Reviewer #1 (Public Review):

The discovery of Homo naledi fossils and the rising star cave system is unquestionably important for paleoanthropology. The fossils themselves hold a wealth of information about the diversity and complexity of morphological and evolutionary change on the hominin family tree. It is a truly amazing find and important site and it is important that information about this site continues to be produced so that more can be known. It is equally important that the papers produced from the site be fully reviewed for scientific rigor. I hope to assist with this to the best of my ability.

In its current form the paper, "Evidence for the deliberate burial of the dead by Homo naledi," does not meet the standards of our field. The paper is hard to follow. It lacks key citations, contextual background information to inform the reader about the geological and depositional structure of the caves, and concise understandable descriptions of the methods and the significance of the results.

The main point of the paper is to describe three possible burial features. The working hypothesis is that the features are intentional burials, and the authors seek to support this hypothesis throughout rather than test it. The authors do this by noting mineralogical differences in sediment and possible bowl-shaped sedimentological distinctions where fossil bones occur. As stated above, this evidence needs to be elaborated on the in text, contextualized, and edited for clarity. In addition, throughout the paper, the authors only consider two depositional scenarios for burial and body decomposition: 1) a body was intentionally buried in a pit that was dug into the cave sediments, and then buried in sediment (without detailing in the main text what sediment was used to backfill the pit); and 2) the body was left in a natural pit and decayed in the open. A major problem with only considering these two scenarios for body decomposition is that previous reports about cave geology and sedimentology show that it is a dynamic system involving erosion, sediment slumping and drainage, and contraction of clay, which is a major component of the sediment, etc. The authors are very clear that flooding is not a viable option for the movement of skeletal elements in the cave. However, they do not mention other processes such as erosion or sediment slumping, that are known to occur and could be responsible for moving sediment and fossils in each chamber of the cave. They also do not consider carnivore involvement which has been suggested by Val (2016) and Egeland et al. (2018). Such processes could naturally transport bodies, shift them around, and sediment erosion could bury them. The articulation of some skeletal elements is a major argument for intentional burial, yet within the cave substructure, articulated bones are often commingled with disarticulated elements from the same or different individuals. This same situation exists in the features included in this paper. It does appear that some skeletal material was covered in sediment before decomposition and remains in articulation, but bodies decompose at different rates, and can decompose slowly, especially in environments that lack insects (see Simmons et al. 2010 Journal of Forensic Sciences https://doi-org.aurarialibrary.idm.oclc.org/10.1111/j.1556-4029.2009.01206.x). Wiersma et al., 2019 describe the cave system as very humid, but dry due to little standing water, mildly acidic, with an average temperature today of 18{degree sign}C and a minimum of 12{degree sign}C over the last million years. The starting null hypothesis should be that the bodies were naturally covered in sediment. Intentional burial requires extraordinary circumstances and requires multiple lines of solid evidence to support the hypothesis. In testing for natural burial processes, the rate of body decomposition should be reconstructed given the environmental parameters of the cave.

In keeping with supporting their starting hypothesis that Homo naledi intentionally buried individuals in the cave, the authors conclude that "A parsimonious explanation for this configuration of skeletal remains is that these remains may be a palimpsest of burials that have sequentially disrupted each other. In this hypothesis, early burials were disturbed when pits were dug for subsequent burials. Other occurrences of remains outside of the Dinaledi Chamber and Hill Antechamber (Hawks et al., 2017; Brophy et al., 2021) are discussed as possible evidence of mortuary practices in SI 4.2. Instances where parts of individuals occur in remote narrow passages cannot be explained as a result of carnivore or water transport (Elliott et al., 2021; Brophy et al., 2021), making it necessary to consider that H. naledi may have placed these partial remains in these locations, possibly representing a form of funerary caching." After reviewing the evidence presented in the current manuscript, it is not clear why this is a parsimonious explanation. The authors have repeatedly described how incredibly challenging it is to get into and out of this cave system and all of its chambers. How could any species, even small bodied species, drag/pull/shove dead bodies through small crevasses, shove or drop them down a narrow shoot, continue to move through the hill antechamber to the Dineledi chamber and bury bodies? It is not impossible but given the previously published descriptions of the dynamic process of sedimentation movement in the cave it is certainly not a parsimonious explanation. To support this will take many more lines of evidence than presented here such as micromorphological analysis of the overall cave system and each feature (discussed in the supplementary information but briefly), full detailed reconstruction of sediment, water, fossil, and debris movement throughout the cave system coupled with reconstructions of body decomposition rates. Scientifically precise computer-generated reconstructions of all of this are possible working with specialists affiliated with National Geographic. An analysis also needs to start by testing a null hypothesis, not deciding on the conclusion and setting out to "prove" it.

Public Review:

In this article, a novel technique allowing the linking of viral transcription levels and progeny virion production is presented. Barcoded libraries of an H1N1 influenza virus (two genes were barcoded near the 3'end) were used to infect cells using an experimental approach ensuring that, in the low multiplicity of infection condition, each cell is infected by one virion and that nearly every virion has a unique barcode. This allows then, upon single-cell RNA sequencing and sequencing of the supernatants, to infer back the cells that were producing certain barcoded viruses. Assessing detection frequencies of barcodes in the single-cell sequencing and in the sequencing of the supernatants allows us to compare the levels of viral transcription and progeny virion production.

Observations that viral transcription levels are very heterogenous at the single-cell level are not novel, but reinforce those from previous studies. The major findings of this study are (i) progeny virion production is also very heterogenous, i.e., a few cells produce most of the progeny virions and (ii) there is a poor correlation between viral transcription levels and progeny virion production at the single-cell level.

Strengths:<br /> The article is very well written, the experimental choices are very well justified and the methods are very detailed, allowing the possibility of reproducing the work performed in this study. The conclusions are very well supported by the data and the limitations of the study and how those might influence the conclusions are also clearly explained. In addition, several experimental caveats, such as PCR cross-overs in next-generation sequencing and cell multiplets in single-cell sequencing, were well accounted for, which is not always the case in studies using these techniques.

Weaknesses:<br /> It seems that the results presented here are from one single experiment. How reproducible are the results?

As explained in the article, it is important that nearly every virion has a unique barcode. This was assessed by sequencing the barcodes in the virus libraries. Between 92% to 96% of the barcodes were unique. With this information, it should be possible to assess whether non-unique barcodes were detected in infected cells, and if yes, remove these from the downstream analysis.

It seems like all the information available in this very rich dataset was not fully exploited. For instance, Figure 5C suggests that cells missing the expression of one viral gene might still be able to produce progeny viruses. It would be interesting to have the information regarding which gene was not expressed in these cells.

The introduction and discussion are rather short and the article could benefit from expanding them. Additional speculations about viral or cellular factors (e.g. differences in innate immune responses, differences in cell division status) that might explain the observed heterogeneity, both at the viral transcription and viral progeny virus production levels, would be interesting.

Reviewer #1 (Public Review):

Gap junctions, formed from connexins, are important in cell communication, allowing ions and small molecules to move directly between cells. While structures of connexins have previously been reported, the structure of Connexin 43, which is the most widely expressed connexin and is important in many physiological processes was not known. Qi et al used cryo-EM to solve the structure of Connexin 43. They then compared this structure to structures of other connexins. Connexin gap junctions are built from two "hemichannels" consisting of hexamers of connexins. Hemichannels from two opposing cells dock together to form a complete channel that allows the movement of molecules between cells. N-terminal helices from each of the 6 subunits of each hemichannel allow control of whether the channels are open or closed. Previously solved structures of Cx26 and Cx46/50 have the N-termini pointing down into the pore of the protein leaving a central pore and so these channels have been considered to be open. The structure that Qi et al observed has the N-termini in a more raised position with a narrower pore through the centre. This led them to speculate whether this was the "closed" form of the protein. They also noted that, if only the protein was considered, there were gaps between the N-terminal helices, but these gaps were filled with lipid-like molecules. They, therefore, speculated that lipids were important in the closure mechanism. To address whether their structure was open or closed with respect to ions they carried out molecular dynamics studies, and demonstrated that under the conditions of the molecular dynamics ions did not traverse the channel when the lipids were present.

Strengths:

The high resolution cryo-EM density maps clearly show the structure of the protein with the N-termini in a lateral position and lipid density blocking the gaps between the neighbouring helices. The conformation that they observe when they have solved the structure from protein in detergent is also seen when they reconstitute the protein into nanodiscs, which is ostensibly a more membrane-like environment. They, therefore, would appear to have trapped the protein in a stable conformational state.<br /> The molecular dynamics simulations are consistent with the channel being closed when the lipid is present and raises the possibility of lipids being involved in regulation.<br /> A comparison of this structure with other structures of connexin channels and hemichannels gives another representation of how the N-terminal helix of connexins can variously be involved in the regulation of channel opening.

Weaknesses:

While the authors have trapped a relatively stable state of the protein and shown that, under the conditions of their molecular dynamics simulations, ions do not pass through, it is harder to understand whether this is physiologically relevant. Determining this would be beyond the scope of the article. To my knowledge there is no direct evidence that lipids are involved in regulation of connexins in this way, but this is also an interesting area for future exploration. It is also possible that lipids were trapped in the pore during the solubilisation process making it non-physiological. The authors acknowledge this and they describe the structure as a "putative" closed state.

The positions of the mutations in disease shown in Figure 4 is interesting. However, the authors don't discuss/speculate how any of these mutations could affect the binding of the lipids or the conformational state of the protein.

It should also be noted that a structure of the same protein has recently been published. This shows a very similar conformation of the N-termini with lipids bound in the same way, despite solubilising in a different detergent.

Reviewer #1 (Public Review):

The authors report a study, where they have sequenced whole genomes of four individuals of an extinct species of butterfly from western North America (Glaucopsyche xerces), along with seven genomes of a closely related species (Glaucopsyche lygdamus), mainly from museum specimens, several to many decades old. They then compare these fragmented genomes to a high-quality, chromosome-level assembly of a genome of a European species in the same genus (Glaucopsyche alexis). They find that the extinct species shows clear signs of declining population sizes since the last glacial period and an increase in inbreeding, perhaps exacerbating the low viability of the populations and contributing to the extinction of the species.

The study really highlights how museum specimens can be used to understand the genetic variability of populations and species in the past, up to a century or more ago. This is an incredibly valuable tool, and can potentially help us to quickly identify whether current populations of rare and declining species are in danger due to inbreeding, or whether at least their genetic integrity is in good condition and other factors need to be prioritised in their conservation. In the case of extinct species, sequencing museum specimens is really our only window into the dynamics of genomic variability prior to extinction, and such information can help us understand how genetic variation is related to extinction.

I think the authors have achieved their goal admirably, they have used a careful approach to mapping their genomic reads to a related species with a high-quality genome assembly. They might miss out on some interesting genetic information in the unmapped reads, but by and large, they have captured the essential information on genetic variability within their mapped reads. Their conclusions on the lower genetic variability in the extinct species are sound, and they convincingly show that Glaucopyche xerces is a separate species to Glaucopsyche lygdamus (this has been debated in the past).

Reviewer #1 (Public Review):

Multiple sclerosis (MS) is a debilitating autoimmune disease that causes loss of myelin in neurons of the central nervous system. MS is characterized by the presence of inflammatory immune cells in several brain regions as well as the brain barriers (meninges). This study aims to understand the local immune hallmarks in regions of the brain parenchyma that are adjacent to the leptomeninges in a mouse model of MS. The leptomeninges are known to be a foci of inflammation in MS and perhaps "bleed" inflammatory cells and molecules to adjacent brain parenchyma regions. To do so, they use novel technology called spatial transcriptomics so that the spatial relationships between the two regions remain intact. The study identifies canonical inflammatory genes and gene sets such as complement and B cells enriched in the parenchyma in close proximity to the leptomeninges in the mouse model of MS but not control. The manuscript is very well written and easy to follow. The results will become a useful resource to others working in the field and can be followed by time series experiments where the same technology can be applied to the different stages of the disease.

Reviewer #1 (Public Review):

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

The analysis procedure depends on the availability of an accurate atlas that serves as a reference map ‎for neural positions. Thus, when imaging a new reporter line without fair prior knowledge of the ‎tagged cells, such an atlas may be very difficult to construct. Moreover, usage of available reference ‎atlases, constructed based on other databases, is not very helpful (as shown by the authors in Fig 3), ‎so for each new reporter line a de-novo atlas needs to be constructed.‎

I have a few comments that may help to better understand the potential of the tool to become handy:

‎1) I wonder the degree by which strain mosaicism affects the analysis (Figs 1-4) as it was performed on ‎a non-integrated reporter strain. As stated, for constructing the reference atlas, the authors used ‎worms in which they could identify the complete set of tagged neurons. But how sensitive is the ‎analysis when assaying worms with different levels of mosaicism? Are the results shown in the paper ‎stem from animals with a full neural set expression? Could the authors add results for which the ‎assayed worms show partial expression where only 80%, 70%, 50% of the cells population are ‎observed, and how this will affect identification accuracy? This may be important as many non-‎integrated reporter lines show high mosaic patterns and may therefore not be suitable for using this ‎analytic method. In that sense, could the authors describe the mosaic degree of their line used for ‎validating the method.‎<br /> ‎<br /> 2) For the gene expression analysis (Fig 5), where was the intensity of the GFP extracted from? As it has ‎no nuclear tag, the protein should be cytoplasmic (as seen in Fig 5a), but in Fig 5c it is shown as if the ‎region of interest to extract fluorescence was nuclear. If fluorescence was indeed extracted from the ‎cytoplasm, then it will be helpful to include in the software and in the results description how this was ‎done, as a huge hurdle in dissecting such multi-cell images is avoiding crossreads between ‎adjacent/intersecting neurons.‎<br /> ‎<br /> 3) In the same matter: In the methods, it is specified that the strain expressing GCAMP was also used ‎in the gene expression analysis shown in Figure 5. But the calcium indicator may show transient ‎intensities depending on spontaneous neural activity during the imaging. This will introduce a ‎significant variability that may affect the expression correlation analysis as depicted in Figure 5.‎

Reviewer #1 (Public Review):

Colin et al demonstrated that condensin is a key factor for the disjunction of sister-telomeres during mitosis and proposed that it is due to that condensin restrains the telomere association of cohesin. The authors first showed that condensin binds telomeres in mitosis evidenced by ChIP-qPCR and calibrated ChIP-seq. They further demonstrated that compromising condensin's activity leads to a failure in the disjunction of telomeres, with convincing cytological and HI-seq evidence. Two telomeric proteins Taz1 and Mit1 were identified to specifically regulate the telomere association of condensin. Deletion of these genes decreased/increased condensin's telomere association and exacerbated/remedied the defected telomere disjunction in a condensin mutant, echoing the role of condensin in telomere disjunction. They proposed that the underlying mechanism is that condensin inhibits cohesin's accumulation at telomeres. However, the evidence for this claim might need to be further strengthened. Nevertheless, this study uncovered a novel role of condensin in the separation of telomeres of sister chromosomes and open a question of how condensin regulates the structure of chromosomal ends.

Reviewer #1 (Public Review):

Summary of the major findings -

1. The authors used saturation mutagenesis and directed evolution to mutate the highly conserved fusion loop (98 DRGWGNGCGLFGK 110) of the Envelope (E) glycoprotein of Dengue virus (DENV). They created 2 libraries with parallel mutations at amino acids 101, 103, 105-107, and 101-105 respectively. The in vitro transcribed RNA from the two plasmid libraries was electroporated separately into Vero and C6/36 cells and passaged thrice in each of these cells. They successfully recovered a variant N103S/G106L from Library 1 in C6/36 cells, which represented 95% of the sequence population and contained another mutation in E outside the fusion loop (T171A). Library 2 was unsuccessful in either cell type.

2. The fusion loop mutant virus called D2-FL (N103S/G106L) was created through reverse genetics. Another variant called D2-FLM was also created, which in addition to the fusion loop mutations, also contains a previously published, evolved, and optimized prM-furin cleavage sequence that results in a mature version of the virus (with lower prM content). Both D2-FL and D2-FLM viruses grew comparably to wild type virus in mosquito (C6/36) cells but their infectious titers were 2-2.5 log lower than wild<br /> type virus when grown in mammalian (Vero) cells. These viruses were not compromised in thermostability, and the mechanism for attenuation in Vero cells remains unknown.

4. Next, the authors probed the neutralization of these viruses using a panel of monoclonal antibodies (mAbs) against fusion loop and domain I, II and III of E protein, and against prM protein. As intended, neutralization by fusion loop mAbs was reduced or impaired for both D2-FL and D2-FLM, compared to wild type DENV2. D2-FLM virus was equivalent to wild type with respect to neutralization by domain I, II, and III antibodies tested (except domain II-C10 mAb) suggesting an intact global antigenic landscape of the mutant virion. As expected, D2-FLM was also resistant to neutralization by prM mAbs (D2-FL was not tested in this batch of experiments).

5. Finally, the authors evaluated neutralization in the context of polyclonal serum from convalescent humans (n=6) and experimentally infected non-human primates (n=9) at different time points (27 total samples). Homotypic sera (DENV2) neutralized D2-FL, D2-FLM, and wild type DENV similarly, suggesting that the contribution of fusion loop and prM epitopes is insignificant in a serotype-specific neutralization response. However, heterotypic sera (DENV4) neutralized D2-FL and D2-FLM less potently than wild type DENV2, especially at later time points, demonstrating the contribution of fusion loop- and prM-specific antibodies to heterotypic neutralization.

Impact of the study-

1. The engineered D2-FL and D2-FLM viruses are valuable reagents to probe antibodies targeting the fusion loop and prM in the overall polyclonal response to DENV.

2. Though more work is needed, these viruses can facilitate the design of a new generation of DENV vaccine that does not elicit fusion loop- and prM-specific antibodies, which are often poorly neutralizing and lead to antibody-dependent enhancement effect (ADE).

3. This work can be extended to other members of the flavivirus family.

4. A broader impact of their work is a reminder that conserved amino acids may not always be critical for function and therefore should not be immediately dismissed in substitution/mutagenesis/protein design efforts.

Evaluating this study in the context of prior literature -

The authors write "Although the extreme conservation and critical role in entry have led to it being traditionally considered impossible to change the fusion loop, we successfully tested the hypothesis that massively parallel directed evolution could produce viable DENV fusion-loop mutants that were still capable of fusion and entry, while altering the antigenic footprint."<br /> ".....Previously, a single study on WNV successfully generated a viable virus with a single mutation at the fusion loop, although it severely attenuated neurovirulence. Otherwise, it has not been generated in DENV or other mosquito-borne flaviviruses"

The above claims are a bit overstated. In the context of other flaviviruses:

- A previous study applied a similar saturation mutagenesis approach to the *full length* E protein of Zika virus and found that while the conserved fusion loop was mutationally constrained, some mutations, including at amino acid residue 106 were tolerated (PMID 31511387).<br /> - The Japanese encephalitis virus (JEV) SA14-14-2 live vaccine strain contains a L107F mutation in the fusion loop (in addition to other changes elsewhere in the genome) relative to the parental JEV SA14 strain (PMID: 25855730).<br /> - For tickborne encephalitis virus (TBEV-DENV4 chimera), H104G/L107F double mutant has been described (PMID: 8331735)

There have also been previous examples of functionally tolerated mutations within the DENV fusion loop:

- Goncalvez et al., isolated an escape variant of DENV 2 using chimpanzee Fab 1A5, with a mutation in the fusion loop G106V (PMID: 15542644). G106 is also mutated in D2-FL clone (N103S/G106L) described in the current study.<br /> - In the context of single-round infectious DENV, mutation at site 102 within the fusion loop has been shown to retain infectivity (PMID 31820734).

Appraisal of the results -

The data largely support the conclusions, but some improvements and extensions can benefit the work.

1. Line 92-93: "This major variant comprised ~95% of the population, while the next most populous variant comprised only 0.25% (Figure 1C)".<br /> What is the sequence of the next most abundant variant?

2. Lines 94-95: "Residues W101, C105, and L107 were preserved in our final sequence, supporting the structural importance of these residues."<br /> L107F is viable in other flaviviruses.

3. Figure 2c: The FLM sample in the western blot shows hardly any E protein, making E/prM quantitation unreliable.

4. Lines 149 -151: "Importantly, D2-FL and D2-FLM were resistant to antibodies targeting the fusion loop. While neutralization by 1M7 is reduced by ~2-logs, no neutralization was observed for 1N5, 1L6, and 4G2 for either variant (Figure 3 A)".

a) Partial neutralization was observed for 1N5, for D2-FL.<br /> b) Do these mAbs cover the full spectrum of fusion loop antibodies identified thus far in the field?<br /> c) Are the epitopes known for these mAbs? It would be useful to discuss how the epitope of 1M7 differs from the other mAbs? What are the critical residues?<br /> d) Maybe the D2-FL mutant can be further evolved with selection pressure with fusion loop mAbs 1M7 +/-1N5 and/or other fusion loop mAbs.

5. It would have been useful to include D2-M for comparison (with evolved furin cleavage sequence but no fusion loop mutations).

6. Data for polyclonal serum can be better discussed. Table 1 is not discussed much in the text. For the R1160-90dpi-DENV4 sample, D2-FL and D2-FLM are neutralized better than wild type DENV2? The authors' interpretation in lines 181-182 is inconsistent with the data presented in Figure 3C, which suggests that over time, there is INCREASED (not waning) dependence on FL- and prM-specific antibodies for heterotypic neutralization.

Suggestions for further experiments-

1. It would be interesting to see the phenotype of single mutants N103S and G106L, relative to double mutant N103S/G106L (D2-FL).<br /> 2. The fusion capability of these viruses can be gauged using liposome fusion assay under different pH conditions and different lipids.<br /> 3. Correlative antibody binding vs neutralization data would be useful.

Reviewer #1 (Public Review):

Masson et al. leveraged the natural genetic diversity presented in a large cohort of the Diversity Outbred in Australia (DOz) mice (n=215) to determine skeletal muscle proteins that were associated with insulin sensitivity. The hits were further filtered by pQTL analysis to construct a proteome fingerprint for insulin resistance. These proteins were then searched against Connectivity Map (CMAP) to identify compounds that could modulate insulin sensitivity. In parallel, many of these compounds were screened experimentally alongside other compounds in the Prestwick library to independently validate some of the compound hits. These two analyses were combined to score for compounds that would potentially reverse insulin resistance. Thiostrepton was identified as the top candidate, and its ability to reverse insulin resistance was validated using assays in L6 myotubes.

Below are several comments made on the original version of this study, addressed by the authors in the current version:

(1) Please describe the rationale of trypsinizing the tissue prior to mitochondrial isolation.

(2) The authors mentioned that the proteomics data were Log2 transformed and median-normalized. Please provide a bit more details on this, including whether the subjects were randomized.

(3) In Figure 1D, please give the numbers of mice the authors used for the CV comparisons in each group, whether they were of similar age and sex, and whether the differences in CV values were statistically significant

(4) The authors stated in lines 155-157 that proteins negatively associated with Matsuda index were further filtered by presence of their cis-pQTLs. Please provide more explanations to justify this filtering criterion.

(5) Please explain why the first half of the paper focused extensively on the authors' discoveries in the mitochondrial proteome, and how proteins involved in mitochondrial processes (such as complex I) were associated with Matsuda Index, but the final fingerprint list of insulin resistance, which contained 76 proteins, only had 7 mitochondrial proteins.

(6) The authors found that thiostrepton-induced insulin resistance reversal effects were not through insulin signalling. Please list the proteins in the fingerprint list that led to identification of thiostrepton on CMAP, and discuss whether you think that thiostrepton directly or indirectly acts on these protein targets.

Reviewer #1 (Public Review):

The authors investigated the molecular correlates in potential neural centers in the Japanese quail brain associated with photoperiod-induced life-history states. The authors simulated photoperiod to attain winter and summer-like physiology and samples of neural tissues at spring, and autumn life-history states, daily rhythms in transcripts in solstices and equinox, and lastly studies FSHb transcripts in the pituitary. The experiments are based on a series of changes in photoperiod and gave some interesting results. The experiment did not have a control for no change in photoperiod so it seems possible that endogenous rhythms could be another aspect of seasonal rhythms that lack in this study. The short-day group does not explain the endogenous seasonal response.

The manuscript would benefit from further clarity in synthesizing different sections. Additionally, there are some instances of unclear language and numerous typos throughout the manuscript. A thorough revision is recommended, including addressing sentence structure for improved clarity, reframing sentences where necessary, correcting typos, conducting a grammar check, and enhancing overall writing clarity.

Data analysis needs more clarity particularly how transcriptome data explains different physiological measures across seasonal life-history states. It seems the discussion is built around a few genes that have been studied in other published literature on quail seasonal response. Extending results on the promotor of DEGs and building discussion is an extrapolating discussion on limited evidence and seems redundant.

Last, I wondered if it would be possible to add an ecological context for the frequent change in the photoperiod schedule and not take account of the endogenous annual response. Adding discussion on ecological relevance would make more sense.

Reviewer #1 (Public Review):

In this study, the authors set out to investigate spatial RNA processing events, specifically alternative splicing and 3' UTR usage, in mouse brain and kidney tissues using ReadZS and SpliZ methodologies on spatial transcriptomics data. The research contributes to understanding tissue-specific gene expression regulation from a spatial perspective. The study introduces a novel approach for analyzing spatial transcriptomics data, allowing for the identification of RNA processing and regulation patterns directly from 10X Visium data. The authors present convincing evidence supporting the identification of novel RNA processing patterns using their methodology, which holds significant implications for researchers in the field of spatial transcriptomics and the study of alternative splicing and 3' UTR usage

The conclusions of the study are mostly well-supported by the data; however, certain aspects could be improved to strengthen the findings.<br /> 1) The conclusions of this study would be strengthened by conducting a more extensive tissue sample analysis and including biological replicates. Additionally, appropriate batch effect corrections should be applied when dealing with biological replicates.<br /> 2) The 3' UTR usage and alternative splicing should be compared among clearly labeled clusters for a more comprehensive analysis.<br /> 3) The authors should clarify their rationale for choosing ReadZS and SpliZ approaches and provide comparisons with other methods to demonstrate the advantages and potential limitations of their chosen methodologies.

Reviewer #1 (Public Review):

Trypanosoma brucei undergoes antigenic variation to evade the mammalian host's immune response. To achieve this, T. brucei regularly expresses different VSGs as its major surface antigen. VSG expression sites are exclusively subtelomeric, and VSG transcription by RNA polymerase I is strictly monoallelic. It has been shown that T. brucei RAP1, a telomeric protein, and the phosphoinositol pathway are essential for VSG monoallelic expression. In previous studies, Cestari et al. (ref. 24) have shown that PIP5Pase interacts with RAP1 and that RAP1 binds PI(3,4,5)P3. RNAseq and ChIPseq analyses have been performed previously in PIP5Pase conditional knockout cells, too (ref. 24). In the current study, Touray et al. did similar analyses except that catalytic dead PIP5Pase mutant was used and the DNA and PI(3,4,5)P3 binding activities of RAP1 fragments were examined. Specifically, the authors examined the transcriptome profile and did RAP1 ChIPseq in PIP5Pase catalytic dead mutant. The authors also expressed several C-terminal His6-tagged RAP1 recombinant proteins (full-length, aa1-300, aa301-560, and aa 561-855). These fragments' DNA binding activities were examined by EMSA analysis and their phosphoinositides binding activities were examined by affinity pulldown of biotin-conjugated phosphoinositides. As a result, the authors confirmed that VSG silencing (both BES-linked and MES-linked VSGs) depends on PIP5Pase catalytic activity, but the overall knowledge improvement is incremental. The most convincing data come from the phosphoinositide binding assay as it clearly shows that N-terminus of RAP1 binds PI(3,4,5)P3 but not PI(4,5)P2, although this is only assayed in vitro, while the in vivo binding of full-length RAP1 to PI(3,4,5)P3 has been previously published by Cestari et al (ref. 24) already. Considering that many phosphoinositides exert their regulatory role by modulating the subcellular localization of their bound proteins, it is reasonable to hypothesize that binding to PI(3,4,5)P3 can remove RAP1 from the chromatin. However, no convincing data have been shown to support the author's hypothesis that this regulation is through an "allosteric switch". Therefore, the title should be revised.

There are serious concerns about many conclusions made by Touray et al., according to their experimental approaches:<br /> 1. The authors have been studying RAP1's chromatin association pattern by ChIPseq in cells expressing a C-terminal HA tagged RAP1. According to data from tryptag.org, RAP1 with an N-terminal or a C-terminal tag does not seem to have identical subcellular localization patterns, suggesting that adding tags at different positions of RAP1 may affect its function. It is therefore essential to validate that the C-terminally HA-tagged RAP1 still has its essential functions. However, this data is not available in the current study. RAP1 is essential. If RAP1-HA still retains its essential functions, cells carrying one RAP1-HA allele and one deleted allele are expected to grow the same as WT cells. In addition, these cells should have the WT VSG expression pattern, and RAP1-HA should still interact with TRF. Without these validations, it is impossible to judge whether the ChIPseq data obtained on RAP1-HA reflect the true chromatin association profile of RAP1.

2. Touray et al. expressed and purified His6-tagged recombinant RAP1 fragments from E. coli and used these recombinant proteins for EMSA analysis: The His6 tag has been used for purifying various recombinant proteins. It is most likely that the His6 tag itself does not convey any DNA binding activities. However, using His6-tagged RAP1 fragments for EMSA analysis has a serious concern. It has been shown that His6-tagged human RAP1 protein can bind dsDNA, but hRAP1 without the His6 tag does not. It is possible that RAP1 proteins in combination with the His6 tag can exhibit certain unnatural DNA binding activities. To be rigorous, the authors need to remove the His6 tag from their recombinant proteins before the in vitro DNA binding analyses are performed. This is a standard procedure for many in vitro assays using recombinant proteins.

3. It is unclear why Nanopore sequencing was used for RNAseq and ChIPseq experiments. The greatest benefit of Nanopore sequencing is that it can sequence long reads, which usually helps with mapping, particularly at genome loci with repetitive sequences. This seems beneficial for RAP1 ChIPseq analysis as RAP1 is expected to bind telomere repeats. However, for ChIPseq, the chromatin needs to be fragmented. Larger DNA fragments from ChIPseq experiments will decrease the accuracy of the final calculated binding sites. Therefore, ChIPseq experiments are not supposed to have long reads to start with, so Nanopore sequencing does not seem to bring any advantage. In addition, compared to Illumina sequencing, Nanopore sequencing usually yields smaller numbers of reads, and the sequencing accuracy rate is lower. The Nanopore sequencing accuracy may be a serious concern in the current study. All telomeres have the perfect TTAGGG repeats, all VSG genes have a very similar 3' UTR, and all 70 bp repeats have very similar sequences. In fact, the active and silent ESs have 90% sequence identity. Are sequence reads accurately mapped to different ESs? How is the sequencing and mapping quality controlled? Furthermore, it is unclear whether the read depth for RNAseq is deep enough.

4. Many statements in the discussion section are speculations without any solid evidence. For example, lines 218 - 219 "likely due to RAP1 conformational changes", no data have been shown to support this at all. In lines 224-226, the authors acknowledged that more experiments are necessary to validate their observations, so it is important for the authors to first validate their findings before they draw any solid conclusions. Importantly, RAP1 has been shown to help compact telomeric and subtelomeric chromatin a long time ago by Pandya et al. (2013. NAR 41:7673), who actually examined the chromatin structure by MNase digestion and FAIRE. The authors should acknowledge previous findings. In addition, the authors need to revise the discussion to clearly indicate what they "speculate" rather than make statements as if it is a solid conclusion.

There are also minor concerns:

1. In the PIP5Pase conditional knockout system, the WT or mutant PIP5Pase with a V5 tag is constitutively expressed from the tubulin array. What's the relative expression level of this allele and the endogenous PIP5Pase? Without a clear knowledge of the mutant expression level, it is hard to conclude whether the mutant has any dominant negative effects or whether the mutant phenotype is simply due to a lower than WT PIP5pase expression level.

2. In EMSA analysis, what are the concentrations of the protein and the probe used in each reaction? The amount of protein used in the binding assay appears to be very high, and this can contribute to the observation that many complexes are stuck in the well. Better quality EMSA data need to be shown to support the authors' claims.

Reviewer #1 (Public Review):

Bierman et al. have developed a set of metrics for measuring the spatial patterning of mRNAs in high-throughput fluorescence in situ hybridisation experiments and applied these to identify a subset of mRNAs whose spatial patterning correlates with 3'UTR length. A strength of the study is the clarity and honesty with which the authors have outlined the strengths and weaknesses of their own approach and reported negative results. A key benefit of the tool is that the methodological choices allow wide applicability to existing datasets. However, these choices also feed into a limitation of the method, which is the difficulty in interpreting the biology underpinning the metrics - raising the question of how users will understand the output of the tool.

Reviewer #1 (Public Review):

This study focuses on molecular and cellular mechanisms underlying the sorting of miRNAs into exosomes originating from multivesicular bodies (MVBs). Following up on their previous work, the authors analysed the biochemical basis of miRNA selection by the RNA-binding protein YBX1 which is known to participate in this sorting. Using electrophoretic mobility shift assays (EMSA) involving a series of YBX1 constructs, they pinpointed the key role of the cold shock domain of YBX1 (supported by the C-terminal domain) in miRNA binding. By comparing a secreted model miRNA (miR223), a control cytoplasmic miRNA that is not enriched in exosomes (miR190), and a series of their swap mutants, the authors identified what could be a sequence motif enabling YBX1 to discriminate - through direct binding - between miRNAs to be secreted or to be retained.

The authors then wondered from which subcellular pool miR223 could be mobilised for secretion. They turned their attention to the mitochondria and found evidence of miR223 association with these organelles. Interestingly, when mitochondria were depleted by Parkin overexpression and CCCP treatment, the cellular level of miR223, but not of miR190, increased, whereas its enrichment in extracellular vesicles dropped. This observation permitted to forward a hypothesis whereby mitochondria could be involved in miR223 mobilisation into exosomes. This process would be mediated by YBX1 which shuttles between mitochondria and endosomes, as was elegantly shown in live imaging experiments.

Finally, the authors provide initial data implicating in this process the mitochondrial matrix protein YBAP1, broadly known as C1QBP, or p32. YBAP1 was found to interact with YBX1 and miR223 in pull-down assays. Moreover, direct and moderately strong miR223 binding by YBAP1 was confirmed by EMSA. Interestingly, just like YBX1, YBAP1 seems to prefer this substrate over miR190, indicating certain binding specificity. The observation that YBAP1 knockout resulted in the decreased association of miR223 with mitochondria, paralleled by its correspondingly better mobilisation into exosomes, enabled the authors to propose that YBAP1 could negatively control miR223 secretion at the level of mitochondria.

Strengths

This is a very interesting study proposing an elegant hypothesis and featuring a creative panel of methods, many of which will certainly be of interest to biochemists and cell biologists working with extracellular RNA and mitochondria (e.g. the Parkin/CCCP-mediated mitochondria depletion and the time-lapse imaging of RNA-binding proteins against cellular organelles).

The authors did a good job of dissecting the YBX1 interaction with miR223 versus miR190. These experiments are performed at a high technical level, and their interpretation is straightforward and convincing. The nearly two orders of magnitude difference in affinity provides a plausible means by which YBX1 could recognise and funnel one, but not the other, miRNA into the secretion pathway.<br /> Another valuable piece of data is related to YBAP1. This important, deeply conserved protein, strongly implicated in severe mitochondrial diseases and cancer, remains poorly understood at the level of basic molecular mechanisms, and even its subcellular localisation is debated. The data presented by the authors reinforce the idea of its primarily mitochondrial localisation, in agreement with earlier studies. They also provided new information about the RNA-binding activity of YBAP1. First proposed to interact with RNA by Yagi et al., Nucleic Acids Res 2012 (doi:10.1093/nar/gks774), YBAP1 is confirmed in the present study as a reasonably affine RNA-binding protein, based on direct EMSA experiments involving a highly purified protein and natural RNAs. These data should encourage the community to explore the full RNA-binding potential of YBAP1/C1QBP/p32 in a wider variety of models, especially in the context of mitochondrial gene expression.

Weaknesses

While the authors might be right about the existence of a sequence motif that specifies miRNAs for exosome sorting by YBX1, it is at present difficult to disentangle the sequence and structure contributions to YBX1 binding within the variants described in the paper. RNA structure predictions, however imperfect, suggest that miR223-3p is a fully single-stranded transcript (ensemble ΔG = -0.33 kcal/mol, RNAfold), while miR190-5p is a tightly base-paired one (ensemble ΔG = -2.85 kcal/mol). This likely explains the differential affinity to YBX1, known to strongly prefer single-stranded RNAs. When mutating the putative sorting motif in miR223 (UCAGU>AGACA), the authors introduced some amount of secondary structure (ΔG = -1.04 kcal/mol), which could have impeded YBX1 binding. By contrast, the mutation of miR190 (AUAUG>UCAGU) significantly weakened the structure (ensemble ΔG = -2.21 kcal/mol), which might explain the improvement in YBX1 interaction.

Mitochondria appear to be a plausible location for mobilisable RNAs, given their multiple associations with ribosomes, RNA-containing condensates, and other organelles. However, the presented evidence of the mitochondrial localisation of miR223 is limited. The colocalisation pattern of the ATTO 647-labelled miR223 with the well-behaved mitochondrial marker Tom22 is remarkable; such a neat overlap has so far only been observed for some abundant mtDNA-encoded transcripts, but not for an extraneous transcript. The interpretation of this result will depend a great deal on experimental details which, unfortunately, are missing for this section. ATTO 647N is known to be quantitatively recruited to mitochondria, producing just the same kind of complete colocalisation, making it a perfect tool to visualise mitochondria in the cell (Han et al., Nat Commun 2017, doi:10.1038/s41467-017-01503-6). There is a worry that the colocalisation observed here might have been driven by the dye alone.

Furthermore, the definition of the topology of RNA localisation with respect to the mitochondrial membranes remains challenging, and a number of more robust methods have been recently proposed to address this contentious issue. At minima, one would expect that the authors would use RNase treatment, with or without Triton X-100 (like they did in the in vitro packaging assay), to see whether miR223 is indeed protected by the mitochondrial membranes and, therefore, resides in the interior of the organelles. As for now, based on the presented data, one can safely conclude that miR223 is associated with the mitochondria, without claiming that it is necessary inside them.

The Parkin/CCCP method is very powerful, which is its strength and weakness at the same time. miR223 secretion does decrease when the mitochondria are depleted. However, it is unclear how direct and specific this effect is. The destruction of mitochondria likely crashed the cellular ATP levels, which could have generally affected vesicular transport, not only miR223 sorting. A more detailed analysis of the overall abundance of extracellular vesicles and their cargo under these conditions could reveal the true scope of the mitochondrial contribution to RNA secretion.

YBAP1 is a difficult, indeed "treacherous", protein to work with. Its strong negative charge (pI = 4) makes it easily stick to positively charged proteins, such as YBX1 (pI = 9.9). Such interactions are routinely observed in pulldown assays from cell lysates, where all components are intermixed (but often cannot be corroborated by in situ or in vivo approaches). The authors carefully showed that YBX1 and YBAP1 do not significantly colocalise in the cell, which makes the interplay between the two proteins in miR223 sorting difficult to stage. They also studied the miR223 distribution between mitochondria and extracellular vesicles using YBAP1 knockout cells. However, such cells are known to be very sick and have an extremely pleiotropic mitochondrial and metabolic phenotype. Therefore, the apparent implication of YBAP1 in miR223 sorting might be less direct than currently envisaged.

Reviewer #1 (Public Review):

This study is based on the hypothesis that tumor treating fields, a form of cancer therapy that exposes tumors to alternating electrical fields, has an effect on tunneling microtubes, fine actin-rich protrusions that connect cancer cells and allow intercellular communication, contributing to the tumor microenvironment and therapeutic resistance. This is an interesting hypothesis and may be of importance. To prove their hypothesis better data presentation and mechanistic studies are needed, as it is not clear based on this study how the proposed effect is working.

Reviewer #1 (Public Review):

In this study, Shin and colleagues investigate the role of the posttranslational modification of the DNA methyltransferase by covalent linkage of the N-Acetylglucosamine (O-GlcNAc).

The authors present compelling evidence showing that a prolonged high fat/sucrose diet causes global protein O-GlcNAcylation in the liver and DNMT1 is among the proteins that increase their O-GlcNAc level. This result is significant because of the paucity of in vivo data addressing the interplay between metabolism and protein O-GlcNAcylation. The paper also shows that DNMT1's O-GlcNAcylation level correlated to the extracellular glucose levels in other cell types.

Using mass spectrometry, the authors identify S878 as the main site for O-GlcNAcylation. It is noteworthy that the mapping was performed with hyper-O-GlcNAcylated cells and may be different in a physiological situation. To investigate how O-GlcNAcylation of S878 of DNMT1 impacts its activity and ultimately DNA methylation patterns, Shin and colleagues mostly use a cellular model of hyper O-GlcNAcylation induced by the combination of high glucose and a chemical inhibitor of OGA (the only enzyme responsible for O-GlcNAc removal). The data shows that increased O-GlcNAcylation resulting from the combination of high glucose and OGA inhibition causes a reduction of DNMT1 activity and local loss of DNA methylation specifically at partially methylated domains.

This study brings completely new knowledge on the regulatory function of glycosylation of DNMT1 and its impact on its methyl-transferase activity and downstream genomic methylation. Furthermore, the manuscript introduces new data on the interplay between cellular metabolism and O-GlcNAcylation on DNMT1 and other proteins. The experiments are well-controlled, and their interpretation is sound. This study should be of special interest to the fields of fundamental and environmental epigenetics, as well as metabolism.

The main limitation of the study is the convolution of the functional experiments where the perturbation is a combination of high glucose and chemical inhibition of OGA. The relative contribution of the two variables is partially addressed in Figure 3-figure supplement 1B which shows that high glucose increases DNMT1 activity (Hep3B cells) while Figure 3D shows that high glucose when combined with OGA inhibitor decreases DNMT1 activity (Hep3B cells). As discussed, the data suggest that high-glucose and OGA inhibition may have an antagonistic effect on DNMT1 activity. An experiment of treatment of the cells with the OGA inhibitor in physiological glucose conditions would address this gap of knowledge.

To understand the impact of the environment (in this study: extracellular glucose level) on the epigenome, one should keep in mind the variation of cytosine methylation patterns between individuals and over time. A recent large-scale profiling of DNA methylation of 137 individuals shows a near absence of individual variation between replicates of the same cell type, suggesting that genomic methylation patterns are largely insensitive to the environment (https://doi.org/10.1038/s41586-022-05580-6).

Comparative methylomes of healthy and diabetic individuals are needed to examine the medical significance of the findings presented here. It is possible that the modulation of DNMT1 activity by O-GlcNAc modification is relevant for a specific cell type or developmental stage that remains to be discovered.

Reviewer #1 (Public Review):

Overall, this manuscript exposes key gaps in patient care resulting from the pandemic, as well as the challenges and unmet needs felt by healthcare workers in cervical cancer screening. The authors' findings on the struggles while regaining screening volume across the nation in a sustainable way, demonstrate that pre-existing weaknesses in the cancer control system were exacerbated by the pandemic and are integral to amend. The authors were able to identify these gaps in care and work environments through their synthesis of qualitative interviews. I applaud the use of such mixed methods, which emphasizes the complementary need for both quantitative and qualitative data. What could be better strengthened in the manuscript is the authors' justification for statistical analyses within the context of the research question, and reporting of survey administration and management.

Reviewer #1 (Public Review):

Flowers et al. studied requirements for the persistence and clearance of mutant mtDNA in C. elegans using the uaDf5 deletion in mtDNA. This mutant mtDNA persists at relatively constant levels, despite clearly having detrimental effects. Surprisingly, no mutations were found in the existing wt copies, which would have otherwise explained the persistence of mutant DNA by complementation. The authors then investigated the potential role of programmed cell death in the removal of mutant mtDNA from the germline using crosses with existing cell death mutants. They observed increased amounts of uaDf5 DNA in 1 day old progeny in strains with mutations in the caspases ced-3 and csp-1 and in several other cell death genes, showing that a significant amount of uaDF5 is removed by PCD in the germline. The authors also observed increased uaDf5 over time in the germline, and effects lifespan mutations on the mount of uaDf5. This was true both for the insulin signaling pathway and the clk-1 pathway, suggesting that both pathways regulate uaDf5 levels consistent with the connection between longevity and mitochondrial homeostasis. Finally, the authors discuss results showing that PCD mutants with high amounts of uaDf5 in the germline, have surprisingly low amounts of uaDf5 in their progeny, which would suggest that PCD can be replaced by another clearance method.

This manuscript is of general interest because it demonstrates the importance of PCD for clearance of mutant mtDNA. The evidence for this mechanism of removal is strong. The effects of the aging mutants are more difficult to understand and the discussion of these effects is therefore somewhat speculative.

Reviewer #1 (Public Review):

The work presented here uses a large collection of PET data to discover the principle axes of neurotransmitter receptor/transporter molecule (NTRM) variation in the human cortex and subcortex. These spatial axes are then systematically annotated for their alignment with diverse other measures of brain organization. The work is valuable for providing a systematic description and annotation of a new "layer" of brain organization that has been relatively poorly integrated with the wider neuroimaging literature to date. The methods used are state-of-the-art and the findings generated by these methods are sound. The discovered NTRM gradients will allow others in the field to more easily incorporate information of neurotransmitter maps in their analyses - helping to advance integration between different views of the human brain. A fundamental challenge to this goal of cross-modal integration, however - which doesn't just impact this work, but the field more broadly - is that we are often left to work with spatial correlations between modalities in humans. The lack of access to experimental methods means that the biological basis for observed spatial correlations between different brain features in humans is typically poorly understood. It is therefore hard to know what newly-reported spatial correlations are telling us about brain organization that was not already captured in prior work. Nevertheless, the new resources and results presented here are important because they can guide the future work needed to unpick the biology behind spatially correlated features of the human brain

Reviewer #1 (Public Review):

The aim of this study was to evaluate the increased prevalence of mental health (MH) disorders such as depression, anxiety, eating disorders, and postpartum depression in patients with polycystic ovary syndrome (PCOS) the most common reproductive disorder affecting about one in seven reproductive-aged women worldwide. The consequences of excess economic burden were estimated.

Meta-analyses were performed using the Der Simonian-Laird random-effects model to compute pooled estimates of prevalence ratios for the associations between PCOS and these MH disorders, and then the excess direct costs in U.S. dollars (USD) for women suffering from PCOS were estimated.

After screening the articles by title/abstract, 25 articles were selected for their quality according to the Newcastle-Ottawa scale. These studies included a control group. The data showed an increase in the prevalence ratios for each of the selected mental health disorder items: anxiety 1.42, depression 1.65, and eating disorders 1.48. The additional direct health care costs associated with these disorders were estimated to be $4.261 billion per year in 2021 USD.

The authors extended their previous report that the total cost of evaluating and providing care to reproductive-aged PCOS women in the United States was $4.36 billion. Interestingly, the cost for diagnostic evaluation including laboratory accounted for a relatively minor part of the total costs (approximately 2%). In the present study, mental health disorders were clearly identified as a part of the excess economic burden. Their cost is estimated at $4.261 billion/year. These results were not anticipated intuitively and are of value for prioritization of the disorder as a public health priority.

Provided that the study is validated for extraction of a meta-analysis, the data are of great interest not only for economic issues but also for early consideration of the mental distress of PCOS patients that has long been underestimated. Several studies have expressed patient resentment of delayed diagnosis and imperfect management, including the physical damage of hyperandrogenism and the associated metabolic syndrome. This medico-economic approach to chronic diseases with a strong impact on quality of life contributes to the global management of PCOS, which is a primary demand of patients.

Reviewer #1 (Public Review):

GSK3 is a multi-tasking kinase that recognises primed (i.e. phosphorylated) substrates. One of the mechanisms by which the activity of GSK3 can be regulated is through N-terminal (pSer9) phosphorylation. In this case, the phosphorylated N-terminus turns into a pseudo-substrate that occupies the substrate binding pocket and thus inhibits the activity of GSK3 towards its real substrates.

One outstanding question is how this autoinhibitory mechanism can affect some, but not all signaling pathways that GSK3 is involved in. One example is WNT/CTNNB1 signaling. Here, GSK3 plays a central role in the turnover of CTNNB1 in the absence of WNT, but this pool of GSK3 is not affected by pSer9 phosphorylation.

Gavagan et al. address this question using an in vitro approach with purified proteins. They identify a role for AXIN1 in protecting the "WNT signaling pool" of GSK3 from the auto-inhibition that occurs upon pSer9 phosphorylation.<br /> Specifically, they show that i) GSK3-pSer9 is less capable of binding and phosphorylating primed CTNNB1 - thus suggesting that GSK3-pSer9 does not contribute to WNT signaling, ii) in the presence of AXIN1, GSK3-pSer9 becomes more capable of binding and phosphorylating CTNNB1 - suggesting that Axin can promote binding of GSK3 and CTNNB1 even when the primed binding pocket on GSK3 is blocked initially, iii) AXIN1 specifically prevents the PKA mediated phosphorylation of GSK3B on pSer9 - while leaving the phosphorylation of other PKA substrates unaffected.

Strengths:<br /> - The authors use an in vitro system in which they can reconstitute different interactions and reactions using purified proteins, thus allowing them to zoom in on specific biochemical events in isolation.<br /> - The authors measure the phosphorylation of primed substrates (pSer45-CTNNB1 or WNT-independent substrates) and quantify specific kinetic parameters (kcat, KM, and kcat/KM) - of wildtype non-phosphorylated GSK3B, pSer9GSK3B, or the non-phosphorylatable S9A-GSK3B, either in the presence or absence of AXIN1 (or an AXIN1 fragment).<br /> - The experiments appear to be well-controlled and the results appear to be interpreted correctly.

Weaknesses:<br /> - Key experiments (e.g. Figures 2 and 3) are described as being performed as n=3 technical replicates rather than independent/biological replicates.<br /> - The validation in a biologically relevant setting (i.e. a cellular context) is limited to Figure 4C, which shows that over-expression of AXIN1 reduces the total levels of pSer9-GSK3.

The authors convincingly show that AXIN1 can play a role in shielding GSK3 from auto-inhibition. As it stands, the impact of this work on the field of WNT/CTNNB1 signaling is likely to remain limited. This is mainly due to the reason that the mechanism by which AXIN1 shields the WNT/CTNNB1 signaling pool of GSK3 from pSer9 inhibition remains unresolved. Based on the fact that a mini AXIN1 (i.e. an AXIN1 fragment) behaves the same as WT AXIN1, the authors conclude that AXIN1 likely causes allosteric changes on GSK3 but is less likely to block PKA from binding. They cannot conclusively show this, however, as they do not have evidence in favour of one or the other explanation.

However, this study does offer more insight into the compartmentalisation of GSK3 and the quantitative parameters may be used in computational models describing the different cellular activities of GSK3.

This work also has conceptual significance: Scaffold proteins are known to promote signal transduction by bringing proteins together (often: kinases and substrates). Here, Gavagan et al. show that AXIN1 also plays a second role, namely in protecting one of its binding kinases (GSK3) from inhibitory signals. This could potentially hold for other scaffolding proteins as well.

Reviewer #1 (Public Review):

In this study, Wang et al performed structural peripheral and central imaging of the auditory pathway using high-resolution MR. For the first time, they evaluated children with congenital severe to profound sensorineural hearing loss with and without cochlear nerve deficiency and cochlear malformations. The authors evaluated 13 children with severe to profound congenital hearing loss (6 with cochlear nerve deficiency) and 10 typically-hearing controls. They found significant differences in the central auditory pathway that were influenced by the status of the peripheral auditory pathway. Determination of outcomes after cochlear implantation or auditory brainstem implantation is critical and we currently have no good methods for this, so this study is very promising in that regard.

The authors have achieved their aim of evaluating these children with high-resolution imaging and identifying differences in auditory pathways. My primary issues are that some of their claims for clinical potential are not justified as of yet and the authors did not determine a diagnosis for the patients' hearing loss.

Reviewer #1 (Public Review):

Various parts of the premotor cortex have been implicated in choices underlying decision-making tasks. Further, norepinephrine has been implicated in modulating behavior during various decision-making tasks. Less work has been done on how noradrenergic modulation would affect M2 activity to alter decision-making, nor is it clear whether noradrenergic modulation effects on activity would differ between the male and female sexes.

This manuscript addresses some of these questions.<br /> - In particular, clear sex differences in task engagement are seen.<br /> - May also show some interesting differences and distributions of β2 adrenergic receptors in M2 between males and females.

Less clear is the specificity of systemic antagonism of β adrenergic receptors on the changes in M2 activity reported. As propranolol was given systemically, changes in M2 firing rates could also be due to broader circuit (indirect) activity changes. As it was not given locally, nor were local receptor populations manipulated, one is unable to make the conclusion that changes in neural activity are due to the direct effects of adrenergic receptors within M2 populations.

Also not clear, is the contribution of M2 to this task, and whether the changes in M2 activity patterns observed are directly responsible for the behavioral disruptions measured.

Reviewer #1 (Public Review):

The authors developed a new approach to enable the reconstruction of fine axonal morphologies based solely on extracellular action potential recordings from in vitro mammalian neurons using a high-density microelectrode array system with an integrated CMOS camera. They provide compelling evidence of fine resolution in mapping functional connections between neurons via very fine axons. The advantage of the approach is that it provides a label-free electrical visualization of axon conduction trajectories as well as the ability to access the AP waveforms. The work may have broad use in neurobiology, bioengineering, stem cell biology, as well as tissue engineering in functional characterization.

Reviewer #1 (Public Review):

Zhou et al. have set up a study to examine how metabolism is regulated across the organism by taking a combined approach looking at gene expression in multiple tissues, as well as analysis of the blood. Specifically, they have created a tool for easily analyzing data from GTEx across 18 tissues in 310 people. In principle, this approach should be expandable to any dataset where multiple tissues of data were collected from the same individuals. While not necessary, it would also raise my interest to see the "Mouse(coming soon)" selection functional, given that the authors have good access to multi-tissue transcriptomics done in similarly large mouse cohorts.

Summary:

The authors have assembled a web tool that helps analyze multiple tissues' datasets together, with the aim of identifying how metabolic pathways and gene regulation are connected across tissues. This makes sense conceptually and the web tool is easy to use and runs reasonably quickly, considering the size of the data. I like the tool and I think the approach is necessary and surprisingly under-served; there is a lot of focus on multi-omics recently, but much less on doing a good job of integrating multi-tissue datasets even within a single omics layer.

What I am less convinced about is the "Research Article" aspect of this paper. Studying circadian rhythm in GTEx data seems risky to me, given the huge range in circadian clock in the sample collection. I also wonder (although this is not even remotely in my expertise) whether the circadian rhythm also gets rather desynchronized in people dying of natural causes - although I suppose this could be said for any gene expression pathway. Similarly for looking at secreted proteins in Figure 4 looking at muscle-hippocampus transcript levels for ADAMTS17 doesn't make sense to me - of all tissue pairs to make a vignette about to demonstrate the method, this is not an intuitive choice to me. The "within muscle" results look fine but panels C-E-G look like noise to me...especially panel C and G are almost certainly noise, since those are pathways with gene counts of 2 and 1 respectively.

I think this is an important effort and a good basis but a significant revision is necessary. This can devote more time and space to explaining the methodology and for ensuring that the results shown are actually significant. This could be done by checking a mix of negative controls (e.g. by shuffling gene labels and data) and a more comprehensive look at "positive" genes, so that it can be clearly shown that the genes shown in Fig 1 and 2 are not cherry-picked. For Figure 3, I suspect you would get almost an identical figure if instead of showing pan-tissue circadian clock correlations, you instead selected the electron transport chain, or the ribosome, or any other pathway that has genes that are expressed across all tissues. You show that colon and heart have relatively high connectivity to other tissues, but this may be common to other pathways as well.

Reviewer #1 (Public Review):

The current manuscript provides a timely contribution to the ongoing discussion about the mechanism of the apical sodium/bile acid transporter (ASBT) transporters. Recent structures of the mammalian ASBT transporters exhibited a substrate binding mode with few interactions with the core domain (classically associated with substrate binding), prompting an unusual proposal for the transport mechanism. Early structures of ASBT homologues from bacteria also exhibit unusual substrate binding in which the core substrate binding domain is less engaged than expected. Due to the ongoing questions of how substrate binding and mechanism are linked in these transporters, the authors set out to deepen our understanding of a model ABST homolog from bacteria N. meningitidis (ABST-NM).

The premise of the current paper is that the bacterial ASBT homologs are probably not physiological bile acid transporters, and that structural elucidation of a natively transported substrate might provide better mechanistic information. In the current manuscript, the authors revisit the first BASS homologue to be structurally characterized, ABST-NM. Based on bacteriological assays in the literature, the authors identify the coenzyme A precursor pantoate as a more likely substrate for ABST-NM than taurocholate, the substrate in the original structure. A structure of ASBT-NM with pantoate exhibits interesting differences in structure. The structures are complemented with MD simulations, and the authors propose that the structures are consistent with a classical elevator transport mechanism.

The structural experiments are generally solid, although showing omit maps would bolster the identification of the substrate binding site. One shortcoming is that, although pantoate binding is observed, the authors do not show transport of this substrate, undercutting the argument that the pantoate structure represents binding of a "better" or more native substrate. Mechanistic proposals, like the proposed role of T112 in unlocking the transporter, would be much better supported by transport data.

Reviewer #1 (Public Review):

The authors set out to use structural biology (cryo-EM), surface plasmon resonance, and complement convertase assays to understand the mechanism(s) by which ISG65 dampens the cytoxicity/cellular clearance to/of trypanosmes opsonised with C3b by the innate immune system.

The cryo-EM structure adds significantly to the author's previous crystallographic data because the latter was limited to the C3d sub-domain of C3b. Further, the in vitro convertase assay adds an additional functional dimension to this study.

The authors have achieved their aims and the results support their conclusions.

The role of complement in immunity to T. brucei (or lack thereof) has been a significant question in molecular parasitology for over 30 years. The identification of ISG65 as the C3 receptor and now this study providing mechanistic insights represents a major advance in the field.

Reviewer #1 (Public Review):

The apicoplast, a non-photosynthetic vestigial chloroplast, is a key metabolic organelle for the synthesis of certain lipids in apicomplexan parasites. Although it is clear metabolite exchange between the parasite cytosol and the apicoplast must occur, very few transporters associated with the apicoplast have been identified. The current study combines data from previous studies with new data from biotin proximity labeling to identify new apicoplast resident proteins including two putative monocarboxylate transporters termed MCT1 and MCT2. The authors conduct a thorough molecular phylogenetic analysis of the newly identified apicoplast proteins and they provide compelling evidence that MCT1 and MCT2 are necessary for normal growth and plaque formation in vitro along with maintenance of the apicoplast itself. They also provide indirect evidence for a possible need for these transporters in isoprenoid biosynthesis and fatty acid biosynthesis within the apicoplast. Finally, mouse infection experiments suggest that MCT1 and MCT2 are required for normal virulence, with MCT2 completely lacking at the administered dose. Overall, this study is generally of high quality, includes extensive quantitative data, and significantly advances the field by identifying several novel apicoplast proteins together with establishing a critical role for two putative transporters in the parasite. The study, however, could be further strengthened by addressing the following aspects:

Main comments<br /> 1. The conclusion that condition depletion of AMT1 and/or AMT2 affects apicoplast synthesis of IPP is only supported by indirect measurements (effects on host GFP uptake or trafficking, possibly due to effects on IPP dependent proteins such as rabs, and mitochondrial membrane potential, possibly due to effects on IPP dependent ubiquinone). This conclusion would be more strongly supported by directly measuring levels of IPP. If there are technical limitations that prevent direct measurement of IPP then the author should note such limitations and acknowledge in the discussion that the conclusion is based on indirect evidence.

2. The conclusion that condition depletion of AMT1 and/or AMT2 affects apicoplast synthesis of fatty acids is also poorly supported by the data. The authors do not distinguish between the lower fatty acid levels being due to reduced synthesis of fatty acids, reduced salvage of host fatty acids, or both. Indeed, the authors provide evidence that parasite endocytosis of GFP is dependent on AMT1 and AMT2. Host GFP likely enters the parasite within a membrane bound vesicle derived from the PVM. The PVM is known to harbor host-derived lipids. Hence, it is possible that some of the decrease in fatty acid levels could be due to reduced lipid salvage from the host. Experiments should be conducted to measure the synthesis and salvage of fatty acids (e.g., by metabolic flux analysis), or the authors should acknowledge that both could be affected.

Reviewer #1 (Public Review):

Metzger et al develop a rigorous method filling an important unmet need in protein evolution - analysis of protein genetic architecture and evolution using data from combinatorially complete 20^N variant libraries. Addressing this need has become increasingly valuable, as experimental methods for generating these datasets expand in scope and scale. Their model incorporates several key features - (1) it reports the effects of mutations relative to the average across all variants, rather than a particular genotype, making it useful for examining genetic architectures and evolution in a less biased way, (2) it infers contributions from both "specific" and "non-specific" epistasis, which is essential for some experimental measurements, and perhaps most importantly (3) it does this for all possible 20 states at each site, in contrast to the binary analyses in prior work. These features are not individually novel but integrating them into a single analysis framework is novel and will be incredibly valuable to the protein evolution community. Using a previously published dataset generated by two of the authors, they conclude that (1) changes in function are largely attributable to pairwise but not higher-order interactions, and (2) epistasis potentiates, rather than constrains, evolutionary paths. These findings are well-supported by the data, though the authors' claim that higher-order epistasis cannot account for the variation they see could be better supported by additional analyses or discussion (as noted in recommendations for authors). Overall, this work has important implications for predicting the relationship between genotype and phenotype, which is of considerable interest to protein biochemistry, evolutionary biology, and numerous other fields.

Reviewer #1 (Public Review):

Here, Ensinck et al. investigated the composition of the yeast mRNA m6A methyltransferase complex required for meiosis. This complex was known to contain three proteins, but is much more complex in mammals, insects and plants. Through IP-MS analysis they identified three more proteins Kar4, Ygl036w and Dyn2. Of these Kar4 and Ygl036w are homologous to Mettl14 and Virma, respectively, and, like the previously described factors are essential for m6A deposition, mating and binding of the reader Pho92 to mRNA during meiosis by evidence acquired with appropriate methodology. Dyn2 is a novel factor not described for any m6A complex and is not essential for m6A deposition, mating and binding of the reader Pho92 to mRNA during meiosis.

In addition, detailed analysis of the Slz1 revealed homology to the mammalian factor m6A complex member ZC3H13 to comprise a conserved complex of five proteins, Mettl3, Mettl14, Mum2/WTAP, Virma and Slz/ZC3H13. When co-expressed in insects cells, they co-purify stoichiometrically and presence of Mum2 as a dimer is also indicated as shown for WTAP.

Complementary to these data they show that stability of the individual complex members is affected in mutants supporting that they are stabilized through complex formation.

Furthermore, the authors then show that kar4 has additional roles in mating that is separable from its role through the m6A complex in meiosis.

The authors employ appropriate methodology throughout to address their aims and present convincing evidence for their claims. The evidence presented here reinforces that the m6A complex is evolutionary highly conserved which has broad scope for its functional analysis in humans and model organisms.

Reviewer #1 (Public Review):

This manuscript compiles the colonization of shrubs during the Late Pleistocene in Northern America and Europe by comparing plant sedimentary ancient DNA (sedaDNA) records from different published lake sediment cores and also adds two new datasets from Island. The major findings of this work aim to illuminate the colonization patterns of woody shrubs (Salicaceae and Betulaceae) in these sediment archives to understand this process in the past and evaluate its importance under future deglaciation and warming of the Arctic.

The strength of evidence is solid as methods (sedimentary DNA) and data analyses broadly support the claims because the authors use an established metabarcoding approach with PCR replicates (supporting the replicability of PCR and thereby proving the occurrence of Salicaeae and Betulaceae in the samples) and quantitative estimation of plant DNA with qPCR (which defines the number of cycles used for each PCR amplification to prevent overamplification). However, the extraction methods need more explanation and the bioinformatic pipeline is not well-known and needs also some further description in the main text (not only referring to other publications).

The authors compare their own data with previously published data to indicate the different timing of shrubification in the selected sites and show that Salicaceae occurs always like a pioneer shrub after deglaciation, followed by Betaluaceae with a various time lag. The successive colonization of Salicaceae followed by Betulaceae is explained by its differences in environmental tolerance, the time lag of colonization in the compared records is e.g. explained by varying distance to source areas.

However, there are some weaknesses in the strength of evidence because full sedaDNA plant DNA assessment, quality of the sedaDNA data (relative abundance and richness of sedaDNA plant composition) and results from Blank controls (for sedaDNA) are not fully provided. I think it is important to show how the plant metabarcoding in general worked out, because it is known that e.g. poor richness can be indicative of less preserved DNA and a full plant assessment (shown in the supplement) would be more comprehensive and would likely attract a larger readership.

Further, it would allow us to see the relative abundance in changes of plants and would make it easier to understand if the families Salicaeae and Betulaceae are a major component of the community signal. Further, the possibility to reach higher taxonomic resolution with sedaDNA compared to pollen or to facilitate a continuous record (which is different from macrofossils) is not discussed in the manuscript but should be added. Also, the taxonomic resolution within these families in the discussed datasets would be of interest, also on the sequence type level if tax. assignments are similar.

Another important aspect is how the abundance/occurrence of Salicaceae is discussed. Many studies on sedaDNA confirm an overrepresentation of this family due to better preservation in the sediment, far-distance transport along rivers, or preferences of primers during amplification etc. As this family is the major objective of this study, such discussion should be added to the manuscript and data should be presented accordingly.

I also miss more clarity about how the authors defined the source areas (refugia) of the shrubs. If these source areas are described in other literature I suggest to show them in a map or so. Further, it should be also discussed and explained more in detail which specific environmental preferences these families have, this is too short in the introduction and too unspecific. Also, it would be beneficial to show relative abundances rather than just highlighted areas in the Figures and it would allow us to see if Salicaeae will be replaced by Betulaceae after colonizing or if both families persist together, which might be important to understand future development of shrubs in these areas.

The author started a discussion about shrubification in the future, but a more defined evaluation and discussion of how to use such paleo datasets to predict future shrubification and its consequences for the Arctic would give more significance to the work.

Reviewer #1 (Public Review):

The current study tests the hypothesis that inhibition of ryanodine receptor 2 (RyR2) in failing arrhythmogenic hearts reduces sarcoplasmic Ca leak, ventricular arrhythmias and improves contractile function. A guinea pig model of nonischemic heart failure (HF) was used and randomized to receive dantrolene (DS) or placebo in early or chronic HF. The authors show that DS treatment prevented ventricular arrhythmias and sudden cardiac death by decreasing dispersion of repolarization. The authors conclude that inhibition of RyR2 hyperactivity with DS mitigates the vicious cycle of sarcoplasmic Ca leak-induced increases in diastolic Ca and reactive oxygen species-mediated RyR2 oxidation. Moreover, the consequent increase in sarcoplasmic Ca2+ load improves contractile function.

In general, the study is well designed and the findings are likely to be of interest to the field. The characterization of the phenotypes is comprehensive, however, the study appears relatively weak in terms of the proposed mechanisms. Only in vivo functional analyses were presented with no in vitro analyses. The rationale for only using the male animals remains unclear. Data presented in Supplemental Figure 1 lacks the HF with DS group. As presented, the manuscript appears relatively descriptive in nature.

Reviewer #1 (Public Review):

This study investigates the structuring of long calls in orangutans. The authors demonstrate long calls are structured around full pulses, repeated following a regular tempo (isochronic rhythm). These full pulses are themselves structured around different sub-pulses, themselves repeated following an isochronic rhythm. The authors argue this patterning is evidence for self-embedded, recursive structuring in orangutang long calls.

The analyses conducted are robust and compelling and they support the rhythmicity the authors argue is present in the long calls. Furthermore, the authors went above and beyond and confirmed acoustically the sub-categories identified were accurate.

However, I believe the manuscript would benefit from a formal analysis of the specific recursive patterning occurring in the long call. Indeed, as of now, it is difficult for the reader to identify what the authors argue to be recursion and distinguish it from simple repetitions of motifs, which is essential. Although the authors already discuss briefly why linear patterning is unlikely, the reader would benefit from expanding on this discussion section and clarifying the argument here (a lay terminology might help). I believe an illustration here might help.<br /> In the same logic, I believe a tree similar to the trees used in linguistics to illustrate hierarchical structuring would help the reader understand the recursive patterning in place here. This would also help get the "big picture", as Fig 1A is depicting a frustratingly small portion of the long call.

Notwithstanding these comments, this paper would provide crucial evidence for recursion in the vocal *production* of a non-human ape species. The implication it would have would represent a key shift in the field of language evolution. The study is very elegant and well-constructed. The paper is extremely well written, and the point of view adopted is original, well-argued and compelling.

Reviewer #1 (Public Review):

In this manuscript, Wang et al. demonstrate that knockdown of DYRK1A results in reduced cell size, which is mediated by mTORC1 activity. They found that DYRK1A interacts with TSC1/TSC2 proteins which leads to the phosphorylation of TSC2 at T1462. Phosphorylation of TSC2 at T1462 inhibits TSC2 activity leading to the activation of mTORC1. The authors complement their findings by demonstrating that overexpression of RHEB (positive regulator of mTORC1) rescues the phenotype of DYRK1A (mnb in flies) mutation in the NMJ.

The authors' findings on the regulation of cell size and mTORC1 activity by DYRK1A reflect the previous findings of Levy et al. (PMID: 33840455) that cortical deletion of Dyrk1a in mice causes decreased neuronal size associated with a decreased activity of mTORC1 that can be rescued by the inhibition of Pten or supplementation of IGF1.

The authors demonstrate that T1462 phospho-site at TSC2 is phosphorylated in response to the overexpression of WT but not kinase-dead DYRK1A. However, the authors do not provide any evidence that the regulation of mTORC1 is mediated via phosphorylation of this site. In addition, T1462 site is known to be phosphorylated by Akt. There is a possibility that Akt was co-purified with TSC1/TSC2 complex and DYRK1A promotes phosphorylation of TSC2 indirectly via the activation of AKT that can be tested by using AKT depleted cells.

RHEB is the most proximal regulator of mTORC1 and can activate mTORC1 even under amino acid starvation. The fact that RHEB overexpression rescues the cell size under DYRK1A depletion or mnb (DYRK1A in Drosophila) mutant phenotype does not prove that DYRK1A regulates the cell size via TSC1 as it would rescue any inhibitory effects upstream to mTORC1.

Reviewer #1 (Public Review):

This work introduces a new method of imaging the reaction forces generated by small crawling organisms and applies this method to understanding locomotion of Drosophila larva, an important model organism. The force and displacement data generated by this method are a qualitative improvement on what was previously available for studying the larva, improving simultaneously the spatial, temporal, and force resolution, in many cases by an order of magnitude. The resulting images and movies are quite impressive.

As it shows the novel application of recent technological innovations, the work would benefit from more detail in the explanation of the new technologies, of the rationales underlying the choice of technology and certain idiosyncratic experimental details, and of the limitations of the various techniques. In the methods, the authors need to be sure to provide sufficient detail that the work can be understood and replicated. The description of the results and the theory of motion developed here focus only on forces generated when the larva pushes against the substrate and ignores the equally strong adhesive forces pulling the larva onto the substrate.

The substrate applies upward, downward, and horizontal forces on the larva, but only upward and downward forces are measured, and only upward forces are considered in the discussions of "Ground Reactive Forces." An apparent weakness of the WARP technique for the study of locomotion is that it only measures forces perpendicular to the substrate surface ("vertical forces" in Meek et al.), while locomotion requires the generation of forces parallel to the substrate ("horizontal forces"). It should be clarified that only vertical forces are studied and that no direct information is provided about the forces that actually move the larva forward (or about the forces which impede this motion and are also generated by the substrate). Along with this clarification, it would be helpful to include a discussion of other techniques, especially micropillar arrays and traction force microscopy, that directly measure horizontal forces and of why these techniques are inappropriate for the motions studied here.

The larvae studied are about 1 mm long and 0.1 mm in cross-section. Their volumes are therefore on order 0.01 microliter, their masses about 0.01 mg, and their weights in the range of 0.1 micronewton. This contrasts with the force reported for a single protpodium of 1 - 7 micronewtons. This is not to say that the force measurements are incorrect. Larvae crawl easily on an inverted surface, showing gravitational forces are smaller than other forces binding the larva to the substrate. The forces measured in this work are also of the same magnitude as the horizontal forces reported by Khare et al. (ref 32) using micropillar arrays.

I suspect that the forces adhering the larva to the substrate are due to the surface tension of a water layer. This would be consistent with the ring of upward stress around the perimeter of the larva visible in S4D, E and in video SV3. The authors remark that upward deflection of the substrate may be due to the Poisson's ratio of the elastomer, but the calibration figure S5 shows that these upward deflections and forces are much smaller than the applied downward force. In any case, there must be a downward force on the larva to balance the measured upward forces and this force must be due to interaction with the substrate. It should be verified that the sum of downward minus upward forces on the gel equals the larva's weight (given the weight is neglible compared to the forces involved, this implies that the upward and downward forces should sum to 0)

Much of the discussion and the model imply that the sites where the larva exerts downward force on the gel are the sites where horizontal propulsion is generated. This assumption should be justified. Can the authors rule out that the larva 'pulls' itself forward using surface tension instead of 'pushing' itself forward using protopodia?

More detail should be provided about the methods, their limitations, and the rationale behind certain experimental choices.

Three techniques are introduced here to study how a crawling larva interacts with the substrate: standard brightfield microscopy of a larva crawling in an agarose capillary, ERISM imaging of an immobilized larva, and WARP imaging of a crawling larva. The authors should make clear why each technique was chosen for a particular study - e.g. could the measurements using brightfield microscopy also be accomplished using WARP? They should also clarify how these techniques relate to and possibly improve on existing techniques for measuring forces organisms exert on a substrate, particularly micropillar arrays and Traction Force Microscopy.

As written, "(ERISM) (19) and a variant, Wavelength Alternating Resonance Pressure microscopy (WARP) (20) enable optical mapping of GRFs in the nanonewton range with micrometre and millisecond precision..." (lines 53-55) may generate confusion. ERISM as described in this work has a much lower temporal resolution (requires the animal to be still for 5 seconds - lines 474-5); In this work, WARP does not appear to have nanonewton precision (judging by noise on calibration figures) and it is not clear that it has millisecond precision (the camera used and its frame rate should be specified in the methods).

It would be helpful to have a discussion of the limits of the techniques presented and tradeoffs that might be involved in overcoming them. For instance, what is the field of view of the WARP microscope, and could it be increased by choosing a lower power objective? What would be required to allow WARP microscopy to measure horizontal forces? Can a crawling larva be imaged over many strides by recentering it in the field of view, or are there only particular regions of the elastomer where a measurement may be made?

Reviewer #1 (Public Review):

This study reports a long-term, multisite study of tropical herbivory on Piper plants. The results are clear that lack of water leads to lower plant survival and altered herbivory. The results varied substantially among sites. The caveats are that ecosystem processes beyond water availability are not investigated although they are brought into play in the title and in the paper, that herbivory beyond leaf damage was not reported (there might be none, the reader needs to be shown the evidence for this), that herbivore diversity is defined by leaf damage (authors need to give evidence that this is a valid inference), that the plots were isolated from herbivores beyond their borders, that the effects of extreme climate events were isolated to Peru, that intraspecific variation in the host plants needs to be explained and interpreted in more detail, the results as reported are extremely complicated, the discussion is overly long and diffuse.

Reviewer #1 (Public Review):

In principle a very interesting story, in which the authors attempt to shed light on an intriguing and poorly understood phenomenon; the link between damage repair and cell cycle re-entry once a cell has suffered from DNA damage. The issue is highly relevant to our understanding of how genome stability is maintained or compromised when our genome is damaged. The authors present the intriguing conclusion that this is based on a timer, implying that the outcome of a damaging insult is somewhat of a lottery; if a cell can fix the damage within the allocated time provided by the "timer" it will maintain stability, if not then stability is compromised. If this conclusion can be supported by solid data, the paper would make a very important contribution to the field.

However, the story in its present form suffers from a number of major gaps that will need to be addressed before we can conclude that MASTL is the "timer" that is proposed here. The primary concern being that altered MASTL regulation seems to be doing much more than simply acting as a timer in control of recovery after DNA damage. There is data presented to suggest that MASTL directly controls checkpoint activation, which is very different from acting as a timer. The authors conclude on page 8 "E6AP promoted DNA damage checkpoint signaling by counteracting MASTL", but in the abstract the conclusion is "E6AP depletion promoted cell cycle recovery from the DNA damage checkpoint, in a MASTL-dependent manner". These 2 conclusions are definitely not in alignment. Do E6AP/MASTL control checkpoint signaling or do they control recovery, which is it?

Also, there is data presented that suggest that MASTL does more than just controlling mitotic entry after DNA damage, while the conclusions of the paper are entirely based on the assumption that MASTL merely acts as a driver of mitotic entry, with E6AP in control of its levels. This issue will need to be resolved.

and finally, the authors have shown some very compelling data on the phosphorylation of E6AP by ATM/ATR, and its role in the DNA damage response. But the time resolution of these effects in relation to arrest and recovery have not been addressed.

Revised manuscript:<br /> I think the authors did a good job in revising the paper, and provide compelling support for a timer function in the checkpoint. I do think they still have missed one important point how MASTL could act as a timer to control recovery. The data clearly show that MASTL somehow controls ATM/ATR activity, whilst their final model (fig.9) places MASTL upstream of CDK activity, without mentioning its feedback on ATM/ATR. I think there are 2 possible explanations for the timer function of MASTL they have discovered here, both may be relevant. The first is enhanced CDK activation by direct control of CDK phosphorylation through MASTL/B55/PP2A. The second is through MASTL-mediated shut-down of ATM/ATR activation (mechanism to be determined) which is also reported here. Their final model and discussion do not display sufficient appreciation for this latter option, and I would argue that the HU-recovery experiment shown in Fig.5B is actually in strong support of the second explanation, rather than the first.

Reviewer #1 (Public Review):

Membrane receptor guanylyl cyclases are important for many physiological processes but their structures in full-length and their mechanism are poorly understood. Caveney et al. determined the cryo-EM structure of a highly engineered GC-C in a complex with endogenous HSP90 and CDC37. The structural work is solid and the structural information will be useful for the membrane receptor guanylyl cyclases field and the HSP90 field.

Reviewer #1 (Public Review):

In this study the authors first perform global knockout of the gene coding for the polarity protein Crumbs 3 (CRB3) in the mouse and show that this leads to perinatal lethality and anopthalmia. Next, they create a conditional knockout mouse specifically lacking CRB3 in mammary gland epithelial cells and show that this leads to ductal epithelial hyperplasia, impaired branching morphogenesis and tumorigenesis. To study the mechanism by which CRB3 affects mammary epithelial development and morphogenesis the authors turn to MCF10A cells and find that CRB3 shRNA-mediated knockdown in these cells impairs their ability to form properly polarized acini in 3D cultures. Furthermore, they find that MCF10A cells lacking CRB3 display reduced primary ciliation frequency compared to control cells, which is in agreement with previous studies implicating CRB3 in primary cilia biogenesis. Using a combination of biochemical, molecular- and imaging approaches the authors then provide some evidence indicating that CRB3 promotes ciliogenesis by mediating Rab11-dependent recruitment of gamma-tubulin ring complex (gamma-TuRC) component GCP6 to the centrosome/ciliary base, and they also show that CRB3 itself is localized to the base of primary cilia. Finally, to assess the functional consequences of CRB3 loss on ciliary signaling function, the authors analyze the effect of CRB3 loss on Hedgehog and Wnt signaling using cell-based assays or a mouse model.

Overall, the described findings are interesting and in agreement with previous studies showing an involvement of CRB3 in epithelial cell biology, tumorigenesis and ciliogenesis. The results showing a role for CRB3 in mammary epithelial development and morphogenesis in vivo seem convincing. Although the authors provide evidence that CRB3 promotes ciliogenesis via (indirect) physical association with Rab11 and gamma-TuRC, the precise mechanism by which CRB3 promotes ciliogenesis remains to be clarified. Specific comments are as follows:

1) For all cell-based assays using shRNA to knock down CRB3, it would be desirable to perform rescue experiments to ensure that the observed phenotype of CRB3 depleted cells is specific and not due to off-target effects of the shRNA.<br /> 2) Figure 3G: it is very difficult to see that the red stained structures are primary cilia.<br /> 3) Figure 5A: it is unfortunate the authors chose not to show the original dataset (Excel file) used for generating this figure; this makes it difficult to interpret the data. It is general policy of the journal to make source data accessible to the scientific community.<br /> 4) The authors have a tendency to overinterpret their data, and not all claims put forth by the authors are fully supported by the data provided.

Reviewer #1 (Public Review):

The authors apply a new approach to monitor brain-wide changes in sensory-evoked hemodynamic activity after focal stroke in fully conscious rats. Using functional ultrasound (fUS), they report immediate and lasting (up to 5 days) depression of sensory-evoked responses in somatosensory thalamic and cortical regions.

Strengths: This a technically challenging and proof-of-concept study that employs new methods to study brain-wide changes in sensory-evoked neural activity, inferred from changes in cerebral blood flow. Despite the minor typos/grammatical errors and small sample size, the authors provide compelling images and rigorous analysis to support their conclusions. Overall, this was a very technically difficult study that was well executed. I believe that it will pave the way for more extensive studies using this methodological approach. Therefore I support this study and my recommendations to improve it are relatively minor in nature and should be simple for the authors to address.

Weaknesses: The primary weakness of this paper is the small sample sizes. Drawing conclusions based on the small sham control group (n=2) or 5-day stroke recovery group (n=2), is rather tenuous. One way to alleviate some uncertainty with regard to the conclusions would be to state in the discussion that the findings (ie. loss of thalamocortical function after stroke) are perfectly consistent with previous studies that examined thalamocortical function after stroke. The authors missed some of these supporting studies in their reference list (see PMID: 28643802, 1400649). A second issue that can easily be resolved is their analysis of the 69 brain regions. This seems like a very important part of the study and one of the primary advantages of employing efUS. As presented, I had difficulty seeing the data. I think it would be worthwhile to expand Fig 3 (especially 3C) into a full-page figure with an accompanying table in the Supplementary info section describing the % change in CBF for each brain region.

Other Recommendations for the authors:.<br /> - Since there is variability in spreading depolarizations, was there any trend in the relationship between # SD's and ischemic volume? I know there are few data points but a scatterplot might be of interest.<br /> - For statistical comparisons of 'response curves' in Fig 3 and 4, what exactly was the primary dependent measure: changes in peak amplitude (%) or area under the curve?<br /> - There are several typos and minor grammatical errors in the manuscript. Some editing is recommended.

Reviewer #1 (Public Review):

Many labs worldwide now use the blind source deconvolution technique to identify the firing patterns of multiple motor units simultaneously in human subjects. This technique has had a truly transformative effect on our understanding of the structure of motor output in both normal subjects and, increasingly, in persons with neurological disorders. The key advance presented here is that the software provides real-time identification of these firing patterns.

The main strengths are the clarity of the presentation and the great potential that real-time decoding will provide. Figures are especially effective and statistical analyses are excellent.

The main limitation of the work is that only male subjects were included in the validation of the software. The reason given - that yield of number of motor units identified is generally larger in males than females - is reasonable in the sense that this is the first systematic test of this real-time approach. At a minimum, however, the authors should clearly commit to future work with female subjects and emphasize the importance of considering sex differences.

A second weakness is that the Introduction does a poor job of establishing the potential importance of the real-time approach.

Reviewer #1 (Public Review):

The main objective of this paper is to report the development of a new intramuscular probe that the authors have named Myomatrix arrays. The goal of the Myomatrix probe is to significantly advance the current technological ability to record the motor output of the nervous system, namely fine-wire electromyography (EMG). Myomatrix arrays aim to provide large-scale recordings of multiple motor units in awake animals under dynamic conditions without undue movement artifacts and maintain long-term stability of chronically implanted probes. Animal motor behavior occurs through muscle contraction, and the ultimate neural output in vertebrates is at the scale of motor units, which are bundles of muscle fibers (muscle cells) that are innervated by a single motor neuron. The authors have combined multiple advanced manufacturing techniques, including lithography, to fabricate large and dense electrode arrays with mechanical features such as barbs and suture methods that would stabilize the probe's location within the muscle without creating undue wiring burden or tissue trauma. Importantly, the fabrication process they have developed allows for rapid iteration from design conception to a physical device, which allows for design optimization of the probes for specific muscle locations and organisms. The electrical output of these arrays are processed through a variety of means to try to identify single motor unit activity. At the simplest, the approach is to use thresholds to identify motor unit activity. Of intermediate data analysis complexity is the use of principal component analysis (PCA, a linear second-order regression technique) to disambiguate individual motor units from the wide field recordings of the arrays, which benefits from the density and numerous recording electrodes. At the highest complexity, they use spike sorting techniques that were developed for Neuropixels, a large-scale electrophysiology probe for cortical neural recordings. Specifically, they use an estimation code called kilosort, which ultimately relies on clustering techniques to separate the multi-electrode recordings into individual spike waveforms.

An account of the major strengths and weaknesses of the methods and results.<br /> The biggest strength of this work is the design and implementation of the hardware technology. It is undoubtedly a major leap forward in our ability to record the electrical activity of motor units. The myomatrix arrays trounce fine-wire EMGs when it comes to the quality of recordings, the number of simultaneous channels that can be recorded, their long-term stability, and resistance to movement artifacts.

The primary weakness of this work is its reliance on kilosort in circumstances where most of the channels end up picking up the signal from multiple motor units. As the authors quite convincingly show, this setting is a major weakness for fine-wire EMG. They argue that the myomatrix array succeeds in isolating individual motor unit waveforms even in that challenging setting through the application of kilosort.

Although the authors call the estimated signals as well-isolated waveforms, there is no independent evidence of the accuracy of the spike sorting algorithm. The additional step (spike sorting algorithms like kilosort) to estimate individual motor unit spikes is the part of the work in question. Although the estimation algorithms may be standard practice, the large number of heuristic parameters associated with the estimation procedure are currently tuned for cortical recordings to estimate neural spikes. Even within the limited context of Neuropixels, for which kilosort has been extensively tested, basic questions like issues of observability, linear or nonlinear, remain open. By observability, I mean in the mathematical sense of well-posedness or conditioning of the inverse problem of estimating single motor unit spikes given multi-channel recordings of the summation of multiple motor units. This disambiguation is not always possible. kilosort's validation relies on a forward simulation of the spike field generation, which is then truth-tested against the sorting algorithm. The empirical evidence is that kilosort does better than other algorithms for the test simulations that were performed in the context of cortical recordings using the Neuropixels probe. But this work has adopted kilosort without comparable truth-tests to build some confidence in the application of kilosort with myomatrix arrays? Furthermore, as the paper on the latest version of kilosort, namely v4, discusses, differences in the clustering algorithm is the likely reason for kilosort4 performing more robustly than kilosort2.5 (used in the myomatrix paper). Given such dependence on details of the implementation and the use of an older kilosort version in this paper, the evidence that the myomatrix arrays truly record individual motor units under all the types of data obtained is under question.

There is an older paper with a similar goal to use multi-channel recording to perform source-localization that the authors have failed to discuss. Given the striking similarity of goals and the divergence of approaches (the older paper uses a surface electrode array), it is important to know the relationship of the myomatrix array to the previous work. Like myomatrix arrays, the previous work also derives inspiration from cortical recordings, in that case it uses the approach of source localization in large-scale EEG recordings using skull caps, but applies it to surface EMG arrays. Ref: van den Doel, K., Ascher, U. M., & Pai, D. K. (2008). Computed myography: three-dimensional reconstruction of motor functions from surface EMG data. Inverse Problems, 24(6), 065010.

The incompleteness of the evidence that the myomatrix array truly measures individual motor units is limited to the setting where multiple motor units have similar magnitude of signal in most of the channels. In the simpler data setting where one motor dominates in some channel (this seems to occur with some regularity), the myomatrix array is a major advance in our ability to understand the motor output of the nervous system. The paper is a trove of innovations in manufacturing technique, array design, suture and other fixation devices for long-term signal stability, and customization for different muscle sizes, locations, and organisms. The technology presented here is likely to achieve rapid adoption in multiple groups that study motor behavior, and would probably lead to new insights into the spatiotemporal distribution of the motor output under more naturally behaving animals than is the current state of the field.

Reviewer #1 (Public Review):

The manuscript by Kadkova et al. describes an electrophysiological analysis of 3 neurodevelopmental disease-causing SNAP-25 mutations in hippocampal neuron autaptic cultures. The work expands on a prior study of these 3 mutations, along with several others in SNAP-25, that was performed in acutely dissociated hippocampal cultures by another group (Alten et al, 2021). Most of the physiology defects found are pretty similar for the 3 mutations the two research groups characterized, with differences largely found in the effects on the size of the readily releasable pool (RRP) of SVs. These differences could be due to technical differences in the approach but are also likely to reflect in part differences in autapses as a model that has been previously described. In addition to the physiological analysis in cultured neurons, the current work extends the analysis beyond the prior study by analyzing the effects of these SNAP-25 mutations in vitro liposome fusion assays with purified proteins, and some modeling of the effects on energy landscapes during priming and fusion.

The authors use lentiviral expression of wildtype or one of the 3 mutants in SNAP-25 autaptic neurons and assay neuronal survival and synaptic output. The authors also combine wildtype with each of the 3 mutants as well, given these diseases manifest as spontaneous mutations in only 1 of the SNAP-25 alleles, suggesting a dominant effect. The authors observe that the V48F and D166Y alleles (that are suggested to disrupt the Syt1-SNAP-25/SNARE interface) result in a very large increase in a spontaneous release that exceeds the Syt1 null mutant alone, suggesting an effect on spontaneous SV release beyond a lack of Syt1 regulation of SNARE-mediated fusion. In contrast, Syt1 nulls have a much more severe loss of evoked release, though both V48F and D166Y also have modest decreases in release. They find both mutants also cause a decrease in the RRP. Applying some modeling for these results, the authors suggest V48F and D166Y lower the energy barrier for fusion, creating enhanced spontaneous release rates and causing a decrease of the RRP. They also find evidence for reduced SV priming. In contrast, a SNAP-25 I167N disease mutation in the SNARE assembly interaction layer causes dramatic decreases in both evoked and spontaneous release, consistent with a disruption to SNARE assembly/stability. In vitro fusion assays with these mutant SNAP-25 alleles were also done and provided supportive evidence for these interpretations for all 3 alleles. The ability to control calcium, Syt1, PIP2, and Complexin levels in the in vitro assays provided additional information on defining the precise steps of the fusion process these mutations disrupt. Together, the study indicates the I167N mutation acts as a dominant-negative allele to block fusion, while the other two alleles have both loss- and gain-of function properties that cause more complex disruptions that decrease evoked release while dramatically enhancing spontaneous fusion.

Overall, these results build on prior work and shed light on how disruptions to the SNAP-25 t-SNARE alter the process of SV priming and fusion.

Reviewer #1 (Public Review):

Valk and Engert et al. examined the potential relations between three different mental training modules, hippocampal structure and functional connectivity, and cortisol levels over a 9-month period. They found that among the three types of mental training: Presence (attention and introspective awareness), Affect (socio-emotional - compassion and prosocial motivation), and Perspective (socio-cognitive - metacognition and perspective taking) modules; Affect training most consistently related to changes in hippocampal structure and function - specifically, CA1-3 subfields of the hippocampus. Moreover, decreases in diurnal cortisol correlated to bilateral increases in volume, and decreases in diurnal and chronic cortisol left CA1-3 functional connectivity. Chronic cortisol levels also related to right CA4/DG volume and left subiculum function. The authors demonstrate that mindfulness training programs impact hippocampus and are a potential avenue for stress interventions, a potential avenue to improve health. The data contribute to the literature on plasticity of hippocampal subfields during adulthood, the impact of mental training interventions on the brain, and the link between CA1-3 and both short- and long-term stress changes. Additional clarification and extension of the methods is needed to strengthen the authors' conclusions.

The authors thoughtfully approached the study of hippocampal subfields, utilizing a method designed for T1w images that outperformed Freesurfer 5.3 and that produced comparable results to an earlier version of ASHS. However, given the use of normalized T1-weighted images to delineate hippocampal subfield volume, some caution may be warranted (Wisse et al. 2020). While the authors note the assessment of quality control processes, the difficulty in ensuring valid measurement is an ongoing conversation in the literature. This also extends to the impact of functional co-registration using segmentations. I appreciate the inclusion of Table 5 in documenting reasons for missing data across subjects. Providing additional details on the distribution of quality ratings across subfields would help contextualize the results and ensure there is equal quality of segmentations across subfields.

Given the consistent pattern of finding results with CA1-3, in contrast to other subfields, it would help to know if the effects of the different training modules on subfields differed from each other statistically (i.e., not just that one is significant, and one is not) to provide an additional context of the strength of results focused on Affect training and CA1-3 (for example, those shown in Figure 3).

Reviewer #1 (Public Review):

In this manuscript, the authors use a large dataset of neuroscience publications to elucidate the nature of self-citation within the neuroscience literature. The authors initially present descriptive measures of self-citation across time and author characteristics; they then produce an inclusive model to tease apart the potential role of various article and author features in shaping self-citation behavior. This is a valuable area of study, and the authors approach it with an appropriate and well-structured dataset.

The study's descriptive analyses and figures are useful and will be of interest to the neuroscience community. However, with regard to the statistical comparisons and regression models, I believe that there are methodological flaws that may limit the validity of the presented results. These issues primarily affect the uncertainty of estimates and the statistical inference made on comparisons and model estimates - the fundamental direction and magnitude of the results are unlikely to change in most cases. I have included detailed statistical comments below for reference.

Conceptually, I think this study will be very effective at providing context and empirical evidence for a broader conversation around self-citation. And while I believe that there is room for a deeper quantitative dive into some finer-grained questions, this paper will be a valuable catalyst for new areas of inquiry around citation behavior - e.g., do authors change self-citation behavior when they move to more or less prestigious institutions? do self-citations in neuroscience benefit downstream citation accumulation? do journals' reference list policies increase or decrease self-citation? - that I hope that the authors (or others) consider exploring in future work.

Statistical comments:

(1) Throughout the paper, the nested nature of the data does not seem to be appropriately handled in the bootstrapping, permutation inference, and regression models. This is likely to lead to inappropriately narrow confidence bands and overly generous statistical inference.

(2) The discussion of the data structure used in the regression models is somewhat opaque, both in the main text and the supplement. From what I gather, these models likely have each citation included in the model at least once (perhaps twice, once for first-author status and one for last-author status), with citations nested within citing papers, cited papers, and authors. Without inclusion of random effects, the interpretation and inference of the estimates may be misleading.

(3) I am concerned that the use of the inverse hyperbolic sine transform is a bit too prescriptive, and may be producing poor fits to the true predictor-outcome relationships. For example, in a figure like Fig S8, it is hard to know to what extent the sharp drop and sign reversal are true reflections of the data, and to what extent they are artifacts of the transformed fit.

(4) It seems there are several points in the analysis where papers may have been dropped for missing data (e.g., missing author IDs and/or initials, missing affiliations, low-confidence gender assessment). It would be beneficial for the reader to know what % of the data was dropped for each analysis, and for comparisons across countries it would be important for the authors to make sure that there is not differential missing data that could affect the interpretation of the results (e.g., differences in self-citation being due to differences in Scopus ID coverage).

Reviewer #1 (Public Review):

This study demonstrates that vitamin D-bound VDR increased the expression of SIRT1 and that vitamin D-bound VDR interacts with SIRT1 to cause auto-deacetylation on Lys610 and activation of SIRT1 catalytic activity. This is an important finding that is relevant to the actions of VDR on colorectal cancer. The data presented to support the presented conclusion is convincing.

A strength of the study is that it is focused on a narrow group of conclusions.

The major weakness of the study is that the site of SIRT1 regulatory lysine acetylation is defined by mutational analysis rather than by direct biochemical analysis. This issue is partially mitigated by previous reports of K610 acetylation using mass spec (https://www.phosphosite.org/proteinAction.action?id=5946&showAllSites=true). However, Fig. 4E is reassuring because it shows that the apparent acetylation of the K610 mutant SIRT1 appears to be lower than WT SIRT1

A second weakness of the study relates to the use of shRNA-mediated knockdown of VDR for some studies in which a previously reported cell line was employed. The analysis presented would be more compelling if similar data was obtained using more than one shRNA. Similarly, only a single siRNA for SIRT1 is presented in Table 1.

A third weakness of the study is that the conclusion that the VDR interaction with SIRT1 is the cause of auto-deacetylation rather than an associated event mediated by another mechanism would be more strongly supported by mutational analysis of SIRT1 and VDR residues required for the binding interaction. Will VDR increase SIRT1 activity when mutations are introduced to block the interaction? While the finding that catalytically inactive SIRT1 does not interact with VDR is helpful, this does not address the role of the binding surface.

A fourth weakness of the study is that it would be improved by testing the proposed hypothesis through in vitro reconstitution with purified proteins. Does VDR cause auto-deacetylation and activation of Sirt1 in vitro?

Reviewer #1 (Public Review):

The objective of this investigation was to determine whether experimental pain could induce alterations in cortical inhibitory/facilitatory activity observed in TMS-evoked potentials (TEPs). Previous TMS investigations of pain perception had focused on motor evoked potentials (MEPs), which reflect a combination of cortical, spinal, and peripheral activity, as well as restricting the focus to M1. The main strength of this investigation is the combined use of TMS and EEG in the context of experimental pain. More specifically, Experiment 1 investigated whether acute pain altered cortical excitability, reflected in the modulation of TEPs. The main outcome of this study is that relative to non-painful warm stimuli, painful thermal stimuli led to an increase on the amplitude of the TEP N45, with a larger increase associated with higher pain ratings. Because it has been argued that a significant portion of TEPs could reflect auditory potentials elicited by the sound (click) of the TMS, Experiment 2 constituted a control study that aimed to disentangle the cortical response related to TMS and auditory activity. Finally, Experiment 3 aimed to disentangle the cortical response to TMS and reafferent feedback from muscular activity elicited by suprathreshold TMS applied over M1. The fact that the authors accompanied their main experiment with two control experiments strengthens the conclusion that the N45 TEP peak could be implicated in the perception of painful stimuli. Perhaps, the addition of a highly salient but non-painful stimulus (i.e. from another modality) would have further ruled out that the effects on the N45 are not predominantly related to intensity/saliency of the stimulus rather than to pain per se.

Public Review:

In countries endemic for P vivax the need to administer a primaquine (PQ) course adequate to prevent relapse in G6PD deficient persons poses a real dilemma. On one hand PQ will cause haemolysis; on the other hand, without PQ the chance of relapse is very high. As a result, out of fear of severe haemolysis, PQ has been under-used.

In view of the above, the Authors have investigated in well-informed volunteers, who were kept under close medical supervision in hospital throughout the study, two different schedules of PQ administration: (1) escalating doses (to a total of 5-7 mg/kg); (2) single 45 mg dose (0.75 mg/kg).

It is shown convincingly that regimen (1) can be used successfully to deliver within 3 weeks, under hospital conditions, the dose of PQ required to prevent P vivax relapse.

As expected, with both regimens acute haemolytic anaemia (AHA) developed in all cases. With regimen (2), not surprisingly, the fall in Hb was less, although it was abrupt. With regimen (1) the average fall in Hb was about 4 G. Only in one subject the fall in Hb mandated termination of the study.

Since the data from the Chicago group some sixty years ago, there has been no paper reporting a systematic daily analysis of AHA in so many closely monitored subjects with G6PD deficiency. The individual patient data in the Supplementary material are most informative and more than precious.

Having said this, I do have some general comments.<br /> 1. Through their remarkable Part 1 study, the Authors clearly wish to set the stage for a revision of the currently recommended PQ regimen for G6PD deficient patients. They have shown that 5-7 mg/kg can be administered within 3 weeks, whereas the currently recommended regimen provides 6 mg/kg over no less than 8 weeks.<br /> 2. Part 2 aims to show that, as was known already, even a single PQ dose of 0.75 mg/kg causes a significant degree of haemolysis: G6PD deficiency-related haemolysis is characteristically markedly dose-dependent. Although they do not state it explicitly in these words (I think they should), the Authors want to make it clear that the currently recommended regimen does cause AHA.<br /> 3. Regulatory agencies like to classify a drug regimen as either SAFE or NOT-SAFE; they also like to decide who is 'at risk' and who is 'not at risk'. A wealth of data, including those in this manuscript, show that it is not correct to say that a G6PD deficient person when taking PQ is at risk of haemolysis: he or she will definitely have haemolysis. As for SAFETY, it will depend on the clinical situation when PQ is started and on the severity of the AHA that will develop.

The above three issues are all present in the discussion, but I think they ought to be stated more clearly.

Finally, by the Authors' own statement on page 15, the main limitation is the complexity of this approach. The authors suggest that blister packed PQ may help; but to me the real complexity is managing patients in the field versus the painstaking hospital care in the hands of experts, of which volunteers in this study have had the benefit. It is not surprising that a fall in Hb of 4 g/dl is well tolerated by most non-anaemic men; but patients with P vivax in the field may often have mild to moderate to severe anaemia; and certainly they will not have their Hb, retics and bilirubin checked every day. In crude approximation, we are talking of a fall in Hb of 4 G with regimen (1), as against a fall in Hb of 2 G with regimen (2), that is part of the currently recommended regimen: it stands to reason that, in terms of safety, the latter is generally preferable (even though some degree of fall in Hb will recur with each weekly dose). In my view, these difficult points should be discussed deliberately.

Reviewer #1 (Public Review):

Sun and colleagues investigated the cross-reactive antibodies between E.Coli and the host in severe alcoholic hepatitis (SAH). The study found that IgA and IgG were deposited in the liver of SAH patients. Complements C3d and C4d were also deposited in the SAH patient's liver. Moreover, they found that the Ig accumulated in the SAH liver, but not in the SAH serum, induced hepatocyte killing, suggesting that liver Ig is important. Then, they found that these Ig can recognize both human and E. Coli antigens. Very interestingly, SAH-derived Ig shows cross-reactivity to both human and E. Coli antigens, suggesting E, Coli-primed Ig in SAH may damage hepatocytes through host antigen recognition. These Ig are not observed in alcoholic cirrhosis patients. The liver RNA-seq data suggested that Ig was also produced in the liver, not only gut-derived Ig. This is a very interesting study showing the novel mechanism of SAH mediated by the Ig with the cross-reactivity with bacteria and host antigens, which is not observed in AC patients. Overall, the study design is reasonable and the data are consistent to support their central hypothesis. There are a few comments.

Specific comments:<br /> 1. Figures 1 and 2 show Ig deposition in the liver (it seems on hepatocytes). Not only Ig reaction to the specific antigen but also non-specific Fc receptor-mediated binding to hepatocytes could be contributed.<br /> 2. Similarly, in Figure 2G Ig-mediated hepatocyte killing, Fc receptor-mediated hepatocyte killing may be involved.<br /> 3. The study examined the possibility of liver resident B cell and plasma cell-mediated Ig. As the authors mentioned in the discussion, B cells may be translocated from the intestine to the liver. Or the resident B cells (not from the gut) are also involved.

Reviewer #1 (Public Review):

The work by Debashish U. Menon, Noel Murcia, and Terry Magnuson brings important knowledge about histone H3.3 dynamics involved in meiotic sex chromosome inactivation (MSCI). MSCI is unique to gametes and failure during this process can lead to infertility. Classically, MSCI has been studied in the context of DNA Damage repair pathways and little is known about the epigenetic mechanisms behind maintenance of the sex body as a silencing platform during meiosis. One of the major strengths of this work is the evidence provided on the role of ARID1A, a BAF subunit, in MSCI through the regulation of H3.3 occupancy in specific genic regions. This is well supported by a combination of immunofluorescence, RNA seq, CUT&RUN and ATAC-seq.<br /> The mouse model in this study is a conditional Stra8 Cre mouse. Loss of ARID1A in this mouse, caused up regulation of XY linked genes in prophase I spermatocytes and ingression of RNA pol II to the sex body, indicating a role for this chromatin remodeller in MSCI. Using RNA seq and CUT&RUN and ATAC-seq, the authors show that ARID1A regulates chromatin accessibility of the sex chromosomes. ARID1A interacts with gene transcription start sites of sex-linked genes, and loss of ARID1A increased promoter accessibility of XY linked genes with concomitant gene up regulation.

This work suggests that ARID1A regulates chromatin composition of the sex body relative to the autosomes. In the absence of ARID1A, spermatocytes show less enrichment of H3.3 in the sex chromosomes and stable levels of the canonical histones H3.1/3.2. By overlapping CUT&RUN and ATAC-seq data, authors show that changes in chromatin accessibility in the absence of ARID1A are given by redistribution of occupancy of H3.3. Gained open chromatin in mutants corresponds to up regulation of H3.3 occupancy at transcription start sites of genes regulated by ARID1A.

Interestingly, ARID1A loss caused increased promoter occupancy by H3.3 in regions usually occupied by PRDM9. PRDM9 is a protein with histone methyltransferase activity that catalyzes histone H3 lysine 4 trimethylation during meiotic prophase I, and positions double strand break (DSB) hotspots. Lack of ARID1A causes reduction in occupancy of DMC1, a recombinase involved in DSB repair, in non-homologous sex regions. These data suggest that ARID1A might indirectly influence DNA DSB repair on the sex chromosomes by regulating the localization of H3.3. This is very interesting given the suggested role for ARID1A in genome instability in cancer cells (Nacarelli et al 2020: 10.1080/23723556.2019.1690923, Zhang et al. 2023: 10.1093/carcin/bgad011 and others). It raises the question of whether this role is also involved in meiotic DSB repair in autosomes and/or how this mechanism differs in sex chromosomes compared to autosomes.

It is worth mentioning that authors show that there are Arid1a transcripts that escape the Cre system. This might mask the phenotype of the Arid1a knockout, given that many of the sequencing techniques used here are done on a heterogeneous population of knockout and wild type spermatocytes. In relation to this, I think that the use of the term "pachytene arrest" might be overstated, since this is not the phenotype truly observed (these mice produce sperm). ARID1A is present throughout prophase I and it might have pre-MSCI roles that impact earlier stages of Meiosis I and cell death might be happening in these earlier stages too.

Overall the research presented here is solid, adds new knowledge on how the sex chromatin is silenced during meiosis and has generated relevant databases for the field.

Reviewer #1 (Public Review):

This work provides a new dataset of 71,688 images of different ape species across a variety of environmental and behavioral conditions, along with pose annotations per image. The authors demonstrate the value of their dataset by training pose estimation networks (HRNet-W48) on both their own dataset and other primate datasets (OpenMonkeyPose for monkeys, COCO for humans), ultimately showing that the model trained on their dataset had the best performance (performance measured by PCK and AUC). In addition to their ablation studies where they train pose estimation models with either specific species removed or a certain percentage of the images removed, they provide solid evidence that their large, specialized dataset is uniquely positioned to aid in the task of pose estimation for ape species.

The diversity and size of the dataset make it particularly useful, as it covers a wide range of ape species and poses, making it particularly suitable for training off-the-shelf pose estimation networks or for contributing to the training of a large foundational pose estimation model. In conjunction with new tools focused on extracting behavioral dynamics from pose, this dataset can be especially useful in understanding the basis of ape behaviors using pose.

Since the dataset provided is the first large, public dataset of its kind exclusively for ape species, more details should be provided on how the data were annotated, as well as summaries of the dataset statistics. In addition, the authors should provide the full list of hyperparameters for each model that was used for evaluation (e.g., mmpose config files, textual descriptions of augmentation/optimization parameters).

Overall this work is a terrific contribution to the field and is likely to have a significant impact on both computer vision and animal behavior.

Strengths:<br /> - Open source dataset with excellent annotations on the format, as well as example code provided for working with it.<br /> - Properties of the dataset are mostly well described.<br /> - Comparison to pose estimation models trained on humans vs monkeys, finding that models trained on human data generalized better to apes than the ones trained on monkeys, in accordance with phylogenetic similarity. This provides evidence for an important consideration in the field: how well can we expect pose estimation models to generalize to new species when using data from closely or distantly related ones?<br /> - Sample efficiency experiments reflect an important property of pose estimation systems, which indicates how much data would be necessary to generate similar datasets in other species, as well as how much data may be required for fine-tuning these types of models (also characterized via ablation experiments where some species are left out).<br /> - The sample efficiency experiments also reveal important insights about scaling properties of different model architectures, finding that HRNet saturates in performance improvements as a function of dataset size sooner than other architectures like CPMs (even though HRNets still perform better overall).

Weaknesses:<br /> - More details on training hyperparameters used (preferably full config if trained via mmpose).<br /> - Should include dataset datasheet, as described in Gebru et al 2021 (arXiv:1803.09010).<br /> - Should include crowdsourced annotation datasheet, as described in Diaz et al 2022 (arXiv:2206.08931). Alternatively, the specific instructions that were provided to Hive/annotators would be highly relevant to convey what annotation protocols were employed here.<br /> - Should include model cards, as described in Mitchell et al (arXiv:1810.03993).<br /> - It would be useful to include more information on the source of the data as they are collected from many different sites and from many different individuals, some of which may introduce structural biases such as lighting conditions due to geography and time of year.<br /> - Is there a reason not to use OKS? This incorporates several factors such as landmark visibility, scale, and landmark type-specific annotation variability as in Ronchi & Perona 2017 (arXiv:1707.05388). The latter (variability) could use the human pose values (for landmarks types that are shared), the least variable keypoint class in humans (eyes) as a conservative estimate of accuracy, or leverage a unique aspect of this work (crowdsourced annotations) which affords the ability to estimate these values empirically.<br /> - A reporting of the scales present in the dataset would be useful (e.g., histogram of unnormalized bounding boxes) and would align well with existing pose dataset papers such as MS-COCO (arXiv:1405.0312) which reports the distribution of instance sizes and instance density per image.

Reviewer #1 (Public Review):

This work investigated Fragile X Messenger Ribonucleoprotein (FMRP) protein impact on neuroblast tangential migration in the postnatal rostral migratory stream (RMS). Authors conducted series of live-imaging on organotypic brain slices from Fmr1-null mice. They continued their analysis silencing Fmr1 exclusively from migrating neuroblasts using electroporation-mediated RNA interference method (MiRFmr1 KD). These impressive approaches show that neuroblasts tangential migration is impaired in Fmr1-null mice RMS and these defects are mostly recapitulated in the MiRFmr1neuroblasts.This nicely supports the idea that FMRP have a cell autonomous function in tangentially migrating neuroblasts. It is an important part of this work as migrating neuroblasts are in contact with each other and surrounding glial cells while migrating towards the olfactory bulb. The authors also confirm that FMRP mRNA target Microtubule Associated Protein 1B (MAP1B) is overexpressed in the Fmr1-null mice RMS. They successfully use electroporation-mediated RNA interference method to silence Map1b in the Fmr1-null mice neuroblasts. This discreet and elaborate experiment rescues most of the migratory defects observed both in Fmr1-null and MiRFmr1neuroblasts. Altogether, these results strongly suggest that FMRP-MAP1B axis has an important role in regulation of the neuroblasts tangential migration in RMS. Neurons move forward in cyclic saltatory manner which includes repeated steps of leading process extension, migration of the cell organelles and nuclear translocation. Authors reveal by analyzing the live-imaging data that FMRP-MAP1B axis is affecting movement of centrosome and nucleus during saltatory migration. An important part of the centrosome and nucleus movement is forces mediated by microtubule dynamics. Authors propose that FMRP regulate tangential migration via microtubule dynamics regulator MAP1B. This work provides valuable new information on regulation of the neuroblasts tangential saltatory migration. These findings also increase and improve our understanding of the issues involved in Fragile X Syndrome (FXS) disorders. The conclusions of this work are mostly supported by the data. However, methods and data analysis would benefit from more careful and comprehensive scrutiny.

1.) It would be beneficial for the detail-oriented readers to have a more comprehensive section of neuronal migration analysis. It would help to understand better the details of the results and analysis. For example, percentage of pausing time. Is neuroblast migration speed and pausing time (%) separated from each other? Does this mean that migration speed is measured from time of the nucleus movement, and it excludes pausing time? Sometimes migration speed refers to the total distance that cells have moved divided by the time between images (e.g., Nam et al. 2007, J Comp Neurol 505: 190-208). This is also important as neuroblasts migration speed fluctuate during their migration in RMS (e.g., Belvindrah et al. 2017, J Cell Biol 216: 2443-2461). It could be useful, for example, to show a plot of total migration distance distribution between controls, Fmr1-null, MiRFmr1 KD and MiRMap1b KD neuroblasts.

2.) The author's claim that Fmr1 interfering RNA (MiFmr1 KD) model "recapitulates the entire migratory phenotype described in Fmr1-null mutants". This is evident from the data analysis for the migration speed, pausing time percentage and NK distance. Parallel, but slightly weaker effect is seen in, NK frequency, CK frequency and CK efficiency. However, interpretation of the directionality analysis causes some concerns.<br /> a) Sinuosity index<br /> Fmr1-null mice (ctr: 1.32{plus minus}0.04; Fmr1-null: 1.93{plus minus}0.16; Figure 2C) and MiFmr1 KD neurons (MiRNEG: 1.5{plus minus}0.12; MiRFmr1 KD 1.62 {plus minus}0.08; Figure S1C). The latter results are significant, but standard error of means (SEM) seems to overlap. In addition, there is only a minor difference between control and MiRFmr1 KD cells sinuosity index.<br /> b) Directionality radar<br /> Migration directionality radar seems to be considerably different between Fmr1-null (Figure 2D) and MiFmr1 KD results (Figure S1D).<br /> It would be beneficial for this article to fully disclose, how these analyses were performed. For example, how sinuosity index was calculated and what does it precisely measure. It would greatly help to understand better the directionality analysis. To make these results more solid authors could have used original migration trajectories in the rose and/or trajectory plots. These plots visualize better the migration directionality results and clarify the changes in the directionality during migration.

3.) Authors claim that "Overall, our results demonstrate that MAP1B is the main FMRP mRNA target involved in the regulation of neuronal migration". Results and analysis show that migration speed, pausing time percentage, NK distance and NK frequency migratory defects are all rescued in Fmr1-null mice when MiRMap1b KD was introduced to the neuroblasts (Figure 4). These results are very interesting, linking FMRP-MAP1B axis to the microtubule dynamics.

4.) Authors could refine in discussion what is known about FRMP in neuronal migration. For example, La Fata et al. 2014 found that N-cadherin protein levels were lower in Fmr1-null mice and reintroducing N-cadherin rescued embryonic radial migration defects. N-cadherin is also expressed in the RMS and its deficiency affects negatively to the neuroblast migration (e.g., Porlan et al. 2014, Nat Cell Biol (7):629-38). This relationship of FMRP and N-cadherin could be discussed and considered in the article more closely. Overall, the article will benefit from clearer writing and more comprehensive discussion.

Reviewer #1 (Public Review):

In their manuscript entitled, "Reward contingency gates selective cholinergic suppression of amygdala neurons," Kimchi and colleagues explore the engagement and consequences of acetylcholine (ACh) signaling in the basolateral amygdala (BLA) using a number of sophisticated methodological approaches.

Perhaps the most compelling new idea in this manuscript is that ACh may have different effects on network activity in the BLA, a conclusion based on the measurement of equivalent photo-stimulated ACh levels in BLA during rewarded vs. unrewarded lick bouts despite increased licking/consumption in the rewarded bouts. The authors hypothesize that, "this could suggest that reward associations may gate post-synaptic responses to photostimulation." The electrophysiological data showing that overall firing of BLA neurons during licking was higher as a result of photostimulation during unreinforced, and lower as a result of photostimulation during reinforced, sessions is intriguing in this context, as is the contrast with the overall ACh-mediated stimulation of firing in dorsomedial prefrontal cortex. The ex-vivo data presented showing that ACh depresses BLA neuron activity via muscarinic ACh receptors on glutamate neurons and nicotinic ACh receptors on GABA neurons, along with previous data in the field suggesting that ACh has divergent effects on neuronal firing rate depending on whether baseline firing is low (tonic) or high (phasic), provides intriguing hints as to the role of ACh in state-dependent modulation of BLA activity.

One of the primary questions that came up while reading this manuscript was what behavioral domains were being measured with the "windows of opportunity" task. As noted by the authors, the cholinergic system has been implicated in arousal, reward thresholds, motivation and many other behaviors that might alter performance in this task, complicating interpretation of the data presented. In addition, some additional details of the task are needed for the field to be able to replicate these experiments.

Reviewer #1 (Public Review):

The manuscript by Hage et al. presents interesting results from a foraging behavior in Marmosets that explores the interactions of saccade and lick vigor with pupil dilation and performance as well as a marginal value theory and foraging theory-inspired value-based decision-making model thereof. The results are generally robust and carefully presented and analyses, particularly of vigor, are carefully executed.

The authors constructed a model that makes two predictions: "In summary, this simple theory made two sets of predictions: in response to an increased cost of harvest, one should work longer, but move with reduced vigor. In response to an increased reward value, as in hunger, one should also work longer, but now move with increased vigor." Their behavioral data meets these predictions. It is not clear if the model was designed and tweaked in order to make those predictions and match the data, or derived from principles. Furthermore, it is not clear what other models would make similar predictions. It would help to assess what is predicted by other simple models, as well as different functional forms for the effort costs in their model.

Line 37 page 6; the link of pupil to NE/LC is tenuous. Other modulators systems and circuits may be equally important and should be mentioned (e.g. Reimer, Jacob, Matthew J. McGinley, Yang Liu, Charles Rodenkirch, Qi Wang, David A. McCormick, and Andreas S. Tolias. "Pupil fluctuations track rapid changes in adrenergic and cholinergic activity in cortex." Nature communications 7, no. 1 (2016): 13289.)<br /> Line 35 page 6-page 7 line 10 emphasizes a cognitive interpretation of the pupil dilations they is emphasized, in relation to effort costs. But there are also concomitant more vigorous movements. Could all of their pupil results be explained by motor correlates? This should be tested and ruled out before making cognitive interpretations.<br /> Page 7, line 37-42: How would the model need to be modified in order to account for this discrepancy with the data? Ideally, this would be tested.<br /> Page 9, line 2-11: In this section, it would help to also consider 'baseline' pupil size (in between trials). This would give a signal that is not 'contaminated' by movements, and may reflect control state. Relatedly, changes in control state may impact and confound the movement-related dilation magnitudes due to e.g. floor and ceiling effects on pupil size, which has a strong tendency for reversion to the mean.<br /> Page 10 line 21-32 presents a dated view of pupil that has/had little data supporting it. They should mention other neuromodulators (Reimer et al., 2016) and related interpretations.<br /> The hunger-related and reward-size related analyses are both heavily confounded since they were not manipulated directly and could co-vary with many latent factors. For example, why might a given Marmoset be lower weight on a given day? Could it affect sleep, stress, activity, or other factors during the preceding 24 hours? If so, could these other variables be driving the results that are interpreted as 'hunger?' Relatedly, since the reward size is determined by the animals behavior on each trial (how much they worked), factors (internal brain state, external noises, etc.) that alter how much they worked will influence the subsequent reward size. Therefore interpretations about reward expectancy are confounded. Both of these issues should be discussed and manipulations of them (different feeding schedules and reward size-work functions proposed, respectively.<br /> A major issue is a lack of alternative models. The authors seem to have constructed a particular model designed to capture the behavioral patterns they observed in the data. The model fails in some instances, as they point out. Even more importantly, there are no results or discussion about how other plausible models could or couldn't fit the data. The lack of model comparisons makes it difficult to interpret the conclusions or put the results in a broader context.

Reviewer #1 (Public Review):

This manuscript by He et al. explores the molecular basis of the different stinging behaviors of two related anemones. The freshwater Nematostella which only stings when a food stimulus is presented with mechanical stimulation and the saltwater Exaiptasia which stings in response to mechanical stimuli. The authors had previously shown that Nematostella stinging is calcium-dependent and mediated by a voltage-gated calcium channel (VGCC) with very pronounced voltage-dependent inactivation, which gets removed upon hyperpolarization produced by taste receptors.

In this manuscript, they show that Exaiptacia and Nematostella differing stinging behavior is near optimal, according to their ecological niche, and conforms to predictions from a Markov decision model.

It is also shown that Exaiptacia stinging is also calcium-dependent, but the calcium channel responsible is much less inactivated at resting potential and can readily induce nematocyte discharge only in the presence of mechanical stimulation. To this end, the authors record calcium currents from Exaipacia nematocysts and discover that the VGCCs in this anemone are not strongly inactivated and thus are easily activated by mechanical stimuli-induced depolarization accounting for the different stinging behavior between species. The authors further explore the role of the auxiliary beta subunit in the modulation of VGCC inactivation and show that different n-terminal splice variants in Exaiptacia produce strong and weak voltage-dependent inactivation.

The manuscript is clear and well-written and the conclusions are in general supported by the experiments and analysis. The findings are very relevant to increase our understanding of the molecular basis of non-neural behavior and its evolutionary basis. This manuscript should be of general interest to biologists as well as to more specialized fields such as ion channel biophysics and physiology.

Some findings need to be clarified and perhaps additional experiments performed.

1) The authors identify by sequencing that the Exaiptacia Cav is a P-type channel (cacna1a). However, the biophysical properties of the nematocyte channel are different from mammalian P-type channels. The cnidarian channel inactivation is exceedingly rapid and activation happens at relatively low voltages. These substantial differences should be mentioned and commented on.

2) The currents from Nematostella in Figure 3d seem to be poorly voltage-clamped. Poor voltage-clamp is also evident in the sudden increase of conductance in Figure 3C and might contribute to incorrect estimation of voltage dependence of activation and if present in inactivation experiments, also to incorrect estimation of the inactivation voltage range. This problem should be reassessed with new data.

3) While co-expression of the mouse Cav channel with the beta1 isoform from Exaiptacia indeed shifts inactivation to more negative voltages, it does not recapitulate the phenotype of the more inactivated Ca-currents in nematocytes (compare Figures 4d and 5d). It should be explained if this might be due to the use of a mammalian alpha subunit. Related to this, did the authors clone the alpha subunit from Exaiptacia? Using this to characterize the effect of beta subunits on inactivation might be more accurate.

4) The in situ shown in Figure 4b are difficult to follow for a non-expert in cnidarian anatomy. Some guidance should be provided to understand the structures. Also, for the left panels, is the larger panel the two-channel image? If so, blue would indicate co-localization of the two isoforms and there seems to be a red mark in the same nematocyte.

Reviewer #1 (Public Review):

In this manuscript, the authors explore the effects of DNA methylation on the strength of regulatory activity using massively parallel reporter assays in cell lines on a genome-wide level. This is a follow-up of their first paper from 2018 that describes this method for the first time. In addition to adding more in-depth information on sequences that are explored by many researchers using two main methods, reduced bisulfite sequencing and sites represented on the Illumina EPIC array, they now show also that DNA methylation can influence changes in regulatory activity following a specific stimulation, even in absence of baseline effects of DNA methylation on activity. In this manuscript, the authors explore the effects of DNA methylation on the response to Interferon alpha (INFA) and a glucocorticoid receptor agonist (dexamethasone). The authors validate their baseline findings using additional datasets, including RNAseq data, and show convergences across two cell lines. The authors then map the methylation x environmental challenge (IFNA and dex) sequences identified in vitro to explore whether their methylation status is also predictive of regulatory activity in vivo. This is very convincingly shown for INFA response sequences, where baseline methylation is predictive of the transcriptional response to flu infection in human macrophages, an infection that triggers the INF pathways. The extension of the functional validity of the dex-response altering sequences is less convincing. Sequences altering the response to glucocorticoids, however, were not enriched in DNA methylation sites associated with exposure to early adversity. The authors interpret that "they are not links on the causal pathway between early life disadvantage and later life health outcomes, but rather passive biomarkers". However, this approach does not seem an optimal model to explore this relationship in vivo. This is because exposure to early adversity and its consequences is not directly correlated with glucocorticoid release and changes in DNA methylation levels following early adversity could be related to many physiological mechanisms, and overall, large datasets and meta-analyses do not show robust associations of exposure to early adversity and DNA methylation changes. Here, other datasets, such as from Cushing patients may be of more interest.

Overall, the authors provide a great resource of DNA methylation-sensitive enhancers that can now be used for functional interpretation of large-scale datasets (that are widely generated in the research community), given the focus on sites included in RBSS and the Illumina EPIC array. In addition, their data lends support that differences in DNA methylation can alter responses to environmental stimuli and thus of the possibility that environmental exposures that alter DNS methylation can also alter the subsequent response to this exposure, in line with the theory of epigenetic embedding of prior stimuli/experiences. The conclusions related to the early adversity data should be reconsidered in light of the comments above.

Reviewer #1 (Public Review):

The studies by Hwangbo et al. diligently attempt to account for many of the typically neglected dietary and non-dietary factors.

Strengths:

• Work addresses many potential artifacts of dietary (e.g., dehydration stress, macronutrient ratios, and protein source) and non-dietary (e.g., leaky expression of S106-GAL4) manipulations-important factors that are too often overlooked.<br /> • Balanced and complementary behavioral, molecular, and bioinformatic experiments<br /> • Show necessity of proteostatic subunits in the fat body for DR-mediated longevity. The findings in the current manuscript lay the ground for future studies that test sufficiency of fat body prosβ3 and rpn7, or necessity of other proteostatic genes in other tissues.

Weaknesses:

• Could the lack of DR response in clock mutants across dietary concentrations be simply because the clock mutants are better at compensatory feeding adjustments to dietary dilutions? If this were the case, there are two major implications to the authors' conclusions:<br /> a) The Clk mutants are differently responding to dietary dilutions, not to dietary restriction, per se.<br /> b) Nutritional intake was unaffected by the dietary manipulations. If the changes in fat body proteostasis and lifespan were due to nourishment, it would be expected that the physiology and lifespan do not change.<br /> Accurate measurements of food consumption and the resulting protein intake could potentially clarify this critical question.

Reviewer #1 (Public Review):

Ciampa et al. investigated the role of the hypoxia-inducible factor 1 (HIF-1) pathway in placental aging. They performed transcriptomic analysis of prior data of placental gene expression over serial timepoints throughout gestation in a mouse model and identified increased expression of senescence and HIF-1 pathways and decreased expression of cell cycle and mitochondrial transcripts with advancing gestational age. These findings were confirmed by RT-PCR, Western blot, and mitochondrial assessment from mouse placental tissues from late gestation time points. Studies of human placental samples at similar late gestational ages showed similar trends in increased HIF-1 targets and decreased mitochondrial abundance with increasing gestation, but were not significantly significant due to the limited availability of uncomplicated preterm placenta samples. The authors demonstrated that stabilization of HIF-1 in vitro using primary trophoblasts and choriocarcinoma cell lines recapitulated the gene and mitochondrial dysfunction seen in the placental tissues and were consistent with senescence. Interestingly, cell-conditioned media from HIF-1 stabilized placenta cell lines induced myometrial cell contractions in vitro and correspondingly, induction of HIF-1 in pregnant mice was associated with preterm labor in vivo. These data support the role of the HIF-1 pathway in the process of placental senescence with increasing gestational age and highlight this pathway as a potentially important contributor to gestational length and a potential target for therapeutics to reduce preterm birth.

Overall, the conclusions of this study are mostly well supported by the data. The concept of placental aging has been controversial, with several prior studies with conflicting viewpoints on whether placental aging occurs at all, is a normal process during gestation, or rather only a pathologic phenomenon in abnormal pregnancies. This has been rather difficult to study given the difficulty of obtaining serial placental samples in late gestation. The authors used both a mouse model of serial placental sampling and human placental samples obtained at preterm, but non-pathologic deliveries, which is an impressive accomplishment as it provides insight into a previously poorly understood timepoint of pregnancy. The data clearly demonstrate changes in the HIF-1 pathway and cellular senescence at increasing gestational ages in the third trimester, which is consistent with the process of aging in other tissues.

Weaknesses of this study are that although the authors attribute alterations in HIF-1 pathways in advanced gestation to hypoxia, there are no experiments directly assessing whether the changes in HIF-1 pathways are due to hypoxia in either in vitro or in vivo experiments. HIF-1 has both oxygen-dependent and oxygen-independent regulation, so it is unclear which pathways contribute to placental HIF-1 activity during late gestation, especially since the third-trimester placenta is exposed to significantly higher oxygen levels compared to the early pregnancy environment. Additionally, the placenta is in close proximity to the maternal decidua, which consists of immune and stromal cells, which are also significantly affected by HIF-1. Although the in vitro experimental data in this study demonstrate that HIF-1 induction leads to a placenta senescence phenotype, it is unclear whether the in vivo treatment with HIF-1 induction acts directly on the placenta or rather on uterine myometrium or decidua, which could also contribute to the initiation of preterm labor.

For any action, habit, and belief you have, ask yourself: "Does this help toward my goals and future self or not?", if the answer is no, it is a distraction and part of the 80% you need to let go in order to reach 10X

Your future self and 10X (or 100X) vision and goals serve as a massive filter for action and belief.

Note: You should not 10X everything! Just 3 priorities.

Counterintuitively, the 10X mindset and goal setting is not about goals. It is about identifying the essential PROCESSES that lead to significant progress.

What is the game you want to play? What is the game you could play? What is a game you could go all in on and succeed at and be really good at?

This defines your pathways and strategies within your 20%

The path can change and adjust over time.

Hope = 1. Clear and Committed Goal 2. Agency Thinking 3. Pathways Thinking

This turns into definite optimism.

To achieve goals, raise the floor, FOCUS on removing bottlenecks. Also create constraints by Schwerpunkt (primary objective), contrary to common wisdom, constraint actually gives freedom, it prevents analysis paralysis.

Reviewer #1 (Public Review):

This manuscript employs a string method with swarms of trajectories to extract a free energy map of KcsA channel inactivation and its model dependence. The approach connects X-ray structures for closed, partially and fully open, and inactivated KcsA through optimisation of a string defined in a collective variable space consisting of distances involving gate size, cavity-filter and filter pinching (as defined in the proposed X-ray structure for an inactivated state). The final trajectory includes pore opening and filter collapse with water penetration behind the filter, via different intermediates depending on the force field. The authors propose a role for residue L81 in controlling water entry in the final stage of this process. The results suggest that KcsA more easily inactivates with the Charmm force field, with lower barrier and direct passage from a partially open state, whereas the pathway for Amber involves transition first to a fully open state with higher barrier, despite not being the dominant open state seen experimentally under activating conditions. The results also suggest that PG lipids help activate the channel within the Amber force field, consistent with experimental evidence. The work represents large-scale advanced MD simulation. Some questions remain, however, such as if the CV space chosen is sufficient to capture all possible slow coordinates in the inactivation process, and how the resultant free energy surfaces may potentially depend on the end structures and initial pulling procedure.

Collective variable choice:

The explanation for the choice of CVs on page 5 is not sufficient to understand the process and its likely success. How were the most important and unimportant CVs identified exactly? Table 2 on page 19 shows only gate distances, cavity-filter distances and a single variable related to filter structure itself (77 CA - 77 CA) representing a pinch. Is that pinching really the only slow variable associated with inactivation changes in the filter? Why are there no variables, say for carbonyl flipping, E71 or D80 movements or even for ion and water occupancy (although water may be sampled with control of other interactions, such as involving L81)? I understand that the X-ray structure is the one source of information used to define an inactivated structure and is one with just a pinch and no complete carbonyl flipping away from the pore, as has been identified in past studies and discussed as being involved by the authors on page 14. Key changes like carbonyl flipping surely are part of the story and may be slow variables. At the very least, if not part of the CV space, could be analysed.

On page 10 the authors discuss possible differences in Amber and Charmm involving the extent to which the 4 subunits change in respect to the L81-W67 water pathway and W67-D80 hydrogen bond, arguing the different results for force field could be to do with different numbers of subunits doing different things. If I understand, the chosen CVs are all tetramer-based distances (including across subunits) and not subunit-based CVs, so that random and incomplete changes may occur to subunits for a given point in CV space. There is thus potential for the string to converge on a local minimum pathway with partial changes to its interactions within and between subunits, and may not be a unique global solution. Can the authors please explain whether or not this is possible and what analysis has been done to check it?

X-ray endpoints and initial pathway:

The string was created from a pulling/steered MD between existing X-ray structures for the closed (5VKH), partially open (3FB5), fully open (5VK6) and finally inactivated (5VKE) states. The authors write on page 12 that "The block of conduction during inactivation appears to result from pinching at the selectivity filter...", but given the end point was forced to be the X-ray structure with pinching, wasn't this outcome predetermined? This raises a significant point of how much has choice of endpoints predetermined the final states of the string? i.e. How much is an end state actually allowed to draft away from the initial Xray structure. Was a bead placed at the very endpoint and allowed to update via swarms, or was it fixed and all beads just interpolate between those fixed end states? The reason this is important is that it is plausible the inactivated crystal structure with pinching but not other changes (such as complete V76 carbonyl flipping or outer filter splaying), may not be the actual free energy minimum structure for that state and that force field.

Another obvious concern is the possible reliance on the initial pulling procedure used before string optimisation began. Fig.2 Supp 1 shows generally that the Amber path stayed pretty close to the initial steered MD path, whereas Charmm drifted downward away from that path. One could justifiably ask, if a very different initial path was chosen, might different local minimum pathways result, including Amber sampling a path like Charmm? How does one test whether or not the final path has not been trapped in some local trough of free energy? e.g. Imagine starting the Amber string using an initial path like the more diagonal Charmm-like path, or even a more extreme unphysiological one, such as a steered trajectory that initially inactivates before opening the gate. Would the final results be the same? I appreciate the simulations are very expensive and such trials may not be possible, but what evidence is there that the final path has not been trapped away from the global minimum?

One test offered by the authors is a set of unbiased MD simulations launched from points on the string. The authors ran 200ns simulations and write on page 5 that "These simulations have the expected stability based on their starting values. This is a good quality test to check the correct estimation of the general features of the free energy surface". While this sounds reasonable, 200ns MD may only be sufficient to begin to explore locally within the solved free energy trough, much like the swarms in the iterations were able to do. My own examination of Fig2 Supp 5 is that some of these simulations linger around the expected states and some drift away within the general trough of sampling, which is a good sign. What those 200ns simulations may not be able to do is escape that trough and see evidence of other possible solutions, beyond what was sampled with the string that was tied to Xray endpoints and trapped in the solution pathway that was already formed after 100-300 iterations. Overall, the string involved 800 iterations of 10ps swarms (80ns around each bead; albeit 32 trajectories in parallel), allowing good local sampling around the beads in the free energy trough, but in terms of ability to diffuse away from that point, only being comparable in contiguous trajectory time to the unbiased MD tests. It therefore would have been interesting to see if longer simulations remain in this trough; though I understand the challenges in running so much MD. Such simulations may, however, lead to exploration beyond what was seen in the string solutions.

Force field effects and origin:

Regarding the effect of the chosen force field, the authors state that "Given that our simulations were conducted under activating conditions, we had expected the open states to be more populated than the closed ones. Simulations carried out at higher pH may be able to resolve this inconsistency". Also running at high pH would be a nice thing to do to prove the method is in fact sensitive to conditions to see a shift in the distribution of states. But the question is why were open states not more occupied under low pH and 50mM K+? From my analysis of the figures, the results show that the Charmm force field tends to allow for opening of the channel somewhat (at least with similar free energy for partially and fully open to closed) whereas Amber tends to close the channel more (with more uphill energy as the channel opens than Charmm; Fig 2). i.e. at low pH and 50 K+, isn't the Amber model incorrectly reporting fairly strong bias against opening? Moreover, regarding the free energy of the inactivated state itself, why should we not expect equilibrated channels under activating conditions to eventually fall into an inactivated state, in which case we should expect low free energy of that state (as found with Charmm and not Amber in Fig2), but with a slow rate. While much discussion in the manuscript appears to discuss limitations in Charmm (although on page 12 discussion leans either way), these factors may seem to favour Charmm over Amber.

On page 12 the authors explain the possible causes for force field dependence, although this seems limited to ion interactions, glutamate charges and dihedrals. But it would be nice to get a bit more insight into what terms may have influenced the pathway, in particular involving interactions between TM2 and the base of the selectivity filter and hydration behind the filter. Regarding ion interactions, is there a good reason to believe ions are key to the difference seen? i.e. How were ions involved differently in the state transitions involving Amber and Charmm? The authors have noted a role for ion-carbonyl interactions. It is important that the authors explain which is the two competing models has been used and why. i.e. Off-the-shelf Charmm36 force field includes strong K+-backbone carbonyl interaction, previously seen to promote high ion occupancy, similar to Amber, whereas Lennard-Jones parameters modified to match N-methyl-acetamide and water partitioning (such as early Berneche, Noskov and Roux work) reduce ion occupancy and increase water content inside the filter.

Public Review:

The primary goal of this paper is to examine microtubule detyrosination as a potential therapeutic target for axon regeneration. Using dimethylamino-parthenolide (DMAPT), this study extensively examines mechanistic links between microtubule detyrosination, hyper-interleukin-6 (hIL-6), and PTEN in neurite outgrowth in retinal ganglion cells in vitro. These findings provide convincing evidence that parthenolide has a synergistic effect on hIL-6- and PTEN-related mechanisms of neurite outgrowth in vitro. The potential efficacy of systemic DMAPT treatment to promote axon<br /> regeneration in mouse models of optic nerve crush and spinal cord injury was also examined.

Strengths:

1) The examination of synergistic activities between parthenolide, hyperIL-6, and PTEN knockout is leveraged not only for potential therapeutic value, but also to validate and delineate mechanism of action.

2) The in vitro studies utilize a multi-level approach that combines cell biology and biochemistry approaches to dissect the mechanistic link from parthenolide to microtubule dynamics.

3) The studies provide a basis for others to test the role of DMAPT in other settings, particularly in the context of other effective pro-regenerative approaches.

Weaknesses:

1) In vivo studies are limited to select outcomes of recovery and do not validate or address mechanism of action in vivo.

2) Known activities of DMAPT beyond microtubule detyrosination, such as oxidative stress, mitochondrial function and NFkB inhibition, are not considered in experimental examinations or in the interpretation of findings.

Reviewer #1 (Public Review):

Using the colon transcriptomes of 52 BXD mouse strains fed either chow or a high-fat diet (HFD), Li et al. present their findings on gene-by-environment interactions underpinning inflammation and inflammatory bowel disease (IBD). They discovered modules that are enriched for IBD-dysregulated genes using co-expression gene networks. They determined Muc4 and Epha6 to be the leading candidates causing variations in HFD-driven intestinal inflammation by using systems genetics in the mouse and integration with external human datasets. In their analysis, they concluded that their strategy "enabled the prioritization of modulators of IBD susceptibility that were generalizable to the human situation and may have clinical value." This dataset is intriguing and generates hypotheses that will be investigated in the future. However, there were no mechanistic or causation-focused investigations; the results were primarily observational and correlative.

Reviewer #1 (Public Review):

Jamge et al. sought to identify the relationships between histone variants and histone modifications in Arabidopsis by systematic genomic profiling of 13 histone variants and 12 histone modifications to define a set of "chromatin states". They find that H2A variants are key factors defining the major chromatin types (euchromatin, facultative heterochromatin, and constitutive heterochromatin) and that loss of the DDM1 chromatin remodeler leads to loss of typical constitutive heterochromatin and replacement of this state with features common to genes in euchromatin and facultative heterochromatin. This study deepens our understanding of how histone variants shape the Arabidopsis epigenome and provides a wealth of data for other researchers to explore.

Strengths:

1. The manuscript provides convincing evidence supporting the claims that: A) Arabidopsis nucleosomes are homotypic for H2A variants and heterotypic for H3 variants, B) that H3 variants are not associated with specific H2A variants, and C) H2A variants are strongly associated with specific histone post-translational modifications (PTMs) while H3 variants show no such strong associations with specific PTMs. These are important findings that contrast with previous observations in animal systems and suggest differences in plant and animal chromatin dynamics.

2. The authors also performed comprehensive epigenomic profiling of all H2A, H2B, and H3 variants and 12 histone PTMs to produce a Hidden Markov Model-based chromatin state map. These studies revealed that histone H2A variants are as important as histone PTMs in defining the various chromatin states, which is unexpected and of high significance.

3. The authors show that in ddm1 mutants, normally heterochromatic transposable element (TE) genes lose H2A.W and gain H2A.Z, along with the facultative heterochromatin and euchromatin signatures associated with H2A.Z at silent and expressed genes, respectively.

Reviewer #1 (Public Review):

In this work, Urbanska and colleagues use a machine-learning based crossing of mechanical characterisations of various cells in different states and their transcriptional profiles. Using this approach, they identify a core set of five genes that systematically vary together with the mechanical state of the cells, although not always in the same direction depending on the conditions. They show that the combined transcriptional changes in this gene set is strongly predictive of a change in the cell mechanical properties, in systems that were not used to identify the genes (a validation set). Finally, they experimentally after the expression level of one of these genes, CAV1, that codes for the caveolin 1 protein, and show that, in a variety of cellular systems and contexts, perturbations in the expression level of CAV1 also induce changes in cell mechanics, cells with lower CAV1 expression being generally softer.

Overall the approach seems accessible, sound and is well described. My personal expertise is not suited to judge its validity, novelty or relevance, so I do not make comments on that. The results it provides seem to have been thoroughly tested by the authors (using different types of mechanical characterisations of the cells) and to be robust in their predictive value. The authors also show convincingly that one of the genes they identified, CAV1, is not only correlated with the mechanical properties of cells, but also that changing its expression level affects cell mechanics. At this stage, the study appears mostly focused on the description and validation of the methodological approach, and it is hard to really understand what the results obtain really mean, the importance of the biological finding - what is this set of 5 genes doing in the context of cell mechanics? Is it really central, or is it just one of the set of knobs on which the cell plays - and it is identified by this method because it is systematically modulated but maybe, for any given context, it is not the dominant player - all these fundamental questions remain unanswered at this stage. On one hand, it means that the study might have identified an important novel module of genes in cell mechanics, but on the other hand, it also reveals that it is not yet easy to interpret the results provided by this type of novel approach.

Reviewer #1 (Public Review):

The authors have approached the study of the mechanism by which the two more antigenic proteins of the influenza A virus, hemagglutinin (HA) and neuraminidase (NA), are expressed later during the infection. For this aim, they set an experimental approach consisting of a 2-hour-long infection at a multiplicity of infection of 2 with the viral strain WSN. They used cells from the lung carcinoma cell line A549. They used the FISH technique to detect the mRNAs in situ and developed an imaging-based assay for mathematically modeling and estimating the nuclear export rate of each of the eight viral segments. They propose that the delay in the expression of HA and NA is based on the retention of their mRNA within the nucleus.

The main strength of this work is the fact that the authors have studied a long-unaddressed mechanism in influenza A virus infectious cycle, as is the late expression of HA and NA, by creating a work flow including mRNA detection (FISH) plus mathematical calculations to arrive at a model, which additionally could be useful for general biological processes where transcription occurs in a burst-like manner. The weakness of this work in its present state is that in order to "quantify" the export rate of the transcripts, several assumptions regarding the viral infection are made without empirical data. It would greatly improve if more precise experiments could be performed and/or include demonstration of the assumptions made (i.e., synchronized infections, empirically demonstrating that cRNA production does not occur within the first 2 hours of infection, and/or separate transcription and replication, inhibiting RNA degradation during viral infection).

Reviewer #1 (Public Review):

This study revealed that one of the mechanisms for iTreg (induced-Treg) lineage instability upon restimulation is through sustained store-operated calcium entry (SOCE), which activates transcription factor NFAT and promotes changes in chromatin accessibility to activated T cell-related genes. The authors revealed that, unlike thymus-derived Tregs (tTreg) with blunted calcium signaling and NFAT activation, iTregs respond to TCR restimulation with fully activated SOCE and NFAT similar to activated conventional T cells. Activated NFAT binds to open chromatin regions in genes related to T helper cells, increases their expression, and leads to the instability of iTreg cells. On the other hand, inhibition of the SOCE/NFAT pathway by chemical inhibitors could partially rescue the loss of Foxp3 expression in iTreg upon restimulation. The conclusion of the study is unexpected since previous studies showed that NFAT is required for Foxp3 induction and iTreg differentiation (Tone Y et al, Nat Immunol. 2008, PMID: 18157133; Vaeth M et al, PNAS, 2012, PMID: 22991461). Additionally, Foxp3 interacts with NFAT to control Treg function (Wu Y et al, Cell, 2006, PMID: 16873067). The data presented in this study demonstrated the complex role NFAT plays in the generation and stability of iTreg cells.

Several concerns are raised from the current study.<br /> 1. Previous studies showed that iTregs generated in vitro from culturing naïve T cells with TGF-b are intrinsically unstable and prone to losing Foxp3 expression due to lack of DNA demethylation in the enhancer region of the Foxp3 locus (Polansky JK et al, Eur J Immunol., 2008, PMID: 18493985). It is known that removing TGF-b from the culture media leads to rapid loss of Foxp3 expression. In the current study, TGF-b was not added to the media during iTreg restimulation, therefore, the primary cause for iTreg instability should be the lack of the positive signal provided by TGF-b. NFAT signal is secondary at best in this culturing condition.

2. It is not clear whether the NFAT pathway is unique in accelerating the loss of Foxp3 expression upon iTreg restimulation. It is also possible that enhancing T cell activation in general could promote iTreg instability. The authors could explore blocking T cell activation by inhibiting other critical pathways, such as NF-kb and c-Jun/c-Fos, to see if a similar effect could be achieved compared to CsA treatment.

3. The authors linked chromatin accessibility and increased expression of T helper cell genes to the loss of Foxp3 expression and iTreg instability. However, it is not clear how the former can lead to the latter. It is also not clear whether NFAT binds directly to the Foxp3 locus in the restimulated iTregs and inhibits Foxp3 expression.

Reviewer #1 (Public Review):

Terzioglu and co-workers tested the provocative hypothesis that mitochondria maintain an internal temperature considerably higher than cytosolic/external environmental temperature due to the inherent thermodynamic inefficiency of mitochondrial oxidative phosphorylation. As a follow-up to a prior paper from some of the same authors, the goal of this study was to conduct additional experiments to assess mitochondrial temperature in cultured cells. Consistent with the prior work, the authors provide consistent evidence that the temperature of mitochondria in four different types of cultured mammalian cells, as well as cells from Drosophila (poikilotherms), is 15oC or more above the external temperature at which cells are maintained (e.g., 37oC). Additional evidence shows that mitochondria maintain higher temperatures under several different types of cellular metabolic stresses predicted to decrease the dependence on OxPhos, adding to the notion that natural thermodynamic inefficiency and heat generation may be an important, and potentially regulated, characteristic of mitochondrial metabolism.

Strengths<br /> Demonstration that both a fluorescent (Mito Thermo Yellow) and a genetic-based (mito-gTEMP) mitochondrial targeted temperature probe elicit similar quantitative changes in mitochondrial temperature under different experimental conditions is a strength. The addition of the genetic probe to the current study supports prior findings using the fluorescent probe and thus achieves a primary objective of the study.

The experiments are well-designed and executed. Specific attention given to potential artifacts affecting probe signal and/or non-specific effects from the different experimental interventions is a strength.

The use of different cultured cell lines from different organisms provides additional evidence of elevated temperature as a general property of functioning mitochondria, representing additional validation.

Weakness:<br /> While the findings and potential interpretations put forward by the authors are intriguing, the severity of the interventions (e.g., mitochondrial complex-specific inhibitors, inhibition of protein synthesis) and the absence of simultaneous or parallel measurements of other key bioenergetic parameters (i.e., membrane potential, oxygen consumption rate, etc.) limits the ability to interpret potential cause and effect - whether the thermogenesis aspect of OxPhos is being sensed and regulated, or whether temperature changes are more of a biproduct of adjustments in OxPhos flux under the experimental circumstances. In other words, the physiological relevance of the findings remains unclear.

Related, several of the interventions are employed to either increase or decrease dependence on OxPhos flux, but no outcome measures are reported to document whether the intended objective was achieved (e.g., increased OxPhos flux in low glucose plus galactose, decreased ATP demand-OxPhos flux with anisomycin, etc.).

Millionairres are just regular people lol. They don't (always) have luxury and other high paid stuff.

Becoming a millionaire takes skillset and effort, not saving.

Becoming wealthy means aiming high. You will not go higher than the goals you set. Aim for a crazy amount, and do the work it takes. It filters behavior down to the essentials.

Success is about delaying gratification and building momentum. Be okay with a big goal taking time. Just work towards it, focus on the process, rather than the goal.

Persistence and perseverance as well.

Perfection is a big limiter. Don't obsess with finding the perfect path before starting. Build the map while going. Of course, starting with a rough idea or sketch for a map is always good.

Advice doesn't matter as much as guidance, this way, not everyone needs to know about your goals. The fewer, the better. Those who do know, let them mentor you (also seek the correct mentors).

To become unstoppable, you must:

1) Outwork everybody

2) Outsmart everybody

3) Hire the greatest talent in the world

The four primary questions to ask yourself for a 100x 10-year vision:

1. What is the commitment I desire to have?
2. What are my hindrances (goal-conflicting actions or inactions)? -- Past-Based Actions/Behaviors
3. What are my hidden commitments of my former self? (things that might've helped my past self but are not as helpful right now.) -- Past-Based Commitments/Identities
4. What are my limiting beliefs or assumptions toward achieving this goal? -- Past-based Beliefs

Step 1 is to fill out these questions. Step 2 is to go backwards, and start identifying what is necessary, so what are the necessary beliefs to achieve this goal, what commitments must I make and thus what actions must I take?

Reviewer #1 (Public Review):

This is an interesting study deploying convergent methodologies to address a timely question: can non-human primates distinguish theory of mind from random behaviours during passive viewing of animated shapes, and what brain regions are implicated? As the authors note, fMRI studies of brain activation in response to the theory of mind stimuli in non-human primates are scarce, and none have explored the processing of abstract stimuli in this context.

The major strengths of the study are the application of the Frith-Happé shapes task in a group of marmosets during fMRI in conjunction with concurrent eye tracking recording. Eye tracking is a very nice addition as it enables the authors to determine the gaze patterns and fixation duration on distinct aspects of the task stimuli (e.g., large triangle versus small triangle) as well as group differences. Overall, the study seems well-designed and technically rigorous, and the major conclusions appear to be supported by the data.

However, there is one aspect I would appreciate some clarity on, namely the failure to include the original "Goal directed condition" from the Frith-Happé task. The authors contrast visuo-oculomotor and fMRI activation between the Random (no discernible interaction or purposeful behaviour) and the ToM (goal-directed behaviours with mental interaction) but neglect the intermediate step of physical interaction between the shapes that the Goal-directed behaviour condition portrays. As such, it is difficult to make clear statements as to what the activation patterns in the ToM condition represent - perhaps this merely reflects the processing of an unfolding narrative rather than random movements.

Reviewer #1 (Public Review):

The study of Aso and colleagues seeks to understand how learned information is steering motor output. Using an artificial training paradigm consisting of odor presentation combined with dopamine neuron activation, they identify upwind orientation as an important parameter of appetitive memory recall (as has been shown before - e.g. Handler 2019). Using the Drosophila genetic targeting library and optogenetic activation, they identify several populations of neurons responsible for upwind orientation by analyzing freely moving animals in an airflow chamber. They concentrate on a specific subset, which they call upwind neurons (UPWINs), and which they can anatomically link downstream to the flies' memory center, the mushroom body (MB), building on the ultrastructural connectome brain atlas. In combination with electrophysiology, in-vivo calcium imaging, and memory assays, they successfully show that (1) UPWINs promote upwind orientation including acceleration of angular speed and bias turning towards the upwind direction, (2) UPWINs receive excitatory and inhibitory input from specific parts of the MB, (3) UPWINs increase odor-evoked activity upon (artificial) appetitive training and (4) appetitive memory recall is impaired when blocking UPWIN neurons only during the memory test.

The authors use state-of-the-art techniques combining tools like optogenetics, connectome analysis as well as electrophysiology, in-vivo calcium imaging, and memory/behavioral assays tracking individual flies. It provides new insights into mushroom body memory retrieval circuits and how they integrate with information from other brain areas. However, some concerns remain regarding some claims of the paper. The timeline of the behavioral and the physiological experiments differ. It is therefore difficult to define the memory phases when upwind orientation is important for recall. Moreover, one main conclusion the authors draw from their data is that upwind orientation is promoted by disinhibition from a specific MB output connection, however, physiological evidence of this effect is missing. The UPWINs seem to have a more complex function in behavioral control beyond memory recall. The fact that optogenetic UPWIN activation is leading to upwind orientation only in starved flies together with the fact that flies show a high returning probability even without any odor present suggests a functional role in state-dependent exploration behavior.

Reviewer #1 (Public Review):

The authors present a PyTorch-based simulator for prosthetic vision. The model takes in the anatomical location of a visual cortical prostheses as well as a series of electrical stimuli to be applied to each electrode, and outputs the resulting phosphenes. To demonstrate the usefulness of the simulator, the paper reproduces psychometric curves from the literature and uses the simulator in the loop to learn optimized stimuli.

One of the major strengths of the paper is its modeling work - the authors make good use of existing knowledge about retinotopic maps and psychometric curves that describe phosphene appearance in response to single-electrode stimulation. Using PyTorch as a backbone is another strength, as it allows for GPU integration and seamless integration with common deep learning models. This work is likely to be impactful for the field of sight restoration.

However, one of the major weaknesses of the paper is its model validation - while some results seem to be presented for data the model was fit on (as opposed to held-out test data), other results lack quantitative metrics and a comparison to a baseline ("null hypothesis") model.<br /> - On the one hand, it appears that the data presented in Figs. 3-5 was used to fit some of the open parameters of the model, as mentioned in Subsection G of the Methods. Hence it is misleading to present these as model "predictions", which are typically presented for held-out test data to demonstrate a model's ability to generalize. Instead, this is more of a descriptive model than a predictive one, and its ability to generalize to new patients remains yet to be demonstrated.<br /> - On the other hand, the results presented in Fig. 8 as part of the end-to-end learning process are not accompanied by any sorts of quantitative metrics or comparison to a baseline model. The results seem to assume that all phosphenes are small Gaussian blobs, and that these phosphenes combine linearly when multiple electrodes are stimulated. Both assumptions are frequently challenged by the field. For all these reasons, it is challenging to assess the potential and practical utility of this approach as well as get a sense of its limitations.

Another weakness of the paper is the term "biologically plausible", which appears throughout the manuscript but is not clearly defined. In its current form, it is not clear what makes this simulator "biologically plausible" - it certainly contains a retinotopic map and is fit on psychophysical data, but it does not seem to contain any other "biological" detail. In fact, for the most part the paper seems to ignore the fact that implanting a prosthesis in one cerebral hemisphere will produce phosphenes that are restricted to one half of the visual field. Yet Figures 6 and 8 present phosphenes that seemingly appear in both hemifields. I do not find this very "biologically plausible".

Review #1 Public Review:

This is an interesting study which attempts to assess the effect of the pandemic on diagnoses of pancreatic cancer. The authors have used a large national database to evaluate this, however, it should be noted that this database only captures 40% of the population in England. The authors have looked at specific parameters including Body Mass Index (BMI) as well as markers of diabetes and liver function. Only BMI had a difference in the frequency of measurements during the pandemic, presumably due to reduced face-to-face visits to allow weight and height to be captured.

Interestingly the authors noticed a reduction in surgery for pancreatic cancer by 25%, yet reported that there were no differences in the frequency of death within 6 months following the diagnosis of pancreatic cancer. The reduction in surgery is likely related at least in part to the loss of operating lists due to pandemic restrictions, however, this paper is not equipped to address another important possibility behind this, which is that pancreatic cancers were presenting too late for surgical intervention. It is not sufficient to comment that pancreatic cancer treatment was not affected by the pandemic based on the data presented on deaths within 6 months of the diagnosis of pancreatic cancer alone, as the median survival of patients diagnosed with pancreatic cancer within the pandemic has not been captured and compared to that of patients diagnosed in the preceding 5 years.

Therefore while the study can conclude no difference in pancreatic cancer diagnoses before and during the pandemic, more work needs to be done to truly assess if the pandemic had any effect on the outcomes from pancreatic cancer for patients diagnosed within this timeframe.

Reviewer #1 (Public Review):

This research aimed to discern the pattern of methylation changes that occur during aging, distinguishing between a unified specific mechanism and stochastic changes. To date, no unified hypothesis exists to guide our understanding of the changes in chromatin geography observed during the aging of cells. This work analysed six different types of purified blood-borne white blood cells allowing comparison across different immune cell subsets to determine if similar patterns occurred in all cell populations. Intriguingly, each subset exhibited its own distinct differential methylation rather than a single program. However, a core set of gene changes close to age-associated CpGs was identified suggesting that a central program existed, but that individual cell type function and metabolism shaped the overall chromatin landscape for the population. These findings establish a new framework for considering the aging process and open new questions about how the individual clocks of different populations might be regulated. While circulating cells are readily accessible for evaluation in humans, the majority of immune cells that regulate immune homeostasis are found within the tissues of the body. Whether these cells exhibit a similar profile to circulating cells or are rather shaped by their tissue or organ-specific ecosystem remains to be determined. In this setting, these tissue-resident cells are exposed to very different oxygen tensions and metabolic substrates. Furthermore, genes identified have been associated with aging, they concurrently appear to be associated with inflammation, thus it is not clear whether aging and low-grade inflammation are inherently linked, or whether these two pathways can be segregated. Thus a number of questions remain warranting further investigation.

Reviewer #1 (Public Review):

The authors convincingly show in this study the effects of the fas5 gene on changes in the CHC profile and the importance of these changes toward sexual attractiveness.

The main strength of this study lies in its holistic approach (from genes to behaviour) showing a full and convincing picture of the stated conclusions. The authors succeeded in putting a very interdisciplinary set of experiments together to support the main claims of this manuscript.

The main weakness stems from the lack of transparency behind the statistical analyses conducted in the study. Detailed statistical results are never mentioned in the text, nor is it always clear what was compared to what. I also believe that some tests that were conducted are not adequate for the given data. I am therefore unable to properly assess the significance of the results from the presented information. Nevertheless, the graphical representations are convincing enough for me to believe that a revision of the statistics would not significantly affect the main conclusions of this manuscript.

The second major problem I had with the study was how it brushes over the somewhat contradicting results they found in males (Fig S2). These are only mentioned twice in the main text and in both cases as being "similarly affected", even though their own stats seem to indicate otherwise for many of the analysed compound groups. This also should affect the main conclusion concerning the effects of fas5 genes in the discussion, a more careful wording when interpreting the results is therefore necessary.

Reviewer #1 (Public Review):

"Melanocortin 1 receptor regulates cholesterol and bile acid metabolism in the liver" by Thapa et al. extends previous findings that MC1R global knockout mice have dysregulated lipid metabolism in APOE KO mice. The authors generated a hepatocyte-specific MC1R KO mouse to assess the hepatic effects of MC1R on the regulation of lipid metabolism. Thapa et al. go on to show that hepatic MC1R deletion leads to dyslipidemia and hepatic steatosis. The authors subsequently show that altered cholesterol homeostasis disrupts bile acid metabolism in hepatic MC1R KO mice. Finally, the authors provide data to suggest a role for AMPK in mediating the effects of MSH on hepatic cholesterol metabolism. The authors designed rigorous experiments using multiple different models (in vivo and in vitro) as well as different approaches (genetic and pharmacological).

The work described herein would have an impact on the field in multiple ways. Firstly, it demonstrates a novel metabolic role for MSH in the regulation of hepatic cholesterol metabolism. This may prove to be a viable therapeutic strategy for the treatment of dyslipidemia. Furthermore, the authors demonstrate an alternative signaling cascade elicited by MSH independent of cAMP, but rather relying on AMPK. This novel interaction between AMPK and MC1R could have more widespread implications beyond the control of hepatic cholesterol metabolism.

For the most part, the conclusions offered by the authors are supported by the data that is presented. There are, however, a number of concerns in the current version of this manuscript detailed below:

1) The authors demonstrate the expression of MC1R in hepatocytes through IHC staining and western blot analysis. Furthermore, the authors show an alteration in systemic bile acid homeostasis in MC1R KO mice. However, no mention of MC1R expression or function in cholangiocytes is discussed. This is important to assess both experimentally and within the discussion given the profound role of the biliary epithelium in modulating bile acid homeostasis. Furthermore, in figure 1 the authors validate the MC1R knockdown only through mRNA expression. Given panels A and C of figure 1 shows there is clearly a functional antibody for MC1R, validation of protein knockdown is needed.

2) Figure 2 demonstrates a steatotic effect of MC1R knockdown in hepatocytes. The authors attempt to provide mechanistic insight into this phenomenon through assessing the mRNA expression of genes involved in cholesterol and fatty acid synthesis. The data provided is modest at the gene level and no protein validation was provided to demonstrate functional alterations of these proteins in MC1R KO mice. Key proteins proposed such as SREBP2 and HMGCR need to be validated via a western blot of IHC analysis.

4) The authors suggest the involvement of AMPK in mediating the cholesterol-lowering effects of MSH. However, MSH is still able to lower free cholesterol levels even in the presence of an AMPK inhibitor. This suggests that MSH does not in fact rely on the activation of AMPK to elicit these cholesterol-lowering effects. The authors' conclusions are stronger than the actual data support. Furthermore, the authors claim LD211 phenocopies the effects of MSH in the presence of an AMPK inhibitor. However, the authors only measured the phosphorylation of Akt as their outcome. This begs the question, does LD211 still lower total cholesterol in the presence of AMPK inhibitors? This experiment is essential to conclude whether or not LD211 phenocopies the effects of MSH.

5) The authors initiate the project by showing high-fat diet disrupts the expression of MC1R. However, all of the subsequent experiments in hepatic MC1R KO mice are performed under normal chow. This begs the question of what is the phenotype of the hepatic MC1R KO mice fed a high-fat diet. Does KO of MC1R in the liver exacerbate HFD-induced obesity, glucose intolerance, and dyslipidemia? Inversely, can WT mice challenged with an HFD be rescued metabolically by treatment with either MSH or LD211? Providing data along these lines of investigation will provide physiological/clinical relevance to their findings.

Reviewer #1 (Public Review):

The authors aim to understand the role of clonal heterogeneity of tumors in immunogenicity of clonally expressed antigens. This is a significant problem with many basic as well as translational implications.

The strength of the manuscript lies in the novel demonstration that a poorly immunogenic tumor antigen, when paired with a stronger tumor antigen, begins to elicit significant immune response. The weakness lies in the fact that the actual mechanism of the key demonstration is never shown. There is a lot of speculation and tangential experimentation, but little actual evidence of a mechanism.

By making the key observation (mentioned in the strength section in the previous paragraph), the authors did achieve their objective albeit very partially. Their observation is based on excellent experimental tools and design. This study will stimulate further experiments in this important field.

Their key observation is somewhat reminiscent of the practice of conjugating small "non-immunogenic" antigens (such as some carbohydrates) to large protein carriers (such as serum albumin) in order to elicit strong antibody response to the weaker antigen. It is interesting to contemplate if the underlying mechanisms have any commonality.

Reviewer #1 (Public Review):

The manuscript described the mechanism of Spermidine modulation of Src kinase on IDO1, accelerating the kinetics of the reaction. Spermidine can act on the backside of the SH2 domain of Src, by the interaction of specific amino acids. Considering the important role of IDO1 in the immune response the results provide proof of principle for the development of molecules that can modulate the kinase activity and the nonenzymatic functions of Src and IDO1 at once. The conclusions of this paper are mostly well supported by data, but some aspects of figure construction and data analysis need to be improved for the sake of clarity.

Reviewer #1 (Public Review):

The authors examine the role of the K700E mutation in the Sf3B1 splicing factor in PDAC and report that this Sf3B1 mutation promotes PDAC by decreasing sensitivity to TGF-b resulting in decreased EMT and decreased apoptosis as a result. They propose that the Sf3b1 K700E mutant causes decreased expression of Map3K7, a known mediator of TGFb signaling and also known to be alternately spliced in other systems by the Sf3b1 K700E mutation. The role of splicing defects in cancer is relatively understudied and could identify novel targets for therapeutic intervention so this work is of potential significance. However, the data is over-interpreted in many instances and it is not clear the authors can make the claims they do based on the data shown. In particular, the data showing that decreased Map3k7 underlies the effects of the Sf3b1K700E mutant is very weak. Does over-expression of Map3k7 promote the EMT signature and induce apoptosis? Do the Map3k7 expressing organoids form tumors more effectively when transplanted into mice? Also, the novelty of the work is a concern since aberrant Map3k7 splicing due to SF3B1 mutation was seen previously in other systems. The authors also do not address the apparent conundrum of Sf3b1 K700E mutation promoting tumorigenesis despite there being less EMT which is also required for progression to metastasis in PDAC.

Major Concerns.<br /> 1. The analysis of the effect of Sf3b1K700E expression on normal pancreas and on PanINs in KC mice and PDAC in KPC mice is superficial and could be enhanced by staining for amylase, cytokeratin-19 and insulin. In particular, the data quantified in figure 1L should be accompanied by staining for CK19, Mucin5AC or some other marker of ductal transformation. Also, are any effects seen at older ages in normal mice?<br /> 2. The invasion assays used are limited and should be complemented by more routine quantification of cell migration and invasion including such assays as a scratch assay, Boyden chamber assays and use of the IncuCyte system to quantify. As it stands the image in Figure 3B is difficult to interpret since it is very poorly described in the figure legend. Additional evidence is needed to make the claims made by the authors.<br /> 3. The authors should show the actual CC3 staining quantified in Suppl. Figure 2G.<br /> 4. The graph in Figure 3L should show WT and Sf3b1K700E expressing organoids number both with and without TGF-b.

Reviewer #1 (Public Review):

This is a nice and elegant genetic study on the role of the Sgs1 and Exo1 factors involved in long DNA resection in the mechanism of double-strand break (DSB) repair by homologous recombination (HR). Most studies have focused on the need for these two factors for the long resection of a DSB to allow efficient HR. Now, this study shows that a major role of the function of long resection mediated by Sgs1 and Exo1 is to activate the DNA damage checkpoint to allow the chromosomal mobility needed to allow the DNA ends to find a distant homologous sequence with which repair via homologous recombination.

Reviewer #1 (Public Review):

The goal of the manuscript is a joint analysis of genetic variation, open chromatin, and gene expression in a genetically diverse population of mouse embryonic stem cells.

This is an important manuscript that links gene expression to genetic variants and regions of open chromatin. The mechanisms of genetic gene regulation are essential to understanding how standing genetic variation translates to function and phenotype. This data set has the ability to add substantial insight into the field. In particular, the authors show how the relationships between variants, chromatin, and genes are spatially constrained by topologically associated domains.

The description of the results is hard to follow, specific terms are not well-defined, and the methods section lacks detail. Several fundamental approaches cannot be understood easily without going to references (particularly #6, #15, and #33). The manuscript will benefit from efforts to improve readability.

In addition, the CTCF binding data (ChIP-seq) gets too little attention. The fundamental question of whether regulatory domains vary between individuals is not directly addressed.

Reviewer #1 (Public Review):

In this manuscript, the authors are building on their previous work showing Delta-Notch regulates the entrance and exit from embryo-larval quiescence of neural stem cells of the central brain (called CB neuroblasts (NB) (PMID: 35112131)). Here they show that continuous depletion of Notch in NBs from early embryogenesis leads to cycling NBs in the adult. This - cycling NBs in the adult - is not seen in controls. The assumption here is that these Notch-RNAi NBs in adults are those that did not undergo terminal differentiation in pupal development. The authors show that Notch is activated by its ligand Delta which is expressed on the GMC daughter cell and on cortex glia. They determine that the temporal requirement for Notch activity is 0-72 hours after larval hatching (ALH) (i.e., 1st instar through mid-3rd instar at 25C). In NBs/GMCs depleted for Notch, early temporal markers were still expressed at time points when they should be off and late markers were delayed in expression. These effects were observed in ~20-40% of NBs (Figures 5 and 6). Through mining existing data sets, they found that the early temporal factor Imp - an RNA binding protein - can bind Delta mRNA. They state that Delta transcripts decrease over time (without any reference to a Figure or to published work), leading to the hypothesis that Delta mRNA is repressed by the late temporal factors. Over-expressing late factors Syp or E93 earlier in development leads to downregulation of a Delta::GFP protein trap. These results lead to a model in which Notch regulates expression of early temporal factors and early temporal factors regulate Notch activity through translation of Delta mRNA.

There are several strengths of this study. The authors report rigorous measurements and statistical analyses throughout the study. Their conclusions are appropriate for the results. Data mining revealed an important mechanism - that Imp binds Delta mRNA - supporting the model that early temporal factors promote Delta expression, which in turn promotes Notch signaling.

There are also several weaknesses:<br /> 1. The activation of Notch in NBs by Delta in GMCs was already shown by this group in their Dev 2022 paper, reducing some of the impact of this study.<br /> 2. The authors do not explain their current results in context of their prior paper (2022 Dev) until the Discussion, but this would be useful to read in the Introduction. Similarly, it would be good to mention that in the 2022 paper, they find a significant number of wor>Notch RNAi NBs at 2 AHL that are cycling. Are the adult Notch RNAi in this study descended from those NBs at 2 hours ALH in the 2022 study? In other words, how does the early requirement for Notch between 0-72 hours ALH reported in the current study relate to the Notch-depleted NBs identified in the 2022 paper?<br /> 3. Most of the experiments rely upon continuous depletion of Notch from embryonic stage 8 until adulthood using the wor-GAL4 driver. There is no lineage tracing of this driver and there is no citation about the published expression pattern of this driver. The inclusion of these details is important for a broad audience journal.<br /> 4. Most of the experiments utilize a single RNAi transgene for Notch, Delta, Imp, Syp, E93. There are no experiments demonstrating the efficacy of the RNAi lines and no references to prior use and/or efficacy of these lines.

An appraisal: The authors use temperature shifts with Gal80TS to show that Notch is required between 0-72 hours ALH. They show with the use of known markers of the temporal factors and Delta protein trap, that Imp promotes Delta protein expression and the later temporal factors reduce Delta, although the molecular mechanisms are not clearly delineated. Overall, these data support their model that the reduction of Delta expression during larval development leads to a loss of Notch activity.

As noted in the Discussion, this study raises many questions about what Notch does in larval CB NBs. For example, does it inhibit Castor or Imp? Is Notch required in certain neural lineages and not others. These studies will be of interest in the community of developmental neurobiologists.

Reviewer #1 (Public Review):

The authors set out to investigate the hypothesis that mirror neurons in ventral premotor area F5 code actions in a common motor representation framework. To achieve this, they trained a linear discriminant classifier on the neural discharge of three types of action trials and test whether the thus trained classifier could decode the same categories of actions when observed. They showed that codes were fully matched for a small subset of neurons during the action epoch, while a wider set of "mirror neurons" showed only poorly matched codes for different epochs.

The authors controlled for potential visual object confounds by having identical objects be manipulated in three different ways and by having the animal carry out the motor execution in the dark. The main strength of the study lies in the clever decoding approach testing the matched tuning to behavioural categories in a model-free way. The central result is in the identification of the small sub-group of mirror neurons that show true matching during the execution epoch, which can dissociate the three types of action almost perfectly. This aligns well with some previous work while offering a novel avenue to identify and investigate those neurons.

The underlying neuronal mechanism and behavioural relevance of these neurons remain an open question. It would have been interesting to understand better whether the specific motor representations at a recording site, for instance identified through microstimulation prior to recording (see Methods), the reaction times on individual trials or the specific gaze targets (object/hand) had a bearing on the decoding performance for a neuron/trial. Ultimately, the uncovered matched mirror representations should in future experiments be tested with causal interventions and linked trial-by-trial to action selection performance.

The authors put the focus of their discussion on the wider, less well-matched neuronal pool to support an action selection framework, which is of course a valid view and well established in motor representations. From a sensory perspective, sparse coding, as suggested by the small group of "true" mirror neurons identified with the decoding approach, should also be considered as the basis for a possible neuronal mechanism. A particular strength of the paper is that it could give new data and impetus to the important discussion about how motor and sensory coding frameworks come together in cortical processing.

Reviewer #1 (Public Review):

Cedillo et al. address the critically important question of how biguanides exert their positive effects on longevity using the powerful C. elegans model. Biguanides metformin and phenformin have been widely prescribed in the clinic to address metabolic challenges of diabetes; more recently the value of metformin in addressing specific cancers has emerged, and testing for impact on healthy human aging is getting underway. The need to understand the mechanism of biguanide action and the metabolic consequences of biguanide administration is clear.

The authors report that three genes that suppress longevity associated with metformin or phenformin treatment affect a common pathway for ether lipid biosynthesis; this ether lipid biosynthesis pathway is required for mitochondrial lifespan extension, eat-2 mediated dietary restriction longevity, and TOR inhibition-associated longevity, but not insulin pathway mediated longevity. Authors document with lipid profiling how ether lipids and some other lipids are impacted by phenformin vs. genetic disruption of ether lipid biosynthesis, define the tissue primarily responsible for the ether lipid biosynthesis, show that over-expression of enzyme fard-1 is sufficient to confer most of the phenformin effect, and implicate conserved stress transcription factor SKN-1 as a downstream outcome of the ether lipid change.

Strengths include the exploitation of the nematode model to address requirements not readily discerned in other models, the rigor of genetic documentation, the inclusion of metabolic profiling, the testing of multiple potential pathways that have been in the general discourse regarding metformin action, and the elaboration of a reasonably supported model that ether lipid biosynthesis is required for phenformin to activate longevity-promoting metabolic defenses downstream of conserved stress-responsive transcription factor SKN-1/NRF2. The novelty includes that ether lipids are directly linked to lifespan, ether lipid biosynthesis is needed for specific longevity pathways, and that ether lipids might play a role in a shift to pro-longevity metabolism.

There are some points that require clarification and could benefit from additional study, some wording and presentation issues, and a few missing points of potential discussion.

Overall, the data reported in this paper contribute a highly valuable advance in the biguanide field and adds stimulating hypotheses to the scientific community for moving forward in this biomedically important area.

Reviewer #1 (Public Review):

The authors expand upon prior findings and show that basolateral amygdala (BLA) activity is necessary for defensive responses elicited by both innate and learned threats. The authors also show that a projection from the auditory thalamus (MGM thalamic nucleus) mediates these effects.

Learned threats were modelled with auditory fear conditioning. The authors finding showing that the MGM-BLA pathway is required for auditory fear learning is largely a replication of prior results.

The novelty in this paper is that the authors show that the auditory MGM-BLA pathway is involved in defense evoked by a visual looming stimulus.

Overall, this is a reasonably designed study. The main weakness is that the loss of function manipulations use either caspase-induced lesions or contralateral chemogenetic disconnection studies, which lack temporal resolution.

Reviewer #1 (Public Review):

It has been shown previously that there are relationships between a transdiagnostic construct of anxious-depression (AD), and average confidence rating in a perceptual decision task. This study sought to investigate these results, which have been replicated several times but only in cross-sectional studies. This work applies a perceptual decision-making task with confidence ratings and a transdiagnostic psychometric questionnaire battery to participants before and after an iCBT course. The iCBT course reduced AD scores in participants, and their mean confidence ratings increased without a change in performance. Participants with larger AD changes had larger confidence changes. These results were also shown in a separate smaller group receiving antidepressant medication. A similar sized control group with no intervention did not show changes.

The major strength of the study is the elegant and well-powered data set. Longitudinal data on this scale is very difficult to collect, especially with patient cohorts, so this approach represents an exciting breakthrough. Analysis is straightforward and clearly presented. However, no multiple comparison correction is applied despite many different tests. While in general I am not convinced of the argument in the citation provided to justify this, I think in this case the key results are not borderline (p<0.001) and many of the key effects are replications, so there are not so many novel/exploratory hypothesis and in my opinion the results are convincing and robust as they are. The supplemental material is a comprehensive description of the data set, which is a useful resource.

The authors achieved their aims, and the results clearly support the conclusion that the AD and mean confidence in a perceptual task covary longitudinally.

I think this study provides an important impact to the project of computational psychiatry.Sspecifically, it shows that the relationship between transdiagnostic symptom dimensions and behaviour is meaningful within as well as across individuals.

Reviewer #1 (Public Review):

The authors consider data by the Heisenberg group on rheological properties of non-confluent tissue in zebrafish embryos. These data had shown a steep increase and subsequent saturation in viscosity with cell density. The authors introduce a physical agent-based model of such tissues that accounts for the dispersion in cell size and the softness of the cells. The model is inspired by previous models to study glassy dynamics and reveals essential physical features that can explain the observed behavior. It goes beyond previous studies that had analysed the observations in terms of a percolation problem. The numerics are thoroughly done and could have a deep impact on how we describe non-confluent tissues.

A major weakness of the manuscript is the way it is written, which gives the impression to have been done rather carelessly. Several quantities are not properly introduced and at places physical jargon is used that makes the work difficult to access for readers without a background in soft matter.

Reviewer #1 (Public Review):

Many drugs have off-target effects on the gut microbiota but the downstream consequences for drug efficacy and side effect profiles remain unclear. Herein, Wang et al. use a mouse model of liver injury coupled to antibiotic and microbiota transplantation experiments. Their results suggest that metformin-induced shifts in gut microbial community structure and metabolite levels may contribute to drug efficacy. This study provides valuable mechanistic insights that could be dissected further in future studies, including efforts to identify which specific bacterial species, genes, and metabolites play a causal role in drug response. Importantly, although some pilot data from human subjects is shown, the clinical relevance of these findings for liver disease remain to be determined.

The major strength of this work is its scope, including detailed mouse phenotyping, inter-disciplinary methods, and numerous complementary experiments. The antibiotic depletion and FMT experiments provide support for a role of the gut microbiota in this mouse model.

A major limitation is the lack of studies narrowing down which microbes are responsible. Sequencing data is shown, but no follow-up studies are done with bacterial isolates or defined communities.

The link to GABA is also somewhat tenuous. While it does match the phenotypic data, there are no targeted experiments in which GABA producing microbial communities/strains are compared to a control community/strain. As such, it seems difficult to know how much of the effects in this model are due to GABA vs. other metabolites.

My major recommendation would be to revise the title, abstract, and discussion to provide more qualification and to consider alternative interpretations.

Some key controls are also missing, which could be addressed by repeat experiments in the mouse model. The antibiotic depletion experiment would be improved by testing the effect of antibiotics in the absence of metformin, to see if the effect is just driven by the model itself as opposed to an interaction between metformin and antibiotics. The FMT experiment lacks a control group and suffers from pseudoreplication: multiple donors from metformin treated and untreated mice could be used to colonize separate groups of recipient mice.

Reviewer #1 (Public Review):

This manuscript provides important evidence on the association between sleep regularity and mortality in the UK Biobank, which is a popular topic in recent sleep and circadian research in population-based studies. The analysis reported robust associations between sleep irregularity and increased total, CVD and cancer mortality, and provided evidence to support the role of sleep and circadian health in disease progression and longevity in human populations. The Sleep Regularity Index (SRI) used in this study is a novel metric that quantifies the consistency in rest-activity rhythms over consecutive 24 hour periods, thus providing objective assessment of potential circadian disruption. The study is based on a large accelerometer study with validated follow-up of incident diseases and deaths. The data quality and large sample size strengthen the credibility of the conclusion. Overall, the analyses are appropriately done and the manuscript is clearly written. Additional justification for the assessment of nonlinearity and further subgroup analyses would further improve the manuscript.

Joint Public Review

In the presence of predators, animals display attenuated foraging responses and increased defensive behaviors that serve to protect them from potential predatory attacks. Previous studies have shown that the basolateral nucleus of the amygdala (BLA) and the periaqueductal gray matter (PAG) are necessary for the acquisition and expression of conditioned fear responses. However, it remains unclear how BLA and PAG neurons respond to predatory threats when animals are foraging for food. The authors employed single-unit recording of BLA and PAG neurons and optogenetic tools to address this question in an 'approach food-avoid predator' paradigm.

The authors observed that rats exhibited a significant increase in the latency to obtain the food pellets and a reduction in the pellet success rate when the predator robot was activated. A subpopulation of PAG neurons showing increased firing rate in response to the robot activation did not change its activity in response to food pellet retrieval during the pre- or post-robot sessions. Optogenetic stimulation of PAG neurons increased the latency to procure the food pellet in a frequency- and intensity-dependent manner, similar to what was observed during the robot test. Combining optogenetics with single-unit recordings, the authors demonstrated that photoactivation of PAG neurons increased the firing rate of 10% of BLA cells. A subsequent behavioral test in three of these same rats demonstrated that BLA neurons responsive to PAG stimulation displayed higher firing rates to the robot than BLA neurons nonresponsive to PAG stimulation. Next, because the PAG does not project monosynaptically to the BLA, the authors used a combination of retrograde and anterograde neural tracing to identify possible regions that could convey robot-related information from PAG to the BLA. They observed that neurons in specific areas of the paraventricular nucleus of the thalamus (PVT) that are innervated by PAG fibers contained neurons that were retrogradely labeled by the injection of CTB in the BLA. In addition, PVT neurons showed increased expression of the neural activity marker cFos after the robot test, suggesting that PVT may be a mediator of PAG signals to the BLA.

Strengths

Overall, the idea that the PAG interacts with the BLA via the midline thalamus during a predator vs. foraging test is new and quite interesting. The authors have used appropriate tools to address their questions. The major impact in the field would be to add evidence to claims that the BLA can be downstream of the dPAG to evoke defensive behaviors. The study also adds to a body of evidence that the PAG mediates primal fear responses.

Weaknesses

The two most significant weaknesses relate to a) anatomical concerns related to the subregions of the BLA and PAG that were targeted by manipulations and analyses and b) the correlational nature of the PVT measures and the lack of any causal role demonstrated. Other concerns are also detailed below.

Anatomical concerns:

1. The authors claim that the recordings were performed in the dorsal PAG (dPAG), but the histological images in Fig. 1B and Supplementary S2 for example show the tip of the electrode in a different subregion of PAG (ventral/lateral). They should perform a more careful histological analysis of the recording sites and explain the histological inclusion and exclusion criteria. Diagrams showing the sites of all PAG and BLA recordings, as well as all fiber optics, would be helpful.<br /> 2. Prior studies investigating the role of BLA neurons during a foraging vs. robot test similar to the one used in this study should be also cited and discussed (e.g., Amir et al 2019, PMID: 30840520; Amir et al 2015, PMID: 26400931). These two studies demonstrated that most neurons in the basal portion of the BLA exhibit inhibitory activity during foraging behavior and only a small fraction of neurons (~4%) display excitatory activity in response to the robot (in contrast to the 25% reported in the present study). A very accurate histological analysis of BLA recording sites should be performed to clarify whether distinct subregions of the BLA encode foraging and predator-related information, as previously shown in the two described studies.<br /> 3. An important claim of this study that the PAG sends predator-related signals to BLA via the PVT (Fig. 4). The authors stated that PVT neurons labeled by intra-BLA injection of the retrograde tracer CTB were activated by the predator, but a proper immunohistochemical quantification with a control group was not provided to support this claim. To provide better support for their claim, the authors should quantify the double-labeled PVT neurons (cFos plus CTB positive neurons) during the robot test.<br /> 4. The AVV anterograde tracer deposit spread to a large part of the PAG, including dorsolateral and lateral PAG, and supraoculomotor regions (Fig. 4B). Is the projection to the PVT from the dPAG or other regions of the PAG?

Concerns about the strength of the evidence supporting a role for the PVT:

5. The authors conclude in the discussion section that the dPAG-amygdala pathway is involved in generating antipredatory defensive behavior. However, the current results are entirely based on correlational analyses of neural firing rate and there is no direct demonstration that the PAG provides information about the robot to the BLA. Therefore, the authors should tone down their interpretation or provide more evidence to support it by performing experiments applying inhibitory tools in the dPAG > PVT > BLA pathway and examining the impact on behavior and downstream neural firing.

Other concerns:

6. One of the main findings of this study is the observation that BLA neurons that are responsive to PAG photostimulation are preferentially recruited during the foraging vs. robot test (Fig. 3). However, the experimental design used to address this question is problematic because the laser photostimulation of PAG neurons preceded the foraging vs. robot test. Prior photoactivation of PAG may have caused indirect short-term synaptic plasticity in BLA cells, which would favor the response of these cells to the robot. Please see Oishi et al, 2019 PMID: 30621738, which demonstrated that 10 trains of 20Hz photoactivation (300 pulses each) was sufficient to induce LTP in brain slices.<br /> 7. The authors should perform a longitudinal analysis of the behavioral responses of the rats across the trials to clarify whether the animals habituate to the robot or not. In Figure 1E, it appears that PAG neurons fire less across the trials, which could be associated with behavioral habituation to the predator robot. If that is the case, the activity of many other PAG and BLA neurons will also most likely vary according to the trial number, which would impact the current interpretation of the results.<br /> 8. In Figure 1, it is unclear why the authors compared the activity of neurons that respond to the robot activation against the activity of the neurons during the retrieval of the food pellets in the pre-robot and post-robot sessions. The best comparison would be aligning the cells that were responsive to the activation of the robot with the moment in which the animals run back to the nest after consuming the pellets during the pre-robot or post-robot sessions. This would enable the authors to demonstrate that the PAG responses are directly associated with the expression of escaping behavior in the presence of the robot rather than associated with the onset of goal-directed movement in direction to the next during the pre- and post-robot sessions. A graphic showing the correlation between PAG firing rate and escape response would be also informative.

Reviewer #1 (Public Review):

The authors performed a comparative study of the effect of the anesthetics isoflurane and ketamine on whole-brain network activation by mapping whole-brain c-fos expression in mice. Principle component analysis on the normalized Fos density showed opposite effects of the 2 anesthetics, consistent with top-down functioning for ketamine and bottom-up functioning for isoflurane. Based on the network analysis the authors suggest that isoflurane mediates anesthesia through a bottom-up mechanism activating subcortical regions and inactivating cortical regions with the locus coeruleus being the most important region while ketamine produced anesthesia through a top-down mechanism activating the cortex and subcortical nuclei with the somatosensory cortex as the most important region. Overall they show that these two anesthetics have two opposite mechanisms to induce unconsciousness, although they also have overlapping coactivation of central sleep-wake, pain, and neuroendocrine regulating areas. This manuscript highlights some interesting findings through interesting analysis. The results are likely to have a significant impact on the field of anesthesia but also on the much larger field of neuropsychopharmacology as the tools and analyses used in this report will be useful for researchers investigating the effects of any psychoactive drugs on the brain. However, there are several issues that should be addressed to support their conclusions. The two main issues of this report are the lack of behavioral/physiological measures of the depth of anesthesia produced by ketamine/isoflurane and inadequate data analysis/interpretations for some of the results.

Strengths<br /> Comparison of two different anesthetics<br /> Use of single-cell whole-brain imaging<br /> Advanced network analysis

Weaknesses<br /> Lack of behavioral/physiological measures<br /> Interpretation of the data is sometimes confusing/unclear<br /> Some statistical tests are missing and others are not controlled for multiple comparisons

Major concerns<br /> 1. The lack of behavioral/physiological measures of the depth of anesthesia (ventilation, heart rate, blood pressure, temperature, O2, pain reflexes, etc...) combined with the lack of dose-response and the use of different routes of administration makes the data difficult to interpret. Sure, there is a clear difference in network activation between KET and ISO, but are those effects due to the depth of the anesthesia, the route of administration, and the dose used? The lack of behavioral/physiological measures prevents the identification of brain regions responsible for some of the physiological effects and different effects of anesthetics.<br /> 2. Under anesthesia there should be an overall reduction of activity, is that the case? There is no mention of significantly downregulated regions. The authors use multiple transformations of the data to interpret the results (%, PC1 values, logarithm) without much explanation or showing the full raw data in Fig 1. It would be helpful to interpret the data to compare the average fos+ neurons in each region between treatment and control for each drug.<br /> 3. I do not understand their interpretation of the PCA analyses. For instance, in Fig 2 they claim that KET is associated with PC1 while ISO is associated with PC2. Looking at the distribution of points it's clear that the KET animals are all grouped at around +2.5 on PC1 and -2.0 on PC2, this means that KET is associated with both PC1 and PC2 to a similar degree (2 to 2.5). Moreover, I'm confused about why they use PCA to represent the animals/group. PCA is a powerful technique to reduce dimensionality and identify groups of variables that may represent the same underlying construct; however, it is not the best way to identify clusters of individuals or groups.<br /> 4. The actual metric used for the first PCA is unclear, is it the FOS density in each of the regions (some of those regions are large and consist of many subregions, how does that affect the analysis) is it the %-fos, or normalized cells? The wording describing this is variable causing some confusion. How would looking at these different metrics influence the analysis?<br /> 5. Based on Fig 3 the authors concludes that ISO activates the hypothalamic regions and inhibits the cortex, however, Fig 1 shows neither an activation of the hypothalamus in the ISO nor an inhibition of the cortex when compared to home cage control. If anything it suggests the opposite.<br /> 6. Control for isoflurane should be air in the induction chamber rather than home cage. It is possible that Fos activation reflects handling/stress pre-anesthesia in the animals, which would increase Fos expression in the stress-related regions such as the BST, striatum (CeA), hypothalamus (PVH) and potentially the LC.<br /> 7. In the Ket network there are a few anticorrelated regions, most of which are amongst the list of the most activated regions, does this mean that the strong correlation results from an overall decreased activation? And if so, is it possible that the ketamine anesthesia was stronger than the isoflurane, causing a more general reduction in activity?<br /> 8. Since they have established networks it would be easy and useful to look at how the different regions identified (sleep, pain, neuroendocrine, motor-related, ...) work together to maintain analgesia, are they within the same module? Do they become functionally connected and is this core network of functional connections similar for KET and ISO?<br /> 9. The naming of the function of some of the regions is very much debatable. For instance, PL/ILA are named "sleep-wakefulness regulation" regions in the paper. I can think of many more important functions of the PL/IL including executive functions, behavioral flexibility, and emotional control. It is unclear how the functions of all the regions were attributed. I am not sure that this biased labeling of structure-function is useful to the reports, it may instead suggest wrong conclusions.<br /> 10. A point of concern and confusion is the number of brain regions analyzed. In the introduction, it is mentioned that 987 brain regions are considered, but this is reduced to 53 selected brain regions in Figure 2, then 201 brain regions in Figure 3, and reduced again to 63 for the network analysis. The rationale for selecting different brain regions is not clear.<br /> 11. The statistical analysis does not seem appropriate considering the high number of comparisons. They use simple t-tests without correction for multiple comparisons.<br /> 12. There is no statistical analysis in Fig 2C,

Reviewer #1 (Public Review):

This EEG study probes the prediction of a mechanistic account of P300 generation through the presence of underlying (alpha) oscillations with a non-zero mean. In this model, the P300 can be explained by a baseline shift mechanism. That is, the non-zero mean alpha oscillations induce asymmetries in the trial-averaged amplitudes of the EEG signal, and the associated baseline shifts can lead to apparent positive (or negative) deflections as alpha becomes desynchronized at around P300 latency. The present paper examines the predictions of this model in a substantial data set (using the typical P300-generating oddball paradigm and careful analyses). The results show that all predictions are fulfilled: the two electrophysiological events (P300, alpha desynchronization) share a common time course, anatomical sources (from inverse solutions), and covariations with behaviour; plus relate (negatively) in amplitude, while the direction of this relationship is determined by the non-zero-mean deviation of alpha oscillations pre-stimulus (baseline shift index, BSI). This is indicative of a tight link of the P300 with underlying alpha oscillations through a baseline shift account, at least in older adults, and hence that the P300 can be explained in large parts by non-zero mean brain oscillations as they undergo post-stimulus changes.

Reviewer #1 (Public Review):

The present study by Berger et al. analyzes to what extent memory formation is dependent on available energy reserves. This has been dealt with extensively in the case of aversive memory formation, but only very sparsely in the case of appetitive memory formation. It has long been known that an appetitive memory in flies can only be formed by starvation. However, the authors here additionally show that not only the duration of starvation plays a role, but also determines which form of memory (short- or long-term memory) is formed. The authors demonstrated that internal glycogen stores play a role in this process and that this is achieved through insulin-like signaling in octopaminergic reward neurons that integrates internal energy stores into memory formation. Here, the authors suggest that octopamine plays a role as a negative regulator of different forms of memory.

The study sheds light on an old question, to what extent the octopaminergic neuronal system plays a role in the formation of appetitive memory, since in recent years only the dopaminergic system has been in focus. Furthermore, the data are an interesting contribution to the ongoing debate whether insulin receptors play a role in neurons themselves or in glial cells. The experiments are very well designed and the authors used a variety of behavioural experiments, genetic tools to manipulate neuronal activity and state-of-the-art imaging techniques. In addition, they not only clearly demonstrated that octopamine is a negative regulator of appetitive memory formation, but also proposed a mechanism by which the insulin receptor in octopaminergic neurons senses the internal energy status and then controls the activity of those neurons. The conclusions are mostly supported by the data, but some aspects related to the experimental design, some explanations and literature references need more clarification and revision.

1. Usually, long-term memory (LTM) is tested 24 hours after training. Here, the authors usually refer to LTM as a memory that is tested 6 hours after training. The addition of a control experiment to show that LTM that the authors observe here lasts longer would increase the power of this study immensely.

2. The authors define here another consolidated memory component as ARM, when they applied a cold-shock 2 hours after training. However, some publications showed that LTM is formed after only one training cycle (Krashes et al 2008, Tempel et al 1983). This makes it difficult to determine, whether appetitive ARM can be formed. Furthermore, one study showed that appetitive ARM is absent after massed training (Colomb et al 2009). Therefore, the conclusion could be also, that different starvation protocols, would lead to different stabilities of LTM. Therefore, additional experiments could help to clarify this opposing explanation. From these results, it can then be concluded either that different stable forms of LTM are formed depending on the starvation state, or that two differently consolidated memory phases (LTM, ARM) are formed, as has already been shown for aversive memory. This is also important for other statements in the manuscript, and therefore the authors should address this. For example, the findings about the insulin receptor (is it two opposing memories or different stabilities of LTM).

Reviewer #1 (Public Review):

She et al studied the evolution of gene expression reaction norms when individuals colonise a new environment that exposes them to physiologically challenging conditions. Their objective was to test the "plasticity first" hypothesis, which suggest that traits that are already plastic (their value changes when facing a new environment compared to the original environment) facilitates the colonisation of novel environments, which, if true, would be predicted to result in the evolution of gene expression values that are similar in the population that colonised the new environment and evolved under these particular selection pressures. To test this prediction, they studied gene expression in cardiac and muscle tissues in individuals originating from three conditions: lowland individuals in their natural environment (ancestral state), lowland individuals exposed to hypoxia (the plastic response state), and a highland population facing hypoxia for several generations (the coloniser state). They classified gene expression patterns as maladaptive or adaptive in lowland individuals responding to short term hypoxia by classifying gene expression patterns using genes that differed between the ancestral state (lowland) and colonised state (highland). Genes expressed in the same direction in lowland individuals facing hypoxia (the plastic state) as what is found in the colonised state are defined as adaptative, while genes with the opposite expression pattern were labelled as maladaptive, using the assumption that the colonised state must represent the result of natural selection. Furthermore, genes could be classified as representing reversion plasticity when the expression pattern differed between the plasticity and colonised states and as reinforcement when they were in the same direction (for example more expressed in the plastic state and the colonised state than in the ancestral state). They found that more genes had a plastic expression pattern that was labelled as maladaptive than adaptive. Therefore, some of the genes have an expression pattern in accordance with what would be predicted based on the plasticity-first hypothesis, while others do not.

As pointed out by the authors themselves, the fact that temperature was not included as a variable, which would make the experimental design much more complex, misses the opportunity to more accurately reflect the environmental conditions that the colonizer individuals face at high altitude. Also pointed out by the authors, the acclimation experiment in hypoxia lasted 4 weeks. It is possible that longer term effects would be identifiable in gene expression in the lowland individuals facing hypoxia on a longer time scale. Furthermore, a sample size of 3 or 4 individuals per group depending on the tissue for wild individuals may miss some of the natural variation present in these populations. Stating that they have a n=7 for the plastic stage and n= 14 for the ancestral and colonized stages refers to the total number of tissue samples and not the number of individuals, according to supplementary table 1.

Impact of the work:

There has been work showing that populations adapted to high altitude environments show changes in their hypoxia response that differs from the short-term acclimation response of lowland population of the same species. For example, in humans, see Erzurum et al. 2007 and Peng et al. 2017, where they show that the hypoxia response cascade, which starts with the gene HIF (Hypoxia-Inducible Factor) and includes the EPO gene, which codes for erythropoietin, which in turns activates the production of red blood cell, is LESS activated in high altitude individuals compared to the activation level in lowland individuals (which gives it its name). The present work adds to this body of knowledge showing that the short-term response to hypoxia and the long term one can affect different pathways and that acclimation/plasticity does not always predict what physiological traits will evolve in populations that colonize these environments over many generations and additional selection pressure (UV exposure, temperature, nutriment availability).

Altogether, this work provides new information on the evolution of reaction norms of genes associated with the physiological response to one of the main environmental variables that affects almost all animals, oxygen availability. It also provides an interesting model system to study this type of question further in a natural population of homeotherms.

Erzurum, S. C., S. Ghosh, A. J. Janocha, W. Xu, S. Bauer, N. S. Bryan, J. Tejero et al. "Higher blood flow and circulating NO products offset high-altitude hypoxia among Tibetans." Proceedings of the National Academy of Sciences 104, no. 45 (2007): 17593-17598.

Peng, Y., C. Cui, Y. He, Ouzhuluobu, H. Zhang, D. Yang, Q. Zhang, Bianbazhuoma, L. Yang, Y. He, et al. 2017. Down-regulation of EPAS1 transcription and genetic adaptation of Tibetans to high-altitude hypoxia. Molecular biology and evolution 34:818-830.

Reviewer #1 (Public Review):

The authors were seeking to improve understanding of how wind and wave action affect the use of energetically demanding wing flapping and running by albatross engaged in takeoff flight. To accomplish this in the complex and challenging environment in which albatross live, the authors sought to use accelerometry and geographic positioning to infer patterns of locomotion, flight orientation relative to the prevailing wind, and wave height during takeoff.

The major strength of the methods and results is that the use of accelerometry and novel interpretations of data from a geographic positioning system provides new insight into the use of waves by albatross and how the effects of wave magnitude interact with wind to modulate energy demands during takeoff. Weaknesses of the approach are due to the challenging environmental conditions in which albatross live. The interpretation of accelerometry data was not validated using a subset of the sample synchronized with video (prior validation was cited for shearwaters). The interpretation of wind direction relative to flight path is based on the behavior of the bird without concurrent measures of local wind velocity.

The authors achieved their aims, and their results support their conclusions.

Although it is generally understood that albatross and many other birds choose to takeoff into the wind to reduce energetic costs, the authors provide novel quantitative data on this behavior. Their results on the effects of wave height and the interactions between wave height and wind provide novel insight into how albatross harvest energy from their complicated and dynamic environment to reduce the energy they must output to get into the air. In particular, the new insight into the effects of wave height should revise understanding among ornithologists, ocean ecologists and those who study the mechanics of animal locomotion. The use of accelerometry and geographic positioning systems to measure flight behavior and ocean ecology should inspire other researchers to adopt similar methods.

Albatross live in a complex and poorly understood environment that is likely to be threatened by climate change. This research provides worthwhile new insight into how wind and wave action affect takeoff in albatross, and can therefore improve insight into how changes in these variables with climate change may affect the distribution of albatross populations.

Reviewer #1 (Public Review):

The manuscript provides analyses on a very complete dataset on weight and length growth, as well as several physiological markers related to growth, in bonobos. Moreover, there is a good overview of the presence of adolescent growth spurts in non-human primates, by reviewing published data, in comparison to their own dataset. They discuss the need to consider scaling laws when interpreting and comparing growth curves of different species and variables.

The manuscript is very well written, the sample is large, and the methods are well explained. It seems they have analyzed a very complete dataset. Also, the discussion and the references supporting the findings are complete.

The main weakness of this manuscript is that they do not provide a direct comparison with previously analyzed datasets in other species, using their own method (in part maybe because there is not available data, but just published figures).

On the other side, conclusions are well supported by the results, and the previously published datasets are discussed in the manuscript, although not in detail.

Reviewer #1 (Public Review):

This paper describes the discovery, functional analysis and structure of TcaP, a protein encoded by the Vibrio phage satellite PLE that forms a size-determining scaffold around PLE procapsids made from helper phage ICP1 structural proteins. The system displays a fascinating similarity to the P2/P4 system, which had previously been unique in its use of a size-determining external scaffolding protein, Sid. The work is interesting, comprehensive and of high quality. The presentation could be improved as listed in the suggestions below.

An interesting observation is that PLE appears to be dependent on small capsids for efficient transduction. This is not completely surprising if the element uses a cos site type mechanism for packaging, since this requires an integer number of genomes to be packaged when the capsid is full, and this might be more difficult to accomplish when the helper capsid is much larger than the satellite, as is the case with ICP1. The authors mention in a few places that this is the first known satellite to have this requirement. However, this is not quite correct: a similar defect was seen in phi12/SaPIbov5, where the large phi12 capsid was not quite the right size for either two or three copies of the wild-type ("unevolved") SaPIbov5 (Carpena et al. 2016).

The authors present several micrographs showing capsids formed in the presence or absence of wildtype or mutant TcaP and CP (Fig. 1, Fig 2., Fig 3). However, each micrograph shows only a handful of particles of the "correct" size, in addition to a few shells that are aberrant or of a different size. I miss a more statistically rigorous enumeration of shells of different size (PLE or ICP1 sized, or different), empty vs. full, aberrant shells etc. This could be presented as a size distribution graph, a histogram or in table form.

In the abstract, the term "divergent satellite P4" is vague and unclear. Divergent from what? Probably they mean distinct from or unrelated to PLE. Please clarify.

How do they know that gp123 is a decoration protein? Was this previously determined, does it have (sequence) similarity to other known decoration proteins, or is it simply the most likely designation based on its position in the genome?

Although the reconstruction and modeling statistics are good, it is difficult to assess the quality of the map and the model from the presented figures. Details of the density and FSC curves (half-map and model-to-map) should be shown. It is also difficult to see the TcaP structure and how it compares to Sid from the figures presented.

Introduction, Paragraph 3: "...which is the number of coat proteins divided by 60" is not strictly speaking the definition of T number. The T number corresponds to the number of subtriangles that one triangular face of the icosahedron is divided into. It corresponds to the number of coat proteins divided by 60 in the canonical case, but in tailed phages, 5 copies are removed to make way for the portal protein. (Other viruses could be described as having architecture corresponding to a specific T number, but with divergent numbers of subunits, e.g. adenoviruses or polyomaviruses.)

Reviewer #1 (Public Review):

This is a paper describing in detail the seasonal movements of a vole-eating raptor, the rough-legged buzzard, from their Arctic breeding areas to the temperate wintering areas and back, in an annual cycle perspective. The basis of the descriptions (using satellite tags) is state of the art, and so are the analyses on aspects of time and space. Of particular relevance is the degree in which this study successfully pinpoints the ecological shaping factors, food availability of course, in this case strongly affected by snow cover (which can be remotely sensed over large areas). The authors claim a new migration pattern called 'foxtrot' with phases with rapid and phases with slower migration movements.

My concern with this paper is the framing. A story on the how and why of these continental movements in response to snow and other habitat features would be a grand contribution.

Reviewer #1 (Public Review):

The authors Wang et al. present a study of a mouse model K74R that they claim can extend the life span of mice, and also has some anti-cancer properties. Importantly, this mechanism seems to be mediated by the hematopoietic system, and protective effects can be transferred with bone marrow transplantation.

The authors need to be more specific in the title and abstract as to what is actually novel in this manuscript (a single tumor model), and what relies on previously published data (lifespan). Because many of these claims derive from previously published data, and the current manuscript is an extension of previously published work. The authors need to be more specific as to the actual data they present (they only use the B16 melanoma model) and the actual novelty of this manuscript.

Especially experiments on life span are published and not sufficiently addressed in this actual paper, as the title would suggest.

Reviewer #1 (Public Review):

In this manuscript, Bockorny, Muthuswamy, and Huang et al. performed proteomics analysis of plasma extracellular vesicles (EVs) from pancreatic ductal adenocarcinoma (PDAC) patients and patients with benign pancreatic diseases (chronic pancreatitis and intraductal papillary mucinous neoplasm, IPMN) to develop a 7-EV protein signature that predicts PDAC. Moveover, the authors identified PSMB4, RUVBL2, and ANKAR as being associated with metastasis. These studies provide important insight into alterations of EVs during PDAC progression and the data supporting predict PDAC with EV protein signatures are solid. However, there are certain concerns regarding the rigor and novelty of the data analysis and interpretation, as well as the clinical implications, as detailed below.

1. Plasma EVs were characterized by transmission electron microscopy and nanoparticle tracking analysis to confirm their morphology and size. The authors should also include an analysis of putative EV markers (e.g., tetraspanins, syntenin, ALIX, etc.) to confirm that the analyzed particles are EVs.

2. The authors identified multiple over-expressed proteins in PDAC based on their fold change and p-value; however, due to the heterogeneity of PDAC, it is necessary to show a heatmap displaying their abundance in all samples. High fold change does not necessarily indicate consistently high abundance in all PDAC samples.

3. PSMB4, RUVBL2, and ANKAR were identified as being associated with metastasis. The authors state that they intended to distinguish early and late-stage cancer samples, but it is unclear why they chose to compare metastatic and non-metastatic samples, as the non-metastatic group also includes late-stage cancer samples. This sentence should be rephrased to more accurately reflect the sample types profiled.

4. Non-metastatic and metastatic patients were separated based on global protein abundance. The samples within each group display significant heterogeneity with, some samples displaying similar patterns although they were classified into different groups (Figure 3A), and the samples within the same group, particularly the metastasis group, did not consistently exhibit similar patterns of protein abundance. The authors should clarify this point.

5. The authors performed the survival analysis on a set of EV proteins but did not specify the origin of these markers or how many markers were examined. The authors should show their abundances across different groups, such as different stages and metastasis status.

6. The classification model yielded a 100% accuracy, which may refer to AUC, in their discovery cohort, but it decreased to 89% in the independent cohort. This suggests that the authors have encountered overfitting issues with their model, where it performed well on the discovery cohort but did not generalize well to the independent cohort. The authors should clarify this point. The AUC score of the 7-EV signature is 0.89 and is not equivalent to prediction accuracy. In order to demonstrate prediction accuracy, the authors should show the confusion matrix of training and testing data as well as other evaluation metrics, such as accuracy, precision, and recall.

7. The authors should include more details of their model and the process of selection of signatures to enhance the reproducibility and transparency of their methods.

Reviewer #1 (Public Review):

The study investigates the nature of "trailblazer" cells in distinct tumor models, including luminal B (MMTV/PyMT) and triple negative (TNBC) tumors (C3-TAg). The authors note that the trailblazer phenotypes in the TNBC model are more complex relative to the Luminal B model and represent distinct EMT programs associated with the expression of distinct EMT-TFs (Zeb1, Zeb2 and Fra-1). They demonstrated that of numerous EMT-TFs, Zeb1 and Fra-1 were required for increased cancer cell migration and invasion. They reveal that TGF-beta and EGF-mediated signaling are required for the diverse EMT states that are required for trailblazer cell activity and increased cell migration/invasion. TGF-beta signaling engaged Zeb 1 and Zeb2 while EGF signaling activated Fra-1. Indeed, inhibitors of either TGF-beta or EGF signaling could impair cell migration/invasion. While both pathways contributed to trailblazer phenotypes, EGF signaling was shown to interfere with certain TGF-beta induced transcriptional response, including the expression of genes encoding extracellular matrix proteins.

One concern was the heavy reliance of the C3-TAg as the sole TNBC model in which the distinct trailblazer phenotypes were described. The data in Fig. 3 of the submission reveals that the phenotypes observed in the C3-TAg model could be recapitulated in a TNBC patient-derived xenograft model (PDX). Using this PDX, the authors were able to show vimentin expression in lung metastatic TNBC cells that were intravascular, those that had extravasated and clusters of cancer cells fully within the lung parenchyma. This was an important addition to the manuscript. The additional experiments to investigate the role of Zeb1 and Zeb1 more fully, beyond the focus on Fra-1 in the initial submission was an additional strength of the new submission. Additional clarifications to the discussion also clarified the concepts articulated in the study. The study employs multiple breast cancer models, utilizes numerous in vitro and in vivo assessments of the trailblazer phenotypes, and the experimental design is rigorous and the interpretation of the data is sound. The manuscript will be of general interest to the research community.

Reviewer #1 (Public Review):

In this manuscript by Wu et al., the authors present the high-resolution cryoEM structures of the WT Kv1.2 voltage-gated potassium channel. Along with this structure, the authors have solved several structures of mutants or experimental conditions relevant to the slow inactivation process that these channels undergo and which is not yet completely understood.

One of the main findings is the determination of the structure of a mutant (W366F) that is thought to correspond to the slow inactivated state. These experiments confirm results in similar mutants in different channels from Kv1.2 that indicate that inactivation is associated with an enlarged selectivity filter.

Another interesting structure is the complex of Kv1.2 with the pore-blocking toxin Dendrotoxin 1. The results show that the mechanism of the block is different from similar toxins, in which a lysine residue penetrates the pore deep enough to empty most external potassium binding sites.

The quality of the structural data presented in this manuscript is very high and allows for the unambiguous assignment of side chains. The conclusions are supported by the data. This is an important contribution that should further our understanding of voltage-dependent potassium channel gating. Specific comments are appended below.

1) In the mains text's reference to Figure 2d residues W18' and S22' are mentioned but are not labeled in the insets.

2) On page 8 there is a discussion of how the two remaining K+ ions in binding sites S3 and S4 prevent permeation K+ in molecular dynamics. However, in Shaker, inactivated W434F channels can sporadically allow K+ permeation with normal single-channel conductance but very reduced open times and open probability at not very high voltages.

3) The structures of WT in the absence of K+ show a narrower selectivity filter, however, Figure 4 does not convey this finding. In fact, the structure in Figure 4B is constructed at such an angle that it looks as if the carbonyl distances are increased, perhaps this should be fixed. Also, it is not clear how the distances between carbonyls given in the text on page 12 are measured. Is it between adjacent or kitty-corner subunits?

4) It would be really interesting to know the authors' opinions on the driving forces behind slow inactivation. For example, potassium flux seems to be necessary for channels to inactivate, which might indicate a local conformational change is the trigger for the main twisting events proposed here.

Reviewer #1 (Public Review):

The authors have previously employed micrococcal nuclease tethered to various Mcm subunits to the cut DNA to which the Mcm2-7 double hexamers (DH) bind. Using this assay, they found that Mcm2-7 DH are located on many more sites in the S. cerevisiae genome than previously shown. They then demonstrated that these sites have characteristics consistent with origins of DNA replication, including the presence of ARS consensus sequences, location of very inefficient sites of initiation of DNA replication in vivo, are free of nucleosomes, they contain a G-C skew and they locate to intergenic regions of the genome. The authors suggest, consistent with published single molecule results, that there are many more potential origins in the S. cerevisiae genome than previously annotated.

The results are convincing and are consistent with prior observations. The analysis of the origin associated features is informative.

Reviewer #1 (Public Review):

The authors investigated the function of BATF in hepatic lipid metabolism. They found BATF alleviated high-fat diet (HFD)-induced hepatic steatosis. In addition, BATF could inhibit programmed cell death protein (PD)1 expression induced by HFD. By using over expression and transcriptional activity analysis, this study confirmed that BATF regulates fat accumulation by inhibiting PD1 expression and promoting energy metabolism. Then, they found PD1 antibodies alleviated hepatic lipid deposition. These data identified the regulatory role of BATF in hepatic lipid metabolism and that PD1 is a target for alleviation of NAFLD.

The conclusions of this manuscript are supported by data, but some remaining concerns need to be addressed.

1. There are different cells in liver tissue, in which BATF protein is expressed most.<br /> 2. The statistical data should be provided to support the liver specific over-expression of BATF.<br /> 3. For in vivo study, food intake is key data to exclude the change of energy intake.<br /> 4. For Fig.6 Since PD1 are also highly expressed in heart and spleen, how to exclude the effect of PD1 antibody on these tissues?