Reviewer #1 (Public Review):

This work by Shen et al. demonstrates a single molecule imaging method that can track the motions of individual protein molecules in dilute and condensed phases of protein solutions in vitro. The authors applied the method to determine the precise locations of individual molecules in 2D condensates, which show heterogeneity inside condensates. Using the time-series data, they could obtain the displacement distributions in both phases, and by assuming a two-state model of trapped and mobile states for the condensed phase, they could extract diffusion behaviors of both states. This approach was then applied to 3D condensate systems, and it was shown that the estimates from the model (i.e., mobile fraction and diffusion coefficients) are useful to quantitatively compare the motions inside condensates. The data can also be used to reconstruct the FRAP curves, which experimentally quantify the mobility of the protein solution.

This work introduces an experimental method to track single molecules in a protein solution and analyzes the data based on a simple model. The simplicity of the model helps a clear understanding of the situation in a test tube, and I think that the model is quite useful in analyzing the condensate behaviors and it will benefit the field greatly. However, the manuscript in its current form fails to situate the work in the right context; many previous works are omitted in this manuscript, exaggerating the novelty of the work. Also, the two-state model is simple and useful, but I am concerned about the limits of the model. They extract the parameters from the experimental data by assuming the model. It is also likely that the molecules have a continuum between fully trapped and fully mobile states, and that this continuum model can also explain the experimental data well.

Reviewer #1 (Public Review):

Kidneys have very high energy needs and they preferentially use lipids as their energy source. Lipid metabolism however poorly understood in the kidney. Lipids accumulate in diseased kidneys, however the mechanism of lipid accumulation is not well understood. The team has studied lipid metabolism in the kidney.

1. The team has first performed some basic lipid accumulation studies in kidneys of healthy mice. Lipid uptake show very significant feeding and fasting differences. It is unclear what stage of the feeding cycle the experiments were performed and whether the team has standardized it.

2. All measurements have been performed using the colorimetric kit, the team should also use mass spec to validate results.

3. The team did not measure lipolysis, fatty acid oxidation or fatty acid synthesis so statements so statements made around these pathways appear to be only speculative. They could measure it or adjust the text.

4. Figure2 is probably the most valuable information in the paper. Lipid accumulation is only measured by staining. The team should also perform Plin stain or other methods to support their statement.

5. The apical vs basal lipid update also seems speculative. I am not sure that we could see these differences. In addition, no quantification is presented to support the findings.

6. For fatty acid uptake the team has analyzed the cd36 KO but not the fatp2 KO mice.

7. The team has analyzed a PT injury model but most proteinuria is the result of glomerular injury. Unclear whether the data is relevant for glomerular disease

8. The human urinary fatty acid quantification would need positive control samples. Clinically we often see lipid droplets in the urine, which is inconsistent with the presented data.

9. No clear conclusion can be drawn from the data

Overall while the project has some interesting elements and the presented data is relatively weak and no clear conclusion can be drawn and the overall message is unclear.

Reviewer #1 (Public Review):

Previous work, much of it by some of the authors, has characterized modification of various ion channels by SUMOYLation. However, there has been relatively little work exploring the effects of such modulation on function of neurons. This manuscript begins by showing quite large reciprocal effects of either enhancing or reducing SUMOYLation in layer 5 pyramidal neurons. One of the effects found is a leftward shift of the voltage-dependence of persistent sodium current. The authors then test the hypothesis that these changes result in part from SUMOYLation of Nav1.2 channels, and using a mouse engineered to eliminate Nav1.2 SUMOYLation, nicely show that modulation of Nav1.2 shifts the voltage-dependence of Nav1.2 and speeds back-propagation of action potentials from the AIS into the soma.

The results in the manuscript are interesting, important, and convincing. Besides being important for describing effects of SUMOYLation on overall neuronal function, the selectivity of SUMOYLation for modifying Nav1.2 but not Nav1.6 channels - and the observation of slowing of back-propagation but not forward propagation from the AIS - adds to the previous data on the distinct functional roles of Nav1.2 and Nav1.6 channels in the AIS.

It is puzzling that the authors focused so much on the effects of SUMOYLation on back-propagation and so little on the large effect on the frequency of firing, which seemed quite dramatic and potentially equally or more important for overall neuronal function than speeding back-propagation. In fact, after introducing the idea that the change in the f-I slope might reflect modulation of Na channels, there is little further discussion of the significance of the changes in firing frequency. Evidently, selective modulation of Nav1.2 channels but not Nav1.6 channels greatly affects firing. Is this explained only by the leftward shift of persistent sodium current from Nav1.2 channels? Or does the leftward shift of Nav1.2 channel gating affect spike threshold? Does modeling of selective modulation of Nav1.2 channels capture these changes in firing frequency and the slope of the f-I curve?

Reviewer #1 (Public Review):

Aguinagalde et al. investigated alternative treatment options for invasive pneumococcal diseases and considered the use of monoclonal antibodies to promote the killing of bacteria. For this to happen, complement deposition and activation are required to occur on the bacterial surface. The authors discovered that hexamerization of IgG strongly augments the recruitment of C1, and specific mutation of the antibodies can support this process and enable efficient phagocytosis and killing of bacteria by neutrophils. Further, sets of in vivo studies support the idea that passive immunization with these modified antibodies improves survival from pneumococcal pneumonia in mice.

Considering vaccine-escape serotypes, the (sometimes) suboptimal vaccine response together with the increased occurrence of strains resistant to antibiotics, the search for alternative treatments is highly warranted, and this study is an excellent example supporting the use of therapeutic antibodies targeting the capsule.

This study is generally very well performed and very well written. The authors conclusively show the importance of mAb hexamerization to augment complement deposition and activation on the surface of pneumococci, which promotes subsequent phagocytosis by neutrophils. Further experiments prove the importance of hexamerization of mAbs and the importance of this complement in the uptake of bacteria by neutrophils. Subsequent in vivo studies showed the therapeutic usefulness of modified antibodies in preventing mortality and bacteremia in female mice. All these data provide strong evidence for the claims of the authors.

I noticed 2 weaknesses, the first related to the killing assays: considering that WT IgG less efficiently promotes complement-mediated phagocytosis of bacteria, one would assume that the ingested bacteria (to be killed) would be lower in neutrophils exposed to this IgG, to begin with - which is not accounted for in the analyses shown.

A second weakness in my mind pertains to the in vivo experiment: the model used obviously requires a very high number of bacteria (the inoculum), somehow indicating that this specific bacterial strain does not lead to progressive infection (i.e. with replicating bacteria) but mice experience a severe acute inflammatory response followed by the rapid elimination of bacteria. This explains the high mortality - and indicates that mice succumb to acute inflammation, rather than the progressive replication of bacteria. To conclusively prove the therapeutic value of those modified antibodies, a clinically more relevant S. pneumoniae model would be helpful.

A third aspect, which should be addressed in the discussion, unless tested and not shown, is how anti-pneumococcal IgM antibodies compare to hexamerized IgGs. Is there any advantage, or do they perform similarly with regards to complement activation?

Reviewer #1 (Public Review):

Histone peptides have been the primary tools for identification and characterization of histone readers. However, in vivo the real substates of histone readers are nucleosomes, in which histone tails exist in dynamic equilibrium between free, accessible state and DNA-bound, inaccessible states. Therefore, other histone modifications, particularly acetylation, impact the accessibility of histone tails to reader proteins. Using modified nucleosomes and known H3K4me3-binding PHD fingers, the authors show that indeed acetylation on nucleosomes has more profound impact than on histone peptide in terms of binding affinity and specificity, likely through increasing H3K4me3 accessibility. The authors further extend the study to investigate the impact of nucleosomal acetylation on H3K4 methyltransferase MLL1C's activity on nucleosome. Surprisingly, MLL1C shows no or very low level of enzymatic activity toward the unacetylated nucleosome, whereas H3 tr-acetylation strongly enhances H3K4 methylation by MLL1C, likely through increasing the accessibility of H3 tail to the enzyme. Consistent with this in vitro data, MS analysis of MCF-7 cells shows that increasing histone acetylation by HDAC inhibitor increases the global levels of H3K4 methylation, particularly on the histone tails with higher levels of acetylation. Together, these findings suggest a model in which acetylation releases nucleosome-bound H3 tails to available H3K4 methyltransferases for subsequent methylation and provide a molecular basis for the long-standing connection between H3 acetylation and H3K4 methylation.

The effect of histone acetylation on histone tail accessibility to reader proteins have been previously reported by several groups, including studies from some of the authors of the current manuscript. The novelty of this study lies on the findings that tail accessibility also affects the enzymatic activity of H3K4 methyltransferases. However, additional evidence is needed to further strengthen the findings.

Reviewer #1 (Public Review):

This manuscript investigates the role of dopamine (DA) release in the dorsal bed nucleus of the stria terminalis (dBNST) investigating sign and goal tracking behavior, response to systemic fentanyl, and to fentanyl self-administration. The studies are largely well conducted and interesting, and the conclusions are justified by the data. The behavioral experiments are elegant and well-conceived, and good thought has been put into how they fit with current theories of learning and reinforcement. As written, however, it is hard to know how much of this data is novel as compared to what is known about DA release in the nucleus accumbens (NAc) which partially comes from similar sources (ventral tegmental area/VTA) but the BNST also receives DA from divergent sources (periaquaductal gray/PAG). The anatomy of DA innervation in the BNST is somewhat distinct and it is doubtful that the optical fiber has the spatial resolution to distinguish between areas that are innervated more by the VTA, which seems more restricted to the juxtacapsular subnucleus, vs. the PAG which more broadly innervates the dlBNST and the oval subnucleus. Further, the release of DA by these two areas may be differentially governed and that is not considered.

Reviewer #1 (Public Review):

Yanis Zekri et al have addressed an important question of the possible role of thyroid hormone (T3) and its nuclear receptor (TR) on local BAT thermogenesis and energy expenditure. In this well-written manuscript and well-carried work, the authors address the above question by A) by generating the BATKO mice by selectively eliminating TR signaling in BAT by knocking-in a TRα1L400R, a dominant negative version of the TRa1 receptor, and by floxing the ThRb gene. They characterized this mouse thoroughly to show that they totally lacked T3 responsiveness. Using qPCR they evaluated the selective abrogation of Thrb and Hr expression in BAT tissue relative to other tissue sites. B) Using time-course transcriptome analysis they then go on to enlist all the T3/TR direct target genes using well-defined criteria and further linking with their ChipSeq data they identified 639 putative target genes which are under the direct control of T3/TR signaling. Interestingly their gene analysis lead them to some target genes directly involved with UCP1 and PGC1α in addition to genes of many other metabolic processes related to BAT thermogenesis. The experiments on denervated BAT on wild-type PTU-fed was a rather neat experiment to eliminate the influence of noradrenergic terminal BAT target genes. Furthermore, the cold exposure experiments and the high-fat diets feeding with the series of complex analyses led them to the conclusion that BAT KO animals suffered from reduced efficiency of BAT adaptive thermogenesis. By comparing the BAT transcriptome of BATKO and CTRL mice after 24h at 4{degree sign}C, the authors further go on to show how BAT TR signaling controls other subsets of genes, especially a wide variety of metabolic regulations, especially lipolysis/fatty acid oxidation. Finally, EdU injection experiments showed a direct effect of T3 on BAT proliferation.

I think it was well thought and well-designed study for understanding the complex action of cell-autonomous T3 regulation of adaptive thermogenesis. The conclusions of this paper are well supported by the data provided.

Reviewer #1 (Public Review):

This work addresses a long-standing question about how tolerance develops at the presynaptic level. That the number of receptors is unchanged following the treatment of animals with opioids was known since the early work using receptor binding assays. The conclusion was that receptor/effector coupling was disrupted was thought to be the primary mechanism underlying tolerance. This work indicates that the location of receptors is critically important in the development of tolerance. This work is groundbreaking and a game changer in the understanding of tolerance at the cellular level.

Reviewer #1 (Public Review):

In this paper, the authors investigate how a large loom-sensitive neuron in grasshoppers becomes sensitive to looming light objects (ON looms) and looming dark objects (OFF looms). They use different visual stimuli, calcium imaging, electrophysiology, and pharmacology to identify how ON and OFF looms each elicit responses in this large neuron.

This topic is important because the segregation of visual signals into ON and OFF channels is fundamental to visual processing, yet these signals must typically be recombined to yield useful visual signals. How and where this happens remains of interest across visual systems. This study finds that, interestingly, ON looms are integrated into the neural response via a pathway that does not retain retinotopic information. The authors suggest potential energetic and functional advantages for the observed arrangement of dendritic integration.

The strength of this paper is in its dissection of the mechanisms of dendritic integration and in its surprising findings. The major weakness in this paper is that when the authors perform detailed modeling of the neural response, they do not provide enough information to evaluate their results. They make some strong arguments about energetic favorability of different synaptic arrangements, which are also not explained in enough detail.

Reviewer #1 (Public Review):

Abdel-Hag, Reem et al. investigated the beneficial effects of a fiber-rich diet in the pathology of α-synuclein overexpressing (ASO) mice, a preclinical model of Parkinson's disease. They found that a prebiotic intervention attenuates motor deficits and reduces microglial reactivity in the substantia nigra and striatum. They extended these findings by doing scRNA sequencing, and they identified the expansion of a protective disease-associated microglia (DAM), a microglial subset previously described during the early stages of disease in several mouse models. Interestingly, the data indicate that microglia do not influence the behavior of ASO mice in the early stages of disease progression. However, microglia are the key mediators of the protective effects of prebiotic treatment in ASO mice. Overall, the conclusions of this paper are well supported by data, but some aspects should be considered to improve the manuscript.

1) Colony-stimulating factor 1 receptor (CSF1R) inhibition has been widely used as a method for microglia depletion, however, the impact of this approach on peripheral immune cells is controversial. The authors elegantly showed that most gut-associated immune cell populations were unaffected by PLX5622. However, CSF1R signaling has been implicated in the maintenance of gut homeostasis. Could it be possible that PLX5622 treatment affects directly the gut microbiome composition? Are the beneficial changes in the gut microbiome composition of a prebiotic diet still maintained in combination with PLX5622? CSF1R inhibitors with low brain penetration such as PLX73086 and therefore unable to deplete resident microglia (Bellver- Landete, Victor et al., Nat Commun, 2019) would be helpful to rule out peripheral off-target effects.

2) The authors claimed that microglial depletion eliminates the protective effects of the prebiotic diet in ASO mice by showing increased levels of aggregated aSyn in the SN (Fig 5G). However, microglial depletion also has the same effect on WT mice. How do authors interpret this result?

3) What is the rationale for doing a long-term (17 weeks) prebiotic intervention? Have the authors considered doing a short-term intervention? The prebiotic diet should change quickly the gut microbiome composition within a few days or weeks.

Reviewer #1 (Public Review):

The software presented in this paper is well documented and represents a significant achievement that breaks new ground in terms of what is possible to render and explore in the web browser. This tool is essential for the exploration of SC2 data, but equally useful for the tree of life and other tree-like data sets.

Reviewer #1 (Public Review):

The paper by Gomez et al. describes investigations employing extracellular recordings of neural spiking in rat pups across different sleep states in primary (M1) and secondary (M2) cortices as well as in the prefrontal cortex (PFC) in response to spontaneous motor activity and tactile stimulation. The authors demonstrate activity across these areas that are associated with active sleep (AS) and identify responses in each region to internally generated movements and external stimuli. Because these results contradict earlier findings in the same areas under anesthesia, they also perform similar recordings in urethane anesthetized animals and show that similar responses are not observed under these conditions. Based on findings from anesthetized and unanesthetized recordings, the authors conclude that early responses associated with AS occur in higher cortical areas, a novel observation for the brain regions studied which had been missed in previous studies because they are absent under anesthesia. Finally, in their discussion, the authors consider the potential roles of state dependent activity in development and contend that differences in activity patterns between motor cortical areas and PFC reflect the diversity or heterogeneity of the inputs they respond to.

The finding presented, particularly the observation of state specific activity in the PFC similar to that observed in more sensory linked areas of the motor cortex, are novel and of interest for their potential relevance to development. However, analysis of the recordings presented in the current manuscript are largely descriptive and do not convey much functional insight beyond simple observation of the phenomenon. In addition, some of the claims made, particularly those related to the differences between PFC and motor cortex, are not clearly supported by the data as currently presented. For these reasons, the study should be revised to including by incorporating additional analysis and potentially further experiments. In addition, clarification of the text and figures is needed to allow the findings to be clearly understood. Resolution of these issues would be important to improve the quality of the manuscript.

Reviewer #1 (Public Review):

Multiple myeloma (MM) is a common often incurable plasma cell disease. Fatty acid binding proteins (FABPs) represent biomarkers for aggressive disease in MM, and pharmacologically inhibiting FABPs kill tumor cells and induce cell cycle arrest. This work demonstrates that targeting FABP5 holds great therapeutic potential for killing diseased cells, with few negative off-target effects on healthy cells.

The authors first found their FABP target by utilizing the Broad Institute's Cancer Dependency Map (DepMap), where only FABP5 exhibited a score in all 20 MM cell lines that demonstrated a strong reliance on FABP5 for survival. To test the effects of FABP5 in MM cell lines they created knock outs, however, the efficacy was relatively low with expression down only 84%. The results demonstrated a reduced proliferation and subsequently, the authors sought to use chemical FABP inhibitors. To understand how FABP5 inhibition could lead to reduced MM cell proliferation, RNAseq was performed. The results analysis demonstrated that MYC, a known oncogene, was found as a central downregulated node. MYC's importance was further confirmed with proteomic analysis.

To help put these findings into clinical context, they investigated the combination of FABP inhibition (FABPi) with dexamethasone, a common therapy for MM patients, where they found that FABPi enhanced dexamethasone's efficacy. Overall, this is an outstanding manuscript that should help advance the overall understanding of MM. The major weakness relates to an unclear mechanism of action (MOA) for FABP5 in MM cells.

Reviewer #1 (Public Review):

Mutations in Doublecortin (DCX), which is a microtubule-binding protein cause lissencephaly. This manuscript by Rao et al. demonstrates the mechanism by which DCX affects retrograde transport in the neurons. Authors show that DCX functions to affect dynein transport in the axon via two different mechanisms - 1. By regulating dynein-microtubule interaction and 2. By regulating the interaction of JIP3 to the dynein motor complex. Interestingly, they have also shown the formation of the dynein-dynactin-JIP3 complex and reconstituted its motility in vitro. Authors demonstrate DCX regulation by affecting the recruitment of the second dynein in the dynein-dynactin-JIP3 complex to affect dynein velocity. Because DCX also regulates Kinesin-3 mediated transport, this work uncovers the role of DCX in regulating opposite polarity motors during neuronal growth. Overall the manuscript is well written, the work is original, experiments are performed carefully and most of the findings justify the conclusions drawn by the authors.

Reviewer #1 (Public Review):

In this manuscript by Kim et al., the authors use live-cell imaging of transcription in the Drosophila blastoderm to motivate quantitative models of gene regulation. Specifically, they focus on the role of repressors and use a 'thermodynamic' model as the conceptual framework for understanding the addition and placement of the repressor Runt, i.e. synthetic insertion of Runt repressor sites into the Bicoid-activated hunchback P2 enhancer. Coupled with kinetic modeling and live-cell imaging, this study is a sort of mathematical enhancer bashing experiment. The overarching theme is measuring the input/output relationship between an activator (bicoid), repressor (runt), and mRNA synthesis. Transcriptional repression is understudied in my opinion. One finding is that the inclusion of cooperativity between trans-acting factors is necessary for understanding transcriptional regulation. Most, if not all, of the tools used in this paper have been published elsewhere, but the real contribution is a deep, quantitative dissection of transcriptional regulation during development. As such, the only real questions for this referee are whether the modeling was done rigorously to produce some general biological conclusions. By and large, I think the answer is yes.

Comments:

Fig. 6 was the most striking figure for this referee, specifically that different placements of Runt molecules on the enhancer lead to distinct higher order interactions. I am wondering if the middle data column in Fig. 6 represents a real difference from the other two, and if so, it seems that the positioning - as opposed to simply the stoichiometry - is essential in cooperativity. This conclusion implies that transcriptional regulation is more precise than what some claim is just a mushy ball of factors close to a promoter. In other words, orientation may matter. Proximity may matter. Interactions in trans matter.

There needs to be at least one prediction which is validated at the level of smFISH / mRNA in the embryo. Without detracting from the effort the authors have expended in looking directly at transcription, if the effects can't be felt by the blastoderm at the level of mRNA/cell, it become difficult to argue for the relevance to development. Also, I feel there is little chance that these measurements can be quantitatively replicated unless translated to the level of total protein or mRNA. Such a measurement (orthogonal quantitative confirmation of the repressor cooperativity result) would also assuage my concern about the time averaging as shown in Fig. S3.

Reviewer #1 (Public Review):

Generally, the strength of this work is the submolecular resolution provided by the MD simulations, while at the same time the weakness is that the results of such simulations are only as good as the force fields used to describe the interactions between gasdermin-D subunits within an assembly and between these subunits and the lipids in the membrane. These simulations yield several interesting results, while also raising various questions, as follows.

The MD simulations are consistent with previous results (e.g., Ding et al., 2016 and Mulvihill et al., 2018, cited in manuscript that gasdermins preferentially bind to anionic lipids, which is not new, but the results are novel here in identifying these interactions at submolecular scale. However, by only showing results for interactions with PI(4,5)P2, without any results for other lipids (if only as a negative control), it remains hard for a reader to assess the relevance and strength of these interactions.

The next result is that the 33-mer "prepore" gasdermin assembly deforms the membrane by just binding - and not inserting into - the membrane. It may seem surprising that such an effect of the membrane may occur without membrane insertion, but it is consistent with previous (experimental) observations for prepore assemblies of the cholesterol dependent cytolysins pneumolysin (Tilley et al., 2005; Faraj et al., Sci Rep 2020) and suilysin (Leung et al., 2014, cited in manuscript). The authors also present data on the 33-mer ring-shaped pore confirmation, not surprisingly finding this pore to be stable.

The more novel results emerge when considering monomers and smaller oligomers. To assess their potential role in pore formation, MD simulations are shown that demonstrate stability of inserted monomers, dimers, etc. of gasdermin-D. Although, as noted by the authors, arc-shaped pores are a common feature for pore forming proteins, it is quite remarkable that a monomer is enough to provide a stable membrane-inserted configuration. The unanswered question, however, is if such smaller gasdermin assemblies will be able to insert into the membrane, presuming that there may be an activation barrier to overcome between prepore (membrane-bound) and pore (membrane-inserted) configuration. That is, while the MD results how that such small oligomers can adopt stable membrane-inserted configurations, they do not justify the authors' claim that such oligomers "create" membrane pores.

The final main and valuable result is about the fate of the lipids in arc-shaped gasdermin assemblies, although the comparison with the ring-shaped pore is lacking (e.g., by initiating the pore assembly with lipids still embedded within the ring). For the arc-shaped pores, the lipids are shown to recede from the inside of the arcs, providing new insight into how the membrane is locally removed. Most intriguingly, the line tension of the lipids appear to "crack" the 16-mer assembly, resulting in a smaller-aperture slit-shaped pores (as have observed by AFM previously). One weakness here is that only a single such cracking event (N=1) is shown to result in the slit-shaped pore.

Another question is how this observation relates to previous MD simulations (by the same lab, Vogele et al, 2019) of pneumolysin pores. Based on MD results and structural details, how do gasdermin-D and pneumolysin compare when viewed through the lens of MD?

Finally, the authors conclude that there are two distinct pathways of membrane pore formation by gasdermin-D (Fig. 5), but do not explain why they exclude formation of larger arc-shaped "adhered prepores" as a pathway of pore formation. Why would larger adhered prepores only insert into the membrane as full rings and not as larger arc-shaped assemblies? That conclusion does not seem justified by the data.

Reviewer #1 (Public Review):

Xu et al show that mutants in three DNA replication proteins, Mcm2, Pole3, and Pole4 have defects in differentiation in a mouse embryonic stem cell (ESC) model. The Mcm2 mutant (called Mcm2-2A), which specifically blocks the interaction of Mcm2 with histones, has defects in multilineage differentiation and neural differentiation, despite having minimal effect on ESC proliferation or gene expression. Mcm2-2A fails to fully silence ESC genes and activate appropriate differentiation genes. Chromatin profiling analyses show Mcm2 binds many promoters. During differentiation, the Mcm2-2 mutant retains K3K27me3 at differentiation gene promoters and reduced accessibility, consistent with the observed defects in gene expression.

The findings that Mcm2-2A has minimal effect on proliferation and gene expression in ESCs, but impairs differentiation are interesting, particularly since this mutant seems to separate the histone binding roles of Mcm2 and its roles in DNA replication. Furthermore, the fact the histone binding function is only necessary when cells exit the pluripotent state is of interest. The studies were reasonably thorough and generally support the conclusions that Mcm2 is important for reshaping histone modifications during differentiation, although the details by which this occurs are not clear. Although the authors used two different strategies for identifying the direct binding sites of Mcm2 on chromatin, Mcm2 enrichment at individual loci was relatively weak, suggesting Mcm2 may localize somewhat diffusely. This somewhat weakens the conclusions about the direct vs indirect effects of Mcm2 on chromatin structure and gene expression.

Overall, this paper reports an interesting set of findings that have a few caveats/limitations regarding how Mcm2 mediates these effects on chromatin during ESC differentiation.

My biggest question is about the Mcm2 CUT&RUN data, which appears to have low signal-to-noise. The authors appear to be aware of this issue, as they also used an Mcm2-FLAG line for CUT&RUN studies, with similarly low signal to noise. To be clear, this may be due to the binding properties of Mcm2, which may bind chromatin relatively broadly, causing few highly enriched peaks to be observed (similar to cohesin complex in the absence of CTCF). However, it makes the Mcm2 binding data difficult to interpret. First, most Mcm2 peaks seem to be near promoters. Promoters often have a small amount of signal in negative control (IgG or irrelevant antibody) CUT&RUN experiments, presumably due to their MNase accessibility. It is not clear to what extent Mcm2 peaks exceed background because no negative control CUT&RUN was performed. The high correlation of FLAG and Mcm2 CUT&RUN libraries might still be evident if some of this signal is due to background at TSSs. Second, the authors call 13,742 peaks, but browser tracks of some example peaks at the Pax6 and Nanog promoters show minimal enrichment relative to surrounding regions (Fig. 5I, 5S1B). I have concerns that some of these peaks called statistically significant are not biologically meaningful.

Reviewer #1 (Public Review):

The sea anemone Nematostella has been previously shown by the authors to exhibit diurnal patterns of movement around their culture dishes -- essentially they move around in darkness and not when in the light. This behaviour is entrained and continues to cycle when animals are kept in constant darkness. In this manuscript the authors test whether temperature cycles can substitute for light cycles in entraining this locomotory behaviour, and it turns out it can. They then test the effects of the two different entraining factors, light and temperature, when applied in phase (aligned exposure cycles) or out of phase (misaligned exposure cycles). The condition the authors call aligned (somewhat arbitrarily) is for the minimal temperature occurring at the beginning of the light cycle. They shift this alignment by 2, 4, 6, 8, 10 and 12 hours; so for example the first altered set has lights on 2 hours after the minimal temperature, and the second set four hours after the minimal temperature. Animals are conditioned to these new entraining cycles for 'at least 2 weeks', then tests applied. The tests are either the behaviour of the animals at a constant temperature in constant darkness, or gene expression under one example of these altered out-of-phase conditions.

The authors view all data within a framework called 'sensory conflict' -- it is even the first two words of the title. This phrase is used in other papers to describe what seems to me an over-simplistic way to view the interactions between different entraining factors. Why is this 'conflict'? If they are two different environmental entraining signals and the cycle of one is shifted relative to the other you are simply changing the alignment of the cycles. By attempting to view the simple change in alignment within the formal framework of 'sensory conflict' the authors are limiting their (and the readers') ability to understand their results; all they see is different levels of conflict, which I would argue are not supported in any way by the results. If you were to shift the alignment of any two in-phase cycles so their peaks were then in anti-phase, you would wipe out any times in which neither of the two cycles was in the negative phase. It would not be 'sensory conflict' if in the anti-phase scenario no clear peak of the behaviour was evident. It would simply be the absence of peaks and troughs of conditions driving the behaviour. The framework used to discuss the data make it difficult to understand.

After exploring impacts on shifting the alignment of light and temperature cycles the authors also examine changes in gene expression patterns in animals entrained to the new regimen. This is a powerful approach as changes in gene expression underlie most of the changes to cellular responses to the environment. The very detailed analysis finds complex changes in transcription patterns. A number of genes associated with biological clocks, or daily cycles of light, have previously been identified in other animals, and a small field has explored them in cnidarians including Nematostella and their relatives, the corals. These cycling genes are found in the results, but temperature was in general a stronger influencer on changes in gene expression. In terms of light, the PAR-bZIP genes once again show up as major responders, and this is strongly supported by the authors' examination of regulatory regions near differentially expressed genes, which are enriched in PAR-bZIP binding sites.

Perhaps the most interesting set of genes identified are those that are only weakly rhythmic when the two entraining factor cycles are aligned, but become sharply rhythmic when they are in antiphase. The new sharp rhythms have an approximately 24 hour periodicity. As mentioned, temperature dominates changes in expression over light. Three core clock genes, Helt, PAR-bZIPa and Clock resist the shift to temperature and maintain their light driven cycles. The clear conclusion is that shifting the two entraining cycles results in large scale shifts in the underlying transcriptome for most rhythmic genes, with temperature dominating light except for core elements of the light responsive clock, and the major shifts are in metabolic processes.

The authors provide a gene level examination of the cellular response to shifting the alignment of two different entraining factors that allows us to view, if not completely understand, how interactions between environmental signals are integrated.

Reviewer #1 (Public Review):

Here, Servello et al explore the role of temperature and the temperature-sensing neuron AFD in promoting protection against peroxide damage. Unlike many other environmental threats, peroxide toxicity is expected to be temperature-dependent, since its chemical reactivity should be enhanced by higher temperatures. The authors convincingly and rigorously show that transient exposure to 25C, a condition of mild heat stress in C. elegans, activates animals' defenses against peroxides but potentially not other agents. Interestingly, this response requires the temperature-sensing AFD neurons, though whether temperature-dependent AFD activity is itself involved in this regulation is not explored. Further, the authors find that temperature regulates AFD's expression of the insulin ins-39 and provide evidence supporting the idea that repression of ins-39 at 25C contributes to enhanced peroxide defense. The authors use transcriptomic approaches to explore gene expression changes in animals in which AFD neurons are ablated, providing evidence that the FoxO-family transcription factor DAF-16 potentiates AFD signaling. However, because AFD ablation triggers effects broader than transient 25C exposure, the significance of these findings for temperature-dependent peroxide defense is somewhat unclear. Additionally, the possibility that DAF-16 (as well as another protective factor, SKN-1) function in parallel to temperature stress is consistent with many of the results shown but is not as thoroughly considered. Together, these studies identify a fascinating example of pre-emptive threat response triggered by the detection of a potentiator of that threat, a phenomenon they term "enhancer sensing." While some predictions of the specificity of this phenomenon remain untested, the paper provides intriguing insight into the potential mechanisms by which it may occur.

Major issues:

The dependence of the enhancer-sensing phenomenon on AFD leads the authors to conclude that the 25C stimulus is sensed by AFD itself, but this needs to be directly tested. To do this, they could ask whether tax-4 function is required in AFD, or use mutants in which AFD's thermosensory function is compromised.

The enhancer-sensing model is fascinating, but as it stands it is somewhat oversold. The authors could tone down the writing, indicating that this model is suggested rather than shown. Alternatively, they could more carefully test some of its predictions - for example by exploring the response to other threats (e.g. some of the toxicants described in Fig. S5) at 20C and 25C in WT and AFD-ablated animals.

The role of ins-39 remains somewhat speculative. Fig 4F shows that ins-39 mutants have a reduced induction of peroxide defense, but it seems that this could be the result of a ceiling effect. The authors' model predicts that overexpression of ins-39, particularly at 25C, should sensitize animals to peroxide damage, a prediction that should be tested directly. Further, the authors seem to assume that AFD is the relevant site of ins-39 function, but this needs to be better supported.

Most of the daf-16 and skn-1 experiments are carried out in AFD-ablated animals, making the relevance of these findings for the 25C-dependent induction of peroxide defense somewhat unclear. As the authors show, AFD ablation causes much more extensive changes than transient 25C exposure, clearly seen in slope of the line in 3C. Further, unlike 25C exposure, AFD ablation is a chronic and non-physiological state. It would be useful for the authors to be cautious in their interpretation of these findings and to be clearer about how strongly they can connect them to the "enhancer sensing" phenomenon. Along these lines, the potentiation idea could be toned down a bit. Much of the data is consistent with parallel function for daf-16 (and skn-1) - for example, Fig 5C indicates additive effects of daf-16 and 25C exposure; 6C shows that AFD ablation still has a clear effect on peroxide sensitivity in the absence of both daf-16 and skn-1; and Fig S8a shows that much of the transcriptional response to AFD ablation (along PC1) is intact in daf-16 animals.

Reviewer #1 (Public Review):

This manuscript investigates the gene regulatory mechanisms that are involved in the development and evolution of motor neurons, utilizing cross-species comparison of RNA-sequencing and ATAC-sequencing data from little skate, chick and mouse. The authors suggest that both conserved and divergent mechanisms contribute to motor neuron specification in each species. They also claim that more complex regulatory mechanisms have evolved in tetrapods to accommodate sophisticated motor behaviors. While this is strongly suggested by the authors' ATAC-seq data, some additional validation would be required to thoroughly support this claim.

Strengths of the manuscript:

1) The manuscript provides a valuable resource to the field by generating an assembly of the little skate genome, containing precise gene annotations that can now be utilized to perform gene expression and epigenetic analyses. The authors take advantage of this novel resource to identify novel gene expression programs and regulatory modules in little skate motor neurons.

2) Cross-species RNA-seq and ATAC-seq data comparisons are combined in a powerful approach to identify novel mechanisms that control motor neuron development and evolution.

Weaknesses:

1) It is surprising that the analysis of RNA-seq datasets between mouse, chick, and little skate only identified 5 genes that are common between the 3 species, especially given the authors' previous work identifying highly conserved molecular programs between little skate and mouse motor neurons, including core transcription factors (Isl1, Hb9, Lhx3), Hox genes and cholinergic transmission genes. This raises some questions about the robustness of the sequencing data and whether the genes identified represent the full transcriptome of these motor neurons.

2) The authors suggest based on analysis of binding motifs in their ATAC-seq data that the greater number of putative binding sites in the mouse MNs allows for a higher complexity of regulation and specialization of putative motor pools. This could certainly be true in theory but needs to be further validated. The authors show FoxP1 as an example, which seems to be more heavily regulated in the mouse, but there is no evidence that FoxP1 expression profile is different between mouse and skate. It is suggested in Fig.5 that FoxP1 might be differentially regulated by SnaiI in mouse and skate but the expression of SnaiI in MNs in either species is not shown.

3) In their discussion section the authors state that they found both conserved and divergent molecular markers across multiple species but they do not validate the expression of novel markers in either category beyond RNA-seq, for example by in situ or antibody staining.

Reviewer #1 (Public Review):

Switching between epithelial and mesenchymal populations is an important stage for cancer growth and metastasis but difficult to study as the cells in this transition are rare. In this study Xu et al investigate changes the splicing regulator environment and changes in specific splice events by monitoring colon cancer cell populations that have epithelial and mesenchymal properties (so are potentially in transition) compared their epithelial partners. Using these potentially transitioning cells should reveal new insights into the causative changes occurring during EMT, a key life threatening step in colon cancer progression, and other cancers too.

The authors were trying to establish if changes in the splicing environment occurred between epithelial and quasi-mesenchymal cells and to what extent this is important for colon cancer in establishing gene expression programmes and cell behaviour related to metastasis. The take home message is that these more "plastic" mesenchymal cells are expressing the mesenchymal transcription factor ZEB1 and reducing expression of the epithelial splicing factor ESRP1 (as well as some other RBPs). The FACS analysis showing that over-expression of ESRP1 alone can switch cell population ratios is very clear and indicates that reduction of this RBP plays a key role in making cells more metastatic. The lentiviral overexpression of CD44s and NUMB2/4 had very dramatic effects on increasing metastatic cellular properties. The clinical stratification analysis of splice isoforms and ZEB1/ESRP1 expression was very informative for understanding what is happening in actual tumours. The methods used and results from these studies are likely to have an impact on understanding the gene expression changes that take place during EMT.

Strengths. The authors have used cell lines that model switching cells between epithelial and quasi-mesenchymal, based on expression of the markers Epcam (epithelial cell adhesion molecule expressed in epithelial cells) and CD44. The study utilises shRNA-mediated knockdown and lentiviral overexpression of ESRP1 and splice isoforms, and monitors endogenous mRNA splice isoforms by RNAseq and qRTPCR, protein isoforms by western, cell surface expression of EpCAM and CD44 using FACS and metastatic potential using a mouse model, and patient gene expression data from TCGA.

Weaknesses: Some of the data here might be novel for colon cancer, but the roles of these RNA binding proteins and ESRP1 target exons are better known in other cancers. Both CD44 and NUMB are known ESRP1 targets already in cells undergoing plasticity (e.g. PMID: 30692202). RBM47 is already known to be downregulated in EMT and quaking upregulated (PMID: 28680090; PMID: 27044866). There is also a lot of literature on ESRP1 expression in cancer and EMT. This should be better discussed.

Reviewer #1 (Public Review):

Detecting a small object is challenging, particularly when the animal is moving. This is because self-generated visual motion interferes with visual perception. Turner et al. established a new method to record neural activity simultaneously from multiple populations of local visual feature detecting neurons (or lobula columnar projection neurons (LCs)) by improving conventional calcium imaging with a new pre-synapse restricted fast calcium indicator and careful image alignment. They found that LCs can be categorized into four types depending on their visual feature selectivity. By simultaneously recording from multi-type LCs, the authors found, for the first time, that several LC types covary their activities, which improves visual feature encoding. Then, the authors performed calcium imaging from walking flies and found that the visual responses are generally suppressed during walking, particularly in small object-detecting populations. Some portion of shared activity among LC populations was explained by the walking-related modulation. Similarly, global visual motion, which is expected from naturalistic fly's walking, suppressed responses to local visual features in a motion coherence-dependent manner. The suppressive effect was prominent when the visual motion was fast and contained low spatial frequency components. Finally, visual and walking-related signals independently suppressed neural responses during saccadic events. These enormous pieces of evidence nicely fit the idea that the fly engages in visual feature processing only during straight walks while the visual inputs are effectively shut down during sharp turns when contamination by self-generated visual motion is non-negligible. On the other hand, responses to important visual stimuli, such as looming produced by predators, are maintained in any conditions. The authors provided a comprehensive view of how a visual circuit operates in a natural condition and further strengthened the growing idea shared across species that sensory perception is dynamically structured during movement.

Joint Public Review:

This manuscript by Harris and Dunn investigates the neurophysiology underlying the optokinetic reflex (OKR), by which image motion on the retina triggers a compensatory eye movement. The strength of the OKR varies with direction of motion, and the present study looks for the origins of that asymmetry in neural signals emerging from the retina, specifically the responses of On-direction-selective retinal ganglion cells (oDSGCs). The authors found that compared to oDSGCs in the inferior retina, superior oDSGCs exhibit higher firing rate and broader tuning width under both high and low contrast conditions. Using whole-cell patch clamp recording, imaging and modeling, they found that the enhanced excitation of superior oDSGCs not only accounts for the higher firing rate of these cells but also broadens their spike tuning curves through spike thresholding. To link these retinal signal to behavior, they used the difference in spike rate between superior and inferior oDSGCs to predict vertical optokinetic responses and show matching results.

This is the first study that systematically compares spiking, synaptic and dendritic properties between superior and inferior oDSGCs. The functional differences between two cell types are interesting and significant, and provide a plausible explanation of OKR. This study also raises the important point that E/I balance is often insufficient to account for the spiking behavior. The data presented are of high quality and comprehensive. Suggestions for revision include clarification of technical issues, and consideration of alternative interpretations. Furthermore, the paper could improve from a better focus on the core results.

Reviewer #1 (Public Review):

The authors sought to establish canine tissue-specific organoids for propagation, storage and potential use in biomedical and translational medicine.

Strengths - The project is ambitious in aim, seeking to raise 6 tissue-specific, stem cell-derived organoid lines.

Weaknesses -

1. While the manuscript refers to stem cell lines, no evidence of progressive organoid morphogenesis has been shown from undifferentiated single stem cells or stem cell clusters. This omission makes it difficult to distinguish true organoids from surviving pieces of parental tissue that the authors actually include within their cultures. The authors infer that high order tissue complexity can be generated within in short term 3D cultures. For example, their kidney organoids contained glomeruli, renal tubules and a Bowman's'capsule. These remarkable findings contrast with a previous study by Chen et al 2019 that showed kidney organoids had restricted morphogenic capacity, forming only simple epithelial dome-like structures (Chen et al 2019). Although the Chen study was cited, the major differences in study findings were not discussed. In the current study, no compelling evidence is provided for the integrated assembly of the glomerular microvascular capillary network, the glomerular epithelial capsule and complex tubular epithelial collecting ducts, during organoid growth.

2. The potential of the organoids for freezing, storage and re-culture is unclear from the data presented.

3. Organoid capacity for regenerative growth in xenograft models has not been tested.

4. Figure 4 lacks appropriate positive and negative tissue controls.

5. Gene expression differences between tissues and organoids are inadequately explained.

6. Methodological detail is sparse. It is not clear how tissue biopsies are obtained, what size they are and how they are processed for organoid preparation.

7. The manuscript as a whole is poorly focussed and difficult to follow. The introduction is repetitive with only weak relevance to the main experiments.

Appraisal - The lack of morphogenesis and xenograft data undermines confidence that the authors have achieved their aims. The above concerns are also likely to hamper utility of the methods for the scientific community.

Reviewer #1 (Public Review):

Voltage-clamp fluorometry combines electrophysiology, reporting on channel opening, with a fluorescence signal reporting on local conformational changes. Classically, fluorescence changes are reported by an organic fluoropohore tethered to the receptor thanks to the cysteine chemistry. However, this classical approach does not allow fluorescent labeling of solvent-inaccessible regions or cytoplasmic regions. Incorporation of the fluorescent unnatural amino acid ANAP directly in the sequence of the protein allows counteracting these limitations. However, expression of ANAP-containing receptors is usually weak, leading to very small ANAP-related fluorescence changes (ΔFs).

In this paper, the authors developed an improved method for expression of full-length, ANAP-mutated proteins in Xenopus oocytes. In particular, they managed to increase the ratio of full-length over truncated proteins for C-terminal ANAP incorporation sites. Since C-terminally truncated P2X receptors are usually functional, it is important to maximize the full-length over truncated protein ratio to have a good correspondence between the observed current and fluorescence. Using their improved strategy, they screened for ANAP incorporation sites and ATP-mediated ANAP ΔFs along the whole structure of the P2X7 receptor: extracellular ligand binding domain (head domain), M2 transmembrane segment (gate), as well as a large extracellular domain specific for the P2X7 subtype, the "ballast" domain. The functional role of this domain and its motions following ATP application are indeed unknown. Monitoring ANAP fluorescence changes in this region following ATP binding provides a unique way to study those questions. By analyzing ATP-induced ΔFs from different parts of the receptors, the authors conclude that the ATP-binding domain mainly follows gating, while intracellular "ballast" motions are largely decoupled from ATP-binding

Strengths of the paper:<br /> This paper provides an improved method for efficient unnatural amino acid incorporation in Xenopus oocytes. Thanks to this technique, they managed to enhance membrane expression of ANAP-mutated P2X7 receptors and observed strong fluorescent changes upon ATP application. The paper furthermore describes an impressive screen of ANAP-incorporation sites along the whole protein sequence, which allows them to monitor conformational changes of solvent-inaccessible regions (transmembrane domains) and cytoplasmic regions that were not accessible to cysteine-reactive fluorophores. This screen was performed in a very thorough manner, each ANAP mutant being characterized biochemically for membrane expression, as well as in term of fluorescence changes. The limitations of the approach -small ΔF upon ATP application on wt receptors, problem of baseline fluorescence variations in presence of calcium- are well explained. Overall, this study should thus not only serve as a guide to anyone willing to perform VCF on P2X7 receptors but it should be useful to the whole community of researchers using unnatural amino acids. Thanks to orthogonal labeling with TMRM and ANAP, the authors managed to simultaneously monitor the motions of the extracellular and intracellular domains of P2X7. Finally, they propose methods to simultaneously monitor intracellular domain motion and downstream signaling.

Weaknesses:<br /> Although the fluorescence screen is impressive and well conducted, the biological conclusions remain superficial at this stage. The paper furthermore lacks quantitative analysis. Finally, the title only reflects a minor part of the paper and is therefore not representative of the paper content.

Reviewer #1 (Public Review):

This study presents a series of experiments that investigate maternal control over egg size in honey bees (Apis mellifera). Honey bees are social insects in which a single reproductive female (the queen) lays all the eggs in the colony. The first set of experiments presented here explore how queens change their egg size in response to changes in colony size. Specifically, they show that queens have relatively larger eggs in smaller colonies, and that egg size changes when queens are transplanted into colonies of a different size (i.e. confirming that egg size is a plastic trait in honey bee queens). The second set of experiments investigates candidate genes involved in egg size determination. Specifically, it shows that Rho1 plays a role in determining egg size in honey bee queens.

A strength of the study is that it combines both manipulative field (apiary) experiments and molecular studies, and therefore attempts to consider broadly the mechanisms of plasticity in egg size. The link between these two types of dataset in the manuscript, however, is not strong. While the two parts are related, the molecular experiments do not follow from the conclusions of the field experiments but rather run in parallel (both using the same initial treatments of queens from large v small colonies).

Another strength of the study is the focus on social cues for egg size control in a social insect. Particularly interesting is data showing that queens suddenly exposed to the cues of a larger colony (even where egg-laying opportunities did not actually increase) will decrease their egg size, in the same way as queens genuinely transplanted to larger colonies. That honey bee queens can control their egg sizes in response to cues in the colony is not unexpected, given that queens are known to vary egg size based on the cell type they are laying into (queen, drone or worker cell). Nevertheless, it is interesting to show that worker egg sizes over time are also mediated by social cues.

A weakness of the study is that the consequence of egg size on egg development and survival in honey bees is not made clear. The assumption is that larger egg size compensates for smaller colonies in some way. Do smaller eggs (i.e. those laid in large colonies) fare worse in smaller colonies than they do in large colonies? Showing that the variation in egg size is biologically relevant to fitness is an important piece of the puzzle.

Also, the relationship between egg number and egg size in honey bees remains rather murky. Does egg size depend at least in part on daily egg laying rate (which is sure to be greater in larger colonies)? The study makes an effort to explore this by preventing queens from laying for two weeks and then comparing their egg size when they resume to those that did not have a pause in laying. Although egg size did not vary between the groups in this case, it is unclear whether the same effect would be seen if queens had simply been restricted from laying at such high rates (e.g. if available empty brood cells had been reduced rather than removed entirely).

Overall this study makes new contributions to our understanding of maternal control over egg size in honey bees. It provides stepping stones for further investigation of the molecular basis for egg size plasticity in insects.

Joint Public Review:

This article reports the results of an observational study in 312 cancer patients to assess post-acute sequelae of SARS-CoV-2 infection (PASC). The descriptive results provide the type of persistant symptoms and their frequency among 188 patients. This information is of interest and adds on to the existing literature.

Strengths:

-The topic is of interest.<br /> -The study has a long-term follow up.<br /> -Data came from both PROs and patients' electronic medical records.

Weaknesses:

-Information about patients' consent and regulatory approval is not provided.<br /> -The relation between the disease stage or anticancer therapy and long covid is missing.<br /> -The impact of long covid on cancer outcome is not shown.

The article describes the main symptoms associated with long covid. However, despite the longitudinal follow-up, a more detailed analysis of the median duration of each symptom is not shown.

Reviewer #1 (Public Review):

The manuscript by Eliazer et al. identifies the Notch ligand Dll4 as a myofiber-derived regulator of muscle stem cells (satellite cells, SCs). The amount of Dll4 surrounding individual SCs on single fiber preparations correlates with the level of Pax7 protein in those cells. Genetic removal of Dll4 from fibers results in: 1) a distribution of Pax7 levels in remaining SCs that skews towards the lower end; and 2) a phenotype similar to, but weaker than, that previously published for removal of the essential Notch pathway transcriptional regulator RBP-J from SCs (including propensity of SCs to spontaneously enter the differentiation program and a deficient regenerative response). Genetic removal of Mib1 from fibers led to loss of Dll4 clustering at SCs and a phenotype similar to loss of Dll4. The authors conclude that Dll4 maintains a continuum of diverse SC states during quiescence, perhaps contributing to which SCs are prone to self-renewal vs. differentiation.

It is accepted that the myofiber is a key niche cell for SCs, but the number of known myofiber-derived niche factors is very small and mechanisms are not well characterized. Furthermore, it is established that Notch signaling in SCs is critical to maintenance of SC quiescence, yet the source and identify of the relevant Notch ligands is not clear. Therefore, the elegant genetic identification of Dll4 as a myofiber niche factor is of high significance. The conclusion about SC states may be somewhat premature, and I have questions about how some of the experiments were performed, but overall this is a very useful paper for the field.

Reviewer #1 (Public Review):

In this study, Sims et al. evaluate how system-level brain functional connectivity is associated with cognitive abilities in a sample of older adults aged > 85 years old. Because the study sample of 146 normal older adults has lived into advanced years of age, the novelty here is the opportunity to validate brain-behavioral associations in aging with a reduced concern of the potential influence of undetected incipient neuropsychological pathology. The participants afforded resting-state functional magnetic resonance imaging (rs-fMRI) data as well as behavioral neuropsychological test assessments of various cognitive abilities. Exploratory factor analysis was applied on the behavioral cognitive assessments to arrive at summary measures of participant ability in five cognitive domains including processing speed, executive functioning, episodic memory, working memory, and language. rsfMRI data were submitted to a graph-theoretic approach that derived underlying functional nodes in brain activity, the membership of these nodes in brain network systems, and indices characterizing the organizational properties of these brain networks. The study applies the classification of the various brain networks into a sensory/motor system of networks and an association system of network, with further sub-systems in the latter that includes the frontoparietal network (FPN), the default-mode network (DMN), the cingulo-opercular network (CON), and the dorsal (DA) and ventral (VA) attention networks. Amongst other graph metrics, the study focused on the extent to which networks in these brain systems were segregated (i.e., separable network communities as opposed to a more singular large community network). Evaluation of the brain network segregation indices and cognitive performance metrics showed that in general higher network functional segregation corresponds with higher cognitive performance ability. In particular, this association was seen between the general association system with overall cognition, and the FPN with overall cognition, and processing speed.

The results worthy of highlighting include the documentation of oldest-old individuals with detectable brain neural network segregration at the level of the association system and its FPN sub-system and the association of this brain functional state notably with general cognition and processing speed and less so with the other specific cognitive domains (such as memory). This finding suggests that (a) apparently better cognitive aging might stem from a specific level of neural network functional segregation, and (b) this linkage applies more specifically to the FPN and processing speed. These specific findings inform the broader conceptual perspective of how human brain aging that is normative vs. that which is pathological might be distinguished.

To show the above result, this study defined functional networks that were driven more by the sample data as opposed to a pre-existing generic template. This approach involves a watershed algorithm to obtain functional connectivity boundary maps in which the boundary brain image voxels separate functionally related voxels from unrelated voxels by virtue of their functional covariance as measured in the immediate data. This is also a notable objective and data-driven approach towards defining functional brain regions-of-interest (ROIs), nodes, and networks that are age-appropriate and configured for a given dataset as opposed to using network definitions based on other datasets used as a generic template.

The sample size of 146 for this age group is generally sufficient.

For the analyses considering the significance of the effect of the brain network metrics on the cognitive variables, the usage of heirarchical regression to evaluate whether the additional variables (in the full model) significantly change the model fit relative to the reduced model with covariates-only (data collection site, cortical thickness), while a possible approach, might be problematic, particularly when the full model uses many more regressors than the reduced model. In general, adding more variables to regression models reduces the residual variance. As such, it is possible that adding more regressors in a full model and comparing that to a reduced model with much fewer regressors would yield significant changes in the R^2 fit index, even if the added regressors are not meaningfully modulating the dependent variable. This may not be an issue for the finding on the FPN segregation effect on overall cognition, but it may be important in interpreting the finding on the association system metrics on overall cognition.

Critically, we should note that the correlation effect sizes (justified by the 0.23 value based on the reported power analyses) were all rather small in size. The largest key brain-behavior correlation effect was 0.273 (between DMN segregation and Processing Speed). In the broader perspective, such effects sizes generally suggest that the contribution of this factor is minimal and one should be careful that the results should be understood in this context.

Overall, the findings based on hierarchical regressions that evaluate the network segregation indices in accounting for cognition and the small correlation magnitudes are basically in line with the notion that more segregated neural networks in the oldest-old support better cognitive performance (particularly processing speed). However, the level of positive support for the notion based on these findings is somewhat moderate and requires further study.

Reviewer #1 (Public Review):

Zukin and colleagues present a high-resolution cryo-EM structure of the yeast histone acetyltransferase complex NuA4, which acetylates histones H4 and H2A. The structural data is of very high quality and was obtained using state-of-the-art methodology. The resulting structural model comprises the rigid "Hub" of the NuA4 complex, consisting of a core module and the Tra1 subunit, while the functional TINTIN and HAT modules remain unresolved, likely due to high flexibility. Nevertheless, the structure provides detailed insights into the architecture of the NuA4 complex and reveals how the subunits in the Hub interact. The authors supplement the structural data with functional characterization of the binding of reconstituted TINTIN and HAT modules to modified nucleosomes, which reveals different specificities of the two. In combination, these data lead to a model for chromatin binding and modification by the NuA4 complex.

Notably, the structural model presented by the authors here differs from a previous structure of the NuA4 core in several key details, including the assignment of densities to subunits (Wang et al., Nat Comm 2018). This is supported by two key lines of evidence. First, the structural data presented here is of higher resolution. Second, the new model presented here is in good agreement with available cross-linking data. Therefore, the revised model presented here is very likely to be more accurate than previous structural models.

One "downside" (if one wishes) of the structural data is the lack of defined density for the HAT and TINTIN modules. However, this is not a shortcoming of the experimental approach employed here but is caused by the inherently flexible nature of this complex. Thus, this is not something that could easily be improved. Indeed, as the authors point out by comparison to the SAGA complex, flexible tethering of the functional modules appears to be common among chromatin-modifying complexes. This issue is elegantly addressed by the authors through a detailed analysis of AlphaFold predicted structures of subcomplexes of the HAT and TINTIN modules, which are in good agreement with previous cross-linking data. This analysis supports the assumption that the poorly defined density observed by the authors originates from these modules.

Taken together, this is a very well-executed study that provides important insights into the molecular basis of chromatin modification. The conclusions drawn by the authors are supported by the structural data. The model for the mechanism of histone acetylation derived by the authors is very plausible based on the available data but remains somewhat speculative in the absence of experimental structural data for the HAT and TINTIN domains in complex with their substrates as well as functional data for the complete NuA4 complex. However, these data provide an important milestone towards a mechanistic understanding of chromatin acetylation and will serve as a framework for addressing the open questions in the future.

Reviewer #1 (Public Review):

The authors present a very nice and timely study detailing how single Pseudomonas aeruginosa cells develop into microcolonies. They demonstrate that motility differences from changes in substrate stiffness are likely responsible for differences in microcolony morphology exhibited at different stiffnesses. The authors further conclude based on modeling data that these motility changes are not due to physiological changes resulting from surface sensing, but rather that mechanical properties of the substrate are responsible for modulating motility differences. However, this conclusion is derived partly from the use of a chpA mutant, which the authors' data demonstrate does not exhibit differences in motility compared to WT. These data are very surprising given that several published studies demonstrate a defect in both pilus synthesis and twitching motility in PilChp mutants (including chpA). It is unclear what the differences are between the presented study and the published literature leading to the disparity in these results.

Major strengths of the manuscript include the detailed analysis of differences in phenotypes on substrates with different rigidities and a link back to changes in motility at the single cell level that could describe these differences.

A weakness of the manuscript is the difference between reported motility phenotypes here and what has been previously published in the literature.

Should the above confounding results be clarified, this work will have a broad impact on the field of microbiology and those studying complex microbial communities as it connects relevant phenotypic differences at the single cell level to mechanical perturbations and multicellular morphologies.

Reviewer #1 (Public Review):

Sukumar et al. examine the orientation selectivity of individual peripheral tactile afferents in humans at the limits of perceptual resolvability. They report that spike rates and similar measures were only moderately informative, while the temporal profile of the spiking responses was highly informative, an effect that was most likely driven by complex sub-field structure of the receptive field itself. Once temporal responses were corrected for scanning speeds, different orientations could be discriminated across a wide range of different scanning speeds.

Strengths: The paper tackles an open question and will inform future research, both electrophysiological and psychophysical. The study is built on high-quality data and the analysis is well described and rigorous.

Weaknesses: The link with the existing psychophysical literature is rather weak, for example there is no discussion on the effects of scanning speeds or other factors that have been described in that literature and that would appear relevant here.

Reviewer #1 (Public Review):

Overall this is an interesting and comprehensive examination of gene expression in Hutchinson-Gilford Progeria using a mix of pre-collected and de novo fibroblast cell lines. Comparisons in expression are made between age groups of Hutchinson-Gilford Progeria patients and with chronological age-matched and "aging" matched normal controls. This work is then extended to explore the impact of the accumulation of progerin on chromosome compartment use and lamina-associated domain distribution. The focus of the remainder of the paper is on the impact of the Hutchinson-Gilford Progeria mutation on signatures reflective of the three cell types that arise from mesenchymal progenitors, namely osteoblasts, chondrocytes, and adipocytes.

Strengths:

This work expands greatly on previous work in this area. Batch smoothing and increased number of cell lines allowed for more power for discovery and for better resolution of the analysis. This powerful data set represents a treasure mine of information that will be of high use to the field.

Weaknesses:

This work is entirely based on fibroblasts. While this weakness is acknowledged by the authors, the validity of the conclusions is not validated in any way to demonstrate that the fibroblast is sufficient in this instance. Rather the authors rely on a series of references from other biological systems. Comparisons are made between a parent and affected offspring, but this is restricted to one pair of samples.

Reviewer #1 (Public Review):

This works makes an important contribution to the study of the cell cycle and the attempt to infer mechanism by studying correlations in division timing between single cells.

Given the importance of circadian rhythms to the ultimate conclusions of the study, I think it would be helpful to clarify the connection between possible oscillatory regulatory mechanisms and the formalism developed in e.g. Equation 3. The treatment appears to be a leading order expansion in stochastic fluctuations of the cell cycle regulators about the mean, but if an oscillatory process is involved, the fluctuations will be correlated in time and need not be small.

Reviewer #1 (Public Review):

In this manuscript the authors describe an approach for controlling cellular membrane potential using engineered gene circuits via ion channel expression. Specifically, the authors use microfluidics to track S. cerevisiae gene expression and plasma membrane potential (PMP) in single cells over time. They first establish a small engineered gene circuit capable of producing excitable gene expression dynamics through the combination of positive and negative feedback, tracking expression using GFP (Figure 1). Though not especially novel or complex, the data quality is high in Figure 1 and the results are convincing. Note that the circuit is excitable and not oscillatory; it is being driven periodically by a chemical inducer. I think the authors could have done a better job justifying the use of an excitable engineered gene circuit system, since you could get a similar result by just driving a promoter with the equivalent time course of inducer. The authors then use a similar approach to produce excitable expression of the bacterial ion channel KcsA, tracking membrane voltage using the voltage-sensitive dye ThT rather than GFP fluorescence (Figure 2). The experimental results in this figure are more novel as the authors are now using the expression of a heterologous ion channel to dynamically control plasma membrane potential. While fairly convincing, I think there are a few experimental controls that would make these results even more convincing. It is also unclear why the authors are now using power spectra to display observed frequencies compared to the much more intuitive histograms used in Figure 1. Finally, the authors move on to use a similar excitable engineered gene circuit approach to produce inducible control of the K1 toxin which influences the native potassium channel TOK1 rather than the heterologous ion channel KcsA (Figure 3). I have a similar reaction to this figure as with Figure 2: the results are novel and interesting but would benefit from more experimental controls. Additionally, the image data shown in Figure 3b is very unclear and could be expanded and improved.

Overall, in my opinion the claims in the abstract and title are a bit strong. I would de-emphasize global coordination and "synchronous electrical signaling" since the authors are driving a global inducer. To make the claim of synchronous signaling I would want to see spatial data for cells near vs. far from K1 toxin producing cells in Figure 3 along with estimates of inducer/flow timescale vs. expression/diffusion of K1 toxin. As I read the manuscript, I see that most of the synchronicity comes from the fact that all cells are experiencing a global inducer concentration.

Reviewer #1 (Public Review):

In this study, Barnes et al. use chronic two-photon imaging of spine calcium in awake mice to examine the functional response types of synapses that undergo homeostatic spine plasticity elicited by sensory deprivation. Spine plasticity is monitored in apical tuft spines of L5 pyramidal cells in the visual or the retrosplenial cortex, following enucleation/visual deprivation or visual and auditory deprivation, respectively. The authors find that spines that convey sensory stimuli, at least those used for testing, do not change but spines whose activity is correlated to intrinsic network activity undergo compensatory strengthening. The experiments are carefully performed, and the writing is clear and concise. The main findings are important in shedding light on the cellular basis by which a network of neurons compensates for the loss of sensory input activity, specifically suggesting a key role of intrinsic network activity. The study is of significant interest to a broad neuroscience readership. Some of the conclusions are not strongly supported by the data as presented, however, and further considerations involving reanalysis of data and/or presentation are warranted.

Reviewer #1 (Public Review):

In this manuscript, the authors explore the mechanisms through which hormone receptors act on their targets to either repress or activate transcription. To do this, they employ a new transgenic tool, a transgenic construct that contains only the ligand binding domain for the ecdysone receptor, EcRLBD, that acts as a sponge for both the steroid hormone ecdysone and for EcR-binding partners. They find that their EcRLBD elicits many of the same phenotypes as other tools used to manipulate the EcR function, suggesting that it acts as a dominant negative. However, it does not elicit all of the same phenotypes as EcR RNAi or overexpression of other dominant negative EcR transgenes (EcRF645A). For example, it interferes with fat body mobilization into the pupal head but does not affect the disintegration of the larval fat body sheets as do the EcR RNAi or EcRF645A.

The authors proceed to provide extensive evidence that the EcRLBD affects both the repression and activation functions of EcR, using EcRE lacz and EcRE GFP transgenes in the developing wing disc. Modifying 20E uptake or metabolism does not affect the ability of EcRLBD to induce precocious de-repression. This is perhaps unsurprising as EcRLBD is proposed to be sponging co-repressors which would be necessary for unliganded EcR repression. However, reducing 20E metabolism does rescue some of the effects of EcRLBD on the activation of gene expression.

The EcRLBD can also induce precocious de-repression of key ecdysone response genes Broad and E93. However, neither of these genes appear to require EcR activation as the later-stage expression is not reduced in EcRLBD larvae. Finally, they demonstrate that the effects they observe when overexpressing EcRLBD in a variety of tissues depend on the ability to bind to a co-repressor Smarter.

There is an impressive amount of work in this manuscript, and the data appears to be of high quality. The experiments are appropriate to the authors' aims, and I feel they will be of broad interest to all those working on developmental physiology and receptor/hormone interactions. Their new transgenic tool is sure to be used by a number of researchers interested in identifying binding partners for EcR across developmental timescales.

I think the most significant weakness of this work is none of the data has been quantified and so it's difficult to judge the extent of variation in samples. Quantification is important, as many of the arguments are based on relative levels of expression. While I feel that the study design supports the authors' aims, the lack of quantitative analysis limits the extent to which the data supports their conclusions.

Reviewer #1 (Public Review):

Dystroglycan, composed of subunits alpha- and beta, is one of the most important non-integrin cellular adhesion complexes, fundamental to establishing a connection between the extracellular matrix and the cytoskeleton in skeletal muscle and in a wide variety of tissues. For a protein that is produced through the ER-Golgi and then trafficked and targeted through exocytosis at the plasma membrane, unraveling the molecular aspects of every step underlining its maturation must be considered to be of utmost importance.

The authors show how the lack of the N-terminal domain of alpha-dystroglycan (aDGN), achieved specifically in the skeletal muscle of model mice, is partially disrupting the decoration with sugars of the central "mucin-like" region of alpha-dystroglycan own central 'mucin like' region. Specifically, it would impact one of the most crucial steps in such a process, i.e. the LARGE1 directed synthesis of matriglycan, with deleterious consequences for dystroglycan function. This is an important work representing another step to drawing a full picture of dystroglycan maturation, with interesting implications for our understanding of dystroglycan biology and pathology.

Strengths:

Arising in part from previous knowledge acquired on the dystroglycan domain organization, a role for the N-terminal of alpha-dystroglycan in the maturation of the full-length subunit could be envisaged. The authors have set a series of experiments whose overall outcome is not in contrast with the hypothesis made (i.e. that of a possible role played by aDGN in matriglycan elongation or modification).

The presence of a link between the molecular structure of matriglycan and the genesis of muscular dystrophy has been further demonstrated.

Weaknesses:

Some of the data, for example, those on the overexpressed aDGN, need to be re-assessed or re-interpreted providing more controls, if possible.

More data should be reported on the histology and biochemistry of different types of muscle from a wider age range of mice. The degree and severeness of the observed muscular dystrophy phenotype remain a bit unclear. Especially, it should be better compared to the one observed in myd mice.

The work does not show how the reaction mediated by LARGE (i.e. the synthesis of matriglycan) would ultimately take place through (or chaperoned by) aDGN, and no clarification is given on whether a direct interaction between aDGN and LARGE1 occurs.

Discussion:

Overall, the results obtained seem to support the conclusions made about the importance of the N-terminal domain of alpha-dystroglycan for the elongation of matriglycan. I feel that there would be an "intrinsic elegance" in a mechanism in which an "internal quality, and length, control" is achieved by means of a protein subdomain belonging to the same protein that needs to be modified, which is processed away once its function is fulfilled. If the data could be further strengthened and opened to some alternative interpretations making the discussion more interesting and stronger, I think that this work can have a high impact in the field of dystroglycan biology and muscular dystrophy.

Reviewer #1 (Public Review):

In this manuscript, Horton et al. seek to define the role of TEs in shaping the murine innate immune regulatory landscape. This work follows previous studies that identified enrichment of RLTR30 elements within STAT1 binding sites in IFN-induced genes. Here, the authors re-analyze previously published transcriptomic and epigenomic datasets to screen for TEs showing signatures of inducible regulatory activity upon IFNG stimulation in mouse macrophages. Data presented in this study provide evidence that a specific B2 SINE subfamily (B2_Mm2) is enriched among regions bound by inducible STAT1 and H3K27ac, which are associated with enhancer activity. Additionally, the authors identify a putative B2_Mm2 derived inducible enhancer for Dicer1 located within its first intron. Cell lines harboring deletions of this element no longer show IFNG-inducible expression of Dicer1 and show a repressive effect on the expression of Serpina genes.

While the data and analyses presented here are of good quality and the authors present some interesting data (specifically that deletion of B2_Mm2.Dicer1 ablates inducible expression of Dicer1), several conclusions drawn by the authors are overstated and not fully supported by the data presented. Furthermore, additional controls are required to firmly establish that B2_Mm2.Dicer1 functions as an inducible enhancer that regulates genes within the Serpina-Dicer1 locus.

Reviewer #1 (Public Review):

This paper proposes a 2D U-Net with attention and adaptive batchnorm modules to perform brain extraction that generalises across species. Generalisation is supported by a semi-supervised learning strategy that leverages test-time monte-carlo uncertainty to integrate the best-predicated labels into the training strategy. Monte-Carlo dropout maps also tend to align with inter-rate disagreement from manual segmentations meaning that they can realistically be used for fast QC. The networks (trained on a range of source domains) have been made publicly available, meaning that it should be relatively simple for users to apply them to their own cohorts, allowing for retraining on a very small number of labelled datasets. Overall the paper is exceptionally well written and validated, and the tool has broad application.

Reviewer #1 (Public Review):

In this paper, the authors use purified Xenopus γ-TuRCs and experiments in cell extract combined with cutting edge imaging techniques to investigate whether binding of the γ-TuNA fragment can activate γ-TuRCs. The authors show that γ-TuNA fragments from both humans and Xenopus are obligate dimers and that dimerization is necessary for γ-TuRC binding. They further show, using direct visualisation of microtubule nucleation from individual purified γ-TuRCs, that γ-TuNA binding increases the nucleation efficiency of γ-TuRCs by ~20 fold, helping to overcome negative regulation by Strathmin.

γ-TuNA, otherwise known as the CM1 domain, CM1 motif or CM1 helix, is well conserved and found within the N-terminal region of proteins across evolution. These proteins bind and recruit γ-TuRCs to MTOCs, such as the centrosome, meaning that γ-TuRC recruitment and activation could be closely linked. Earlier studies had provided strong evidence that binding of γ-TuNA activated γ-TuRCs, hence the name "γ-TuRC mediated nucleation activator" (Choi et al., 2010), and this claim was supported by similar work a few years later (Muroyama et al. 2016). Moreover, several other studies showed that expressing in cells γ-TuNA, or equivalent protein fragments, led to ectopic microtubule nucleation in the cytoplasm, with some of the studies showing that mutations preventing the binding of these fragments to γ-TuRCs ablated this effect (Choi et al., 2010; Lynch et al., 2014; Hanafusa et al., 2015; Cota et al., 2016; Tovey et al., 2021). Collectively, therefore, it was accepted that binding of these fragments somehow activated γ-TuRCs. More recent data, however, including from the authors themselves, had provided evidence that γ-TuNA binding did not activate γ-TuRCs (Liu et al., 2019; Thawani et al., 2020). A major objective of this paper was therefore to help resolve this controversy. The author's data suggest that the ability of these fragments to activate γ-TuRCs depends upon the type and position of tag attached to the N-terminus of the γ-TuNA fragment, with large tags seemingly turning γ-TuNA into a γ-TuRC inhibitor (although they also note that one of the previous studies, which concluded γ-TuNA was an activator, had also used fragments with large N-terminal tags). The authors also insist that the new results benefit from a much-improved γ-TuRC purification protocol that results in higher yield and purity. This purification approach uses the affinity of the γ-TuNA fragment and so could be adopted by others in the field.

The major strength of this paper is directly showing, using very powerful single molecule imaging and their improved protocols, that γ-TuNA is a γ-TuRC activator, thus resolving the controversy that has existed for the last few years. The weakness is that we still don't learn how γ-TuNA binding activates γ-TuRCs (this has been proposed to be via structural changes but other mechanisms can be considered), and thus there is little conceptual advance from the original Choi et al. 2010 paper, which had already concluded that γ-TuNA binding increased the nucleation efficiency of γ-TuRCs. Moreover, the authors do not include experiments with the other proposed γ-TuRC activator, XMAP215, which they have investigated previously (Thawani et al., 2020), and so we are left wondering whether γ-TuNA and XMAP215 work together or as part of separate activation pathways.

Overall, this paper is timely as it finally resolves the controversy over γ-TuNA and it is admirable that the authors are willing to directly address and correct their previous conclusion. The data is solid and well-presented and the text is clear and has appropriate citations. In my opinion, papers that clarify the literature are just as important as those that make conceptual advances.

Reviewer #1 (Public Review):

In this study Zhao et al. investigated the effect of defective R loop removal during Class Switch Recombination (CSR). Using conditional deletion of RNaseH2b in combination with a Senataxin germline knock-out, the authors showed that combined loss of these enzymes, which participate in R loop removal in mouse B cells, is accompanied by an increase of RNA:DNA hybrid formation at the Sµ region and results in AID-dependent Igh locus instability. No changes were detected in germline transcription, AID expression or recruitment, and surprisingly CSR efficiency was unaffected in these cells. Altogether, these observations led the authors to conclude that persistent R loop formation predisposes B cells to increased genome instability at the Igh locus without affecting CSR. In addition, the authors reported that ablation of Senataxin, individually or in combination with RNaseH2, correlates with an increase in insertional/deletional repair at CSR junctions at the expense of blunt joining events. Based on these findings, they suggested a potential link between AID-induced lesions in the absence of efficient R loop removal and the use of A-EJ repair during CSR.

Overall, the study contains many interesting observations in reference to AID-induced DNA damage, Igh locus instability, and S region break processing and repair under conditions of persistent R loop formation. As such, the manuscript has the potential to contribute insights to the biology of R loops' metabolism and their contribution to CSR. However, there are major conceptual and technical concerns in reference to the data and their interpretation:

Key experiments in reference to R loop formation, AID and RNA-Pol II recruitments show high inter-experimental variability. Because of this point, and the unexpected finding of increased AID-dependent Igh genomic instability and mutational load in the absence of any effect on GL transcriptional status, AID recruitment and CSR, the model put forward by the authors is speculative in its current form.

The proposed link between persistent R loop formation and insertional/deletional repair is somewhat not supported by the fact that R loop phenotype is only detected in the double-KO cells, but altered junction profiles are observed in both Setx-/- and double-KO cells.

The involvement of the A-EJ pathway is postulated only on the basis of the analysis of CSR junctions, but no evidence is provided regarding the recruitment (or lack of) of key A-EJ and cNHEJ factors. This is one of the most interesting points of the study but it has not been fully developed.

Reviewer #1 (Public Review):

Here, Canetta and collaborators tackled the following question: is PV cell activity during a sensitive period of neurodevelopment a critical factor in determining prefrontal cortex function in adult mice? To address this question, the authors used viral-based approaches to express chemogenetic molecules specifically in prefrontal cortical PV cells. PV cell activity was then reduced either between P14-50 or P94-P130, by daily CNO injections. To evaluate the effect of PV cell inhibition on prefrontal cortex function, the authors used two readouts: an odor- and texture-based attentional set-shifting task and an EEG-based analysis of brain oscillations. The authors report that mice experiencing PV cell inhibition between P14-50 show impaired responses in the extra-dimensional set-shifting part of the behavioral test and in the task-induced increase in the 65 Hz range power as adults. More in detail, the authors reported a statistically significant difference in the 65 Hz range power in the choice period of correct trials relative to baseline in both control and treated groups. However, the 65Hz frequency range power differed between the choice period of correct and incorrect trials in the naïve mice, but not in those that experienced PV cell inhibition between P14-50. None of these effects were present in mice that experienced PV cell inhibition as adults (P94-130). Of note, optogenetic mediated acute activation in adult mice improved both phenotypes in mice that experienced PV cell inhibition in the juvenile-adolescent periods, while it appeared to have no effects in naïve mice. Overall, these data sets demonstrate that prefrontal cortex PV cell activity during a sensitive period strongly modulates cognitive flexibility and network activity in the adult mouse. This set of carefully designed experiments in vivo constitutes a strength of the study.

In parallel, Canetta et al showed that spontaneous inhibitory postsynaptic currents (sIPSC) and PV cell-evoked IPSC are reduced in adult mice that experienced PV cell inhibition as juveniles. These alterations were not due to PV cell loss or PV cell synapse alterations. The authors reported a trend, which did not reach significance, towards lower PV expression levels. Overall, the cellular and molecular bases of PV cell deficits in adult mice following juvenile PV cell inhibition remain to be explored.

The major implication of this finding is that genetic and/or environmental risk factors affecting PV cell activity in juvenile/adolescent individuals likely contribute to the long term cognitive deficits that have been associated with neurodevelopmental disorders.

Reviewer #1 (Public Review):

In this manuscript, the authors analyze the impact of thymic selection on the TCR repertoire in Nur77-mice by studying the repertoire at different developmental stages using high-throughput sequencing. In combination with different statistical methods and analytical approaches, they searched for specific TCR patterns that could be characteristic for different stages of T cell differentiation. Based on their methods and analyses, they found that there was no evidence for a selection of specific sequences at different stages of development, proposing that negative selection to avoid self-recognition is mainly performed on the collective level rather than at the single-cell level.

The authors use a very interesting and reasonable set of analytical approaches to compare the TCR repertoires at different stages of development. The performed analyses lead to the conclusion that there is no specific pattern of sequences or sequence motifs that are suppressed by negative selection. Their comparisons are valid, but as the authors already point out in their discussion, there could be some aspects that could mask the ability to detect characteristic signatures of TCR repertoires with regard to developmental stage. This includes the separate analyses of alpha- and beta-chain repertoires without considering their combination, as well as the selected experimental system that could affect identification of clearly non-selected cell populations, but also potentially the pooling of the read outs from several mice that could mask signatures on an individual level. The authors provide reasonable arguments for the performed approaches, but some additional analyses might be helpful to corroborate the claims put forward within this manuscript.

Reviewer #1 (Public Review):

This is a very interesting study examining the possibility that high incidence of SARS-CoV2 reactive T cells in apparently COVID 19 naive individuals. While it has been assumed that these emerge as a consequence of cross-reactivity with other coronavirus, this study investigates an alternate possibility, that they may arise through cross-reactivity with unrelated viruses. The authors demonstrate that cross-reactivity between the two viruses is dictated by shared public TCR. This is broadly of interest to the field in understanding how T cell populations emerge in the context of different viral infections.

Reviewer #1 (Public Review):

The study by Ding et al. reports a new mechanism concerning the deletion of bone osteocytes and changes of MSC and HSC linkages in bone marrow. A heterozygous mouse model by DMP-1-driven DTA expression was used to remove osteocytes given that homozygous mice are embryonic lethal. The authors showed that partial deletion cause severe bone loss in both trabecular and cortical bone as well as sarcopenia, osteoporosis and degenerative kyphosis. With single cell RNA seq, they found that osteocyte deletion affects both MSC and HSC lineage commitment. Consistent with bone phenotypes, MSC showed reduced osteogenic differentiation. scRNA seq of HSC showed an increase of myeloid progenitors, neutrophils and monocytes, but a decrease of lymphopoiesis. Interestingly, multiple senescence genes are upregulated, implying premature aging in bone marrow.

Strengths:<br /> The paper shows a new mechanism that numbers of healthy osteocytes have a major impact on differentiation and commitment of bone marrow MSC and HSC progenitor cells and bone marrow cell senescence.<br /> Various thorough techniques (microCT, bone formation and bone histomorphometry, histochemistry) were used to study the bone properties. The data convincing show that partial ablation of osteocytes leads to severe bone loss and compromised bone structure. Interestingly, it does not just affect bone, but has a major effect on muscle. The consist phenotypes are manifested in both male and female mice.<br /> In addition to expected MSC data, they further conducted scRNA seq with HSC and showed the increased cell population in myeloid, monocyte linkage, but reduce in lymphogenic progenitors. Further analysis revealed increased gene expression related to senescence-associated secretory phenotype (SASP). All evidence including reduced viable osteocytes, reduced bone mass and osteogenic commitment of MSC, and increased myeloid and senescence is related to premature aging.<br /> The experiments in general were well designed and conducted with compressive characterization, and the data supported the conclusions. The paper is also logically written and figures were well presented providing clear graphic illustrations.

Minor weaknesses:<br /> The previous study as the authors stated showed a weaker expression of DMP1 in skeletal muscle. The authors provide a clear justification that sarcopenia-like phenotype was unlikely caused by DMP1-cre expression in muscle cells given there is no change of muscle cell numbers. It would be helpful to provide some quantification data of muscle cells to further preclude this possibility.<br /> The underlying molecular mechanism is not shown in the current study, but it might be worthwhile to provide some more-depth discussions and hypotheses concerning how osteocytes could influence cell lineage commitment in bone marrow.

Reviewer #1 (Public Review):

Probably the shortest review I've ever written! Most birds today can lift the upper beak independently of the brain case. This is made possible by a series of mobile joints and bending zones in the skull. To investigate the evolution of this phenomenon, the authors successfully CT-scanned the thoroughly squished skull of the Early Cretaceous stem-bird Yuanchuavis. The detailed description and illustration of the shapes and positions of the skull bones leave no doubt about the conclusion that the toothed snout was unable to move independently of the brain case. They also show, however, that the loss of a few extensions from specific skull bones would have made mobility possible. This plugs a major gap in our understanding of the evolution of mobility within the skull in birds (and by extension elsewhere, notably in the similarly diverse lizards & snakes).

Reviewer #1 (Public Review):

The majority of genetic variants associated with complex human traits reside in the non-coding genome, leading to the assumption that they act through transcriptional regulation. In this work, Connally et al. set to challenge this widespread assumption by showing that genes with plausible links to both severe/familial and common complex forms of the same traits show limited evidence of colocalization with eQTLs or TWAS signals.

More specifically, they first establish that putatively causative genes for severe or familial forms of human traits are enriched for nearby non-coding variants associated with common complex forms of the same traits. Next, using colocalization in tissues related to these traits, they show that only for 7% of these genes the same variant is driving the trait and gene expression associations. In addition, only 6% of these genes are TWAS hits with correct effect direction. Finally, they provide a thorough discussion of possible causes for lack of colocalization and TWAS hits. Among others, the possibility of the incorrect assumption that underlying biological causes of an extreme phenotypic presentation are similar to the causes of the polygenic form, the lack of statistical power of GWAS, eQTL, and/or colocalization analyses, the lack of the right biological context for the eQTL effect, and alternative regulatory mechanisms.

The main conclusion of this work, i.e., that the mechanism by which our genes influence complex traits is generally not their baseline expression, is partly justified by the data and results presented here for the seven traits which show a significant overlap of severe/familial and common complex trait genes. The paper introduces a very useful framework to test this hypothesis by leveraging the joint signals from extreme and polygenic forms of disease to build some form of a set of true positive cases, in which the gene driving trait variation is known. The study also opens up a lot of interesting discussions about alternative hypotheses to fill this gap of 'missing regulation'.

However, the very limited number of traits studied, and the possible alternative explanation of their results, especially by the combination of lack of power and the right biological context, severely limit the generalization of their main conclusion across all/most complex human traits. Adding more traits would be needed to increase confidence in and generalizability of the results supporting the main conclusion. In addition, the study is testing for colocalization with eQTLs identified in bulk post-mortem adult tissues. However, several studies have shown that cell type-dependent/specific eQTLs (Westra et al PLoS Genet 2015, Zhernakova et al. NatGen 2017, Lu et al BioRxiv 2021), as well as response eQTLs (Moyerbrailean et al Genome Res 2016), are particularly enriched in disease association. Due to the limited number of well-powered response or single-cell eQTL studies, it is yet unclear how many of these eQTLs are captured by steady-state bulk tissue eQTLs. This, in combination with the low power of colocalization analyses (Barbeira et al. BioRxiv 2020), is also a very likely explanation of the lack of colocalization of (putatively causative) genes reported here. A better understanding of the degree to which these findings are driven by a lack of sufficiently granular eQTLs is needed.

Reviewer #1 (Public Review):

In this paper, Prince and colleagues present a new toolbox, GLMsingle, for estimating single-trial BOLD responses from fMRI data. This is an important problem, since fMRI is the most used human neuroimaging method, but the signal it yields is notoriously noisy and autocorrelated. This is especially problematic when one is interested in the response to events that occur only a few times, rather than being repeated many times. This is an increasingly common scenario with the increase in complexity of cognitive neuroscience studies, which for instance consist of the presentation of many naturalistic images or videos that are only repeated a few times in the scanner, or studies aimed at studying the response to surprising events, which by definition cannot be repeated many times.

Prince and colleagues convincingly show that their GLMsingle toolbox, which combines three techniques that have been previously used independently but not studied together, strongly improves the reliability of single trial BOLD estimates in two different fMRI datasets. Furthermore, the estimated responses were not only more reliable, they also contained more information about stimulus identity, indicating that the estimates yielded more information about the underlying neural representations. This is an exciting development, and suggests that this toolbox will be useful to many if not most human neuroimaging scientists.

Some questions remain. For instance, would the reduction in autocorrelation in estimates achieved by ridge regression, while desirable when it comes to removing the confounding autocorrelation due to the slow haemodynamics underlying the BOLD signal, also remove genuine neural autocorrelation caused by for instance stimulus-specific adaptation and serial dependence? Also, the two datasets used here had fixed intertrial intervals, would the benefits be as significant for studies employing jittered intertrial intervals?

Overall, the GLMsingle method presented here promises to be of great benefit to human neuroimaging researchers interested in studying infrequent or rare events.

Reviewer #1 (Public Review):

In this manuscript, Kowalczyk and colleagues report on identifying coding and non-coding genetic determinants of hairlessness in mammals using an approach they developed called RER-converge. The approach has previously been employed to examine several different traits in previous publications from this group. The authors determine that hairlessness is associated with relaxed evolutionary constraint at genetic loci and identify both coding genes and non-coding sequencing associated with this phenotype. Several known-hair-associated and novel genes and microRNAs are observed.

This is a strong manuscript with interesting results. It is remarkable how robust this method is. There are a few places where I was not fully convinced of the choice to highlight a gene as "significant" however.

In Figure 4 and the associated text and figure legend the claim is made that non-coding regions exhibit accelerated evolution of matrix and dermal papilla elements. However, the enrichment, even prior to multiple testing correction is not significant. Should this be reported on?

Related to the above, Table 1 includes just one 'significant gene,' with the remainder of the genes highlighted because they have a Bayes Factor ratio >5. Should a gene with a BF HvM be highlighted as a gene "whose evolutionary rates are significantly associated with the hairless phenotype?" Perhaps I am incorrect, but the hypothesis that is being tested by this approach seems distinct from "is the gene associated with hair loss."

Slightly more description of the Bayes factor calculation would be beneficial to the supplement. e.g. is the R package BayesFactor package being used here... or something else?

Why are the qq-plot distributions of non-coding elements so distinct compared to coding? Some comment on this would be appreciated in the main text, even if briefly.

Reviewer #1 (Public Review):

In this manuscript, Chou-Zheng and Hatoum-Aslan follow up on their previous studies that have characterized the collaborations between the type III-A CRISPR-Cas10 Csm complex and various cellular housekeeping nucleases. The authors have previously demonstrated that the Csm complex associates with several nucleases that are implicated in RNA degradation via pulldown and mass spectrometry analysis. They also previously showed that some of these enzymes, including PNPase, are important for CRISPR RNA (crRNA) maturation and for robust anti-phage defense. They now show that a second housekeeping enzyme, RNase R, is required for crRNA maturation. PNPase and RNase R act in concert to produce the mature crRNA. The authors also analyze the interactions between Csm5 and both housekeeping proteins. Finally, they demonstrate that PNPase and RNase R are important for robust anti-plasmid activity when using crRNAs that are complementary to low-abundance transcripts.

This is a well-written paper with clear figures and well-described experiments and results. The experiments in Figures 1 and 2 demonstrating the importance of RNase R for crRNA maturation are excellent. The biochemistry experiments in Figure 2 are especially convincing, in which the authors were able to reconstitute the concerted activities of RNase R and PNPase for crRNA biogenesis. The experiments in Figure 5 implicating PNPase and RNase R in robust anti-plasmid activity when targeting low-abundance transcripts are also clear and convincing, and the result is intriguing. Overall, these experiments provide a new example in a growing list of co-opted host proteins that are important for crRNA biogenesis and CRISPR-mediated defense.

I do have some concerns about experiments in Figures 3 and 4 analyzing interactions between PNPase or RNase R and the Csm5 subunit of the Csm complex, and I believe that some of the authors' conclusions are not fully supported by the evidence presented in these experiments. These concerns, along with a question about their model, are detailed below.

1. The authors used the structure of S. thermophilus Csm5 to guide their design of truncations to probe potential intrinsically disordered regions (IDR1 and IDR2) that may be sites of interaction with PNPase or RNase R. Since the authors submitted their manuscript, an AlphaFold predicted structure of the S. epidermidis Csm5 has been released on the AlphaFold Protein Structure Database. In this model, the IDR2 region is predicted by AlphaFold to be a beta strand at the center of a beta sheet, rather than a disordered region. If the prediction is accurate, deletion of this strand could cause Csm5 to misfold, making it difficult to interpret what causes loss of interaction with PNPase (i.e. deletion of a specific interaction surface versus misfolding of the overall tertiary structure). In light of this, the discussion surrounding these experiments should be altered to include more caveats about the truncations, and conclusions based on this experiment should be softened.

2. The native gels testing interactions between Csm5 and RNase R show a slight change in mobility of RNase R upon the addition of Csm5. Although I agree with the authors' interpretation that this shift could be due to transient interactions between Csm5 and RNase R, it is also possible that the mobility of RNase R is affected simply based on the addition of a large excess of a second protein, even without a specific interaction between the two proteins. As a result, the evidence for direct interaction with Csm5 is limited. Discussion of how RNaseR is recruited by the Csm complex could contain more possible explanations. For example, it is possible that the interaction between RNase R and the Csm complex is mediated by another protein (e.g. PNPase could bridge interaction between the two) or that such an interaction could be stabilized by intermediate crRNA or target RNA binding by the Csm complex.

3. On lines 367-391, the authors propose a model for how PNPase and RNase R may contribute to defense against foreign DNA through their recruitment by the Csm complex to the target transcript. However, their experiments do not test whether PNPase and RNase R must interact with the Csm complex to support anti-plasmid activity. Indeed, it may make more sense for free RNase R to be involved in defense, similar to how free activated Csm6 degrades transcripts non-specifically, rather than only cleaving transcripts in close proximity to the Csm complex. The authors could expand their discussion to mention the possibility that free RNase R or PNPase are acting in anti-plasmid defense.

Reviewer #1 (Public Review):

As an m6A reader, YTHDC1 is known to affect the processing of RNA post-transcriptionally and this article attempted to relate this function in splicing and nuclear export to defects in muscle regeneration after acute injury using LACE-seq. Mechanistically, they provided evidence on m6A-YTHDC1 participation in modulating splicing and target export in myoblast. Additionally, the authors preliminarily confirmed the interaction of YTHDC1 with several key RNA processing factors such as hnRNPG1 to suggest a possible mechanism for m6A-YTHDC1 regulating splicing. Overall it provides new insight into YTHDC1 function in regulating SC activation/proliferation, although some of the data could be improved to fully support the conclusions.

1. The title "Nuclear m6A Reader YTHDC1 Promotes Muscle Stem Cell Activation/Proliferation by Regulating mRNA Splicing and Nuclear Export" seems a bit overstated. Their data are not sufficient to show YTHDC1 regulating nuclear export. From figure 6 we could see some mRNAs export was inhibited upon YTHDC1 loss but intron retention also occurs on these mRNAs, for example, Dnajc14. Since intron retention could lead to mRNA nuclear retention, the mRNA export inhibition may be caused by splicing deficiency. From the data they provided we could not draw the conclusion that YTHDC1 directly affects mRNA export. I think they should not emphasize this point in the title.

2. The mechanism of YTHDC1 promoting muscle stem cell activation/proliferation is not solidified. The authors could strengthen their evidence through bioinformatics analysis or give more discussion. Besides, the previous work done by Zhao and colleagues (Zhao et al., Nature 542, 475-478 (2017).) reported another m6A reader Ythdf2 promotes m6A-dependent maternal mRNA clearance to facilitate zebrafish maternal-to-zygotic transition. Does YTHDC1 regulate mRNA clearance during SC activation/proliferation? The authors should explore this possibility by deep-seq data analysis and provide some discussion.

Reviewer #1 (Public Review):

The group of Ueno also describes that the pkd2l1:cre line previously used to study CSF-cNs was not specific in the adult mouse as it labels olig2+ glial cells in the gray matter. Ueno's group in this study has discovered a method to target mouse CSF-cNs via intracerebroventricular injection of adeno-associated virus (AAV) with a neuron-specific promoter, which enabled them to introduce any genes into CSF-cNs. By doing so, the authors established the structure, connectivity, and function of mouse CSF-cNs in locomotion, recapitulating the findings obtained in zebrafish and lamprey, and extending the recent observations in mice. This study is very conclusive and important for the sensorimotor field in vertebrates.

Ueno's group showed using a set of sophisticated and versatile approaches that :<br /> 1) Most CSF-cNs conserve in mouse the ipsilateral and ventral ascending morphology and are inhibitory neurons;<br /> 2) CSF-cNs project onto axial motor neurons of the neck and back and premotor excitatory neurons in addition to themselves via recurrent connections;<br /> 3) CSF-cNs contribute to enhancing speed during movement on a treadmill, similarly to what has been observed in zebrafish. Chemogenetic inhibition of CSF-cNs reduce speed and increase stride length, indicating that CSF-cNs control body-limb coordination. This exciting finding had been missed in previous investigation.

Reviewer #1 (Public Review):

This manuscript investigates the assembly and function of BUB-1, HCP-1/2 (CenpF) and CLS-2 (CLASP), which they call the BHC module, at the kinetochores. The experiments were executed at a high standard. The data is clearly presented and generally support the conclusions.

Independency of Bub-1 kinetochore localization from Bub-3 is novel and different from humans or yeast. Detailed analysis of interaction domains among the BHC module is well carried out. Showing the redundant roles of the kinetochore and ring localizations of CLS-2 is an interesting result. They further present in vivo and in vitro evidences that Bub-1 and HCP-1/2 are not simply recruiting CLS-2 to the kinetochores, but also enhance the activity of CLS-2. These findings provide a significant insight into how the BHC module functions together. Why the kinetochore and ring localizations of CLS-2 are redundant and how regulation of single microtubules is linked to the overall spindle assembly have not been addressed, but I think that they are beyond the scope of this manuscript.

Reviewer #1 (Public Review):

This manuscript explores how biliary epithelial cells respond to excess dietary lipids, an important area of research given the increasing prevalence of NAFLD. The authors utilize in vivo models complemented with cultured organoid systems. Interesting, E2F transcription factors appear important for BEC glycolytic activation and proliferation.

Much of the work utilizes the BEC-organoid model, which is complicated by the fact that liver cell organoid models often fail to maintain exclusive cell identity in culture. The method used by the authors (Broutier et al., 2016) can lead to organoids with a mixture of ductal and hepatocyte markers. It would be helpful for the authors to further demonstrate the cholangiocyte identity of the organoid cells.

The authors suggest that BECs form lipid droplets in vivo by detecting BODIPY immunofluorescence of liver cryosections. While confocal microscopy would ensure that the BODIPY fluorescence signal is within the same plane as the cell of interest, the authors use a virtual slide microscope that cannot exclude fluorescence from a different focal plane. The conclusion that BECs accumulate lipids does not seem to be fully supported by this analysis.

Several mouse experiments rely heavily on rare BEC proliferation events with the median proliferation event per bile duct being 0-1 cell. While the proliferative effect appears consistent across experiments, a more quantitative approach, such as performing Epcam+ BEC FACS and flow cytometry-based cell cycle analyses, would be helpful.

Finally, it is not yet clear how relevant the findings in this study are to ductular reaction, which is a non-specific histopathologic indicator of liver injury in the context of severe liver disease. In NAFLD, the ductular reaction is uncommon in benign steatosis, and if seen at all, occurs in the setting of substantial liver inflammation and fibrosis (Gadd et al., Hepatology 2014). The authors use a dietary model containing 60 kcal% fat, which causes adipose lipid accumulation as well as subsequent liver lipid accumulation. This diet does not cause overt inflammation or fibrosis that would represent experimental NASH, which typically requires the addition of cholesterol in dietary lipid NASH models (Farrell et al., Hepatology, 2019). While the E2F-driven proliferation may be important for physiologic bile duct function in the setting of obesity, the claim that E2Fs mediate DR initiation would require an additional pathophysiologic model or human data to demonstrate relevance. The authors could clarify this point in their discussion.

Reviewer #1 (Public Review):

In this study, the authors seek to determine the potential role of aging-induced iron accumulation on the effects of hormone replacement therapy (HRT) on atherosclerosis in late postmenopausal women. The authors are commended for the novel and relevant line of investigation and the many complex experiments that they performed. Central to the manuscript, the authors find that high iron levels, in late postmenopausal women as well as in ApoE-/- mice, are associated with reduced expression of the estrogen receptor (ERα). Also, estradiol (E2) treatment in ApoE-/- mice further downregulated ERα expression, but the authors have not sufficiently demonstrated that this occurs in an iron-dependent manner, as the authors have concluded in section 3.3.

The data showed that high iron and E2 treatment trigger the ubiquitin proteasome degradation pathway to degrade ERα via Mdm2-mediated degradation. Interestingly, iron chelation therapy restored ERα expression and attenuated E2-triggered atherosclerosis in late postmenopausal mice. Overall, the authors have concluded that in late postmenopause, iron accumulation prevents the HRT benefit through negative regulation of ERα expression via Mdm2-mediated proteolysis. However, important control groups in the in vivo experiments need to be included to support the conclusions made by the authors and variability in the in vitro experiments diminishes enthusiasm for the findings. Furthermore, in the in vitro experiments, the predominant reduction in ERalpha expression appears to be driven by iron even in the absence of E2, thus making it uncertain how specific these findings are to menopause.

Reviewer #1 (Public Review):

The manuscript by Kado and coworkers investigates the mechanism underlying the partitioning of the cytoplasmic domain of Mycobacteria in specific domains. Earlier work from the authors' laboratories has shown that the membrane consists of two different domains, the intracellular membrane domain (IMD) which is enriched at the pole, and the 'conventional' plasma membrane (termed PM-CW). In work published last year (García-Heredia et al., 2021) the authors described experiments that implicated both an intact cell wall and the polar localizing protein DivIVA in the promotion of membrane compartmentalization in M. smegmatis. The current work provides insight into what it is about the intact cell wall that is really required to facilitate partitioning, by looking at the recovery of membrane partitioning after disruption the membrane fluidizer benzyl alcohol.

In a clever Tn-seq experiment, various genes are identified that are associated with recovery, notably PonA2, a non-essential peptidoglycan synthase. This experiment, and the subsequent characterization of PonA2 as a factor that is important for membrane partitioning, is convincing.

The paper describes an important finding with implications for microbiologists interested in the interplay between peptidoglycan and the membrane, and membrane biologists in general. As the authors state, the study of physical connections between the membrane and matrix or cell wall in live cells is complicated and this study shows how a minimal disruption in peptidoglycan synthesis can affect membrane organization.

However, given the additional role of DivIVA in this process, the claims that the cell wall polymer is "critical" (summary) or "initiates" (title) membrane partitioning are not sufficiently supported by the data.

Strengths:

- The Tn seq experiment and the subsequent confirmation that PonA2 is required for recovery of cells after treatment with benzylalcohol.

- The genetic dissection of PonA2's functionalities, showing that the transglycosylase activity is required for the synthesis of peptidoglycan that allows repartitioning is convincing.

Weakness:

- Since the authors have previously shown a contribution of DivIVA to membrane partitioning (García-Heredia et al., 2021), the authors should consider this in the current work. It is very well possible that, as the authors stated previously, both the PonA2 synthesized cell wall and DivIVA contribute to effective recovery. Given that DivIVA is essential, the Tn-seq experiment will not identify it as a factor contributing to recovery. It is clear that over time, dPonA2 cells recover from the benzyl-alcohol treatment (Fig. 3 - it would also be interesting to see a 12h polarity calculation for dPonA2 as for wild-type in F4C). It is also clear that PonA2 is not the only factor contributing to membrane partitioning, as dPonA2 cells show partitioning before disruption. Importantly, DivIVA does not delocalize upon benzyl alcohol treatment so it is also present at the right location from the moment of wash out to act as a reorganizing factor for membrane partitioning. A recovery experiment in a strain with a DivIVA depletion allele could be very informative and should be included.

- On the one hand, the authors argue that it is "the pre-existing cell wall polymer, rather than active cell wall polymerization" (line 335-6) that is required for membrane partitioning, on the other hand, the authors argue that "PonA2 does not affect membrane-cell wall interaction under basal conditions" (line 215, 378-9). This is counterintuitive - if a priori PonA2 synthesized cell wall is required for recovery (active cell wall polymerization is not needed) one would expect that in a PonA2 knockout strain the interaction between wall and membrane is also (slightly) altered. In fact, several experiments in the paper provide, in my view, an indication for the latter:<br /> o The propidium iodide experiment clearly shows that dPonA2 cells have a membrane that is much more susceptible to damage upon benzyl alcohol treatment. Also, when examining the data, it may be that the TG domain of PonA2 plays a critical role in this process: although the difference in PI positive cells in the population of dponA2 cells compared to TP- ponA2 is non-significant, the difference between the wild-type complementation and the TP- ponA2 allele should also be tested for significance. The distribution in the TP- ponA2 population between the six experiments is rather large and one could argue, looking at the graph, that there is at least a subpopulation in the TP- ponA2 allele that does complement. If so, this would be important as this experiment was done immediately after benzyl alcohol washout and thus does not depend on ongoing activity by the ponA2 mutants.<br /> o There is no comparison between the polarity of marker proteins between wild-type and dponA2 cells. The single cell image in Fig. 4A suggests that Ppm1 is more distributed throughout the dPonA2 cell, whereas in the wild type cell PPm1 is really focused. This would be similar to the slight distribution change in peptidoglycan synthesis (F6C) between wild-type and dPonA2 cells, which is significant.<br /> o The PM-CW fraction (Fig4D) of wild-type cells before benzyl alcohol treatment shows a more focused zone compared to the dPonA2 sample - the latter more resembles the PM-CW fractions of cells treated with benzyl-alcohol.<br /> o The hyperosmotic shock experiment (Fig 4 supplement 2) - it seems that wild-type cells have a larger number of cells that have two plasmolysis bays - 9% with 'subpolar & midcell' bays vs 1% in dPonA2 cells. Also, there is a fraction of cells that have no plasmolysis bay. To me, it would seem better to determine first the % of cells with one or more bays vs cells with no bays, and then compare the number of bays per cell only for the population of cells that show plasmolysis.

Reviewer #1 (Public Review):

The present study was concerned with examining the crucial question of how the brain compensates for its own neural transmission delays, such that representations of moving objects bare some resemblance to their real position rather than their position several hundred ms ago (the time for the retinal input to be transmitted to various points in the hierarchy). It asked whether such compensation may all be seen in early visual nodes of the hierarchy (e.g., V1), with any apparent compensation in subsequent nodes (e.g., V4) generated by simply receiving its information from earlier regions, or whether there is evidence for compensation generated in later nodes.

The authors used a decoding approach to examine this question, where they trained binary classifiers on static locations within a grid and tested on moving stimuli that involved wave-like motion across this grid. One element that makes this study interesting is that it provides a novel demonstration that one can effectively train on static locations and exhibit above-chance performance at tests with moving stimuli. This suggests a common representation of static and moving events. When training and testing on matching timepoints (training=static and test=moving) the classifier performed above chance between 102 and 180 ms after stimulus onset.

They used different epochs to train (static events) to approximate different stages of neural processing and different test epochs (moving events) to examine when the static location representation was predominantly active during motion. The position of a moving object was represented at a time 70 ms shorter than a static object in the same location. For training times 70-80 ms (likely corresponding to V1-V3 activation) the latency of neural activation for moving stimuli approximately corresponded to its real-time position, unlike on static trials. The authors state that subsequent cortical areas do not implement further compensation for neural delays. The claims appear supported by the data.

I found this manuscript exceptionally well-written - well-tuned, clear, and interesting. I also find the patterns in the data interesting and believe this view would be shared by other visual neuroscientists as well as neuroscientists from other fields - where neural delays are likely to prove a universal sticking point to many theories.

Reviewer #1 (Public Review):

Here the authors show using T cells that nuclear and cytoplasmic actin polymerization is differentially mediated by distinct Arp2/3 complexes containing ARPC5L or ARPC5. They further show that nuclear F-actin formation in T cells differs during calcium-mediated signaling versus replication stress response. They also provide information on molecular players mediating these 2 responses in T cells.

All in all this is an interesting study and provides valuable insights into the growing evidence of nuclear actin polymerisation for cell physiology and further highlights an intriguing importance of the composition of a given Arp2/3 complex via incorporation of different Arp subunits.

Reviewer #1 (Public Review):

The authors set out to study the evolutionary origins of acid-sensing ion channels (ASICs) using phylogenetic analysis of hundreds of ASIC related genes from dozens of diverse organisms. Using subsequent gene expression and biophysical characterization, they provide evidence that ASICs evolved far earlier than previously thought. Based on observation that Cndiaria lack ASICs yet all major forms of Bilaterians possess ASICs, they conclude that ASICs emerged after these two branches diverged, approximately 680 million years ago.

Furthermore, Bilaterians are divided into three groups: Deuterostomes (which include chordates), Xenacoelomorpha and Protostomes. All three of these groups contain functional ASIC sequences. However, the Protostomes are more complicated. These are further subdivided into Spiralla and Ecdysozoa (which include arthropods). The Spiralla possess functional ASIC sequences while the Ecdysozoa seem to have lost them. The authors suggest this maybe because ASICs were initially expressed in ectodermal ciliated cells where they helped drive locomotion in response to environmental cues and other behaviors. However, when ecdysozoa lost the ectodermal ciliated cells, they also were able to dispense with ASICs, hence modern ecdysoza such as drosophila do not have ASIC sequences.

This work combines several disparate techniques to supply insight into the history of ASIC evolution. The core findings are well supported by the data and will be of general interest to the ASIC community as well as the ligand-gated channel field more broadly. However, the main weakness of the paper is the author's limited discussion of what part(s) of the receptor, if any, are preserved or altered between groups. This work could be much more impactful if the sequences were more thoroughly explored and tied to functional and structural differences.

Reviewer #1 (Public Review):

In this paper, Quiniou and colleagues show via orthogonal methods human thymopoiesis releases a large population of CD8+ T cells harboring a/b paired TCRs that (i) have high generation probabilities and (ii) a preferential usage of some V and J genes, (iii) are shared between individuals and (iv) can each recognize and be activated by multiple unrelated viral peptides, notably from EBV, CMV and influenza.

Major strengths of the paper:

Quiniou et al. generated single-cell sequencing datasets of the earliest stages of TCR beta chain gene recombination. And then showed that a subset of them is highly clustered also having high generation probability.

They show that these T cells can bind multiple antigens, both via the use of public antigen-specific datasets as well as corroborating experimental TCR expression and binding essays.

Minor weaknesses:

To what extent is TCR clustering and high pgen and cross-individual sharing correlated? What is the pgen of the sequences clustered with the high pgen cells? Can you comment on the correlation between these three phenomena? In other words, to what extent is this surprising to see that highly clustered TCRs have higher pgen and are more shared?

Potential Impact of the paper:

This work highlights an intrinsic property of the adaptive immune response: to generate TCRs with high generation probability that can efficiently bind multiple antigens. This finding has, therefore important impact on drug discovery and vaccine design.

Reviewer #1 (Public Review):

This is a very interesting manuscript by Pan et al. The authors focused their analysis on TCR degeneracy and systematically characterized it in single TCR antigen sequencing data, bulk TCR sequencing data, and single-cell RNA sequencing data and obtained unexpected results. Later, authors also used TCR degeneracy as a metric to predict the immunotherapy outcomes. The analyses are sound, and the manuscript is well written.

Reviewer #1 (Public Review):

This study investigates how pathogens might shape animal societies by driving the evolution of different social movement rules. The authors find that higher disease costs induce shifts away from positive social movement (preference to move towards others) to negative social movement (avoidance from others). This then has repercussions on social structure and pathogen spread.

Overall, the study comprises a good mixture of intuitive and less intuitive results. One major weakness of the work, however, is that the model is constructed around one pathogen that repeatedly enters a population across hundreds of generations. While the authors provide some justification for this, it does not capture any biological realism in terms of the evolution of the pathogen itself, which would be expected. The lack of co-evolution in the model substantially limits the generality of the results. For example, a number of recent studies have reported that animals might be expected to become very social when pathogens are very infectious, because if the pathogen is unavoidable they may as well gain the benefits of being social. The authors make some arguments about being focused on introduction events, but this does not really align well with their study design that carries through many generations after the introduction. Given the rapid evolutionary dynamics, perhaps the study could have a more focused period immediately after the initial introduction of the pathogen to look at rapid evolutionary responses (albeit this may need some sensitivity analyses around the parameters such as the mutation rates).

A final, and much more minor comment is whether this is really a paper about movement. The model does not really look at evolutionary changes in how animals move, but rather at where they move. How important is the actual movement process under this model? For example, would the results change if the model was constructed without explicit consideration of space and resources, but instead simply modelled individuals' decisions to form and break ties? (Similar to the recent paper by Ashby & Farine https://onlinelibrary.wiley.com/doi/full/10.1111/evo.14491). It might help to provide more information about how putting social decisions into a spatially explicit framework is expected to extend studies that have not done so (e.g.., because they are analytical).

Reviewer #1 (Public Review):

In this manuscript, the authors investigated the role of a long noncoding RNA VPS9D1-AS1(VPS) in colorectal cancer (CRC). They found that a high level of VPS was negatively associated with T cell infiltration in CRC patients; in cell line-derived xenograft models or a conditional knock-in mouse model, VPS overexpression enhanced tumor growth and suppressed the infiltration of CD8+ T cells, which was reverted by VPS antisense oligonucleotide (ASO) treatment. They also investigated the molecular mechanisms underlying VPS function and revealed a VPS/TGF-β/ISG signaling cascade in tumor cells and crosstalk between tumors and T cells depending on IFNAR1 level.

The authors had performed extensive analyses on the functions of VPS using patient samples, CRC cell lines, xenograft tumors, and drug-induced tumors, and the data were of relatively good quality; they targeted VPS overexpression in cell line-derived xenografts or mouse tumors by ASO treatment as potential therapeutics, although the overexpression level may not be physiologically relevant.

The authors also made great efforts to explore the mechanisms in vitro and proposed a very interesting model of ribosomes/VPS/TGF-β/ISG signaling axis in tumor cells and opposing regulation on IFNAR1 in tumor and T cells; however, the mechanistic model was tested in vitro, not in cell line-derived xenografts or mouse tumors used in the study, which undermined the authors' claims.

Reviewer #1 (Public Review):

Harkin and colleagues present a very interesting study in utilizing cell-specific excitability properties of identified 5-HT raphe neurons and SOM-interneurons to specify computational integrate and fire neuronal models. In turn, they explore the resulting properties of these biophysically augmented GIF (aGIF) populations. While their electrophysiological characterization of firing properties is mainly confirmatory and their characterization of relevant conductances limited to fast-inactivating A-type currents, the identified features of the aGIF models are highly relevant for a better understanding of neuromodulatory systems. In particular the link between the strong spike firing adaptation of 5-HT neurons and the associated ability to detect transient changes in synaptic input are important. However, the biophysical mechanisms of adaptation (e.g. SK channels) and its variability across 5-HT neurons has not been experimentally explored.

Reviewer #1 (Public Review):

Overall this is an interesting study of the function of ATP6AP2 in the osteoblastic lineage. This gene is unstudied in the osteoblast, despite its known role in WNT signaling. In this study, the authors first show that loss of this gene in mature osteoblasts results in a strong cortical bone phenotype, with reduced osteocyte numbers and disorganized collagen. This phenotype is not present at birth but progressively worsens as the animals reach weaning age. In the compact bone, they show that loss of ATP6AP2 results in osteocytes largely devoid of dendritic processes. Loss of this gene starting at the osteocyte stage results in a milder phenotype. They then show that the osteocytes presenting have reduced MMP14 and that partial restoration of MM14 attenuates the severity of the cortical phenotype.

Strengths

This study uses cutting-edge microscopy to thoroughly characterize how and where the loss of ATP6AP2 in either the mature osteoblast or the osteocyte results in disorganized bone. Innovative proteomics techniques are used to identify cell surface proteins, including MMP14 that may mediate this phenotype. Two cre-drivers are used to determine when in the osteoblast-osteocyte lineage this gene has the maximum effect. Lastly, in vivo lentivirus replacement is used to test if the replacement of MMP14 can rescue the phenotype. This latter experiment solidifies the importance of MMP14 as a major player in the downstream sequela of ATP6AP2 action.

Weaknesses

Unfortunately, all of the histology is conducted on demineralized bone, and counts of osteoblasts and osteoclasts on the bone surface are not presented. This reduces the ability to interpret all downstream work. As such, the extent of the mineralization defects is difficult to interpret. Much of this paper is focused on the osteocyte, which is curious as the phenotype of the mature osteoblasts ATP6AP2 knockout mice is so much more severe than that of the osteocyte ATP6AP2 knockout mice. While it is clear how MMP14 was identified as being deficient in the mature osteoblasts ATP6AP2 knockout cells, it is not obvious how this gene became the sole focus of the remainder of this paper. This phenotype progresses as the mice become ambulatory and therefore weight bearing on their limbs. This could partially explain the presentation of the mouse phenotype, but this is not discussed.

Reviewer #1 (Public Review):

In this work, Bentley et al. describe the development and use of a novel microfluidic platform to study motility of green algae. By confining algae to circular corrals of various diameters (and with a height that renders the system quasi-two-dimensional), the authors gather extremely long time series of the swimming trajectories under various degrees of lateral confinement, in the presence of several different kinds of perturbations.

The data is presented in a number of ways, most importantly by means of transitions between the three characteristic states of motion for these algae. This allows contact to be made with ideas from nonequilibrium dynamical systems by examining the transition probabilities between those states and identifying nonequilibrium characteristics of the fluxes between them.

Overall the work is extremely impressive in terms of the data acquisition and careful time series analysis. The work falls short though in not following through on the many interesting observations that can be deduced from the data to come to precise conclusions about the biology and physics. For example, we see in Figs. 2 and 3 the effects of confinement on the trajectories, leading to clearly chiral motion at the strongest confinement. I would have expected the next step of the analysis to be a study of this problem in the context of, say, a Fokker-Planck equation for the probability distribution function for orientations, complete with boundary conditions that encode the scattering laws that we know from prior work by Kantsler et al. and others. Similar comments can be made about the other observations, which are followed up with any clear mechanistic analysis or comparison with theory.

The example above suggests that this paper, in its current form, is more akin to a "Methodology" paper than one that discovers new phenomena and explains them.

Reviewer #1 (Public Review):

The clearly stated authors' aims were to test the association of a type 2 diabetes polygenic risk score (PRS) generated from a multiethnic GWAS with GDM and related traits (Fasting glucose and 2 hour post load glucose, and area under the curve glucose) in pregnant South Asian women from two large well characterized cohorts. Also to test the population attributable fraction of the PRS on GDM and to determine whether the effect of the PRs is modulated by other GDM risk factors including age, BMI, diet quality, birth country, education and parity.

Major strengths are the large and well characterized populations used for generation of the PRS and GDM data and for testing the performance of the PRS thus providing clear results.

The authors achieved their aims and the results support their conclusions. The work provides insight into which South Asian women are predisposed to GDM.

Who develops GDM and which of these women then develop T2DM later are major research questions of public health importance. This study provides important insight into the first question and provides a hypothesis for the second question but the PRS won't be available for clinical use in the near future.

Ultimately the aim is to find practical ways to direct resources to those at highest risk in detecting and managing GDM and its complications and in identifying and intervening in those at highest risk of developing T2DM later on.

Reviewer #1 (Public Review):

In one of the most creative eDNA studies I have had the pleasure to review, the authors have taken advantage of an existing program several decades old to address whether insect declines are indeed occurring - an active area of discussion and debate within ecology. Here, they extracted arthropod environmental DNA (eDNA) from pulverized leaf samples collected from different tree species across different habitats. Their aim was to assess the arthropod community composition within the canopies of these trees during the time of collection to assess whether arthropod richness, diversity, and biomass were declining. By utilizing these leaf samples, the greatest shortcoming of assessing arthropod declines - the lack of historical data to compare to - was overcome, and strong timeseries evidence can now be used to inform the discussion. Through their use of eDNA metabarcoding, they were able to determine that richness was not declining, but there was evidence of beta diversity loss due to biotic homogenization occurring across different habitats. Furthermore, their application of qPCR to assess changes in eDNA copy number temporally and associate those changes with changes to arthropod biomass provided support to the argument that arthropod biomass is indeed declining. Taken together, these data add substantial weight to the current discussion regarding how arthropods are being affected in the Anthropocene.

I find the conclusions of the paper to be sound and mostly defensible, though there are some issues to take note of that may undermine these findings.

Firstly, I saw no explanation of the requisite controls for such an experiment. An experiment of this scale should have detailed explanations of the field/equipment controls, extraction controls, and PCR controls to ensure there are no contamination issues that would otherwise undermine the entirety of the study. At one point in the manuscript the presence of controls is mentioned just once, so I surmise they must exist. Trusting such results needs to be taken with caution until such evidence is clearly outlined. Furthermore, the plate layout which includes these controls would help assess the extent of tag-jumping, should the plate plan proposed in Taberlet et al., 2018 be adopted.

Second, without the presence of adequate controls, filtering schemes would be unable to determine whether there were contaminants and also be unable to remove them. This would also prevent samples from being filtered out should there be excessive levels of contamination present. Without such information, it makes it difficult to fully trust the data as presented.

Finally, there is insufficient detail regarding the decontamination procedures of equipment used to prepare the samples (e.g., the cryomil). Without clear explanations of the steps the authors took to ensure samples were handled and prepared correctly, there is yet more concern that there may be unseen problems with the dataset.

Reviewer #1 (Public Review):

Causality is important and desired but usually difficult to establish. In this work, Park et al. conducted a comprehensive phenome-wide, two-sample Mendelian randomization analysis to infer the casual effects of plasma triglyceride (TG) levels on 2,600 disease traits. They identified causal associations between plasma TG levels and 19 disease traits, related to both atherosclerotic cardiovascular diseases (ASCVD) and non-ASCVD diseases. They used biobank-scale data in both discovery analysis and replication analysis.

The conclusions of this work are mostly supported by the data and analysis, but some aspects need to be clarified and extended.

(1) The datasets used in this study may not be very consistent. For example, UKB participants are aged 40-69 years old at recruitment. In addition, UKB is United Kingdom-based and FinnGen is Finland-based. So the definition of outcomes may not be identical. The authors should discuss the differences between the datasets and their potential effects.<br /> (2) The discovery analysis and replication analysis are not completely independent because data from UKB have been used in both analyses. Although in discovery, the data were used for association with outcomes; while in replication, the data were used for association with exposure. The authors may want to explain if this may cause problems.<br /> (3) As stated in the manuscript, there are three assumptions for MR analysis. The validity of the results depends on the validity of the assumptions. The last two assumptions are usually difficult to validate. To the authors' credit, they conducted sensitivity analyses addressing horizontal pleiotropy, which is related to assumption 3. It would be helpful if the authors can discuss those assumptions explicitly.

Reviewer #1 (Public Review):

This study investigates and compares spontaneous turn-taking behavior in pairs of macaque monkeys and human participants. The authors use a transparent, bi-directional touch screen to facilitate visual interactions while pairs performed a task in which payouts increased with coordinated responses. They found that most human pairs converged on a turn-taking strategy without verbal communication or instruction from experimenters, but monkeys coordinated with simpler strategies that did not involve turn-taking and could be accomplished without attending to the partner's responses. Interestingly, the monkeys could learn to attend to a partner's responses with explicit training but used this to interact competitively, where the faster animal led and the slower followed his responses. Together, the results suggest differences between species in their tendencies toward cooperative, mutually beneficial behaviors.

Specific strengths of the study include the novel task apparatus and experimental design, and unique cross-species comparisons. In addition, the analyses are appropriate and results are compelling. The approach that tests monkeys with a human "confederate" is clever, and clarified that their performance with other monkeys did not result from an inability to coordinate with another agent. While the species differences are compelling, the main weakness is that there are different potential explanations that can't be teased apart by the present study. On one hand, different behaviors may arise from qualitatively different social motivations, such as humans placing value on equity or their partner's success that monkeys do not. On the other hand, the rewards the monkeys were working for are qualitatively different and potentially more motivating, and this could drive them toward a more competitive approach to the task, even if they could, under other circumstances, display the same social motivations as humans. More in-depth consideration of how different task strategies relate to earned rewards could provide some insights on this confound.

Reviewer #1 (Public review):

The manuscript by Foster et al. details how PEP cycling and specific pyruvate kinase isoforms impact beta-cell ATP/ADP levels, KATP activity, calcium handling, and insulin secretion. The manuscript clearly illuminates the beta-cell specific roles of PKm1, PKm2, and mitochondrial PEP carboxykinase. The manuscript finds that beta-cell PEP production leads to KATP inhibition via ATP produced by PKm1 and PKm2. The manuscript also finds that amino acid induced closure of KATP channels depends on mitochondrial PEP production but not elevations in cytoplasmic ATP/ADP. Finally, the manuscript suggests that the PEP cycle is also involved in KATP activation, but the mechanism remains to be determined. The manuscript is well written and easy to follow. Overall, this is an excellent manuscript that will be very useful to the diabetes research community.

Reviewer #1 (Public Review):

Laurent et al. generate genotyping data from 259 individuals from Cabo Verde to investigate the histories and patterns of admixture in the set of islands that make up Cabo Verde. The authors had previously studied admixture in an earlier study but in a smaller set of individuals from two cities on one island (from Santiago) in Cabo Verde. Here, the authors sample from all the islands of Cabo Verde to study admixture in these islands and reveal that there is a varied picture of admixture in that the demographic histories are distinct amongst this set of islands.

I found the article interesting and clearly written, and I like that it highlights that admixture is a dynamic process that has manifested differently in distinct geographical regions, which will be of broad interest. It also highlights how genetic ancestry patterns are correlated with the populations that were in power/enslaved during colonial times and proposes that certain social practices (e.g. legally enforced segregation) might have affected the distribution/length of runs of homozygosity.

My main suggestion is that the authors provide a set of hypotheses regarding admixture that may explain their observations, and it would be nice to see if at least one of these has some support using simulations. Could the authors run simulations under their proposed demographic model for populations in Cabo Verde vs what we would expect in a pseudo-panmictic population with two sources of admixture? The authors probably already have simulations they could use. And then see how pre/post admixture founding events change patterns of ancestry.

Reviewer #1 (Public Review):

Bustion and colleagues outline the creation and testing of an in-silicon method to query gut microbiome databases for genes encoding enzymes predicted to catalyze a reaction of interest, which is provided by the user. Strengths of the tool include attempts to examine nearly 9,000 MetaCyc reactions in a pre-calculated fashion and to rank order enzymes based on their likelihood of catalyzing a reaction. Substrates, products, and even cofactors, if known, are employed to strengthen the power of the search algorithm, which also employs a hidden Markov model to improve the selection of putative hit enzymes. The authors outline high success rates with examples presented and compare those results with other extant methods, which are reported to perform in a less robust manner. Weaknesses include lack of evidence of success on a more difficult "real world" example. However, the tool outlined is a clear advance over existing methods and will be useful to explore the diversity of chemical transformation performed by commensal microbiota.

Reviewer #1 (Public Review):

This manuscript argues that populations of descending neurons, connecting the fly's brain to its ventral nerve cord, encode high-level behaviors (resting, walking, or grooming) rather than specific limb movements or joint positions. This argument is supported by correlations between activity shown by functional imaging using a genetically-encoded calcium indicator expressed in many neurons passing through the neck connective and simultaneous measurements of the fly's behaviors in a tethered preparation.

Reviewer #1 (Public Review):

This paper investigates waves in embryonic mouse retinas. These stage 1 waves have been studied less than the post-natal (stage 2) waves. The paper uses calcium imaging in whole retinas to determine the properties of the waves and their dependence on cholinergic and electrical synapses. A strength of the work is the ability to monitor waves over the entire retina at high resolution and weaknesses include reliance on pharmacology and some missing details in analysis.

Reliance on pharmacology<br /> The results in the paper depend largely on pharmacological manipulations. Not enough consideration is given to the possible unintended effects of those manipulations. This is particularly true for the gap junction inhibitors. The Discussion brings up the possibility of such effects, but they need to come up much earlier. Is there anything else that can be done to mitigate concerns about the drugs - e.g. does MFA affect waves in Cx36 KO mice?

Comparison of ACh receptor block and knockout mice<br /> The ACh receptor knockout mouse provides a useful alternative to the pharmacological block of ACh receptors. But different features are described in Figures 2 and 3, preventing direct comparison of the two. A related point is the apparent increased role of gap junctions in mediating waves in the absence of ACh receptors. On this point, the description of the effect of MFA (page 8, second paragraph, 3rd sentence) was confusing. It looks to me like MFA almost completely eliminates waves in both WT and KO - so the connection to an altered role of gap junctions was not clear.

ipRGC densities<br /> The goal of the measurements of ipRGC densities was not entirely clear. Why are ipRGCs a reasonable way to determine the importance of waves for development? For example, the introduction raises the issue that changes in RGC proliferation depend on RGC type. Is there reason to think ipRGCs are "special" or, alternatively, that they are following the same developmental trajectory as other RGCs? Is it possible to track another RGC type (e.g. using SMI32 staining)? Related to this general point, page 9 (top) sets up the need to identify the mechanism of RGC cell death but then jumps to waves without a clear connection between the two. It would also be good to mention early that the measurements include multiple ipRGC types, so that issue does not come up only as an explanation for why the ipRGCs are not organized spatially (page 10 top).

Analysis<br /> Quantitative analysis of the calcium measurements relies on the discretization of the signals measured in small ROIs. It was not clear how closely the discretized signals represented the original recordings. The traces illustrated in Figures 1 and 2, for example, appear to be measured in larger ROIs. Two things would be helpful here: (1) an illustration of several original recorded traces in the small ROIs plotted with the discretized versions of those traces; (2) a determination of how sensitive the results are to specifics of the discretization process.

Reviewer #1 (Public Review):

This manuscript provided descriptive information about the development of endothelial cells and co-developing mural cells. The researcher induced hiPSCs into mesodermal cells before furthermore differentiating the cells into endothelial cells by chemical induction. To investigate in vitro neovascularization, the researchers employed single-cell RNA sequencing to analyze the differentiation process of hiPSCs into endothelial cells under various 3D microenvironments at several time points. Utilization of single cell RNA sequencing in this manuscript provides insight about signature process and molecules in various time points and conditions, where cell-cell interaction between endothelial cells and pericytes, angiogenic signaling and roles of cytokine signaling during tubulogenesis are highlighted. This study also compared the 2D environment previously employed by another study to the 3D microenvironment and found interesting differences in endothelial cells proliferation pattern, extracellular matrix production, and expression of mesenchyme-angioblast markers. In conclusion, this study gave us a lot of important data for improving strategies for future vascular tissue engineering.

Strength<br /> This study is well designed with a robust method, convincing data, and in-depth analysis of various possible conditions for endothelial cells differentiation. This study could provide additional information about the dynamics of vascularization and serve as a benchmark for future hIPSC-derived endothelial cells-based experiments.

Weakness<br /> As several studies have been published before about single-cell sequencing profiling of endothelial cell derived from hIPSCs, the novelty and merit of this study are impacted accordingly.

Joint Public Review:

This study investigates whether the compression of time cells in the hippocampus follows the Weber-Fechner Law, using a hierarchical Bayesian model that simultaneously accounts for the firing pattern at the trial, cell, and population levels. Recording where performed in dorsal CA1 of rats (N = 4) performing a delayed paired-associate task that required rats to run on a treadmill between sample and test, during which time delay-dependent firing could be assessed (N = 131 neurons). The authors highlight three novel observations: 1) Simultaneously recorded CA1 neurons showed consistent deviations in their trial-to-trial variability in the timing of peak activity, 2) After controlling for trial-to-trial variability, time field width increased linearly with delay, and 3) The number of neurons with time field peaks observed at each delay was logarithmically allocated across the treadmill interval. The findings are related to the authors' broader theory of how hippocampal firing provides a continuous, scale-free temporal context that defines the backbone of episodic memory.

The findings in this paper are interesting, timely, and the data generally support the conclusions, though some technical revisions would be beneficial. The strong theoretical basis of this line of inquiry from both a physiological and cognitive perspective is appreciated. The hierarchical Bayesian analysis is a major strength of the paper and a relatively novel contribution to the hippocampal field. The main concern about this study is that all the conclusions of the paper are based on the results of the hierarchical Bayesian model, urging for alternative analytical accounts for the increase in time field width as a function of delay. In addition, more can be done to address the observed variability in time field activity and whether behavioral changes explain apparent changes in the receptive field properties for late firing neurons.

Reviewer #1 (Public Review):

The manuscript by Chen et al., describes an in vivo zebrafish assay to test the impact of human tp53 variants on rhabdomyosarcoma tumor pathogenesis. There are a number of novel observations in this study. First, tumor onset is significantly enhanced when the zebrafish rhabdomyosarcoma tumor model is in a tp53 null background, leading to >97% of animals developing tumors, most within the first 20 days of life. Second, introduction of wildtype human tp53 cDNA suppresses tumor initiation, establishing a rapid and effective in vivo assay to examine the activity of tp53 variants on tumor induction and growth. Three tp53 variants identified in human rhabdomyosarcoma patients were tested in standard in vitro assays, but none of the variants demonstrated wild type tp53 activity such as suppression of tumor cell growth or activation of gene expression. In contrast, all three variants were able to suppress tumor onset and initiation in the in vivo zebrafish rhabdomyosarcoma model. The ability of the variants to suppress tumor initiation was attributed to retention of some level of activity, or novel activity, in vivo. Additional molecular data would strengthen the conclusion that the different tp53 variants function as hypomorph or gain of function alleles.

Reviewer #1 (Public Review):

This paper primarily assessed the host/phage interactions for bacteria in the order of Cornyebacteriales to identify novel host factors necessary for phage infection, in regards to genes responsible for bacterial envelope assembly. Bacteria in this order, such as Mycobacterium tuberculosis and Corynebacterium diphtheriae have unique, complex envelopes composed of peptidoglycan, arabinogalactan, and mycolic acids. This barrier is a potent protector against the therapeutic effects of antibiotics. Phages can be used to discover novel aspects of this bacterial envelope assembly because they engage with cell surface receptors. To uncover new factors, the researchers challenged a high-density transposon library of Corynebacterium glutamicum (called Cglu in the paper) with phages, Cog, and CL31. Results by transposon sequencing identified loci that were interrupted, leading to phage resistance. This study implicated the importance of Cglu genes, ppgS, cgp_0658, cgp_0391, and cgp_0393. They also identified a new gene called cgp_0396 necessary for arabinogalactan modification and recognized a conserved host factor called Ahfa (Cpg_0475) that plays a crucial role in Cglu mycolic acid synthesis. Ultimately, this work implicated the importance of mycomembrane porins, arabinogalactan, and mycolic acid synthesis pathways in the assembly of the Cornyebacteriales envelope.

Strengths of the research:<br /> - Language choice: A major strength of the paper is that this could easily be given to an undergraduate student with introductory knowledge of biology and they would still be able to get the gist of this paper. The language is written in a clear, concise fashion with explanations of terms not everyone would immediately know unless they worked in the field specifically.<br /> - These figures are generally explained in a direct manner, clearly stating the major conclusions the reader should get after carefully analyzing the presented data

How the research could be strengthened:<br /> - It could be worthwhile to describe some of your results mathematically. For example, the differences you see in your phage infections relating to the differences in logs, etc. Bar graphs also should be described in mathematical terms, when "something is lower compared to the WT," how much is lower, etc?<br /> - There were no p values relating to the statistical significance of any of the data presented, which should be changed for the final manuscript implicating the importance of this work.<br /> - Figure 8 was not entirely supported by the data, especially Figure 8A which either could be improved with better images that support the author's claims, etc.

Reviewer #1 (Public Review):

Exploiting a previously established fluorescence complementation microscopy-based approach, Castro and colleagues screen a large library of fluorescently tagged proteins for their potential localization and regulation of MCS sites. This approach pinpointed known as well as previously unidentified components of MCS. Based on the homology models and structural prediction in silico, new classes of potential lipid transfer proteins were identified. While more work will be necessary, these are important results to the field. In a screen for components of the ER-LD MCS the authors identified ypr097W (re-named Lec1). This protein, which distributes to the cytosol, plasma membrane and upon overexpression also to LDs, appears to affect the distribution of ergosterol between mother and daughter cells. While these are all interesting results, in its current form, the manuscript presents some shortcomings, as described below.

Main concerns:<br /> 1) Validation of the MCS reporters is not shown. This is particularly important for pCLIP and GoPo, which have not been reported before. Fluorescence complementation between two proteins that normally localize to different organelles is far from demonstrating the existence of a MCS between those organelles. It would be important to demonstrate using marker proteins and ideally electron microscopy/CLEM the existence of the mentioned MCS and the suitability of the fluorescent reporter.

2) As pointed out above, the identification of a phenotype in ergosterol distribution for Ypr097W/Lec1 is very interesting. However, it is unclear how this observation relates with the localization of Lec1 to LDs, which is observed only upon over-expression. Instead, further characterization of Ypr097w phenotype (via mutagenesis, modulation of ergosterol biosynthetic pathway, test ability to bind ergosterol, etc) in ergosterol distribution would be a plus.

Reviewer #1 (Public Review):

This well-written manuscript presents a technically impressive and carefully controlled biophysical study of the nature of the mechanosensitivity of voltage-gated Na+ (Nav) channels. As a simplified model system, the study employs an inactivation-deficient bacterial Nav channel that appears to respond to mechanical stimulation in a similar fashion to mammalian Nav1.5. The results provide strong evidence that a step in the activation path which has little intrinsic voltage dependence is mechanosensitive. The manuscript proposes that the mechanosensitive step is pore opening. While this seems the most parsimonious explanation, it still seems possible that a conformational change other than the opening of the pore could be the voltage-independent mechanosensitive step. The swinging door model presented here seems apt and is conceptually valuable. However, it is not clear that the I228G hinge mutagenesis provides strong support for the swinging door model. Overall, the conclusion that a voltage-insensitive step of the bacterial channel is mechanosensitive is well-founded. The additional proposal that this mechanosensitive step is the opening of the intracellular S6 pore gate is best-considered speculation.

Reviewer #1 (Public Review):

Cryo-EM structure determination is reported for S. cerevisiae Yta7, a type II AAA+ ATPase that is known to disassemble nucleosomes by unfolding histone H3. The structures determined in the presence of ADP or ATPgS plus histone H3 peptide show a three-layer homo-hexameric architecture in which the top BRD-BIM layer is followed by AAA1 and AAA2 layers. At the base of the structure, the AAA2 ring comprises inactive ATPase cassettes, and is planar. The AAA1 layer forms a spiral structure when bound to ADP, and this spiral is more pronounced when bound to both ATPgS and an H3 peptide. The H3-bound AAA1 spiral resembles the structure of multiple other AAA unfoldases, and seems to be consistent with the proposal that Yta7 utilizes a hand-over-hand sequential model of substrate translocation/unfolding. These findings were anticipated from published structural studies on the S.pombe homolog, Abo1 which had reported a very similar structure, albeit with a non-H3 peptide.

In addition to verifying expected findings with a scientifically important homolog, the main advance in the current study is the assembly of BRD domains. They are partially ordered above the AAA1 ring in the absence of bound H3 but are disordered in the presence of bound H3. To some extent, this was also anticipated from the earlier studies on Abo1, although there are some notable differences and additional insights. In Yta7, only three of the six BRD domains are visible, and they form a spiral assembly with the lowest subunit blocking the substrate translocation pore. One important observation is that the BRD domains, which are encoded in a loop of the AAA2 cassette, bind to a BIM sequence in the long and inherently flexible N-terminal sequences that project above the AAA1 ring. This explains how BRDs are located to the top layer where they both regulate access to the substrate translocation pore and help recruit substrate.

Another important finding comes from the low-resolution structure reported for a Yta7-nucleosome complex. Although this structure is only at 14Å resolution, it is apparent that the BRD domains are in a flattened rather than spiral configuration, and that the substrate translocation pore is open. Thus, the current paper verifies expectations of overall architecture of AAA cassettes and substrate engagement, and explains how the substrate recognition BRD domains are located to the top surface of the complex and block substrate engagement in the absence of nucleosome binding but are displaced to a substrate-accepting conformation upon association with the nucleosome.

The work seems to be performed well. Although multiple aspects of the mechanistic analysis/discussion should be clarified.

Reviewer #1 (Public Review):

This manuscript presents a fast, and cost-effective multicolor 3D imaging system termed translational rapid ultraviolet-excited sectioning tomography (TRUST). TRUST combined sub-systems of sectioning, staining, and imaging to take the complexity out of preparation workload. The integrated system can provide high-contrast fluorescence images from the cellular level to the organ level in an automated series of protocols. Although the authors have well described the systematic feasibility of TRUST, its novelty is ambiguous from a biological point of view.

Reviewer #1 (Public Review):

In this manuscript, the authors use experimental evolution in C. elegans to ask whether evolution in locomotor traits in a high salt environment can be predicted using the Roberston-Price identity and whether the evolutionary response to the salt environment is due to direct selection on locomotor phenotypes or indirect selection via an unmeasured trait. The authors also examine the alignment between phenotypic plasticity in the ancestral environment aligns with G.

The experimental evolution system in C. elegans is a powerful model system to test these types of questions, in particular, because it is possible to resurrect ancestral populations and compare them contemporaneously to evolved lines. It is also possible to estimate broad sense genetic covariance matrices from inbred lines, as the authors have done here.

The introduction does not clearly set up the background for the key questions that the manuscript addresses. One of the key parts of the manuscript is to attempt to determine whether locomotory behaviour evolves because of direct or indirect selection of the traits. However, the authors don't provide an argument for why a salty environment would select for locomotory traits. Indeed, in the discussion, the authors point out that it is likely an unmeasured trait (body size) correlated with locomotory traits that are under selection. They present arguments for why this might be the case and point to un-included data that show body size significantly genetically covaries with all of the traits studied. Since the authors appear to have these data, and one of their key questions is comparing direct vs. indirect responses to selection, it would be more powerful to include the body size data and estimate selection on all traits together.

Phenotypic plasticity was estimated from a series of univariate models, with estimates arranged in a vector. As the authors point out in the manuscript, traits that are not included in a model but covary with traits that are can largely bias estimates of the traits that are included. For this reason, it would make sense to estimate phenotypic plasticity using a multivariate model, as has been done for G matrices.

The estimation and interpretation of G matrices are a critical part of the manuscript. The authors state that broad sense estimates of G are a good proxy for additive genetic variation in this system, but in the Discussion they also state that overdominance was likely important during evolution to the salt environment, leading to some lack of clarity on whether dominance is important or not. It is also unclear how uncertainty in estimated G matrices was assessed. Showing that G differs from noise is critical to the majority of the results presented. The authors cite Morrissey and Bonnet (2019) as providing the method for generating the null distribution of G, however, this paper does not appear to propose or describe a method to do this.

Although the figure captions state that they are showing estimates of genetic variances, it appears to be heritability (bounded between 0 and 1). Whether the authors are studying heritability or genetic variance is an important difference, particularly in the context of a changing environment and phenotypic plasticity, where environmental variation is important and expected to change. For example, the result that G is smaller in evolved populations could simply be due to their being larger environmental variance in the salt environment (as you would expect). This is unrelated to an evolutionary response.

It seems that comparisons to the ancestral population were done for A160, not the founding population for each evolved line at G0. It is not clear whether the founder effects of each replicate are important and if this is the most appropriate comparison (the Discussion suggests that founder effects are important).

Overall, there is much interesting data collected and analysed in this manuscript, addressing a valuable question. However, it is not obvious whether the estimates of G matrices are different from noise, and heritability may not be the most appropriate scale to ask questions about phenotypic plasticity and evolution in a novel stressful environment that may affect levels of environmental variation.

Reviewer #1 (Public Review):

The manuscript is written clearly and places appropriate emphasis on the strengths and weaknesses of the new approach. In Figure 1, the authors aggregate a large body of data from 126 previously performed CRISPRi screens as well as previously vetted computational predictions to create dual sgRNA libraries that can be used for succinct CRISPRi screens in human cells. They validate the functionality of their dual sgRNA library using Perturb-seq. They also note that one particular weakness of the dual sgRNA system is sgRNA recombination between lentiviruses, resulting in chimeric delivery of sgRNAs to cells. Quantitative analyses estimate that in K562 cells, recombination frequency in their new system is ~30%. In Figure 2, the authors use RNA-seq to demonstrate that in K562 cells, certain dCas9-repressors have greater off target effects than others; this was particularly striking for the SID-dCas9-Kox1 construct, which previously had been used for a handful of CRISPRi screens. In Figure 3 the authors compare the ability of different dCas9-repressors to reduce expression of several target genes, and from these experiments, identify Zim3-dCas9 as the most effective in K562 cells. In Figure 4 the authors show that Zim3-dCas9 is effective in additional human cell lines that have been used for CRISPRi screens. By my evaluation, all conclusions are well supported and justified by the data. CRISPR screens are labor and cost intensive endeavors. With this work, Replogle et al. present a validated set of novel reagents that will enable more effective and efficient CRISPR screens. The manuscript also highlights certain limitations and caveats of different forms of CRISPR screens whose clear explanation here will also benefit the community.

Reviewer #1 (Public Review):

In this study, Pajevic and colleagues devised a mathematical model to study the biophysical properties of oligodendrocytes (OLs) in mediating myelin plasticity in the brain. The authors suggest that the OLs can sense the neuronal firing and in a calculated manner release a factor (M), which can locally regulate myelin sheath addition or removal at a given axonal segment. As far as my expertise goes, the modeling work seems quite sound and robust. But, the critical issue is that many parameters the authors chose for generating the oligodendrocyte-mediated myelin plasticity (OMP) model are hypothetical from a physiological point of view. For several modeling parameters, it was challenging to relate to the 'real' electrophysiology quantities recorded for OLs. For example, it is unclear what the ultra-fast signaling factor (G) could be at OLs/myelin segments, which can act at a speed of 40ms. Another assumption is that OLs release a potent factor called M, which can instantaneously promote the formation of new myelin internodes or stabilize the existing node. In addition, the release of such factor locally by OLs to self-maintain the myelin sheath has not been experimentally demonstrated yet. Throughout the manuscript, I felt that OLs were morphed into neuron-like cells exhibiting fast responsive electrophysiological properties. But, the actual experimental electrophysiological recordings or Ca2+ imaging data suggest that OLs operate in seconds rather than milliseconds. Hence, the study requires selecting physiological parameters that are more "realistic" for OLs and are guided by the rich sets of published experimental data.

Reviewer #1 (Public Review):

In this work, Meisler and Gabrieli investigated whole-brain white matter fixel-based analysis and their relationship with reading skills for the first time. They used data from the Healthy Brain Network including children and adolescents with typical reading, reading disability, or unclassified status, however the main focus was on continuous Test of Word Reading Efficiency (TOWRE) measures.

The authors found that the product of fibre density and cross-section, denoted as FDC, was related to TOWRE scores. In particular, higher FDC in bilateral temporal-parietal and cerebellar white matter were most strongly related to better reading measures, with a slight left lateralization. These two main clusters likely involved sections of the arcuate, superior longitudinal, and middle longitudinal fasciculi that overlap in that region, and the superior cerebellar peduncle.

This represents the first investigation of fixel-based analysis related to measures of reading, and the large sample allowed a whole-brain analysis approach and reaffirmation of the importance of the temporal and cerebellar white matter that support reading. The authors also included a supplementary analysis that will be of interest to researchers in the field, where they also investigated other white matter measures and their relationships with reading, where orientation dispersion index was inversely related to reading skills in the same regions.

This paper opens the door for further exploration using fixel-based analysis approaches to study reading, but more widespread development. Though it was not explored here, it would be interesting to see if tracts derived using this analysis approach can aid in understanding regions with crossing fibres, and disentangle the role of specific white matter pathways that support specific reading skills and other cognitive abilities.

Reviewer #1 (Public Review):

The authors report the bone phenotype of the global Ksr2 null mouse and find that Ksr2 negatively regulates cortical (femur) but not cancellous (vertebra) bone mass. The paralog Ksr1, in contrast, is not required for bone mass regulation. They also show that KSR2 regulates bone formation by shifting adipocyte differentiation at the expense of osteoblasts in the bone marrow. This is a very well-performed study with clear results that have been appropriately interpreted and discussed. The overall impact of understanding the mechanistic switch between the adipocyte and osteoblast is significant. The identification of an actionable target that is validated through prior human studies is also of importance. Weaknesses are minor.

Good question.

Reviewer #1 (Public Review):

The authors use their expertise in live-cell imaging and mathematical modeling to further explore the relationship between chromatin structure, gene positioning and transcriptional co-regulation. One of the strengths of the manuscript arises from the authors analysis of two publicly available datasets encompassing chromatin tracing and transcriptional activity. Using spatial analysis and modeling, the authors have impressively extended the findings of Su et. al, Cell 2020, who generated the analyzed dataset. A number of important concepts were explored including 1.) do genes re-position upon activation and 2.) can spatial proximity be correlated with transcriptional co-regulation. In general the authors conclusions are supported by their findings and should provide a blueprint for analysis of additional related big imaging datasets in the future.

However there are a number of weaknesses including lack of statistical analysis or incomplete description (e.g. bootstrapping parameters, statistical methods, number of genes/cells/measurements, etc.) on some figures that make it difficult to interpret the significance of the trends. In addition, the modeling using live-cell studies is generalized based on a behavior (e.g. diffusion) of a single gene. The manuscript is densely written in a way that may be inaccessible for non-specialists. A final schematic model that summarizes biological findings would help alleviate this weakness.

Reviewer #1 (Public Review):

The finding that taste memory formation follows the same or highly similar logic and mechanisms as olfactory memory is very interesting. In particular, the new approach to use an operant learning assay developed by the authors to address this outstanding question in the field is very impressive. The shown data are of high quality and very convincing.

While the current version will be of clear interest to fly people dissecting memory formation, it might be less accessible outside this immediate field. Below I list my suggestions, questions and criticisms.

You have developed an operant assay and stress this in the introduction. This is important because it allows you to gain much better inside into how memory is formed and how it is recalled. Nevertheless, I was somewhat disappointed that you did not exploit that aspect more in your study. First, I suggest showing, at least for the initial figures, the traces (e.g. Fig 1D) not only for the test phase but also for the training phase. As you also mention in your discussion, the extent of memory formation will depend critically on the number of pairings during training. And perhaps not only on their number but also on their evolution/change over time. Second, you only show preference indices. I suggest showing the number of actual interactions with the food source in addition. In my opinion and experience, the preference index can be misleading or at least the interpretation might be questioned if the number of actual choices is very low or very high compared to controls or other groups. Third, regarding the same point, you show traces for test phases, but you do not comment or discuss why they might look the way they look. For instance, it appears that in some cases it takes a while to see an actual difference in the preference index while at other times it seems more instantaneously etc.

Along the same lines, I am wondering why you do not observe extinction. Frequently if the CS is re-experienced without the US over several trials, you start to see memory fade. The preference traces as well as the actual interactions might help to explain this.

You use salt as a negative US. I suggest showing at least one experiment with bitter taste (e.g. quinine) to show how general your finding is to negative conditioning. Your optogenetic data suggests it is.

You analyze the role of energy state in memory formation. This is very interesting. In light of the importance of feeding state, it would be very helpful to include starvation/metabolic state information not only in the methods but also in the results section (at least briefly).

Your data convincingly shows that taste memory is formed in the mushroom body. For instance, you show that inhibition of KCs prevents the change in preference. KC inhibition was done during the entire experiment (training and test). Thus, it's important to show how KC inhibition affects (or does not) training vs. test.

Along the same lines, how do you envision this memory formation to happen at the circuit level? KCs and DANs are likely activated by CS and US. It would be important to at least include a paragraph in the discussion to clarify this.

Reviewer #1 (Public Review):

The authors have employed a variety of techniques (single-molecule fluorescence kinetic and steady state measurements, cryo-EM structure determination, and in vivo measurements of protein synthesis and cell proliferation) to investigate the mechanism of action of two molecule products: Didemnin B and Ternatin-4. Both molecules have previously shown to target eEF1A and have potential as cancer therapeutics. In addition, the structure of Didemnin B, bound to eEIF1A and to an elongation complex, have previously been solved.

The authors here show that both compounds disrupt the dynamic accommodation of tRNA driven by eEF1A and its activation by the GTPase activation center of the ribosomal large subunit, relying on previous assignment of the FRET intensities observed in pre-steady state single-molecule fluorescence experiments in which peptide-tRNA and incoming aminoacylated tRNA are labeled with donor and acceptor dyes, respectively. They further show that this inhibition is dose dependent for both compounds and sensitive to the A399V eEF1A mutant, which creates a steric clash with didemnin B in its usual binding site. Subsequent analysis of steady-state single-molecule FRET experiments shows that didemin B more strongly inhibits transitions between the intermediate (0.45) FRET state and the high (0.8) FRET states (though the authors choose to focus only on the effect of transitions from 0.45 to 0.8) previously assigned to the GTPase activated and fully accommodated conformations of the ternary complex, respectively. Further single-molecule experiments provide initial evidence that Didemnin B remains more stably bound to elongation complexes than does Ternatin-4.

The authors then turn to cryo-EM structures of each compound bound to elongation complexes purified either from lysate or assembled from purified components. The structure of the Ternatin-4 complex shows additional density in the same binding cleft observed for Didemnin B in a prior structure reported elsewhere, with which the Didemnin B structures reported here also agree. This binding location provides structural evidence for both compounds effects on ternary complex dynamics, as well as their previously described effects on tRNA accommodation and elongation. Further comparison of the Didemnin B and Ternatin-4 structures reveals decreased electron density in the Ternatin-4 structure for elements of eEF1A (switch loops 1 and 2 and helix alpha2) , as compared to the Didemnin B structures. The authors interpret this as evidence for greater mobility of these elements, which might explain the more modest restriction of A-site tRNA dynamics they observe in the presence of Ternatin-4 (as opposed to Didemnin B). Certainly this decreased density (which might be more convincingly demonstrated using difference maps of the two structures) is consistent with that interpretation. That said, it is certainly not a smoking gun.

Finally, the authors turn to in vivo measurements of protein synthesis and effects on cellular proliferation or survival in the presence of both compounds. Consistent with their single-molecule experiments, they observe more severe and durable inhibition of protein synthesis in the presence of Didemnin B, whereas Ternatin-4 exhibits more modest effects that are more rapidly restored upon removal of the drug in solution. Interestingly, Ternatin-4 appears to elicit similar, and perhaps more rapid, effects on cellular survival, increasing apoptosis more rapidly than Didemnin B, though these effects (like those on protein synthesis rates) are once again more sensitive to removal of the drug. The authors describe these results as evidence that Didemin-B "irreversibly inhibits" protein synthesis in cells. I find this assertion strange, given that the authors have previously measured a dissociation rate for this molecule from elongation complexes and they have not performed measurements to ensure that activity is not simply restored at timescales longer than their initial measurements. That said, I concede that this might be a semantic distinction if the vast majority of cells perish prior to dissociation of the drug. In either case, I would suggest the authors apply a somewhat more nuanced interpretation of these results lest they be misunderstood.

Overall, this is a rigorous and well reasoned study that employs multiple complementary techniques to investigate the mechanism of action of compounds of potential therapeutic interest. In places, the higher order interpretation of the experimental data leaks into the results section (as opposed to being fully explored in the discussion) and is at times somewhat aggressive. Nonetheless, the results presented here illuminate important questions at the intersection of translational mechanism, cell proliferation, and cancer.

Reviewer #1 (Public Review):

This study investigates the psychological and neurochemical mechanisms of pain relief. To this end, 30 healthy human volunteers participated in an experiment in which tonic heat pain was applied. Three different trial types were applied. In test trials, the volunteers played a wheel of fortune game in which wins and losses resulted in decreases and increases of the stimulation temperature, respectively. In control trials, the same stimuli were applied but the volunteers did not play the game so that stimulation decreases and increases were passively perceived. In neutral trials, no changes of stimulation temperature occurred. The experiment was performed in three conditions in which either a placebo, or a dopamine-agonist or an opioid-antagonist was applied before stimulations. The results show that controllability, surprise, and novelty-seeking modulate the perception of pain relief. Moreover, these modulations are influenced by the dopaminergic but not the opioidergic manipulation.

Strengths:

• The mechanisms of pain relief is a timely and relevant basic science topic with potential clinical implications.

• The experimental paradigm is innovative and well-designed.

• The analysis includes advanced assessments of reinforcement learning.

Weaknesses:

• There is no direct evidence that the opioidergic manipulation has been effective. This weakens the negative findings in the opioid condition and should be directly demonstrated or at least critically discussed.

• The negative findings are exclusively based on the absence of positive findings using frequentist statistics. Bayesian statistics could strengthen the negative findings which are essential for the key message of the paper.

• The effects were found in one (pain intensity ratings) but not the other (behaviorally assessed pain perception) outcome measure. This weakens the findings and should at least be critically discussed.

• The instructions given to the participants should be specified. Moreover, it is essential to demonstrate that the instructions do not yield differences in other factors than controllability (e.g., arousal, distraction) between test and control trials. Otherwise, the main interpretation of a controllability effect is substantially weakened.

• The blinding assessment does not rule out that the volunteers perceived the difference between placebo on the one hand and levodopa/naltrexone on the other hand. It is essential to directly show that the participants were not aware of this difference.

• The effects of novelty seeking have been assessed in the placebo and the levodopa but not in the naltrexone conditions. This should be explained. Assessing novelty seeking effects also in the naltrexone condition might represent a helpful control condition supporting the specificity of the effects in the naltrexone condition.

• The writing of the manuscript is sometimes difficult to follow and should be simplified for a general readership. Sections on the information-processing account of endogenous modulation in the introduction (lines 78-93), unpredictability and endogenous pain modulation in the results (lines 278-331) are quite extensive and add comparatively little to the main findings. These sections might be shortened and simplified substantially. Moreover, providing a clearer structure for the discussion by adding subheadings might be helpful.

• Effect sizes are generally small. This should be acknowledged and critically discussed. Moreover, effect sizes are given in the figures but not in the text. They should be included to the text or at least explicitly referred to in the text.

• The directions of dopamine and opioid effects on pain relief should be discussed.

Reviewer #1 (Public Review):

This study by Wild et al. investigates mechanisms of resistance to therapy in triple-negative breast cancer (TNBC) as a result of tumor heterogeneity. They perform clonal transcriptomics in TNBC cell using WILD-seq in tumors undergoing BET inhibition or taxane-based chemotherapy. They identify that increased NRF2 activation as a major mechanism of taxane resistance. Importantly, they observe that as a result of NRF2 activation, the resistant cells become dependent on exogenous asparagine which renders them sensitive to asparagine deprivation therapy. Their observations in the animal models are also validated in patient specimens. This is a very elegant study that identifies mechanisms of resistance in TNBC and a new therapeutic approach to overcome the resistance. The combination of models and approaches used are highly innovative. The authors describe a new barcoding system that allows them to simultaneously define clonal lineage and determine gene expression changes, termed WILD-seq.

Reviewer #1 (Public Review):

Summary: In this manuscript the authors report an RNAi screen to identify proteins that, when depleted, alter nuclear shape and/or nuclear size. They demonstrate that the changes cannot be solely attributed to changes in the expression of nuclear lamins. Many of the hits are factors that impinge on histone modifications and chromatin biology. Comparing hits between fibroblasts and an epithelial cell type (MCF10) demonstrated relatively little overlap. The authors then relate their observations to a potential direct interaction between lamin A and histone H3 that, using histone peptide arrays, may be modulated by the methylation status. Last, the authors find that over-expression of some histone H3 variants/oncohistones alters nuclear appearance.

Overall assessment: The screening effort has revealed a number of interesting and novel suggestions of new modulators of nuclear appearance that are exciting and have the potential to be of value to the field. Unfortunately, the remainder of the manuscript is largely descriptive and rather superficial; perhaps most importantly validation experiments to rigorously confirm the screen "hits" are lacking. There are also concerns about the interpretation of biochemical experiments into lamin A-histone H3 binding although there are also some promising hints into the histone modification-dependence of lamin A binding to H3 that, if more fully investigated, would be an important contribution.

Major Points:

1. The discussion of the screen hits and prior knowledge key to their interpretation is lacking. For example, the authors only report on the purported localization of the hits without an unbiased analysis of their function(s). As a sole example, multiple members of the condensin complex are hits in Fig.1 while multiple members of the cohesin complex are hits in Fig. 2 - but there are many more factors worthy of further discussion. Moreover, the authors need to provide more information on the data used to assign the localization of the hits and how rigorous these assignments may be. For example, multiple CHMP proteins (ESCRTs) are listed - indeed CHMP4B is the highest scoring hit in Fig.1 - but this protein does not reside at the nuclear envelope at steady-state; rather, it is specifically recruited at mitotic exit to drive nuclear envelope sealing. Moreover, there are many hits for which there is prior published evidence of a connection to nuclear shape or size that are ignored: examples include BANF1, CHMP7, Nup155 (and likely far more that I am not aware of). This is a missed opportunity to put the findings into context and to provide a more mechanistic interpretation of the type of effects that lead to the observed changes in nuclear appearance. For example, there is already hints as to whether the effects occur as a mitotic exit defect versus an interphase defect, but conceptually this is not addressed.

2. Validation of the screen is lacking. There appears to be no evidence that the authors validated the initial screen hits by addition siRNA experiments in which the levels of the knock-down could be assessed. As an example: do nucleoporin hits decrease in their abundance at the nuclear envelope in these conditions? This validation is absolutely essential.

3. Differences in cell type - the authors' interpretation that a lack of overlap in the hits across cell types reveals that there are fundamentally cell type-specific mechanisms at play is a stretch. This could also reflect a lack of robustness in the screen, which should be addressed by directly testing the knock-down of the hits from one cell line in the other. Even if this approach reinforces the cell type specificity, the differences in the biology beyond the nucleus itself - an obvious example being the mechanical state of the cell - organization of the cytoskeleton, adhesions, etc that influence forces exerted on the nucleus are different rather than the nuclear response is different. These caveats needs to be explicitly acknowledged.

4. There are major issues with the interpretation of the presented biochemistry. For example, the basis for the supposed effect of monomer/dimer state of lamin is confusing and likely misinterpreted. It is well established that GST imposes dimerization on proteins expressed as GST fusions independent of cysteines. Any effect of DDT would have to manifest through some other mechanism (disulfides between the lamin domains - assumedly what the authors are thinking). Further, GST will impose dimerization of lamin A and lamin C in the co-incubation experiments. It is therefore entirely expected that if lamin A binds H3 and lamin C does not that the mixed dimers will bind H3 with lower affinity. Critically, this does not, however, address how full-length lamin C influences binding of lamin A to H3 in vivo. Last, how an effect of lamin C on lamin A would manifest through a disulfide bond in the nucleus, which has a reducing environment, is entirely unclear.

5. It is important for the authors to address the concept of nuclear size changes versus changes in the nuclear to cell volume ratio - biologically these could be quite different conditions, but obviously these cannot be distinguished by measuring nuclear volume alone. Addressing this experimentally would be best (to provide more depth to the size measurements).

6. There are important caveats to the approach of using the nuclear area as proxy measurement for nuclear size, most prominently that it is highly responsive to changes in nuclear height that can occur for a multitude of reasons (increased height = small radius and decreased height = larger radius), particularly given the different cell types. This needs to be acknowledged directly.

7. What is the evidence that the H3 effects manifest through lamins rather than directly?

8. Context is needed for the "methyl-methyl" histone states described as being the highest binders in the peptide array experiments. Are these states commonly found? Where in the genome? Does this match any DamID data? Again - more depth of investigation is required.

9. That oncohistones induce changes in nuclear shape or size does not mean that this is related to the mechanism in cancer. Also - how over-expression of H3 without its obligate partner H4 could disrupt the cell or an assessment of the extent of the oncohistone incorporation into chromatin achieved in these experiments makes it challenging to interpret.

10. Throughout the manuscript it would be helpful to the reader if the author would provide at minimum a brief statement on the previously identified functions of the hits that are explicitly discussed beyond their localization (membrane versus chromatin). References would also be helpful (for example, again - what is the evidence that SLC27A3 resides at the nuclear envelope?).

Reviewer #1 (Public Review):

The authors have used computational models and protein design to enhance antibody binding, which should have broad applications pending a few additional controls.

The authors' new method could have a broad and immediate impact on a variety of diagnostic procedures that use antibodies as sensitivity is often an issue in these kinds of experiments and the sensitivity enhancement achieved in the two test cases is substantial. Affinity maturation is a viable approach, but it is laborious and expensive. If the catenation method is generalizable, it will open up opportunities for antibody optimization for cases where affinity maturation is either not feasible or otherwise impractical. Less clear is how this method might enhance therapeutic potency. Issues that arise when using therapeutic antibodies are often multifactorial and vary depending on the target and disease state. Many issues that occur with antibody-based therapies will not be rectified with affinity enhancement.

Reviewer #1 (Public Review):

In this paper the authors reconstruct the ancestral states of the body sizes and genomes of Xenarthrans (sloths, armadillos, anteaters) to understand whether duplications of genes that reduce cancer risk are associated with large body size, as has previously been found in Proboscideans (elephants and mammoths). The strong points of the paper are that Xenarthran cells are particularly sensitive to DNA damage, which induces cell death, and that nine-banded armadillos have strikingly little cancer. They also present comparative evidence on the doubling times of cells in culture, but their conclusions are compromised by the small sample size - one individual - for sloths. The data on which their inferences are based do not yet allow them to decide whether large body size evolved before the gene duplications that reduce cancer risk, or vice versa. The main impact of this work is likely to be additional focus on nine-banded armadillos in cancer research, with an emphasis on the discovery of the mechanisms mediating the effects. It should also spur attempts to recover genomes from soft tissues of large-bodied Xenarthran fossils; that may be possible for giant ground sloths.

Reviewer #1 (Public Review):

In this manuscript, Smith et al. evaluate whether phenotypic plasticity and/or species sorting (changes in community composition) occur during a four-week-long incubation of a single soil at a range of temperatures. By using a relatively simple setup and restricting their cultivation to relatively fast-growing taxa well-suited to growth in the lab, the authors were able to measure a commendable number of traits and successfully evaluate the thermal niche of the majority of organisms tested. The authors did a very thorough job of explaining how they came to the conclusion that species sorting is the dominant driver of community-level adaptation to temperature in their experiment, and they do an outstanding job using other literature to support and contextualize these conclusions. I also commend the authors for not overstating the relevance of their results and sticking to the conclusion that this is a possible range of responses rather than concluding that the patterns observed for these taxa are representative of how dominant soil bacteria are responding. Overall this is a very good paper and sets the stage well for future work in, for instance, constraining community turnover vs. acclimation in trait-based carbon cycling models.

Reviewer #1 (Public Review):

This is a well performed study to demonstrate the antiviral function and viral antagonism of the dynein activating adapter NINL. The results are clearly presented to support the conclusions.

This reviewer has only one minor suggestion to improve the manuscript.

Add a discussion (1) why the folds of reduction among VSV, SinV and CVB3 were different in the NINL KO cells and (2) why the folds of reduction of VSV in the NINL KO A549 and U-2 OS cells.

Reviewer #1 (Public Review):

In this study, the authors performed scRNA-seq analysis of iNKT cells from spleen and adipose tissue at steady state and after short-term, long-term, and repetitive ⍺-GalCer stimulation in vivo. They found iNKT cells undergo rapid and extensive transcriptional remodeling during activation. By reanalyzing published scRNA-Seq data of human iNKT cells, the authors found transcriptional signatures of iNKT cell activation are conserved across species. in addition, they showed, adipose NKT10 cells, had blunted response to ⍺-GalCer and expressed markers associated with Tr1 cells. Furthermore, they demonstrated two memory-like iNKT cell populations, expressing immunoregulatory cytokines and maf (cMAF+ iNKT cells) or cytotoxic markers and klrg1 (KLRG1+ iNKT), were constitutively present in adipose tissue, and were induced in the spleen following ⍺-GalCer challenge. Overall, this study provides novel insights into the transcriptional program of activated iNKT cells and the phenotype of regulatory iNKT cells. The bioinformatic aspect of this study is well performed, but the immunology and T cell biology aspects could be strengthened.

Reviewer #1 (Public Review):

Champer et al. evaluate two homing drives that have been developed in the Anopheles mosquito. Variants of one of these (zpg) are possibly being further investigated for an eventual release. Work with the other has seemingly been discontinued because of unintended fitness costs. The authors argue that this second drive may be in fact better if the experimental results are interpreted more favourably. An important point if true, but somewhat separate from the findings in the paper. To a large extent, this point could be made without any of the results in the paper. However, the authors do show through modelling that this difference may in fact be relevant.

This careful justification of the model parameters increases its relevance to the evaluation of those specific gene drives. The zpg drive will likely be extensively investigated and the specific relevance of this work is a valuable contribution. While a range of parameters is tested for each expression pattern, there are no step-by-step investigations of how the drive outcomes are effect by changes to the underlying DNA-repair/deposition/fitness parameters. So while a reader may learn one drive is better than the other, the ability to get a deeper understanding of the underlying relationship is limited. This means this work has a more limited scope and relies on the relevance of the chosen parameters. In that regard, there may be room for improvement. The chosen parameters for zpg and nos may not be completely fair in regards to the target site and I believe this needs to be addressed.

The second aspect of this paper is the comparison between the commonly used panmictic modelling approach and spacial models. This also somewhat relies on the drive parameters being chosen well, as a more comprehensive evaluation of the spacial approach has been done in prior work by this group. However, showing that these particular extremely efficient drives may still struggle when additional spacial factors are considered is useful and relevant. That a second Anopheles-specific spacial model further reduces the drive performance is a relevant finding. This is helped by a specific analysis of the effect of changes to the migration rates and the low-density growth rate. This spacial modelling also has relevant findings for the homing X-shredder design.

Reviewer #1 (Public Review):

The molecular mechanism of articular chondrocyte differentiation has not been fully revealed. In this study, the authors identified NFATc1 as a key regulator of articular chondrocyte differentiation during early mouse development. At E13.5, NFATc1-expressing cells were mainly located in the flanking region of the joint interzone. With cartilage development, NFATc1-expressing cells generated most of articular chondrocytes. Through transcriptome analyses the authors showed a set of genes expressed in NFATc1-enriched articular cartilage progenitor cells. The expression of NFATc1 was diminished during articular chondrocyte differentiation. The authors found that suppression of NFATc1 expression in articular cartilage progenitor cells is sufficient to induce spontaneous chondrogenesis; while overexpressing NFATc1 suppresses chondrogenesis. They further demonstrated that NFATc1 negatively regulated Col2a1 gene transcription. This is an important study since it will help us understand the regulatory mechanism of articular chondrocyte differentiation and pathological mechanisms of joint diseases, such as osteoarthritis.

Reviewer #1 (Public Review):

The authors show that LPS activates Pink1-Parkin-dependent mitophagy in lung endothelial cells, which is mediated by TNFα. Endothelial-specific Pink1-KO improves LPS-induced inflammatory responses in mice and reduces the release of one of the mitochondrial formylated proteins, ND6. These results suggest that PINK1-dependent endothelial mitophagy is pro-inflammatory and increases the release of mitochondrial formyl peptides, one of the mitochondrial DAMPs. The identification of the origin of serum formyl proteins and the elucidation of the release mechanism would be important.

The data are generally clear and convincing. The authors' conclusion would be strengthened if the authors could show that serum ND6 levels during inflammation is indeed regulated by endothelial mitophagy using their endothelial-specific Pink1-KO mouse model.

Reviewer #1 (Public Review):

This study sets out to decipher whether the eDNA that promotes biofilm dispersal in Caulobacter crescentus biofilms is released when a random portion of cells lyse within biofilms, or whether eDNA release is a regulated process. They start by investigating whether any of the C. crescentus TA systems contribute to biofilm-associated cell death, and find that one of the systems, ParDE4 is responsible for cell death and eDNA release. They go on to show that this system is O2-regulated and thus contributes to cell death in particular in the oxygen limited interior regions of biofilms. These findings contribute significantly to our understanding of the biological functions of toxin-antitoxin systems, mechanisms of bacterial programmed cell death, and biofilm growth. The notion that TA systems function in cell death in particular has been controversial, and often based on overexpression of the toxin component, therefore the fact that this study uses a TA system in its native genomic context is notable. The authors also show clearly the somewhat counterintuitive result that the cell death (and presumably, toxin activity) is negatively correlated with transcription of the TA system. This is consistent with what is known about TA biology (but not with many past TA papers, which often correlated TA transcription with toxin activation). The study also provides a logical rationale for how ParDE4 mediated cell death ultimately contributes to bacterial fitness. The paper is well written and figures are clear and easy to follow.

There are two relatively minor shortcomings of the paper, both acknowledged as caveats by the authors in their discussion. First, while the authors do include one experiment that addresses whether the toxin is responsible for the cell death (Fig 3), they do not show direct evidence of the activity of the toxin other than cell death/eDNA release. Second, the authors do not address whether the reduced TA transcription they observe is what causes the release of the toxin and thus the cell death phenotype. This seems likely to be the case based on previous studies of other TA systems (e.g. TA systems involved in plasmid segregation, most clearly shown for CcdAB, or more recently the ToxIN system during phage infection). Connecting this directly would be a very valuable addition to this study.

Reviewer #1 (Public Review):

The findings reported in this paper demonstrate that integration of Wnt and BMP signaling by Axin1 is crucial for lower limb development, and defects in Axin1 and Axin2 signaling could lead to the development of FH disease.

Reviewer #1 (Public Review):

The manuscript by Dolan et al. presents a high-resolution structure of the SARS-CoV-2 M (membrane) protein, determined by cryo-electron microscopy. Despite the protein's small size (50 kDa as a homodimer), the structure is well-determined and of sufficient resolution to build a confident model for the vast majority of the protein chain (missing only a short disordered N-terminal tail). The protein forms a homodimer with each protomer possessing three transmembrane helices and a beta-strand rich C-terminal domain. The overall structure of M is similar to that of ORF3a, a viral-encoded pore protein. The cytosolic surface of M's C-terminal domain is highly positively charged, and the authors propose that this charge mediates interactions with the N protein, S protein, and possibly viral RNA. Finally, the authors perform molecular dynamics simulations that demonstrate that the M dimer is relatively stable, at least over the time-frame of the simulation (1.6 microseconds).

Overall, this is a straightforward work that describes the structure of an important protein in the life cycle of SARS-CoV-2. As such, it is important and timely, and will be of interest to a broad set of readers. The work suggests many directions for future experiments.

Reviewer #1 (Public Review):

This paper follows several innovative articles from the authors exploring the molecular mechanisms of insulin and IGF1 receptors activation by their ligands using cryo-electron microscopy. Here the authors explore the role of an alpha helical C-terminal segment (called the alpha-CT motif) of a disordered disulfide-linked insert domain in the FnIII-2 module of the insulin and IGF1 receptors (at the end of the alpha subunit), in the mechanism of ligand binding, negative cooperativity and receptor activation.

Biochemical data gathered over several decades have suggested that insulin and IGF1 use two separate binding sites, site 1 and site 2, to bind to two distinct domains (sites 1 and 2, and 1'and 2') on each protomer of the homodimeric receptors, disposed in an antiparallel symmetry. This disposition was corroborated by the early x-ray crystallographic studies of the unliganded insulin receptor ectodomain (apo-receptor). A subsequent somewhat surprising finding was that the insulin receptor site 1 is in fact a composite, made of the beta surface of the L1 module of one protomer, and of the alpha-CT motif of the other protomer which binds perpendicularly to the L1 surface (a "tandem binding element"), with insulin binding more to the alpha-CT motif than to L1.

Previous work from the authors showed that the subsaturated insulin receptor has an asymmetric configuration while the receptor saturated with 4 insulins has a symmetric T-shaped configuration. In contrast, the IGF1R shows only one IGF1 bound to an asymmetric configuration, indicating according to the authors a stronger negative cooperativity. This is attributed to a more rigid and elongated conformation of the alpha-CT motives that restricts the structural flexibility of the alternate binding site.

To test this hypothesis, the authors determined the cryo-EM structure of IGF1 bound to IGF1R with a mutated alpha-CT motif elongated by four glycine residues. Strikingly, a portion of these constructs adopt a T-shaped symmetric structure.

Conversely, they show that the cryo-EM structure of insulin bound to an insulin receptor with non-covalently bound alpha-CTs insert domains by mutation of the cysteines to serine adopts asymmetric conformations even at saturated insulin concentrations. They conclude that the alpha-CTs in disulfide-linked insert domains of the insulin receptor play an important role in the structural transition from asymmetric to symmetric during the insulin-induced insulin receptor activation.

All in all, this is a very interesting and well-designed study that represents an advance in the knowledge of the insulin/IGF1 receptor systems, although the details of the structural interpretations deserve some discussion.

Joint Public Review:

As demonstrated in alpha genus human papillomavirus (HPV) 16 positive head and neck squamous cell carcinoma, Hu et al. report that the E6 protein of beta genus HPV8, which is implicated in the development of skin cancer, promotes genomic instability by increasing use of error-prone alternative end-joining repair. Liu et al. (2018) showed that loss of TGFβ signaling compromised HR in HPV+ head and neck squamous cell carcinomas (HNSCC) and shifted repair to alt-EJ. A follow up paper in 2021 showed that cancers with an alt-EJ gene signature have high frequency of microhomology flanked indels, pathognomonic for alt-EJ repair. Leeman et al. (2019) demonstrated that HPV16 E7 promotes error-prone, alt-EJ and suppresses NHEJ. Hence the finding that E6 from beta HVP8 does so as well is not unexpected but it is important to provide convincing experimental evidence as is done in this manuscript. Here the authors confirm and extend earlier studies (Hu et al., 2020; Hu and Wallace, 2022; Wallace et al., 2015) showing that loss of p300, a transcription factor necessary for robust expression of key DNA repair genes, impairs execution of HR and NHEJ, which is a prerequisite for driving up use of alt-EJ as a salvage path. Both pathways are inhibited past the initiation step. During NHEJ 8E6 allows DNA-PKcs assembly and activation at DSBs but attenuates downstream steps, Likewise, during HR 8E6 allows assembly of RAD51 foci but blocks their processing. As 8E6 does not block but rather delays DSB repair, this manuscript provides convincing evidence that alternative end-joining of DSBs is increased in 8E6 expressing cells. The study employs gamma-H2AX foci as a surrogate for DSB levels and employs appropriate reporter assays to monitor HR and alt-EJ. Genomic DNA sequencing of 8E6 expressing human foreskin keratinocytes and control cells documents the typical genome scars of elevated alt-EJ including small deletions flanked by microhomology and small templated insertions. The results contribute to our understanding of DSB repair pathway control and may explain the association of HPV8 with nonmelanoma skin cancer, although this tumor type specificity remains unclear. A key limitation of the study that it is not established which alt-EJ pathway is active in 8E6 expressing cells, in particular whether DNA polymerase theta (POLθ) is involved. The mechanism by which p300 favors NHEJ and HR and its absence favors alt-EJ remains to be determined.

Reviewer #1 (Public Review):

This manuscript investigates the KOW domain of the fold switching, NusG protein family. E. coli RfaH-KOW is known to form an a-helical hairpin when docked onto the NGN domain and generate an auto-inhibited conformation, which blocks access to the RNA polymerase binding site. Upon activation, this helical KOW subdomain can refold into a beta-barrel structure that then is important for making contacts to ribosomes. In this manuscript the authors identify and structurally characterize RfaH protein from Vibrio cholerae, which has only ~36% sequence identity with EcRfaH, yet displays similar fold switching ability. In addition, the authors provide thermodynamic measurements and some structural information on 4 KOW domains from different organisms, and show that it is the instability in their β-barrel structure that enables the conformational plasticity and fold switching ability of these proteins.

Reviewer #1 (Public Review):