and while the authors did a fine job of trying to synthesize their results and situate them in the context of the field in 2 short paragraphs, I honestly think there should be more discussion of the findings and their implications. In sum, however, we think that this is a solid paper that we found very exciting.

    3. Reviewer #3 (Public Review):

      The aim of this paper is to investigate whether internally driven changes in mental context involve similar neural mechanisms as externally driven changes. In particular, the paper investigates whether there are consistent neural responses that align with the transition between stimuli when watching or recalling movies. The authors show that there is a consistent pattern of neural responses, particularly in precuneus and angular gyrus, that is evoked by the offset of a movie during movie watching and by the self-generated transitions between movies during movie-recall. Their results suggest that self-generated shifts in mental context involve similar neural processes as externally-generated shifts.

      The paper is well written and the results are interesting. The analyses and methods are thoughtful and rigorous and provide an interesting new perspective on internally driven changes in mental context that (as far as I know) have not been investigated before in this way. It is clear that the authors spend a lot of time and effort on additional analyses to understand in detail what is going on and which factors might be driving the observed differences.

      I do have a couple of concerns about the interpretation of the findings. In the abstract the authors state that the findings reflect: 'a cognitive state related to the flushing and reconfiguration of situation models'. If the between-movie activity patterns reflect the flushing/reconfiguration of the prior context, it is very surprising that there is a negative correlation to within-movie boundary patterns. The premise of event segmentation theory is that event boundaries (within a given context/movie) result in a reset of the event model, therefore also resulting in a 'flushing' of the prior context. I have three main concerns related to this point:

      1. To what extent can the similarity between recall and encoding be driven by the (sudden lack of) external input that occurs at transitions between movies? The authors already investigated the role of auditory input, but of course there is also a sudden lack/reduction of visual input during the boundaries between movies at encoding. The authors state that: "Visual features (i.e., black screen) or pauses in speech cannot explain boundary-specific similarity between encoding and recall phases, because boundary and non-boundary periods were identical in terms of visual input during recall and speech generation during movie watching." I do not agree with this statement. When looking at the similarity between encoding and recall during boundaries, the characteristics of the input are more similar (absent visual and auditory input) than when looking at the similarity between encoding and recall in the middle of movies (present vs. absent visual and auditory input). This confound should be taken into account in the analyses.

      2. Could the negative correlation between within-movie boundary patterns and between-movie boundary patterns be due to the long time-window that is averaged (15 seconds)? Both within and between-movie boundaries might result in a similar transient activity patterns, which persists longer for the between-movie boundaries possibly due to the 6 seconds of black/title screen at movie offset.

      3. In addition to these two concerns, it is unclear to me at this point to what extent these findings can be related to a reset of context that might occur in a real-life setting or if it is specific to the current experimental setup. Do the authors think that subtle shifts in context (e.g. within a given movie/context) involve fundamentally different mechanisms as compared to more stark transitions that occur between contexts (e.g. between movies)?

    1. Reviewer #1 (Public Review):

      The work, mostly performed in yeast S. cerevisiae, shows that the knockout of DIP2 leads to accumulation in cells of some DAG subspecies (36:0 and 36:1), and also a deficit of similar TAG subspecies (something which mostly occurs, as they showed, in early to mid log growth phase). Accordingly, over-expression of DIP2 leads to the opposite outcome (lower DAG and higher TAG subspecies levels). ∆DIP2 cells showed increased ER stress and UPR, which can be counterbalanced by incubating cells with oleic acid. Moreover, the authors show that the absence of DIP2 causes vacuole fusion defects, which they ascribe to a localization of the protein in the vacuole and possibly to the fact that enhanced levels of DAG in the vacuole membrane can promote vacuole fusion. Although it is true that neither of these claims are fully supported by the experimental results, the data that the authors show serves as a starting point for future, more robust studies to test those claims. Finally, the authors show that the DBD1 domain is not necessary and that the two FLD domains are key for the observed lipid metabolism induced by DIP2 expression. Altogether this manuscript presents interesting new data on an uncharacterized protein that seems to be regulating the metabolism of relatively low abundant DAG/TAG subspecies in cells, and by doing so possibly control cell homeostasis.

    2. Reviewer #2 (Public Review):

      In this manuscript the authors study the role of the protein DIP2 which contains two fatty acyl-AMP ligase (FAAL)-like domains in lipid metabolism. They find that deletion of DIP2 in yeast, drosophila and mouse cells results in the increase of specific diacylglycerol species which is often coupled to a reduction in triglycerides. Overexpression studies in yeast corroborate this evidence. The DIP2 KO induced lipid deregulation correlates with a moderate induction of ER stress that can be rescued by promoting diacylglycerol conversion to triglycerides through the administration of oleic acid. The authors also show that DIP2 expression in yeast is maximal in the exponential growth phase where the effects of its KO on lipid metabolism are more sustained. The authors report that DIP2 in yeast is localized at the vacuole and at mitochondria and that manipulation of DIP2 levels impair the normal vacuolar response to osmotic stresses. They conclude by demonstrating that the (FAAL)-like domains of DIP2 are necessary and sufficient to sustain the function of DIP2 in regulating diacylglycerol levels and ER stress, with mutations in specific amino acids possibly required for the FAAL enzymatic activity rendering DIP2 inactive. While the manuscript is compelling in many respects and most of the conclusions drawn by the authors are well supported by their data, this paper does not contain a clear proof of the enzymatic activity of DIP2, nor a molecular explanation for the substrate specificity and mode of action of DIP2.

    3. Reviewer #3 (Public Review):

      This study examines a family of poorly defined enzymes that contain fatty acyl-AMP ligase like domains (FAALs). The study reveals that these DISCO-interacting protein 2 (DIP2) enzymes are required to maintain a specific pool of diacylglycerol (DAG) lipids containing primarily C36 acyl chain lengths in budding yeast. Using primarily yeast, the study shows that deletion of ScDIP2 significantly increases C36 DAG pools while leaving the more abundant C32 and C34 DAG pools generally unaltered. Triglyceride (TAG) is also reduced in this deletion. Conversely, ScDIP2 over-expression promotes C36 inclusion in TAG. The ScDIP2 KO yeast manifests ER stress that can be relieved by the addition of oleic acid, but not other fatty acids. In the last section of the study, ScDIP2 is proposed to localize to the vacuole and mitochondria, where it maintains a specific DAG pool to enable proper vacuole morphology and fusion, as well as proper osmoregulation of the vacuole.

      This is a well executed study that begins to characterize a conserved and generally poorly understood family of enzymes. However, questions still remain about some of the conclusions of the study. There are two general issues with the study. The first is the specificity of the effect of loss of ScDIP2. The study beautifully shows that loss of ScDIP2 (or its over-expression) affects a specific sub-pool of DAG (mainly the C36 species). TAG levels are also somewhat lower. However, how ScDIP2 impacts other lipid precursors to DAG synthesis such as PA and lyso-PA is under-examined, and should be looked at as they can also affect ER stress. Whether the change in DAG/TAG is primarily driven by decreased synthesis versus increased lipolysis also required additional analysis.

      The second issue relates to how ScDIP2 relates to the yeast vacuole. It is proposed that some of the ScDIP2 enzyme is vacuole localized, and influences vacuole morphology. The evidence presented here does not strongly support that model. From imaging at least, it appears that ScDIP2 is primarily mitochondria localized. It is therefore possible that it influences vacuole lipid composition and morphology distally from the mitochondria. Resolving ScDIP2's native subcellular localization would strengthen the manuscript.

  2. May 2022
    1. Reviewer #1 (Public Review):

      Mitra et al. extensively utilized the publicly available pan-cancer multi-omics datasets including CCLE, TCGA, RNAseq, and ChIPseq datasets from GEO, and conducted impressive computational analysis work to discover the potential regulatory functions of lncRNA at the pan-cancer level. The idea of using co-essential modules generated by Wainberg et al. 2021 is very interesting and was important to leverage the genome-wide set of functional modules to identify the new lncRNA functions. The overall statistical analyses are rigorous, and the evidence in this paper is logical and solid, especially given the additional RNAseq/ChIPseq data analysis. The validation experiments using cell lines were also appropriate. Overall, this is an excellent paper that combines both dry and wet lab experiments to systematically discover unknown functions of lncRNAs in cancer.

    2. Reviewer #2 (Public Review):

      Mitra and colleagues performed statistical analyses to evaluate associations between lncRNAs and mRNAs, using transcriptome data generated in tumor tissue samples in multiple cancer types from both CCLE and TCGA projects. They further integrated the association results into previously well-characterized co-essential pathways/modules (Wainberg et al., 2021), together with additional pathway/Hallmark genesets annotations, aiming to explore function potential for lncRNAs. Based on these analyses, they characterized 30 high-confidence pan-cancer proliferation/growth-regulating lncRNAs. Importantly, they provided in vitro functional evidence to verify potential tumor-suppressive roles of two prioritized lncRNAs (PSLR-1 and PSLR-2) in proliferation and growth in two lung adenocarcinoma cell models. Overall, this is a well-motivated and conducted study, especially given the large number of lncRNAs that currently have poor-characterized functions. The findings in this manuscript could advance the overall understanding of the roles of lncRNAs in cancer formation and progression.

    1. Reviewer #1 (Public Review):

      The work by Flores-Kim et al. reports the identification and characterisation of WhyD, an enzyme involved in the hydrolysis of wall teichoic acids (WTAs) in S. pneumoniae. They explore the role of WhyD in autolysis control and cell growth and division. This study reveals that WhyD controls the abundance and localization of WTAs, which in turn inhibits autolytic activities.

      The methods used convincingly address the questions asked by the authors and overall, the data are robust and support the conclusions drawn, providing experimental evidence for a mechanism proposed a long time ago but that remained poorly characterised. This work provides a convincing model to explain how the enzymatic activity of WhyD contributes to control peptidoglycan hydrolysis in the context of actively growing and dividing S. pneumoniae cells. It also highlights that WhyD activity is not sufficient to prevent autolysis and cell death during late stationary phase, indicating that this process awaits further analysis.

    2. Reviewer #2 (Public Review):

      Authors identified the gene whyD, an essential factor for the bacterial human pathogen Streptococcus pneumoniae (Sp) survival. Surprisingly, WhyD importance is reversed in the absence of LytA, making cells' survival dependent on the simultaneous deletion of both genes. The authors investigated the relationship between these 2 proteins and the production and localization of peptidoglycan (PG), teichoic acids (WTAs), and lipoteichoic acids (LTAs) in the cell wall. Combining genetically engineered strains, in vivo tagging of proteins, chemical probes, and purification of recombinant proteins, authors concluded that more than regulating levels of WTA and LTA, WhyD acts as a topological factor, supporting the spatio-temporal synthesis and degradation of cell wall necessary for cell elongation.


      Working with Streptococcus, as with any less-studied bacterial model compared to B. subtilis and E. coli, is challenging but increasingly important to understand human pathogens and their interactions with antibiotic drugs. This work represents a tour de force and joins a relatively small collection of state-of-the-art studies by combining genetics, biochemistry, and cell biology approach to solve a specific problem. The questions asked in each experiment are clear and the performed experiments were, in the majority, well designed with proper controls. The paper is well-written and accessible to the general scientific community.

      One of the highlights of this study is the development of a good proxy for WTA localization - something not trivial - providing the field with endless possibilities to study the immediate and lasting effects of antibiotic resistance and other genetic pathways in this model. A second important development - not so obvious as the first one - is the successful use of purified hydrolases from one species (in this case, LytA from S. pneumoniae) in orthogonal organisms (here, B. subtilis). This is an elegant assay that can be useful to study the function of proteins from challenging model systems.


      Although this is certainly a technically difficult goal, the paper does not show a direct interaction between WhyD (or its GlpQ sub-domain) with WTAs. While the effect of WhyD over WTA levels showed here is undeniable, and the proposed interaction is the simplest explanation, it's not possible to assert whether this is the case without a crosslink co-purification using an inactive mutant of WhyD.

      Another aspect the paper could improve is the explanation of the labeled cell-wall analogs, very well established in the cell-wall field but likely obscure to other biologists. Especially on figures that nothing at all is said about the data (Figures 4 and 5). The microscopy data, despite evidently being well-performed, begs for better quantitation and visualization. For example, it's not clear whether there were replicates, the sample size (informing that at least 300 cells were used is not enough information to inform on sample size effects). Sub-panels where no signal is apparently detected (e.g. Figure 7 and supplements) should be clarified and the background should be displayed.

    3. Reviewer #3 (Public Review):

      In this study, the authors aim at identifying new factors regulating the activity of bacterial cell wall hydrolases using Streptococcus pneumoniae as a model. Based on previous Tn-Seq screens, they showed that the gene whyD becomes non essential in a ∆lytA genetic background and that this protein restrains the activity of LytA. To explain this observation, they provide in vivo and in vitro evidence showing that WhyD specifically hydrolyses wall teichoic acids. A series of experiments is then achieved to demonstrate that WhyD regulates the activity of the cell wall hydrolase LytA to prevent cell lysis and that of other cell wall hydrolases to allow the insertion of new peptidoglycan in the preexisting polymer, promoting thus cell elongation. Notably, it is shown that WhyD localizes at mid-cell and together with FDAA labelling and the use of an inactive form of LytA as a WTA localization probe, it is further shown that WhyD co-localizes with nascent peptidoglycan, while WTA are more abundant in the peripheral cell wall, during cell elongation. By contrast, WTA localization changes in cells nearing cell constriction, co-localizing with WhyD and nascent peptidoglycan. It is concluded that WhyD modulates the presence of wall teichoic acids in the PG layer in the course of the cell cycle, modulating in time and space their availability for choline-binding hydrolases in order to prevent aberrant cell lysis, proper cell elongation and final separation. The methodology used is logical, appropriate and well-executed. The data are clear and the interpretations are reasonable. This work represents a nice contribution to our understanding of the regulation of cell wall hydrolysis in bacterial growth and morphogenesis.

    1. Reviewer #1 (Public Review):

      1. In terms of the prior hypothesis here I think the authors justify a prior with respect to striatum and I think the most principled analysis of their hypothesis would be based on volumes of interest in striatum. Figure 1 does show difference in MTsat in striatum between neurotypicals and DLDs but the changes are all in the caudate I think- I cannot see anything in putamen. The authors actually describe changes in only one part of anterior caudate. The authors do describe a number of previous conflicting studies that examine caudate structural changes but that is not their hypothesis. The discussion goes into developmental changes affecting striatum at different times that might be relevant and would require a longitudinal study for a definitive study - as the authors acknowledge.<br /> 2. There is a lot of overlap between the caudate signal in the two groups - although the correlation of individual differences is reasonable. The caudate signal would not allow group classification.<br /> 3. Outside of the caudate they do show changes in left IFG and auditory cortex that are hypothesised. But there is a lot else going on - I was struck by occipital changes in figure 1 which are only mentioned once in the manuscript.<br /> 4. Should I be concerned by i) apparent signal changes in right anterior lateral ventricle from group comparison in figure 1 ii) signal change correlation in right anterior lateral ventricle in figure 4 (slice 22) and iii) signal change outside the pial surface of the occipital lobe in figure 1?

    2. Reviewer #2 (Public Review):

      This work demonstrates the value that multiparameter mapping imaging protocols can have in uncovering microstructural neural differences in populations with atypical development. Previous studies looking at differences in brain structure have typically used voxel based morphometry (VBM) approaches where differences in volumes can be hard to interpret due to complex tissue compositions. The imaging protocol outlined in this paper can specifically index different tissue properties e.g. myelin, giving a much more sensitive and interpretable measure of structural brain differences. This paper applies this methodology to a population of adolescents with developmental language disorder (DLD). Previous evidence of structural brain differences in DLD is very inconsistent and, indeed, using traditional VBM the authors do not find a difference between children with DLD and those with typical language development. However, they provide convincing evidence that despite no macrostructural differences, children with DLD show clear differences in levels of myelin in the dorsal striatum and in brain regions in the wider speech and language network. This can help to reconcile previous inconsistent findings and provide a useful springboard for both theoretical and empirical work uncovering the nature of the brain bases of language disorders.


      The imaging protocol is robust and is explained very clearly by the authors. It has been used before in other populations so is an established method but has not been applied to populations of children with DLD before, yielding novel and very interesting results. The authors demonstrate that this is a methodology which could have great value in other populations that display atypical development, increasing the impact of these findings.

      The sample size is large for research in this area which increases confidence in the results and the conclusions.

      Rather than relying solely on group differences in brain microstructure to draw conclusions about neural bases of language development, the authors correlated brain microstructural measures with performance on standardised language tests, allowing stronger inferences to be drawn about the relationships between structure and function. This is often an important omission from developmental neuroimaging work. It gave increased confidence in the finding that alterations in striatal myelin are linked to language difficulties.


      The authors rightly use the CATALISE definition of developmental language disorder, which differs from much of the previous literature by not requiring that children with language difficulties have nonverbal ability that is in the normal range. As can be common when using this definition of DLD, the group with DLD have significantly weaker nonverbal ability than the typically developing group. The authors show that brain microstructural differences correlate with language ability but they don't rule out a correlation with nonverbal or wider cognitive skills. Given the widespread differences in myelination across areas of the brain, including those that weren't predicted e.g. medial temporal lobe, it is plausible that perhaps some of the brain microstructural differences are not linked directly to language impairment but a broader constellation of difficulties. Some of the arguments in the paper would be strengthened if this interpretation could be ruled out.

      The authors acknowledge in the limitations section that their data cannot speak to whether brain differences are a cause or consequence of language impairment. However, there are some implied assumptions throughout the discussion of the results that brain differences in myelination have functional consequences for language learning. A correlation between structure and function does not indicate this level of causality, particularly in an adolescent population - function could just as easily have had structural consequences or environmental differences could have influenced both structure and function. In my view, the speculations about functional consequences of myelin differences are not fully supported by the data collected.

      The data suggest that there is much greater variability in left caudate nucleus MTsat values for the DLD group than the other two groups. The impact this may have on the results is not discussed in the interpretation and it is unclear whether this greater variability occurs throughout all of the key MPM measures for the DLD group.

    3. Reviewer #3 (Public Review):

      Developmental Language Disorder (DLD) is observed in children who struggle to learn and use oral language despite no obvious cause. It is extremely wide-spread affecting 7-10% of children, and extremely consequential as it persists throughout life and has downstream effects on reading, academic outcomes, and career success. A large number of prior studies have attempted to identify the structural neural differences that are associated with DLD. These have generally shown mixed results, but support a number of candidate regions including left hemisphere language areas (particularly the inferior frontal gyrus), and striatal regions that are possibly linked to learning. However, these studies have suffered from small sample sizes and conflicting results. Part of this may be their reliance on traditional voxel-based-morphometric techniques which estimate cortical thickness and gray matter density. The authors argue that these measures are biologically imprecise; gray matter can be thinner for example, due to synaptic pruning or increased mylenation.

      The authors of this study offer a powerful new tool for understanding these differences. Multi-Parameter Mapping (MPM) is based on standard MRI techniques but offers several measures with much greater biological precision that can be tied specifically to myelination, a key marker of efficient neural transmission. The test a very large number of children (>150) with and without DLD using MPM and show strong evidence for fundamental biological differences in these children.

      This study features a number of key strengths. First, at the level of neuro-imaging, the MPM technique is new in this population and offers fundamental insight that cannot be obtained by other measures. Indeed, the authors wisely use a traditional gray matter approach (voxel based morphometry) and find few if any differences between children with DLD and typical development. This offers a powerful proof of the sensitivity of this approach. Moreover, the authors analyze their data comprehensively, looking at two measures of myelin (MTsat and R1) and their convergence.

      However, at the most important level, I think structural approaches (like MPM, diffusion weighted imaging and so forth) offer tremendous promise for dealing with this as they avoid the ambiguity associated with interpreting functional MRI. Are children showing reduced BOLD because they are less good at language processing? Or do the differences in brain function cause poorer language processing? Structural approaches - and MPM in particular - offer tremendous promise as they unambiguously assess the fundamental neuro-biology.

      Beyond the neuro-imaging this study is also strong in their sample and the measurements of language. The sample size is very large and an order of magnitude larger than existing studies. It is well characterized, and the authors use a large set of well-motivated measures that capture the relevant dimensionality of language. Moreover, the authors treat language both as a clinical category and a continuous measure which is consistent with current thinking on the nature of DLD as potentially the low end of a continuous scale rather than a discrete disorder.

      Finally, the discussion of this paper for the most part does a good job of fitting these neurobiological findings into our broader understanding of DLD. It does an excellent job of mapping the observed brain differences onto functional differences in the child. Importantly, in doing this it does a very good job of avoiding the common trope of assuming neural differences play a causal role in DLD (when in fact, reduced atypical development could cause neural differences).

      Despite these strengths, I have a number of substantive concerns that if addressed will improve the overall impact of this paper.

      First, as the authors are aware, trhere is a long running and active debate in DLD as to whether DLD is the tail end of continuous distribution of children or a unique disorder (Leonard, 1987, 1991; Tomblin, 2011; Tomblin & Zhang, 1999). The results here offer great promise for informing that debate. And in that vein the authors quite appropriately analyze their data in two ways: once using DLD as a categorical variable and once using continuous measures of language. However, they don't really attempt to wrestle with the differences between the model.

      Second, I was a little surprised to see the authors highlight left IFG in the discussion to the degree they did. While there was clear evidence for reduced myelin there in the MTsat analysis, this did not hold up in R1 analysis, and even in the MTsat, IFG was clearly not the primary locus. Rather the areas of differences seemed to be centered at Pre- and Post-Central gyrus and extending ventrally (to IFG) and posteriorly from there. Given debate on the role of IFG in language specific processing in general (Diachek, Blank, Siegelman, Affourtit, & Fedorenko, 2020; Fedorenko, Duncan, & Kanwisher, 2013), it was not immediately clear to me why that area was important to highlight. For example, some of the posterior temporal areas (and motor areas) that were found were equally important for perceptual, lexical and phonological processing that are important for other theories of DLD.

      The authors rightly point to their differences in the striatum as supporting theories of DLD centered around differences learning. However, as they discuss, there are also large differences throughout the brain in both perceptual, motor and language areas. These would seem to support theories of DLD centered around processing and representation. In particular, the differences in myelination likely are linked to differences in the efficiency of neural coding. This would seem to favor two theoretical views that might be worth mentioning - speed of processing (Miller, Kail, Leonard, & Tomblin, 2001), and approaches based on lexical processing (McMurray, Klein-Packard, & Tomblin, 2019; McMurray, Samelson, Lee, & Tomblin, 2010; Nation, 2014). I was surprised these were not mentioned, given the clear link to the timecourse of processing. Does then suggest that these theories might complement each other? It would be useful to see some more discussion of the implications of these findings for broader theories.

    1. Reviewer #1 (Public Review):

      In this work the authors study biofilm growth and wrinkling in a controlled microfluidic setup. They argue that previous work involving such growth on agar substrates are complicated by the nature of transport within the agar and that the present arrangement simplifies the approach. Growth-induced wrinkling is a commonly observed phenomenon, and it stands to reason that the biofilm problem may be amenable to the kinds of theoretical approaches already developed.

      Using confocal imaging to study the folding and wrinkling of confined, growing biofilms, the authors find a number of very interesting results, including insights into the role of compressive forces and of adhesion with the substrate in the instability. Their imaging also reveals the generation of fluid-filled channels within the biofilm complex that contain motile bacteria.

      Existing theory from many years ago on growth-induced buckling and delamination (from the metallurgical literature) is used to rationalize the experimental observations, particularly with regard to the role of adhesion.

      Overall this work provides an impressive array of results that provide real insight into the basic problem of biofilm wrinkling. Yet, a quantitative connection between the experimental observations and theories of wrinkling is absent. Although there is a discussion of the prior theory, it is not presented in a manner that significantly enhances the discussion, as there are parameters undefined/unexplained (toughness, for example) and no attempt is made to connect the observed wavelength with that arising from theory.

    2. Reviewer #2 (Public Review):

      In this paper, the authors study the buckling instability and wrinkling of a Pseudomonas aeruginosa biofilm growing on a PDMS substrate inside a rectangular microfluidic channel in the presence of fluid flow. Overall, this paper addresses a topic of growing interest - the mechanical aspects of biofilm growth - and some of its results are clearly novel, namely: (i) the control of biofilm wrinkling and the subsequent formation of a network of channels by means of the flow of nutrients and the substrate surface properties, (ii) disentangling the main driving mechanisms for wrinkling and their interplay, and (iii) the presence of swimming bacteria inside channels formed within wrinkles, whose swimming speed remains unaffected by the external flow outside the biofilm. Nonetheless, the paper contains a few shortcomings, in particular regarding the underdeveloped connection to theory and a lack of detail regarding quantitative measurements, that need to be addressed.

    1. Reviewer #1 (Public Review):

      This manuscript presents predictions of COVID-19 cases, hospitalizations, and deaths over July-December 2021 in the US using data up to July 2021, and combining the predictions from nine different models. The predicted resurgence in cases during late summer has been largely borne out by the data that has come out since. The results have some important implications for public health policy, as discussed below.

      Unsurprisingly, the models find that outcomes over the next six months are highly dependent on prior and future vaccination coverage by state, reinforcing the importance of increasing vaccination coverage. The authors also theorize some of the variation in model predictions may be due to non-pharmaceutical intervention adherence, however they do not explore this relationship in this paper. It would have been interesting to see if there was also a correlation between the predicted number of cases per state and non-pharmaceutical intervention adherence across states in July, but this information is possibly more difficult to acquire.<br /> The combination of predictions from different models is a strength, as it allows to assess future uncertainty related to different model structures and parameter assumptions. However, it is disappointing that this paper does not capitalize on this strength by also showing a comparison of individual model predictions, other than by showing the prediction intervals. The authors mentioned the models varied both in the magnitude of COVID-19 incidence, but also in the timing of the peak of infection. It would have been interesting to see a figure showing a cross-model comparison of predictions for a given scenario (the most suitable scenario given the data would have likely been the worst-case low vaccination-high variant transmissibility scenario). This would have allowed a better understanding of uncertainty related to different model assumptions, and would have allowed the reader to assess whether the median captures well the central tendency across models.

      An interesting feature of this paper is the comparison of model predictions against prospective data which was not used to fit the model. This comparison shows that reported cases up to July 31 exceeded those projected in all scenarios. This discrepancy should not be interpreted as indicating the models' predictions are invalid, but rather as a healthy and important exercise in reassessing our assumptions regarding the spread of SARS-CoV-2. It suggests that current SARS-CoV-2 variants in the US might for example be more transmissible than what was assumed in the models, or that adherence to non-pharmaceutical interventions is lower than anticipated. It would have been useful if the authors could have further explored which models with which assumptions were most closely able to match the earlier surge in cases. Nonetheless, even though the models may have underestimated the speed at which the cases would start to increase in July, there is a very strong correlation between the observed and predicted number of cases by state during July. This suggests that though the absolute number of COVID-19 cases and deaths the models predict over the next 6 months could be be an underestimate, the models do capture the major drivers of epidemic surges and are able to predict with good accuracy which states are likely to be most impacted by the pandemic over the next few months.

    2. Reviewer #2 (Public Review):

      The paper is well written, and data and codes have been made publicly available.

      The Delta wave in the US has been descending since early September. So it looks like two scenarios (i.e., high/low vaccination + high variant transmissibility) in Figure 1 predicted the timing of the decline quite well.

    3. Reviewer #3 (Public Review):

      This paper reports on rounds 6 and 7 of the COVID-19 scenario modelling hub projections for the US COVID-19 epidemic. The specific focus of this round is the impact of the more transmissible Delta variant during the second half of 2021, for a range of vaccine uptake and virus transmissibility assumptions.

      The ensemble models show logical relative differences in the likely magnitude of impacts of COVID over the period of interest given the various paired assumptions. The uncertainty in these estimates is very high and observed data following the period of projection up the time of submission are tracking along the 95% prediction interval.

      Looking at what's happened since, my sense is that the models assuming high transmissibility have captured the time course reasonably well but vastly underestimated magnitude. Given all of the very reasonably acknowledged uncertainties, it's not surprising that projections beyond a few weeks are rarely informative, but the conclusions regarding the suitability of this approach to inform response should be tempered. Forecasts beyond more than a few weeks are unlikely to be sufficiently valid for public health and clinical preparedness, which these findings clearly show.

      The ensemble modelling exercise is very useful, but there's a lot of information lost in merging the outputs of models that make different critical assumptions about VE against infectiousness and age distribution of uptake, which were allowed to vary between models, with other assumptions. I would be very interested to hear more about the variation in outputs that went into the ensemble and which aggregate assumptions have resulted in trajectories that have been more accurately predictive of the observed epidemic. This process is important for validation and improvement of future rounds.

      At the level of states there is clear heterogeneity in coverage and mandated or spontaneous behaviours that drive variation in outcomes. The models seem to capture those rankings well, even if the absolute values prove to be underestimates. It would be interesting to understand how heterogeneity below the level of states contributes to the overestimation of vaccine impact and whether this is also more marked in some states than others.

      There's no mention of waning immunity but given the time at which the US rollout commenced, one anticipates declining effectiveness against transmission in this observation window for early uptake cohorts.

    1. Reviewer #1 (Public Review):

      In this manuscript from the Crump laboratory, the authors use zebrafish histological staining, lineage tracing, enhancer transgenes, and mutants to find evidence for the gill origin of vertebrate jaws hypothesis. The data are mostly strong and convincing. I have only a few comments on some of the data and some suggestions for strengthening the discussion.

      • This study nicely integrates into the literature on the origins of the mandibular jaw including discussion of fossil evidence and work in chondrichthyans. However, there is no discussion of jawless fish and how the considerable literature from species such as lamprey relates to the findings presented here. Do agnathans develop a pseudobranch?

      • Are there gill-like structures derived from the second arch in teleosts? The authors mention the hemibranch derived from the hyoid arch in cartilaginous fishes. Is there a similar structure in any teleost? If there are no arch 2 gill-like structures, how this fits into the gill origin of jaw hypothesis should be discussed.

      • The first sentence of the discussion is not fully supported by the data. More species need to be included to make this claim. This statement would need to be tempered.

      • I like that the authors mention the alternative hypothesis in their discussion that "the pseudobranch arose independently from the jaw by co-option of a gill filament developmental program".

      • More care needs to be taken with the word homology and to make the distinction between serial homology vs historical or functional homology. This manuscript would be stronger if it were to include a definition of these terms (or at least serial homology because that's the argument here). To avoid confusion, the authors should also specifically state serial homology when that's what the authors mean, for example in the second sentence of the discussion.

    2. Reviewer #2 (Public Review):

      Thiruppathy and colleagues investigate pseudobranch development in a teleost fish, the zebrafish. The pseudobranch has long been hypothesized to be a vestigial mandibular arch gill. The classic model of jaw evolution in vertebrates posits that the gnathostome jaw evolved from modifications of an anterior gill-bearing segment. Here the authors were trying to test the hypotheses that (1) the pseudobranch is derived from the mandibular arch, and (2) that the pseudobranch is a segmental homolog of the gills.

      One major strength of the methods and results is that they strongly support both hypotheses. The authors use cutting-edge fate mapping methods involving photoconversion of transgene-driven fluorophores as well as Cre-mediated lineage tracing to fate map mandibular arch mesenchyme and epithelium. By fate mapping both neural crest-derived mesenchyme (by photoconverting sox10: kikGR, as well as labeling crest with a sox10:Cre excision of a stop cassette) and pharyngeal endoderm (by photoconverting fgf10b:nEOS) in the mandibular segment, the authors test for mandibular origins of two different pseudobranch progenitors (mesenchyme and epithelium). Another strength is including the enhancer activity of three different enhancers as a character to assess the serial homology of the pseudobranch and gills. A third strength is using gene function to test for serial homology by assessing pseudobranch and gill development in gata3 mutants.

      One weakness is that the resolution of the photoconverted kikGR cells within the pseudobranch is low and visualizing individual cells is difficult in Figure 1e. However, the resolution of labeled crest cells in Fig. S1A is higher, and convincingly shows labeled neural crest cells in the center of the forming pseudobranch. A second weakness is that the fgf10b:nEOS expression, although strongest in the endodermal pouches, appears to also be more lowly expressed in non-endodermal cells.

      Overall, the authors have achieved their goals and their conclusions of a first arch origin of the pseudobranch and likely serial homology between the pseudobranch and gills are both well supported.

      The likely impact of this work, together with the cited 2022 bioRxiv paper (in which Hirschberger and Gillis show convincingly that the pseudobranch in a chondrichthyan is derived in part from mandibular arch pharyngeal endoderm and expresses similar gene expression patterns as gills during development) is high. Together the papers argue strongly that the pseudobranch is a mandibular arch-derived segmental homolog of a gill. These are exactly the kinds of data needed from extant gnathostomes to critically test the classic theory of a gill arch origin of the gnathostome jaw. As chondrichthyans are derived relative to the gnathostome stem ancestor, one possibility could be that the condition in chondrichthyans is derived and does not reflect the state of the gnathostome ancestor. This paper, in adding teleosts, argues against that scenario, as the more parsimonious explanation is that derived teleosts and chondrichthyans inherited a mandibular arch serial homolog of a gill from their common ancestor. This paper will likely have an impact on the field of developmental biology in that the fate mapping methods used are generally applicable to a range of other lineage questions. But the likely biggest impact will be on the field of vertebrate evolutionary biology, as together these two papers will be landmark studies arguing for a gill arch origin of the jaw, and strong motivations for paleontologists to look harder for fossils that can be evaluated for having a gill in the mandibular segment.

    1. Reviewer #1 (Public Review):

      The authors use smFISH to study the activation dynamics of the ventrally-expressed T48 gene. They observe that different concentrations of Dorsal (in a gradient with peak levels in the ventral-most nuclei) lead to 1) different probabilities of "priming" and thus timing of activation, and 2) RNA polymerase loading. Together these define a dual mechanism of T48 mRNA accumulation in a graded manner. The experiments are elegant and the data well presented. The issue I have is that the idea of graded activation by Dorsal is not new. It is known that Dl and Twi regulate T48 (as hypothesized on lines 204 and 214), and that the T48 enhancer contains both TF binding sites, as this was published by the Lim lab in PNAS - Keller et al, 2020. This paper reported on the dynamics of transcriptional activation of T48, using live imaging to examine the effects of Dorsal (by removing binding sites) on transcriptional activation timing and transcriptional output (similar to priming and Pol II loading rate in this study). The Keller et al. paper concluded that T48 "exhibits a dynamic expression pattern, where nuclei along the ventral midline are the first to begin transcription, with more lateral nuclei becoming active as NC14 progresses." It further showed that "Dl site modulations change spatial boundaries of t48, mostly by affecting the timing of activation and bursting frequency rather than transcriptional amplitude or bursting duration." Thus, the Keller et al. paper came to basically the same conclusion about the effects of Dl on T48 transcription. Even the experiment utilizing constitutively-active Dl had a similar effect on T48 as the Lim experiment optimizing Dl binding sites in the T48 enhancer.

    2. Reviewer #2 (Public Review):

      The manuscript by Carmon et al investigates the mechanisms underlying the graded response to a morphogen gradient. Specifically they monitor the transcriptional response of two Dorsal targets, T48 and mist during mesoderm formation in Drosophila early embryos. For this, they employ single molecule FISH (smFISH) on wild type and ventralized embryos at various timing of mesoderm induction.<br /> The major finding of this manuscript is that the gradual response of T48/mist relies on two complementary mechanisms:<br /> 1) the priming of these genes by Dorsal<br /> 2) the loading rate of Pol II, also dependent on Dorsal nuclear levels.

      The data are of high quality and represent solid quantitative information.

      A weakness is that I find is the lack of functional analysis to validate some of the conclusions made from the quantitative analysis of the transcriptional response.<br /> For example, it would be exciting to assess the functional consequences of an intronless T48 gene by CRISPR gene editing. Phenotypes in terms of timing/coordination in the constriction of the actomyosin network would greatly improve the biological significance of the transcriptional findings of this manuscript.

      The overall conclusions of this paper are interesting and supported by their data.

      Specific comments:

      1-The word 'priming' is used in various instances (text and title of figures) but might be an over-interpretation. The results show the timing of transcriptional activation in various domains. A gradual activation could be interpreted as a gradual priming by a TF, but this is an interpretation.<br /> In particular, while the pioneering action of Zelda has been demonstrated and provides its 'priming' capacity, a similar scenario has not yet been shown for Dorsal. Thus, priming should be discussed with respect to a coordinated action between Dorsal and Zelda. Alternatively, if the authors believe that it's an action solely based on Dorsal, then this should be demonstrated (e.g. RNAi Zelda) or at least discussed.

      By the way, manipulation of Dorsal binding sites or Zelda binding site at T48 mesodermal enhancer has already been performed (albeit in a transgenic context). The authors should discuss and cite this important work: Keller et al 2021, DOI: 10.1073/pnas.1917040117 .

      The differential response to the Dorsal gradient shown for twi (switch like) compared to T48 (gradual) is an interesting paradigm. It would have been interesting to discuss the potential mechanisms supporting this differential response. For example, the contribution of Dorsal binding sites types and arrangement (grammar) and that of pioneering activity of Zelda. Many papers determined the contribution of Zelda binding to fostering the response to Dorsal gradient (with static approaches Crocker et al, 2017; Foo et al, Sun et al., or with live imaging Yamada et al. 2019, Dufourt et al. 2018). It would be interesting to discuss these results.

      2-It is well known that expression is extremely dynamic during the course of nc14. The authors usually show two images, and state that one is an earlier stage. I could not find how embryonic staging was performed, with the exception of the statement line 205 'monitoring embryos of different ages (as defined by the number of T48 TSs)'.<br /> A more quantitative assessment of embryonic timing by examining the level of membrane invagination for the embryo treated by smFISH and quantification would be preferable.<br /> Moreover, it would be exciting to compare the quantifications of %TS activation from smFISH with those already published, from MS2-tagged lines (Lim et al. 2017).

    3. Reviewer #3 (Public Review):

      This is an interesting study addressing an overlooked feature of morphogen gradient interpretation. By studying transcriptional activation of early zygotic genes during early Drosophila development the authors' findings suggest that morphogens, which are known to pattern tissues in distinct domain of gene expression, can also induce a graded transcriptional response within these domains. They speculate this might help orchestrating the spatiotemporal organization of downstream morphogenetic movements. While the results presented convincingly demonstrate graded activation of target genes, the underlying mechanisms remain unclear and additional information are needed to link the expression of these genes to the activity of Dorsal. Furthermore, whether the graded transcription of the genes analysed is translated in corresponding gradients of protein activity and the impact on morphogenesis remain to be investigated.

    1. Reviewer #2 (Public Review):

      The manuscript of Sadhukhan and Nandi presents a theoretical study of the shape fluctuations in a confluent epithelial monolayer. The theory which is following the lines of what has been done for 2 dimensional foams is knowledge new and original and the derivation of the results looks sound. It leads to the surprising result that the fluctuations in shape are "almost" universal and the distribution of the rescaled area depends only on a single parameter. The results are obtained with a series of approximations that would need to be discussed more extensively.

    1. Reviewer #1 (Public Review):

      Xiong and colleagues use an elegant combination of theory development, simulations, and empirical population genomics to interrogate a largely unexplored phenomenon in speciation/ hybridization genomics: the consequences and implications of admixture between species with differing substitution rates. The work presented in this well-written manuscript is thorough, thought provoking, and represents an important advancement for the field. However, there are a few instances where I feel the strength of the conclusions drawn is not fully supported.

      The authors begin by presenting evidence based on whole genome sequencing that the two focal species, P. syfanius and P. maackii, are highly diverged despite ongoing hybridization. Though the discussion of remarkable mitochondrial sequence similarity is underdeveloped. I do not understand how such a pattern is not most likely the result of introgression from one species to the other given the relatively high FST across much of the nuclear genome coupled with the generally higher mitochondrial mutation rate in animals.

      Next, they posit that barrier loci are likely to exist. To support this assertion, the authors use a combination of parental population genetic diversity and divergence comparisons and ancestry pattern analysis in hybrid populations. They show that there is a strong correlation between divergence across pure species and within species diversity across the autosomes. Then using four hybrid individuals they show that low ancestry randomness, as quantified estimates of between group and within group entropy, is associated with genomic region of reduced within group diversity and elevated between group divergence. The use of entropy estimates as a stand-in for admixture proportions and ancestry block analysis when sample size is severely limited is particularly clever. Though I must admit, I do not fully understand the derivations of the two entropy measures, it seems to me that relatedness might have a strong effect on the interpretability of between individual entropy estimates (Sb). With very small population sizes this may be a real issue. A brief discussion of potential caveats in using the new method developed here seems warranted given its potential usefulness to the population genomics field more broadly. One plausible but less likely alternative interpretation of these patterns is briefly discussed.

      The authors then move on to evidence for divergent substitution rates. Analysis of both D3 and D4 statistics using several different outgroups and a series of progressively stringent FST thresholds shows that site patterns between the two species are highly asymmetrical with P. maackii lineage harboring more substitutions than P. syfanius. The authors offer two possible explanations for this finding and then test both hypotheses.

      First, they use a comparative tree-based method to show that there is little phylogenetic evidence for lineage biased hybridization from outgroups into either of the focal lineages. Further, the range overlaps of the study species do not correspond with the inferred direction of allele sharing from the Dstat analysis. This is a good argument against contemporary gene flow between the outgroups and P. syfanius, but I am not convinced that ancient gene flow that could have occurred when, say, species distributions may have been different, can be ruled out using this analysis.

      To test whether this asymmetry can be explained by a difference in substitution rate between the two species the authors show that observed D3 increases and D4 decreases with increasingly divergent outgroups as predicted by theory developed here. The authors take this as evidence supporting the divergent substitution rates. Though they claim only that existence such rate divergence is likely. The unfortunately limited samples sizes seem to preclude attaining more certainty than this. Interestingly, as a byproduct of using D4 as an extended measure of site pattern asymmetry the authors highlight one way in which the ABBA-BABA test can give false positives for introgression. This is an important contribution to the field.

      Finally, the authors observe a monotonic relationship substitution rate ratio and relative genetic divergence across the genome which is in line with their theoretical predictions for differential substitution rates in the face of gene flow. From this they infer an 80% increase in substitution rate from P. syfanius to P. maackii. It is remarkable to be able to extract these substitution rates from genomic regions with the least gene flow. However the veracity of these estimates relies on the assumptions I have highlighted above and should be presented with appropriate caution.

    2. Reviewer #2 (Public Review):

      In their manuscript ("Admixture of evolutionary rates across a hybrid zone"), Xiong et al. use whole genome resequencing data to assess rates of genome evolution between two species of butterflies and determine whether putative barrier loci between the species are also those that evolve at asymmetric rates between them. This work presents a novel hypothesis and rigorously tests these ideas using a combination of empirical and theoretical work. I think the authors could more formally link loci that are evolving at highly asymmetric rates with those that are most likely to be barrier loci by evaluating the relationship between ancestry entropy and ratios of substitution rates between species. Additionally, clarifying the relationship between barrier loci and asymmetric evolution would be beneficial (i.e. are loci that we typically envision to be barrier loci, such as loci involved in reproductive isolation, evolving at asymmetric rates or do asymmetrically evolving loci represent a new type of barrier loci?).

    1. Reviewer #1 (Public Review):

      In this paper, the authors address the underlying processes that lead to cell fate specification in the early mouse embryo. They explore the role of laminin-integrin signaling in specifying the fate of the inside cells to become the Inner Cell Mass and the subsequent separation of the epiblast and the primitive endoderm. They provide a nice description of the expression patterns of components of the integrin signaling pathway in early development, which adds to previously published work. Then using a combination of gain of function and loss of function experiments, they provide some evidence for integrin signaling playing a role in both lineage segregation events. Gain of function experiments involved culturing inside cells from morulae in the presence of Matrigel as a source of laminins, while loss of function experiments involved the use of blocking antibodies to integrinb1 and genetic studies with Itgb1-/- and Lamc1-/- embryos. They show that culture in Matrigel blocked the ability of inside cells to repolarize and form trophectoderm (TE) and that this was mediated by the integrin receptor. However, loss of function of both integrin and laminin had no effect on the formation of ICM and TE in the intact embryo, diminishing the impact of these results. The clearer results came from examining the formation and organization of the primitive endoderm where loss of function of both integrin and laminin led to a failure of the primitive endoderm to organize into a single epithelial layer.

      Overall the claim that integrin signaling plays an active role in inducing ICM specification is not strongly supported. Rather, the role of Matrigel/integrin activation can be interpreted as blocking repolarization and thus blocking TE specification. They do not present any evidence of active induction of inside fate. Second, as they admit in the discussion, the culture of inside cells in Matrigel is an artificial situation, which may provide a non-physiological level of ECM/integrin activation. They might make a stronger case by testing directly the role of exogenous Laminin, rather than the complex Matrigel. Exogenous Laminin has been shown to rescue some phenotypic defects in laminin-null EBs (Li et al. 2002 J Cell Biol.). Given that embryos mutant for integrin or laminins (not shown) do not show any defects in ICM/TE specification at the blastocyst stage, the importance of integrin signaling for ICM/TE cell fate specification cannot be considered as a predominant influence on early patterning.

      However both integrin and laminin mutant embryos do show defects during the next lineage differentiation event separating Epiblast (EPI) and primitive endoderm (PrE). The primitive endoderm and epiblast are formed and specified but PrE fails to form an organized epithelial layer overlying the EPI, suggestive of a role for the ECM in this process. This observation is interesting but not novel- similar defects in laminin mutant embryos and embryoid bodies have been previously reported. There is no new experimental insight into the actual mechanism of action of this ECM/integrin interaction.

      Although this study provides some new information, the underlying mechanisms of action are not sufficiently explored and the study will be primarily of interest to specialists in mouse development.

    2. Reviewer #2 (Public Review):

      This manuscript from the Hiragii Lab identifies two new roles for Integrins and Laminins in the preimplantation mouse embryo.

      One of these roles is to influence trophectoderm-regenerating ability following removal of native trophectoderm by immunosurgery. In this context, the authors show that integrin/laminin can repress Hippo signaling in isolated inner cell masses.

      The other role reported for Integrin/Laminin is to ensure that primitive endoderm cells form a monolayer after sorting out from epiblast cells within the inner cell mass. Unfortunately, however, there does not appear to be a role for integrin/laminin in regulating Hippo signaling or trophectoderm fate specification during normal development.

      The evidence for the two roles for integrin/laminin comes from immunofluorescence (evaluation of integrin/laminin and cell fate markers), loss of function studies (blocking antibodies and null alleles), and gain of function studies (where Matrigel contains laminins, among other things...).

      Ultimately, this paper might interest preimplantation devotees, but will be less interesting to the broader scientific community in its current form. This is because the most compelling observations revolve around immunosurgery/regeneration, where the biological relevance is uncertain, while the embryo development phenotypes are minor and their analysis relatively superficial. If the study had revealed the mechanism by which integrin/laminin "talk to" the Hippo signaling pathway, it might have been more impactful.

    3. Reviewer #3 (Public Review):

      Kim at al., proposed a model in which the extracellular matrix, a fibrous environment, serves as a niche for inner cell mass specification in the preimplantation mouse embryo. The presence of extracellular matrix components within the preimplantation embryo was demonstrated a few decades ago. However, our current knowledge on their role during early mammalian develpoment still remains limited to processes during peri- and postimplantation embryogenesis.

      To simulate the in vivo environment, the authors removed the surrounding outer cells of the embryo by enzymatic digestion, called immunosurgery, and embedded the uncovered inner cell mass into Matrigel. Matrigel-embedded inner cell mass from embryos undergoing the first cell fate decision maintained their inner fate, shown by Sox2-positive immunostaining. In contrast, inner cell mass soaked in culture media (KSOM) developed into a proper blastocyst embryo with Sox2-positive inner and oblong CDX2-positive outer cells, and a blastocoel. The authors then underpinned their hypothesis by adding blocking antibodies against integrin beta1 and alpha6 into Matrigel or using embryos from integrin beta1-knockout mice. The manuscript takes then a sudden and contradictory twist. The authors proceed to perform similar experiments with inner cell mass isolated from blastocyst embryos to investigate the extracellular matrix-dependent processes on the second cell fate decision. At this developmental stage, the cells showed no altered cell fates but instead spatial mis-positioning. The authors finished with an interesting signalling mechanism but supported solely by co-expression analysis of Talin, Laminin gamma1 and integrin beta1 using immunostainings.

      Overall, the strength of the paper is diminished by describing too many different aspects, but none of them in sufficient detail.

      1) Sox2 has been extensively described to be involved in ICM specification. While the authors claim it as "the earliest marker of ICM specification", they then use it to visualise final internalised cells, several hours after the onset of internalisation. Therefore, the authors used a previously published Sox2-GFP mouse line. When checking the original paper on the generation of this Sox2-GFP line, it becomes clear that this is a reporter line in which GFP is under the regulatory elements of the Sox2 gene but not a Sox2-GFP fusion gene. Thus, measuring Sox2 expression levels as stated by the authors is not correct and it is questionable if the cytoplasmic GFP levels exactly reflect the physiological nuclear Sox2 expression levels.

      2) The presence of extracellular matrix components, including integrins and laminins, have been shown in previous studies as cited by the authors in the introduction. Thus, the novelty of result chapter 2 is unclear.

      3) It is an interesting approach to remove the outer cells from the morula embryos, unknown however if 16- or 32-cell stage embryos, by enzymatic digestion, allowing to study and manipulate the "naked" inner cell mass. The authors show one inner cell mass in which all cells maintained their inner cell fate. However, due to the lack of control experiments, the author's statement that "During the first lineage segregation, Matrigel is sufficient to drive ICM specification in an integrin-dependent manner,..." is not adequately supported. It is stated several times throughout the manuscript that the "extracellular matrix induces/drives ICM specification", contradictory to the subsequent chapter headed "integrin b1 is not required for initial ICM specification...".

      4) The strength of the paper is a more comprehensible effect on the spatial organisation of epiblast and primitive endoderm cells in the more advanced blastocyst. A more in-depth analysis on this phenomenon, including the signalling mechanism, would have helped to substantiate the author's claims. The sorting of epiblast and primitive endoderm cells is position-independent as stated by the authors. Thus, the title of the manuscript is not in accordance with this finding.

      5) A major drawback is the presentation of all imaging results in 2D. The preimplantation mouse embryo is a 3D object. If presented in 2D there is no reliable presentation of cell number of the embryos shown, no reliable quantification of expression intensities as it depends if cells are cut in the centre or at the edges and cropped outer cells can be mistaken by inner cells.

      The study needs to be put into context with more recent embryo-related publications. Currently over 80% of all cited references in introduction and discussion are >10 years old. For instance, previous studies demonstrating the necessity of Sox2 in epiblast formation (Avilion et al., 2003) and Sox2-DNA dynamics rather than expression levels (Goolam et al., 2016; White et al., 2016) were not taken into consideration. Major advances in the molecular mechanisms of inner cell allocation have been achieved, including Yap signalling, E-cadherin, cytoskeleton-dependent tension, but neglected by the authors.