Reviewer #2 (Public Review):

Summary

Faress et al. address how synaptic plasticity (i.e. potentiation induced by high frequency stimulation, HFS) induced at different time points and pathways relative to those active during initial learning can transform memories. They adopt an experimental design developed by Nabavi et al, 2014 to optogenetically induce a weak fear memory by pairing an optical conditioned stimulus (CS) at thalamo-LA synapses with a footshock unconditioned stimulus (US) in male mice. Homosynaptic HFS delivered in the same pathway before or after conditioning transforms the weak memory into a stronger one. Leveraging a new dual wavelength optogenetic approach in vivo, they also show that heterosynaptic (cortico-LA) HFS directly following the opto-conditioning can transform the thalamo-LA induced fear memory, or create a memory when directly delivered after unpaired conditioning. Lastly, they demonstrate that heterosynaptic potentiation of the thalamo-LA pathway accompanies the strengthening of fear memory in freely moving mice. The authors conclude that a transient experience (i.e. weak memory) can be transformed into a stable one by non-Hebbian forms of plasticity.

Strengths

This study uses well-defined and elegant optogenetic manipulations of distinct neural pathways in awake behaving mice combined with in vivo recordings, which allows to directly manipulate and monitor synaptic strength and memory. It addresses an interesting, timely, and important question.

Weaknesses

A key experiment with in vivo monitoring of LFPs and behavior (Fig. 5a-c) seems a bit underpowered and input-output curves (extended data 5c) not entirely convincing.<br /> Ex vivo slice experiments (Fig. 5d-f) are not well aligned with in vivo experimental conditions. While they provide proof of principle, this is not entirely novel (see Fonseca et al, 2013).

Significance and impact

The conclusions are well supported by the data. The significance of the study lies in showing in vivo, that plasticity induced at different times or synaptic pathways than those engaged during learning can modify a memory and the synaptic strength in the neural pathway related to that memory. While heterosynaptic and timing-dependent effects in synaptic plasticity have been described largely ex vivo on shorter time scales, the discovery of lasting behavioral effects on memory is novel. The study was enabled by a combination of clever approaches: creation of a "synthetic" pathway-specific association and a novel dual opsin approach in vivo to probe the role of plasticity in a converging second pathway at the same time.<br /> This work broadens our understanding of how Hebbian and non-Hebbian forms of plasticity shape neural activity and associative memory and is of broad interest to the neuroscience community.

Reviewer #2 (Public Review):

Summary:

The authors investigated how predicted stimuli are represented in posterior regions of the brain by recording electroencephalography during a visual sequence learning task. After learning the spatial order in which four stimuli were presented within a fixed sequence, participants were shown partial sequences - i.e., sequences in which only one element of the sequence was presented. By examining the decoding accuracy of the omitted stimuli, the authors aimed to investigate whether anticipated stimuli are (pro)actively represented in the expected spatial location at the expected time.

Strengths and Weaknesses:

The study successfully replicated previous findings on omitted stimuli within a predicted sequence (Ekman et al., 2023), while providing novel information regarding the temporal dynamics of predictive representation. Nevertheless, this outcome is not entirely surprising, as similar temporal dynamics were observed in a previous study employing a different task (Kok et al., 2017). The high level of scientific rigor is evident, as demonstrated by the numerous control analyses. Additionally, the results are particularly intriguing in terms of discerning the nature of the prepared representation, spanning from early perceptual to late attentional representations. Unfortunately, this distinction is not investigated in detail, thus allowing for alternative interpretations of the results.

The connection between the findings and the literature on priority maps could benefit from further clarification. There is room for a clearer delineation of how much of the representation can be ascribed to a perceptual prediction mechanism versus an attentional (post-perceptual) spatial cueing process. Although the latter can be readily connected to the concept of a priority map guiding spatial attention, the relationship between the priority map and perceptual prediction remains somewhat ambiguous. Noteworthy, an explanation of the results in terms of spatial cueing does not necessarily require a perceptual predictive mechanism. The significant decoding of the location of the omitted stimulus might be attributed to the preceding stimulus orienting attention towards the following location. While this potential explanation was not explicitly addressed in the study, it presents an intriguing avenue for further investigation.

The study provides valuable insight into how omitted, yet predicted stimuli are represented in the brain and its dynamics. While the research is commendable, addressing the outlined limitations would enhance its impact in the field. Specifically, the spatial location decoding results do not disentangle between perceptual prediction (i.e., the features of the expected stimulus) and attentional processes (i.e., the cueing of the to-be-attended location),

Reviewer #2 (Public Review):

Summary:

The authors investigate axonal and synapse development in two distinct visual feature-encoding neurons (VPN), LC4 and LPLC2. They first show that they occupy distinct regions on the GF dendrites, and likely arrive sequentially. Analysis of the VPNs' morphology throughout development, and synaptic gene and protein expression data reveals the temporal order of maturation. Functional analysis then shows that LPLC2 occupancy of the GF dendrites is constrained by LC4 presence.

Strengths:

The authors investigate an interesting and very timely topic, which will help to understand how neurons coordinate their development. The manuscript is very well written, and data are of high quality, that generally support the conclusions drawn (but see some comments for Fig. 2 below). A thorough descriptive analysis of the LC4/LPLC2 to GF connectivity is followed by some functional assessment showing that one neuron's occupancy of the GF dendrite depend on another.<br /> The manuscripts uses versatile methods to look at membrane contact, gene and protein expression (using scRNAseq data and state-of-the art genetic tools) and functional neuronal properties. I find it especially interesting and elegant how the authors combine their findings to highlight the temporal trajectory of development in this system.

Weaknesses:

After reading the summary, I was expecting a more comprehensive analysis of many VPNs, and their developmental relationships. For a better reflection of the data, the summary could state that the authors investigate *two* visual projection neurons (VPNs) and that ablation *of one cell type of VPNs* results in the expansion of the remaining VPN territory.

The manuscript is falling a bit short of putting the results into the context of what is known about synaptic partner choice/competition between different neurons during neuronal or even visual system development. Lots of work has been done in the peripheral the visual system, from the Hiesinger lab and others. Both the introduction and the discussion section should elaborate on this.

The one thing that the manuscript does not unambiguously show is when the connections between LC4 and LPLC2 become functional.

Figure 2:<br /> Figure 2A-C: I found the text related to that figure hard to follow, especially when talking about filopodia. Overall, life imaging would probably clarify at which time point there really are dynamic filopodia. For this study, high magnification images of what the authors define as filopodia would certainly help.<br /> L137ff: This section talks about filopodia between 24-48 hAPF, but only 36h APF is shown in A, where one could see filopodia. The other time points are shown in B and C, but number of filopodia is not quantified.<br /> L143: "filopodia were still present, but visibly shorter": This is hard to see, and again, not quantified.<br /> L144f: "from 72h APF to eclosion, the volume of GF dendrites significantly decreased": this is not actually quantified, comparisons are only done to 24, 36 and 48 h APF.<br /> Furthermore, 72h APF is not shown here, but in Figure 2D, so either show here, or call this figure panel already?

Figure 2D/E: to strengthen the point that LC4 and LPLC2 arrive sequentially, it would help to show all time points analyzed in Figure D/E.

L208: "significant increase ... from 60h APF to 72h APF": according to the figure caption, this comparison is marked by "+" but there is no + in the figure itself.

Figure 3:<br /> A key point of the manuscript is the sequential arrival of different VPN classes. So then why is the scRNAseq analysis in Figure 3 shown pooled across VPNs? Certainly, the reader at this point is interested in temporal differences in gene expression. The class-specific data are somewhat hidden in Supp. Fig. 9, and actually do not show temporal differences. This finding should be presented in the main data.

L438: "silencing LC4 by expressing Kir2.1... reduced the GF response": Is this claim backed by some quantification?

Figure 4K: Do the control data have error bars, which are just too small to see? And what is tested against what? Is blue vs. black quantified as well? What do red, blue, and black asterisks indicate? Please clarify in figure caption.

Optogenetics is mentioned in methods (in "fly rearing", in the genotypes, and there is an extra "Optogenetics" section in methods), but no such data are shown in the manuscripts. (If the authors have those data, it would be great to know when the VPN>GF connections become functional!)

Methods:

Antibody concentrations are not given anywhere and will be useful information for the reader

Could the authors please give more details on the re-analysis of the scRNAseq dataset? How did you identify cell type clusters in there, for example?

L785 and L794: I am curious. Why is it informative to mention what was *not* done?

Custom-written analysis code is mentioned in a few places. Is this code publicly available?

Reviewer #2 (Public Review):

Summary:

This manuscript by Poltavski and colleagues explores the relative contributions of Pax2- and Wnt1- lineage-derived cells in the enteric nervous system (ENS) and how they are each affected by disruptions in Ret and Endrb signaling. The current understanding of ENS development in mice is that vagal neural crest progenitors derived from a Wnt1+ lineage migrate into and colonize the developing gut. The sacral neural crest was thought to make a small contribution to the hindgut in addition but recent work has questioned that contribution and shown that the ENS is entirely populated by the vagal crest (PMID: 38452824). GDNF-Ret and Endothelin3-Ednrb signaling are both known to be essential for normal ENS development and loss of function mutations are associated with a congenital disorder called Hirschsprung's disease. The transcription factor Pax2 has been studied in CNS and cranial placode development but has not been previously implicated in ENS development. In this work, the authors begin with the unexpected observation that conditional knockout of Ednrb in Pax2-expressing cells causes a similar aganglionosis, growth retardation, and obstructed defecation as conditional knockout of Ednrb in Wnt1-expressing cells. The investigators then use the Pax2 and Wnt1 Cre transgenic lines to lineage-trace ENS derivatives and assess the effects of loss of Ret or Ednrb during embryonic development in these lineages. Finally, they use explants from the corresponding embryos to examine the effects of GDNF on progenitor outgrowth and differentiation.

Strengths:

- The manuscript is overall very well illustrated with high-resolution images and figures. Extensive data are presented.

- The identification of Pax2 expression as a lineage marker that distinguishes a subset of cells in the ENS that may be distinct from cells derived from Wnt1+ progenitors is an interesting new observation that challenges the current understanding of ENS development.

- Pax2 has not been previously implicated in ENS development - this manuscript does not directly test that role but hints at the possibility.

- Interrogation of two distinct signaling pathways involved in ENS development and their relative effects on the two purported lineages.

Weaknesses:

- The major challenge with interpreting this work is the use of two transgenic lines, rather than knock-ins, Wnt1-Cre and Pax2-Cre, which are not well characterized in terms of fidelity to native gene expression and recombination efficiency in the ENS. If 100% of cells that express Wnt1 do not express this transgene or if the Pax2 transgene is expressed in cells that do not normally express Pax2, then these observations would have very different interpretations and not support the conclusions made. The two lineages are never compared in the same embryo, which also makes it difficult to assess relative contributions and renders the evidence more circumstantial than definitive.

- Visualization of the Pax2-Cre and Wnt-1Cre induced recombination in cross-sections at postnatal ages would help with data interpretation. If there is recombination induced in the mesenchyme, this would particularly alter the interpretation of Ednrb mutant experiments, since that pathway has been shown to alter gut mesenchyme and ECM, which could indirectly alter ENS colonization.

- No consideration of glia - are these derived from both lineages?

- No discussion of how these observations may fit in with recent work that suggests a mesenchymal contribution of enteric neurons (PMID: 38108810).

Reviewer #2 (Public Review):

Summary:

Developing neuronal models that are shareable, reproducible, and interoperable allows the neuroscience community to make better use of published models and to collaborate more effectively. In this manuscript, the authors present a consolidated overview of the NeuroML model description system along with its associated tools and workflows. They describe where different components of this ecosystem lay along the model development pathway and highlight resources, including documentation and tutorials, to help users employ this system.

Strengths:

The manuscript is well-organized and clearly written. It effectively uses the delineated model development life cycle steps, presented in Figure 1, to organize its descriptions of the different components and tools relating to NeuroML. It uses this framework to cover the breadth of the software ecosystem and categorize its various elements. The NeuroML format is clearly described, and the authors outline the different benefits of its particular construction. As primarily a means of describing models, NeuroML also depends on many other software components to be of high utility to computational neuroscientists; these include simulators (ones that both pre-date NeuroML and those developed afterwards), visualization tools, and model databases.

Overall, the rationale for the approach NeuroML has taken is convincing and well-described. The pointers to existing documentation, guides, and the example usages presented within the manuscript are useful starting points for potential new users. This manuscript can also serve to inform potential users of features or aspects of the ecosystem that they may have been unaware of, which could lower obstacles to adoption. While much of what is presented is not new to this manuscript, it still serves as a useful resource for the community looking for information about an established, but perhaps daunting, set of computational tools.

Weaknesses:

The manuscript in large part catalogs the different tools and functionalities that have been produced through the long development cycle of NeuroML. As discussed above, this is quite useful, but it can still be somewhat overwhelming for a potential new user of these tools. There are new user guides (e.g., Table 1) and example code (e.g. Box 1), but it is not clear if those resources employ elements of the ecosystem chosen primarily for their didactic advantages, rather than general-purpose utility. I feel like the manuscript would be strengthened by the addition of clearer recommendations for users (or a range of recommendations for users in different scenarios).

For example, is the intention that most users should primarily use the core NeuroML tools and expand into the wider ecosystem only under particular circumstances? What are the criteria to keep in mind when making that decision to use alternative tools (scale/complexity of model, prior familiarity with other tools, etc.)? The place where it seems most ambiguous is in the choice of simulator (in part because there seem to be the most options there) - are there particular scenarios where the authors may recommend using simulators other than the core jNeuroML software?

The interoperability of NeuroML is a major strength, but it does increase the complexity of choices facing users entering into the ecosystem. Some clearer guidance in this manuscript could enable computational neuroscientists with particular goals in mind to make better strategic decisions about which tools to employ at the outset of their work.

Reviewer #2 (Public Review):

Summary:

This study employed voltage imaging in the CA1 region of the mouse hippocampus during the exploration of a novel environment. The authors report synchronous activity, involving almost half of the imaged neurons, occurred during periods of immobility. These events did not correlate with SWRs, but instead, occurred during theta oscillations and were phased-locked to the trough of theta. Moreover, pairs of neurons with high synchronization tended to display non-overlapping place fields, leading the authors to suggest these events may play a role in binding a distributed representation of the context.

Strengths:

Technically this is an impressive study, using an emerging approach that allows single-cell resolution voltage imaging in animals, that while head-fixed, can move through a real environment. The paper is written clearly and suggests novel observations about population-level activity in CA1.

Weaknesses:

The evidence provided is weak, with the authors making surprising population-level claims based on a very sparse data set (5 data sets, each with less than 20 neurons simultaneously recorded) acquired with exciting, but less tested technology. Further, while the authors link these observations to the novelty of the context, both in the title and text, they do not include data from subsequent visits to support this. Detailed comments are below:

(1) My first question for the authors, which is not addressed in the discussion, is why these events have not been observed in the countless extracellular recording experiments conducted in rodent CA1 during the exploration of novel environments. Those data sets often have 10x the neurons simultaneously recording compared to these present data, thus the highly synchronous firing should be very hard to miss. Ideally, the authors could confirm their claims via the analysis of publicly available electrophysiology data sets. Further, the claim of high extra-SWR synchrony is complicated by the observation that their recorded neurons fail to spike during the limited number of SWRs recorded during behavior- again, not agreeing with much of the previous electrophysiological recordings.

(2) The authors posit that these events are linked to the novelty of the context, both in the text, as well as in the title and abstract. However, they do not include any imaging data from subsequent days to demonstrate the failure to see this synchrony in a familiar environment. If these data are available it would strengthen the proposed link to novelty if they were included.

(3) In the discussion the authors begin by speculating the theta present during these synchronous events may be slower type II or attentional theta. This can be supported by demonstrating a frequency shift in the theta recording during these events/immobility versus the theta recording during movement.

(4) The authors mention in the discussion that they image deep-layer PCs in CA1, however, this is not mentioned in the text or methods. They should include data, such as imaging of a slice of a brain post-recording with immunohistochemistry for a layer-specific gene to support this.

Reviewer #2 (Public Review):

Summary:

This manuscript describes P. falciparum population structure in Zanzibar and mainland Tanzania. 282 samples were typed using molecular inversion probes. The manuscript is overall well written and shows clear population structure. It follows a similar manuscript published earlier this year, which typed a similar number of samples collected mostly in the same sites around the same time. The current manuscript extends this work by including a large number of samples from coastal Tanzania, and by including clinical samples, allowing for a comparison with asymptomatic samples.

The two studies made overall very similar findings, including strong small-scale population structure, related infections on Zanzibar and the mainland, near-clonal expansion on Pemba, and frequency of markers of drug resistance.

Strengths:

The overall results show a clear pattern of population structure. The finding of highly related infections detected in close proximity shows local transmission and can possibly be leveraged for targeted control.

Comments on revised version:

The authors have addressed my comments.

Reviewer #2 (Public Review):

Summary:

This study proposed a new mechanism by which TGF-beta signaling pathway promotes contacts between oocyte and the surrounding somatic cells in mouse, by regulating the numbers of transzonal projections (TZPs).

Strengths:

The conditional Smad4 knockout and three-dimensional observation of transzonal projections are solid and sufficiently support the major conclusions.

Comments on revised version:

The authors have adequately addressed the reviewers' questions and comments.

Reviewer #2 (Public Review):

Summary:

In this paper, Chamness and colleagues make a pioneering effort to map epistatic interactions among mutations in a membrane protein. They introduce thousands of mutations to the mouse GnRH Receptor (GnRHR), either under wild-type background or two mutant backgrounds, representing mutations that destabilize GnRHR by distinct mechanisms. The first mutant background is W107A, destabilizing the tertiary fold, and the second, V276T, perturbing the efficiency of cotranslational insertion of TM6 to the membrane, which is essential for proper folding. They then measure surface expression of these three mutant libraries, using it as a proxy for protein stability, since misfolded proteins do not typically make it to the plasma membrane. The resulting dataset is then used to shed light on how diverse mutations interact epistatically with the two genetic background mutations. Their main conclusion is that epistatic interactions vary depending on the degree of destabilization and the mechanism through which they perturb the protein. The mutation V276T forms primarily negative (aggravating) epistatic interactions with many mutations, as is common to destabilizing mutations in soluble proteins. Surprisingly, W107A forms many positive (alleviating) epistatic interactions with other mutations. They further show that the locations of secondary mutations correlate with the types of epistatic interactions they form with the above two mutants.

Strengths:

Such a high throughput study for epistasis in membrane proteins is pioneering, and the results are indeed illuminating. Examples of interesting findings are that: (1) No single mutation can dramatically rescue the destabilization introduced by W107A. (2) Epistasis with a secondary mutation is strongly influenced by the degree of destabilization introduced by the primary mutation. (3) Misfolding caused by mis-insertion tends to be aggravated by further mutations. The discussion of how protein folding energetics affects epistasis (Fig. 7) makes a lot of sense and lays out an interesting biophysical framework for the findings.

Weaknesses:

The major weakness comes from the potential limitations in the measurements of surface expression of severely misfolded mutants. It seems that only about 5% of the W107A makes it to the plasma membrane compared to wild-type. This point is discussed quite fairly in the paper. (Figures 2 and 3). This might be a low starting point from which to accurately measure the effects of secondary mutations. I am concerned about the extent to which surface expression can report on protein stability, especially when it comes to double mutants where each mutation alone severely decreases surface expression. It is possible that in these cases, both the single and double mutants are completely misfolded, beyond repair. The surface-expressed proteins in such mutants may not be stable, folded or active at all, and the authors do not provide any indication that the combined effects of the mutations are derived from effects on folding stability or misfolding. Therefore, the reason for the epistatic effects of these mutations is hard to interpret, leaving a notable gap in our understanding. However, I find that this point is discussed much more fairly in the current manuscript.

With that said, I believe that the results regarding the epistasis of V276T with other mutations are strong and very interesting on their own.

Another concern relates to the measurements of the epistatic effects of mutations in the background of the V107A mutation. I am concerned about their measurement accuracy. Firstly, the authors note that the surface immunostaining measurements of these mutants are on average only 2-fold above background, which is quite a low signal-to-noise regimen. Secondly, I believe that the authors still haven't demonstrated the reproducibility of their surface expression measurements. To showcase the reproducibility, the authors show the correlation of two biological replicates in Figure S3. However, these are shown only for the 251 mutations that passed a reproducibility filter, after the authors "discarded variant scores for which the difference in percentile rank across the two replicates was greater than 25%. " . this means that all mutations that showed irreproducible results were filtered out before the analysis in Figure S3. It is, therefore, no surprise that the remaining mutations are highly reproducible, and such an analysis cannot serve as an indication of the reproducibility. It remains possible that a large fraction of the surface immunostaining scores of the V107A variants are dominated by noise and that their correlation in these two replicates might be random and may not necessarily be reproduced in a third replicate, for example.

Reviewer #2 (Public Review):

Summary:

This is an interesting work where Wen et al. aimed to shed light on the mechanisms driving the protective role of soluble uric acid (sUA) toward avoiding excessive inflammation. They present biochemical data to support that sUA inhibits the enzymatic activity of CD38 (Figures 1 and 2). In a mouse model of acute response to sUA and using mice deficient in CD38, they find evidence that sUA increases the plasma levels of nicotinamide nucleotides (NAD+ and NMN) (Figure 3) and that sUA reduces the plasma levels of inflammasome-driven cytokines IL-1b and IL-18 in response to endotoxin, both dependent on CD38 (Figure 4). Their work is an important advance in the understanding of the physiological role of sUA, with mechanistic insight that can have important clinical implications.

Strengths:

The authors present evidence from different approaches to support that sUA inhibits CD38, impacts NAD+ levels, and regulates inflammatory responses through CD38.

Weaknesses:

The authors investigate macrophages as the cells impacted by sUA to promote immunoregulation, proposing that inflammasome inhibition occurs through NAD+ accumulation and sirtuin activity due to sUA inhibition of CD38. Unfortunately, the study still lacks data to support this model, as they could not replicate their in vivo findings using murine bone marrow-derived macrophages, a standard model to assess inflammasome activation. Without an alternative approach, the study lacks data to establish in vitro that sUA inhibition of CD38 reduces inflammasome activation in macrophages - consequently, they cannot determine yet if both NAD+ accumulation and sirtuin activity in macrophages is a mechanism leading to sUA role in vivo.

Reviewer #2 (Public Review):

In the manuscript, the authors combine SARS-CoV-2 sequence data from a state in Germany and mobility data to help in understanding the movement of the virus and the potential to help decide where to focus sequencing. The global expansion in sequencing capability is a key outcome of the public health response. However, there remains uncertainty about how to maximise the insights the sequence data can give. Improved ability to predict the movement of emergent variants would be a useful public health outcome. Also knowing where to focus sequencing to maximising insights is also key. The presented case study from one State in Germany is therefore a useful addition to the literature. Nevertheless, I have a few comments.

One of the key goals of the paper is to explore whether mobile phone data can help predict the spread of lineages. However, it appears unclear whether this was actually addressed in the analyses. To do this, the authors could hold out data from a period of time, and see whether they can predict where the variants end up being found.

The abstract presents the mobility-guided sampling as a success, however, the results provide a much more mixed result. Ultimately, it's unclear what having this strategy really achieved. In a quickly moving pandemic, it is unclear what hunting for extra sequences of a specific, already identified, variant really does. I'm not sure what public health action would result, especially given the variant has already been identified.

Relatedly, it is unclear to me whether simply relying on spatial distance would not be an alternative simpler approach than mobile phone data. From Figure 2, it seems clear that a simple proximity matrix would work well at reconstructing viral flow. The authors could compare the correlation of spatial, spatial proximity, and CDR data.

Reviewer #2 (Public Review):

Summary:

This research involves conducting experiments to determine the role of Fmnl2 during oocyte meiosis I.

Strengths:

Identifying the role of Fmnl2 during oocyte meiosis I is significant.

Weaknesses:

The quantitative analysis and the used approach to perturb FMNL2 function would benefit from more confirmatory approaches and rigorous analysis.

Comments on revised version:

The authors addressed most of my comments. However, some comments were not addressed convincingly.

My concern is still valid. The authors used only one approach to knockdown FMNL2 which is "siRNA-mediated knockdown". Using an additional approach to inhibit FMNL2 (Trim-Away or morpholino,..) would be beneficial to confirm that the effect of siRNA-mediated knockdown of FMNL2 is specific.

Response 1: In the author's response, they mentioned that successful migration was quantified based on the contact between the spindle pole and the oocyte cortex.<br /> After spindle migration, it is very common for the spindle to be close to (but not in contact with) the cortex for a considerable time. The spindle pole comes in contact with the cortex later (just before anaphase onset and polar body extrusion). Fig. 3A shows an example where at 9 h, the spindle is already migrated but did not come in contact with the cortex until 9:30 h. Based on Fig. 3B,C, the authors assessed spindle migration in fixed oocytes, making it impossible to fix all oocytes at the time of spindle contact with the cortex. Also,<br /> the representative images in Fig. 3C do not show spindle staining to assess the contact between the spindle and the cortex.<br /> Overall, I still believe that the distance between the spindle and the cortex is more accurate for quantifying spindle migration.

Response 2: The authors mentioned, "we made appropriate modifications to the relevant descriptions of immunoprecipitation experiments". I can't find these modifications in the manuscript. The authors need to state clearly that the immunoprecipitation results do not necessarily reflect meiotic oocytes specifically because these experiments were done using the whole ovary which contains both somatic cells and oocytes.

Response 5: The authors mentioned that "Based on our observations, during the extrusion of the first polar body in oocytes, there is a temporary occurrence of cellular morphological fragmentation due to cortical reorganization". Unfortunately, this means that the live imaging system in the authors' laboratory is not ideal for oocyte maturation. Several publications show normal oocyte morphology during cytokinesis. Please delete or replace Fig. 2E.

Reviewer #2 (Public Review):

Summary:

Numerous studies by the authors and other groups have demonstrated an important role for HIV gene expression kidney cells in promoting progressive chronic kidney disease, especially HIV associated nephropathy. The authors had previously demonstrated a role for protein kinase R (PKR) in a non-HIV transgenic model of kidney disease (Okamoto, Commun Bio, 2021). In this study, the authors used innovative techniques including bulk and single nuclear RNAseq to demonstrate that mice expressing a replication-incompetent HIV transgene have prominent dysregulation of mitochondrial gene expression and activation of PKR and that treatment of these mice with a small molecule PKR inhibitor ameliorated the kidney disease phenotype in HIV-transgenic mice. They also identified STAT3 as a key upstream regulator of kidney injury in this model, which is consistent with previously published studies. Other important advances include identifying the kidney cell types that express the HIV transgene and have dysregulation of cellular pathways.

Strengths:

Major strengths of the study include the use of a wide variety of state-of-the-art molecular techniques to generate important new data on the pathogenesis of kidney injury in this commonly used model of kidney disease and the identification of PKR as a potential druggable target for the treatment of HIV-induced kidney disease. The authors also identify a potential novel cell type within the kidney characterized by high expression of mitochondrial genes.

Weaknesses:

Though the HIV-transgenic model used in these studies results in a phenotype that is very similar to HIV-associated nephropathy in humans, the model has several limitations that may prevent direct translation to human disease, including the fact that mice lack several genetic factors that are important contributors to HIV and kidney pathogenesis in humans. Additional studies are therefore needed to confirm these findings in human kidney disease.

Reviewer #2 (Public Review):

Summary:

In this manuscript "Immunogenicity and safety of a live-attenuated SARS-CoV-2 vaccine candidate based on multiple attenuation mechanisms" by Suzuki-Okutani et al., the authors evaluate the attenuation, immunogenicity, and protection efficacy of a live-attenuated SARS-CoV-2 vaccine candidate (BK2102) against SARS-CoV-2.

Strengths:

The authors demonstrate that intranasal inoculation of BK2102 is safe and able to induce humoral and cellular immune responses in hamsters, without apparent signs of damage in the lungs, that protects against homologous SARS-CoV-2 and Omicron BA.5 challenge. Safety of BK2102 was further confirmed in a new hACE2 transgenic mouse model generated by the authors.

Weaknesses:

No major weaknesses were identified, however, this reviewer notes the following:

The authors missed the opportunity to include a mRNA vaccine to demonstrate that the immunity and protection efficacy of their live attenuated vaccine BK2102 is better than a mRNA vaccine.

One of the potential advantages of live-attenuated vaccines is their ability to induce mucosal immunity. It would be great if the authors included experiments to assess the mucosal immunity of their live-attenuated vaccine BK2102.

Reviewer #2 (Public Review):

Summary:

Drawing from tools of synthetic biology, Mihajlovic et al. use a cleverly designed experimental system to dissect Ohno's hypothesis, which describes the evolution of functional novelty on the gene-level through the process of duplication & divergence.

Ohno's original idea posits that the redundancy gained from having two copies of the same gene allows one of them to freely evolve a new function. To directly test this, the authors make use of a fluorescent protein with two emission maxima, which allows them to apply different selection regimes (e.g. selection for green AND blue, or, for green NOT blue). To achieve this feat without being distracted by more complex evolutionary dynamics caused by the frequent recombination between duplicates, the authors employ a well-controlled synthetic system to prevent recombination: Duplicates are placed on a plasmid as indirect repeats in a recombination-deficient strain of E.coli. The authors implement their directed evolution approach through in vitro mutagenesis and selection using fluorescent-activated cell sorting. Their in-depth analysis of evolved mutants in single-copy versus double-copy genotypes provides clear evidence for Ohno's postulate that redundant copies experience relaxed purifying selection. In contrast to Ohno's original postulate, however, the authors go on to show that this does not in fact lead to more rapid phenotypic evolution, but rather, the rapid inactivation of one of the copies.

Strengths:

This paper contributes with great experimental detail to an area where the literature predominantly leans on genomics data. Through the use of a carefully designed, well-controlled synthetic system the authors are able to directly determine the phenotype & genotype of all individuals in their evolving populations and compare differences between genotypes with a single or double copy of coGFP. With it they find clear evidence for what critics of Ohno's original model have termed "Ohno's dilemma", the rapid non-functionalization by predominantly deleterious mutations.

Including an expressed but non-functional coGFP in (phenotypically) single copy genotypes provides an especially thoughtful control that allows determining a baseline dN/dS ratio in the absence of selection. All in all the study is an exciting example of how the clever use of synthetic biology can lead to new insights.

Weaknesses:

The major weakness of the study is tied to its biggest strength (as often in experimental biology there is a trade-off between 'resolution' and 'realism').

The paper ignores an important component of the evolutionary process in favour of an in-depth characterization of how two vs one copy evolve. Specifically, by employing a recombination-deficient strain and constructing their duplicates as inverted repeats their experimental design completely abolishes recombination between the two copies.

This is problematic for two reasons:

i) In nature, new duplicates do not arise as inverted, but rather as direct (tandem) repeats and - as the authors correctly point out - these are very unstable, due to the fact that repeated DNA is prone to recA-dependent homologous recombination (which arise orders of magnitude more frequently than point mutations).

ii) This instability often leads to further amplification of the duplicates under dosage selection both in the lab and in the wild (e.g. Andersson & Hughes, Annu. Rev. Genet. 2009), and would presumably also be an outcome under the current experimental set-up if it was not prevented from happening?

So in sum, recombination between duplicate genes is not merely a nuisance in experiments, but occurring at extremely high frequencies in nature (such that the authors needed to devise a clever engineering solution to abolish it), and is often observed in evolving populations, be it in the laboratory or the wild.

The manuscript sells controlling of copy number as a strength. And clearly, without it, the same insights could not be gained. However, if the basis for the very process of what Ohno's model describes is prevented from happening for the process to be studied, then, for reasons of clarity and context this needs pointing out, especially, to readers less familiar with the principles of molecular evolution.

Connected to this, there are several places in the introduction and the discussion where I feel that the existing literature, in particular models put forward since Ohno that invoke dosage selection (such as IAD) end up being slightly misrepresented.

My point is best exemplified in line 1 of Discussion: "To test Ohno's hypothesis and to distinguish its predictions from those of competing hypotheses, it is necessary to maintain a constant and stable copy number of duplicated genes during experimental evolution."

I think this statement is simply not true and might be misleading. To take the exaggerated position of a devil's advocate, the goal of evolutionary biology should be to find out how evolution actually proceeds in nature most of the time, rather than creating laboratory systems that manage to recapitulate influential ideas.

While fixing copy number may be a necessary step to understand how one copy evolves if a second one is present, it seems that if Ohno's hypothesis only works out in recA-deficient bacterial strains and on engineered inverted repeats, that Ohno might have missed one crucial aspect of how paralogs evolve. The mentioned competing hypotheses have been put forward to (a) address Ohno's dilemma (which the present study beautifully demonstrates exists under their experimental conditions) and (b) to reflect a commonly observed evolutionary process in bacteria (dosage gain in response to selection, e.g. a classic way of gaining antibiotic resistance). Fixing the copy number allowed the authors to show which predictions of Ohno's model hold up and which don't (under these specific conditions). But they do so without even preventing the processes described by alternative models from happening, so the experimental system is hardly appropriate to distinguish between Ohno & alternatives. Therefore, I think it could be made clearer that the experimental system is great to look at certain aspects Ohno's hypothesis in detail, but it can only inform us about a universe without recombination.

(1) Citing the works by ref 8, 26, 27 to merely state that "in some copies were gained and some were lost (ref 6, ref 25)" makes it seem as if fixing at 2 copies is some sort of sensible average. Yet ref 6 (Dhar et al.) specifically states that dosage is the most important response. Moreover, in this study gene copies are lost, but plasmid copies are gained instead. In Holloway et al. 2007 (ref 25), the 2 copies resided on different plasmids, so entirely different underlying molecular genetics might be at work (high cost of plasmid maintenance, and competitive binding on both proteins onto the respective (off)-target, where either way selection favored a single copy, so a different situation altogether). In both cited studies, fixing the copy would have prohibited learning something about the process of duplication & divergence.

Hence this statement seems to distract the readers from the main message, which seems that preventing recombination experimentally allows to follow the divergence of each copy and studying a response that does not involve dosage-increase.

(2) "These studies highlighted the importance of gene duplication in providing fast adaptation under changing environmental conditions but they focused on the importance of gene dosage." I think this constructs a false dichotomy. Instead, these studies pointed out that dosage (and with it, selection for dosage) is an important part of the equation that might have been missed by Ohno.

(3) "Such models are also easier to test experimentally, because they do not require precise control of gene copy number. The necessary tests can even benefit from massive gene amplifications8. Although Ohno's hypothesis is more difficult to test experimentally (...)" - again, I feel the wording is slightly misleading. The point is not that IAD is easier to test and Ohno's model is harder to test in laboratory experiments, rather, experiments (and some more limited observations of naturally evolving populations) seem to suggest that in reality evolution proceeds (more often?) according to IAD rather than Ohno's neofunctionalization hypothesis. However, as the authors point out, it will be exciting to see their clever experimental system used to test other genes and conditions to get a more comprehensive understanding of what gene- and selection- parameter values would overcome Ohno's dilemma.

Reviewer #2 (Public Review):

Centrosomes are an integral part of cell division in most animal cells. There are notable exceptions, however, such as oocytes and plants. In addition, some animal cells can be engineered to lack centrosomes yet they can still manage to complete mitosis. This paper uses a couple of methods (PLK4 inhibition and deletion of SASS6) to remove centrosomes from an immortalized cell line. Indeed, a strength of the paper is that similar results are obtained using both protocols to generate acentrosomal cells. The authors find acentrosomal cells take longer to divide, mostly due to a longer metaphase. The paper is based on the finding that inhibition of MPS1 results in a failure to divide, and the hypothesis that a SAC - dependent delay is required for these acentrosomal cells to divide.

The finding that MPS1 inhibition results in a failure to division is interesting. This is investigated by analyzing cells where AurB, APC or Eg5 (to generate monastral spindles) have been inhibited. My concerns are that the results are not conclusive that the effect of MPS1 is on cell cycle regulation. There is not enough data to make a conclusion as to why inhibition of MPS1 results in cell division failure.

1) An example is how to interpret the effect of Aurora B inhibition, which does not block acentrosomal cell division. If Aurora B is required for SAC activity, it suggests this effect of MPS1 may be a function other than SAC. Given the complexity of the SAC, it would be informative to test other SAC components. Instead, the authors conclude that the mitotic delay caused by MPS is required for acentrosomal cell division. I don't think they have ruled out, or even addressed other functions of MPS1.

2) The authors find that when both the APC and MPS1 are inhibited, the cells eventually divide. These results are intriguing, but hard to interpret. The authors suggest that the failure to divide in MPS1-inhibited cells is because they enter anaphase, and then must back out. This is hard to understand and there is not data supporting some kind of aborted anaphase. Is the division observed with double inhibition some sort of bypass of the block caused by MPS1 inhibition alone? It is not clear why inhibition of APC causes increased cell division when MPS1 is inhibited.

3) The authors characterize MTOC formation in these cells, which is also interesting. MTOCs are established after NEB in acentrosomal cells. Indeed, forming these MTOCs is probably a key mechanism for how these cells complete a division, like mouse oocytes.

Following this, the results with inhibiting Eg5 are interesting. The double inhibition of MPS1 and Eg5 results in division failure, like MPS1 inhibition in acentrosomal cells. Thus, there is a cell division block when the centrioles fail to divide. This result raises the possibility that failure to make a bipolar spindle, or the presence of a monopolar spindle, is the problem. In the absence of a bipolar spindle, a SAC induced delay is required for spindle assembly. This is a possibility but there are multiple interpretations of these results. Primarily, these results do not show the MPS1 effect on acentrosomal cells is SAC related. That a SAC mediated delay is required for acentrosmomal spindle assembly is not the only conclusion.

Comments on revised version:

It appears that very few changes have been made. These are all suggestions in the writing and interpretation.

It's disappointing the most of the readouts are cell division success. There is a lack of data about what happens in the MPS1 knockdowns, such as microtubule attachment to KTs and localization/ activity of checkpoint proteins or downstream factors. More mechanistic insights may be found by testing other checkpoint proteins or assaying more metrics for spindle assembly and cell cycle progression. Or if inducing cell cycle delay suppresses the MPS1 effect. These experiments would implicate cell cycle factors as being required for acentrosomal spindle assembly while ruling out a requirement for MPS1 in spindle assembly.

The paper is well written. But some of the terminology could be improved and some descriptions of the cytology are confusing. Some clear definitions of terms may help. The authors refer to an "extended mitosis" (line 73) and then "exit in the absence of cell division" (line 96) when MPS1 is inhibited. Both are misleading and don't tell the full story. These cells attempt to divide and then fail, resulting in one cell. Use of terms like "spread back out", "rounding up", and "sitting down" seems like jargon and should at least be defined. The term "timely two-ness" (line 23-24) is also not helpful. A brief discussion of data on MPS1 function in mouse and fly acentrosomal meiosis might be appropriate. A comparison would be interesting since loss of MPS1 in acentrosomal oocytes does not have such a drastic block in cell division.

Reviewer #2 (Public Review):

Summary:

This study reveals that short-term social isolation increases social behavior at a reunion, and a population of hypothalamic preoptic area neurons become active after social interaction following short-term isolation (POAiso neurons). Effectively utilizing a TRAP activity-dependent labeling method, the authors inhibit or activate the POAiso neurons and find that these neurons are involved in controlling various social behaviors, including ultrasonic vocalization, investigation, and mounting in both male and female mice. This work suggests a complex role for the POA in regulating multiple aspects of social behavior, beyond solely controlling male sexual behaviors.

Strengths:

A few studies have shown that optogenetic activation of the POA in females promotes vocalization and mounting behavior, similar to the effects observed in males. However, those were the results of artificially stimulating POA neurons, and it was unknown whether POA neurons play a role in naturally occurring female social behaviors. This paper clearly demonstrates that there exists a population of POA neurons that are necessary for naturally evoked female social vocalizations and mounting behaviors.

Weaknesses:

The authors conclude that "In the current study, we identify and characterize a population of preoptic hypothalamic neurons that contribute to the effects of short-term social isolation on the social behaviors of mice." This is an interesting hypothesis, but in my opinion, critical control experiments are missing to support this claim.

All the activity-dependent labeling experiments with TRAP mice, including the subsequent neural activity manipulation experiments (Figures 2, 3, 4, 5E-F), were conducted by labeling neurons only in socially isolated animals, not group-housed animals. The authors labeled neurons after 30-minute social interactions, raising the possibility that the labeled neurons simply represent a "social interaction/behavior population" (mediating mounting and USVs in females and males) rather than a set of neurons specific to social isolation.

I strongly recommend including experimental groups that involve labeling neurons after 30-minute social interactions in group-housed female or male mice and inhibit TRAPed neurons after social isolation or activate TRAPed neurons after group housing. If manipulating the group-housed TRAP neurons has similar effects to manipulating the isolated TRAP neurons, it would suggest the current labeling paradigm is not isolating neurons specific to the effect of social isolation per se. Rather, the neurons may mediate more general social interaction or motivation-related activities. Given the known role of POA in male mating behavior, a group-housed TRAP experiment in males with a female visitor is especially important for understanding the selectivity of the labeled cells.

Without proper controls, referring to the labeled neurons as "POAiso" neurons is potentially misleading. The data thus far suggests these neurons may predominantly reflect a "POA social behavior" population rather than a set of cells distinctly responsive to isolated housing.

Overall, this paper is well-written and provides valuable new data on the neural circuit for female social behaviors and the potentially complex role of POA in social behavior control.

Reviewer #2 (Public Review):

The paper has two main merits. Firstly, it documents a new and important characteristic of the re-organization of the brains of the deaf, namely its variability. The search for a well-defined set of functions for the deprived auditory cortex of the deaf has been largely unsuccessful, with several task-based approaches failing to deliver unanimous results. Now, one can understand why this was the case: most likely there isn't a fixed one well-defined set of functions supported by an identical set of areas in every subject, but rather a variety of functions supported by various regions. In addition, the paper extends the authors' previous findings from blind subjects to the deaf population. It demonstrates that the heightened variability of connectivity in the deprived brain is not exclusive to blindness, but rather a general principle that applies to other forms of deprivation. On a more general level, this paper shows how sensory input is a driver of the brain's reproducible organization.

The method and the statistics are sound, the figures are clear, and the paper is well-written. The sample size is impressively large for this kind of study.

The main weakness of the paper is not a weakness, but rather a suggestion on how to provide a stronger basis for the authors' claims and conclusions. I believe this paper could be strengthened by including in the analysis at least one of the already published deaf/hearing resting-state fMRI datasets (e.g. Andin and Holmer, Bonna et al., Ding et al.) to see if the effects hold across different deaf populations. The addition of a second dataset could strengthen the evidence and convincingly resolve the issue of whether delayed sign language acquisition causes an increase in individual differences in functional connectivity to/from Broca's area. Currently, the authors may not have enough statistical power to support their findings.

Secondly, the authors could more explicitly discuss the broad implications of what their results mean for our understanding of how the architecture of the brain is determined by the genetic blueprint vs. how it is determined by learning (page 9). There is currently a wave of strong evidence favoring a more "nativist" view of brain architecture, for example, face- and object- sensitive regions seem to be in place practically from birth (see e.g. Kosakowski et al., Current Biology, 2022). The current results show what is the role played by experience.

Reviewer #2 (Public Review):

Summary:

In the present work, the authors present an engineering solution to sample preparation in 96-well plates for high-throughput super-resolution microscopy via Expansion Microscopy. This is not a trivial problem, as the well cannot be filled with the gel, which would prohibit the expansion of the gel. A device was engineered that can spot a small droplet of hydrogel solution and keep it in place as it polymerizes. It occupies only a small portion of space at the center of each well, the gel can expand into all directions, and imaging and staining can proceed by liquid handling robots and an automated microscope.

Strengths:

In contrast to Reference 8, the authors' system is compatible with standard 96 well imaging plates for high-throughput automated microscopy and automated liquid handling for most parts of the protocol. They thus provide a clear path towards high-throughput exM and high-throughout super-resolution microscopy, which is a timely and important goal.

Weaknesses:

The assay they chose to demonstrate what high-throughput ExM could be useful for, is not very convincing. But for this reviewer that is not important.

Reviewer #2 (Public Review):

Summary:

This paper evaluates the effect of COVID-19 booster vaccination on reinfection in Shanghai, China among individuals who received primary COVID-19 vaccination followed by initial infection, during an Omicron wave.

Strengths:

A large database is collated from electronic vaccination and infection records. Nearly 200,000 individuals are included in the analysis and 24% became reinfected.

Weaknesses:

The article is difficult to follow in terms of the objectives and individuals included in various analyses. There appear to be important gaps in the analysis. The electronic data are limited in their ability to draw causal conclusions.

More detailed comments:

In multiple places (abstract, introduction), the authors frame the work in terms of understanding the benefit of booster vaccination among individuals with hybrid immunity (vaccination + infection). However, their analysis population does not completely align with this framing. As best as I can tell, only individuals who first received COVID-19 vaccination, and subsequently experienced infection, were included. Why the analysis does not also consider individuals who were infected and then vaccinated is not clear.

In vaccine effectiveness analyses, why was time since initial infection not examined as a modifier of the booster effect? Time since the onset of the Omicron wave is only loosely tied to the immune status of the individual.

The effect of booster vaccination on preventing symptomatic vs. asymptomatic reinfection does not appear to have been evaluated; this is a key gap in the analysis and it would seem the data would support it.

In lines 105-108, the demographic description of the analysis population is incomplete. Is sex or gender identity being described? Are any individuals non-binary? What is the age distribution? (Only the proportions 20-39 and under 6 are stated.)

Figure 1 consort diagram is confusing. In the last row, are the two boxes independent or overlapping sets of individuals? Are all included in secondary analyses?

Reviewer #2 (Public Review):

Summary:

Birnavirus replication factories form alongside early endosomes (EEs) in the host cell cytoplasm. Previous work from the Delgui lab has shown that the VP3 protein of the birnavirus strain infectious bursal disease virus (IBDV) interacts with phosphatidylinositol-3-phosphate (PI3P) within the EE membrane (Gimenez et al., 2018, 2020). Here, Zanetti et al. extend this previous work by biochemically mapping the specific determinants within IBDV VP3 that are required for PI3P binding in vitro, and they employ in silico simulations to propose a biophysical model for VP3-PI3P interactions.

Strengths:

The manuscript is generally well-written, and much of the data is rigorous and solid. The results provide deep knowledge into how birnaviruses might nucleate factories in association with EEs. The combination of approaches (biochemical, imaging, and computational) employed to investigate VP3-PI3P interactions is deemed a strength.

Weaknesses:

(1) Concerns about the sources, sizes, and amounts of recombinant proteins used for co-flotation: Figures 1A, 1B, 1G, and 4A show the results of co-flotation experiments in which recombinant proteins (control His-FYVE v. either full length or mutant His VP3) were either found to be associated with membranes (top) or non-associated (bottom). However, in some experiments, the total amounts of protein in the top + bottom fractions do not appear to be consistent in control v. experimental conditions. For instance, the Figure 4A western blot of His-2xFYVE following co-flotation with PI3P+ membranes shows almost no detectable protein in either top or bottom fractions. Reading the paper, it was difficult to understand which source of protein was used for each experiment (i.e., E. coli or baculovirus-expressed), and this information is contradicted in several places (see lines 358-359 v. 383-384). Also, both the control protein and the His-VP3-FL proteins show up as several bands in the western blots, but they don't appear to be consistent with the sizes of the proteins stated on lines 383-384. For example, line 383 states that His-VP3-FL is ~43 kDa, but the blots show triplet bands that are all below the 35 kDa marker (Figures 1B and 1G). Mass spectrometry information is shown in the supplemental data (describing the different bands for His-VP3-FL) but this is not mentioned in the actual manuscript, causing confusion. Finally, the results appear to differ throughout the paper (see Figures 1B v. 1G and 1A v. 4A).

(2) Possible "other" effects of the R200D mutation on the VP3 protein. The authors performed mutagenesis to identify which residues within patch 2 on VP3 are important for association with PI3P. They found that a VP3 mutant with an engineered R200D change (i) did not associate with PI3P membranes in co-floatation assays, and (ii) did not co-localize with EE markers in transfected cells. Moreover, this mutation resulted in the loss of IBDV viability in reverse genetics studies. The authors interpret these results to indicate that this residue is important for "mediating VP3-PI3P interaction" (line 211) and that this interaction is essential for viral replication. However, it seems possible that this mutation abrogated other aspects of VP3 function (e.g., dimerization or other protein/RNA interactions) aside from or in addition to PI3P binding. Such possibilities are not mentioned by the authors.

(3) Interpretations from computational simulations. The authors performed computational simulations on the VP3 structure to infer how the protein might interact with membranes. Such computational approaches are powerful hypothesis-generating tools. However, additional biochemical evidence beyond what is presented would be required to support the authors' claims that they "unveiled a two-stage modular mechanism" for VP3-PI3P interactions (see lines 55-59). Moreover, given the biochemical data presented for R200D VP3, it was surprising that the authors did not perform computational simulations on this mutant. The inclusion of such an experiment would help tie together the in vitro and in silico data and strengthen the manuscript.

Reviewer #2 (Public Review):

In the manuscript, the authors highlighted the importance of T-cell receptor (TCR) analysis and the lack of amino acid embedding methods specific to this domain. The authors proposed a novel bi-directional context-aware amino acid embedding method, catELMo, adapted from ELMo (Embeddings from Language Models), specifically designed for TCR analysis. The model is trained on TCR sequences from seven projects in the ImmunoSEQ database, instead of the generic protein sequences. They assessed the effectiveness of the proposed method in both TCR-epitope binding affinity prediction, a supervised task, and the unsupervised TCR clustering task. The results demonstrate significant performance improvements compared to existing embedding models. The authors also aimed to provide and discuss their observations on embedding model design for TCR analysis: 1) Models specifically trained on TCR sequences have better performance than models trained on general protein sequences for the TCR-related tasks; and 2) The proposed ELMo-based method outperforms TCR embedding models with BERT-based architecture. The authors also provided a comprehensive introduction and investigation of existing amino acid embedding methods. Overall, the paper is well-written and well-organized.

The work has originality and has potential prospects for immune response analysis and immunotherapy exploration. TCR-epitope pair binding plays a significant role in T cell regulation. Accurate prediction and analysis of TCR sequences are crucial for comprehending the biological foundations of binding mechanisms and advancing immunotherapy approaches. The proposed embedding method presents an efficient context-aware mathematical representation for TCR sequences, enabling the capture and analysis of their structural and functional characteristics. This method serves as a valuable tool for various downstream analyses and is essential for a wide range of applications.

Reviewer #2 (Public Review):

Summary:

Franke et al. characterize the representation of color in the primary visual cortex of mice, highlighting how this changes across the visual field. Using calcium imaging in awake, head-fixed mice, they characterize the properties of V1 neurons (layer 2/3) using a large center-surround stimulation where green and ultra-violet colors were presented in random combinations. Clustering of responses revealed a set of functional cell-types based on their preference to different combinations of green and UV in their center and surround. These functional types were demonstrated to have different spatial distributions across V1, including one neuronal type (Green-ON/UV-OFF) that was much more prominent in the posterior V1 (i.e. upper visual field). Modelling work suggests that these neurons likely support the detection of predator-like objects in the sky.

Strengths:

The large-scale single-cell resolution imaging used in this work allows the authors to map the responses of individual neurons across large regions of the visual cortex. Combining this large dataset with clustering analysis enabled the authors to group V1 neurons into distinct functional cell types and demonstrate their relative distribution in the upper and lower visual fields. Modelling work demonstrated the different capacity of each functional type to detect objects in the sky, providing insight into the ethological relevance of color opponent neurons in V1.

Weaknesses:

While the study presents convincing evidence about the asymmetric distribution of color-opponent neurons in V1, the paper would greatly benefit from a more in-depth discussion of the caveats related to the conclusions drawn about their origin. This is particularly relevant regarding the conclusion drawn about the contribution of color opponent neurons in the retina. The mismatch between retinal color opponency and V1 color opponency could imply that this feature is not solely inherited from the retina, however, there are other plausible explanations that are not discussed here. Direct evidence for this statement remains weak.

In addition, the paper would benefit from adding explicit neuron counts or percentages to the quadrants of each of the density plots in Figures 2-5. The variance explained by the principal components does not capture the percentage of color opponent cells. Additionally, there appear to be some remaining errors in the figure legend and labels that have not been addressed (e.g. '??' in Fig 2 legend).

Overall, this study will be a valuable resource for researchers studying color vision, cortical processing, and the processing of ethologically relevant information. It provides a useful basis for future work on the origin of color opponency in V1 and its ethological relevance.

Reviewer #2 (Public Review):

The question of whether the neural mechanisms for reward and punishment learning are similar has been a constant debate over the last two decades. Numerous studies have shown that the midbrain dopamine neurons respond to both negative and salient stimuli, some of which can't be well accounted for by the classic RL theory (Delgado et al., 2007). Other research even proposed that aversive learning can be viewed as reward learning, by treating the omission of aversive stimuli as a negative PE (Seymour et al., 2004).

Although the current study took an axiomatic approach to search for the PE encoding brain regions, which I like, I have major concerns regarding their experimental design and hence the results they obtained. My biggest concern comes from the false description of their task to the participants. To increase the number of "valid" trials for data analysis, the instructed and actual probabilities were different. Under such a circumstance, testing axiom 2 seems completely artificial. How does the experimenter know that the participants truly believe that the 75% is more probable than, say, the 25% stimulation? The potential confusion of the subjects may explain why the SCR and relief report were rather flat across the instructed probability range, and some of the canonical PE encoding regions showed a rather mixed activity pattern across different probabilities. Also for the post-hoc selection criteria, why pick the larger SCR in the 75% compared to the 25% instructions? How would the results change if other criteria were used?

To test axiom 3, which was to compare the 100% stimulation to the 0% stimulation conditions, how did the actual shock delivery affect the fMRI contrast result? It would be more reasonable if this analysis could control for the shock delivery, which itself could contaminate the fMRI signal, with extra confound that subjects may engage certain behavioral strategies to "prepare for" the aversive outcome in the 100% stimulation condition. Therefore, I agree with the authors that this contrast may not be a good way to test axiom 3, not only because of the arguments made in the discussion but also the technical complexities involved in the contrast.

Comments on revised version:

I want to thank the authors for their thorough and comprehensive work in revising this manuscript. I agree with the authors that learning paradigms might not be a necessity when it comes to study the PE signals, but I don't particularly agree with some of the responses in the rebuttal letter ("Furthermore, conditioning paradigms generally only include one level of aversive outcome: the electrical stimulation is either delivered or omitted."). This is of course correct description for the conditioning paradigm, but the same can be said for an instructed design: the aversive outcome was either delivered or not. That being said, adopting the instructed design itself is legitimate in my opinion.

My main concern, which the authors spent quite some length in the rebuttal letter to address, still remains about the validity for different instructed probabilities. Although subjects were told that the trials were independent, the big difference between 75% and 25% would more than likely confuse the subjects, especially given that most of us would fall prey to the Gambler's fallacy (or the law of small numbers) to some degree. When the instruction and subjective experience collides, some form of inference or learning must have occurred, making the otherwise straightforward analysis more complex. Therefore, I believe that a more rigorous/quantitative learning modeling work can dramatically improve the validity of the results. Of course, I also realize how much extra work is needed to append the computational part but without it there is always a theoretical loophole in the current experimental design.

As the authors mentioned in the rebuttal letter, "selecting participants only if their anticipatory SCR monotonically increased with each increase in instructed probability 0% < 25% < 50% < 75% < 100%, N = 11 participants", only ~1/3 of the subjects actually showed strong evidence for the validity of the instructions. This further raises the question of whether the instructed design, due to the interference of false instruction and the dynamic learning among trials, is solid enough to test the hypothesis.

Reviewer #2 (Public Review):

Summary:

To identify sugar receptors and assess the capacity of these genes the authors first set out to identify behavioral responses in larva and adult as well as physiological response. They used phylogenetics and gene expression (RNAseq) to identify candidates for sugar reception. Using first an in vitro oocyte system they assess the responses to distinct sugars. A subsequent genetic analysis shows that the Gr10 and Gr6 genes provide stage specific functions in sugar perception.

Strengths:

A clear strength of the manuscript is the breadth of techniques employed allowing a comprehensive study in a non-canonical model species.

Weaknesses:

There are no major weaknesses in the study for the current state of knowledge in this species. Since it is much basic work to establish a broader knowledge, context with other modalities remain unknown. It might have been possible to probe certain context known from the fruit fly, which would have strengthened the manuscript.

Reviewer #2 (Public Review):

Summary:

In this manuscript, Shen and collaborators described the generation of conditional double knockout (cDKO) mice lacking LRRK1 and LRRK2 selectively in DAT-positive dopaminergic neurons. The Authors asked whether selective deletion of both LRRK isoforms could lead to a Parkinsonian phenotype, as previously reported by the same group in germline double LRRK1 and LRRK2 knockout mice (PMID: 29056298). Indeed, cDKO mice developed a late reduction of TH+ neurons in SNpc that partially correlated with the reduction of NeuN+ cells. This was associated with increased apoptotic cell and microglial cell numbers in SNpc. Unlike the constitutive DKO mice described earlier, cDKO mice did not replicate the dramatic increase in autophagic vacuole numbers. The study supports the authors' hypothesis that loss of function rather than gain of function of LRRK2 leads to Parkinson's Disease.

Strengths:

For the first time, the study described a model in which both the Parkinson's disease-associated gene LRRK2 and its homolog LRRK1 are deleted selectively in dopaminergic neurons. This offers a new tool to understand the physiopathological role of LRRK2 and the compensating role of LRRK1 in modulating dopaminergic cell function.

Weaknesses:

The model has no construct validity since loss of function mutations of LRRK2 are well tolerated in humans and do not lead to Parkinson's disease. The evidence of a Parkinsonian phenotype in these conditional knockout mice is limited and should be considered preliminary.

Reviewer #2 (Public Review):

In this manuscript, the authors set out to identify transporters that must exist in Stramenophiles due to the fact that the second half of glycolysis appears to be conducted in the mitochondria. They hypothesize that a Stramenophile-specific clade of transporters related to the dicarboxylate carriers are likely the relevant family and then go on to test two proteins from Blastocystis due to the infectious disease relevance of this organism. They show rather convincingly that these two proteins are expressed and are localized to the mitochondria in the native organism. The purified proteins bind to glycolytic intermediates and one of them, GIC-2, transports several glycolytic intermediates in vitro. This is a very solid and well-executed study that clearly demonstrates that bCIC-2 can transport glycolytic intermediates.

(1) The major weakness is that the authors aren't able to show that this protein actually has this function in the native organism. This could be impossible due to the lack of genetic tools in Blastocystis, but it leaves us without absolute confidence that bGIC-2 is the important glycolytic intermediate mitochondrial transporter (or even that it has this function in vivo).

(2) My impression is that the authors under-emphasize the fact that the hDIC also binds (and is stabilized by) glycolytic intermediates (G3P and 3PG). In the opinion of this reviewer, this might change my interpretation about the uniqueness of the bGIC proteins. They act on additional glycolytic intermediates, but it's not unique.

Reviewer #2 (Public Review):

The authors aim to better understand the neural responses to Shepard tones in auditory cortex. This is an interesting question as Shepard tones can evoke an ambiguous pitch that is manipulated by a proceeding adapting stimulus, therefore it nicely disentangles pitch perception from simple stimulus acoustics.

The authors use a combination of computational modelling, ferret A1 recordings of single neurons, and human EEG measurements.

Their results provide new insights into neural correlates of these stimuli. However, the manuscript submitted is poorly organized, to the point where it is near impossible to review. We have provided Major Concerns below. We will only be able to understand and critique the manuscript fully after these issues have been addressed to improve the readability of the manuscript. Therefore, we have not yet reviewed the Discussion section.

Major concerns

Organization/presentation<br /> The manuscript is disorganized and therefore difficult to follow. The biggest issue is that in many figures, the figure subpanels often do not correspond to the legend, the main body, or both. Subpanels described in the text are missing in several cases. Many figure axes are unlabelled. There is an inconsistent style of in-text citation between figures and the main text. The manuscript contains typos and grammatical errors. My suggestions for edits below therefore should not be taken as an exhaustive list. I ask the authors to consider the following only a "first pass" review, and I will hopefully be able to think more deeply about the science in the second round of revisions after the manuscript is better organized.

Frequency and pitch<br /> The terms "frequency" and "pitch" seem to be used interchangeably at times, which can lead to major misconceptions in a manuscript on Shepard tones. It is possible that the authors confuse these concepts themselves at times (e.g. Fig 5), although this would be surprising given their expertise in this field. Please check through every use of "frequency" and "pitch" in this manuscript and make sure you are using the right term in the right place. In many places, "frequency" should actually be "fundamental frequency" to avoid misunderstanding.

Insufficient detail or lack of clarity in descriptions<br /> There seems to be insufficient information provided to evaluate parts of these analysis, most critically the final pitch-direction decoder (Fig 6), which is a major finding. Please clarify.

Reviewer #2 (Public Review):

Summary:

In this manuscript, Peng et al., reported that 36THz high-frequency terahertz stimulation (HFTS) can suppress the activity of pyramidal neurons by enhancing the conductance of voltage-gated potassium channel. The authors also demonstrated the effectiveness of using 36THz HFTS for treating neuropathic pain.

Strengths:

The manuscript is well written and the conclusions are supported by robust results. This study highlighted the potential of using 36THz HFTS for neuromodulation.

Weaknesses:

More characterization of HFTS is needed, so the readers can have a better assessment of the potential usage of HFTS in their own applications.

(1) It would be very helpful to estimate the volume of tissue that can be influenced by HFTS. It is not clear how 15 mins HFTS was chosen for this functional study. Does a longer time have a stronger effect? A better characterization of the relationship between the stimulus duration of HFTS and its beneficial effects would be very useful.

(2) How long does the behavioral effect last after 15 minutes of HFTS? Figure 5b only presents the behavioral effect for one hour, but the pain level is still effectively reduced at this time point. The behavioral measurement should last until pain sensitization drops back to pre-stim level.

(3) Although the manuscript only tested in ACC, it will also be useful to demonstrate the neural modulation effect on other brain regions. Would 36THz HFTS also robustly modulate activities in other brain regions? Or are different frequencies needed for different brain regions?

Reviewer #2 (Public Review):

Summary:

The manuscript by Hou et al is a short technical report which details the potential use of a recently developed FRET based biosensor for gamma-secretase activity (Houser et al 2020) for in vivo imaging in the mouse brain. Gamma-secretase plays a crucial role in Alzheimer disease pathology and therefore developing methodologies for precise in vivo measurements would be highly valuable to better understand AD pathophysiology in animal models.

The current version of the sensor utilizes a pair of far-red fluorescent proteins fused to a substrate of the enzyme. Using live imaging, it was previously demonstrated it is possible to monitor gamma-secretase activity in cultured cells. Notably, this is a variant of a biosensor that was previously described using CFP-YFP variants FRET pair (Maesako et al, iScience. 2020). The main claim and hypothesis for the MS is that IR excitation and emission has considerable advantages in terms of depth of penetration, as well as reduction in autofluorescence. These properties would make this approach potentially suitable to monitor cellular level dynamics of Gama-secretase in vivo.

The authors use confocal microscopy and show it is possible to detect fluorescence from single cortical cells. The paper described in detail technical information regarding imaging and analysis. The data presented in figures 5-8 details analysis of FRET ratio (FR) measurements within populations of cells. The authors claim it is possible to obtain reliable measurements at the level of individual cells. They compare the FR values across cells and mice and find a spatial correlation among neighboring cells. This is compared with data obtained after inhibition of endogenous gamma-secretase activity, which abolishes this correlation.

Strengths:

The authors describe in detail their experimental design and analysis for in vivo imaging of the reporter. The idea of using a far-red FRET sensor for in vivo imaging is novel and potentially useful to circumvent many of the pitfalls associated with intensity-based FRET imaging in complex biological environments (such as autofluorescence and scattering).

Weaknesses:

There are several critical points regarding validation of this approach and concerns with the data presented that must be addressed:

(1) Regarding the variability and spatial correlation- the dynamic range of the sensor previously reported in vitro is in the range of 20-30% change (Houser et al 2020) whereas the range of FR detected in vivo is between cells is significantly larger (Fig. 3). This raises considerable doubts for specific detection of cellular activity (see point 3).<br /> (2) One direct way to test the dynamic range of the sensor in vivo, is to increase or decrease endogenous gamma-secretase activity and to ensure this experimental design allows to accurately monitor gamma-secretase activity. In the previous characterization of the reporter (Hauser et al 2020), DAPT application and inhibition of gamma-secretase activity results in increased FR (Figures 2 and 3 of Houser et al). This is in agreement with the design of the biosensor, since FR should be inversely correlated with enzymatic activity. Here, while the authors repeat the same manipulation and apply DAPT to block gamma-secretase activity, it seems to induce the opposite effect and reduces FR (comparing figures 8 with figures 5,6,7). First, there is no quantification comparing FR with and without DAPT. Moreover, it is possible to conduct this experiment in the same animals, meaning comparing FR before and after DAPT in the same mouse and cell populations. This point is absolutely critical- if indeed FR is reduced following DAPT application, this needs to be explained since this contradicts the basic design and interpretation of the biosensor.<br /> (3) For further validation, I would suggest including in vivo measurements with a sensor version with no biological activity as a negative control, for example, a mutation that prevents enzymatic cleavage and FRET changes. This should be used to showcase instrumental variability and would help to validate the variability of FR is indeed biological in origin. This would significantly strengthen the claims regarding spatial correlation within population of cells.<br /> (4) In general, confocal microcopy is not ideal for in vivo imaging. Although the authors demonstrate data collected using IR imaging increases penetration depth, out of focus fluorescence is still evident (Figure 4). Many previous papers have primarily used FLIM based analysis in combination with 2p microscopy for in vivo FRET imaging (Some examples: Ma et al, Neuron, 2018; Massengil et al, Nature methods, 2022; DIaz-Garcia et al, Cell Metabolism, 2017; Laviv et al, Neuron, 2020). This technique does not rely on absolute photon number and therefore has several advantage sin terms of quantification of FRET signals in vivo.<br /> It is therefore likely that use of previously developed sensors of gamma-secretase with conventional FRET pairs, might be better suited for in vivo imaging. This point should be at least discussed as an alternative.

Reviewer #2 (Public Review):

Summary:

Naïve CD4 T cells in CD11c-Cre p28-floxed mice express highly elevated levels of proinflammatory IFNg and the transcription factor T-bet. This phenotype turned out to be imposed by thymic dendritic cells (DCs) during CD4SP T cell development in the thymus [PMID: 23175475]. The current study affirms these observations, first, by developmentally mapping the IFNg dysregulation to newly generated thymic CD4SP cells [PMID: 23175475], second, by demonstrating increased STAT1 activation being associated with increased T-bet expression in CD11c-Cre p28-floxed CD4 T cells [PMID: 36109504], and lastly, by confirming IL-27 as the key cytokine in this process [PMID: 27469302]. The authors further demonstrate that such dysregulated cytokine expression is specific to the Th1 cytokine IFNg, without affecting the expression of the Th2 cytokine IL-4, thus proposing a role for thymic DC-derived p28 in shaping the cytokine response of newly generated CD4 helper T cells. Mechanistically, CD4SP cells of CD11c-Cre p28-floxed mice were found to display epigenetic changes in the Ifng and Tbx21 gene loci that were consistent with increased transcriptional activities of IFNg and T-bet mRNA expression. Moreover, in autoimmune Aire-deficiency settings, CD11c-Cre p28-floxed CD4 T cells still expressed significantly increased amounts of IFNg, exacerbating the autoimmune response and disease severity. Based on these results, the investigators propose a model where thymic DC-derived IL-27 is necessary to suppress IFNg expression by CD4SP cells and thus would impose a Th2-skewed predisposition of newly generated CD4 T cells in the thymus, potentially relevant in autoimmunity.

Strengths:

Experiments are well-designed and executed. The conclusions are convincing and supported by the experimental results.

Weaknesses:

The premise of the current study is confusing as it tries to use the CD11c-p28 floxed mouse model to explain the Th2-prone immune profile of newly generated CD4SP thymocytes. Instead, it would be more helpful to (1) give full credit to the original study which already described the proinflammatory IFNg+ phenotype of CD4 T cells in CD11c-p28 floxed mice to be mediated by thymic dendritic cells [PMID: 23175475], and then, (2) build on that to explain that this study is aimed to understand the molecular basis of the original finding.

In its essence, this study mostly rediscovers and reaffirms previously reported findings, but with different tools. While the mapping of epigenetic changes in the IFNg and T-bet gene loci and the STAT1 gene signature in CD4SP cells are interesting, these are expected results, and they only reaffirm what would be assumed from the literature. Thus, there is only incremental gain in new insights and information on the role of DC-derived IL-27 in driving the Th1 phenotype of CD4SP cells in CD11c-p28 floxed mice.

Altogether, the major issues of this study remain unresolved:

(1) It is still unclear why the p28-deficiency in thymic dendritic cells would result in increased STAT1 activation in CD4SP cells. Based on their in vitro experiments with blocking anti-IFNg antibodies, the authors conclude that it is unlikely that the constitutive activation of STAT1 would be a secondary effect due to autocrine IFNg production by CD4SP cells. However, this possibility should be further tested with in vivo models, such as Ifng-deficient CD11c-p28 floxed mice. Alternatively, is this an indirect effect by other IFNg producers in the thymus, such as iNKT cells? It is necessary to explain what drives the STAT1 activation in CD11c-p28 floxed CD4SP cells in the first place.

(2) It is also unclear whether CD4SP cells are the direct targets of IL-27 p28. The cell-intrinsic effects of IL-27 p28 signaling in CD4SP cells should be assessed and demonstrated, ideally by CD4SP-specific deletion of IL-27Ra, or by establishing bone marrow chimeras of IL-27Ra germline KO mice.

Reviewer #2 (Public Review):

Summary:

This valuable study characterizes the requirement for individual let-7 clusters to limit the generation of IL-17 producing CD8 T cells and the severity of emphysema in mouse models. Mature let-7 family miRNAs originate from multiple loci, several of which have been reported and/or are reported here to be downregulated in emphysematous lung tissue and/or lung T cells. The results provided are convincing but incomplete, as the let-7 cluster with the most convincing effects on T cell cytokine production is not tested for effects on disease pathogenesis.

Let-7 family miRNAs are largely redundant in function and originate from multiple genomic loci ("clusters"). Erice et al demonstrate that two individual clusters (let7afd and let7bc2) in mice regulate the generation of IL-17 producing CD8 T cells in vitro and in vivo in a model of emphysema. These cells also express higher levels of the IL-17-inducing transcription factor RORgt, encoded by Rorc, which the authors demonstrate to be a direct target of let-7. Since multiple let-7 family miRNAs are downregulated in T cells and lung tissue in emphysema, these data support a model in which reduced let-7 allows increased IL-17 production by T cells, contributing to disease pathogenesis.

Strengths:

The inclusion of miRNA and pri-miRNA expression data from sorted human lung T cells as well as mouse T cells from an emphysema model is a strength.

The study includes complementary loss of function and gain of function experimental systems to test the effect of altered let-7 function, though it should be noted that these involved different let-7 family members and did not yield simple, complementary results for all experimental outcomes.

The most important finding is that deletion of just one let-7 cluster ("Let7bc2") is sufficient to exacerbate emphysema in the nCB and CS models.

Weaknesses:

The human miRNA expression data that motivate functional analyses used sorted CD4+ T cells. The authors note that prior work on let-7 showed that it regulates Th17 (CD4) responses, yet this study's functional analyses are all focused on Tc17 (CD8) T cells. Data in this paper show that Tc17 cells are far less numerous than Th17 cells in the nCB and CS models of emphysema.

Compared with Let7bc2 deletion, Let7afd deletion had a much larger effect on IL17 production by CD8 T cells in vitro, and it also had a larger effect on RORgt expression in untreated mice in vivo, especially in the lung. In the revised manuscript, the authors show that let7afdLOF mice have normal numbers of CD4 and CD8 T cells in the thymus and peripheral lymphoid organs and do not exhibit lung histopathology or inflammatory changes at baseline at least up to 6 months of age. As such, they are set up perfectly to test the requirement for Let7afd in the nCB and/or CS models. These experiments would add strength to the core novelty of this work - demonstration of the functional importance of individual let-7 clusters.

The authors could do more to explain the complexity of the let7 miRNA family and the genomic clusters examined in this study. In particular, it would help to know the relationship between mouse Let7bc2 and corresponding human Let7 clusters. It would also be very helpful to know the relative expression of each mature let-7 family member in Tc17 cells. Are mature miRNAs derived from the Let7afd cluster more or less abundant?

The provided evidence for the effect of Let7GOF has an important caveat that came to light during review. Let7g overexpression caused a marked reduction in Rorgt expression in T cells at baseline and in the setting of nCB challenge, and it reduced the frequency of IL17+ producing CD8 T cells in the lung to baseline levels. Yet there was no change in the MLI measurement of histopathology. However, the responses in the experiment shown in Fig. 6C-D are quite muted compared to those shown in Figure 2. In the response to reviewers, the authors speculate that an anti-inflammatory of doxycycline, required for induction of Let7g in this model, "could account for the differences in the magnitude of emphysemic response".

Although RORgt is a great candidate to have direct effects on IL-17 expression, the mechanistic understanding of let-7 action on T cell differentiation and cytokine production is limited to this single target. As noted in the discussion, others have identified cytokine receptor targets that may play a role, but it is also likely others among the many targets of let-7 also contribute.

Reviewer #2 (Public Review):

The manuscript investigates the connections between the ubiquitin ligase protein deltex and the wingless pathway. Two different connections are proposed, one is function of deltex to modulate the gradient of wingless diffusion and hence modulate the spatial patter of wingless pathway targets, which regulate at different thresholds of wingless concentration. The second is a direct interaction between deltex and armadillo, a downstream component of the wingless pathway. Deltex is proposed to cause the degradation of armadillo resulting in suppression of wingless pathway activity. The results and conclusions of the manuscript are interesting and for the most part novel, although previously published work linking Notch and deltex to wingless signal regulation, and endocytosis to wingless gradient formation could be more extensively discussed. However neither of the two parts to the manuscript seem, in themselves sufficiently complete, and combining both parts together therefore seems to lack focus.

The main issue with the manuscript is that much of the conclusions are inferred from genetic interactions in vivo between loss of function mutants and overexpression. While providing useful in vivo physiological context, this type of approach struggles to be able to make definitive conclusions on whether an interaction is due to direct or indirect mechanism, as the authors themselves conclude at the end of section 2.3. The problem is confounded by the fact that there is already documented much cross talk between the Notch signaling pathway and wingless at the transcriptional level, and deltex is already a Notch modulator that can alter wingless mRNA expression (See Hori et al 2004). Deltex in addition to promoting a ligand-independent Notch signal can also induce expression of Notch ligand, allowing further non-autonomous Notch activation and subsequent cell autonomous cis-inhibition of the initial deltex-induced signal. The dynamics and outcomes of the Notch signal response to deltex in vivo is therefore already very complicated to interpret before even considering to unravel indirect (via Notch) and direct interactions with wingless, although the two possibilities are not mutually exclusive. Whilst the revised manuscript does not completely overcome these limitations, further data and quantification have improved the support for the conclusions and there is a wider discussion of the relevant literature. The conclusions are interesting and add significantly to our understanding of the intersections between Wingless, Notch and trafficking regulators in an in vivo context.

Reviewer #2 (Public Review):

Summary:

The study by Algranati et al. introduces an exciting and promising therapeutic approach for the treatment of H3-K27M pediatric gliomas, a particularly aggressive brain cancer predominantly affecting children. By exploring the dual targeting of histone deacetylases (HDACs) and MYC activation, the research presents a novel strategy that significantly reduces cell viability and tumor growth in patient-derived glioma cells and xenograft mouse models. This approach, supported by transcriptomic and epigenomic profiling, unveils the potential of combining Sulfopin and Vorinostat to downregulate oncogenic pathways, including the mTOR signaling pathway. While the study offers valuable insights, it would benefit from additional clarification on several points, such as the rationale behind the dosing decisions for the compounds tested, the specific contributions of MYC amplification and H3K27me3 alterations to the observed therapeutic effects, and the details of the treatment protocols employed in both in-vitro and in-vivo experiments.

Clarification is needed on how doses were selected for the compounds in Figure S2A and throughout the study. Understanding the basis for these choices is crucial for interpreting the results and their potential clinical relevance. IC50s are calculated for specific patient derived lines, but it is not clear how these are used for selecting the dose.

The introduction mentions MYC amplification in high-grade gliomas. It would be beneficial if the authors could delineate whether the models used exhibit varying degrees of MYC amplification and how this factor, alongside differences in H3K27me3, contributes to the observed effects of the treatment.

In Figure 2A, the authors outline an optimal treatment timing for their in vitro models, which appears to be used throughout the figure. It would be helpful to know how this treatment timing was selected and also why Sulfopin is dosed first (and twice) before the vorinostat. Was this optimized?

It should be clarified whether the dosing timeline for the combination drug experiments in Figure 3 aligns with that of Figure 2. This information is also important for interpreting the epigenetic and transcriptional profiling and the timing should be discussed if they are administered sequentially (also shown in Figure 2A).I have the same question for the mouse experiments in Figure 4.

The authors mention that the mice all had severe dehydration and deterioration after 18 days. It would be helpful to know if there were differences in the side effects for different treatment groups? I would expect the combination to be the most severe. This is important in considering the combination treatment.

Minor Points:

(1) For Figure 1F, reorganizing the bars to directly compare the K27M and KO cell lines at each dose would improve readability of this figure.

(2) In Figure 4D, it would be helpful to know how many cells were included (or a minimum included) to calculate the percentages.

Reviewer #3 (Public Review):

In this study Gray and coworkers use a transposon mutant library in order to define: (i) essential genes for K. pneumoniae growth in LB medium, (ii) genes required for growth in urine, (iii) genes required for resistance to serum and complement mediated killing. Although there are previous studies, using a similar strategy, to describe essential genes for K. pneumoniae growth and genes required for serum resistance, this is the first work to perform such a study in urine. This is important because these types of pathogens can cause urinary tract infections. Moreover, the authors performed the work using a highly saturated library of mutants, which makes the results more robust, and used a clinically relevant strain from a pathotype for which similar studies have not been performed yet. Besides applying the transposon mutant library coupled with high-throughput sequencing, the authors validate some of the most relevant genes required for each condition using targeted mutagenesis. This is an important step to confirm that the results obtained from the library are reliable. Although this was done for only a small subset of the most significant genes. In addition, in vitro experiments involving complementation of urine with iron provide additional support to the results obtained with the mutants suggesting the importance of genes required for iron acquisition in a limiting-iron environment such as urine. Overall, the study is well-designed and written, and the methodology and analysis performed are adequate. The study would have benefited from in vivo experiments, including a mouse model of bacterial sepsis or urinary tract infections which could have demonstrated the role of some of the identified genes in the infection process. Nevertheless, the results obtained are informative for the scientific community since they pinpoint genes potentially more relevant in infections caused by K. pneumoniae. The identified genes could represent future targets for developing new therapies against a type of pathogen that is acquiring resistance to all available antibiotics. Although, as mentioned above, these potential targets should be confirmed using in vivo models.

One potential weakness of the work is that the TnSeq analysis only included two replicates per condition, thus it is possible that some of the differences detected may not be reproducible in future studies, first of all those that are less significant. In this sense, hundreds of genes were detected to be theoretically relevant for bacterial resistance to complement in serum. It is possible that some of these genes represent false positives. Thus, confirmation of the relevance of these genes in resistance to complement should be performed in future studies.

Reviewer #3 (Public Review):

Summary:

The receptor tyrosine kinase Anaplastic Lymphoma Kinase (ALK) in humans is nervous system expressed and plays an important role as an oncogene. A number of groups have been studying ALK signalling in flies to gain mechanistic insight into its various roles. In flies, ALK plays a critical role in development, particularly embryonic development and axon targeting. In addition, ALK was also shown to regulate adult functions including sleep and memory. In this manuscript, Sukumar et al., used a suite of molecular techniques to identify downstream targets of ALK signalling. They first used targeted DamID, a technique that involves a DNA methylase to RNA polymerase II, so that GATC sites in close proximity to PolII binding sites are marked. They performed these experiments in wild type and ALK loss of function mutants (using an Alk dominant negative ALkDN), to identify Alk responsive loci. Comparing these loci with a larval single cell RNAseq dataset identified neuroendocrine cells as an important site of Alk action. They further combined these TaDa hits with data from RNA seq in Alk Loss and Gain of Function manipulations to identify a single novel target of Alk signalling - a neuropeptide precursor they named Sparkly (Spar) for its expression pattern. They generated a mutant allele of Spar, raised an antibody against Spar, and characterised its expression pattern and mutant behavioural phenotypes including defects in sleep and circadian function.

Strengths:

The molecular biology experiments using TaDa and RNAseq were elegant and very convincing. The authors identified a novel gene they named Spar. They also generated a mutant allele of Spar (using CrisprCas technology) and raised an antibody against Spar. These experiments are lovely, and the reagents will be useful to the community. The paper is also well written, and the figures are very nicely laid out making the manuscript a pleasure to read.

Weaknesses:

The manuscript has improved very substantially in revision. The authors have clearly taken the comments on board in good faith. Yet, some small concerns remain around the behavioural analysis.

In Fig. 8H and H' average sleep/day is ~100. Is this minutes of sleep? 100 min/day is far too low, is it a typo?

The numbers for sleep bouts are also too low to me e.g. in Fig 9 number of sleep bouts avg around 4.

In their response to reviewers the authors say these errors were fixed, yet the figures appear not to have been changed. Perhaps the old figures were left in inadvertently?

The circadian anticipatory activity analyses could also be improved. The standard in the field is to perform eduction analyses and quantify anticipatory activity e.g. using the method of Harrisingh et al. (PMID: 18003827). This typically computed as the ratio of activity in the 3hrs preceding light transition to activity in the 6hrs preceding light transition.

In their response to reviewers, the authors have revised their anticipation analyses by quantifying the mean activity in the 6 hrs preceding light transition. However, in the method of Harrisingh et al., anticipation is the ratio of activity in the 3hrs preceding light transition to activity in the 6hrs preceding light transition. Simply computing the activity in the 6hrs preceding light transition does not give a measure of anticipation, determining the ratio is key.

Reviewer #2 (Public Review):

Summary:

In this study, Gularte-Merida et al investigate the occurrence of transgenerational effects of non-transmitted parental alleles outside of the well-described effect of "genetic nurture." To achieve this they employed consomic male mice to generate an N2 and N3 population, allowing for the observation of effects due to non-transmitted paternal alleles while controlling for maternal care by using isogenic B6 dams. The authors conduct RNAseq, qPCR validation, and anatomical phenotyping measures to investigate the presence of non-genetic nurture TGE. The author's findings challenge the frequency of non-genetic nurture TGE, a meaningful contribution to the field. Overall, this is an ambitious study with important negative data. The authors are to be commended on this. This greatly strengthens the negative findings within the paper.

The paper, however, is written extremely technically, with little detail, and is not currently suitable for the lay audience. The authors need to greatly increase the clarity of the writing and data presentation.

Strengths:

Elegant experimental design using consomic mouse populations.

The use of a second replication cohort using the same genetic founders as the first study.

Weaknesses:

While much of the explanation of the methods is understandable by geneticists, the paper has implications outside of the genetics field. Overall, I suggest expanding the explanation and language for non-geneticists. This will allow the paper to reach a wider audience.

Reviewer #2 (Public Review):

Summary:

The Authors identified miR-199b-5p is a potential OA target gene using serum exosomal small RNA-seq from human healthy and OA patients. Their RNA-seq results were further compared with publicly available datasets to validate their finding of miR-199b-5p. In vitro chondrocyte culture with miR-199b-5p mimic/inhibitor and in vivo animal models were used to evaluate the function of miR-199b-5p in OA. The possible genes that were potentially regulated by miR-199b-5p were also predicted (i.e., Fzd6 and Gcnt2) and then validated by using Luciferase assays.

Strengths:

(1) Strong in vivo animal models including pain tests.<br /> (2) Validate the binding of miR-199b-5p with Fzd6 and binding of miR-199b-5p with Gcnt2

The authors have addressed my concerns.

Reviewer #2 (Public review):

Summary:

This manuscript aimed to investigate the non-genetic impact of KHDC3 mutation on the liver metabolism. To do that they analyzed the female liver transcriptome of genetically wild type mice descended from female ancestors with a mutation in the Khdc3 gene. They found that genetically wild type females descended from Khdc3 mutants have hepatic transcriptional dysregulation which persist over multiple generations in the progenies descended from female ancestors with a mutation in the Khdc3 gene. This transcriptomic deregulation was associated with dysregulation of hepatically-metabolized molecules in the blood of these wild type mice with female mutational ancestry. Furthermore, to determine whether small non-coding RNA could be involved in the maternal non-genetic transmission of the hepatic transcriptomic deregulation, they performed small RNA-seq of oocytes from Khdc3-/- mice and genetically wild type female mice descended from female ancestors with a Khdc3 mutation and claimed that oocytes of wild type female offspring from Khdc3-null females has dysregulation of multiple small RNAs.

Finally, they claimed that their data demonstrates that ancestral mutation in Khdc3 can produce transgenerational inherited phenotypes.

However, at this stage and considering the information provided in the paper, I think that these conclusions are too preliminary. Indeed, several controls/experiments need to be added to reach those conclusions.

Additional context you think would help readers interpret or understand the significance of the work<br /> • Line 25: this first sentence is very strong and needs to be documented in the introduction.<br /> • Line 48: Reference 5 is not appropriate since the paper shows the remodeling of small RNA during post-testicular maturation of mammalian sperm and their sensibility to environment. Please, change it<br /> • Line 51: "implies" is too strong and should be replaced by « suggests »<br /> • Line 67: reference is missing<br /> Database, the accession numbers are lacking.<br /> • References showing the maternal transmission of non-genetically inherited phenotypes in mice via small RNA need to be added<br /> • Line 378: All RNA-Seq and small RNA-Seq data are available in the NCBI GEO

Reviewer #2 (Public Review):

Summary:

The authors have addressed the role of S1086 in the FMNL1beta DAD domain in F-actin dynamics, MVB polarization, and exosome secretion, and investigated the potential implication of PKCdelta, which they had previously shown to regulate these processes, in FMNL1beta S1086 phosphorylation. This is based on:<br /> (1) the documented role of FMNL1 proteins in IS formation;<br /> (2) their ability to regulate F-actin dynamics;<br /> (3) the implication of PKCdelta in MVB polarization to the IS and FMNL1beta phosphorylation;<br /> (4) the homology of the C-terminal DAD domain of FMNL1beta with FMNL2, where a phosphorylatable serine residue regulating its auto-inhibitory function had been previously identified.

They demonstrate that FMNL1beta is indeed phosphorylated on S1086 in a PKCdelta-dependent manner and that S1086-phosphorylated FMNL1beta acts downstream of PKCdelta to regulate centrosome and MVB polarization to the IS and exosome release. They provide evidence that FMNL1beta accumulates at the IS where it promotes F-actin clearance from the IS center, thus allowing for MVB secretion.

Strengths

The work is based on a solid rationale, which includes previous findings by the authors establishing a link between PKCdelta, FMNL1beta phosphorylation, synaptic F-actin clearance, and MVB polarization to the IS. The authors have thoroughly addressed the working hypotheses using robust tools. Among these, of particular value is an expression vector that allows for simultaneous RNAi-based knockdown of the endogenous protein of interest (here all FMNL1 isoforms) and expression of wild-type or mutated versions of the protein as YFP-tagged proteins to facilitate imaging studies. The imaging analyses, which are the core of the manuscript, have been complemented by immunoblot and immunoprecipitation studies, as well as by the measurement of exosome release (using a transfected MVB/exosome reporter to discriminate exosomes secreted by T cells).

Weaknesses

The data on F-actin clearance in Jurkat T cells knocked down for FMNL1 and expressing wild-type FMNL1 or the non-phosphorylatable or phosphomimetic mutants thereof would need to be further strengthened, as this is a key message of the manuscript. Also, the entire work has been carried out on Jurkat cells. Although this is an excellent model easily amenable to genetic manipulation and biochemical studies, the key finding should be validated on primary T cells.

Reviewer #2 (Public Review):

The first portion of the manuscript centered on identifying and confirming the ATHB2 microprotein (ATHB2miP), which constitutes the core message of this study. Overall, I find no issue with the selection criteria employed for identifying alternative microprotein mRNA transcripts. However, I do have some queries that I hope the authors can address for clarity.

(1) Upon reviewing the supplemental dataset where the authors listed the 377 unique novel miPs, along with those specifically in WL or shade treatments, I sought to comprehend the rationale behind focusing on ATHB2. Have the authors examined the shade response of all 377 potential microprotein candidates? Readers may be intrigued to learn how many of these candidates exhibit induction or repression under shade conditions, and whether such changes correlate positively or negatively with alterations in the full-length TSSs in response to shade. Essentially, I aim to discern the prevalence of microprotein production during shade responses and any shared characteristics among these microprotein transcripts. This inquiry also aims to uncover the existence of a common mechanism regulating microprotein transcription.

(2) To confirm that ATHB2miP stems from an independent transcription event, the authors sequenced full-length cDNAs using PacBio isoseq. However, I find the information regarding isoseq missing from the manuscript. My assumption is that the full-length cDNAs were reverse transcribed from mRNAs isolated from whole seedlings, where mature mRNAs in the cytoplasm predominate, making it challenging to evaluate whether a specific mRNA undergoes post-transcriptional processing. One approach to confirming ATHB2miP as a product of independent transcription involves examining nascent mRNA produced in the nucleus. The authors may need to isolate nascent mRNAs associated with RNA Polymerase II in the nucleus from seedlings treated with shade for 45 min, and then perform reverse transcription and PacBio isoseq.

(3) The authors noted the identification of two potential start codons, TTG and CTG, in the alternative TSS of ATHB2 using TISpredictor. Yet, it's imperative to identify the actual translation initiation site and the full-length sequence of ATHB2miP. I suggest the authors fuse an epitope tag (e.g., 3xFLAG) to the C-terminus of ATHB2 (utilizing the genomic sequence of ATHB2) and generate transgenic lines to be treated with shade to induce ATHB2miP-3xFLAG production. Affinity purification (anti-FLAG beads) and mass spectrometry can then identify the actual start site of ATHB2miP. This step is crucial, as the current ATHB2miP used may not be the exact sequence, and any observed phenotype could be artifacts arising from these lines.

(4) My confusion arose when analyzing the results in Figures 1E - G. The authors didn't specify whether these plants were subjected to shade treatment. What are the sequences within the second intron and third exon excluded from pATHB2control::GUS that promote transcription and translation? Have the authors examined the sequence features? This information is pivotal and related to the above question #1 because it may tell us whether the sequence feature is shared by other miP candidates.

The latter part of the manuscript focused on the functional characterization of ATHB2miP. The approaches adopted by the authors resemble those used in studying antimorphic (dominant negative) alleles. However, I have several concerns regarding the approaches and conclusions.

(5) Firstly, as mentioned in question #3, the authors did not map the actual translation initiation site of ATHB2miP. Therefore, all constructs involving ATHB2miP, such as eGFP-ATHB2miP, BD-ATHB2miP, and mCherry-ATHB2miP in Figure 2, and 35S::miP in Figures 3-5, may contain extra amino acids in the N-terminus, given that epitope tags were all added to the N terminus. These additional amino acids could potentially impact the behavior of ATHB2miP and lead to artifacts. Identifying the translation initiation site in ATHB2miP would facilitate the development of tools to disrupt ATHB2miP expression without affecting full-length ATHB2 expression. For instance, if the "CTG" before the leucine zipper domain is confirmed as the translation initiation site, mutating it to another Leu codon (e.g., TTA) could generate transgenic lines using the genomic sequence of ATHB2, including this mutation, to evaluate the impact of losing ATHB2miP on shade responses.

(6) Another concern pertains to the 35S::miP line utilized in Figures 3-5. The authors only presented results from one 35S::miP line, raising the possibility of T-DNA insertion disrupting an endogenous gene in the transgenic plant genome. It is essential to clarify how many individual T1 plants were generated and how many of them showed the same phenotype as the line used in the manuscript. Additionally, the use of the constitutive CaMV35S promoter could generate artifacts akin to neomorphic mutations. For example, the authors identified Cluster 1 genes that were only induced in 35S::miP, but not in t-athb2 or WT plants (Figure 3B); moreover, they found an overrepresentation of genes involved in root development in this cluster. This observation correlated well with the root phenotype of 35S::miP under the proximity shade (Figure 4D), in which the short-root phenotype was only observed in lines expressing 35S::miP. These data could be artifacts due to the constitutive expression of ATHB2miP in roots but didn't necessarily reflect the natural function of ATHB2miP.

(7) Furthermore, I seek clarification regarding the rationale behind employing different shade conditions, including deep shade, canopy shade, and proximity shade, and the significance of treating plants with these conditions. The results were challenging to interpret, and I have reservations about some statements made. The authors claimed that ATHB2 acts as a growth repressor in deep shade but a growth promoter in the canopy and proximity shade (Lines 366-368). However, it appears that regardless of the shade conditions, most mutant and transgenic lines were not significantly different from WT (Figure 4C). Additionally, the definition of proximity shade in this manuscript (R:FR = 0.06) differs from that in Roig-Villanova & Martinez-Garcia (Front. Plant Sci., 2016; R:FR, 0.5-0.3). Clarity on this disparity would be appreciated.

(8) In Figure 5, no statistical analyses were presented in Figure 5C. It remains unclear whether the differences observed are statistically significant. Moreover, the values appear quite similar among all three genotypes. Even if statistically significant, do these minor differences in Fe concentrations significantly impact plant physiology? Additionally, some statements related to Figure 5 do not align with the data presented. For instance, claims about longer hypocotyls in t-athb2, athb2∆, and atbh2∆LZ mutants compared to wild type under shade conditions on high iron media (lines 453-455) were not supported by the data in Figure 5D. Similarly, statements about the differences between mutants (lines 458-460) were not substantiated by the data.

Reviewer #2 (Public Review):

In this study, Kavaklıoğlu et al. investigated and presented evidence for the role of domesticated transposon protein L1TD1 in enabling its ancestral relative, L1 ORF1p, to retrotranspose in HAP1 human tumor cells. The authors provided insight into the molecular function of L1TD1 and shed some clarifying light on previous studies that showed somewhat contradictory outcomes surrounding L1TD1 expression. Here, L1TD1 expression was correlated with L1 activation in a hypomethylation-dependent manner, due to DNMT1 deletion in the HAP1 cell line. The authors then identified L1TD1-associated RNAs using RIP-Seq, which displays a disconnect between transcript and protein abundance (via Tandem Mass Tag multiplex mass spectrometry analysis). The one exception was for L1TD1 itself, which is consistent with a model in which the RNA transcripts associated with L1TD1 are not directly regulated at the translation level. Instead, the authors found the L1TD1 protein associated with L1-RNPs, and this interaction is associated with increased L1 retrotransposition, at least in the contexts of HAP1 cells. Overall, these results support a model in which L1TD1 is restrained by DNA methylation, but in the absence of this repressive mark, L1TD1 is expressed and collaborates with L1 ORF1p (either directly or through interaction with L1 RNA, which remains unclear based on current results), leads to enhances L1 retrotransposition. These results establish the feasibility of this relationship existing in vivo in either development, disease, or both.

Reviewer #2 (Public Review):

Summary:

The authors describe a "beads-on-a-string" (BOAS) immunogen, where they link, using a non-flexible glycine linker, up to eight distinct hemagglutinin (HA) head domains from circulating and non-circulating influenzas and assess their immunogenicity. They also display some of their immunogens on ferritin NP and compare the immunogenicity. They conclude that this new platform can be useful to elicit robust immune responses to multiple influenza subtypes using one immunogen and that it can also be used for other viral proteins.

Strengths:

The paper is clearly written. While the use of flexible linkers has been used many times, this particular approach (linking different HA subtypes in the same construct resembling adding beads on a string, as the authors describe their display platform) is novel and could be of interest.

Weaknesses:

The authors did not compare to individuals HA ionized as cocktails and did not compare to other mosaic NP published earlier. It is thus difficult to assess how their BOAS compare.

Other weaknesses include the rationale as to why these subtypes were chosen and also an explanation of why there are different sizes of the HA1 construct (apart from expression). Have the authors tried other lengths? Have they expressed all of them as FL HA1?

Reviewer #2 (Public Review):

Summary:

The study by Rößling et al. addresses the link between the biochemical constitution of the cell wall, in particular the methylesterification state of pectin with signalling induced by the extracellular RALF peptide. The work suggests that only in the presence of demethylesterifies pectin, RALF is able to trigger activation of its receptor FERONIA (FER).

Remarkably, the application of RALF peptides leads to rather dramatic FER-dependent changes in wall integrity and plasma membrane invaginations not observed before. Interestingly, RALF can be out-titrated from the wall by short pectin fragments. In addition, the study provides further evidence for multiple FER-dependent pathways by showing the presence of LRX proteins is not required for the pectin/RALF mediated signalling.

Strengths:

This work provides fundamental insight into a complex emerging pathway, or perhaps several pathways, linking pectin sensing, pectin structure and RALF/FER signalling. The study provides convincing evidence that pectin methylesterase activity is required for RALF sensing, indicating that the physical interaction of RALFs with the cell wall is important for signalling. Beyond that, the study documents very clearly how profoundly RALF signalling can affect cell wall integrity and membrane topology.

Weaknesses:

The genetic material used by the authors to strengthen the connection of RALF signalling and PME activity might not be as suitable as an acute inhibition of PME activity.

The PMEI3ox line generated by Peaucelle et al., 2008 is alcohol-inducible. Was expression of the PMEI induced during the experiments? As ethanol inducible systems can be rather leaky, it would not be surprising if PME activity would be reduced even without induction, but maybe this would warrant testing whether PMEI3 is actually overexpressed and/or whether PME activity is decreased. On a similar note, the PMEI5ox plants do not appear to show the typical phenotype described for this line. I personally don't think these lines are necessary to support the study. Short-term interference with PME activity (such as with EGCG) might be more meaningful than life-long PMEI overexpression, in light of the numerous feedback pathways and their associated potential secondary effects. This might also explain why EGCG leads to an increase in pH, as one would expect from decreased PME activity, while PMEI expression (caveats from above apply) apparently does not (Fig 3A-D).

At least at first sight, the observation that OGs are able to titrate RALF from pectin binding seems at odds with the idea of cooperative binding with low affinity, leading to high avidity oligomers. Perhaps the can provide a speculative conceptual model of these interactions?

I could not find a description of the OG treatment/titration experiments, but I think it would be important to understand how these were performed with respect to OG concentration, timing of the application, etc.

Reviewer #2 (Public Review):

Summary:

The manuscript by Zhang et al. explores the effect of autophagy regulator ATG6 on NPR1-mediated immunity. The authors propose that ATG6 directly interacts with NPR1 in the nucleus to increase its stability and promote NPR1-dependent immune gene expression and pathogen resistance. This novel role of ATG6 is proposed to be independent of its role in autophagy in the cytoplasm. The authors demonstrate through biochemical analysis that ATG6 interacts with NPR1 in yeast and very weakly in vitro. They further demonstrate using overexpression transgenic plants that in the presence of ATG6-mcherry the stability of NPR1-GFP and its nuclear pool is increased.

However, the overall conclusions of the study are not well supported experimentally. The significance of the findings is low because of their mostly correlational nature, and lack of consistency with earlier reports on the same protein.

Based on the integrity and quality of the data as well as the depth of analysis, it is not yet clear if ATG6 is a specific regulator of NPR1 or if it is affecting NPR1's stability indirectly, through inducing an elevation of SA levels in plants. As such, the current study demonstrates a correlation between overexpression of ATG6, SA accumulation, and NPR1 stability, however, whether and how these components work together is not yet demonstrated.

Based on the provided biochemical data, it is not yet clear if the ATG6 functions specifically through NPR1 or through its paralogs NPR3 and NPR4, which are negative regulators of immunity. It is quite possible that interaction with NPR1 (or any NPR) is not the major regulatory step in the activity of ATG6 in plant immunity. The effect of ATG6 on NPR1 could well be indirect, through a change in the SA level and redox environment of the cell during the immune response. Both SA level and redox state of the cell were reported to induce accumulation of NPR1 in the nucleus and increase in stability.

Another major issue is the poor quality of the subcellular analyses. In contradiction to previous studies, ATG6 in this study is not localized to autophagosome puncta, which suggests that the soluble localization pattern presented here does not reflect the true localization of ATG6. Even if the authors propose a novel, non-canonical nuclear localization for ATG6, they still should have detected the canonical autophagy-like localization of this protein.

Reviewer #2 (Public Review):

In this study, the authors report that both mice and human patients carrying function-disrupting mutations in the OFD1 gene exhibited ectopic brown adipose tissue formation in the malformed tongue. The OFD1 gene is located on the X-chromosome and encodes a protein product required for the formation and function of the primary cilium, which is required for cells to properly receive and activate several signaling pathways, particularly the hedgehog signaling pathway. Loss of OFD1 function causes prenatal lethality of male fetuses and mosaic disruption of tissues in females due to random inactivation of the X-chromosome carrying either the mutant or wildtype allele. Using cell type-specific gene inactivation and genetic lineage labeling, the manuscript shows that the ectopic brown adipose tissue in the mutant tongue was not derived from cranial neural crest cells (CNCCs). Additional genetic and embryological studies led to the conclusion that loss of Ofd1 function in the CNCC cells in the embryonic hypoglossal cord, via which the tongue myoblast precursor cells migrate from anterior somites to the tongue primordia, caused disruption of cell-cell interactions between the CNCCs and migrating muscle precursor cells, resulting in altered differentiation of those myoblast precursor cells into brown adipocytes. The authors provided data that disruption of Smo in a subset of CNCCs also resulted in ectopic adipose tissue formation in the tongue, indicating that this phenotype in the Ofd1 mutant mice was likely caused by disruption of hedgehog signaling in CNCCs. However, no experimental evidence is provided to support a major conclusion of the manuscript regarding altered differentiation of the tongue myoblast precursor cells into brown adipocytes in the Ofd1 mutant mice. Since it is well established that hedgehog signaling in the CNCCs is required for them to direct tongue myoblast cell migration as well as for tongue muscle differentiation/organization after the myoblasts arrived in the tongue primordia, the finding of tongue muscle defects in the Ofd1 mutant mice is not surprising. However, if proven true that disruption of Ofd1 function in CNCCs caused tongue myoblast precursor cells to alter their fate and differentiate into brown adipocytes, it would be an interesting new finding. Further identification of the signals produced by the Ofd1 mutant CNCCs for directing the cell fate switch will be a highly significant new advance in understanding the cellular and molecular mechanisms regulating tongue morphogenesis.

Reviewer #2 (Public Review):

Summary<br /> This study by Knop, et al. defines two different developmental roles for the conserved SEL-5/AAK1 protein kinase in Caenorhabditis elegans. In other organisms, AAK1 was known to promote the recycling of the Wntless sorting receptor and endocytosis of Wnt receptors. This study establishes that SEL-5 acts in two roles in C. elegans: in Wnt-producing cells, a role that promotes migration of a neuroblast termed QL.d, and in Wnt-receiving cells, a role that promotes outgrowth of the excretory cell (EXC). Before this study, SEL-5/AAK1 was thought to regulate endocytosis through phosphorylation of AP2M1 and other endocytic adaptor proteins. This study shows convincing data that the SEL-5 makes a partial contribution to AP2M1 phosphorylation, and more surprisingly, that its roles in Wnt-producing and Wnt-receiving cells of C. elegans do not require SEL-5 catalytic activity. Human AAK1 was previously suggested to be a target of drug design efforts due to its roles in neuropathic pain, viral infection, and Alzheimer's disease. The discovery that some roles for SEL-5/AAK1 are independent of catalytic activity will be of broad interest to cell biologists and biochemists.

Strengths<br /> (1) The data establishing the requirement for SEL-5 in QL.d migration and EXC outgrowth (Fig. 1 and Fig. 4) is rigorous and convincing. My assessment of the rigor is based on the following: First, the authors show that two independently derived sel-5 deletion mutations result in defects in QL.d and EXC. Second, the authors show that providing wild-type, GFP-tagged SEL-5 results in significant rescue of both phenotypes. Importantly, they use tissue-specific transgenes to show that the requirement for SEL-5 in QL.d migration is non-cell-autonomous, and the requirement for SEL-5 in EXC outgrowth is cell-autonomous (Fig. 2). For rescue experiments, they show that each tissue-specific transgene is indeed expressed strongly in the tissue of interest. This establishes the roles for SEL-5 in two different roles, in Wnt-producing and Wnt-receiving cells.

(2) The authors present three lines of convincing biochemical and genetic evidence that SEL-5 kinase catalytic activity is not important for its roles in Wnt-producing and Wnt-receiving cells.

Taking a biochemical approach, they use quantitative Westerns to assess the degree of AP2M1 phosphorylation in sel-5 mutants (Fig. 3). Their results show that AP2M1 phosphorylation is diminished, but not absent in mutants. Their results are convincing because they make use of GFP-tagged AP2M1 to probe for total and phospho-AP2M1. I note that they included uncropped Western blots in supplemental data. Furthermore, they make use of a GFP-tagged AP2M1 mutant (T160A) to confirm which residue is phosphorylated. Their results suggest that some mechanism other than AP2M1 phosphorylation may account for the sel-5 mutant phenotypes.

Taking a genetic approach, they make use of a unique allele, dpy-23(mew25), that alters the known AP2M1 phosphorylation site. They show that animals carrying this allele do not display the QL.d and EXC phenotypes (Fig. 3 and Fig. 5). Finally, in a more direct test of whether SEL-5 requires catalytic activity, they make use of GFP-tagged SEL-5 forms mutated at either the active site or the ATP-binding site of the SEL-5 kinase domain. They show that either SEL-5 mutant form successfully rescues the QL.d and EXC defects seen in sel-5 mutants (Fig. 3), suggesting that SEL-5 catalytic activity is unnecessary.

(3) The authors have produced an elegant GFP knock-in allele of the sel-5 gene, allowing analysis of expression and localization in living animals (Fig. 2).

(4) The authors make use of genetic interactions with Wnt signaling mutants to show that SEL-5 acts in a role that promotes Wnt signaling for the QL.d cell (Fig. 1) and counteracts Wnt signaling for the EXC (Fig. 5).

Weaknesses<br /> (1) Some changes to statistical analyses are needed in this study.

Fig. 1B, 1D, 2A, 3E, and 3F report the QL.d phenotype as a percentage of animals scored that were defective in migration. The methods make it clear this data is categorical rather than quantitative. Therefore, a t-test or any test designed for quantitative data is not appropriate. I suggest that the authors should investigate using a chi-squared or Fisher's exact test.

For the reasons mentioned above, the calculation of standard deviation (as shown in error bars) is also not appropriate for Fig. 1B, 1D, 2A, 3E, and 3F. Of course, it is excellent that the authors scored multiple trials. For experiments with mutants, I suggest the authors might combine these trials or show separate results of each trial. For experiments using RNAi (Fig. 1B), each trial should be plotted separately because RNAi effectiveness can vary. If there is not enough space to show multiple trials, then I would ask that a representative trial be shown in the main figure and additional trials in a supplement.

In Fig. 1, 2, 3, and 5, it is not specified whether/how p-values were adjusted for multiple tests.

(2) I felt the author's interpretation of the sel-5 mutant phenotypes in EXC, and the genetic interactions with Wnt signaling mutants, might be improved. The authors show convincing data that the sel-5 mutants display a shortened EXC outgrowth phenotype. Conversely, mutants with reduced Wnt signaling, such as the lin-17 or lin-44 mutants, displayed lengthened EXC outgrowth. The authors show that in double mutants, loss of sel-5 partially suppressed the EXC overgrowth defects of lin-17 or lin-44 mutants (Fig. 5). In my opinion, this data is consistent with a model where SEL-5 acts to inhibit Wnt signaling in EXC. An inhibitory role in a Wnt-receiving cell would be consistent with the known activity for human AAK1 in promoting negative feedback and endocytosis of LPR6. Interestingly, the authors mention in their discussion that a mutant of plr-1, which acts in the internalization of Frizzled receptors, has a shortened EXC phenotype similar to that of sel-5 mutants. These observations all seem consistent with an inhibitory role, yet the authors do not state this as their conclusion. A clarification of their interpretation is needed.

Impact/significance<br /> (1) Among researchers using C. elegans, this study provides a foundation for further investigation of the role of endocytosis, SEL-5, and the retromer, in Wnt trafficking. It is particularly useful that the authors define two different phenotypes that arise from Wnt-producing and Wnt-receiving cells.

(2) Among a broader community of cell biologists and biochemists, this study will be of interest in its finding that SEL-5/AAK1 kinase catalytic activity is unnecessary for the regulation of Wnt signaling.

Reviewer #2 (Public Review):

Urea is widely utilized in agriculture. In this study, the authors the mechanism underlying the adverse impact of urea on seed germination and seedling growth under salt stress conditions. The results show that salt stress induces a pronounced hydrolysis of urea, resulting in an elevation of cytoplasmic pH and subsequent inhibition of seed germination. These findings challenge the previous notion that ammonium accumulation is the primary cause of salt-induced inhibition of germination, thereby offering novel insights into this process.

The authors have provided well-organized genetic or biochemical evidence to support most of their conclusions.

Reviewer #2 (Public Review):

The manuscript entitled " Multimodal HLA-I genotypes regulation by human cytomegalovirus US10 and resulting surface patterning" by Gerke et al describes the biochemical analysis of US10-mediated down regulation of HLA-I molecules. The authors systemically examine the surface expression of different HLA-I alleles in cells expressing US10 and interactions of US10 with HLA-I and antigen presentation machinery. Further, studies examined genotypic and allotypic differences during expression of US10/US11 transcripts suggest a different allelic class I downregulation. In general, the authors have included data supporting the major claims. Yet, the conclusions and findings of the study only marginally advance the overall understanding of HCMV viral evasion and the mechanism of US10 function.

Strengths:<br /> The studies are well characterized and the studies utilize diverse HLA-I and HCMV viral molecules. The biochemistry is excellent and is of high quality. Importantly, the study describes HLA-I allelic specific HCMV down regulation at the cell surface and molecular levels.

Weaknesses:<br /> (1) The authors use over expressive language such as "strong binding" that does not have a quantitative value and it is relative to the specific assay with only small differences among the factors.<br /> (2) The US10 binding to the HLA-I did not correlate with class I surface levels suggesting that binding to the APC machinery (Figure 1); hence, why does the binding of US10 to the APC define its mechanism of action.<br /> (3) The innovative and significant aspects of the study are limited. The study does not delineate the US10 mechanism of action or show data in which US10-mediated MHC class I down regulation impacts adaptive or innate immune function.

Reviewer #2 (Public Review):

Summary:

The interplay between environmental factors and cognitive performance has been a focal point of neuroscientific research, with illuminance emerging as a significant variable of interest. The hypothalamus, a brain region integral to regulating circadian rhythms, sleep, and alertness, has been posited to mediate the effects of light exposure on cognitive functions. Previous studies have illuminated the role of the hypothalamus in orchestrating bodily responses to light, implicating specific neural pathways such as the orexin and histamine systems, which are crucial for maintaining wakefulness and processing environmental cues. Despite advancements in our understanding, the specific mechanisms through which varying levels of light exposure influence hypothalamic activity and, in turn, cognitive performance, remain inadequately explored. This gap in knowledge underscores the need for high-resolution investigations that can dissect the nuanced impacts of illuminance on different hypothalamic regions. Utilizing state-of-the-art 7 Tesla functional magnetic resonance imaging (fMRI), the present study aims to elucidate the differential effects of light on the hypothalamic dynamics and establish a link between regional hypothalamic activity and cognitive outcomes in healthy young adults. By shedding light on these complex interactions, this research endeavors to contribute to the foundational knowledge necessary for developing innovative therapeutic strategies aimed at enhancing cognitive function through environmental modulation.

Strengths:

(1) Considerable Sample Size and Detailed Analysis:<br /> The study leverages a robust sample size and conducts a thorough analysis of hypothalamic dynamics, which enhances the reliability and depth of the findings.

(2) Use of High-Resolution Imaging:<br /> Utilizing 7 Tesla fMRI to analyze brain activity during cognitive tasks offers high-resolution insights into the differential effects of illuminance on hypothalamic activity, showcasing the methodological rigor of the study.

(3) Novel Insights into Illuminance Effects:<br /> The manuscript reveals new understandings of how different regions of the hypothalamus respond to varying illuminance levels, contributing valuable knowledge to the field.

(4) Exploration of Potential Therapeutic Applications:<br /> Discussing the potential therapeutic applications of light modulation based on the findings suggests practical implications and future research directions.

Weaknesses:

(1) Foundation for Claims about Orexin and Histamine Systems:<br /> The manuscript needs to provide a clearer theoretical or empirical foundation for claims regarding the impact of light on the orexin and histamine systems in the abstract.

(2) Inclusion of Cortical Correlates:<br /> While focused on the hypothalamus, the manuscript may benefit from discussing the role of cortical activation in cognitive performance, suggesting an opportunity to expand the scope of the manuscript.

(3) Details of Light Exposure Control:<br /> More detailed information about how light exposure was controlled and standardized is needed to ensure the replicability and validity of the experimental conditions.

(4) Rationale Behind Different Exposure Protocols:<br /> To clarify methodological choices, the manuscript should include more in-depth reasoning behind using different protocols of light exposure for executive and emotional tasks.

Reviewer #2 (Public Review):

Summary:

In this work, using in-depth computational analysis, Bell et al. explore the diverse repertoire of type IV McrBC modification dependent restriction systems. The prototypical two-component McrBC system has been structurally and functionally characterised and is known to act as a defence by restricting phage and foreign DNA containing methylated cytosines. Here, the authors find previously unanticipated complexity and versatility of these systems and focus on detailed analysis and classification of a distinct branch, the so-called CoCoNut, named after its composition of coiled-coil structures and tandem nucleases. These CoCoNut systems are predicted to target RNA as well as DNA and to utilise defence mechanisms with some similarity to type III CRISPR-Cas systems.

Strengths:

This work is enriched with a plethora of ideas and a myriad of compelling hypotheses that now will await experimental verification. The study comes from the group that was amongst the first to describe, characterise, and classify CRISPR-Cas systems. By analogy, the findings described here can similarly promote ingenious experimental and conceptual research that could further drive technological advances. It could also instigate vigorous scientific debates that will ultimately benefit the community.

Weaknesses:

The multi-component systems described here function in the context of large oligomeric complexes similarly to the prototypical McrBC system. While the AlphaFold2 (AF2) multimer predictions are provided in this work, these are not compared with the known McrBC structures. These comparisons could have been helpful not only for providing insights into these multimeric protein systems but also for giving more sound explanations of the differences observed amongst different McrBC types.

Reviewer #2 (Public Review):

Summary:

Key findings of this research include the sequencing of the wasp's genome, identification of venom constituents and teratocytes, and examination of Trichopria drosophilae (Td)'s ecology and parasitic strategies. It was observed that Td doesn't distinguish between hosts based on age but can recognize previously parasitized hosts. The study also explored whether multiple parasitisms by Td improved outcomes, which indeed it did, possibly by increasing venom and teratocyte levels. Utilizing Drosophila ectopic expression tools, the authors functionally characterized venom components, specifically tissue inhibitors of metalloproteinases (Timps), which were found to cause delays in host development. Additionally, experiments revealed that teratocytes produce numerous proteases, aiding in the digestion of host tissues for parasite consumption. The discussion suggests that genes involved in different aspects of parasitism may arise from gene duplication and shifts in tissue expression to venom glands or teratocytes.

Strengths:

This manuscript provides an in-depth and detailed depiction of the parasitic strategies employed by Td wasps, spanning both molecular and behavioral aspects. It consolidates a significant amount of research that, in the past, might have been distributed across multiple papers. By presenting all this data in a single manuscript, it delivers a comprehensive and engaging study that could help future developments in the field of biological control against a major insect pest.

Weaknesses:

While none of the findings are particularly groundbreaking, as similar results have been reported for other parasitoid species in prior research, the integration of these results into one comprehensive overview offers valuable biological insights into an interesting new potential biocontrol species.

Reviewer #2 (Public Review):

Summary:

Pancreatic ductal adenocarcinoma is generally considered a "cold" tumor type with little T cell infiltration. This group demonstrated previously that deletion of the PIK3CA isoform of PI3K in the orthotopic pancreatic ductal adenocarcinoma KPC mouse tumor model led to the elimination of tumors by T cells. Here they performed a genome-wide gene-deletion screen in this tumor using CRISPR to determine what was required for this T cell-mediated infiltration and tumor rejection. Deletion of Pccb in the tumors, which encodes propionyl-CoA carboxylase subunit B, allowed for the outgrowth of the PIK3CA-deleted KPC tumors. This was confirmed with the specific deletion of Pccb in the tumor cells. Demonstrating a likely role in tumor progression in human patients as well, high expression of PCCB in pancreatic ductal adenocarcinoma correlated with lower patient survival. T cells still infiltrated these tumors, but had much higher expression of exhaustion markers. Blockade of PD-1 signaling allowed for the rejection of these tumors. While these are intriguing data demonstrating that loss of PCCB by pancreatic ductal adenocarcinoma is a mechanism to escape T cell immunity, the mechanism by which this occurs is not determined. In addition, there are a few issues that suggest the conclusions of the manuscript should be tempered.

Strengths:

In vivo analysis of tumor CRISPR deletion screen.

The study describes a possible novel mechanism by which a tumor maintains a "cold" microenvironment.

Weaknesses:

(1) A major issue is that it seems these data are based on the use of a single tumor cell clone with PIK3CA deleted. Therefore, there could be other changes in this clone in addition to the deletion of PIK3CA that could contribute to the phenotype.

(2) The conclusion that the change in the PCCB-deficient tumor cell line is unrelated to mitochondrial metabolic changes may be incorrect based on the data provided. While it is true that in the experiments performed, there was no statistically significant change in the oxygen consumption rate or metabolite levels, this could be due to experimental error. There is a trend in the OCR being higher in the PCCB-deficient cells, although due to a high standard deviation, the change is not statistically significant. There is also a trend for there being more aKG in this cell line, but because there were only 3 samples per cell line, there is no statistically significant difference.

(3) More data are required to make the authors' conclusion that there are myeloid changes in the PCCB-deficient tumor cells. There is only flow data from shown from one tumor of each type.

(4) The previous published study demonstrated increased MHC and CD80 expression in the PIK3CA-deficient tumors and these differences were suggested to be the reason the tumors were rejected. However, no data concerning the levels of these proteins were provided in the current manuscript.

Reviewer #2 (Public Review):

Summary:

This study was designed to test the hypothesis that motor neurons play a causal role in circuit assembly of the vestibulo-ocular reflex circuit, which is based on the retrograde model proposed by Hans Straka. This circuit consists of peripheral sensory neurons, central projection neurons, and motor neurons. The authors hypothesize that loss of extraocular motor neurons, through CRISPR/Cas9 mutagenesis of the phox2a gene, will disrupt sensory selectivity in presynaptic projection neurons if the retrograde model is correct.

Account of the major strengths and weaknesses of the methods and results:

The work presented is impressive in both breadth and depth, including the experimental paradigms. Overall, the main results were that the loss of function paradigm to eliminate extraocular motor neurons did not 1) alter the normal functional connections between peripheral sensory neurons and central projection neurons, 2) affect the position of central projection neurons in the sensorimotor circuit, or 3) significantly alter the transcriptional profiles of central projection neurons. Together, these results strongly indicate that retrograde signals from motor neurons are not required for the development of the sensorimotor architecture of the vestibulo-ocular circuit.

Appraisal of whether the authors achieved their aims, and whether the results support their conclusions:

The results of this study showed that extraocular motor neurons were not required for central projection neuron specification in the vestibulo-ocular circuit, which countered the prevailing retrograde hypothesis proposed for circuit assembly. A concern is that the results presented may be limited to this specific circuit and may not be generalizable to other circuit assemblies, even to other sensorimotor circuits.

Discussion of the likely impact of the work on the field, and the utility of the methods and data to the community:

As mentioned above, this study sheds valuable new insights into the developmental organization of the vestibulo-ocular circuit. However, different circuits likely utilize various mechanisms, extrinsic or intrinsic (or both), to establish proper functional connectivity. So, the results shown here, although begin to explain the developmental organization of the vestibulo-ocular circuit, are not likely to be generalizable to other circuits; though this remains to be seen. At a minimum, this study provides a starting point for the examination of patterning of connections in this and other sensorimotor circuits.

Reviewer #2 (Public Review):

This is an interesting study of high quality with important and novel findings. Bruguera et al. report a biochemical and structural analysis of the Tspan12 co-receptor for norrin. Major findings are that Norrin directly binds Tspan12 with high affinity (this is consistent with a report on BioRxiv: Antibody Display of cell surface receptor Tetraspanin12 and SARS-CoV-2 spike protein) and a predicted structure of Tspan12 alone or in complex with Norrin. The Norrin/Tspan12 binding interface is largely verified by mutational analysis. An interaction of the Tspan12 large extracellular loop (LEL) with Fzd4 cannot be detected and interactions of full-length Tspan12 and Fzd4 cannot be tested using nano-disc based BLI, however, Fzd4/Tspan12 heterodimers can be purified and inserted into nanodiscs when aided by split GFP tags. An analysis of a potential composite binding site of a Fzd4/Tspan12 complex is somewhat inconclusive, as no major increase in affinity is detected for the complex compared to the individual components. A caveat to this data is that affinity measurements were performed for complexes with approximately 1 molecule Tspan12 and FZD4 per nanodisc, while the composite binding site could potentially be formed only in higher order complexes, e.g., 2:2 Fzd4/Tspan12 complexes. Interestingly, the authors find that the Norrin/Tspan12 binding site and the Norrin/Lrp6 binding site partially overlap and that the Lrp6 ectodomain competes with Tspan12 for Norrin binding. This result leads the authors to propose a model according to which Tspan12 captures Norrin and then has to "hand it off" to allow for Fzd4/Lrp6 formation. By increasing the local concentration of Norrin, Tspan12 would enhance the formation of the Fzd4/Lrp5 or Fzd4/Lrp6 complex.

The experiments based on membrane proteins inserted into nano-discs and the structure prediction using AlphaFold yield important new insights into a protein complex that has critical roles in normal CNS vascular biology, retinal vascular disease, and is a target for therapeutic intervention. However, it remains unclear how Norrin would be "handed off" from Tspan12 or Tspan12/Fzd4 complexes to Fzd4/Lrp6 complexes, as the relatively high affinity of Norrin to Fzd4/Tspan12 dimers likely does not favor the "handing off" to Fzd4/Lrp6 complexes.

Areas that would benefit from further experiments, or a discussion, include:

- The authors test a potential composite binding site of Fzd4/Tspan12 heterodimers for norrin using nanodiscs that contain on average about 1 molecule Fzd4 and 1 molecule Tspan12. The Fzd4/Tspan12 heterodimer is co-inserted into the nanodiscs supported by split-GFP tags on Fzd4 and Tspan12. The authors find no major increase in affinity, although they find changes to the Hill slope, reflecting better binding of norrin at low norrin concentrations. In 293F cells overexpressing Fzd4 and Tspan12 (which may result in a different stoichiometry) they find more pronounced effects of norrin binding to Fzd4/Tspan12. This raises the possibility that the formation of a composite binding requires Fzd4/Tspan12 complexes of higher order, for example, 2:2 Fzd4/Tspan12 complexes, where the composite binding site may involve residues of each Fzd4 and Tspan12 molecule in the complex. This could be tested in nanodiscs in which Fzd4 and Tspan12 are inserted at higher concentrations or using Fzd4 and Tspan12 that contain additional tags for oligomerization.

- While Tspan12 LEL does not bind to Fzd4, the successful reconstitution of GFP from Tspan12 and Fzd4 tagged with split GFP components provides evidence for Fzd4/Tspan12 complex formation. As a negative control, e.g., Fzd5, or Tspan11 with split GFP tags (Fzd5/Tspan12 or Fzd4/Tspan11) would clarify if FZD4/Tspan12 heterodimers are an artefact of the split GFP system.

- Fzd4/Tspan12 heterodimers stabilized by split GFP may be locked into an unfavorable orientation that does not allow for the formation of a composite binding site of FZD4 and Tspan12, this is another caveat for the interpretation that Fzd4/Tspan12 do not form a composite binding site. This is not discussed.

- Mutations that affect the affinity of norrin/fzd4 are not used to further test if Fzd4 and Tspan12 form a composite binding site. Norrin R41E or Fzd4 M105V were previously reported to reduce norrin/frizzled4 interactions and signaling, and both interaction and signaling were restored by Tspan12 (Lai et al. 2017). Whether a Fzd4/Tspan12 heterodimer has increased affinity for Norrin R41E was not tested. Similarly, affinity of FZD4 M105V vs a Fzd4 M105V/Tspan12 heterodimer were not tested.

- An important conclusion of the study is that Tspan12 or Lrp6 binding to Norrin is mutually exclusive. This could be corroborated by an experiment in which LRP5/6 is inserted into nanodiscs for BLI binding tests with Norrin, or Tspan12 LEL, or a combination of both. Soluble LRP6 may remove norrin from equilibrium binding/unbinding to Tspan12, therefore presenting LRP6 in a non-soluble form may yield different results.

- The authors use LRP6 instead of LRP5 for their experiments. Tspan12 is less effective in increasing the Norrin/Fzd4/Lrp6 signaling amplitude compared to Norrin/Fzd4/Lrp5 signaling, and human genetic evidence (FEVR) implicates LRP5, not LRP6, in Norrin/Frizzled4 signaling. The authors find that Norrin binding to LRP6 and Tspan12 is mutually exclusive, however this may not be the case for Lrp5.

- The biochemical data are largely not correlated with functional data. The authors suggest that the Norrin R115L FEVR mutation could be due to reduced norrin binding to tspan12, but do not test if Tspan12-mediated enhancement of the norrin signaling amplitude is reduced by the R115L mutation. Similarly, the impressive restoration of binding by charge reversal mutations in site 3 is not corroborated in signaling assays.

Reviewer #2 (Public Review):

Summary:

Karim et al investigated the regulation of ACSS2 by SIRT2. The authors identified a previously undescribed acetylation that they then show is important for the regulation and stability of ACSS2 in cells. The authors show that ACSS2 ubiquitination and degradation by the proteasome is regulated by SIRT2-mediated deacetylation of ACSS2 and that stabilizing ACSS2 by blocking SIRT2 can alter lipid accumulation in adipocytes.

Strengths:

Identification of a novel acetylation site on ACSS2 that regulates its protein stability and that has consequences on its activity in adipocytes. Multiple standard approaches were used to manipulate the expression and function of SIRT2 and ACSS2 (i.e., overexpression, knockdown, inhibitors).

Weaknesses:

The authors do not show direct deacetylation of ACSS2 by SIRT2 in an in vitro biochemical assay.

It would have been nice to have included a bona-fide SIRT2 target as a control throughout the study.

Throughout the manuscript, normalizing the data to 1 and then comparing the fold-change using a t-test is not the best statistical approach in that situation since every normalized value for control is 1 with zero standard deviation. The authors should consider an alternative statistical approach.

Though not necessary, using 13C-acetate or D3-acetate tracing would be better for understanding the impact of acetylation on the activity of ACSS2 and its impact on lipogenesis.

Did the authors also consider investigating SIRT1 in their assays? SIRT1 activates ACSS2 while SIRT2 leads to degradation of ACSS2. They should at least discuss these seemingly opposing roles of SIRT1 and SIRT2 in the regulation of ACSS2 and acetate metabolism in more depth, particularly as it concerns situations (i.e., diseases, pathologies) where either SIRT1, SIRT2, or both sirtuins, are active. This would enhance the significance of the findings to the broader research community.

In Figure 3, the authors should consider immunoblotting for endogenous ACSS2 throughout the differentiation and lipogenesis study since the total ACSS2 levels is the crucial aspect to affecting acetate-dependent promotion of lipogenesis in adipocytes, and to confirm TM-dependent stabilization of ACSS2 in that assay.

Do the authors have any data proving the K271 mutants of ACSS2 are still functional? Or that K271 ACSS2 protein is folded correctly?

Reviewer #2 (Public Review):

Summary:

In this study by Bendzunas et al, the authors show that the formation of intra-molecular disulfide bonds involving a pair of Cys residues near the catalytic HRD motif and a highly conserved T-Loop Cys with a BRSK-specific Cys at an unusual CPE motif at the end of the activation segment function as repressive regulatory mechanisms in BSK1 and 2. They observed that mutation of the CPE-Cys only, contrary to the double mutation of the pair, increases catalytic activity in vitro and drives phosphorylation of the BRSK substrate Tau in cells. Molecular modeling and molecular dynamics simulations indicate that oxidation of the CPE-Cys destabilizes a conserved salt bridge network critical for allosteric activation. The occurrence of spatially proximal Cys amino acids in diverse Ser/Thr protein kinase families suggests that disulfide-mediated control of catalytic activity may be a prevalent mechanism for regulation within the broader AMPK family. Understanding the molecular mechanisms underlying kinase regulation by redox-active Cys residues is fundamental as it appears to be widespread in signaling proteins and provides new opportunities to develop specific covalent compounds for the targeted modulation of protein kinases.

The authors demonstrate that intramolecular cysteine disulfide bonding between conserved cysteines can function as a repressing mechanism as indicated by the effect of DTT and the consequent increase in activity by BSK-1 and -2 (WT). The cause-effect relationship of why mutation of the CPE-Cys only increases catalytic activity in vitro and drives phosphorylation of the BRSK substrate Tau in cells is not clear to me. The explanation given by the authors based on molecular modeling and molecular dynamics simulations is that oxidation of the CPE-Cys (that will favor disulfide bonding) destabilizes a conserved salt bridge network critical for allosteric activation. However, no functional evidence of the impact of the salt-bridge network is provided. If you mutated the two main Cys-pairs (aE-CHRD and A-loop T+2-CPE) you lose the effect of DTT, as the disulfide pairs cannot be formed, hence no repression mechanisms take place, however when looking at individual residues I do not understand why mutating the CPE only results in the opposite effect unless it is independent of its connection with the T+2residue on the A-loop.

Strengths:

This is an important and interesting study providing new knowledge in the protein kinase field with important therapeutic implications for the rationale design and development of next-generation inhibitors.

Comments on revised version:

I have one remark related to question number 5 (my question was not clear enough). I meant if the authors did look at the functional relevance of the residues implicated in the identified salt-bridge network/tethers. What happens to the proteins functionally when you mutate those residues? (represented on Fig. 8).

Otherwise, the authors have satisfactorily addressed my concerns.

Reviewer #2 (Public Review):

Summary:

This manuscript describes the analysis of blood transcriptomic data from patients with TB meningitis, with and without HIV infection, with some comparison to those of patients with pulmonary tuberculosis and healthy volunteers. The objectives were to describe the comparative biological differences represented by the blood transcriptome in TBM associated with HIV co-infection or survival/mortality outcomes, and to identify a blood transcriptional signature to predict these outcomes. The authors report an association between mortality and increased levels of acute inflammation and neutrophil activation, but decreased levels of adaptive immunity and T/B cell activation. They propose a 4-gene prognostic signature to predict mortality.

Strengths:

Biological evaluations of blood transcriptomes in TB meningitis and their relationship to outcomes have not been extensively reported previously.<br /> The size of the data set is a major strength and is likely to be used extensively for secondary analyses in this field of research.<br /> The addition of a new validation cohort to evaluate the generalisability of their prognostic model in the revised manuscript is welcome.

Weaknesses:

The bioinformatic analysis is limited to a descriptive narrative of gene-level functional annotations curated in GO and KEGG databases. This analysis cannot be used to make causal inferences. In addition the functional annotations are limited to 'high-level' terms that fail to define the biology very precisely. As a result, the conclusions about the immunopathogenesis of TBM are not adequately substantiated.<br /> The lack of AUROC confidence intervals and direct comparison to the reference prognostic model in the validation cohort undermines confidence in their conclusion that their new prognostic model combing gene expression data and clinical variables performs better than the reference model.

Reviewer #2 (Public Review):

Summary:

The endocannabinoid system (ECS) regulates many critical functions, including reproductive function. Recent evidence indicates that dysregulated ECS contributes to endometriosis pathophysiology and the microenvironment. Therefore, the authors further examined the dysregulated ECS and its mechanisms in endometriosis lesion establishment and progression using two different endometrial sources of mouse models of endometriosis with CNR1 and CNR2 knockout mice. The authors presented differential gene expressions and altered pathways, especially those related to the adaptive immune response in CNR1 and CNR2 ko lesions. Interstingly, the T-cell population was dramatically reduced in the peritoneal cavity lacking CNR2, and the loss of proliferative activity of CD4+ T helper cells. Imaging mass cytometry analysis provided spatial profiling of cell populations and potential relationships among immune cells and other cell types. This study provided fundamental knowledge of the endocannabinoid system in endometriosis pathophysiology.

Strengths:

Dysregulated ECS and its mechanisms in endometriosis pathogenesis were assessed using two different endometrial sources of mouse models of endometriosis with CNR1 and CNR2 knockout mice. Not only endometriotic lesions, but also peritoneal exudate (and splenic) cells were analyzed to understand the specific local disease environment under the dysregulated ECS.

Providing the results of transcriptional profiles and pathways, immune cell profiles, and spatial profiles of cell populations support altered immune cell population and their disrupted functions in endometriosis pathogenesis via dysregulation of ECS.

In line 386: Role of CNR2 in T cells. The finding that nearly absent CD3+ T cells in the peritoneal cavity of CNR2 ko mice is intriguing.

The interpretation of the results is well-described in the Discussion.

Weaknesses:

The study was terminated and characterized 7 days after EM induction surgery without the details for selecting the time point to perform the experiments.

The authors also mentioned that altered eutopic endometrium contributes to the establishment and progression of endometriosis. This reviewer agrees with lines 324-325. If so, DEGs are likely identified between eutopic endometrium (with/without endometriosis lesion induction) and ectopic lesions. It would be nice to see the data (even though using publicly available data sets).

Figure 7 CDEF. The results of the statistical analyses and analyzed sample numbers should be added. Lines 444-450 cannot be reviewed without them.

This reviewer agrees with lines 498-500. In contrast, retrograded menstrual debris is not decidualized. The section could be modified to avoid misunderstanding.

Reviewer #2 (Public Review):

Liu et al., by focusing on the regulation of G protein-signaling 10 (RGS10), reported that RGS10 expression was significantly lower in patients with breast cancer, compared with normal adjacent tissue. Genetic inhibition of RGS10 caused epithelial-mesenchymal transition, and enhanced cell proliferation, migration, and invasion, respectively. These results suggest an inhibitory role of RGS10 in tumor metastasis. Furthermore, bioinformatic analyses determined signaling cascades for RGS10-mediated breast cancer distant metastasis. More importantly, both in vitro and in vivo studies evidenced that alteration of RGS10 expression by modulating its upstream regulator miR-539-5p affects breast cancer metastasis. Altogether, these findings provide insight into the pathogenesis of breast tumors and hence identify potential therapeutic targets in breast cancer.

The conclusions of this study are mostly well supported by data. However, there is a weakness in the study that needs to be clarified.

In Figure 2A, although some references supported that SKBR3 and MCF-7 possess poorly aggressive and less invasive abilities, examining only RGS10 expression in those cells, it could not be concluded that 'RGS10 acts as a tumor suppressor in breast cancer'. It would be better to introduce a horizontal comparison of the invasive ability of these 3 types of cells using an invasion assay.

Reviewer #2 (Public Review):

Summary:

Weinberg et al. show that spike LCB minibinders can be used as the extracellular domain for SynNotch, SNIPR, and CAR. They evaluated their designs against cells expressing the target proteins and live virus.

Strengths:

This is a good fundamental demonstration of alternative use of the minibinder. The results are unsurprising but robust and solid in most cases.

Weaknesses:

The manuscript would benefit from better descriptions of the study's novelty. Given that LCB previously worked in SynNotch, what unexpected finding was uncovered by this study? It is well known that the extracellular domain of CAR is amendable to different types of binding domains (e.g., scFv, nanobody, DARPin, natural ligands). So, it is not surprising that a minibinder also works with CAR. We don't know if the minibinders are more or less likely to be compatible with CAR or SNIPR.

The demonstrations are all done using just 1 minibinder. It is hard to conclude that minibinders, as a unique class of protein binders, are generalizable in different contexts. All it can conclude is that this specific Spike minibinder can be used in synNotch, SNIPR, and CAR. The LCB3 minibinder seems to be much weaker.

The sensing of live viruses is interesting, but the output is very weak. It is difficult to imagine a utility for such a weak response.

Reviewer #2 (Public Review):

Summary:

In this new paper, the authors used biochemical, structural, and biophysical methods to elucidate the mechanisms by which IP4, the PIP3 headgroup, can induce an autoinhibit form of P-Rex1 and propose a model of how PIP3 can trigger long-range conformational changes of P-Rex1 to relieve this autoinhibition. The main findings of this study are that a new P-Rex1 autoinhibition is driven by an IP4-induced binding of the PH domain to the DH domain active site and that this autoinhibit form stabilized by two key interactions between DEP1 and DH and between PH and IP4P 4-helix bundle (4HB) subdomain. Moreover, they found that the binding of phospholipid PIP3 to the PH domain can disrupt these interactions to relieve P-Rex1 autoinhibition.

Strengths:

The study provides good evidence that binding of IP4 to the P-Rex1 PH domain can make the two long-range interactions between the catalytic DH domain and the first DEP domain, and between the PH domain and the C-terminal IP4P 4HB subdomain that generate a novel P-Rex1 autoinhibition mechanism. This valuable finding adds an extra layer of P-Rex1 regulation (perhaps in the cytoplasm) to the synergistic activation by phospholipid PIP3 and the heterotrimeric Gβγ subunits at the plasma membrane. Overall, this manuscript's goal sounds interesting, the experimental data were carried out carefully and reliably.

Weakness:

The set of experiments with the disulfide bond S235C/M244C caused a bit of confusion for interpretation, it should be moved into the supplement, and the text and Figure 4 were altered accordingly.

Reviewer #2 (Public Review):

This manuscript describes an adaptive laboratory evolution (ALE) study with a previously constructed genome-reduced E. coli. The growth performance of the end-point lineages evolved in M63 medium was comparable to the full-length wild-type level at lower cell densities.

Subsequent mutation profiling and RNA-Seq analysis revealed many changes on the genome and transcriptomes of the evolved lineages. The authors did a great deal on analyzing the patterns of evolutionary changes between independent lineages, such as the chromosomal periodicity of transcriptomes, pathway enrichment analysis, weight gene co-expression analysis, and so on. They observed a striking diversity in the molecular characteristics amongst the evolved lineages, which, as they suggest, reflect divergent evolutionary strategies adopted by the genome-reduced organism.

As for the overall quality of the manuscript, I am rather torn. The manuscript leans towards elaborating observed findings, rather than explaining their biological significance. For this reason, readers are left with more questions than answers. For example, fitness assay on reconstituted (single and combinatorial) mutants was not performed, nor any supporting evidence on the proposed contributions of each mutants provided. This leaves the nature of mutations - be them beneficial, neutral or deleterious, the presence of epistatic interactions, and the magnitude of fitness contribution, largely elusive. Also, it is difficult to tell whether the RNA-Seq analysis in this study managed to draw biologically meaningful conclusions, or instill insight into the nature of genome-reduced bacteria. The analysis primarily highlighted the differences in transcriptome profiles among each lineage based on metrics such as 'DEG counts' and the 'GO enrichment'. However, I could not see any specific implications regarding the biology of the evolved minimal genome drawn. In their concluding remark, 'Multiple evolutionary paths for the reduced genome to improve growth fitness were likely all roads leading to Rome,' the authors observed the first half of the sentence, but the distinctive characteristics of 'all roads' or 'evolutionary paths', which I think should have been the key aspect in this investigation, remains elusive.

Comments on revised version:

I appreciate the author's responses. They responded to most of the comments, but I still think that there is room for improvement. Please refer to the following comments. Quoted below are the author's responses.

"We agree that our study leaned towards elaborating observed findings rather than explaining the detailed biological mechanisms."<br /> - Comment: I doubt if there are scientific merits in merely elaborating observed findings. The conclusion of this study suggests that evolutionary paths in reduced genomes are highly diverse. But if you think about the nature of adaptive evolution, which relies upon the spontaneous mutation event followed by selection, certain degree of divergence is always expected. The problem with current experimental setting is that there are no ways to quantitively assess whether the degree of evolutionary divergence increases as the function of genome reduction, as the authors claimed. In addition, this notion is in direct contradiction to the prediction that genome reduction constraints evolution by reducing the number of solution space. It is more logical to think and predict that genome reduction would, in turn, lead to the loss of evolutionary divergence. We are also interested to know whether solution space to the optimization problem altered in response to the genome reduction. In this regard, a control ALE experiment on non-reduced wild-type seems to be a mandatory experimental control. I highly suggest that authors present a control experiment, as it was done for "JCVI syn3.0B vs non-minimal M. mycoides" (doi: 10.1038/s41586 023 06288 x) and "E. coli eMS57 vs MG1655" (doi: 10.1038/s41467 019 08888 6).<br /> "We focused on the genome wide biological features rather than the specific biological functions."<br /> - Comment: The 'biological features' delivered in current manuscript does not give insight as to which genomic changes translated into strain fitness improvement. Rather than explaining the genotype-phenotype relationships and/or the mechanistic basis of fitness improvement, authors merely elaborated on the observed phenotypes. I question the scientific merits of such 'findings'.<br /> "Although the reduced growth rate caused by genome reduction could be recovered by experimental evolution, it remains unclear whether such an evolutionary improvement in growth fitness was a general feature of the reduced genome and how the genome wide changes occurred to match the growth fitness increase."<br /> - Comment: This response is very confusing to understand. "it remains unclear whether such an evolutionary improvement in growth fitness was a general feature of the reduced genome" - what aspects remain unclear?? What assumption led the authors to believe that reduced genome's fitness cannot be evolutionarily improved?<br /> - Comment: "and how the genome wide changes occurred to match the growth fitness increase" - this is exactly the aspect that authors should deliver, instead of just elaborating the observed findings. Why don't authors select one or two fastest-growing (or the fittest) lineages and specifically analyze underlying adaptive changes (i.e. genotype-phenotype relationships)?

Reviewer #2 (Public Review):

Summary:

In this manuscript, the authors provide evidence that posttranslational modification of synuclein by N-acetylation increases clustering of synaptic vesicles in vitro. When using liposomes the authors found that while clustering is enhanced by the presence of either lysophosphatidylcholine (LPC) or phosphatidylcholine in the membrane, N-acetylation enhanced clustering only in the presence of LPC. Enhancement of binding was also observed when LPC micelles were used, which was corroborated by increased intra/intermolecular cross-linking of N-acetylated synuclein in the presence of LPC.

Strengths:

It is known for many years that synuclein binds to synaptic vesicles but the physiological role of this interaction is still debated. The strength of this manuscript is clearly in the structural characterization of the interaction of synuclein and lipids (involving NMR-spectroscopy) showing that the N-terminal 100 residues of synuclein are involved in LPC-interaction, and the demonstration that N-acetylation enhances the interaction between synuclein and LPC.

Weaknesses:

Lysophosphatides form detergent-like micelles that destabilize membranes, with their steady-state concentrations in native membranes being low, questioning the significance of the findings. Oddly, no difference in binding between the N-acetylated and unmodified form was observed when the acidic phospholipid phosphatidylserine was included. It remains unclear to which extent binding to LPC is physiologically relevant, particularly in the light of recent reports from other laboratories showing that synuclein may interact with liquid-liquid phases of synapsin I that were reported to cause vesicle clustering.

Reviewer #2 (Public Review):

In this study, Badugu et al investigate the Rev7 roles in regulating the Mre11-Rad50-Xrs2 complex and in the metabolism of G4 structures. The authors also try to make a conclusion that REV7 can regulate the DSB repair choice between homologous recombination and non-homologous end joining.

The major observations of this study are:

(1) Rev7 interacts with the individual components of the MRX complex in a two-hybrid assay and in a protein-protein interaction assay (microscale thermophoresisi) in vitro.<br /> (2) Modeling using AlphaFold-Multimier also indicated that Rev7 can interact with Mre11 and Rad50.<br /> (3) Using a two-hybrid assay, a 42 C terminal domain in Rev7 responsible for the interaction with MRX was identified.<br /> (4) Rev7 inhibits Mre11 nuclease and Rad50 ATPase activities in vitro.<br /> (5) Rev 7 promotes NHEJ in plasmid cutting/relegation assay.<br /> (6) Rev7 inhibits recombination between chromosomal ura3-1 allele and plasmid ura3 allele containing G4 structure.<br /> (7) Using an assay developed in V. Zakian's lab, it was found that rev7 mutants grow poorly when both G4 is present in the genome and yeast are treated with HU.<br /> (8) In vitro, purified Rev7 binds to G4-containing substrates.

In general, a lot of experiments have been conducted, but the major conclusion about the role of Rev7 in regulating the choice between HR and NHEJ is not justified.

(1) Two stories that do not overlap (regulation of MRX by Rev7 and Rev7's role in G4 metabolism) are brought under one umbrella in this work. There is no connection unless the authors demonstrate that Rev7 inhibits the cleavage of G4 structures by the MRX complex.

(2) The authors cannot conclude based on the recombination assay between G4-containing 2-micron plasmid and chromosomal ura3-1 that Rev7" completely abolishes DSB-induced HR". First of all, there is no evidence that DSBs are formed at G4. Why is there no induction of recombination when cells are treated with HU? Second, as the authors showed, Rev7 binds to G4, therefore it is not clear if the observed effects are the result of Rev7 interaction with G4 or its impact on HR. The established HO-based assays where the speed of resection can be monitored (e.g., Mimitou and Symington, 2010) have to be used to justify the conclusion that Rev7 inhibits MRX nuclease activity in vivo.

Reviewer #2 (Public Review):

Summary:

In their manuscript "Released Bacterial ATP Shapes Local and Systemic Inflammation during Abdominal Sepsis", Daniel Spari et al. explored the dual role of ATP in exacerbating sepsis, revealing that ATP from both host and bacteria significantly impacts immune responses and disease progression.

Strengths:<br /> The study meticulously examines the complex relationship between ATP release and bacterial growth, membrane integrity, and how bacterial ATP potentially dampens inflammatory responses, thereby impairing survival in sepsis models. Additionally, this compelling paper implies a concept that bacterial OMVs act as vehicles for the systemic distribution of ATP, influencing neutrophil activity and exacerbating sepsis severity.

Weaknesses:

(1) The researchers extracted and cultivated abdominal fluid on LB agar plates, then randomly picked 25 colonies for analysis. However, they did not conduct 16S rRNA gene amplicon sequencing on the fluid itself. It is worth noting that the bacterial species present may vary depending on the individual patients. It would be beneficial if the authors could specify whether they've verified the existence of unculturable species capable of secreting high levels of Extracellular ATP.

(2) Do mice lacking commensal bacteria show a lack of extracellular ATP following cecal ligation puncture?

(3) The authors isolated various bacteria from abdominal fluid, encompassing both Gram-negative and Gram-positive types. Nevertheless, their emphasis appeared to be primarily on the Gram-negative E. coli. It would be beneficial to ascertain whether the mechanisms of Extracellular ATP release differ between Gram-positive and Gram-negative bacteria. This is particularly relevant given that the Gram-positive bacterium E. faecalis, also isolated from the abdominal fluid, is recognized for its propensity to release substantial amounts of Extracellular ATP.

(4) The authors observed changes in the levels of LPM, SPM, and neutrophils in vivo. However, it remains uncertain whether the proliferation or migration of these cells is modulated or inhibited by ATP receptors like P2Y receptors. This aspect requires further investigation to establish a convincing connection.

(5) Additionally, is it possible that the observed in vivo changes could be triggered by bacterial components other than Extracellular ATP? In this research field, a comprehensive collection of inhibitors is available, so it is desirable to utilize them to demonstrate clearer results.

(6) Have the authors considered the role of host-derived Extracellular ATP in the context of inflammation?

(7) The authors mention that Extracellular ATP is rapidly hydrolyzed by ectonucleotases in vivo. Are the changes of immune cells within the peritoneal cavity caused by Extracellular ATP released from bacterial death or by OMVs?

(8) In the manuscript, the sample size (n) for the data consistently remains at 2. I would suggest expanding the sample size to enhance the robustness and rigor of the results.

Reviewer #2 (Public Review):

Summary:

This work has an important and ambitious goal: understanding the effects of drugs, in this case antimicrobial molecules, from a holistic perspective. This means that the effect of drugs on a group of genes and whole metabolic pathways is unveiled, rather than its immediate effect on a protein target only. To achieve this goal the authors successfully implement the PISA-Express method (Protein Integral Solubility Alteration), using combined transcriptomics, proteomics, and drug-induced changes in protein stability to retrieve a large number of genes and proteins affected by the used compounds. The compounds used in the study (compound IVa, IVb, IVj, and IVk) were all derived from the precursors compound IV, they are effective against Helicobacter pylori, and their mode of action on clusters of genes and proteins has been compared to the one of the known pylori drug metronidazole (MNZ). Due to this comparison, and confirmed by the diversity of responses induced by these very similar compounds, it can be understood that the approach used is reliable and very informative. Notably, although all compound IV derivatives were designed to target pylori Flavodoxin (Fld), only one showed a statistically significant shift of Fld solubility (compound IVj, FIG S11). For most other compounds, instead, the involvement of other possible targets affecting diverse metabolic pathways was also observed, notably concerning a series of genes with other important functions: CagA (virulence factor), FtsY/FtsA (cell division), AtpD (ATP-synthase complex), the essential GTPase ObgE, Tig (protein export), as well as other proteins involved in ribosomal synthesis, chemotaxis/motility and DNA replication/repairs. Finally, for all tested molecules, in vivo functional data have been collected that parallel the omics predictions, comforting them and showing that compound IV derivatives differently affect cellular generation of reactive oxygen species (ROS), oxygen consumption rates (OCR), DNA damage, and ATP synthesis.

Strengths:

The approach used is very potent in retrieving the effects of chemically active molecules (in this case antimicrobial ones) on whole cells, evidencing protein and gene networks that are involved in cell sensitivity to the studied molecules. The choice of these compounds against H. pylori is perfect, showcasing how different the real biological response is, compared to the hypothetical one. In fact, although all molecules were retrieved based on their activity on Fld, the authors unambiguously show that large unexpected gene clusters may, and in fact are, affected by these compounds, and each of them in different manners.

Impact:

The present work is the first report relying on PISA-Express performed on living bacterial cells. Because of its findings, this work will certainly have a high impact on the way we design research to develop effective drugs, allowing us to understand the fine effects of a drug on gene clusters, drive molecule design towards specific metabolic pathways, and eventually better plan the combination of multiple active molecules for drug formulation. Beyond this, however, we expect this article to impact other related and unrelated fields of research as well. The same holistic approaches might also allow gaining deep, and sometimes unexpected, insight into the cellular targets involved in drug side effects, drug resistance, toxicity, and cellular adaptation, in fields beyond the medicinal one, such as cellular biology and environmental studies on pollutants.

Reviewer #2 (Public Review):

The manuscript by Ma et al. reports the identification of three unrelated people who are heterozygous for de novo missense variants in PLCG1, which encodes phospholipase C-gamma 1, a key signaling protein. These individuals present with partially overlapping phenotypes including hearing loss, ocular pathology, cardiac defects, abnormal brain imaging results, and immune defects. None of the patients present with all of the above phenotypes. PLCG1 has also been implicated as a possible driver for cell proliferation in cancer.

The three missense variants found in the patients result in the following amino acid substitutions: His380Arg, Asp1019Gly, and Asp1165Gly. PLCG1 (and the closely related PLCG2) have a single Drosophila ortholog called small wing (sl). sl-null flies are viable but have small wings with ectopic wing veins and supernumerary photoreceptors in the eye. As all three amino acids affected in the patients are conserved in the fly protein, in this work Ma et al. tested whether they are pathogenic by expressing either reference or patient variant fly or human genes in Drosophila and determining the phenotypes produced by doing so.

Expression in Drosophila of the variant forms of PLCG1 found in these three patients is toxic; highly so for Asp1019Gly and Asp1165Gly, much more modestly for His380Arg. Another variant, Asp1165His which was identified in lymphoma samples and shown by others to be hyperactive, was also found to be toxic in the Drosophila assays. However, a final variant, Ser1021Phe, identified by others in an individual with severe immune dysregulation, produced no phenotype upon expression in flies.

Based on these results, the authors conclude that the PLCG1 variants found in patients are pathogenic, producing gain-of-function phenotypes through hyperactivity. In my view, the data supporting this conclusion are robust, despite the lack of a detectable phenotype with Ser1021Phe, and I have no concerns about the core experiments that comprise the paper.

Figure 6, the last in the paper, provides information about PLCG1 structure and how the different variants would affect it. It shows that the His380, Asp1019, and Asp1165 all lie within catalytic domains or intramolecular interfaces and that variants in the latter two affect residues essential for autoinhibition. It also shows that Ser1021 falls outside the key interface occupied by Asp1019, but more could have been said about the potential effects of Ser1021Phe.

Overall, I believe the authors fully achieved the aims of their study. The work will have a substantial impact because it reports the identification of novel disease-linked genes, and because it further demonstrates the high value of the Drosophila model for finding and understanding gene-disease linkages.

Reviewer #2 (Public Review):

Summary:

Casas-Tinto et al., provide new insight into glial plasticity using a crush injury paradigm in the ventral nerve cord (VNC) of adult Drosophila. The authors find that both astrocyte-like glia (ALG) and ensheating glia (EG) divide under homeostatic conditions in the adult VNC and identify ALG as the glial population that specifically ramps up proliferation in response to injury, whereas the number of EGs decreases following the insult. Using lineage-tracing tools, the authors interestingly observe the interconversion of glial subtypes, especially of EGs into ALGs, which occurs independent of injury and is dependent on the availability of the transcription factor Prospero in EGs, adding to the plasticity observed in the system. Finally, when tracing the progeny of differentiated glia, Casas-Tinto and colleagues detect cells of neuronal identity and provide evidence that such glia-derived neurogenesis is specifically favored following ventral nerve cord injury, which puts forward a remarkable way in which glia can respond to neuronal damage.

Strengths:

This study highlights a new facet of adult nervous system plasticity at the level of the ventral nerve cord, supporting the view that proliferative capacity is maintained in the mature CNS and stimulated upon injury.

The injury paradigm is well chosen, as the organization of the neuromeres allows specific targeting of one segment, compared to the remaining intact, and with the potential to later link observed plasticity to behavior such as locomotion.

Numerous experiments have been carried out in 7-day-old flies, showing that the observed plasticity is not due to residual developmental remodeling or a still immature VNC.

By elegantly combining different genetic tools, the authors show glial divisions with mitotic-dependent tracing and find that the number of generated glia is refined by apoptosis later on.

The work identifies Prospero in glia as an important coordinator of glial cell fate, from development to the adult context, which draws further attention to the upstream regulatory mechanisms.

Weaknesses:

Although the authors do use a variety of methods to show glial proliferation, the EdU data (Figure 1B) could be more informative (Figure 1B) by displaying images of non-injured animals and providing quantifications or the mention of these numbers based on results previously acquired in the system.

The experiments relying on the FUCCI cell cycle reporter suggested considerable baseline proliferation for EGs and ALGs, but when using an independent method (Twin Spot MARCM), mitotic marking was only detected for ALGs. This discrepancy could be addressed by assessing the co-localization of the different glia subsets using the identified driver lines with mitotic markers such as PH3.

The data in Figure 1C would be more convincing in combination with images of the FUCCI Reporter as it can provide further information on the location and proportion of glia that enter the cell cycle versus the fraction that remains quiescent.

The analyses of inter-glia conversion in Figure 3 are complicated by the fact that Prospero RNAi is both used to suppress EG - to ALG conversion and as a marker to establish ALG nature. Clarifications if the GFP+ cells still expressed Pros or were classified as NP-like GFP cells are required here.

The conclusion that ALG and EG glial cells can give rise to cells of neuronal lineage is based on glial lineage information (GFP+ cells from glial G-trace) and staining for the neuronal marker Elav. The use of other neuronal markers apart from Elav or morphological features would provide a more compelling case that GFP+ cells are mature neurons.

Although the text discusses in which contexts, glial plasticity is observed or increased upon injury, the figures are less clear regarding this aspect. A more systematic comparison of injured VNCs versus homeostatic conditions, combined with clear labelling of the injury area would facilitate the understanding of the panels.

Context/Discussion

The study finds that glia in the ventral cord of flies have latent neurogenic potential. Such observations have not been made regarding glia in the fly brain, where injury is reported to drive glial divisions or the proliferation of undifferentiated progenitor cells with neurogenic potential.

Discussing this different strategy for cell replacement adopted by glia in the VNC and pointing out differences to other modes seems fascinating. Highlighting differences in the<br /> the reactiveness of glia in the VNC compared to the brain also seems highly relevant as they may point to different properties to repair damage.

Based on the assays employed, the study points to a significant amount of glial "identity" changes or interconversions, which is surprising under homeostatic conditions. The significance of this "baseline" plasticity remains undiscussed, although glia unarguably show extensive adaptations during nervous system development.

It would be interesting to know if the "interconversion" of glia is determined by the needs in the tissue or would shift in the context of selective ablation/suppression of a glial type.

This one was wrong in the reference transcript too. It should be Phycoerythrin.

Reviewer #2 (Public Review):

Summary:

The authors provide strong evidence that bacteria, such as E. coli, compete with tumor cells for iron resources and consequently reduce tumor growth. When sequestration between LCN2 and bacterobactin is blocked by upregulating CDG(DGC-E. coli) or salmochelin(IroA-E.coli), E. coli increase iron uptake from the tumor microenvironment (TME) and restrict iron availability for tumor cells. Long-term remission in IroA-E.coli treated mice is associated with enhanced CD8+ T cell activity. Additionally, systemic delivery of IroA-E.coli shows a synergistic effect with chemotherapy reagent oxaliplatin to reduce tumor growth.

Strengths:

It is important to identify the iron-related crosstalk between E. coli and TME. Blocking lcn2-bacterobactin sequestration by different strategies consistently reduce tumor growth.

Weaknesses:

As engineered E.coli upregulate their function to uptake iron, they may increase the likelihood of escaping from nutritional immunity (LCN2 becomes insensitive to sequester iron from the bacteria). Would this raise the chance of developing sepsis? Do authors think that it is safe to administrate these engineered bacteria in mice or humans?

Reviewer #2 (Public Review):

The goal of the present study is to better understand the 'control objectives' that subjects adopt in a video-game-like virtual-balancing task. In this task, the hand must move in the opposite direction from a cursor. For example, if the cursor is 2 cm to the right, the subject must move their hand 2 cm to the left to 'balance' the cursor. Any imperfection in that opposition causes the cursor to move. E.g., if the subject were to move only 1.8 cm, that would be insufficient, and the cursor would continue to move to the right. If they were to move 2.2 cm, the cursor would move back toward the center of the screen. This return to center might actually be 'good' from the subject's perspective, depending on whether their objective is to keep the cursor still or keep it near the screen's center. Both are reasonable 'objectives' because the trial fails if the cursor moves too far from the screen's center during each six-second trial.

This task was recently developed for use in monkeys (Quick et al., 2018), with the intention of being used for the study of the cortical control of movement, and also as a task that might be used to evaluate BMI control algorithms. The purpose of the present study is to better characterize how this task is performed. What sort of control policies are used. Perhaps more deeply, what kind of errors are those policies trying to minimize? To address these questions, the authors simulate control-theory style models and compare with behavior. They do in both in monkeys and in humans.

These goals make sense as a precursor to future recording or BMI experiments. The primate motor-control field has long been dominated by variants of reaching tasks, so introducing this new task will likely be beneficial. This is not the first non-reaching task, but it is an interesting one and it makes sense to expand the presently limited repertoire of tasks. The present task is very different from any prior task I know of. Thus, it makes sense to quantify behavior as thoroughly as possible in advance of recordings. Understanding how behavior is controlled is, as the authors note, likely to be critical to interpreting neural data.

From this perspective - providing a basis for interpreting future neural results - the present study is fairly successful. Monkeys seem to understand the task properly, and to use control policies that are not dissimilar from humans. Also reassuring is the fact that behavior remains sensible even when task-difficulty become high. By 'sensible' I simply mean that behavior can be understood as seeking to minimize error: position, velocity, or (possibly) both, and that this remains true across a broad range of task difficulties. The authors document why minimizing position and minimizing velocity are both reasonable objectives. Minimizing velocity is reasonable, because a near-stationary cursor can't move far in six seconds. Minimizing position error is reasonable, because the trial won't fail if the cursor doesn't stray far from the center. This is formally demonstrated by simulating control policies: both objectives lead to control policies that can perform the task and produce realistic single-trial behavior. The authors also demonstrate that, via verbal instruction, they can induce human subjects to favor one objective over the other. These all seem like things that are on the 'need to know' list, and it is commendable that this amount of care is being taken before recordings begin, as it will surely aid interpretation.

Yet as a stand-alone study, the contribution to our understanding of motor control is more limited. The task allows two different objectives (minimize velocity, minimize position) to be equally compatible with the overall goal (don't fail the trial). Or more precisely, there exists a range of objectives with those two at the extreme. So it makes sense that different subjects might choose to favor different objectives, and also that they can do so when instructed. But has this taught us something about motor control, or simply that there is a natural ambiguity built into the task? If I ask you to play a game, but don't fully specify the rules, should I be surprised that different people think the rules are slightly different?

The most interesting scientific claim of this study is not the subject-to-subject variability; the task design makes that quite likely and natural. Rather, the central scientific result is the claim that individual subjects are constantly switching objectives (and thus control policies), such that the policy guiding behavior differs dramatically even on a single-trial basis. This scientific claim is supported by a technical claim: that the authors' methods can distinguish which objective is in use, even on single trials. I am uncertain of both claims.

Consider Figure 8B, which reprises a point made in Figure 1&3 and gives the best evidence for trial-to-trial variability in objective/policy. For every subject, there are two example trials. The top row of trials shows oscillations around the center, which could be consistent with position-error minimization. The bottom row shows tolerance of position errors so long as drift is slow, which could be consistent with velocity-error minimization. But is this really evidence that subjects were switching objectives (and thus control policies) from trial to trial? A simpler alternative would be a single control policy that does not switch, but still generates this range of behaviors. The authors don't really consider this possibility, and I'm not sure why. One can think of a variety of ways in which a unified policy could produce this variation, given noise and the natural instability of the system.

Indeed, I found that it was remarkably easy to produce a range of reasonably realistic behaviors, including the patterns that the authors interpret as evidence for switching objectives, based on a simple fixed controller. To run the simulations, I made the simple assumption that subjects simply attempt to match their hand position to oppose the cursor position. Because subjects cannot see their hand, I assumed modest variability in the gain, with a range from -1 to -1.05. I assumed a small amount of motor noise in the outgoing motor command. The resulting (very simple) controller naturally displayed the basic range of behaviors observed across trials (see Image 1)

Image 1.

Some trials had oscillations around the screen center (zero), which is the pattern the authors suggest reflects position control. In other trials the cursor was allowed to drift slowly away from the center, which is the pattern the authors suggest reflects velocity control. This is true even though the controller was the same on every trial. Trial-to-trial differences were driven both by motor noise and by the modest variability in gain. In an unstable system, small differences can lead to (seemingly) qualitatively different behavior on different trials.

This simple controller is also compatible with the ability of subjects to adapt their strategy when instructed. Anyone experienced with this task likely understands (or has learned) that moving the hand slightly more than 'one should' will tend to shepherd the cursor back to center, at the cost of briefly high velocity. Using this strategy more sparingly will tend to minimize velocity even if position errors persist. Thus, any subject using this control policy would be able to adapt their strategy via a modest change in gain (the gain linking visible cursor position to intended hand position).

This model is simple, and there may be reasons to dislike it. But it is presumably a reasonable model. The nature of the task is that you should move your hand opposite where the cursor is. Because you can't see your hand, you will make small mistakes. Due to the instability of the system, those small mistakes have large and variable effects. This feature is likely common to other controllers as well; many may explicitly or implicitly blend position and velocity control, with different trials appearing more dominated by one versus the other. Given this, I think the study presents only weak evidence that individual subjects are switching their objective on individual trials. Indeed, the more parsimonious explanation may be that they aren't. While the study certainly does demonstrate that the control policy can be influenced by verbal instructions, this might be a small adjustment as noted above.

I thus don't feel convinced that the authors can conclusively tell us the true control policy being used by human and monkey subjects, nor whether that policy is mostly fixed or constantly switching. The data are potentially compatible with any of these interpretations, depending on which control-style model one prefers.

I see a few paths that the authors might take if they chose.<br /> --First, my reasoning above might be faulty, or there might be additional analyses that could rule out the possibility of a unified policy underlying variable behavior. If so, the authors may be able to reject the above concerns and retain the present conclusions. The main scientifically novel conclusion of the present study is that subjects are using a highly variable control policy, and switching on individual trials. If this is indeed the case, there may be additional analyses that could reveal that.<br /> --Second, additional trial types (e.g., with various perturbations) might be used as a probe of the control policy. As noted below, there is a long history of doing this in the pursuit system. That additional data might better disambiguate control policies both in general, and across trials.<br /> --Third, the authors might find that a unified controller is actually a good (and more parsimonious) explanation. Which might actually be a good thing from the standpoint of future experiments. Interpretation of neural data is likely to be much easier if the control policy being instantiated isn't in constant flux.

In any case, I would recommend altering the strength of some conclusions, particularly the conclusion that the presented methods can reliably discriminate amongst objectives/policies on individual trials. This is mentioned as a major motivation on multiple occasions, but in most of these instances, the subsequent analysis infers the objective only across trial (e.g., one must observe a scatterplot of many trials). By Figure 7, they do introduce a method for inferring the control policy on individual trials, and while this seems to work considerably better than chance, it hardly appears reliable.

In this same vein I would suggest toning down aspects of the Introduction and Discussion. The Introduction in particular is overly long, and tries to position the present study as unique in ways that seem strained. Other studies have built links between human behavior, monkey behavior, and monkey neural data (for just one example, consider the corpus of work from the Scott lab that includes Pruszynski et al. 2008 and 2011). Other studies have used highly quantitative methods to infer the objective function used by subjects (e.g. Kording and Wolpert 2004). The very issue that is of interest in the present study - velocity-error-minimization versus position-error-minimization - has been extensively addressed in the smooth pursuit system. That field has long combined quantitative analyses of behavior in humans and monkeys, along with neural recordings. Many pursuit experiments used strategies that could be fruitfully employed to address the central questions of the present study. For example, error stabilization was important for dissecting the control policy used by the pursuit system. By artificially stabilizing the error (position or velocity) at zero, or at some other value, one can determine the system's response. The classic Rashbass step (1961) put position and velocity errors in opposition, to see which dominates the response. Step and sinusoidal perturbations were useful in distinguishing between models, as was the imposition of artificially imposed delays. The authors note the 'richness' of the behavior in the present task, and while one could say the same of pursuit, it was still the case that specific and well-thought through experimental manipulations were pretty critical. It would be better if the Introduction considered at least some of the above-mentioned work (or other work in a similar vein). While most would agree with the motivations outlined by the authors - they are logical and make sense - the present Introduction runs the risk of overselling the present conclusions while underselling prior work.

Reviewer #2 (Public Review):

Summary

The paper concerns the phenomenon of continuous flash suppression (CFS), relevant to questions about the extent and nature of subconscious visual processing. Whereas standard CFS studies only measure the breakthrough threshold-the contrast at which an initially suppressed target stimulus with steadily increasing contrast becomes visible-this study also measures the re-suppression threshold, the contrast at which a visible target with decreasing contrast becomes suppressed. Thus, the authors could calculate suppression depth, the ratio between the breakthrough and re-suppression thresholds. To measure both thresholds, the study introduces the tracking-CFS method, a continuous-trial design that results in faster, better controlled, and lower-variance threshold estimates compared to the discrete trials standard in the literature. The study finds that suppression depths are similar for different image categories, providing an interesting contrast to previous results that breakthrough thresholds differ for different image categories. The new finding calls for a reassessment of interpretations based solely on the breakthrough threshold that subconscious visual processing is category-specific.

Strengths

(1) The tCFS method quickly estimates breakthrough and re-suppression thresholds using continuous trials, which also better control for slowly varying factors such as adaptation and attention. Indeed, tCFS produces estimates with lower across-subject variance than the standard discrete-trial method (Fig. 2). The tCFS method is straightforward to adopt in future research on CFS and binocular rivalry.

(2) The CFS literature has lacked re-suppression threshold measurements. By measuring both breakthrough and re-suppression thresholds, this work calculated suppression depth (i.e., the difference between the two thresholds), which warrants different interpretations from the breakthrough threshold alone.

(3) The work found that different image categories show similar suppression depths, suggesting some aspects of CFS are not category-specific. This result enriches previous findings that breakthrough thresholds vary with image categories. Re-suppression thresholds vary symmetrically, such that their differences are constant.

Weakness

I do not follow the authors' reasoning as to why the suppression depth is a better (or fuller, superior, more informative) indication of subconscious visual processing than the breakthrough threshold alone. To my previous round of comments, the authors replied that 'breakthrough provides only half of the needed information.' I do not understand this. One cannot infer the suppression depth from the breakthrough threshold alone, but *one cannot obtain the breakthrough threshold from the suppression depth alone*, either. The two measures are complementary. (To be sure, given *both* the suppression depth and the re-suppression threshold, one can recover the breakthrough threshold. The discussion concerns the suppression depth *alone* and the breakthrough threshold *alone*.) I am fully open to being convinced that there is a good reason why the suppression depth may be more informative than the breakthrough threshold about a specific topic, e.g., inter-ocular suppression or subconscious visual processing. I only request that the authors make such an argument explicit. For example, in the significance statement, the authors write, 'all images show equal suppression when both thresholds are measured. We *thus* find no evidence of differential unconscious processing and *conclude* reliance on breakthrough thresholds is misleading' (emphasis added). Just what supports the 'thus' and the 'conclude'? Similarly, at the end of the introduction, the authors write, '[...] suppression depth was constant for faces, objects, gratings and visual noise. *In other words*, we find no evidence to support differential unconscious processing among these particular, diverse categories of suppressed images' (emphasis added). I am not sure the statements in the two sentences are equivalent.

The authors' reply included a discussion of neural CRFs, which may explain why the bCFS thresholds differ across image categories. A further step seems necessary to explain why CRFs do not qualify as a form of subconscious processing.

Reviewer #2 (Public Review):

Summary:

This study aims to address existing differences in the literature regarding the extent of reward versus aversive dopamine signaling in the prefrontal cortex. To do so, the authors chose to present mice with both a reward and an aversive stimulus during different trials each day. The authors used high spatial resolution two-photon calcium imaging of individual dopaminergic axons in the medial PFC to characterize the response of these axons to determine the selectivity of responses in unique axons. They also paired the reward (water) and an aversive stimulus (tail shock) with auditory tones and recorded across 12 days of associative learning.

The authors find that some axons respond to both reward and aversive unconditioned stimuli, but overall, there is a preference to respond to aversive stimuli consistent with expectations from prior studies that used other recording methods. The authors find that both of their two auditory stimuli initially drive responses in axons, but that with training axons develop more selective responses for the shock associated tone indicating that associative learning led to changes in these axon's responses. Finally, the authors use anticipatory behaviors during the conditioned stimuli and facial expressions to determine stimulus discrimination and relate dopamine axons signals with this behavioral evidence of discrimination. This study takes advantage of cutting-edge imaging approaches to resolve the extent to which dopamine axons in PFC respond appetitive or aversive stimuli. They conclude that there is a bias to respond to the aversive tail shock in most axons and weaker more sparse representation of water reward.

Strengths:

The strength of this study is the imaging approach that allows for investigation of the heterogeneity of response across individual dopamine axons unlike other common approaches such as fiber photometry which provide a measure of the average population activity. The use of appetitive and aversive stimuli to probe responses across individual axons is another strength as it reveals response diversity that is often overlooked in reward-only studies.

Weaknesses:

A weakness of this study is the design of the associative conditioning paradigm. The use of only a single reward and single aversive stimulus makes it difficult to know whether these results are specific to the valence of the stimuli versus the specific identity of the stimuli. Further, the reward presentations are more numerous than the aversive trials making it unclear how much novelty and habituation account for results. Moreover, the training seems somewhat limited by the low number of trials and did not result in strong associative conditioning. The lack of omission responses reported may reflect weak associative conditioning. Finally, the study provides a small advance in our understanding of dopamine signaling in the PFC and lacks evidence for if and what might be the consequence of these axonal responses on PFC dopamine concentrations and PFC neuron activity.

Reviewer #2 (Public Review):

Summary:

The authors investigate replay (defined as sequential reactivation) and clustered reactivation during retrieval of an abstract cognitive map. Replay and clustered reactivation were analysed based on MEG recordings combined with a decoding approach. While the authors state to find evidence for both, replay and clustered reactivation during retrieval, replay was exclusively present in low performers. Further, the authors show that reactivation strength declined with an increasing graph distance.

Strengths:

The paper raises interesting research questions, i.e., replay vs. clustered reactivation and how that supports retrieval of cognitive maps. The paper is well written, well structured and easy to follow. The methodological approach is convincing and definitely suited to address the proposed research questions.

The paper is a great combination between replicating previous findings (Wimmer et al. 2020) with a new experimental approach but at the same time presenting novel evidence (reactivation strength declines as a function of graph distance).

What I also want to positively highlight is their general transparency. For example, they pre-registered this study but with a focus on a different part of the data and outlined this explicitly in the paper.

The paper has very interesting findings. However, there are some shortcomings especially in the experimental design. These are shortly outlined below but are also openly and in detail discussed by the authors.

Weaknesses:

The individual findings are interesting. However, due to some shortcomings in the experimental design they cannot be profoundly related to each other. For example, the authors show that replay is present in low but not in high performers with the assumption that high performers tend to simultaneously reactivate items. But then, the authors do not investigate clustered reactivation (= simultaneous reactivation) as a function of performance due to a low number of retrieval trials and ceiling performance in most participants.<br /> As a consequence of the experimental design, some analyses are underpowered (very low number of trials, n = ~10, and for some analyses, very low number of participants, n = 14).

Reviewer #2 (Public Review):

Summary:

Jellinger, Suthard, et al. investigated the transcriptome of positive and negative valence engram cells in the ventral hippocampus, revealing anti- and pro-inflammatory signatures of these respective valences. The authors further reactivated the negative valence engram ensembles to assay the effects of chronic negative memory reactivation in young and old mice. This chronic re-activation resulted in differences in aspects of working memory, and fear memory, and caused morphological changes in glia. Such reactivation-associated changes are putatively linked to GABA changes and behavioral rumination.

Strengths:

Much of the content of this manuscript is of benefit to the community, such as the discovery of differential engram transcriptomes dependent on memory valence. The chronic activation of neurons, and the resultant effects on glial cells and behavior, also provide the community with important data. Laudable points of this manuscript include the comprehensiveness of behavioral experiments, as well as the cross-disciplinary approach.

Weaknesses:

There are several key claims made that are unsubstantiated by the data, particularly regarding the anthropomorphic framing of "rumination" on a mouse model and the role of GABA. The conclusions and inferences in these areas need to be carefully considered.

(1) There are many issues regarding the arguments for the behavioural data's human translation as "rumination." There is no definition of rumination provided in the manuscript, nor how rumination is similar/different to intrusive thoughts (which are psychologically distinct but used relatively interchangeably in the manuscript), nor how rumination could be modelled in the rodent. The authors mention that they are attempting to model rumination behaviours by chronically reactivating the negative engram ("To understand if our experimental model of negative rumination..."), but this occurs almost at the very end of the results section, and no concrete evidence from the literature is provided to attempt to link the behavioural results (decreased working memory, increased fear extinction times) to rumination-like behaviours. The arguments in the final paragraph of the Discussion section about human rumination appear to be unrelated to the data presented in the manuscript and contain some uncited statements. Finally, the rumination claims seem to be based largely upon a single data figure that needs to be further developed (Figure 6, see also point 2 below).

(2) The staining and analysis in Figure 6 are challenging to interpret, and require more evidence to substantiate the conclusions of these results. The histological images are zoomed out, and at this resolution, it appears that only the pyramidal cell layer is being stained. A GABA stain should also label the many sparsely spaced inhibitory interneurons existing across all hippocampal layers, yet this is not apparent here. Moreover, both example images in the treatment group appear to have lower overall fluorescence intensity in both DAPI and GABA. The analysis is also unclear: the authors mention "ROIs" used to measure normalized fluorescence intensity but do not specify what the ROI encapsulates. Presumably, the authors have segmented each DAPI-positive cell body and assessed fluorescence - however, this is not explicated nor demonstrated, making the results difficult to interpret.

(3) A smaller point, but more specific detail is needed for how genes were selected for GSEA analysis. As GSEA relies on genes to be specified a priori, to avoid a circular analysis, these genes need to be selected in a blind/unbiased manner to avoid biasing downstream results and conclusions. It's likely the authors have done this, but explicitly noting how genes were selected is an important context for this analysis.

Reviewer #2 (Public Review):

Summary:

This series of experiments studied the involvement of PVN OT neurons and their projection to the mPFC in pup-care and attack behavior in virgin male and female Mandarin voles. Using Fos visualization, optogenetics, fiber photometry, and IP injection of OT the results converge on OT regulating caregiving and attacks on pups. Some sex differences were found in the effects of the manipulations.

Strengths:

Major strengths are the modern multi-method approaches and involving both sexes of Mandarin vole in every experiment.

Weaknesses:

Weaknesses include the lack of some specific details in the methods that would help readers interpret the results. These include:

(1) No description of diffusion of centrally injected agents.

(2) Whether all central targets were consistent across animals included in the data analyses. This includes that is not stated if the medial prelimbic mPFC target was in all optogenetic study animals as shown in Figure 4 and if that is the case, there is no discussion of that subregion's function compared to other mPFC subregions.

(3) How groups of pup-care and infanticidal animals were created since there was no obvious pre-test mentioned so perhaps there was the testing of a large number of animals until getting enough subjects in each group.

(4) The apparent use of a 20-minute baseline data collection period for photometry that started right after the animals were stressed from handling and placement in the novel testing chamber.

(5) A weakness in the results reporting is that it's unclear what statistics are reported (2 x 2 ANOVA main effect of interaction results, t-test results) and that the degrees of freedom expected for the 2 X 2 ANOVAs in some cases don't appear to match the numbers of subjects shown in the graphs; including sample sizes in each group would be helpful because the graph panels are very small and data points overlap.

The additional context that could help readers of this study is that the authors overlook some important mPFC and pup caregiving and infanticide studies in the introduction which would help put this work in better context in terms of what is known about the mPFC and these behaviors. These previous studies include Febo et al., 2010; Febo 2012; Peirera and Morrell, 2011 and 2020; and a very relevant study by Alsina-Llanes and Olazábal, 2021 on mPFC lesions and infanticide in virgin male and female mice. The introduction states that nothing is known about the mPFC and infanticide. In the introduction and discussion, stating the species and sex of the animals tested in all the previous studies mentioned would be useful. The authors also discuss PVN OT cell stimulation findings seen in other rodents, so the work seems less conceptually novel. Overall, the findings add to the knowledge about OT regulation of pup-directed behavior in male and female rodents, especially the PVN-mPFC OT projection.

Reviewer #2 (Public Review):

The paper "The Value of Livestock Abortion Surveillance in Tanzania: Identifying Disease Priorities and Informing Interventions" provides a comprehensive analysis of the importance of livestock abortion surveillance in Tanzania. The authors aim to highlight the significance of this surveillance system in identifying disease priorities and guiding interventions to mitigate the impact of livestock abortions on both animal and human health.

Summary:

The paper begins by discussing the context of livestock farming in Tanzania and the significant economic and social impact of livestock abortions. The authors then present a detailed overview of the livestock abortion surveillance system in Tanzania, including its objectives, methods, and data collection process. They analyze the data collected from this surveillance system over a specific period to identify the major causes of livestock abortions and assess their public health implications.

Evaluation:

Overall, this paper provides valuable insights into the importance of livestock abortion surveillance as a tool for disease prioritization and intervention planning in Tanzania. The authors effectively demonstrate the utility of this surveillance system in identifying emerging diseases, monitoring disease trends, and informing evidence-based interventions to control and prevent livestock abortions.

Strengths:

(1) Clear Objective: The paper clearly articulates its objective of highlighting the value of livestock abortion surveillance in Tanzania.

(2) Comprehensive Analysis: The authors provide a thorough analysis of the surveillance system, including its methodology, data collection process, and findings as seen in the supplementary files.

(3) Practical Implications: The paper discusses the practical implications of the surveillance system for disease control and public health interventions in Tanzania.

(4) Well-Structured: The paper is well-organized, with clear sections and subheadings that facilitate understanding and navigation.

Suggestions for Improvement:

(1) Data Presentation: While the analysis is comprehensive, the presentation of data could be enhanced with the use of more visual aids such as tables, graphs, or charts to illustrate key findings.

(2) Discussion Section: The paper could benefit from a more in-depth discussion of the implications of the findings for disease control strategies and policy formulation in Tanzania.

(3) Future Directions: Including recommendations for future research or areas for further investigation would add depth to the paper.

Summary:

This paper contains thorough analysis and valuable insights. Overall, it makes a significant contribution to the literature on livestock abortion surveillance and its implications for disease control in Tanzania.

Reviewer #2 (Public Review):

The authors have proposed a computational algorithm to identify runs of homozygosity (ROH) segments in a generally outbred population and then study the association of ROH with self-reported disorders in the UK biobank. The algorithm certainly identifies such segments. However, more work is needed to justify the importance of ROH.

Reviewer #2 (Public Review):

Summary:

The manuscript by Liu et al. reports a task that is designed to examine the extent to which "past" and "future" information is encoded in working memory that combines a retrocue with rules that indicate the location of an upcoming test probe. An analysis of microsaccades on a fine temporal scale shows the extent to which shifts of attention track the location of the encoded item (past) and the location of the future item (test probe). The location of the encoded grating and test probe were always on orthogonal axes (horizontal, vertical) so that biases in microsaccades could be used to track shifts of attention to one or the other axis (or mixtures of the two). The overall goal here was then to (1) create a methodology that could tease apart memory for the past and future, respectively, (2) to look at the time-course attention to past/future, and (3) to test the extent to which microsaccades might jointly encode past and future memoranda. Finally, some remarks are made about the plausibility of various accounts of working memory encoding/maintenance based on the examination of these time-courses.

Strengths:

This research has several notable strengths. It has a clear statement of its aims, is lucidly presented, and uses a clever experimental design that neatly orthogonalized "past" and "future" as operationalized by the authors. Figure 1b-d shows fairly clearly that saccade directions have an early peak (around 300ms) for the past and a "ramping" up of saccades moving in the forward direction. This seems to be a nice demonstration that the method can measure shifts of attention at a fine temporal resolution and differentiate past from future oriented saccades due to the orthogonal cue approach. The second analysis shown in Figure 2, reveals a dependency in saccade direction such that saccades toward the probe future were more likely also to be toward the encoded location than away from the encoded direction. This suggests saccades are jointly biased by both locations "in memory". The "central contribution" (as the authors characterize it) is that "the brain simultaneously retains the copy of both past and future-relevant locations in working memory, and (re)activates each during mnemonic selection", and that: "... while it is not surprising that the future location is considered, it is far less trivial that both past and future attributes would be retained and (re)activated together. This is our central contribution." The authors provide a nuanced analysis that offers persuasive evidence that past and future representations are jointly maintained in memory.

Reviewer #2 (Public Review):

Summary:

Jones et al. extend their previous work on the translation machinery in Dinoflagellate. In particular, they study the species Amphidium carterae. They characterize the type of cap structure mRNAs possess in this species, as well as the eight eIF4E family members A. carterae possesses and their affinity to the mRNA cap. They also establish the leader sequences of the transpliced mRNAs that A. carterae generates during gene expression.

Strengths:

The authors performed a solid phylogenetic and biochemical study to understand the structure of Dinoflagellate mRNAs at the 5'-UTR as well as the divergence and biochemical features of eIF4Es across Dinoflagellate. They also establish eIF4E-1a as the prototypical paralog of the eIF4E family of proteins. The scientific questions they ask are very relevant to the gene expression field across eukaryotes. The experiments and the phylogenetic analysis are performed with a very high quality. They perform a wide spectrum of experimental approaches and techniques to answer the questions.

Weaknesses:

The authors assume all eIF4E from Dinoflagellate are involved in translation, i.e., mRNA recruitment to the ribosome. Indeed, they think that the diverse biochemical features of all eIF4E in A. carterae have to do with the possible recruitment of different subsets of mRNAs to the ribosome for translation. I think that the biochemical differences among all paralogs also might be due to the involvement of some of them in different processes of RNA metabolism, other than translation. For instance, some of them could be involved only in RNA processing in the nucleus or mRNA storage in cytoplasmic foci.

Reviewer #2 (Public Review):

Summary:

The specific objective of this study was to determine the role of the large apical hook on the head of mouse sperm (Mus musculus) in sperm migration through the female reproductive tract. The authors used a custom-built two-photon microscope system to obtain digital videos of sperm moving within the female reproductive tract. They used sperm from genetically modified male mice that produce fluorescence in the sperm head and flagellar midpiece to enable visualization of sperm moving within the tract. Based on various observations, the authors concluded that the hook serves to facilitate sperm migration by hooking sperm onto the lining of the female reproductive tract, rather than by hooking sperm together to form a sperm train that would move them more quickly through the tract. The images and videos are excellent and inspirational to researchers in the field of mammalian sperm migration, but interpretations of the behaviors are highly speculative and not supported by controlled experimentation.

Strengths:

The microscope system developed by the authors could be of interest to others investigating sperm migration.

The new behaviors shown in the images and videos could be of interest to others in the field, in terms of stimulating the development of new hypotheses to investigate.

Weaknesses:

The authors stated several hypotheses about the functions of the sperm behaviors they saw, but the hypotheses were not clearly stated or tested experimentally.

The hypothesis statements were weakened by the use of hedge words, such as "may".

Reviewer #2 (Public Review):

Summary:

This is an interesting manuscript that describes a series of molecular dynamics studies on the peptide transporter PepT2 (SLC15A2). They examine, in particular, the effect on the transport cycle of protonation of various charged amino acids within the protein. They then validate their conclusions by mutating two of the residues that they predict to be critical for transport in cell-based transport assays. The study suggests a series of protonation steps that are necessary for transport to occur in Petp2. Comparison with bacterial proteins from the same family shows that while the overall architecture of the proteins and likely mechanism are similar, the residues involved in the mechanism may differ.

Strengths:

This is an interesting and rigorous study that uses various state-of-the-art molecular dynamics techniques to dissect the transport cycle of PepT2 with nearly 1ms of sampling. It gives insight into the transport mechanism, investigating how the protonation of selected residues can alter the energetic barriers between various states of the transport cycle. The authors have, in general, been very careful in their interpretation of the data.

Weaknesses:

Interestingly, they suggest that there is an additional protonation event that may take place as the protein goes from occluded to inward-facing but they have not identified this residue. Some things are a little unclear. For instance, where does the state that they have defined as occluded sit on the diagram in Figure 1a? - is it truly the occluded state as shown on the diagram or does it tend to inward- or outward-facing? The pKa calculations and their interpretation are a bit unclear. Firstly, it is unclear whether they are using all the data in the calculations of the histograms, or just selected data and if so on what basis was this selection done. Secondly, they dismiss the pKa calculations of E53 in the outward-facing form as not being affected by peptide binding but say that E56 is when there seems to be a similar change in profile in the histograms.

Reviewer #2 (Public Review):

Summary:

In this article, Kumar et al., report on a previously unappreciated mechanism of translational regulation whereby p130Cas induces LLPS condensates that then traffic out from focal adhesion into the cytoplasm to modulate mRNA translation. Specifically, the authors employed EGFP-tagged p130Cas constructs, endogenous p130Cas, and p130Cas knockouts and mutants in cell-based systems. These experiments in conjunction with various imaging techniques revealed that p130Cas drives assembly of LLPS condensates in a manner that is largely independent of tyrosine phosphorylation. This was followed by in vitro EGFP-tagged p130Cas-dependent induction of LLPS condensates and determination of their composition by mass spectrometry, which revealed enrichment of proteins involved in RNA metabolism in the condensates. The authors excluded the plausibility that p130Cas-containing condensates co-localize with stress granules or p-bodies. Next, the authors determined mRNA compendium of p130Cas-containing condensates which revealed that they are enriched in transcripts encoding proteins implicated in cell cycle progression, survival, and cell-cell communication. These findings were followed by the authors demonstrating that p130Cas-containing condensates may be implicated in the suppression of protein synthesis using puromycylation assay. Altogether, it was found that this study significantly advances the knowledge pertinent to the understanding of molecular underpinnings of the role of p130Cas and more broadly focal adhesions on cellular function, and to this end, it is likely that this report will be of interest to a broad range of scientists from a wide spectrum of biomedical disciplines including cell, molecular, developmental and cancer biologists.

Strengths:

Altogether, this study was found to be of potentially broad interest inasmuch as it delineates a hitherto unappreciated link between p130Cas, LLPS, and regulation of mRNA translation. More broadly, this report provides unique molecular insights into the previously unappreciated mechanisms of the role of focal adhesions in regulating protein synthesis. Overall, it was thought that the provided data sufficiently supported most of the authors' conclusions. It was also thought that this study incorporates an appropriate balance of imaging, cell and molecular biology, and biochemical techniques, whereby the methodology was found to be largely appropriate.

Weaknesses:

Two major weaknesses of the study were noted. The first issue is related to the experiments establishing the role of p130Cas-driven condensates in translational suppression, whereby it remained unclear whether these effects are affecting global mRNA translation or are specific to the mRNAs contained in the condensates. Moreover, some of the results in this section (e.g., experiments using cycloheximide) may be open to alternative interpretation. The second issue is the apparent lack of functional studies, and although the authors speculate that the described mechanism is likely to mediate the effects of focal adhesions on e.g., quiescence, experimental testing of this tenet was lacking.

Reviewer #2 (Public Review):

Summary:

In this study, the authors study how the deubiquitinase USP8 regulates endosome maturation in C. elegans and mammalian cells. The authors have isolated USP8 mutant alleles in C. elegans and used multiple in vivo reporter lines to demonstrate the impact of USP8 loss-of-function on endosome morphology and maturation. They show that in USP8 mutant cells, the early endosomes and MVB-like structures are enlarged while the late endosomes and lysosomal compartments are reduced. They elucidate that USP8 interacts with Rabx5, a guanine nucleotide exchange factor (GEF) for Rab5, and show that USP8 likely targets specific lysine residue of Rabx5 to dissociate it from early endosomes. They also find that the localization of USP8 to early endosomes is disrupted in Rabx5 mutant cells. They observe that in both Rabx5 and USP8 mutant cells, the Rab7 GEF SAND-1 puncta which likely represents late endosomes are diminished, although Rabex5 is accumulated in USP8 mutant cells. The authors provide evidence that USP8 regulates endosomal maturation in a similar fashion in mammalian cells. Based on their observations they propose that USP8 dissociates Rabex5 from early endosomes and enhances the recruitment of SAND-1 to promote endosome maturation.

Strengths:

The major highlights of this study include the direct visualization of endosome dynamics in a living multi-cellular organism, C. elegans. The high-quality images provide clear in vivo evidence to support the main conclusions. The authors have generated valuable resources to study mechanisms involved in endosome dynamics regulation in both the worm and mammalian cells, which would benefit many members of the cell biology community. The work identifies a fascinating link between USP8 and the Rab5 guanine nucleotide exchange factor Rabx5, which expands the targets and modes of action of USP8. The findings make a solid contribution toward the understanding of how endosomal trafficking is controlled.

Weaknesses:

- The authors utilized multiple fluorescent protein reporters, including those generated by themselves, to label endosomal vesicles. Although these are routine and powerful tools for studying endosomal trafficking, these results cannot tell whether the endogenous proteins (Rab5, Rabex5, Rab7, etc.) are affected in the same fashion.

- The authors clearly demonstrated a link between USP8 and Rabx5, and they showed that cells deficient in both factors displayed similar defects in late endosomes/lysosomes. However, the authors didn't confirm whether and/or to which extent USP8 regulates endosome maturation through Rabx5. Additional genetic and molecular evidence might be required to better support their working model.

Reviewer #2 (Public Review):

Summary:

The manuscript "Formation of a giant unilocular vacuole via macropinocytosis-like process confers anoikis resistance" describes an interesting observation and provides initial steps towards understanding the underlying molecular mechanism.

The manuscript describes that the majority of non-tumorigenic mammary gland epithelial cells (MCF-10A) in suspension initiate entosis. A smaller fraction of cells form a single giant unilocular vacuole (hereafter referred to as a GUVac). GUVac appeared to be empty and did not contain invading (entotic) cells. The formation of GUVac could be promoted by disrupting actin polymerisation with LatB and CytoD. The formation of GUVacs correlated with resistance to anoikis. GUVac formation was detected in several other epithelial cells from secretory tissues.

The authors then use electron microscopy and super-resolution imaging to describe the biogenesis of GUVac. They find that GUVac formation is initiated by a micropinocytosis-like phenomenon (that is independent of actin polymerisation). This process leads to the formation of large plasma membrane invaginations, that pinch off from the PM to form larger vesicles that fuse with each other into GUVacs.

Inhibition of actin polymerisation in suspended MCF-10a leads to the recruitment of Septin 6 to the PM via its amphipathic helix. Treatment with FCF (a septin polymerisation inhibitor) blocked GUVac biogenesis, as did pharmacological inhibition of dynamin-mediated membrane fission. The fusion of these vesicles in GUVacs required (perhaps not surprisingly) PI3P.

Strengths:

The authors have made an interesting and potentially important observation. They describe the formation of an endo-lysosomal organelle (a giant unilocular vacuole - GUVac) in suspended epithelial cells and correlate the formation of GUVacs with resistance to aniokis.

Weaknesses:

My major concern is the experimental strategy that is used throughout the paper to induce and study the formation GUVac. Almost every experiment is conducted in suspended cells that were treated with actin depolymerising drugs (e.g. LatB) and thus almost all key conclusions are based on the results of these experiments. I only have a few suggestions that would improve these experiments or change their outcome and interpretation.

Yet, I believe it is essential to identify the endogenous pathway leading to the actin depolymerisation that drives the formation of GUVacs in detached epithelial cells (or alternatively to figure out how it is suppressed in most detached cells). A first step in that direction would be to investigate the polymerization status of actin in MCF-10a cells that 'spontaneously' form GUVacs and to test if these cells also become resistant to anoikis.

Also, it would be great (and I believe reasonably easy) to better characterise molecular markers of GUVacs (LAMP's, Rab's, Cathepsins, etc....) to discriminate them from other endosomal organelles

Reviewer #2 (Public Review):

Summary:

In the manuscript 'Structural analysis of the dynamic ribosome-translocon complex' Lewis and Hegde present a structural study of the ribosome-bound multipass translocon (MPT) based on re-analysis of cryo-EM single particle data of ribosome-MPTs processing the multipass transmembrane substrate RhoTM2 from a previous publication (Smalinskaité et al, Nature 2022) and AlphaFold2 multimer modeling. Detailed analysis of the laterally open Sec61 is obtained from PAT-less particles.

The following major claims are made:

- TMs can bind similarly to the Sec61 lateral gate as signal peptides.

- Ribosomal H59 is in immediate proximity to basic residues of TMs and signal peptides, suggesting it may contribute to the positive-inside rule.

- RAMP4/SERP1 binds to the Sec61 lateral gate and the ribosome near 28S rRNA's helices 47, 57, and 59 as well as eL19, eL22, and eL31.

- uL22 C-terminal tail binds H24/47 blocking a potential escape route for nascent peptides to the cytosol.

- TRAP and BOS compete for binding to Sec61 hinge.

- Calnexin TM binds to TRAPg.

- NOMO wedges between TRAP and MPT.

Strengths:

The manuscript contains numerous novel new structural analyses and their potential functional implications. While all findings are exciting, the highlight is the discovery of RAMP4/SERP1 near the Sec61 lateral gate. Overall, the strength is the thorough and extensive structural analysis of the different high-resolution RTC classes as well as the expert bioinformatic evolutionary analysis.

Weaknesses:

A minor downside of the manuscript is the sheer volume of analyses and mechanistic hypotheses, which makes it sometimes difficult to follow. The authors might consider offloading some analyses based on weaker evidence to the supplement to maximize impact.

Reviewer #2 (Public Review):

Summary:

This combined experimental-theoretical paper introduces a novel two-domain statistical thermodynamic model (primarily Equation 1) to study allostery in generic systems but focusing here on the tetracycline repressor (TetR) family of transcription factors. This model, building on a function-centric approach, accurately captures induction data, maps mutants with precision, and reveals insights into epistasis between mutations.

Strengths:

The study contributes innovative modeling, successful data fitting, and valuable insights into the interconnectivity of allosteric networks, establishing a flexible and detailed framework for investigating TetR allostery. The manuscript is generally well-structured and communicates key findings effectively.

Comments on revised version:

I am happy with the changes made by the authors

Reviewer #2 (Public Review):

Original review:<br /> Summary:<br /> In this paper, Zambo and coworkers use a powerful technique, called native holdup, to measure the affinity of the SH3 domain of BIN1 for cellular partners. Using this assay, they combine data using cellular proteins and proline-containing fragments in these proteins to identify 97 distinct direct binding partners of BIN1. They also compare the binding interactome of the BIN1 SH3 domain to the interactome of several other SH3 domains, showing varying levels of promiscuity among SH3 domains. The authors then use pathway analysis of BIN1 binding partners to show that BIN1 may be involved in mitosis. Finally, the authors examine the impact of clinically relevant mutations of the BIN1 SH3 domain on the cellular interactome. The authors were able to compare the interactome of several different SH3 domains and provide novel insight into the cellular function of BIN1. Generally, the data supports the conclusions, although the reliance on one technique and the low number of replicates in each experiment is a weakness of the study.

Strengths:<br /> The major strength of this paper is the use of holdup and native holdup assays to measure the affinity of SH3 domains to cellular partners. The use of both assays using cell-derived proteins and peptides derived from identified binding partners allows the authors to better identify direct binding partners. This assay has some complexity but does hold the possibility of being used to measure the affinity of the cellular interactome of other proteins and protein domains. Beyond the utility of the technique, this study also provides significant insight into the cellular function of BIN1. The authors have strong evidence that BIN1 might have an undiscovered function in cellular mitosis, which potentially highlights BIN1 as a drug target. Finally, the study provides outstanding data on the cellular binding properties and partners of seven distinct SH3 domains, showing surprising differences in the promiscuity of these proteins.

Weaknesses:<br /> There are three major weaknesses of the study. First, the authors rely completely on a single technique to measure the affinity of the cellular interactome. The native holdup is a relatively new technique that is powerful yet relatively unproven. However, it appears to have the capacity to measure the relative affinity of proteins. Second, the authors appear to use a relatively small number of replicates for the holdup assays. There is no information in the legends about the number of replicates but the materials and methods suggest the native holdup data is from a single experimental replicate with multiple technical replicates. Finally, the authors' data using cellular proteins and fragments show that the affinity of the whole proteins is 5-20 fold lower than individual proline-containing fragments. The authors state that this difference suggests that there is cooperativity between different proline-rich sites of the binding partners of BIN1, yet BIN1 only has one SH3 domain. It is unclear what the molecular mechanism of the cooperative interaction would be exactly since there would be only one SH3 domain to bind the partner. An alternative interpretation would be that the BIN 1 SH3 domain requires sequences outside of the short proline-rich regions for high-affinity interactions with cellular partners, a hypothesis that is supported by other studies.

Comments on revision:<br /> I thank the authors for their thoughtful response. I have additional comments.

I appreciate that this is not a techniques paper and that the authors have done more detailed work in a separate publication. It would be helpful to readers not familiar with this new method to more fully describe this technique in this manuscript.

I also thank the authors for their description of why they performed only 1 biological replicate of the experiment. However, I still believe that multiple biological replicates will provide more rigorous and reproducible data. The data the authors provide actually argues for the inclusion of more biological replicates. They state they performed 2 separate nHU replicates using different mass spectrometers. It is unclear if this data uses the same lysates and protein preparations, but by the data, the two methods detected a total of 207 distinct binding partners. Only 29 of these were significant binders in both replicates and only 90 were detected binders in both replicates. 117 binding partners were found in only one replicate suggesting a significant differences between replicates. Different batches of SH3 domains can have different activities and different replicates of cell lysates can vary, even when made from the same cell line. Thus, there can still be significant differences between replicates in this method. I appreciate the difficulty of performing and analyzing multiple biological replicates, but it is the most rigorous way to identify potential cellular partners.

I also thank the author for including the mechanistic discussion about the differences between peptides and whole proteins. There is literature showing that regions outside of the short PxxP regions drive binding to SH3 domains, especially for the GRB2 family of adaptor proteins.

Reviewer #2 (Public Review):

Summary:

The manuscript by Brotherton et al. describes a structural study of connexin-26 (Cx26) gap junction channel mutant K125E, which is designed to mimic the CO2-inhibited form of the channel. In the wild-type Cx26, exposure to CO2 is presumed to close the channel through carbamylation of the redeye K125. The authors mutated K125 to a negatively charged residue to mimic this effect and observed by cryo-EM analysis of the mutated channel that the pore of the channel is constricted. The authors were able to observe conformations of the channel with resolved density for the cytoplasmic loop (in which K125 is located). Based on the observed conformations and on the position of the N-terminal helix, which is involved in channel gating and in controlling the size of the pore, the authors propose the mechanisms of Cx26 regulation.

Strengths:

This is a very interesting and timely study, and the observations provide a lot of new information on connexin channel regulation. The authors use the state of the art cryo-EM analysis and 3D classification approaches to tease out the conformations of the channel that can be interpreted as "inhibited", with important implications for our understanding of how the conformations of the connexin channels controlled.

Weaknesses:

The revised version of the manuscript is improved, and the authors have addressed the review comments/criticisms in a satisfactory manner.

Reviewer #2 (Public Review):

This model of skeletal muscle includes springs and dampers which aim to capture the effect of crossbridge and titin stiffness during the stretch of active muscle. While both crossbridge and titin stiffness have previously been incorporated, in some form, into models, this model is the first to simultaneously include both. The authors suggest that this will allow for the prediction of muscle force in response to short-, mid- and long-range stretches. All these types of stretch are likely to be experienced by muscle during in vivo perturbations, and are known to elicit different muscle responses. Hence, it is valuable to have a single model which can predict muscle force under all these physiologically relevant conditions. In addition, this model dramatically simplifies sarcomere structure to enable this muscle model to be used in multi-muscle simulations of whole-body movement.

In order to test this model, its force predictions are compared to 3 sets of experimental data which focus on short-, mid- and long-range perturbations, and to the predictions of a Hill-type muscle model. The choice of data sets is excellent and provide a robust test of the model's ability to predict forces over a range of length perturbations. However, I find the comparison to a Hill-type muscle model to be somewhat limiting. It is well established that Hill-type models do not have any mechanism by which they can predict the effect of active muscle stretch. Hence, that the model proposed here represents an improvement over such a model is not a surprise. Many other models, some of which are also simple enough to be incorporated into whole-body simulations, have incorporated mechanistic elements which allow for the prediction of force responses to muscle stretch. And it is not clear from the results presented here that this model would outperform such models.

The paper begins by outlining the phenomenological vs mechanistic approaches taken to muscle modelling, historically. It appears, although is not directly specified, that this model combines these approaches. A somewhat mechanistic model of the response of the crossbridges and titin to active stretch is combined with a phenomenological implementation of force-length and force-velocity relationships. This combination of approaches may be useful improving the accuracy of predictions of muscle models and whole-body simulations, which is certainly a worthy goal. However, it also may limit the insight that can be gained. For example, it does not seem that this model could reflect any effect of active titin properties on muscle shortening. In addition, it is not clear to me, either physiologically or in the model, what drives the shift from the high stiffness in short-range perturbations to the somewhat lower stiffness in mid-range perturbations.

Reviewer #2 (Public Review):

The authors (Yang et al.) present a well-executed study of a mouse model of Bmpr1a focusing on microtia development and pathogenesis.

The authors report that the generation of the Bmpr1a in Prrx1+ cells in adult mice helps characterize the developmental progression of the external ear.

The authors explain how auricular chondrocytes differ from growth plates or other chondrocytes and BMP-Smd1/5/9 activation, which is required to maintain chondrocyte fate in the distal part of the ear. The authors explain with evidence how BMP signaling actively maintains auricle cartilage in the post-developmental stage.

Elegant immunofluorescence staining, excellent histology preparations and dissections, excellent microscopy, sufficient experimental sample size, and good statistical analyses support the results. The study is well grounded in extensively reviewed and cited existing literature. This report sets the stage for a comprehensive interrogation of Bmpr1a deficiency and ear defects.

Reviewer #2 (Public Review):

Summary:

The authors describe a form of synaptic plasticity at synapses from granule cells onto Purkinje cells in the mouse cerebellum, which is specific to synapses proximal to the cell body but not to distal ones. This plasticity is induced by the paired or associative stimulation of the two types of synapses because it is not observed with stimulation of one type of synapse alone. In addition, this form of plasticity is dependent on the order in which the stimuli are presented, and is dependent on NMDA receptors, metabotropic glutamate receptors and to some degree on GABAA receptors. However, under all experimental conditions described, there is a progressive weakening or run-down of synaptic strength. Therefore, plasticity is not relative to a stable baseline, but relative to a process of continuous decline that occurs whether or not there is any plasticity-inducing stimulus.

Strengths:

The focus of the authors on the properties of two different synapse-types on cerebellar Purkinje cells is interesting and relevant, given previous results that ascending and parallel fiber synapses might be functionally different and undergo different forms of plasticity. In addition, the interaction between these two synapse types during plasticity is important for understanding cerebellar function. The demonstration of timing and order-dependent potentiation of only one pathway, and not another, after associative stimulation of both pathways, changes our understanding of potential plasticity mechanisms. In addition, this observation opens up many new questions on underlying intracellular mechanisms as well as on its relevance for cerebellar learning and adaptation.

Weaknesses and suggested improvements:

A concern with this study is that all recordings demonstrate "rundown", a progressive decrease in the amplitude of the EPSC, starting during the baseline period and continuing after the plasticity-induction stimulus. In the absence of a stable baseline, it is hard to know what changes in strength actually occur at any set of synapses. Moreover, the issues that are causing rundown are not known and may or may not be related to the cellular processes involved in synaptic plasticity. This concern applies in particular to all the experiments where there is a decrease in synaptic strength.<br /> The authors should consider changes in the shape of the EPSC after plasticity induction, as in Fig 1 (orange trace) as this could change the interpretation.<br /> In addition, the inconsistency with previous results is surprising and is not explained; specifically, that no PF-LTP was induced by PF-alone repeated stimulation.<br /> The authors test the role of NMDARs, GABAARs and mGluRs in the phenotype they describe. The data suggest that the form of plasticity described here is dependent on any one of the three receptors. However, the location of these receptors varies between the Purkinje cells, granule cells and interneurons. The authors do not describe a convincing hypothetical model in which this dependence can be explained. They suggest that there is crosstalk between AA and PF synapses via endocannabinoids downstream of mGluR or NO downstream of NMDARs. However, it is not clear how this could lead to the long-term potentiation that they describe. Also, there is no long-lasting change in paired-pulse ratio, suggesting an absence of changes in presynaptic release.<br /> Is the synapse that undergoes plasticity correctly identified? In this study, since GABAergic inhibition is not blocked for most experiments, PF stimulation can result in both a direct EPSC onto the Purkinje cell and a disynaptic feedforward IPSC. The authors do address this issue with Supplementary Fig 3, where the impact of the IPSC on the EPSC within the EPSC/IPSC sequence is calculated. However, a change in waveform would complicate this analysis. An experiment with pharmacological blockade will make the interpretation more robust. The observed dependence of the plasticity on GABAA receptors is an added point in favor of the suggested additional experiments.<br /> A primary hypothesis of this study is that proximal, or AA, and distal, or PF, synapses are different and that their association is specifically what drives plasticity. The alternative hypothesis is that the two synapse-types are the same. Therefore, a good control for pairing AA with PF would be to pair AA with AA and PF with PF, thereby demonstrating that pairing with each other is different from pairing with self.<br /> It is hypothesized that the association of a PF input with an AA input is similar to the association of a PF input with a CF input. However, the two are very different in terms of cellular location, with the CF input being in a position to directly interact with PF-driven inputs. Therefore, there are two major issues with this hypothesis: 1) how can sub-threshold activity at one set of synapses affect another located hundreds of micrometers away on the same dendritic tree? 2) There is evidence that the CF encodes teaching/error or reward information, which is functionally meaningful as a driver of plasticity at PF synapses. The AA synapse on one set of Purkinje cells is carrying exactly the same information as the PF synapses on another set of Purkinje cells further up and down the parallel fiber beam. It is suggested that the two inputs carry sensory vs. motor information, which is why this form of plasticity was tested. However, the granule cells that lead to both the AA and PF synapses are receiving the same modalities of mossy fiber information. Therefore, one needs to presuppose different populations of granule cells for sensory and motor inputs or receptive field and contextual information. As a consequence, which granule cells lead to AA synapses and which to PF synapses will change depending on which Purkinje cell you're recording from. And that's inconsistent with there being a timing dependence of AA-PF pairing in only one direction. Overall, it would be helpful to discuss the functional implications of this form of plasticity.

Reviewer #2 (Public Review):

Summary:

This paper presents an automated method to track individual mammalian cells as they progress through the cell cycle using the FUCCI system and applies the method to look at different tumor cell lines that grow in suspension and determine their cell cycle profile and the effect of drugs that directly affect the cell cycles, on progression through the cell cycle for a 72 hour period.

Strengths:

This is a METHODS paper. The one potentially novel finding is that they can identify cells that are at the G1-S transition by the change in color as one protein starts to go up and the other one goes down, similar to the change seen as cells enter G2/M.

Weaknesses:

They did not clearly indicate whether the G1/S cells are identified automatically or need to be identified by the person reviewing the data. In Figures 1 and S1, the movie shows cells with no color at a time corresponding to what is about the G1/S transition. Their assigned cell cycle phase is shown in Figure 1 but not in Figure S1. None of these pictures show the G1/S cells that they talk about being able to detect with a different color.

Reviewer #2 (Public Review):

In this study, Wang et al., report the significance of XAP5L and XAP5 in spermatogenesis, involved in transcriptional regulation of the ciliary gene in testes. In previous studies, the authors demonstrate that XAP5 is a transcription factor required for flagellar assembly in Chlamydomonas. Continuing from their previous study, the authors examine the conserved role of the XAP5 and XAP5L, which are the orthologue pair in mammals.

XAP5 and XAP5L express ubiquitously and testis specifically, respectively, and their absence in the testes causes male infertility with defective spermatogenesis. Interestingly, XAP5 deficiency arrests germ cell development at the pachytene stage, whereas XAP5L absence causes impaired flagellar formation. RNA-seq analyses demonstrated that XAP5 deficiency suppresses ciliary gene expression including Foxj1 and Rfx family genes in early testis. By contrast, XAP5L deficiency abnormally remains Foxj1 and Rfx genes in mature sperm. From the results, the authors conclude that XAP5 and XAP5L are the antagonistic transcription factors that function upstream of Foxj1 and Rfx family genes.

This reviewer thinks the overall experiments are performed well and that the manuscript is clear. However, the current results do not directly support the authors' conclusion. For example, the transcriptional function of XAP5 and XAP5L requires more evidence. In addition, this reviewer wonders about the conserved XAP5 function of ciliary/flagellar gene transcription in mammals - the gene is ubiquitously expressed despite its functional importance in flagellar assembly in Chlamydomonas. Thus, this reviewer thinks authors are required to show more direct evidence to clearly support their conclusion with more descriptions of its role in ciliary/flagellar assembly.

Reviewer #2 (Public Review):

Summary:

This work follows previous work from the group where they have demonstrated the role of TASK1 in the regulation of glucose stimulated insulin secretion. Moreover, a recent study links a mutation in KCNK16, the gene encoding TALK-1 channels to MODY. Here the authors have constructed a mouse model with the specific mutation (TALK-1 L114P mutation) and investigated the phenotype. They have to perform a couple of breeding tricks to find a model that is lethal in adult which might complicate the conclusions, however, the phenotype of the heterozygote model used have a MODY-like phenotype. The study is convincing and solid.

Strengths:

(1) The work is a natural follow-up from previous studies from the groups.<br /> (2) The authors present convincing and solid data that in the long perspective will help patients with this mutations.<br /> (3) Both in vivo and in vitro data are presented to give the full picture of the phenotype.<br /> (4) Data from both female and male mice are presented.

Weaknesses:

The authors have answered all my comments in the revised version and I find no more weaknesses. Some questions still remain but have been clearly discussed in the new version of the manuscript.

Reviewer #3 (Public Review):

The paper aims to investigate the relationship between anti-S protein antibody titers with the phenotypes & clonotypes of S-protein-specific T cells in people who receive SARS-CoV2 mRNA vaccines. The paper recruited a cohort of COVID-19 naive individuals who received the SARS-CoV2 mRNA vaccines and collected sera and PBMCs samples on different time points. Then, three sets of data were generated: 1). Anti-S protein antibody titers on all time points. 2) Single-cell RNAseq/TCRseq analysis for divided T cells after in vitro stimulation by S-protein. 3) Peptide epitopes for each expanded TCR clone. Based on these, the paper reports two major findings: A) Individuals having more sustained anti-S protein antibody response also have more Tfh-featured S-specific cells in their blood after 2nd-dose vaccination. B). S-specific cross-reactive T cells exist in COVID-19 naive individuals, but most of these T cell clones are not expanded after SARS-CoV-2 vaccination.

The paper's strength is that it uses a very systemic strategy trying to dissect the relationship between antibody titers, T cell phenotypes, TCR clonotypes and corresponding epitopes. The conclusion is solid in general. However, the weaknesses include the relatively small sample size (4 sustainers vs. 4 decliners) and the use of in vitro stimulated cells for analysis, which may 'blur' the classification of T cell subsets. Nevertheless, it may have great impact on future vaccine design because it demonstrated that promoting Tfh differentiation is crucial for the longevity of antibody response. Additionally, this paper nicely showed that most cross-reactive clones that are specific to environmental/symbiotic microbes did not expand post- vaccination, providing important fundamental insights into the establishment of T-cell responses after SARS-CoV-2 vaccination.

Reviewer #2 (Public Review):

Summary:

A deletion analysis of the MSL1 gene to assess how different parts of the protein product interact with the MSL2 protein and roX RNA to affect the association of the MSL complex with the male X chromosome of Drosophila was performed.

Strengths:

The deletion analysis of the MSL1 protein and the tests of interaction with MSL2 are adequate.

Weaknesses:

This reviewer does not adhere to the basic premise of the authors that the MSL complex is the primary mediator of dosage compensation of the X chromosome of Drosophila. Several lines of evidence from various laboratories indicate that it is involved in sequestering the MOF histone acetyltransferase to the X chromosome but there is a constraint on its action there. When the MSL complex is disrupted, there is no overall loss of compensation but there is an increase in autosomal expression. Sun et al (2013, PNAS 110: E808-817) showed that ectopic expression of MSL2 does not increase expression of the X and indeed inhibits the effect of acetylation of H4Lys16 on gene expression. Aleman et al (2021, Cell Reports 35: 109236) showed that dosage compensation of the X chromosome can be robust in the absence of the MSL complex. Together, these results indicate that the MSL complex is not the primary mediator of X chromosome dosage compensation. The authors use sex-specific lethality as a measure of disruption of dosage compensation, but other modulations of gene expression are the likely cause of these viability effects.

A detailed explanation was provided by Birchler and Veitia (2021, One Hundred Years of Gene Balance: How stoichiometric issues affect gene expression, genome evolution, and quantitative traits. Cytogenetics and Genome Research 161: 529-550). The relevant portions of that article that pertain to Drosophila are quoted below. The cited references can be found in that publication.

"In Drosophila, the sex chromosomes consist of an X and a Y. The Y in this species contains only a few genes required for male fertility (Zhang et al., 2020). The X consists of approximately 20% of the genome. Thus, females have two X chromosomes and males have one. Muller (1932) found that the expression of genes between the two sexes was similar but when individual genes on the X were varied in dosage they exhibited a proportional dosage effect. Each copy in a male was expressed at about twice the level as each copy in a female. Females with three X chromosomes are highly inviable but when they do survive to the adult stage, Stern (1960) found that they too exhibited dosage compensation in that the expression in the triple X genotype was similar to normal females and males. Studies in triploid flies found that dosage compensation also occurred among X; AAA, XX; AAA, and XXX; AAA genotypes via upregulation of the Xs, where X indicates the dosage of the X and A indicates the triploid nature of the autosomes (see Birchler, 2016 for further discussion). Diploid and triploid females have a similar per-gene expression but the other five genotypes each must modulate gene expression by different amounts equivalent to an inverse relationship between the X versus autosomal dosage to achieve a balanced expression between the X and the A (Birchler, 1996).

Some years ago, mutations were sought in Drosophila that were lethal to males but viable in females. A number of such mutations were found and termed Male Specific Lethal (MSL) loci (Belote and Lucchesi, 1980). Once the products of these genes were identified, they were found to be at high concentrations on the male X chromosome (Kuroda et al., 1991). One of these genes encodes a histone acetyltransferase that acetylates Lysine16 of Histone H4 (Bone et al., 1994; Hilfiker et al., 1997). The recognition of the MSL complex and its association with the male X was an important set of contributions to an understanding of sex chromosome evolution in Drosophila (Kuroda et al., 2016). Thus, the hypothesis arose that the MSL complex accumulated this chromatin modifier on the male X to activate the expression about two-fold to bring about dosage compensation. Other data that contributed to this hypothesis were that when autoradiography of nascent transcription on salivary gland polytene chromosomes was examined in the MSL maleless mutation, the ratio of the number of grains over the X versus an autosomal region was reduced compared to the normal ratio (Belote and Lucchesi, 1980).

It has been pointed out (Hiebert and Birchler, 1994; Bhadra et al., 1999; Pal Bhadra et al., 2005; Sun et al., 2013a; Birchler, 2016), however, that the grain counts over the X and the autosomes when considered in absolute terms rather than as a ratio show that the X more or less retained dosage compensation and the autosomal numbers are about doubled, i.e. exhibit an inverse dosage effect. The same situation occurs with the msl3 mutation (Okuno et al., 1984), another MSL gene, in that the autoradiographic grain numbers as an absolute measure show retention of X dosage compensation and an autosomal increase. The data treatment to produce an X to A ratio seemed reasonable in the context of the time when all regulation in eukaryotes was considered positive. However, when studies were conducted in such a manner as to assay the absolute effect on gene expression in the maleless mutation, in adults (Hiebert and Birchler, 1994), larvae (Hiebert and Birchler, 1994; Bhadra et al., 1999; 2000; Pal Bhadra et al., 2005), and embryos (Pal Bhadra et al., 2005), the trend was for retention of dosage compensation of X linked genes and an increase in expression of autosomal genes.

In global studies, if the X to autosomal expression does not change between mutant and normal, one can conclude that dosage compensation is operating. However, a lower X to A ratio could be a loss of compensation or an increased transcriptome size from the increase of the autosomes, as suggested by the absolute data of Belote and Lucchesi (1980) and Okuno et al (1984) and was visualized directly in embryos (Pal Bhadra et al., 2005). The transcriptome size in aneuploids can change, which cannot be detected in RNA-seq analyses alone (Yang et al., 2021), so it is an important consideration for studies of dosage compensation. It was recently acknowledged that in MSL2 knockdowns the relative X expression is decreased and a moderate autosomal increase is found (Valsecchi et al., 2021b). A similar trend is evident in the microarray data on MSL2 knockdown in SL2 tissue culture cells (Hamada et al., 2005) and in the roX RNA (noncoding RNAs essential for MSL localization on the male X) mutants (Deng and Meller, 2006). This trend is in fact consistent with the absolute data that suggest an increase in the transcriptome size (Figure 7). A global change in transcriptome size can cause a generalized dosage compensation of a single chromosome to appear as a proportional dosage effect (loss of compensation) to some degree (Figure 7).

Examination of expression in triple X metafemales, where there is no MSL complex, found that X-linked genes generally show dosage compensation but there is a generalized inverse effect on the autosomes, which could account for the detrimental effects of metafemales (Birchler et al., 1989; Sun et al., 2013b). An examination in metafemales of alleles of the white eye color gene that do or do not exhibit dosage compensation in males, showed the same response, namely, increased expression if there was no dosage compensation in males and no difference from normal females for the male dosage-compensated alleles (Birchler, 1992). This experiment demonstrated a relationship between the mechanism of dosage compensation in males and metafemales and implicated the inverse dosage effect in both. An involvement of the inverse effect in Drosophila dosage compensation provides an explanation for how the five levels of gene expression can be explained (Birchler, 1996), whereas an all-or-none presence of a complex on the X does not. The stoichiometric relationship of regulatory gene products provides a means to read the relative dosage at multiple doses to produce the appropriate inverse level.

What then is the function of the MSL complex? It was discovered that the MSL complex will actually constrain the effect of H4 lysine16 acetylation to prevent it from causing overexpression of genes (Bhadra et al., 1999; 2000; Pal Bhadra et al., 2005; Sun and Birchler 2009; Sun et al., 2013a). Indeed, in the chromatin remodeling Imitation Switch (ISWI) mutants, the male X chromosome was specifically overexpressed suggesting that its normal function is needed for the constraint to occur (Pal Bhadra et al., 2005). Independently, the Mtor nuclear pore component shows a similar specific male X upregulation when Mtor is knocked down and this effect was shown to operate on the transcriptional level (Aleman et al., 2021). Interestingly, the increased expression of the X in the Mtor knockdown is accompanied by an inverse modulation of a substantial subset of autosomal genes, illustrating why the constraining process evolved to counteract male X overexpression. The constraining effect might involve a number of gene products (Birchler, 2016) and is an interesting direction for further study.

Furthermore, when the H4Lys16 acetylase was individually targeted to reporter genes, there was an increase in expression (Sun et al., 2013a). However, when other members of the MSL complex were present in normal males or ectopically expressed, this increase did not occur (Sun et al., 2013a). It thus appears that the function of the MSL complex is to sequester the acetylase from the autosomes and constrain it on the X (Bhadra et al., 1999; 2000; Pal Bhadra et al., 2005; Sun and Birchler, 2009; Sun et al., 2013a). Indeed, in the Mtor knockdowns, the X-linked genes with the greatest upregulation were those with the greatest association with the acetylase and the H4K16ac histone mark (Aleman et al 2021), supporting the idea of a constraining activity that becomes released in the Mtor knockdown. When the MSL complex is disrupted, there is an inverse effect on the autosomes that occurs but in normal circumstances the sequestration mutes this effect. The MSL complex disruption releases the acetylase to be uniformly distributed across all chromosomes as determined cytologically (Bhadra et al., 1999) or via ChIPseq for H4Lys16ac (Valsecchi et al., 2021a). Indeed, the quantity of the H4Lys16ac mark only has a proportional effect on gene expression when the constraining activity is disrupted (Aleman et al., 2021) or when the MSL complex is not present (Sun et al., 2013a). Thus, in normal flies, there is a more or less equalized expression of the X and autosomes despite the monosomy for 20% of the genome.

The component of the complex that is expressed in males and thought to organize the complex to the male X, MSL2, was recently found to also be associated with autosomal dosage-sensitive regulatory genes (Valsecchi et al., 2018). MSL2 was found to modulate these autosomal dosage-sensitive genes in various directions, which illustrates that MSL2 has a role in dosage balance that goes beyond the X chromosome. This finding is consistent with the evolutionary scenario that the initial attraction of the complex to the X chromosome was to upregulate dosage-sensitive genes in hemizygous regions as the progenitor Y became deleted for them, with the constraining activity evolving to prevent an overexpression as the amount of acetylase on the male X increased with time (Birchler, 2016).

The MSL hypothesis takes an X-centric view that does not accommodate what is now known about dosage effects across the whole genome. The idea that dissolution of the MSL complex would cause a reduction in expression of the male X-linked genes without any consequences for the autosomes is not consistent with current knowledge of gene regulatory networks and their dosage sensitivity. Indeed, the finding of dosage compensation in large autosomal aneuploids that operates on the transcriptional level (Devlin et al., 1982; 1984; Birchler et al., 1990; Sun et al., 2013c), as well as a predominant inverse effect by the same (Devlin, et al., 1988; Birchler et al., 1990), argues that one must consider the inverse effect for an understanding of the evolution of dosage compensation in Drosophila (and other species). Further discussion of models of Drosophila compensation has been published (Birchler, 2016).

What is likely to be the most critical issue with sex chromosome evolution is the consequences for dosage-sensitive regulatory genes. This fact is nicely illustrated by the retention of these types of genes in different independent vertebrate sex chromosome evolutions (Bellott and Page, 2021). In Drosophila, by contrast, dosage compensation is more of a blanket effect on most but not all X-linked genes despite the fact that many genes on the X are unlikely to have dosage detrimental effects, although dosage-sensitive genes might have played a role as noted above. The particularly large size of the X in Drosophila compared to the whole genome is potentially a contributing factor because such a large genomic imbalance is likely to modulate most genes across the genome. Also, there is no evidence of a WGD in Drosophila as there is in other species for which the inverse effect has been documented (maize, Arabidopsis, yeast, mice, human). These other species have various numbers of retained duplicate dosage-sensitive regulatory genes from WGDs. Thus, the relative change of regulatory genes in aneuploids in these species will not be as great compared to some of their interactors in the remainder of the genome, which could result in lesser magnitudes of some trans-acting effects, similar to how aneuploids in ascending ploidies have fewer effects as described above. The absence of duplicate regulatory genes in Drosophila would predict a stronger inverse effect in general and that could have been capitalized upon to produce dosage compensation of most genes on the X chromosome despite many of them not being dosage critical. While sex chromosome evolution must accommodate dosage-sensitive genes for proper development and viability, it could also be capitalized upon to evolve sexual dimorphisms in expression (Sun et al., 2013c)."

Reviewer #2 (Public Review):

Summary:

In the manuscript, Zhao et al. have carried out a thorough examination of the effects of targeted ablation of resident astrocytes on behavior, cellular responses, and gene expression after spinal cord injury. Employing transgenic mice models alongside pharmacogenetic techniques, the authors have successfully achieved the selective removal of these resident astrocytes. This intervention led to a notable reduction in neuropathic pain and induced a shift in microglial cell reactivation states within the spinal cord, significantly altering transcriptome profiles predominantly associated with interferon (IFN) signaling pathways.

Strengths:

The findings presented add considerable value to the current understanding of the role of astrocyte elimination in neuropathic pain, offering convincing evidence that supports existing hypotheses and valuable insights into the interactions between astrocytes and microglial cells, likely through IFN-mediated mechanisms. This contribution is highly relevant and suggests that further exploration in this direction could yield meaningful results.

Weaknesses:

The methodology and evidence underpinning the study are solid, yet some areas would benefit from further clarification, particularly concerning methodological details and the choice of statistical analyses. Additionally, the manuscript's organization and clarity could be improved, as certain figures and schematics appear inconsistent or misleading.

Reviewer #2 (Public Review):

Summary:

This study used a novel diffusion-weighted pseudo-continuous arterial spin labelling (pCASL) technique to simultaneously explore age- and sex-related differences in brain tissue perfusion (i.e., cerebral blood flow (CBF) & arterial transit time (ATT) - a measure of CBF delivery to brain tissue) and blood-brain barrier (BBB) function, measured as the water exchange (kw) across the BBB. While age- and sex-related effects on CBF are well known, this study provides new insights to support the growing evidence of these important factors in cerebrovascular health, particularly in BBB function. Across the brain, the decline in CBF and BBB function (kw) and elevation in ATT were reported in older adults, after the age of 60, and more so in males compared to females. This was also evident in key cognitive regions including the insular, prefrontal, and medial temporal regions, stressing the consideration of age and sex in these brain physiological assessments.

Strengths:

Simultaneous assessment of CBF with BBB along with transit time and at the voxel-level helped elucidate the brain's vulnerability to age and sex-effects. It is apparent that the investigators carefully designed this study to assess regional associations of age and sex with attention to exploring potential non-linear effects.

Weaknesses:

It appears that no brain region showed concurrent CBF and BBB dysfunction (kw), based on the results reported in the main manuscript and supplemental information. Was an association analysis between CBF and kw performed? There is a potential effect of the level of formal education on CBF (PMID: 12633147; 15534055), which could have been considered and accounted for as well, especially for a cohort with stated diversity (age, race, sex).

Reviewer #3 (Public Review):

Summary:

This manuscript characterizes a chemoattractant response to human serum by pathogenic bacteria, focusing on pathogenic stratins of Salmonella enterica Se. The researchers conduct the chemotaxis assays using a micropipette injection method that allows real-time tracking of bacterial population densities. They found that clinical isolates of several Se strains present a chemoattractant response to human serum. The specific chemoattractant within the serum is identified as L-serine, a highly characterized and ubiquitous chemoattractant, that is sensed by the Tsr receptor. They further show that chemoattraction to serum is impaired with a mutant strain devoid of Tsr. X-ray crystallography is then used to determine the structure of L-serine in the Se Tsr ligand binding domain, which differs slightly from a previously determine structure of a homologous domain. They went on to identify other pathogens that have a Tsr domain through a bioinformatics approach and show that these identified species also present a chemoattractant response to serum.

Strengths and Weaknesses:

This study is well executed and the experiments are clearly presented. These novel chemotaxis assays provide advantages in terms of temporal resolution and ability to detect responses from small concentrations. That said, it is perhaps not surprising these bacteria respond to serum as it is known to contain high levels of known chemoattractants, serine certainly, but also aspartate. In fact, the bacteria are shown to respond to aspartate and the tsr mutant is still chemotactic. The authors do not adequately support their decision to focus exclusively on the Tsr receptor. Tsr is one of the chemoreceptors responsible for observed attraction to serum, but perhaps, not the receptor. Furthermore, the verification of chemotaxis to serum is a useful finding, but the work does not establish the physiological relevance of the behavior or associate it with any type of disease progression. I would expect that a majority of chemotactic bacteria would be attracted to it under some conditions. Hence the impact of this finding on the chemotaxis or medical fields is uncertain.

The authors also state that "Our inability to substantiate a structure-function relationship for NE/DHMA signaling indicates these neurotransmitters are not ligands of Tsr." Both norepinephrine (NE) and DHMA have been shown previously by other groups to be strong chemoattractants for E. coli (Ec), and that this behavior was mediated by Tsr (e.g. single residue changes in the Tsr binding pocket block the response). Given the 82% sequence identity between the Se and Ec Tsr, this finding is unexpected (and potentially quite interesting). To validate this contradictory result the authors should test E. coli chemotaxis to DHMA in their assay. It may be possible that Ec responds to NE and DHMA and Se doesn't. However, currently the data is not strong enough to rule out Tsr as a receptor to these ligands in all cases. At the very least the supporting data for Tsr being a receptor for NE/DHMA needs to be discussed.

The authors also determine a crystal structure of the SeTsr periplasmic ligand binding domain bound to L-Ser and note that the orientation of the ligand is different than that modeled in a previously determined structure of lower resolution. I agree that the SeTsr ligand binding mode in the new structure is well-defined and unambiguous, but I think it is too strong to imply that the pose of the ligand in the previous structure is wrong. The two conformations are in fact quite similar to one another and the resolution of the older structure, is, in my view, insufficient to distinguish them. It is possible that there are real differences between the two structures. The domains do have different sequences and, moreover, the crystal forms, and cryo-cooling conditions are different in each case. It's become increasingly apparent that temperature, as manifested in differential cooling conditions here, can affect ligand binding modes. It's also notable that full-length MCPs show negative cooperativity in binding ligands, which is typically lost in the isolated periplasmic domains. Hence ligand binding is sensitive to the environment of a given domain. In short, the current data is not convincing enough to say that a previous "misconception" is being corrected.

Reviewer #2 (Public Review):

Summary:

The authors generate an optimized small molecule inhibitor of SMARCA2/4 and test it in a panel of cell lines. All uveal melanoma (UM) cell lines in the panel are growth-inhibited by the inhibitor making the focus of the paper. This inhibition is correlated with the loss of promoter occupancy of key melanocyte transcription factors e.g. SOX10. SOX10 overexpression and a point mutation in SMARCA4 can rescue growth inhibition exerted by the SMARCA2/4 inhibitor. Treatment of a UM xenograft model results in growth inhibition and regression which correlates with reduced expression of SOX10 but not discernible toxicity in the mice. Collectively the data suggest a novel treatment of uveal melanoma.

Strengths:

There are many strengths of the study including the strong challenge of the on-target effect, the assays used, and the mechanistic data. The results are compelling as are the effects of the inhibitor. The in vivo data is dose-dependent and doses are low enough to be meaningful and associated with evidence of target engagement.

Weaknesses:

The authors introduce the field stating that SMARCA4 inhibitors are more effective in SMARCA2 deficient cancers and the converse. Since the desirable outcome of cancer therapy would be synthetic lethality it is not clear why a dual inhibitor is desirable. Wouldn't this be associated with more side effects? It is not known how the inhibitor developed here impacts normal cells, in particular T cells which are essential for any durable response to cancer therapies in patients. Another weakness is that the UM cell lines used do not molecularly resemble metastatic UM. These UM most frequently have mutations in the BAP1 tumor suppressor gene. It is not clear if the described SMARCA2/4 inhibitor is efficacious in BAP1 mutant UM cell lines in vitro or BAP1 mutant patient-derived xenografts in vivo.

Reviewer #2 (Public Review):

Summary:

The authors investigated systemic inflammation induced by LPS in various tissues and also examined immune cells of the mice using tight junction protein-based PDZ peptide. They explored the mechanism of anti-systemic inflammatory action of PDZ peptides, which enhanced M1/M2 polarization and induced the proliferation of M2 macrophages. Additionally, they insisted on the physiological mechanism that inhibited the production of ROS in mitochondria, thereby preventing systemic inflammation.

Strengths:<br /> In the absence of specific treatments for septic shock or sepsis, the study demonstrating that tight junction-based PDZ peptides inhibit systemic inflammation caused by LPS is highly commendable. Whereas previous research focused on antibiotics, this study proves that modifying parts of intracellular proteins can significantly suppress symptoms caused by septic shock. The authors expanded the study of localized inflammation caused by LPS or PM2.5 in the respiratory tract, to systemic inflammation, presenting promising results. They not only elucidated the physiological mechanism by identifying the transcriptome through RNA sequencing but also demonstrated that PDZ peptides inhibit the production of ROS in mitochondria and prevent mitochondrial fission. This research is highly regarded as an excellent study with potential as a treatment for septic shock or sepsis.

Weaknesses<br /> (1) The authors focused intensively on acute inflammation for a short duration instead of chronic inflammation.<br /> (2) LPS was used to induce septic shock, but administrating actual microbes such as E.coli would yield more accurate results.<br /> (3) The authors used pegylated peptides, but future research should utilize the optimized peptides to derive the optimal peptide, and further, PK/PD studies are also necessary.

Reviewer #2 (Public Review):

Summary:

This work proposes a synaptic plasticity rule that explains the generation of learned stochastic dynamics during spontaneous activity. The proposed plasticity rule assumes that excitatory synapses seek to minimize the difference between the internal predicted activity and stimulus-evoked activity, and inhibitory synapses try to maintain the E-I balance by matching the excitatory activity. By implementing this plasticity rule in a spiking recurrent neural network, the authors show that the state-transition statistics of spontaneous excitatory activity agree with that of the learned stimulus patterns, which are reflected in the learned excitatory synaptic weights. The authors further demonstrate that inhibitory connections contribute to well-defined state transitions matching the transition patterns evoked by the stimulus. Finally, they show that this mechanism can be expanded to more complex state-transition structures including songbird neural data.

Strengths:

This study makes an important contribution to computational neuroscience, by proposing a possible synaptic plasticity mechanism underlying spontaneous generations of learned stochastic state-switching dynamics that are experimentally observed in the visual cortex and hippocampus. This work is also very clearly presented and well-written, and the authors conducted comprehensive simulations testing multiple hypotheses. Overall, I believe this is a well-conducted study providing interesting and novel aspects of the capacity of recurrent spiking neural networks with local synaptic plasticity.

Weaknesses:

This study is very well-thought-out and theoretically valuable to the neuroscience community, and I think the main weaknesses are in regard to how much biological realism is taken into account. For example, the proposed model assumes that only synapses targeting excitatory neurons are plastic, and uses an equal number of excitatory and inhibitory neurons.

The model also assumes Markovian state dynamics while biological systems can depend more on history. This limitation, however, is acknowledged in the Discussion.<br /> Finally, to simulate spontaneous activity, the authors use a constant input of 0.3 throughout the study. Different amplitudes of constant input may correspond to different internal states, so it will be more convincing if the authors test the model with varying amplitudes of constant inputs.

Reviewer #2 (Public Review):

Summary:

Cell intrinsic signaling pathways controlling the function of macrophages in inflammatory processes, including in response to infection, injury or in the resolution of inflammation are incompletely understood. In this study, Rosell et al. investigate the contribution of RAS-p110α signaling to macrophage activity. p110α is a ubiquitously expressed catalytic subunit of PI3K with previously described roles in multiple biological processes including in epithelial cell growth and survival, and carcinogenesis. While previous studies have already suggested a role for RAS-p110α signaling in macrophages function, the cell intrinsic impact of disrupting the interaction between RAS and p110α in this central myeloid cell subset is not known.

Strengths:

Exploiting a sound previously described genetically mouse model that allows tamoxifen-inducible disruption of the RAS-p110α pathway and using different readouts of macrophage activity in vitro and in vivo, the authors provide data consistent with their conclusion that alteration in RAS-p110α signaling impairs the function of macrophages in a cell intrinsic manner. The study is well designed, clearly written with overall high-quality figures.

Weaknesses:

My main concern is that for many of the readouts, the difference between wild-type and mutant macrophages in vitro or between wild-type and Pik3caRBD mice in vivo is rather modest, even if statistically significant (e.g. Figure 1A, 1C, 2A, 2F, 3B, 4B, 4C). In other cases, such as for the analysis of the H&E images (Figure 1D-E, S1E), the images are not quantified, and it is hard to appreciate what the phenotype in samples from Pik3caRBD mice is or whether this is consistently observed across different animals. Also, the authors claim there is a 'notable decrease' in Akt activation but 'no discernible chance' in ERK activation based on the western blot data presented in Figure 1A. I do not think the data shown supports this conclusion.

To further substantiate the extent of macrophage function alteration upon disruption of RAS-p110α signaling, the manuscript would benefit from testing macrophage activity in vitro and in vivo across other key macrophage activities such as bacteria phagocytosis, cytokine/chemokine production in response to titrating amounts of different PAMPs, inflammasome function, etc. This would be generally important overall but also useful to determine whether the defects in monocyte motility or macrophage lysosomal function are selectively controlled downstream of RAS-p110α signaling.

Furthermore, given the key role of other myeloid cells besides macrophages in inflammation and immunity it remains unclear whether the phenotype observed in vivo can be attributed to impaired macrophage function. Is the function of neutrophils, dendritic cells or other key innate immune cells not affected?

Compelling proof of concept data that targeting RAS-p110α signalling constitutes indeed a putative approach for modulation of chronic inflammation is lacking. Addressing this further would increase the conceptual advance of the manuscript and provide extra support to the authors' suggestion that p110α inhibition or activation constitute promising approaches to manage inflammation.

Finally, the analysis by FACS should also include information about the total number of cells, not just the percentage, which is affected by the relative change in other populations. On this point, Figure S2B shows a substantial, albeit not significant (with less number of mice analysed), increase in the percentage of CD3+ cells. Is there an increase in the absolute number of T cells or does this apparent relative increase reflect a reduction in myeloid cells?

Reviewer #2 (Public Review):

Summary:

This paper introduces a novel approach for improving personalized cancer immunotherapy by integrating TCR profiling with traditional pHLA binding predictions, addressing the need for more precise neoantigen CRC patients. By analyzing TCR repertoires from tumor-infiltrating lymphocytes and applying machine learning algorithms, the authors developed a predictive model that outperforms conventional methods in specificity and sensitivity. The validation of the model through ELISpot assays confirmed its potential in identifying more effective neoantigens, highlighting the significance of combining TCR and pHLA data for advancing personalized immunotherapy strategies.

Strengths:

(1) Comprehensive Patient Data Collection: The study meticulously collected and analyzed clinical data from 27 CRC patients, ensuring a robust foundation for research findings. The detailed documentation of patient demographics, cancer stages, and pathology information enhances the study's credibility and potential applicability to broader patient populations.

(2) The use of machine learning classifiers (RF, LR, XGB) and the combination of pHLA and pHLA-TCR binding predictions significantly enhance the model's accuracy in identifying immunogenic neoantigens, as evidenced by the high AUC values and improved sensitivity, NPV, and PPV.

(3) The use of experimental validation through ELISpot assays adds a practical dimension to the study, confirming the computational predictions with actual immune responses. The calculation of ranking coverage scores and the comparative analysis between the combined model and the conventional NetMHCpan method demonstrate the superior performance of the combined approach in accurately ranking immunogenic neoantigens.

(4) The use of experimental validation through ELISpot assays adds a practical dimension to the study, confirming the computational predictions with actual immune responses.

Weaknesses:

(1) While multiple advanced tools and algorithms are used, the study could benefit from a more detailed explanation of the rationale behind algorithm choice and parameter settings, ensuring reproducibility and transparency.

(2) While pHLA-TCR binding displayed higher specificity, its lower sensitivity compared to pHLA binding suggests a trade-off between the two measures. Optimizing the balance between sensitivity and specificity could be crucial for the practical application of these predictions in clinical settings.

(3) The experimental validation was performed on a limited number of patients (four), which might affect the generalizability of the findings. Increasing the number of patients for validation could provide a more comprehensive assessment of the model's performance