Reviewer #3 (Public Review):

Summary:

This study used prolonged stimulation of a limb to examine possible plasticity in somatosensory evoked potentials induced by the stimulation. They also studied the extent that the blood brain barrier (BBB) was opened by the prolonged stimulation and whether that played a role in the plasticity. They found that there was potentiation of the amplitude and area under the curve of the evoked potential after prolonged stimulation and this was long-lasting (>5 hrs). They also implicated extravasation of serum albumin, caveolae-mediated transcytosis, and TGFb signalling, as well as neuronal activity and upregulation of PSD95. Transcriptomics was done and implicated plasticity related genes in the changes after prolonged stimulation, but not proteins associated with the BBB or inflammation. Next, they address the application to humans using a squeeze ball task. They imaged the brain and suggest that the hand activity led to an increased permeability of the vessels, suggesting modulation of the BBB.

Strengths:

The strengths of the paper are the novelty of the idea that stimulation of the limb can induce cortical plasticity in a normal condition, and it involves opening of the BBB with albumin entry. In addition, there are many datasets and both rat and human data.

Weaknesses:

The conclusions are not compelling however because of a lack of explanation of methods. The explanation of why prolonged stimulation in the rat was considered relevant to normal conditions should be as clear in the paper as it is in the rebuttal. The authors need to ensure other aspects of the rebuttal are as clear in the paper as in the rebuttal too. The only remaining concern that is significant is that it is hard to understand the figures.

Reviewer #3 (Public Review):

A brain region called the retrotrapezoid nucleus (RTN) regulates breathing in response to changes in CO2/H+, a process termed central chemoreception. A transcription factor called PHOX2B is important for RTN development and mutations in the PHOX2B gene result in a severe type of sleep apnea called Congenital Central Hypoventilation Syndrome. PHOX2B is also expressed throughout life, but its postmitotic functions remain unknown. This study shows that knockdown of PHOX2B in the RTN region in adult rats decreased expression of Task2 and Gpr4 in Nmb-expressing RTN chemoreceptors and this corresponded with a diminished ventilatory response to CO2 but did not impact baseline breathing or the hypoxic ventilatory response. These results provide novel insight regarding the postmitotic functions of PHOX2B in RTN neurons.

Main issues:<br /> 1) The experimental approach was not targeted to Nmb+ neurons and since other cells in the area also express Phox2b, conclusions should be tempered to focus on Phox2b expressing parafacial neurons NOT specifically RTN neurons

2) It is not clear whether PHOX2B is important for the transcription of pH sensing machinery, cell health, or both. If knockdown of PHOX2B knockdown results in loss of RTN neurons this is also expected to decrease Task2 and Gpr4 levels, albeit by a transcription-independent mechanism.

Reviewer #3 (Public Review):

Summary:<br /> The study claims to investigate trunk representations in elephant trigeminal nuclei located in the brainstem. The researchers identified large protrusions visible from the ventral surface of the brainstem, which they examined using a range of histological methods. However, this ventral location is usually where the inferior olivary complex is found, which challenges the author's assertions about the nucleus under analysis. They find that this brainstem nucleus of elephants contains repeating modules, with a focus on the anterior and largest unit which they define as the putative nucleus principalis trunk module of the trigeminal. The nucleus exhibits low neuron density, with glia outnumbering neurons significantly. The study also utilizes synchrotron X-ray phase contrast tomography to suggest that myelin-stripe-axons traverse this module. The analysis maps myelin-rich stripes in several specimens and concludes that based on their number and patterning they likely correspond with trunk folds; however, this conclusion is not well supported if the nucleus has been misidentified.

Strengths:<br /> The strength of this research lies in its comprehensive use of various anatomical methods, including Nissl staining, myelin staining, Golgi staining, cytochrome oxidase labeling, and synchrotron X-ray phase contrast tomography. The inclusion of quantitative data on cell numbers and sizes, dendritic orientation and morphology, and blood vessel density across the nucleus adds a quantitative dimension. Furthermore, the research is commendable for its high-quality and abundant images and figures, effectively illustrating the anatomy under investigation.

Weaknesses:<br /> While the research provides potentially valuable insights if revised to focus on the structure that appears to be the inferior olivary nucleus, there are certain additional weaknesses that warrant further consideration. First, the suggestion that myelin stripes solely serve to separate sensory or motor modules rather than functioning as an "axonal supply system" lacks substantial support due to the absence of information about the neuronal origins and the termination targets of the axons. Postmortem fixed brain tissue limits the ability to trace full axon projections. While the study acknowledges these limitations, it is important to exercise caution in drawing conclusions about the precise role of myelin stripes without a more comprehensive understanding of their neural connections.

Second, the quantification presented in the study lacks comparison to other species or other relevant variables within the elephant specimens (i.e., whole brain or brainstem volume). The absence of comparative data for different species limits the ability to fully evaluate the significance of the findings. Comparative analyses could provide a broader context for understanding whether the observed features are unique to elephants or more common across species. This limitation in comparative data hinders a more comprehensive assessment of the implications of the research within the broader field of neuroanatomy. Furthermore, the quantitative comparisons between African and Asian elephant specimens should include some measure of overall brain size as a covariate in the analyses. Addressing these weaknesses would enable a richer interpretation of the study's findings.

Reviewer #3 (Public Review):

Summary: This study investigated the role of mTORC1 and 2 in a mouse model of developmental epilepsy which simulates the epilepsy in cortical malformations. Given activation of genes such as PTEN activate TORC1, and this is considered to be excessive in cortical malformations, the authors asked whether inactivating mTORC1 and 2 would ameliorate the seizures and malformation in the mouse model. The work is highly significant because a new mouse model is used where Raptor and Rictor, which regulate mTORC1 and 2 respectively, were inactivated in one hemisphere of the cortex. The work is also significant because the deletion of both Raptor and Rictor improved the epilepsy and malformation. In the mouse model, the seizures were generalized or there were spike wave discharges (SWD). They also examined the interictal EEG. The malformation was manifested by increased cortical thickness and soma size.

Strengths: The presentation and writing is strong. Quality of data are strong. The data support the conclusions for the most part. The results are significant: Generalized seizures and SWDs were reduced when both Torc1 and 2 were inactivated but not when one was inactivated.

Weaknesses: One of the limitations is a somewhat small sample size. Another is that there was hippocampal expression. A third is that recordings of seizures were not continuous and different for each mouse. Another concern is they only measured layer II/III neurons.

Reviewer #3 (Public Review):

Summary:<br /> This work examined efference copy related to eye movements in healthy adults who have high autistic traits. Efference copies allow the brain to make predictions about sensory outcomes of self-generated actions, and thus serve important roles in motor planning and maintaining visual stability. Consequently, disrupted efference copies have been posited as a potential mechanism underlying motor and sensory symptoms in psychopathology such as Autism Spectrum Disorder (ASD), but so far very few studies have directly investigated this theory. Therefore, this study makes an important contribution as an attempt to fill in this knowledge gap. The authors conducted two eye-tracking experiments examining the accuracy of motor planning and visual perception following a saccade and found that participants with high autistic traits exhibited worse task performance (i.e., less accurate second saccade and biased perception of object displacement), consistent with their hypothesis of less impact of efference copies on motor and visual updating. Moreover, the motor and visual biases are positively correlated, indicative of a common underlying mechanism. These findings are promising and can have important implications for clinical intervention if they can be replicated in a clinical sample.

Strengths:<br /> The authors utilized well-established and rigorously designed experiments and sound analytic methods. This enables easy translations between similar work in non-human primates and humans and readily points to potential candidates for underlying neural circuits that could be further examined in follow-up studies (e.g., superior colliculus, frontal eye fields, mediodorsal thalamus). The finding of no association between initial saccade accuracy and level of autistic trait in both experiments also serves as an important control analysis and increases one's confidence in the conclusion that the observed differences in task performance were indeed due to disrupted efference copies, not confounding factors such as basic visual/motor deficits or issues with working memory. The strong correlation between the observed motor and visual biases further strengthens the claim that the findings from both experiments may be explained by the same underlying mechanism - disrupted efference copies. Lastly, the authors also presented a thoughtful and detailed mechanistic theory of how efference copy impairment may lead to ASD symptomatology, which can serve as a nice framework for more research into the role of efference copies in ASD.

Weaknesses:<br /> Although the paper has a lot of strengths, the main weakness of the paper is that a direct link with ASD symptoms (i.e., sensory overload and motor inflexibility as the authors suggested) cannot be established. First of all, the participants are all healthy adults who do not meet the clinical criteria for an ASD diagnosis. Although they could be considered a part of the broader autism phenotype, the results cannot be easily generalized to the clinical population without further research. Secondly, the measure used to quantify the level of autistic traits, Autistic Quotient (AQ), does not actually capture any sensory or motor symptoms of ASD. Therefore, it is unknown whether those who scored high on AQ in this study experienced high, or even any, sensory or motor difficulties. In other words, more evidence is needed to demonstrate a direct link between disrupted efference copies and sensory/motor symptoms in ASD.

Reviewer #3 (Public Review):

Observers make judgements about expected stimuli faster and more accurately. How expectations facilitate such perceptual decisions remains an ongoing area of investigation, however, as expectations may exert their effects in multiple ways. Expectations may directly influence the encoding of sensory signals. Alternatively (or additionally), expectations may influence later stages of decision-making, such as motor preparation, when they bear on the appropriate behavioral response.

In the present study, Walsh and colleagues directly measured the effect of expectations on sensory and motor signals by making clever use of the encephalogram (EEG) recorded from human observers performing a contrast discrimination task. On each trial, a predictive cue indicated which of two superimposed stimuli would likely be higher contrast and, therefore, whether a left or right button press was likely to yield a correct response. Deft design choices allowed the authors to extract both contrast-dependent sensory signals and motor preparation signals from the EEG. The authors provide compelling evidence that, when predictive cues provide information about both a forthcoming stimulus and the appropriate behavioral response, expectation effects are immediately manifest in motor preparation signals and only emerge in sensory signals after extensive training.

Future work should attempt to reconcile these results with related investigations in the field. As the authors note, several groups have reported expectation-induced modulation of sensory signals (using both fMRI and EEG/MEG) on shorter timescales (e.g. just one or two sessions of a few hundred trials, versus the intensive multi-session study reported here). One interesting possibility is that perceptual expectations are not automatic but demand the deployment of feature-based attention, while motor preparation is comparatively less effortful and so dominates when both sources of information are available, as in the present study. This hypothesis is consistent with the authors' thoughtful analysis showing decreased neural signatures of attention over posterior electrodes following predictive cues. Therefore, observing the timescale of sensory effects using the same design and methods (facilitating direct comparison with the present work), but altering task demands slightly such that cues are no longer predictive of the appropriate behavioral response, could be illuminating.

Reviewer #3 (Public Review):

Summary:<br /> This paper explores the relationships among evolutionary and epidemiological quantities in influenza, using a wide range of datasets and features, and using both correlations and random forests to examine, primarily, what are the drivers of influenza epidemics. It's a strong paper representing a thorough and fascinating exploration of potential drivers, and it makes a trove of relevant data readily available to the community.

Strengths:<br /> This paper makes links between epidemiological and evolutionary data for influenza. Placing each in the context of the other is crucial for understanding influenza dynamics and evolution and this paper does a thorough job of this, with many analyses and nuances. The results on the extent to which evolutionary factors relate to epidemic burden, and on interference among influenza types, are particularly interesting. The github repository associated with the paper is clear, comprehensive, and well-documented.

Weaknesses:<br /> The format of the results section can be hard to follow, and we suggest improving readability by restructuring and simplifying in some areas. There are a range of choices made about data preparation and scaling; the authors could explore sensitivity of the results to some of these.

Reviewer #3 (Public Review):

In this study, Ruan et al. investigate the role of the IQCH gene in spermatogenesis, focusing on its interaction with calmodulin and its regulation of RNA-binding proteins. The authors examined sperm from a male infertility patient with an inherited IQCH mutation as well as Iqch CRISPR knockout mice. The authors found that both human and mouse sperm exhibited structural and morphogenetic defects in multiple structures, leading to reduced fertility in Ichq-knockout male mice. Molecular analyses such as mass spectrometry and immunoprecipitation indicated that RNA-binding proteins are likely targets of IQCH, with the authors focusing on the RNA-binding protein HNRPAB as a critical regulator of testicular mRNAs. The authors used in vitro cell culture models to demonstrate an interaction between IQCH and calmodulin, in addition to showing that this interaction via the IQ motif of IQCH is required for IQCH's function in promoting HNRPAB expression. In sum, the authors concluded that IQCH promotes male fertility by binding to calmodulin and controlling HNRPAB expression to regulate the expression of essential mRNAs for spermatogenesis. These findings provide new insight into molecular mechanisms underlying spermatogenesis and how important factors for sperm morphogenesis and function are regulated.

The strengths of the study include the use of mouse and human samples, which demonstrate a likely relevance of the mouse model to humans; the use of multiple biochemical techniques to address the molecular mechanisms involved; the development of a new CRISPR mouse model; ample controls; and clearly displayed results. There are some minor weaknesses in that more background details could be provided to the reader regarding the proteins involved; some assays could benefit from more rigorous quantification; some of the mouse testis images and analyses could be improved; and larger sample sizes, especially for the male mouse breeding tests, could be increased. Overall, the claims made the authors in this manuscript are well-supported by the data provided, but there some technical issues that, if addressed, could increase the robustness and rigor of the study.

1. More background details are needed regarding the proteins involved, in particular IQ proteins and calmodulin. The authors state that IQ proteins are not well-represented in the literature, but do not state how many IQ proteins are encoded in the genome. They also do not provide specifics regarding which calmodulins are involved, since there are at least 5 family members in mice and humans. This information could help provide more granular details about the mechanism to the reader and help place the findings in context.

2. The mouse fertility tests could be improved with more depth and rigor. There was no data regarding copulatory plug rate; data was unclear regarding how many WT females were used for the male breeding tests and how many litters were generated; the general methodology used for the breeding tests in the Methods section was not very explicitly or clearly described; the sample size of n=3 for the male breeding tests is rather small for that type of assay; and, given that ICHQ appears to be expressed in testicular interstitial cells (Fig. S10) and somewhat in other organs (Fig. S2), another important parameter of male fertility that should be addressed is reproductive hormone levels (e.g., LH, FSH, and testosterone). While normal epididymal size in Fig. S3 suggests that hormone (testosterone) levels are normal, epididymal size and/or weight were not rigorously quantified.

3. The Western blots in Figure 6 should be rigorously quantified from multiple independent experiments so that there is stronger evidence supporting claims based on those assays.

4. Some of the mouse testis images could be improved. For example, the PNA and PLCz images in Figure S7 are difficult to interpret in that the tubules do not appear to be stage-matched, and since the authors claimed that testicular histology is unaffected in knockout testes, it should be feasible to stage-match control and knockout samples. Also, the anti-ICHQ and CaM immunofluorescence in Figure S10 would benefit from some cell-type-specific co-stains to more rigorously define their expression patterns, and they should also be stage-matched.

Reviewer #3 (Public Review):

In this study, the authors investigate the role of the Notch signalling regulator RBP-J on Ly6Clow monocyte biology starting with the observation that RBP-J-deficient mice have increased circulating Ly6low monocytes. Using myeloid specific conditional mouse models, the authors investigate how RBP-J deficiency effects circulating monocytes and lung interstitial macrophages.<br /> A major strength of this study is that it provides compelling evidence that RBP-J is a novel, critical factor regulating Ly6Clow monocyte cell frequency in the blood. The authors demonstrate that RBP-J deficiency leads to increased Ly6Clow monocytes in the blood and lung and CD16.2+ interstitial macrophages in steady state. The authors use a number of different techniques to confirm this finding including bone marrow transplantation experiments and parabiosis.

The main conclusion of the paper is that RBP-J controls the fate of Ly6ClowCCR2hi monocytes in a cell-intrinsic manner. This conclusion is strongly supported by the data provided. However, this paper is predominantly descriptive and further research is required to fully uncover the mechanisms by which RBP-J deficiency leads to Ly6Clo monocyte numbers increasing specifically in the blood and lungs and the consequence of RBP-J deficiency on Ly6C-low monocyte functionality.

The authors have performed RNA-seq and more in-depth analysis of this sequencing may provide clues for uncovering the thus far elusive mechanism.

Reviewer #3 (Public Review):

In this study, the authors utilized mass spectrometry-based quantification of polar metabolites and lipids in normal and cancerous tissue interstitial fluid and plasma. This showed that nutrient availability in tumor interstitial fluid was similar to that of interstitial fluid in adjacent normal kidney tissue, but that nutrients found in both interstitial fluid compartments were different from those found in plasma. This suggests that the nutrients in kidney tissue differ from those found in blood and that nutrients found in kidney tumors are largely dictated by factors shared with normal kidney tissue. Those data could be useful as a resource to support further study and modeling of the local environment of RCC and normal kidney physiology.

In Figures 1D and 1E, there were about 30% of polar metabolites and 25% of lipids significantly different between TIF and KIF, which could be key factors for RCC tumors. This reviewer considers that the authors should make comments on this.

https://www.levenger.com/products/vintage-library-catalog-cards-set-of-50?variant=43444256243861

These don't have the pre-drilled holes, but at least are still offered, though at the price gouging cost of $14.50 for 50 (in 2024).

Cost per card: $0.29

https://www.levenger.com/products/card-catalog-box?variant=43007544066197

A bit on the small side, but has a built in pen holder:

Reviewer #3 (Public Review):

Mohammed et al perform functional follow-up studies on the single nucleotide polymorphism rs6740960 located on chromosome 2p21 that was previously linked to lower jaw and chin shape variation and an increased risk of non-syndromic orofacial clefting. Through a combination of in silico multi-species alignment, in vitro enhancer marks, and finally in vivo data the team could confirm that the SNP is located in an active enhancer element driving transgene expression in the upper and lower jaw. The team tested the human and chimp orthologs in transgenic mice. Interestingly the mouse ought to look did not show any active enhancer activity in the LacZ reporter assay. Next, the authors could show a selective interaction of the enhancer element with the neighboring gene PKDCC in chondrocytes using H3K27ac HiChIP. Deletion of this enhancer in vitro led to an allele specific reduction of PKC expression. Finally, the authors aimed at evaluating the effect of rs6740960 in vivo using a mouse model. Since the enhancer sequence of the mouse did not show any positive reporter activity, the authors decided to use previously described Pkdcc full knockout mouse model (Kinoshita et al. 2009). Using sophisticated imaging technologies the authors were able to show that in mice several facial bones are Pkdcc dose sensitive.

Overall this is an extremely exciting manuscript that addresses one of the key challenges in the post GWAS time: the functional connection of lead SNPs to their target genes and a detailed evaluation of the biological and morphological consequences.<br /> The manuscript is well written, and the conclusions are completely supported by the evidence provided. I really think this is a great paper, however I have several major concerns with the manuscript and its current format.

Major comments:

1: My main concern about the manuscript in its current format is the disconnection between the beautiful work of linking rs6740960 to Pkdcc in the first part of the manuscript and the investigation of dose sensitivity of Pkdcc itself in end of the manuscript. While I realized that this is because the enhancer itself is not conserved between humans and mice, in my opinion it still weakens the novelty of the finding of the second part of the manuscript quite significantly. The Pkdcc knockout has been well described and that the authors now present evidence that also heterozygous knockouts show a minimal phenotype in the facial bones is really not surprising. More importantly it doesn't show how the rs6740960 influences Pkdcc expression in vivo.

A rather straightforward and very interesting experimental approach would be to replace the mouse enhancer sequence with the human or chimp enhancer carrying the risk allele or the wild type. In the last figure the authors have nicely shown that the entire experimental setup for the functional analysis of even minor changes to the facial bones caused by the SNP are available to the team. Even if the result was negative this experiment would significantly enhance the scientific impact of the paper.

2: Another option would be to repeat the LacZ reporter essay with the human wild type and the risk allele in direct comparison. A beautiful example of such an experiment was recently shown by Yanchuset et al (A noncoding single-nucleotide polymorphism at 8q24 drives IDH1-mutant glioma formation, Yanchuset al.,Science378,68-78 2022)

3: It is unclear how the H3K27ac HiChIP signal looks like at the Pkdcc locus in H9 ESC. What is the naïve interaction profile?

Reviewer #3 (Public Review):

This study represents a useful addition to the authors' previous study examining the effects of paternal high-fat diet on offspring metabolism and gene expression in offspring (PMID: 35183795). It differs from the previous study in some of the details of the experimental model (age of sire when exposed to the diet manipulation, mouse substrain, and the nature of the control diet) and the results are largely in line with previous findings. The major finding is that many genes at which sperm H3K4me3 signal is altered also have altered expression in the placenta; some of these genes are paternally imprinted, providing a paternal-specific epigenetic signature. Strengths of the study include establishment of an important dataset correlating the sperm epigenome with gene expression in placental tissue, leading to an interesting and provocative conclusion. Weaknesses include a relatively superficial analysis of the dataset, revealing broad patterns but few specific conclusions, reliance on correlative analysis to draw conclusions, and absence of validation studies. Deconvolution analysis of bulk RNA-seq data helps to account for differences in cell composition between placental datasets, but does not add additional insight toward the central question of how sperm epigenetic state contributes to offspring gene expression. Overall the advance over previous work is relatively small.

Specific points:

1) The analysis as it stands is limited. To compare sperm H3K4me3 and placental expression, numbers of overlapping genes are provided, but no statistical analysis is done to indicate the significance of the overlap.

2) There is little direct connection to biological systems or validation of differential enrichment/expression analysis. Gene ontology enrichments for genes differentially enriched for H3K4me3 in sperm or differentially expressed in placenta (broken up by sex) are performed, but the biological significance of these categories is not clear.

3) The overall effect size is small. In most cases the magnitude of differences is minor, and it is not clear which of these changes are significant over noise. For example, the y-axis for the metagene plots in Figure 2B does not start at zero, so the total range of the difference in H3K4me3 is small. In Figure 6C, DEGs detected in hypoxic placenta after deconvolution analysis do not look very different compared to control.

4) Deconvolution analysis was done on bulk RNA-seq data from placenta, and the numbers of DEGs identified with this analysis compared to the original analysis are shown, but is not clear how the deconvolution analysis changes the specific biological conclusions. In addition, the reference dataset for deconvolution is a published dataset generated in another lab, and it is unclear how comparable the reference sample is to the samples analyzed in this study, or how robust this analysis is when using a dataset generated under different conditions.

Reviewer #3 (Public Review):

Summary:<br /> In this manuscript, Verma et al. set out to visualize cytoplasmic dynein in living cells and describe their behaviour. They first generated heterozygous CRISPR-Cas9 knock-ins of DHC1 and p50 subunit of dynactin and used spinning disk confocal microscopy and TIRF microscopy to visualize these EGFP-tagged molecules. They describe robust localization and movement of DHC and p50 at the plus tips of MTs, which was abrogated using SiR tubulin to visualize the pool of DHC and p50 on the MTs. These DHC and p50 punctae on the MTs showed similar, highly processive movement on MTs. Based on comparison to inducible EGFP-tagged kinesin-1 intensity in Drosophila S2 cells, the authors concluded that the DHC and p50 punctae visualized represented 1 DHC-EGFP dimer+1 untagged DHC dimer and 1 p50-EGFP+3 untagged p50 molecules.

Strengths:<br /> The idea and motivation behind this work are commendable.

Weaknesses:<br /> There are several major issues with the characterization of the knock-in lines generated, the choice of imaging and analysis methods, and inadequate discussion of prior findings.

The specific points are below:

1. CRISPR-edited HeLa clones:<br /> (i) The authors indicate that both the DHC-EGFP and p50-EGFP lines are heterozygous and that the level of DHC-EGFP was not measured due to technical difficulties. However, quantification of the relative amounts of untagged and tagged DHC needs to be performed - either using Western blot, immunofluorescence or qPCR comparing the parent cell line and the cell lines used in this work.<br /> (ii) The localization of DHC predominantly at the plus tips (Fig. 1A) is at odds with other work where endogenous or close-to-endogenous levels of DHC were visualized in HeLa cells and other non-polarized cells like HEK293, A-431 and U-251MG (e.g.: OpenCell (https://opencell.czbiohub.org/target/CID001880), Human Protein Atlas (https://www.proteinatlas.org/ENSG00000197102-DYNC1H1/subcellular#human), https://www.biorxiv.org/content/10.1101/2021.04.05.438428v3). The authors should perform immunofluorescence of DHC in the parental cells and DHC-EGFP cells to confirm there are no expression artifacts in the latter. Additionally, a comparison of the colocalization of DHC with EB1 in the parental and DHC-EGFP and p50-EGFP lines would be good to confirm MT plus-tip localisation of DHC in both lines.<br /> (iii) It would also be useful to see entire fields of view of cells expressing DHC-EGFP and p50-EGFP (e.g. in Spinning Disk microscopy) to understand if there is heterogeneity in expression. Similarly, it would be useful to report the relative levels of expression of EGFP (by measuring the total intensity of EGFP fluorescence per cell) in those cells employed for the analysis in the manuscript.<br /> (iv) Given that the authors suspect there is differential gene regulation in their CRISPR-edited lines, it cannot be concluded that the DHC-EGFP and p50-EGFP punctae tracked are functional and not piggybacking on untagged proteins. The authors could use the FKBP part of the FKBP-EGFP tag to perform knock-sideways of the DHC and p50 to the plasma membrane and confirm abrogation of dynein activity by visualizing known dynein targets such as the Golgi (Golgi should disperse following recruitment of EGFP-tagged DHC-EGFP or p50-EGFP to the PM), or EGF (movement towards the cell center should cease).

2. TIFRM and analysis:<br /> (i) What was the rationale for using TIRFM given its limitation of visualization at/near the plasma membrane? Are the authors confident they are in TIRF mode and not HILO, which would fit with the representative images shown in the manuscript?<br /> (ii) At what depth are the authors imaging DHC-EGFP and p50-EGFP?<br /> (iii) The authors rely on manual inspection of tracks before analyzing them in kymographs - this is not rigorous and is prone to bias. They should instead track the molecules using single particle tracking tools (eg. TrackMate/uTrack), and use these traces to then quantify the displacement, velocity, and run-time.<br /> (iv) It is unclear how the tracks that were eventually used in the quantification were chosen. Are they representative of the kind of movements seen? Kymographs of dynein movement along an entire MT/cell needs to be shown and all punctae that appear on MTs need to be tracked, and their movement quantified.<br /> (v) What is the directionality of the moving punctae?<br /> (vi) Since all the quantification was performed on SiR tubulin-treated cells, it is unclear if the behavior of dynein observed here reflects the behavior of dynein in untreated cells. Analysis of untreated cells is required.

3. Estimation of stoichiometry of DHC and p50<br /> Given that the punctae of DHC-EGFP and p50 seemingly bleach on MT before the end of the movie, the authors should use photobleaching to estimate the number of molecules in their punctae, either by simple counting the number of bleaching steps or by measuring single-step sizes and estimating the number of molecules from the intensity of punctae in the first frame.

4. Discussion of prior literature<br /> Recent work visualizing the behavior of dyneins in HeLa cells (DOI: 10.1101/2021.04.05.438428), which shows results that do not align with observations in this manuscript, has not been discussed. These contradictory findings need to be discussed, and a more objective assessment of the literature in general needs to be undertaken.

Reviewer #3 (Public Review):

Summary:<br /> Using a protein degradation approach, Eaton et al show that INST11 can terminate the sense and anti-sense transcription but higher activity of CDK9 in the sense direction protects it from INS11-dependent termination. They developed sPOINT-seq that detects nascent 5'-capped RNA. The technique allowed them to reveal robust transcription initiation of sense-RNA as compared to anti-sense.

Strengths:<br /> The strength of the paper is the acute degradation of proteins, eliminating the off-target effects. Further, the paper uses elegant approaches such as POINT and sPOINT-seq to measure nascent RNA and 5'-capped short RNA. Together, the combination of these three allowed the authors to make clean interpretations of data.

Weaknesses:<br /> While the manuscript is well written, the details on the panel are not sufficient. The methods could be elaborated to aid understanding. Additional discussion on howthe authors' findings contradict the existing model of anti-sense transcription termination should be added.

Reviewer #3 (Public Review):

Summary:

The manuscript by Flaherty III S.E. et al identified SPAG7 gene in their forward mutagenetic screening and created the germline knockout and inducible knockout mice. The authors reported that the SPAG7 germline knockout mice had lower birth weight likely due to intrauterine growth restriction and placental insufficiency. The SPAG7 KO mice later developed obesity phenotype as result of reduced energy expenditure. However, the inducible SPAG7 knockout mice had normal body weight and composition.

Strengths:

In this reviewer's opinion, this study has high significance in the field of metabolic research for the following reasons.

1) The authors' findings are significant in the field of obesity research, especially from the perspective of maternal-fetal medicine. The authors created and analyzed the SPAG7 KO mice and found that the KO mice had a "thrifty phenotype" and developed obesity.

2) SPAG7 gene function hasn't been thoroughly studied. The reported phenotype will fill the gap of knowledge.

Overall, the authors have presented their results in a clear and logically organized structure, clearly stated the key question to be addressed, used the appropriate methodology, produced significant and innovative main findings.

Comments on revised version:

The authors have satisfactorily addressed my previous concerns.

Reviewer #3 (Public Review):

Summary:<br /> Radial muscle growth involves an increase in overall muscle cross-sectional area. For decades this process has been described as the splitting of myofibrils to produce more myofibrils during the growth process. However, a closer look at the original papers shows that the evidence underlying this description was incomplete. In this paper, the authors have developed a novel method using fluorescence microscopy to directly measure myofibril size and number. Using a mouse model of mechanical loading and a human model of resistance exercise they discovered that myofibrillogenesis is playing a key role in the radial growth of muscle fibers.

Strengths:<br /> 1. Well-written and clear description of hypothesis, background, and experiments.<br /> 2. Compelling series of experiments.<br /> 3. Different approaches to test the hypothesis.<br /> 4. Rigorous study design.<br /> 5. Clear interpretation of results.<br /> 6. Novel findings that will be beneficial to the muscle biology field.<br /> 7. Innovative microscopy methods that should be widely available for use in other muscle biology labs.

Reviewer #3 (Public Review):

Summary:<br /> Previous research on the Drosophila mushroom body (MB) has made this structure the best-understood example of an associative memory center in the animal kingdom. This is in no small part due to the generation of cell-type specific driver lines that have allowed consistent and reproducible genetic access to many of the MB's component neurons. The manuscript by Shuai et al. now vastly extends the number of driver lines available to researchers interested in studying learning and memory circuits in the fly. It is an 800-plus collection of new cell-type specific drivers target neurons that either provide input (direct or indirect) to MB neurons or that receive output from them. Many of the new drivers target neurons in sensory pathways that convey conditioned and unconditioned stimuli to the MB. Most drivers are exquisitely selective, and researchers will benefit from the fact that whenever possible, the authors have identified the targeted cell types within the Drosophila connectome. Driver expression patterns are beautifully documented and are publicly available through the Janelia Research Campus's Flylight database where full imaging results can be accessed. Overall, the manuscript significantly augments the number of cell type-specific driver lines available to the Drosophila research community for investigating the cellular mechanisms underlying learning and memory in the fly. Many of the lines will also be useful in dissecting the function of the neural circuits that mediate sensorimotor circuits.

Strengths:<br /> The manuscript represents a huge amount of careful work and leverages numerous important developments from the last several years. These include the thousands of recently generated split-Gal4 lines at Janelia and the computational tools for pairing them to make exquisitely specific targeting reagents. In addition, the manuscript takes full advantage of the recently released Drosophila connectomes. Driver expression patterns are beautifully illustrated side-by-side with corresponding skeletonized neurons reconstructed by EM. A comprehensive table of the new lines, their split-Gal4 components, their neuronal targets, and other valuable information will make this collection eminently useful to end-users. In addition to the anatomical characterization, the manuscript also illustrates the functional utility of the new lines in optogenetic experiments. In one example, the authors identify a specific subset of sugar reward neurons that robustly promotes associative learning.

Weaknesses:<br /> While the manuscript succeeds in making a mass of descriptive detail quite accessible to the reader, the way the collection is initially described - and the new lines categorized - in the text is sometimes confusing. Most of the details can be found elsewhere, but it would be useful to know how many of the lines are being presented for the first time and have not been previously introduced in other publications/contexts. And where can the lines be found at Flylight? Are they listed as one collection or as many? Also, the authors say that some of the lines were included in the collection despite not necessarily targeting the intended type of neuron (presumably one that is involved in learning and memory). What percentage of the collection falls into this category? And what about the lines that the authors say they included in the collection despite a lack of specificity? How many lines does this represent?

Reviewer #3 (Public Review):

Summary:

Te Rietmolen et al., investigated the selectivity of cortical responses to speech and music stimuli using neurosurgical stereo EEG in humans. The authors address two basic questions: 1. Are speech and music responses localized in the brain or distributed; 2. Are these responses selective and domain-specific or rather domain-general and shared? To investigate this, the study proposes a nomenclature of shared responses (speech and music responses are not significantly different), domain selective (one domain is significant from baseline and the other is not), domain preferred (both are significant from baseline but one is larger than the other and significantly different from each other). The authors employ this framework using neural responses across the spectrum (rather than focusing on high gamma), providing evidence for a low level of selectivity across spectral signatures. To investigate the nature of the underlying representations they use encoding models to predict neural responses (low and high frequency) given a feature space of the stimulus envelope or peak rate (by time delay) and find stronger encoding for both in the low-frequency neural responses. The top encoding electrodes are used as seeds for a pair-wise connectivity (coherence) in order to repeat the shared/selective/preferred analysis across the spectra, suggesting low selectivity. Spectral power and connectivity are also analyzed on the level of the regional patient population to rule out (and depict) any effects driven by a select few patients. Across analyses the authors consistently show a paucity of domain selective responses and when evident these selective responses were not represented across the entire cortical region. The authors argue that speech and music mostly rely on shared neural resources.

Strengths:

I found this manuscript to be rigorous providing compelling and clear evidence of shared neural signatures for speech and music. The use of intracranial recordings provides an important spatial and temporal resolution that lends itself to the power, connectivity, and encoding analyses. The statistics and methods employed are rigorous and reliable, estimated based on permutation approaches, and cross-validation/regularization was employed and reported properly. The analysis of measures across the entire spectra in both power, coherence, and encoding models provides a comprehensive view of responses that no doubt will benefit the community as an invaluable resource. Analysis of the level of patient population (feasible with their high N) per region also supports the generalizability of the conclusions across a relatively large cohort of patients. Last but not least, I believe the framework of selective, preferred, and shared is a welcome lens through which to investigate cortical function.

Weaknesses:

I did not find methodological weaknesses in the current version of the manuscript. I do believe that it is important to highlight that the data is limited to passively listening to naturalistic speech and music. The speech and music stimuli are not completely controlled with varying key acoustic features (inherent to the different domains). Overall, I found the differences in stimulus and lack of attentional controls (passive listening) to be minor weaknesses that would not dramatically change the results or conclusions.

Reviewer #3 (Public Review):

Summary:<br /> This paper explores the cost of toxin resistance in snakes that prey on newts defended by highly potent TTX. Two species of garter snakes, T. atratus and T. sirtalis, are examined. Both species have resistant and sensitive populations. Resistance is achieved by substitutions in the voltage-gated sodium channels, which block TTX binding. Resistant T. atratus carry the triple substitutions EPN while resistant T. sirtalis carry the quadruple LVNV. These substitutions occur on the third and fourth intracellular domains of the voltage-gated sodium-channel gene Nav1.4, which is the paralog found in skeletal muscle. EPN and LVNV have been previously attributed to conferring resistance to TXX through target-site insensitivity of the channel. Previous work has also shown that snakes from resistant populations have reduced locomotor capabilities compared to their non-resistant counterparts.

The authors systematically test the hypothesis that the resistance-conferring substitutions affect other phenotypes related to the function of the voltage-gated sodium channel, which is, in turn, responsible for the reduced locomotor capabilities. First, they compare the effects of EPN and LVNV on recombinantly expressed rat Nav1.4 with and without EPN and LVNV (in vitro). They find that both EPN and LVNV significantly reduce the channel's conductance. On top of that, LVNV also causes premature deactivation of the channel, thus reducing the current passing through the membrane. Next, they compare muscle tissue function between resistant and non-resistant populations of T. atratus and T. sirtalis (ex vivo). They find that both resistant populations have reduced twitch force (with T. sirtalis, carrying LVNV, having an even stronger reduction), reduced peak rate of force development, and overall reduced force. In addition, T. sirtalis (LVNV) muscle also has reduced peak tetanic force. Finally, they compare the biophysical effects of EPN and LVNV through homology modeling of Nav1.4 to explain the in vitro and in vivo results (in silico). They found that E1248 (of EPN) has a counteracting effect on the destabilizing effect of N1539, shared by both species. T. sirtalis (LVNV) lacks such a counteracting mutation, which could explain the stronger negative effects observed in LVNV channels and muscles.

Strengths:<br /> A particular strength of this paper is the multi-level approach used to tease apart the negative pleiotropic effects of resistance-conferring substitutions. Each level of experiments informed the next, creating a focused comprehensive analysis of the costs associated with this specialized dietary adaptation in snakes. The results make an important contribution to our understanding of the role of negative pleiotropy in adaptive evolution and would be of broad interest to readers. The paper is well-written, and the data and analyses are clearly presented.

Weaknesses:<br /> The sheer size of the Nav1.4 gene makes it difficult to clone into an expression vector and that's probably why an already cloned rat Nav1.4 was selected for the in vitro experiments. It would be great if the authors could comment on how the level of resistance produced by mutations on the rat Nav1.4 compared to the garter snake Nav1.4s. Are there previous data on tissue-isolated T. sirtalis and T. atratus channels? Is it possible that the snake mutations have slightly different effects on the rat genetic background due to epistatic interactions with sites beyond the 3rd and 4th domains?

Following up on the first comment, sometimes negative pleiotropic effects are mitigated by compensatory mutations in other regions of the protein. This reviewer would recommend that the authors comment on this possibility. Are there substitutions beyond the 3rd and 4th domains that could potentially play a role in this adaptation?

Based on the results, it seems that resistant T. sirtalis got the shorter end of the stick concerning negative pleiotropic effects, despite having similar (the same?) levels of resistance to TTX. Does this difference/disadvantage scale up to locomotor performance as well?

It would be great if the authors could comment on how these resistant populations have persisted despite the locomotor/muscular disadvantages. Are there known differences in predation rates between the populations? The benefit must have outweighed the cost in these cases.

Reviewer #3 (Public Review):

Summary:<br /> The authors of this study aim to develop OpenNucleome, a computational tool designed to simulate the structure and dynamics of the human nucleus. This software models nuclear components like chromosomes and nuclear bodies, leveraging GPU acceleration for improved performance. The key objective is to enhance our understanding of nuclear organization, providing a tool that aligns with experimental data and is accessible to the genome architecture scientific community.

Strengths:<br /> OpenNucleome offers a detailed and dynamic model of the nucleus, a significant step forward in computational biology.

The integration of GPU acceleration with the OpenMM package is a good technical advancement, potentially enhancing performance.

The comparison with experimental data adds credibility to the tool's accuracy and relevance.

Weaknesses:<br /> The lack of comprehensive tutorials and clear documentation on the OpenNucleome GitHub page is a considerable barrier to accessibility and user-friendliness.

The process for generating necessary input files is not adequately explained, which could hinder the tool's practical application.

The paper could benefit from more explicit explanations on the standardization of practices and cross-validation with existing tools like OpenMiChroM.

Reviewer #3 (Public Review):

Summary:<br /> In the manuscript "Overflow metabolism originates from growth optimization and cell heterogeneity" the author Xin Wang investigates the hypothesis that the transition into overflow metabolism at large growth rates actually results from an inhomogeneous cell population, in which every individual cell either performs respiration or fermentation.

Weaknesses:<br /> The paper has several major flaws. First, and most importantly, it repeatedly and wrongly claims that the origins of overflow metabolism are not known. The paper is written as if it is the first to study overflow metabolism and provide a sound explanation for the experimental observations. This is obviously not true and the author actually cites many papers in which explanations of overflow metabolism are suggested (see e.g. Basan et al. 2015, which even has the title "Overflow metabolism in E. coli results from efficient proteome allocation"). The paper should be rewritten in a more modest and scientific style, not attempting to make claims of novelty that are not supported. In fact, all hypotheses in this paper are old. Also the possiblility that cell heterogeneity explains the observed 'smooth' transition into overflow metabolism has been extensively investigated previously (see de Groot et al. 2023, PNAS, "Effective bet-hedging through growth rate dependent stability") and the random drawing of kcat-values is an established technique (Beg et al., 2007, PNAS, "Intracellular crowding defines the mode and sequence of substrate uptake by Escherichia coli and constrains its metabolic activity"). Thus, in terms of novelty, this paper is very limited. It reinvents the wheel and it is written as if decades of literature debating overflow metabolism did not exist.

Moreover, the manuscript is not clearly written and is hard to understand. Variables are not properly introduced (the M-pools need to be discussed, fluxes (J_E), "energy coefficients" (eta_E), etc. need to be more explicitly explained. What is "flux balance at each intermediate node"? How is the "proteome efficiency" of a pathway defined? The paper continues to speak of energy production. This should be avoided. Energy is conserved (1st law of thermodynamics) and can never be produced. A scientific paper should strive for scientific correctness, including precise choice of words.

The statement that the "energy production rate ... is proportional to the growth rate" is, apart from being incorrect - it should be 'ATP consumption rate' or similar (see above), a non-trivial claim. Why should this be the case? Such statements must be supported by references. The observation that the catabolic power indeed appears to increase linearly with growth rate was made, based on chemostat data for E.coli and yeast, in a recent preprint (Ebenhöh et al, 2023, bioRxiv, "Microbial pathway thermodynamics: structural models unveil anabolic and catabolic processes").

All this criticism does not preclude the possibility that cell heterogeneity plays a role in overflow metabolism. However, according to Occam's razor, first the simpler explanations should be explored and refuted before coming up with a more complex solution. Here, it means that the authors first should argue why simpler explanations (e.g. the 'Membrane Real Estate Hypothesis', Szenk et al., 2017, Cell Systems; maximal Gibbs free energy dissipation, Niebel et al., 2019, Nature Metabolism; Saadat et al., 2020, Entropy) are not considered, resp. in what way they are in disagreement with observations, and then provide some evidence of the proposed cell heterogeneity (are there single-cell transcriptomic data supporting the claim?).

Reviewer #3 (Public Review):

The manuscript by Agha et al. explores mechanisms of rhythmicity in V2a neurons in larval zebrafish. Two subpopulations of V2a neurons are distinguishable by anatomy, connectivity, level of GFP, and speed-dependent recruitment properties consistent with V2a neurons involved in rhythm generation and pattern formation. The descending neurons proposed to be consistent with rhythm-generating neurons are active during either slow or fast locomotion, and their firing frequencies during current steps are well matched with the swim frequency they firing during. The bifurcating (patterning neurons) are active during a broader swim frequency range unrelated to their firing during current steps. All of the V2a neurons receive strong inhibitory input but the phasing of this input is based on neuronal type and swim speed when the neuron is active, with prominent in-phase inhibition in slow descending V2a neurons and bifurcating V2a neurons active during fast swimming. Antiphase inhibition is observed in all V2a neurons but it is the main source of rhythmic inhibition in fast descending V2a neurons and bifurcating neurons active during slow swimming. The authors suggest that properties supporting rhythmic bursting are not directly related to locomotor speed but rather to functional neuronal subtypes.

This is a well-written paper with many strengths including the rigorous approach. Many parameters, including projection pattern, intracellular properties, inhibition received, and activity during slow/fast swimming were obtained from the same neuron. This links up very well with prior data from the lab on cell position, birth order, morphology/projections, and control of MN recruitment to provide a comprehensive overview of the functioning of V2a interneuronal populations in the larval zebrafish. The overall conclusions are well supported by the data. Weaknesses are relatively minor and were largely related to terminology for some of the secondary conclusions.

1. The assumption is made that all in-phase inhibition is recurrent and out-of-phase inhibition is reciprocal. The latter is likely true but the definition of recurrent may be a bit loose as could be multisegmental feed-forward inhibition as well.

2. In a few places, it is mentioned that the properties of the V2a-D neurons are consistent with pacemakers. This could be true of both the V2a-D and -B neurons that burst in response to depolarizing steps but the properties of the remaining (fast) V2a-D neurons do not seem to be consistent with pacemakers, based on the properties shown. Tonic firing at a frequency related to the locomotor speed the neuron is active during and strong antiphase inhibition may instead suggest a stronger network component driving the rhythmicity.

Reviewer #3 (Public Review):

This manuscript presents a comprehensive investigation into the mechanisms that explain the presence of TADs (P-TADs) in cells where cohesin has been removed. In particular, to study TADs in wildtype and cohesin depleted cells, the authors use a combination of polymer simulations to predict whole chromosome structures de novo and from Hi-C data. Interestingly, they find that those TADs that survive cohesin removal contain a switch in epigenetic marks (from compartment A to B or B to A) at the boundary. Additionally, they find that the P-TADs are needed to retain enhancer-promoter and promoter-promoter interactions.

Overall, the study is well-executed, and the evidence found provides interesting insights into genome folding and interpretations of conflicting results on the role of cohesin on TAD formation.

Reviewer #3 (Public Review):

Summary: The manuscript by Erice et al describes let-7 miRNA promotes Tc17 differentiation and emphysema by repressing the transcription factor RORgt. The authors found that overall expression of the let-7 miRNA clusters, let-7b/let-7c2 and let-7a1/let-30 7f1/let-7d are reduced in the lungs and T cells of mice with cigarette smoke-induced emphysema. They also found that the loss of the let-7b/let-7c2-cluster in T cells exaggerated cigarette smoke-induced emphysema. It appears that deletion of the let-7b/let-7c2-cluster lead to enhancement of IL-17-secreting CD8+ T cells (Tc17) in mice with emphysema. The opposite phenotype was observed when let-7 was overexpressed in T cells. They found a potential let-7 binding site in the 3' UTR of RORgt. They demonstrated a direct effect of let-7 on RORgt expression using let-7 mimic in a RORgt luciferase reporter assay. They have done an outstanding job of translating the finding of reduced let-7 expression in emphysema patients to a thorough delineation of its mechanism in a mouse model. Together, this study suggests an important role for let-7 miRNA in Tc17 cells in emphysema which appears to be mediated via repression of RORgt.

Strengths: This well written manuscript flows logically and the data supports the overall claim let-7 miRNA promotes Tc17 differentiation during emphysema. There are several strengths to this study including the use of conditional let-7 knock out animals to decipher the role of this miRNA in Tc17 cells in emphysema.

Weaknesses: There are no major weaknesses in this study. It would be interesting to see if knockdown RORgt could rescue enhanced Tc17 differentiation seen in let-7b/let-7c2-cluster-deficient T cells. The authors show no change in frequencies of Treg cells in let-7bc2LOF mice exposed to nCB. Do these Treg cells also express higher levels of RORgt and IL-17? The major question that was not addressed in this study is how let-7 expression is regulated in emphysema. The other recommendation is that the authors include the sequences of the let-7 mimic oligos used in the luciferase assay.

Reviewer #3 (Public Review):

Summary:<br /> Adamic and colleagues present fMRI data from ADE patients and a healthy control group acquired during two interoceptive tasks (attention and perturbation) from the same session. They report convergent activity within the granular and dysgranular insular cortex during both tasks, with a patient group-specific lateralisation effect. Furthermore, insular functional connectivity was found to be linked to disease severity.

Strengths:<br /> The study is well-designed and - despite some limitations noted by the authors - provides much-needed insight into the functional pathways of interoceptive processing in health and disease. The manuscript is clear, concise, and well-written so that I only have a few comments I would mostly regard as minor points.

Weaknesses:<br /> There are a few instances where it is not entirely clear whether the authors' claims are fully supported by the underlying statistics.

Reviewer #3 (Public Review):

Summary:<br /> Here the authors show global synchronization of cerebral blood flow (CBF) induced by oscillating visual stimuli in the mouse brain. The study validates the use of endogenous autofluorescence to quantify the vessel "shadow" to assess the magnitude of frequency-locked cerebral blood flow changes. This approach enables straightforward estimation of artery diameter fluctuations in wild-type mice, employing either low magnification wide-field microscopy or deep-brain fibre photometry. For the visual stimuli, awake mice were exposed to vertically oscillating stripes at a low temporal frequency (0.25 Hz), resulting in oscillatory changes in artery diameter synchronized to the visual stimulation frequency. This phenomenon occurred not only in the primary visual cortex but also across a broad cortical and cerebellar surface. The induced CBF changes adapted to various stimulation parameters, and interestingly, repeated trials led to plastic entrainment. The authors control for different artefacts that may have confounded the measurements such as light contamination and eye movements but found no influence of these variables. The study also tested horizontally oscillating visual stimuli, which induce the horizontal optokinetic response (HOKR). The amplitude of eye movement, known to increase with repeated training sessions, showed a strong correlation with CBF entrainment magnitude in the cerebellar flocculus. The authors suggest that parallel plasticity in CBF and neuronal circuits is occurring. Overall, the study proposes that entrained "vasomotion" contributes to meeting the increased energy demand associated with coordinated neuronal activity and subsequent neuronal circuit reorganization.

Strengths:<br /> -The paper describes a simple and useful method for tracking vasomotion in awake mice through an intact skull.<br /> -The work controls for artefacts in their primary measurements.<br /> -There are some interesting observations, including the nearly brain-wide synchronization of cerebral blood flow oscillations to visual stimuli and that this process only occurs after mice are trained in a visual task.<br /> -This topic is interesting to many in the CBF, functional imaging, and dementia fields.

Weaknesses:<br /> -I have concerns with the main concepts put forward, regarding whether the authors are actually studying vasomotion as they state, as opposed to functional hyperemia which is sensory-induced changes in blood flow, which is what they are actually doing. I recommend several additional experiments/analyses for them to explore. This is mostly further characterizing their effect which will benefit the interpretations.

-Neuronal calcium imaging would also benefit the study and improve the interpretations.

-The plastic effects in vasomotion synchronization that occur with training are interesting but they could use an additional control for stress. Is this really a plastic effect, or is it caused by progressively decreasing stress as trials and progress? I recommend a habituation control experiment.

Appraisal<br /> I think the authors have an interesting effect that requires further characterization and controls. Their interpretations are likely sound and additional experiments will continue to support the main hypothesis. If brain-wide synchrony of blood flow can be trained and entrained by external stimuli, this may have interesting therapeutic potential to help clear out toxic proteins from the brain as seen in several neurodegenerative diseases.

Reviewer #3 (Public Review):

Summary:<br /> The study provides a detailed analysis of the chromosomal rearrangements related to the deletions of histidine-rich protein 2 (pfhrp2) and pfhrp3 genes in P. falciparum that have clinical significance since malaria rapid diagnostic tests detect these parasite proteins. A large number of publicly available short sequence reads for the whole genome of the parasite were analyzed, and data on coverage and discordant mapping allowed the authors to identify deletions, duplications, and chromosomal rearrangements related to pfhrp3 deletions. Long-read sequences showed support for the presence of a normal chromosome 11 and a hybrid 13-11 chromosome lacking pfhrp3 in some of the pfhrp3-deleted parasites. The findings support that these translocations have repeatedly occurred in natural populations. The authors discuss the implications of these findings and how they do or do not support previous hypotheses on the emergence of these deletions and the possible selective pressures involved.

Strengths:<br /> The genomic regions where these genes are located are challenging to study since they are highly repetitive and paralogous and the use of long-read sequencing allowed to span the duplicated regions, giving support to the identification of the hybrid 13-11 chromosome.

All publicly available whole-genome sequences of the malaria parasite from around the world were analysed which allowed an overview of the worldwide variability, even though this analysis is biased by the availability of sequences, as the authors recognize.

Despite the reduced sample size, the detailed analysis of haplotypes and identification of the location of breakpoints gives support to a single origin event for the 13-5++ parasites.

The analysis of haplotype variation across the duplicated chromosome-11 segment identified breakpoints at varied locations that support multiple translocation events in natural populations. The authors suggest these translocations may be occurring at high frequency in meiosis in natural populations but are strongly selected against in most circumstances, which remains to be tested.

Weaknesses:<br /> Relying on sequence data publicly available, that were collected based on diagnostic test positivity and that are limited by sequencing availability, limits the interpretation of the occurrence and relative frequency of the deletions. In the discussion, caution is needed when identifying the least common and most common mechanisms and their geographical associations. The identification of only one type of deletion pattern for Pfhrp2 may be related to these biases.

The specific objectives of the study are not stated clearly, and it is sometimes difficult to know which findings are new to this study. Is it the first study analyzing all the worldwide available sequences? Is it the first one to do long-read sequencing to span the entire duplicated region?

Another aspect that should be explained in the introduction is that there was previous information about the association of the deletions to patterns found in chromosomes 5 and 11. In the short-read sequences results, it is not clear if these chromosomes were analysed because of the associations found in this study (and no associations were found to putative duplications or deletions in other chromosomes), or if they were specifically included in the analysis because of the previous information (and the other chromosomes were not analysed).

An interesting statement in the discussion is that existing pfhrp3 deletions in a low-transmission environment may provide a genetic background on which less frequent pfhrp2 deletion events can occur. Does it mean that the occurrence of pfhrp3 deletions would favor the pfhrp2 deletion events? How, and is there any evidence for that?

Reviewer #3 (Public Review):

This neuroimaging study investigated how brain activity related to visual pattern-based reasoning changes over the adult lifespan, addressing the topic of functional compensation in older age. To this end, the authors employed a version of the Cattell task, which probes visual pattern recognition for identifying commonalities and differences within sets of abstract objects in order to infer the odd object among a given set. Using a state-of-the-art univariate analysis approach on fMRI data from a large lifespan sample, the authors identified brain regions in which the activation contrast between hard and easy Cattell task conditions was modulated by both age and performance. Regions identified comprised prefrontal areas and bilateral cuneus. Applying a multivariate decoding approach to activity in these regions, the authors went on to show that only in older adults, the cuneus, but not the prefrontal regions, carried information about the task condition (hard vs. easy) beyond that already provided by activity patterns of voxels that showed a univariate main effect of task difficulty. This was taken as compelling evidence for task-specific compensatory activity in the cuneus in advanced age.

The study is well-motivated and well-written. The authors used appropriate, rigorous methods that allowed them to control for a range of possible confounds or alternative explanations. Laudable aspects include the large sample with a wide and even age distribution, the validation of the in-scanner task performance against previous results obtained with a more standard version outside the scanner, and the control for vascular age-related differences in hemodynamic activity via a BOLD signal amplitude measure obtained from a separate resting-state fMRI scan. Overall, the conclusions are well-supported by the data.

In the following, I list some points of discussion that I would like to see addressed by the authors in a revision:

1) I don't quite follow the argumentation that compensatory recruitment would need to show via non-redundant information carried by any given non-MDN region (cf. p14). Wouldn't the fact that a non-MDN region carries task-related information be sufficient to infer that it is involved in the task and, if activated increasingly with increasing age, that its stronger recruitment reflects compensation, rather than inefficiency or dedifferentiation? Put differently, wouldn't "more of the same" in an additional region suffice to qualify as compensation, as compared to the "additional information in an additional region" requirement set by the authors? As a consequence, in my honest opinion, showing that decoding task difficulty from non-MDN ROIs works better with higher age would already count as evidence for compensation, rather than asking for age-related increases in decoding boosts obtained from adding such ROIs. It would be interesting to see whether the arguably redundant frontal ROI would satisfy this less demanding criterion. At any rate, it seems useful to show whether the difference in log evidence for the real vs. shuffled models is also related to age.

2) Relatedly, does the observed boost in decoding by adding the cuneal ROI (in older adults) really reflect "additional, non-redundant" information carried by this ROI? Or could it be that this boost is just a statistical phenomenon that is obtained because the cuneus just happens to show a more clear-cut, less noisy difference in hard vs. easy task activation patterns than does the MDN (which itself may suffer from increased neural inefficiency in older age), and thus the cuneaus improves decoding performance without containing additional (novel) pieces of information (but just more reliable ones)? If so, the compensation account could still be maintained by reference to the less demanding rationale for what constitutes compensation laid out above.

3) On page 21, the authors state that "...traditional univariate criteria alone are not sufficient for identifying functional compensation." To me, this conclusion is quite bold as I'd think that this depends on the unvariate criterion used. For instance, it could be argued that compensation should be more clearly indicated by an over additive interaction as observed for the relationship of cuneal activity with age and performance (i.e., the activity increase with better performance becomes stronger with age), rather than by an additive effect of age and performance as observed for the prefrontal ROI (see Fig. 2C). In any case, I'd appreciate it if the authors discussed this issue and the relationship between univariate and multivariate results in more detail (e.g. how many differences in sensitivity between the two approaches have contributed), in particular since the sophisticated multivariate approach used here is not widely established in the field yet.

4) As to the exclusion of poorly performing participants (see p24): If only based on the absolute number of errors, wouldn't you miss those who worked (overly) slowly but made few errors (possibly because of adjusting their speed-accuracy tradeoff)? Wouldn't it be reasonable to define a criterion based on the same performance measure (correct - incorrect) as used in the main behavioural analyses?

5) Did the authors consider testing for negative relationships between performance and brain activity, given that there is some literature arguing that neural efficiency (i.e. less activation) is the hallmark of high intelligence (i.e. high performance levels in the Cattell task)? If that were true, at least for some regions, the set of ROIs putatively carrying task-related information could be expanded beyond that examined here. If no such regions were found, it would provide some evidence bearing on the neural efficiency hypothesis.

Reviewer #3 (Public Review):

In this manuscript, Sperry and colleagues identify SNC80 as a compound that can slow metabolism and mimic hibernation, thereby prolonging tissue viability in organ transplantation and cardiovascular disease settings. Overall, the use of varied and relevant model systems is a strength of this study.

The authors perform a literature search to identify SNC80 as a promising hit. However, the details of the literature search, a list of other potential hits, and the criteria for identification of SNC80 are not described. The hypometabolic effect of SNC80 exposure is well-characterized in the Xenopus model. Furthermore, the authors show that SNC80 localises to the brain, but do not discuss several studies that have pointed to convulsions induced by exposure to high doses of SCN80, and whether this would be apparent in the Xenopus studies. The authors have promising data on the WB3 morpholino that retains or even improves on the hypometabolism phenotype of SCN80 while likely not retaining delta opioid activity. However, this is not functionally demonstrated. Moreover, WB3 is not used in any of the other assays and models used in the study. In the setting of cardiac transplant surgery, co-administration of SNC80 reduces metabolic activity and inflammation, although it is unclear if there is an improvement in recovery of organ function due to SCN80. The reversible induction of hypometabolic status is also demonstrated in two different organ chips. These models could identify the differential response of epithelial cells and vascular cells to drug perfusion, but the authors have mostly focused on the former. Finally, the authors identify specific targets for the hypometabolic effect of SNC80, which is a valuable resource for other screening studies and can form the basis for future work.

Reviewer #3 (Public Review):

Summary:

The authors are trying to find a vaccine solution for invasive candidiasis.

Strengths:

The testing of the antifungal activity of EDTA on Candida is not new as many other papers have examined this effect. The novelty here is the use of this EDTA-treated strain as a vaccine to protect against a secondary challenge with wild-type Candida.

Weaknesses:

However, data presented in Figure 5 and Figure 6 are not convincing and need further experimental controls and analysis as the authors do not show a time-dependent effect on the CFU of their vaccine formulation.

The methodology used is also an issue. As it stands, the impact is minor.

Reviewer #3 (Public Review):

Summary:<br /> These studies focus on a very interesting, understudied phenomenon in vascular development - the formation of pial collaterals between cerebral arteries. Understanding the mechanism(s) that regulates this process during normal development could provide important insights for the treatment of adult stroke patients, for which repair is highly dependent on collateral formation. Insights may also be relevant to other collateral-dependent diseases, such as heart disease and chronic peripheral ischemia.

Strengths:<br /> The investigators use lineage tracing and 3D imaging to show that, in mouse embryos, endothelial cells (ECs) predominantly from Bmx+ arteries and some from the Vegfr3+ microvasculature, invade pre-existing pre-collateral vascular structures in a process they termed "mosaic colonization", and arterialization of the vessel segments is said to occur concurrently with colonization, although details about EC phenotypes are lacking. Growth of the collaterals in response to ischemic injury relies on local replication of the ECs within the collaterals and not further recruitment from veins and the microvasculature. Although detailed molecular mechanisms are not provided, demonstration of the "cellular mechanism" of pial collateral vascularization is novel.

Weaknesses:<br /> Nonetheless, there are some issues that should be addressed, particularly to clarify the phenotype of the ECs forming the collaterals and expanding in response to injury; only their "origin" was traced and not their identity/growth after labeling in Bmx+ vessels.

https://www.demco.com/demco-reg-permalife-catalog-cards

The old school Library of Congress card catalog cards with the red ruling.

Reviewer #3 (Public Review):

Summary:

Machhua et al. in their work focused on unravelling the molecular mechanism of daptomycin binding and interaction with bacterial cell membranes. Daptomycin (Dap) is an acidic, cyclic lipopeptide composed of 13 amino acids, known for preferential binding to anionic lipids, particularly phosphatidylglycerol (PG), which are prevalent components in the membranes of Gram-positive bacteria. The process of binding and antimicrobial efficacy of Dap is significantly influenced by the ionic composition of the surrounding environment, especially the presence of Ca2+ ions. The authors underscore the presence of significant knowledge gaps in our understanding of daptomycin's mode of action. Several critical questions remain unanswered, including the basis for selective recognition and accumulation in membranes of Gram-positive strains, the specific role of Ca2+ ions in this process, and the mechanisms by which daptomycin binds to and inserts into the cell membrane.

Dap is intrinsically fluorescent due to its kynurenine residue (Kyn-13) and this property allows direct imaging of Dap binding to model cell membranes without the need for additional labeling. Taking advantage of this Dap autofluorescence, authors monitored the emission intensity of micelles, composed of varying DMPG content upon their exposure to Dap and compared it with the kinetics of fluorescence observed for zwitterionic DMPC and other negatively charged lipids such as cardiolipin (CA), POPA and POPS. The authors noted that the linear relationship between DMPG content and Dap fluorescence is strongly lipid-specific, as it was not observed for other anionic lipids. The manuscript sheds light on the specificity of Dap's interaction with CA and DMPG lipids. Through Ca2+ sequestration with EGTA, the authors demonstrated that the binding of Dap with CA is reversible, while its interaction with DMPG results in the irreversible insertion of Dap into the lipid membrane structure, caused by the significant conformational change of this lipopeptide. The formation of a stable DMPG-Dap complex was also verified in bacterial cells isolated from Gram-positive bacteria B. subtilis, where Dap exhibited a permanent binding to PG lipids.

Altogether, the authors endeavored to illuminate novel insights into the molecular basis of Dap binding, interaction, and the mechanism of insertion into bacterial cell membranes. Such understanding holds promise for the development of innovative strategies in combating drug resistance and the emergence of the so-called superbugs.

Strengths:

- The manuscript by Machhua et al. provides a comprehensive analysis of the Dap mechanism of binding and interaction with the membrane. It discusses various aspects of this, only apparently trivial interactions such as the importance of PG presence in the membrane, the impact of Ca2+ ions, and different mechanisms of Dap binding with other negatively charged lipids.

- The authors focused not only on model membranes (micelles) but also extended their research to bacterial cell membranes obtained from B. subtilis.

- The research is not only a report of the experimental findings but tries to give potential hypotheses explaining the molecular mechanisms behind the observed results.

Weaknesses:

- The authors overestimate their findings, stating that they propose a novel mechanism of Dap interaction with bacterial cell membranes. In fact, they rather extend the already reported hypotheses.

- The literature study was not done as thoroughly as it should be. Many publications discussing the importance and mechanism of action of Ca2+ ions or conformational changes of daptomycin were not cited.

Reviewer #3 (Public Review):

Summary:

Yamada et al. build on classic and more recent studies (Chen et al., 2023; Lemmon et al., 1992; Nichol et al., 2016; Zheng et al., 1994; Schense and Hubbell, 2000) to better understand the relationship between substrate adhesion and neurite outgrowth.

Strengths:

The primary strength of the manuscript lies in developing a method for investigating the role of adhesion in axon outgrowth and traction force generation using a femtosecond laser technique. The most exciting finding is that both outgrowth and traction force generation have a biphasic relationship with laminin concentration.

Weaknesses:

The primary weaknesses are a lack of discussion of prior studies that have directly measured the strength of growth cone adhesions to the substrate (Zheng et al., 1994) and traction forces (Koch et al., 2012), the inverse correlation between retrograde flow rate and outgrowth (Nichol et al., 2016), and prior studies noting a biphasic effect of substrate concentration of neurite outgrowth (Schense and Hubbell, 2000).

Overall, the claims and conclusions are well justified by the data. The main exception is that the data is more relevant to how the rate of neurite outgrowth is controlled rather than axonal guidance.

This manuscript will help foster interest in the interrelationship between neurite outgrowth, traction forces, and substrate adhesion, and the use of a novel method to study this problem.

Reviewer #3 (Public Review):

Summary:<br /> This work addresses an important question of how Gtr1/2 small GTPases and Pib2, two major regulators of the TORC1 cell growth controller, differentially operate in yeast. They found not all the TORC1 downstream targets respond to Gtr1/2 and Pib2 equally. In fact, they demonstrate that TORC1-dependent phosphorylation of Ser33, a 3-phosphoglycerate dehydrogenase, is responsive to only Pib2. They attributed this specificity to the physical interaction between Ser33 and Pib2. This part is novel and important, revising the canonical view in the field that Gtr1/2 and Pib2 branches act towards the same TORC1 downstream targets. Of note, this claim largely agrees with a recent independent study (PMID: 38127619).

Moving on, the authors describe different behaviors of TORC1 downstream readouts in intermediate nutrient conditions with a poor nitrogen source, with some readouts still active while others inactive. They argue that selective activation of certain TORC1 downstream targets reflects the "Gtr1/2 off, Pib2 on" state. However, this claim is not sufficiently supported by the presented data.

Strengths:<br /> The data presented in this paper has high value to the TOR community. In particular, a rigorous and comprehensive phospho-proteomic dataset that compares the Gtr1/2- and Pib2-dependency of diverse TORC1 downstream targets is very informative, potentially stimulating follow-up studies on each target.

Identification of Ser33 as a Pib2-specific TORC1 downstream is important and convincing (although whether Ser33 is a direct substrate of TORC1 was not addressed in this work). Physical interaction between Ser33 and Pib2 could represent a novel layer of TORC1 signaling regulation, in line with the mammalian Rag-TFEB interaction model, as discussed by the authors.

Weaknesses:<br /> The authors' three-state model, particularly the claim that cells are in the "Gtr1/2 off, Pib2 on" state in a poor nitrogen condition (e.g., proline medium), is not convincing enough because of the following reasons.

1) The "Pib2 on" claim contradicts with the observation that Ser33, Pib2-specific readout, is hypo-phosphorylated in proline medium (Fig 5F).

2) In the genetic experiments (Figure 8), the authors compare pib2D with Gtr1/2OFF. This is not appropriate, because GTR1/2OFF (GTR1-GDP and Gtr2-GTP) actively inhibits TORC1, differing from the null nature of pib2D. pib2D should be compared with gtr1/2D instead.

3) In general, diverse behaviors of TORC1 targets are not unexpected because their phosphorylation levels should have different dynamic ranges depending on how "good" they are as TORC1 substrates, with some requiring a higher TORC1 activity than others to be detectably phosphorylated. Although this aspect can be physiologically meaningful, and it is indeed important to look at multiple substrates as the authors suggest, this approach does not inform whether the signal is coming from Gtr1/2 or Pib2. An informative way in this context would be to look at the Gtr1/2- or Pib2-specific targets, but the former has not been identified, and observations on the latter, Ser33, do not support the "Pib2 on" claim as mentioned in the above 1).

4) In addition, comparisons made between direct TORC1 substrates (e.g., Sch9) and indirect downstream targets (e.g., Rps6 and Par32) are not very informative, because indirect targets can be impacted by TORC1-independent regulation of the mediating factors (e.g., Ypk3 for Rps6 and Npr1 for Par32).

In summary, the presented data do not tell us which of the two branches (Gtr1/2 or Pib2) is "more active" in the poor nitrogen condition. Their observations do not necessarily prefer their 3-state on/off model (Figure 8) over the more natural assumption that both branches have the gradation of activity depending on the nutrient status.

Reviewer #3 (Public Review):

Bing et al. attempt to address fundamental mechanisms of TAD formation in Drosophila by analyzing gene expression and 3D conformation within the vicinity of the eve TAD after insertion of a transgene harboring a Homie insulator sequence 142 kb away in different orientations. These transgenes along with spatial gene expression analysis were previously published in Fujioka et al. 2016, and the underlying interpretations regarding resulting DNA configuration in this genomic region were also previously published. This manuscript repeats the expression analysis using smFISH probes in order to achieve more quantitative analysis, but the main results are the same as previously published. The only new data are the Micro-C and an additional modeling/analysis of what they refer to as the 'Z3' orientation of the transgenes. The rest of the manuscript merely synthesizes further interpretation with the goal of addressing whether loop extrusion may be occurring or if boundary:boundary pairing without loop extrusion is responsible for TAD formation. The authors conclude that their results are more consistent with boundary:boundary pairing and not loop extrusion; however, most of this imaging data seems to support both loop extrusion and the boundary:boundary models. This manuscript lacks support, especially new data, for its conclusions. Furthermore, there are many parts of the manuscript that are difficult to follow. There are some minor errors in the labelling of the figures that if fixed would help elevate understanding. Lastly, there are several major points that if elaborated on, would potentially be helpful for the clarity of the manuscript.

Major Points:<br /> 1. The authors suggest and attempt to visualize in the supplemental figures, that loop extrusion mechanisms would appear during crosslinking and show as vertical stripes in the micro-C data. In order to see stripes, a majority of the nuclei would need to undergo loop extrusion at the same rate, starting from exactly the same spots, and the loops would also have to be released and restarted at the same rate. If these patterns truly result from loop extrusion, the authors should provide experimental evidence from another organism undergoing loop extrusion.<br /> 2. On lines 311-314, the authors discuss that stem-loops generated by cohesin extrusion would possibly be expected to have more next-next-door neighbor contacts than next-door neighbor contacts and site their models in Figure 1. Based on the boundary:boundary pairing models in the same figure would the stem-loops created by head-to-tail pairing also have the same phenotype? Making possible enrichment of next-next-door neighbor contacts possible in both situations? The concepts in the text are not clear, and the diagrams are not well-labeled relative to the two models.<br /> 3. The authors appear to cite Chen et al., 2018 as a reference for the location of these transgenes being 700nM away in a majority of the nuclei. However, the exact transgenes in this manuscript do not appear to have been measured for distance. The authors could do this experiment and include expression measurements.<br /> 4. The authors discuss the possible importance of CTCF orientation in forming the roadblock to cohesin extrusion and discuss that Homie orientation in the transgene may impact Homie function as an effective roadblock. However, the Homie region inserted in the transgene does not contain the CTCF motif. Can the authors elaborate on why they feel the orientation of Homie is important in its ability to function as a roadblock if the CTCF motif is not present? Trans-acting factors responsible for Homie function have not been identified and this point is not discussed in the manuscript.<br /> 5. The imaging results seem to be consistent with both boundary:boundary interaction and loop extrusion stem looping.<br /> 6. The authors suggest that the eveMa TAD could only be formed by extrusion after the breakthrough of Nhomie and several other roadblocks. Additionally, the overall long-range interactions with Nhomie appear to be less than the interactions with endogenous Homie (Figures 7, 8, and supplemental 5). Is it possible that in some cases boundary:boundary pairing is occurring between only the transgenic Homie and endogenous Homie and not including Nhomie?<br /> 7. In Figure 4E, the GFP hebe expression shown in the LhomieG Z5 transgenic embryo does not appear in the same locations as the LlambdaG Z5 control. Is this actually hebe expression or just a background signal?<br /> 8. Figure 6- The LhomieG Z3 late-stage embryo appears to be showing the ventral orientation of the embryo rather than the lateral side of the embryo as was shown in the previous figure. Is this for a reason? Additionally, there are no statistics shown for the Z3 transgenic images. Were these images analyzed in the same way as the Z5 line images?<br /> 9. Do the Micro-C data align with the developmental time points used in the smFISH probe assays?

Reviewer #3 (Public Review):

Summary:<br /> The manuscript by Wang et. al. described the crystal structures of the N-terminal fragments of Scavenger receptor class F member 1 (SCARF1) ectodomains. SCARF1 recognizes modified LDLs, including acetylated LDL and oxidized LDL, and it plays an important role in both innate and adaptive immune responses. They characterized the dimerization of SCARF1 and the interaction of SCARF1 with modified lipoproteins by mutational and biochemical studies. The authors identified the critical residues for dimerization and demonstrated that SCARF1 may function as homodimers. They further characterized the interaction between SCARF1 and LDLs and identified the lipoprotein ligand recognition sites, the highly positively charged areas. Their data suggested that the teichoic acid inhibitors may interact with SCARF1 in the same areas as LDLs.

Strengths:<br /> The crystal structures of SCARF1 were high quality. The authors performed extensive site-specific mutagenesis studies using soluble proteins for ELISA assays and surface-expressed proteins for flow cytometry.

Weaknesses:<br /> 1. The schematic drawing of human SCARF1 and SCARF2 in Fig 1A did not show the differences between them. It would be useful to have a sequence alignment showing the polymorphic regions.<br /> 2. The description of structure determination was confusing. The f1 crystal structure was determined by SAD with Pt derivatives. Why did they need molecular replacement with a native data set? The f2 crystal structure was solved by molecular replacement using the structure of the f1 fragment. Why did they need to use EGF-like fragments predicted by AlphaFold as search models?<br /> 3. It's interesting to observe that SCARA1 binds modified LDLs in a Ca2+-independent manner. The authors performed the binding assays between SCARF1 and modified LDLs in the presence of Ca2+ or EDTA on Page 9. However, EDTA is not an efficient Ca2+ chelator. The authors should have performed the binding assays in the presence of EGTA instead.<br /> 4. The authors claimed that SCARF1Δ353-415, the deletion of a C-terminal region of the ectodomain, might change the conformation of the molecule and generate hinderance for the C-terminal regions. Why didn't SCARF1Δ222-353 have a similar effect? Could the deletion change the interaction between SCARF1 and the membrane? Is SCARF1Δ353-415 region hydrophobic?<br /> 5. What was the point of having Figure 8? Showing the SCARF1 homodimers could form two types of dimers on the membrane surface proposed? The authors didn't have any data to support that.

DOI: 10.1016/j.neuron.2024.01.001

Resource: (ZFIN Cat# ZDB-ALT-140811-3,RRID:ZFIN_ZDB-ALT-140811-3)

Curator: @evieth

SciCrunch record: RRID:ZFIN_ZDB-ALT-140811-3

What is this?

Reviewer #3 (Public Review):

Cheng et al. studied if and how blood flow regulates the differentiation of vascular smooth muscle cells (VSMC) in the Circle of Willis (CW) in zebrafish embryos. They show that CW vessels gradually acquire an arterial identity. VSMCs also undergo gradual differentiation, which correlates with blood flow velocity. Using cell culture they show that pulsatile blood flow promotes pericyte differentiation into smooth muscle cells. They further identify transcription factor klf2a as differentially regulated by blood flow, and show that klf2a inhibition results in VSMC differentiation. The authors conclude that pulsatile flow promotes VSMC differentiation through klf2a activation.

Overall this is an important study, because VSMC differentiation in CW has not been previously studied, although analogous observations regarding the role of blood flow and klf2 involvement have been previously made in other systems and other vascular beds, for example, mouse klf2 mutants, which have deficient VSMC coverage of the dorsal aorta (Wu et al., 2008, JBC 283: 3942-50). The results convincingly show that VSMC differentiation in CW depends on the blood flow and that klf2a flow-dependent function regulates VSMC differentiation.

Reviewer #3 (Public Review):

Summary:<br /> It has been proposed in the literature, that the ATP release channel Panx1 can be activated in various ways, including by tyrosine phosphorylation of the Panx1 protein. The present study reexamines the commercial antibodies used previously in support of the phosphorylation hypothesis and the presented data indicate that the antibodies may recognize proteins unrelated to Panx1. Consequently, the authors caution about the use and interpretation of results obtained with these antibodies.

Strengths:<br /> The manuscript by Ruan et al. addresses an important issue in Panx1 research, i.e. the activation of the channel formed by Panx1 via protein phosphorylation. If the authors' conclusions are correct, the previous claims for Panx1 phosphorylation on the basis of the commercial anti-phospho-Panx1 antibodies would be in question.

This is a very detailed and comprehensive analysis making use of state-of-the-art techniques, including mass spectrometry and phos-tag gel electrophoresis.

In general, the study is well-controlled as relating to negative controls.

The value of this manuscript is, that it could spawn new, more function-oriented studies on the activation of Panx1 channels.

Weaknesses:<br /> Although the manuscript addresses an important issue, the activation of the ATP-release channel Panx1 by protein phosphorylation, the data provided do not support the firm conclusion that such activation does not exist. The failure to reproduce published data obtained with commercial anti-phospho Panx1 antibodies can only be of limited interest for a subfield.

1. The title claiming that "Panx1 is NOT phosphorylated..." is not justified by the failure to reproduce previously published data obtained with these antibodies. If, as claimed, the antibodies do not recognize Panx1, their failure cannot be used to exclude tyrosine phosphorylation of the Panx1 protein. There is no positive control for the antibodies.

2. The authors claim that exogenous SRC expression does not phosphorylate Y198. DeLalio et al. 2019 show that Panx1 is constitutively phosphorylated at Y198, so an effect of exogenous SRC expression is not necessarily expected.

3. The authors argue that the GFP tag of Panx1at the COOH terminus does not interfere with folding since the COOH modified (thrombin cleavage site) Panx1 folds properly, forming an amorphous glob in the cryo-EM structure. However, they do not show that the COOH-modified Panx1 folds properly. It may not, because functional data strongly suggest that the terminal cysteine dives deep into the pore. For example, the terminal cysteine, C426, can form a disulfide bond with an engineered cysteine at position F54 (Sandilos et al. 2012).

4. The authors dismiss the additional arguments for tyrosine phosphorylation of Panx1 given by the various previous studies on Panx1 phosphorylation. These studies did not, as implied, solely rely on the commercial anti-phospho-Panx1 antibodies, but also presented a wealth of independent supporting data. Contrary to the authors' assertion, in the previous papers the pY198 and pY308 antibodies recognized two protein bands in the size range of glycosylated and partial glycosylated Panx1.

5. A phosphorylation step triggering channel activity of Panx1 would be expected to occur exclusively on proteins embedded in the plasma membrane. The membrane-bound fraction is small in relation to the total protein, which is particularly true for exogenously expressed proteins. Thus, any phosphorylated protein may escape detection when total protein is analyzed. Furthermore, to be of functional consequence, only a small fraction of the channels present in the plasma membrane need to be in the open state. Consequently, only a fraction of the Panx1 protein in the plasma membrane may need to be phosphorylated. Even the high resolution of mass spectroscopy may not be sufficient to detect phosphorylated Panx1 in the absence of enrichment processes.

6. In the electrophysiology experiments described in Figure 7, there is no evidence that the GFP-tagged Panx1 is in the plasma membrane. Instead, the image in Figure 7a shows prominent fluorescence in the cytoplasm. In addition, there is no evidence that the CBX-sensitive currents in 7b are mediated by Panx1-GFP and are not endogenous Panx1. Previous literature suggests that the hPanx1 protein needs to be cleaved (Chiu et al. 2014) or mutated at the amino terminus (Michalski et al 2018) to see voltage-activated currents, so it is not clear that the currents represent hPANX1 voltage-activated currents.

Reviewer #3 (Public Review):

This is a manuscript that attempts to validate Plasmodium M1 alanyl aminopeptidase as a target for antimalarial drug development. The authors provide evidence that MIPS2673 inhibits recombinant enzymes from both Pf and Pv and is selective over other proteases. There is in vitro antimalarial activity. Chemoproteomic experiments demonstrate selective targeting of the PfA-M1 protease.

This is a continuation of previous work focused on designing inhibitors for aminopeptidases by a subset of these authors. Medicinal chemistry explorations resulted in the synthesis of MIPS2673 which has improved properties including potent inhibition of PfA-M1 and PvA-M1 with selectivity over a closed related peptidase. The compound also demonstrated selectivity over several human aminopeptidases and was not toxic to HEK293 cells at 40 uM. The activity against P. falciparum blood-stage parasites was about 300 nM.

Thermal stability studies confirmed that PfA-M1 was a binding target, however, there were other proteins consistently identified in the thermal stability studies. This raises the question as to their potential role as additional targets of this inhibitor. The authors dismiss these because they are not metalloproteases, but further analysis is warranted. This is particularly important as the authors were not able to generate mutants using in vitro evolution of resistance strategies. This often indicates that the inhibitor has more than one target.

The next set of experiments focused on a limited proteolysis approach. Again several proteins were identified as interacting with MIPS2673 including metalloproteases. The authors go on to analyze the LiP-MS data to identify the peptide from PfA-M1 which putatively interacts with MIPS2673. The authors are clearly focused on PfA-M1 as the target, but a further analysis of the other proteins identified by this method would be warranted and would provide evidence to either support or refute the authors' conclusions.

The final set of experiments was an untargeted metabolomics analysis. They identified 97 peptides as significantly dysregulated after MIPS2673 treatment of infected cells and most of these peptides were derived from one of the hemoglobin chains. The accumulation of peptides was consistent with a block in hemoglobin digestion. This experiment does reveal a potential functional confirmation, but questions remain as to specificity.

Overall, this is an interesting series of experiments that have identified a putative inhibitor of PfA-M1 and PvA-M1. The work would be significantly strengthened by structure-aided analysis. It is unclear why putative binding sites cannot be analyzed via specific mutagenesis of the recombinant enzyme. In the thermal stability and LiP -MS analysis, other proteins were consistently identified in addition to PfA-M1 and yet no additional analysis was undertaken to explore these as potential targets. The metabolomics experiments were potentially interesting, but without significant additional work including different lengths of treatment and different stages of the parasite, the conclusions drawn are overstated. Many treatments disrupt hemoglobin digestion - either directly or indirectly and from the data presented here it is premature to conclude that treatment with MIPS2673 directly inhibits hemoglobin digestion. Finally, the potency of this compound on parasites grown in vitro is 300 nM - this would need improvements in potency and demonstration of in vivo efficacy in the SCID mouse model to consider this a candidate for a drug.

Summary:<br /> Overall, this is an interesting series of experiments that have identified a putative inhibitor of the Plasmodium M1 alanyl aminopeptidases, PfA-M1 and PvA-M1.

Strengths:<br /> The main strengths include the synthesis of MIPS2673 which is selectively active against the enzymes and in whole-cell assay.

Weaknesses:<br /> The weaknesses include the lack of additional analysis of additional targets identified in the chemoproteomic approaches.

Reviewer #4 (Public Review):

I am a new reviewer for this manuscript, which has been reviewed before. The authors provide a variational autoencoder that has three objectives in the loss: linear reconstruction of behavior from embeddings, reconstruction of neural data, and KL divergence term related to the variational model elements. They take the output of the VAE as the "behaviorally relevant" part of neural data and call the residual "behaviorally irrelevant". Results aim to inspect the linear versus nonlinear behavior decoding using the original raw neural data versus the inferred behaviorally relevant and irrelevant parts of the signal.

Overall, studying neural computations that are behaviorally relevant or not is an important problem, which several previous studies have explored (for example PSID in (Sani et al. 2021), TNDM in (Hurwitz et al. 2021), TAME-GP in (Balzani et al. 2023), pi-VAE in (Zhou and Wei 2020), and dPCA in (Kobak et al. 2016), etc). However, this manuscript does not properly put their work in the context of such prior works. For example, the abstract states "One solution is to accurately separate behaviorally-relevant and irrelevant signals, but this approach remains elusive", which is not the case given that these prior works have done that. The same is true for various claims in the main text, for example "Furthermore, we found that the dimensionality of primary subspace of raw signals (26, 64, and 45 for datasets A, B, and C) is significantly higher than that of behaviorally-relevant signals (7, 13, and 9), indicating that using raw signals to estimate the neural dimensionality of behaviors leads to an overestimation" (line 321). This finding was presented in (Sani et al. 2021) and (Hurwitz et al. 2021), which is not clarified here. This issue of putting the work in context has been brought up by other reviewers previously but seems to remain largely unaddressed. The introduction is inaccurate also in that it mixes up methods that were designed for separation of behaviorally relevant information with those that are unsupervised and do not aim to do so (e.g., LFADS). The introduction should be significantly revised to explicitly discuss prior models/works that specifically formulated this behavior separation and what these prior studies found, and how this study differs.

Beyond the above, some of the main claims/conclusions made by the manuscript are not properly supported by the analyses and results, which has also been brought up by other reviewers but not fully addressed. First, the analyses here do not support the linear readout from the motor cortex because i) by construction, the VAE here is trained to have a linear readout from its embedding in its loss, which can bias its outputs toward doing well with a linear decoder/readout, and ii) the overall mapping from neural data to behavior includes both the VAE and the linear readout and thus is always nonlinear (even when a linear Kalman filter is used for decoding). This claim is also vague as there is no definition of readout from "motor cortex" or what it means. Why is the readout from the bottleneck of this particular VAE the readout of motor cortex? Second, other claims about properties of individual neurons are also confounded because the VAE is a population-level model that extracts the bottleneck from all neurons. Thus, information can leak from any set of neurons to other sets of neurons during the inference of behaviorally relevant parts of signals. Overall, the results do not convincingly support the claims, and thus the claims should be carefully revised and significantly tempered to avoid misinterpretation by readers.

Below I briefly expand on these as well as other issues, and provide suggestions:

1) Claims about linearity of "motor cortex" readout are not supported by results yet stated even in the abstract. Instead, what the results support is that for decoding behavior from the output of the dVAE model -- that is trained specifically to have a linear behavior readout from its embedding -- a nonlinear readout does not help. This result can be biased by the very construction of the dVAE's loss that encourages a linear readout/decoding from embeddings, and thus does not imply a finding about motor cortex.

2) Related to the above, it is unclear what the manuscript means by readout from motor cortex. A clearer definition of "readout" (a mapping from what to what?) in general is needed. The mapping that the linearity/nonlinearity claims refer to is from the *inferred* behaviorally relevant neural signals, which themselves are inferred nonlinearly using the VAE. This should be explicitly clarified in all claims, i.e., that only the mapping from distilled signals to behavior is linear, not the whole mapping from neural data to behavior. Again, to say the readout from motor cortex is linear is not supported, including in the abstract.

3) Claims about individual neurons are also confounded. The d-VAE distilling processing is a population level embedding so the individual distilled neurons are not obtainable on their own without using the population data. This population level approach also raises the possibility that information can leak from one neuron to another during distillation, which is indeed what the authors hope would recover true information about individual neurons that wasn't there in the recording (the pixel denoising example). The authors acknowledge the possibility that information could leak to a neuron that didn't truly have that information and try to rule it out to some extent with some simulations and by comparing the distilled behaviorally relevant signals to the original neural signals. But ultimately, the distilled signals are different enough from the original signals to substantially improve decoding of low information neurons, and one cannot be sure if all of the information in distilled signals from any individual neuron truly belongs to that neuron. It is still quite likely that some of the improved behavior prediction of the distilled version of low-information neurons is due to leakage of behaviorally relevant information from other neurons, not the former's inherent behavioral information. This should be explicitly acknowledged in the manuscript.

4) Given the nuances involved in appropriate comparisons across methods and since two of the datasets are public, the authors should provide their complete code (not just the dVAE method code), including the code for data loading, data preprocessing, model fitting and model evaluation for all methods and public datasets. This will alleviate concerns and allow readers to confirm conclusions (e.g., figure 2) for themselves down the line.

5) Related to 1) above, the authors should explore the results if the affine network h(.) (from embedding to behavior) was replaced with a nonlinear ANN. Perhaps linear decoders would no longer be as close to nonlinear decoders. Regardless, the claim of linearity should be revised as described in 1) and 2) above, and all caveats should be discussed.

6) The beginning of the section on the "smaller R2 neurons" should clearly define what R2 is being discussed. Based on the response to previous reviewers, this R2 "signifies the proportion of neuronal activity variance explained by the linear encoding model, calculated using raw signals". This should be mentioned and made clear in the main text whenever this R2 is referred to.

7) Various terms require clear definitions. The authors sometimes use vague terminology (e.g., "useless") without a clear definition. Similarly, discussions regarding dimensionality could benefit from more precise definitions. How is neural dimensionality defined? For example, how is "neural dimensionality of specific behaviors" (line 590) defined? Related to this, I agree with Reviewer 2 that a clear definition of irrelevant should be mentioned that clarifies that relevance is roughly taken as "correlated or predictive with a fixed time lag". The analyses do not explore relevance with arbitrary time lags between neural and behavior data.

8) CEBRA itself doesn't provide a neural reconstruction from its embeddings, but one could obtain one via a regression from extracted CEBRA embeddings to neural data. In addition to decoding results of CEBRA (figure S3), the neural reconstruction of CEBRA should be computed and CEBRA should be added to Figure 2 to see how the behaviorally relevant and irrelevant signals from CEBRA compare to other methods.

References:

Kobak, Dmitry, Wieland Brendel, Christos Constantinidis, Claudia E Feierstein, Adam Kepecs, Zachary F Mainen, Xue-Lian Qi, Ranulfo Romo, Naoshige Uchida, and Christian K Machens. 2016. "Demixed Principal Component Analysis of Neural Population Data." Edited by Mark CW van Rossum. eLife 5 (April): e10989. https://doi.org/10.7554/eLife.10989.

Sani, Omid G., Hamidreza Abbaspourazad, Yan T. Wong, Bijan Pesaran, and Maryam M. Shanechi. 2021. "Modeling Behaviorally Relevant Neural Dynamics Enabled by Preferential Subspace Identification." Nature Neuroscience 24 (1): 140-49. https://doi.org/10.1038/s41593-020-00733-0.

Zhou, Ding, and Xue-Xin Wei. 2020. "Learning Identifiable and Interpretable Latent Models of High-Dimensional Neural Activity Using Pi-VAE." In Advances in Neural Information Processing Systems, 33:7234-47. Curran Associates, Inc. https://proceedings.neurips.cc/paper/2020/hash/510f2318f324cf07fce24c3a4b89c771-Abstract.html.

Hurwitz, Cole, Akash Srivastava, Kai Xu, Justin Jude, Matthew Perich, Lee Miller, and Matthias Hennig. 2021. "Targeted Neural Dynamical Modeling." In Advances in Neural Information Processing Systems. Vol. 34. https://proceedings.neurips.cc/paper/2021/hash/f5cfbc876972bd0d031c8abc37344c28-Abstract.html.

Balzani, Edoardo, Jean-Paul G. Noel, Pedro Herrero-Vidal, Dora E. Angelaki, and Cristina Savin. 2023. "A Probabilistic Framework for Task-Aligned Intra- and Inter-Area Neural Manifold Estimation." In . https://openreview.net/forum?id=kt-dcBQcSA.

Reviewer #3 (Public Review):

Summary:<br /> Object classification serves as a vital normative principle in both the study of the primate ventral visual stream and deep learning. Different models exhibit varying classification performances and organize information differently. Consequently, a thriving research area in computational neuroscience involves identifying meaningful properties of neural representations that act as bridges connecting performance and neural implementation. In the work of Lindsey and Issa, the concept of factorization is explored, which has strong connections with emerging concepts like disentanglement [1,2,3] and abstraction [4,5]. Their primary contributions encompass two facets: (1) The proposition of a straightforward method for quantifying the degree of factorization in visual representations. (2) A comprehensive examination of this quantification through correlation analysis across deep learning models.

To elaborate, their methodology, inspired by prior studies [6], employs visual inputs featuring a foreground object superimposed onto natural backgrounds. Four types of scene variables, such as object pose, are manipulated to induce variations. To assess the level of factorization within a model, they systematically alter one of the scene variables of interest and estimate the proportion of encoding variances attributable to the parameter under consideration.

The central assertion of this research is that factorization represents a normative principle governing biological visual representation. The authors substantiate this claim by demonstrating an increase in factorization from macaque V4 to IT, supported by evidence from correlated analyses revealing a positive correlation between factorization and decoding performance. Furthermore, they advocate for the inclusion of factorization as part of the objective function for training artificial neural networks. To validate this proposal, the authors systematically conduct correlation analyses across a wide spectrum of deep neural networks and datasets sourced from human and monkey subjects. Specifically, their findings indicate that the degree of factorization in a deep model positively correlates with its predictability concerning neural data (i.e., goodness of fit).

Strengths:<br /> The primary strength of this paper is the authors' efforts in systematically conducting analysis across different organisms and recording methods. Also, the definition of factorization is simple and intuitive to understand.

Weaknesses:<br /> This work exhibits two primary weaknesses that warrant attention: (i) the definition of factorization and its comparison to previous, relevant definitions, and (ii) the chosen analysis method.

Firstly, the definition of factorization presented in this paper is founded upon the variances of representations under different stimuli variations. However, this definition can be seen as a structural assumption rather than capturing the effective geometric properties pertinent to computation. More precisely, the definition here is primarily statistical in nature, whereas previous methodologies incorporate computational aspects such as deviation from ideal regressors [1], symmetry transformations [3], generalization [5], among others. It would greatly enhance the paper's depth and clarity if the authors devoted a section to comparing their approach with previous methodologies [1,2,3,4,5], elucidating any novel insights and advantages stemming from this new definition.

Secondly, in order to establish a meaningful connection between factorization and computation, the authors rely on a straightforward synthetic model (Figure 1c) and employ multiple correlation analyses to investigate relationships between the degree of factorization, decoding performance, and goodness of fit. Nevertheless, the results derived from the synthetic model are limited to the low training-sample regime. It remains unclear whether the biological datasets under consideration fall within this low training-sample regime or not.

[1] Eastwood, Cian, and Christopher KI Williams. "A framework for the quantitative evaluation of disentangled representations." International conference on learning representations. 2018.<br /> [2] Kim, Hyunjik, and Andriy Mnih. "Disentangling by factorising." International Conference on Machine Learning. PMLR, 2018.<br /> [3] Higgins, Irina, et al. "Towards a definition of disentangled representations." arXiv preprint arXiv:1812.02230 (2018).<br /> [4] Bernardi, Silvia, et al. "The geometry of abstraction in the hippocampus and prefrontal cortex." Cell 183.4 (2020): 954-967.<br /> [5] Johnston, W. Jeffrey, and Stefano Fusi. "Abstract representations emerge naturally in neural networks trained to perform multiple tasks." Nature Communications 14.1 (2023): 1040.<br /> [6] Majaj, Najib J., et al. "Simple learned weighted sums of inferior temporal neuronal firing rates accurately predict human core object recognition performance." Journal of Neuroscience 35.39 (2015): 13402-13418.

Reviewer #3 (Public Review):

Summary:<br /> Ai et al. studied texture, color, and disparity selectivity in the human visual cortex at the mesoscale level using high-resolution fMRI. They reproduced earlier monkey and human studies showing interdigitated color-selective and disparity-selective sub-compartments within area V2, likely corresponding to thin and thick stripes, respectively. At least with the stimuli used, no clear evidence for texture-selective mesoscale activations was observed in area V2. The most interesting and novel part of this study focused on cortical-depth-dependent connectivity analyses across areas. The data suggest feedback and feedforward functional connectivity between V1 and V3A for disparity signals and feedback from V4 to the deep layers of V2 for textures.

Strengths:<br /> High-resolution fMRI and highly interesting layer-specific informational connectivity analyses.

Weaknesses:<br /> The authors tend to overclaim their results.

Reviewer #3 (Public Review):

Summary:<br /> In this work, Duan and Curtis addressed an important issue related to the nature of working memory representations. This work is motivated by findings illustrating that orientation decoding performance for perceptual representations can be biased by the stimulus aperture (modulator). Here, the authors examined whether the decoding performance for working memory representations is similarly influenced by these aperture biases. The results provide convincing evidence that working memory representations have a different representational structure, as the decoding performance was not influenced by the type of stimulus aperture.

Strengths:<br /> The strength of this work lies in the direct comparison of decoding performance for perceptual representations with working memory representations. The authors take a well-motivated approach and illustrate that perceptual and working memory representations do not share a similar representational structure. The authors test a clear question, with a rigorous approach and provide convincing evidence. First, the presented oriented stimuli are carefully manipulated to create orthogonal biases introduced by the stimulus aperture (radial or angular modulator), regardless of the stimulus carrier orientation. Second, the authors implement advanced methods to decode the orientation information present, in visual and parietal cortical regions, when directly perceiving or holding an oriented stimulus in memory. The data illustrates that working memory decoding is not influenced by the type of aperture, while this is the case in perception. In sum, the main claims are important and shed light on the nature of working memory representations.

Weaknesses:<br /> I have a few minor concerns that, although they don't affect the main conclusion of the paper, should still be addressed.

1. Theoretical framing in the introduction: Recent work has shown that decoding of orientation during perception does reflect orientation selectivity, and it is not only driven by the stimulus aperture (Roth, Kay & Merriam, 2022).

2. Figure 1C illustrates the principle of how the radial and angular modulators bias the contrast energy extracted by the V1 model, which in turn would influence orientation decoding. It would be informative if the carrier orientations used in the experiment were shown in this figure, or at a minimum it would be mentioned in the legend that the experiment used 3 carrier orientations (15{degree sign}, 75{degree sign}, 135{degree sign}) clockwise from vertical. Related, when trying to find more information regarding the carrier orientation, the 'Stimuli' section of the Methods incorrectly mentions that 180 orientations are used as the carrier orientation.

3. The description of the image computable V1 model in the Methods is incomplete, and at times inaccurate. i) The model implements 6 orientation channels, which is inaccurately referred to as a bandwidth of 60{degree sign} (should be 180/6=30). ii) The steerable pyramid combines information across phase pairs to obtain a measure of contrast energy for a given stimulus.<br /> Here, it is only mentioned that the model contains different orientation and spatial scale channels. I assume there were also 2 phase pairs, and they were combined in some manner (squared and summed to create contrast energy). Currently, it is unclear what the model output represents. iii) The spatial scale channel with the maximal response differences between the 2 modulators was chosen as the final model output. What spatial frequency does this channel refer to, and how does this spatial frequency relate to the stimulus?

4. It is not clear from the Methods how the difficulty in the perceptual control task was controlled. How were the levels of task difficulty created?

Reviewer #3 (Public Review):

Summary:<br /> The study conducted by Pisanski et al investigates the role of the lateral parafacial area (pFL) in controlling active expiration. Stereotactic injections of bicuculline were utilized to map various pFL sites and their impact on respiration. The results indicate that injections at more rostral pFL locations induce the most robust changes in tidal volume, minute ventilation, and combined respiratory responses. The study indicates that the rostrocaudal organization of the pFL and its influence on breathing is not simple and uniform.

Strengths:<br /> The data provide novel insights into the importance of rostral locations in controlling active expiration. The authors use innovative analytic methods to characterize the respiratory effects of bicuculline injections into various areas of the pFL.

Weaknesses:<br /> Bicuculline injections increase the excitability of neurons. Aside from blocking GABA receptors, bicuculline also inhibits calcium-activated potassium currents and potentiates NMDA current, thus insights into the role of GABAergic inhibition are limited.

Increasing the excitability of neurons provides little insights into the activity pattern and function of the activated neurons. Without recording from the activated neurons, it is impossible to know whether an effect on active expiration or any other respiratory phase is caused by bicuculline acting on rhythmogenic neurons or tonic neurons that modulate respiration. While this approach is inappropriate to study the functional extent of the conditional "oscillator" for active expiration, it provides valuable insights into this region's complex role in controlling breathing.

Reviewer #3 (Public Review)

Summary<br /> This is an important study that tests the hypothesis that Cav1.4 calcium channels do more than provide a voltage-dependent influx of Ca2+ into photoreceptors. The relevant background can be divided into two tranches. First, deletion of Cav1.4 channels (Cav1.4 knock-out) disrupts rod and cone photoreceptors and their synapses in the outer plexiform layer. Second, knock-in of a non-conducting Cav1.4 channel (Cav1.4 knock-in) partially spares the organization of the outer plexiform layer and photoreceptor synapses (Maddox et al., eLife 2020), which is remarkable considering the disruption of the outer plexiform layer in the Cav1.4 knock-out. In addition, phototransduction, assessed by scotopic and phototopic electroretinography (a-wave amplitude) in the Cav1.4 knock-in retina was partially spared for rods and only slightly impaired for cones. However, the non-conducting Cav1.4 channel of the Cav1.4 knock-in failed to rescue synaptic transmission across the outer retina (electroretinography: b-wave amplitude, Maddox et al., eLife 2020). The 2020 Maddox et al. (eLife) focused more on the rod pathway, while the current work addressed the cone pathway.

Strengths<br /> The study addresses the important question of how disruption of Cav1.4 function in both rod and cone photoreceptors leads to impairment primarily of the rod pathway for scotopic vision. This is clinically relevant as human mutations lead to stationary night blindness rather than blindness. The work relevance provides excellent single-cell electrophysiological recordings of Ca2+ currents from cones of wild-type, Cav1.4 knock-out, and Cav1.4 knock-in mice and, in addition, from ground squirrel and monkey cones. To make these recordings successfully in the various species and the compromised retinas (Cav1.4 knock-out and Cav1.4 knock-in) is very impressive. The findings clearly advance our understanding of Ca2+ channel function in cones. In addition, the study presents high-quality electron microscopy reconstructions of cones and further physiological and behavioral data related to the cone pathway.

Weaknesses<br /> The major critiques are related to the description of the Cav1.4 knock-in mouse as "sparing" function, which can be remedied in part by a simple rewrite, and in certain places, the data may need to be examined more critically. In particular, the authors should address features in the data presented in Figures 6 and 7 that seem to indicate that the retina of the Cav1.4 knock-in is not intact, but the interpretation given by the authors as "intact" is not appropriate and made without rigorous statistical testing.

Reviewer #3 (Public Review):

Summary:<br /> The authors propose to invert a mechanistic model of phototransduction in mouse and rod photoreceptors to derive stimuli that compensate for nonlinearities in these cells. They fit the model to a large set of photoreceptor recordings and show in additional data that the compensation works. This can allow the exclusion of photoreceptors as a source of nonlinear computation in the retina, as desired to pinpoint nonlinearities in retinal computation. Overall, the recordings made by the authors are impressive and I appreciate the simplicity and elegance of the idea. The data support the authors' conclusions but the presentation can be improved.

Strengths:<br /> - The authors collected an impressive set of recordings from mouse and primate photoreceptors, which is very challenging to obtain.<br /> - The authors propose to exploit mechanistic mathematical models of well-understood phototransduction to design light stimuli that compensate for nonlinearities.<br /> - The authors demonstrate through additional experiments that their proposed approach works.

Weaknesses:<br /> - The authors use numerical optimization for fitting the parameters of the photoreceptor model to the data. Recently, the field of simulation-based inference has developed methods to do so, including quantification of the uncertainty of the resulting estimates. Since the authors state that two different procedures were used due to the different amounts of data collected from different cells, it may be worthwhile to rather test these methods, as implemented e.g. in the SBI toolbox (https://joss.theoj.org/papers/10.21105/joss.02505). This would also allow them to directly identify dependencies between parameters, and obtain associated uncertainty estimates. This would also make the discussion of how well constrained the parameters are by the data or how much they vary more principled because the SBI uncertainty estimates could be used.

- In several places, the authors refer the reader to look up specific values e.g. of parameters in the associated MATLAB code. I don't think this is appropriate, important values/findings/facts should be in the paper (lines 142, 114, 168). I would even find the precise values that the authors measure interesting, so I think the authors should show them in a figure/table. In general, I would like to see also the average variance explained by different models summarized in a table and precise mean/median values for all important quantities (like the response amplitude ratios in Figures 6/9).

- If the proposed model is supposed to model photoreceptor adaptation on a longer time scale, I fail to see why this can be an invertible model. Could the authors explain this better? I suspect that the model is mainly about nonlinearities as the authors also discuss in lines 360ff.

- The important Figures 6-8 are very hard to read, as it is not easy to see what the stimulus is, the modified stimulus, the response with and without modification, what the desired output looks like, and what is measured for part B. Reworking these figures would be highly recommended.

- If I understand Figure 6 correctly, part B is about quantifying the relative size of the response to the little first flash to the little second flash. While clearly, the response amplitude of the second flash is only 50% for the second flash compared to the first flash in primate rod and cones in the original condition, the modified stimulus seems to overcompensate and result in 130% response for the second flash. How do the authors explain this? A similar effect occurs in Figure 9, which the authors should also discuss.

Noise is anything interfering with clear communication; distractions that occur during communication that resolves in misinterpretation.

Internal noise: psychological or physical problem someone is dealing with during communication

External noise : Environmental components that cause distraction

Semantic noise: a disturbance in the transmission of a message; interferes with interpretation of the message due to words having more than one meaning.

A connotation: positive or negative emotional connection to a definition. connotative semantic noise is more of an emotional issue with the intended use of words.

Denotation: the literal meaning of a word; denotative semantic noise: when we hear or see language we cannot define therefore we cannot interpret.

Presuming my 1956 dating for my 4181-3 (SN 004365) slide rule is correct, the 1956 price list has the 10 inch ivorite 4181-3 model for $15.00.

https://www.mccoys-kecatalogs.com/KECatalogs/1954/1956Price/1956kecatprice43.htm

see: https://hypothes.is/a/SmzszMBcEe6Ug29KK3b93A

My 4181-3 slide rule was likely manufactured in 1956 as it has the SRT scale initiated in 1955 and has a serial number 004365 which is in the series 3 segment which reset in 1956 and ran from 0 to 88,500 that year.

https://www.mccoys-kecatalogs.com/keserialnumbers/Dating-2.htm

https://www.mccoys-kecatalogs.com/KECollection/4081-3/ke4181-3_s5.htm

My 4181-3 was likely made in 1955 or after as it has the SRT and not the ST scale. (It has a 1947 copyright mark on it.)

These are the scale sets on my K+E 4181-3 slide Rule

The Keuffel & Esser 4181-3 was part of the 4081-3 family and was described in their catalogs as a 10 inch Log Log Duplex Decitrig Plastic slide rule.

via https://www.mccoys-kecatalogs.com/KEModels/kexrefmain.htm

Reviewer #3 (Public Review):

Summary:<br /> Aubert et al investigated the role of PENK in regulatory T cells. Through the mining of publicly available transcriptome data, the authors confirmed that PENK expression is selectively enriched in regulatory but not conventional T cells. Further data mining suggested that OX40, 4-1BB as well as BATF, can regulate PENK expression in Tregs. The authors generated fate-mapping mice to confirm selective PENK expression in Tregs and activated effector T cells in the colon and spleen. Interestingly, transgenic mice with conditional deletion of PENK in Tregs resulted in hypersensitivity to heat, which the authors attributed to heat hyperalgesia.

Strengths:<br /> The generation of transgenic mice with conditional deletion of PENK in foxp3 and PENK fate-mapping is novel and can potentially yield significant findings. The identification of upstream signals that regulate PENK is interesting but unlikely to be the main reason why PENK is predominantly expressed in Tregs as both BATF and TNFR are expressed in effector T cells.

Weaknesses:<br /> There is a lack of direct evidence and detailed analysis of Tregs in the control and transgenic mice to support the authors' hypothesis. PENK was previously reported to be expressed in skin Tregs and play a significant role in regulating skin homeostasis: this should be considered as an alternative mechanism that may explain the changed sensitivity to heat observed in the paper.

Reviewer #3 (Public Review):

The introduction/background is excellent. It reviews evidence showing that extinction of conditioned responding is regulated by noradrenaline and suggesting that the locus coeruleus (LC) may be a critical locus of this regulation. This naturally leads to the aim of the study: to determine whether the locus coeruleus is involved in extinction of an appetitive conditioned response. Overall, the study is well designed, nicely conducted and the results advance our understanding of the role of the LC in extinction of conditioned behaviour. Future studies may provide more fine-grained analyses of behavioral data to clarify the impact of the LC manipulations (stimulation and inhibition) on performance in the task.

Reviewer #3 (Public Review):

Summary:<br /> Medina and colleagues explored transcriptional kinetics during SARS-CoV-2 between non-hospitalized and hospitalized cohorts and identified that early NK signaling may be responsible for less severe disease.

Strengths:<br /> The paper includes extremely detailed analyses and makes an interesting attempt to link innate and adaptive responses. The analyses are appropriate for the data and described in clear language. The inclusion of late time points is interesting and potentially relevant to long COVID studies. Most findings were compatible with other detailed immune mapping during severe COVID-19.

Weaknesses:<br /> 1. The authors claim to be looking at the earliest stages of infection but this is not true as all patients enrolled are already symptomatic. The time points selected are unlikely to be useful clinically for biomarker selection as they are too late, and are likely beyond the point when the immune responses between severe and mild infection start to diverge.<br /> 2. The comparator timepoints between mild and severe cases do not match. The most comparison would be between day 7 of mild versus day 0 of severe which is already fairly late during infection.<br /> 3. The authors mention viral clearance but I see no evidence of viral loads measured in these individuals.<br /> 4. The cohort is quite small to draw definitive conclusions.<br /> 5. It is uncertain whether the results are applicable to current conditions as most infected people are immune experienced.<br /> 6. I found the discussion to be a bit too detailed and dense. I would suggest editing to make it more streamlined.

Reviewer #3 (Public Review):

Summary:<br /> The authors have initiated studies to understand the molecular mechanisms underlying the devolvement of multi-drug resistance in clinical Mtb strains. They demonstrate the association of isoniazid-resistant isolates by rifampicin treatment supporting the idea that selection of MDR is a microenvironment phenomenon and involves a group of isolates.

Strengths:<br /> The methods used in this study are robust and the results support the authors' claims to a major extent.

Weaknesses:<br /> The manuscript needs a thorough vetting of the language. At present, the language makes it very difficult to comprehend the methodology and results.

Reviewer #3 (Public Review):

Summary:<br /> Chen et al. are addressing a fundamental question in mammalian gut biology, namely how the host controls a mutualistic host-microbiota symbiosis. The authors focus on a protein called Chitinase 3-like protein 1 (Ch3l1) and its interaction with the protective colonic mucus layer. The rationale for the study comes from previous work showing that microbial-associated molecular patterns (MAMPs) can induce Ch3l1 in vitro, but its biological functions in the colon are unknown. In this study, the authors provide evidence supporting the claim that the gut microbiota induces the expression of Ch3l1 in vivo, mainly in mucus-producing goblet cells. Insightfully, the authors note that Ch3l1, although it lacks enzyme (chitinase) activity, still binds Chitin, a glycan that has structural similarity to bacterial cell wall peptidoglycan. This leads the authors to hypothesize that Ch3l1 binds microbial cell walls, particularly those of peptidoglycan-rich Gram-positive probiotic bacteria within the mucus, to promote their retention in the colon. Using a combination of in vivo work with mice conditionally lacking Ch3l1 in gut epithelium (IEC Ch3l1 KO); microbiota profiling; imaging of host-microbiota interactions with labeled microbes; and fecal transplants, the authors provide compelling evidence that Ch3l1 is secreted into the gut mucus layer and that the presence of Ch3l1 is associated with increased levels of beneficial Gram+ bacteria, including Lactobacillus spp. In turn, using a well-characterized colitis model, the authors show that Ch3l1 is associated with protection from intestinal injury caused by Dextran Sodium Sulfate. While these studies are novel and informative, there are several issues that undermine the authors' conclusions.

Strengths:<br /> The authors nicely link microbial induction of Ch3l1 to mucosal protection from intestinal injury. This is done through the use of germ-free and ex-germ-free studies and by comparing Ch3l1 expression in situ between them; microbial sequencing between Control and IEC Ch3l1 KO mice, and clinical and histological injury metrics between these strains. The authors convincingly demonstrate the presence of Ch3l1 in the gut mucus through imaging, and that the deletion of this protein in mice alters the microbiota by reducing the relative abundance of Gram-positive species.

The study employs a technically diverse set of analyses to address their hypothesis, including fluorescent labelling of microbial species for add-back studies, fecal transplants to distinguish the role(s) of the microbiota vs. host in the IEC Ch3l1 KO phenotypes in the intestinal challenge models.

Weaknesses:<br /> The claim that mucus-associated Ch3l1 controls colonization of beneficial Gram-positive species within the mucus is not conclusive. The study should take into account recent discoveries on the nature of mucus in the colon, namely its mobile fecal association and complex structure based on two distinct mucus barrier layers coming from proximal and distal parts of the colon (PMID: ). This impacts the interpretation of how and where Ch3l1 is expressed and gets into the mucus to promote colonization. It also impacts their conclusions because the authors compare fecal vs. tissue mucus, but most of the mucus would be attached to the feces. Of the mucus that was claimed to be isolated from the WT and IEC Ch3l1 KO, this was not biochemically verified. Such verification (e.g. through Western blot) would increase confidence in the data presented. Further, the study relies upon relative microbial profiling, which can mask absolute numbers, making the claim of reduced overall Gram-positive species in mice lacking Ch3l1 unproven. It would be beneficial to show more quantitative approaches (e.g. Quantitative Microbial Profiling, QMP) to provide more definitive conclusions on the impact of Ch3l1 loss on Gram+ microbes.

Other weaknesses lie in the execution of the aims, leaving many claims incompletely substantiated. For example, much of the imaging data is challenging for the reader to interpret due to it being unfocused, too low of magnification, not including the correct control, and not comparing the same regions of tissues among different in vivo study groups. Statistical rigor could be better demonstrated, particularly when making claims based on imaging data. These are often presented as single images without any statistics (i.e. analysis of multiple images and biological replicates). These images include the LTA signal differences, FISH images, Enterococcus colonization, and mucus thickness.

Reviewer #3 (Public Review):

This is an interesting manuscript that addresses a longstanding debate in evolutionary biology - whether social or ecological factors are primarily responsible for the evolution of the large human brain. To address this, the authors examine the relationship between the size of two prefrontal regions involved in metacognition and working memory (DLPFC and FP) and socioecological variables across 16 primate species. I recommend major revisions to this manuscript due to: 1) a lack of clarity surrounding model construction; and 2) an inappropriate treatment of the relative importance of different predictors (due to a lack of scaling/normalization of predictor variables prior to analysis).

Reviewer #3 (Public Review):

The authors have made simultaneous recordings of the responses of large numbers of neurons from the primary visual cortex using optical two-photon imaging of calcium signals from the superficial layers of the cortex. Recordings were made to compare the responses of the cortical neurons under normal binocular viewing of a flat screen with both eyes open and monocular viewing of the same screen with one eye's view blocked by a translucent filter. The screen displayed visual stimuli comprising small contrast patches of Gabor function distributions of luminance, a stimulus that is known to excite cortical neurons.

This is an important data set, given the large numbers of neurons recorded. The authors present a simple model to explain the binocular combination of neuronal signals from the right and left eyes.

The limitations of the paper as written are as follows. These points can be addressed with some additional analysis and rewriting of sections of the paper. No new experimental data need to be collected.

1) The authors should acknowledge the fact that these recordings arise from neurons in the superficial layers of the cortex. This limitation arises from the usual constraints on optical imaging in the macaque cortex. This means that the sample of neurons forming this data set is not fully representative of the population of binocular neurons within the visual cortex. This limitation is important in comparing the outcome of these experiments with the results from other studies of binocular combination, which have used single-electrode recording. Electrode recording will result in a sample of neurons that is drawn from many layers of the cortex, rather than just the superficial layers.

2) Single-neuron recording of binocular neurons in the primary visual cortex has shown that these neurons often have some spontaneous activity. Assessment of this spontaneous level of firing is important for accurate model fitting [1]. The paper here should discuss the level of spontaneous neuronal firing and its potential significance.

3) The arrangements for visual stimulation and comparison of binocular and monocular responses mean that the stereoscopic disparity of the binocular stimuli is always at zero or close to zero. The animal's fixation point is in the centre of a single display that is viewed binocularly. The fixation point is, by definition, at zero disparity. The other points on the flat display are also at zero disparity or very close to zero because they lie in the same depth plane. There will be some small deviations from exactly zero because the geometry of the viewing arrangements results in the extremities of the display being at a slightly different distance than the centre. Therefore, the visual stimulation used to test the binocular condition is always at zero disparity, with a slight deviation from zero at the edges of the display, and never changes. [There is a detail that can be ignored. The experimenters tested neurons with visual stimulation at different real distances from the eyes, but this is not relevant here. Provided the animals accurately converged their eyes on the provided binocular fixation point, then the disparity of the visual stimuli will always be at or close to zero, regardless of viewing distance in these circumstances.] However, we already know from earlier work that neurons in the visual cortex exhibit a range of selectivity for binocular disparity. Some neurons have their peak response at non-zero disparities, representing binocular depths nearer than the fixation depth or beyond it. The response of other neurons is maximally suppressed by disparities at the depth of the fixation point (so-called Tuned Inhibitory [TI] neurons). The simple model and analysis presented in the paper for the summation of monocular responses to predict binocular responses will perform adequately for neurons that are tuned to zero disparity, so-called tuned excitatory neurons [TE], but is necessarily compromised when applied to neurons that have other, different tuning profiles. Specifically, when neurons are stimulated binocularly with a non-preferred disparity, the binocular response may be lower than the monocular response[2, 3]. This more realistic view of binocular responses needs to be considered by the authors and integrated into their modelling.

4) The data in the paper show some features that have been reported before but are not captured by the model. Notably for neurons with extreme values of ocular dominance, the binocular response is typically less than the larger of the two monocular responses. This is apparent in the row of plots in Figure 2D from individual animals and in the pooled data in Figure 2E. Responses of this type are characteristic of tuned inhibitory [TI] neurons[2]. It is not immediately clear why this feature of the data does not appear in the summary and analysis in Figure 3. The paper text states that the responses were "first normalized by the median of the binocular responses". This will certainly get rid of this characteristic of the data, but this step needs better justification, or an amendment to the main analysis is needed. In the present form, the model and analysis do not appear to fit the data in Figure 2 as accurately as needed. The authors should address the discrepancy between the data as presented in Figures 2D, E, and Figure 3.

Citations<br /> 1. Prince, S.J.D., Pointon, A.D., Cumming, B.G., and Parker, A.J., (2002). Quantitative analysis of the responses of V1 neurons to horizontal disparity in dynamic random-dot stereograms. Journal of Neurophysiology, 87: 191-208.<br /> 2. Prince, S.J.D., Cumming, B.G., and Parker, A.J., (2002). Range and mechanism of encoding of horizontal disparity in macaque V1. Journal of Neurophysiology, 87: 209-221.<br /> 3. Poggio, G.F. and Fischer, B., (1977). Binocular interaction and depth sensitivity in striate and prestriate cortex of behaving rhesus monkey. Journal of Neurophysiology, 40: 1392-1405 doi 10.1152/jn.1977.40.6.1392.

Reviewer #3 (Public Review):

Summary:<br /> This paper presents a new formulation of a computational model of adaptive learning amid environmental volatility. Using a behavioral paradigm and data set made available by the authors of an earlier publication (Gagne et al., 2020), the new model is found to fit the data well. The model's structure consists of three weighted controllers that influence decisions on the basis of (1) expected utility, (2) potential outcome magnitude, and (3) habit. The model offers an interpretation of psychopathology-related individual differences in decision-making behavior in terms of differences in the relative weighting of the three controllers.

Strengths:<br /> The newly proposed "mixture of strategies" (MOS) model is evaluated relative to the model presented in the original paper by Gagne et al., 2020 (here called the "flexible learning rate" or FLR model) and two other models. Appropriate and sophisticated methods are used for developing, parameterizing, fitting, and assessing the MOS model, and the MOS model performs well on multiple goodness-of-fit indices. The parameters of the model show decent recoverability and offer a novel interpretation for psychopathology-related individual differences. Most remarkably, the model seems to be able to account for apparent differences in behavioral learning rates between high-volatility and low-volatility conditions even with no true condition-dependent change in the parameters of its learning/decision processes. This finding calls into question a class of existing models that attribute behavioral adaptation to adaptive learning rates.

Weaknesses:<br /> 1. Some aspects of the paper, especially in the methods section, lacked clarity or seemed to assume context that had not been presented. I found it necessary to set the paper down and read Gagne et al., 2020 in order to understand it properly.

2. There is little examination of why the MOS model does so well in terms of model fit indices. What features of the data is it doing a better job of capturing? One thing that makes this puzzling is that the MOS and FLR models seem to have most of the same qualitative components: the FLR model has parameters for additive weighting of magnitude relative to probability (akin to the MOS model's magnitude-only strategy weight) and for an autocorrelative choice kernel (akin to the MOS model's habit strategy weight). So it's not self-evident where the MOS model's advantage is coming from.

3. One of the paper's potentially most noteworthy findings (Figure 5) is that when the FLR model is fit to synthetic data generated by the expected utility (EU) controller with a fixed learning rate, it recovers a spurious difference in learning rate between the volatile and stable environments. Although this is potentially a significant finding, its interpretation seems uncertain for several reasons:

- According to the relevant methods text, the result is based on a simulation of only 5 task blocks for each strategy. It would be better to repeat the simulation and recovery multiple times so that a confidence interval or error bar can be estimated and added to the figure.

- It makes sense that learning rates recovered for the magnitude-oriented (MO) strategy are near zero, since behavior simulated by that strategy would have no reason to show any evidence of learning. But this makes it perplexing why the MO learning rate in the volatile condition is slightly positive and slightly greater than in the stable condition.

- The pure-EU and pure-MO strategies are interpreted as being analogous to the healthy control group and the patient group, respectively. However, the actual difference in estimated EU/MO weighting between the two participant groups was much more moderate. It's unclear whether the same result would be obtained for a more empirically plausible difference in EU/MO weighting.

- The fits of the FLR model to the simulated data "controlled all parameters except for the learning rate parameters across the two strategies" (line 522). If this means that no parameters except learning rate were allowed to differ between the fits to the pure-EU and pure-MO synthetic data sets, the models would have been prevented from fitting the difference in terms of the relative weighting of probability and magnitude, which better corresponds to the true difference between the two strategies. This could have interfered with the estimation of other parameters, such as learning rate.

- If, after addressing all of the above, the FLR model really does recover a spurious difference in learning rate between stable and volatile blocks, it would be worth more examination of why this is happening. For example, is it because there are more opportunities to observe learning in those blocks?

4. Figure 4C shows that the habit-only strategy is able to learn and adapt to changing contingencies, and some of the interpretive discussion emphasizes this. (For instance, line 651 says the habit strategy brings more rewards than the MO strategy.) However, the habit strategy doesn't seem to have any mechanism for learning from outcome feedback. It seems unlikely it would perform better than chance if it were the sole driver of behavior. Is it succeeding in this example because it is learning from previous decisions made by the EU strategy, or perhaps from decisions in the empirical data?

5. For the model recovery analysis (line 567), the stated purpose is to rule out the possibility that the MOS model always wins (line 552), but the only result presented is one in which the MOS model wins. To assess whether the MOS and FLR models can be differentiated, it seems necessary also to show model recovery results for synthetic data generated by the FLR model.

6. To the best of my understanding, the MOS model seems to implement valence-specific learning rates in a qualitatively different way from how they were implemented in Gagne et al., 2020, and other previous literature. Line 246 says there were separate learning rates for upward and downward updates to the outcome probability. That's different from using two learning rates for "better"- and "worse"-than-expected outcomes, which will depend on both the direction of the update and the valence of the outcome (reward or shock). Might this relate to why no evidence for valence-specific learning rates was found even though the original authors found such evidence in the same data set?

7. The discussion (line 649) foregrounds the finding of greater "magnitude-only" weights with greater "general factor" psychopathology scores, concluding it reflects a shift toward simplifying heuristics. However, the picture might not be so straightforward because "habit" weights, which also reflect a simplifying heuristic, correlated negatively with the psychopathology scores.

Reviewer #3 (Public Review):

Summary:<br /> The manuscript from Tariq and Maurici et al. presents important biochemical and biophysical data linking protein phosphorylation to phase separation behavior in the repressive arm of the Neurospora circadian clock. This is an important topic that contributes to what is likely a conceptual shift in the field.

Reviewer #3 (Public Review):

Summary<br /> Pham, Pahuja, Hagenbeek, et al. have conducted a comprehensive range of assays to biochemically and genetically determine TEAD degradation through RNF146 ubiquitination. Additionally, they designed a PROTAC protein degrader system to regulate the Hippo pathway through TEAD degradation. Overall, the data appears robust. However, the manuscript lacks detailed methodological descriptions, which should be addressed and improved. For instance, the methods used to analyze the K48 ubiquitination site on TEAD and the gene expression analysis of Hippo Signaling are unclear. Furthermore, the multiple proteomics, RNA-seq, and ATAC-seq data must be made publicly available upon publication to ensure reproducibility. Most of the main figures are of low resolution, which needs addressing.

Strengths:<br /> - A broad range of assays was used to robustly determine the role of RNF146 in TEAD degradation.<br /> - Development of novel PROTAC for degrading TEAD.

Weaknesses:<br /> - An orthogonal approach is needed (e.g., PARP1 inhibitor) to demonstrate PARP1's dependency in TEAD ubiquitination.

- The data from Table 2 is unclear in illustrating the association of identified K48 ubiquitination with TEAD4, especially since the experiments were presumably to be conducted on whole cell lysates with KGG enrichment. This raises the possibility that the K48 ubiquitination could originate from other proteins. Alternatively, if the authors performed immunoprecipitation on TEAD followed by mass spectrometry, this should be explicitly described in the text and materials and methods section.

- Figure 2D: The methodology for measuring the Hippo signature is unclear, as is the case for Figures 7E and F regarding the analysis of Hippo target genes.

- Figure S3F requires quantification with additional replicates for validation.

- There is a misleading claim in the discussion stating "TEAD PARylation by PAR-family members (Figure 3)"; however, the demonstration is only for PARP1, which should be corrected.

Reviewer #3 (Public Review):

Summary:<br /> In this paper, Curran et al investigate the role of Ntn, Slit1, and Slit 2 in the axon patterning of DRG neurons. The paper uses mouse genetics to perturb each guidance molecule and its corresponding receptor. Cre-based approaches and immunostaining of DRG neurons are used to assess the phenotypes. Overall, the study uses the strength of mouse genetics and imaging to reveal new genetic modifiers of DRG axons. The conclusions of the experiments match the presented results. The paper is an important contribution to the field, as evidence that dorsal funiculus formation is impacted by Ntn and Slit signaling. However, there are some potential areas of the manuscript that should be edited to better match the results with the conclusions of the work.

Strengths:<br /> The manuscript uses the advantage of mouse genetics to investigate the axon patterning of DRG neurons. The work does a great job of assessing individual phenotypes in single and double mutants. This reveals an intriguing cooperative and independent function of Ntn, Slit1, and Slit2 in DRG axon patterning. The sophisticated triple mutant analysis is lauded and provides important insight.

Weaknesses:<br /> Overall, the manuscript is sound in technique and analysis. However, the majority of the manuscript is about the dorsal funiculus and not the bifurcation of the axons, as the title would make a reader believe. Further, the manuscript would provide a more scholarly discussion of the current knowledge of DRG axon patterning and how their work fits into that knowledge.

Reviewer #3 (Public Review):

In this work, the authors shed light onto the structures of Fusarium oxysporum f.sp. lycopersici proteins involved in the infection of tomato. They unravelled several new secreted effector protein structures and additionally used computational approaches to structurally classify the remaining effectors known from this pathogen. Through this they uncovered a new and unique structural class of proteins which they found to be present and widely distributed in fungal plant pathogens and plant symbiotic fungi. The authors further predicted structural models for the full SIX effector set revealing that genome-proximal effector pairs share similar structural classes. Building on their Avr1 structure, the authors also define the C-terminal domain and specific amino acid residues that are essential to Avr1 detection by its cognate immune receptor.

A major strength of this work is a portfolio of several (Avr1, Avr3, SIX6, SIX8) new structurally resolved proteins which led to the discovery that several of them fall into the same structural class. These findings are supported by strong results.

The experiments addressing the structure-function relationship of Avr1's avirulence activity are highly relevant to our understanding of disease resistance mechanisms against Fusarium. Additional controls would allow for better support of the conclusions to be drawn. An example is FonSIX4's cell death activity in N.benthamiana leaves and whether FonSIX4 cdll death is indeed dependent on the tomato I receptor. Complementary work in Fusarium mutants lacking Avr1 and expressing chimeric versions would document that the observations from transient expressions in Nicotiana benthamiana are relevant in the biological context of a Fusarium/tomato interaction.

The discovered solvent-exposed residues conditioning Avr1 recognition by the I receptor seem to be positioned in an area of the protein which had previously been highlighted as being highly variable in FOLD proteins of symbiotic fungi but it is not clear from the work whether this is indeed the case or whether Avr1 differs significantly in its structure from FOLD proteins found in other fungi.<br /> It remains to be tested whether the residues conditioning avirulence activity are also crucial for virulence activity in Fusarium.

This work uncovered a new structural class of proteins with critical roles in plant-pathogen interactions. Structure-based predictions and genome-wide comparisons have emerged as a new approach enabling the identification of similar proteins with divergent sequences. The work undertaken by the authors adds to a growing body of work in plant-microbe research, predominantly from pathogenic organisms, and more recently in symbiotic fungi.

Reviewer #3 (Public Review):

Summary:<br /> In this study, the authors investigated the potential of nifuroxazide to enhance responsiveness to radiotherapy, employing both an in vitro cell culture system and an in vivo syngeneic mouse tumor model.

Strengths:<br /> The researchers conducted a series of experiments to elucidate the role of nifuroxazide in facilitating the radiotherapy-induced reduction of proliferation, migration, and invasion of HepG2 cells.

Weaknesses:<br /> The evidence supporting the claim that nifuroxazide contributes to the degradation of radiotherapy-induced upregulation of PD-L1 via the ubiquitin-proteasome pathway is still relatively weak.

Reviewer #3 (Public Review):

Summary:<br /> The manuscript introduces a new computational framework for choosing 'the best method' according to the case for getting the best possible structural prediction for the CDR-H3 loop. The authors show their strategy improves on average the accuracy of the predictions on datasets of increasingly difficulty in comparison to several state-of-the-art methods. They also show the benefits of improving the structural predictions of the CDR-H3 in the evaluation of different properties that may be relevant for drug discovery and therapeutics design.

Strengths:<br /> Authors introduce a novel framework, which can be easily adapted and improved. Authors use a well defined dataset to test their new method. A modest average accuracy gain is obtained in comparison to other state-of-the art methods for the same task, while avoiding for testing different prediction approaches. Although the accuracy gain is mainly ascribed to easy cases, the accuracy and precision for moderate to challenging cases is comparable to the best PLM methods (see Fig. 4b and Extended Data Fig. 2), reflecting the present methodological limit in the field.

Weaknesses:<br /> The proposed method lacks of a confidence score or a warning to help guiding the users in moderate to challenging cases.

Reviewer #3 (Public Review):

Summary:<br /> This study examines the impact of CTRP10/C1QL2 absence on obesity and metabolic health in mice. Female mice lacking CTRP10 tend to develop obesity, particularly on a high-fat diet. Surprisingly, they do not display the typical metabolic traits associated with obesity, like fatty liver or glucose intolerance. This indicates a disconnection between weight gain and metabolic issues in these female mice. The research underscores the need to understand sex-specific factors in how obesity influences metabolic health.

Strengths:<br /> The study provides compelling evidence regarding Ctrp10's role in female-specific metabolic regulation in mice, shedding light on its potential significance in metabolically healthy obese (MHO) individuals.

Weaknesses:<br /> -The analysis and description of sex-specific human data require more details to highlight the relevance of Ctrp10 mouse data and the analysis of differentially expressed genes in humans.<br /> -There's a lack of analysis regarding secreted Ctrp10 under various dietary conditions.<br /> -The study didn't assess adipose tissue function to evaluate metabolic health.

Reviewer #3 (Public Review):

Summary:

Bartolome et al. present ProteasomeID, a novel method to identify components, interactors, and (potentially) substrates of the proteasome in cell lines and mouse models. As a major protein degradation machine that is highly conserved across eukaryotes, the proteasome has historically been assumed to be relatively homogeneous across biological scales (with few notable exceptions, e.g., immunoproteasomes and thymoproteasomes). However, a growing body of evidence suggests that there is some degree of heterogeneity in the composition of proteasomes across cell tissues, and can be highly dynamic in response to physiologic and pathologic stimuli. This work provides a methodological framework for investigating such sources of variation. The authors start by adapting the increasingly popular biotin ligation strategy for labelling proteins coming into close proximity with one of three different subunits of the proteasome, before proceeding with PSMA4 for further development and analysis based on their preliminary labelling data. In a series of well-constructed and convincing validation experiments, the authors go on to show that the tagged PSMA4 construct can be incorporated into functional proteasomes, and is able to label a broad set of known proteasome components and interacting proteins in HEK293T cells. They also attempt to identify novel proteasomal degradation substrates with ProteasomeID; while this was convincing for known substrates with particularly short half-lives (exemplified by the transcription factor c-myc), follow-up validation experiments with other substrates were less clear. One of the most compelling results was from a similar experiment to confirm proteasomal degradation induced by a BRD-targeting PROTAC, which I think is likely to be of keen interest to the targeted degradation community. Finally, the authors establish a ProteasomeID mouse model, and demonstrate its utility across several tissues.

Strengths:

1) ProteasomeID itself is an important step forward for researchers with an interest in protein turnover across biological scales (e.g., in sub-cellular compartments, in cells, in tissues, and whole organisms). I especially see interest from two communities: those studying fundamental proteostasis in physiological and pathologic processes (e.g., ageing; tissue-specific protein aggregation diseases), and those developing targeted protein degradation modalities (e.g., PROTACs; molecular glues). All the datasets generated and deposited here are likely to provide a rich resource to both. The HEK293T cell line data are a valuable proof-of-concept to allow expansion into more biologically-relevant cell culture settings; however, I envision the greatest innovation here to be the mouse model. For example, in the targeted protein degradation space, two major hurdles in early-stage pre-clinical development are (i) evaluation of degradation efficacy across disease-relevant tissues, and (ii) toxicity and safety implications caused by off-target degradation, e.g., of newly-identified molecular glues and/or in particularly-sensitive tissues. The ProteasomeID mouse allows early in vivo assessment of both these questions. The results of the BRD PROTAC experiment in 293T cells provides an excellent in vitro proof-of-concept for this approach.

2) The mass spectrometry-based proteomics workflows used and presented throughout the manuscript are robust, rigorous, and convincing. For example, the algorithm the authors use for defining enrichment score cut-offs are logical and based on rational models, rather than on arbitrary cut-offs that are common for similar proteomics studies. The construction (and subsequent validation) of both BirA*- and miniTurbo- tagged PSMA4 variants also increases the utility of the method, allowing researchers to choose the variant with the labelling time-scale required for their particular research question.

3) The optimised BioID and TurboID protocol the authors develop (summarised in Fig. S2A) and validate (Fig. S2B-D) is likely to be of broad interest to cell and molecular biologists beyond the protein degradation field, given that proximity labelling is a current gold-standard in global protein:protein interaction profiling.

Limitations:

I think the authors do an excellent job in highlighting the limitations of ProteasomeID throughout the Results and Discussion. I do have some specific comments that might provide additional context for the reader.

1) The authors do a good job in showing that a substantial proportion of PSMA4-BirA* is incorporated into functional proteasome particles; however, it is not immediately clear to me how much background (false-positive IDs) might be contributed by the ~40 % of PSMA4-BirA* that is not incorporated into the mature core particle (based on the BirA* SEC-MS traces in Fig. 2b and S3b, i.e., the large peak ~ fraction 20). Are there any bands lower down in the native gel shown in Fig. 2c, i.e., corresponding to lower molecular weight complexes or monomeric PSMA4-BirA*? The enrichment of proteasome assembly factors in all the ProteasomeID experiments might suggest the presence of assembly intermediates, which might themselves become substrates for proteasomal degradation (as has been shown for other incompletely-assembled protein complexes, e.g., the ribosome, TRiC/CCT).

2) Although the authors attempt to show that BirA* tagging of PSMA4 does not interfere with proteasome activity (Fig. 2e-f), I think the experimental evidence for this is incomplete. They show that the overall chymotrypsin-like activity (attributable to PSMB5) in cells expressing PSMA4-BirA* is not markedly reduced compared with control BirA*-expressing cells. However, they do not show that the activity of the specific proteasome sub-population that contains PSMA4-BirA* is unaffected (e.g., by purifying this sub-population via the Flag tag). The proteasome activity of the sub-population of wild-type proteasome complexes that do not contain the PSMA4-BirA* (~50%, based on the earlier immunoblots) could account for the entire chymotrypsin-like activity-especially in the context of HEK293T cells, where steady-state proteasome levels are unlikely to be limiting. It would also be useful to assess any changes in tryspin- and caspase- like activities, especially as tagging of PSMA4 could conceivably interfere with the activity of some PSMB subunits, but not others.

3) I was left unsure of the general utility of ProteasomeID for identifying novel proteasomal substrates in homeostatic or stressed conditions. The immunoblots for the two candidates the authors follow up in Fig. 4g was not especially clear; the reduction in the bands are modest, at best. Furthermore, classifying candidates based on enrichment following proteasome inhibition with MG-132 have the potential to lead to a high number of false positives. ProteasomeID's utility in identifying potential substrates in more targeted settings (e.g., molecular glues, off-target PROTAC substrates) is far more apparent.

Reviewer #3 (Public Review):

Summary:<br /> The manuscript by Zhang et al. analyzes the function of the centrosomal protein CEP44 in centriole duplication and in the formation of the mitotic spindle. The first part addresses the role of CEP44 at centrioles. Using mostly RNAi-mediated depletion in cell lines and in some cases KO cells, the authors find increased centriole numbers in depleted cells and, based on quantification of centrioles stained with various centriole markers as well as live imaging, conclude that this is due to premature centriole disengagement and overduplication. The second part, which is largely independent of the first, focuses on the role of CEP44 in the mitotic spindle. The authors find that CEP44 is phosphorylated in mitosis in an Aurora A-dependent manner and identify the phosphorylation site, which controls CEP44 spindle localization and functions in maintaining spindle integrity.

Strength:<br /> The manuscript makes the interesting observation that reduced expression of CEP44 is observed in breast cancer and correlated with poor survival in patients.<br /> The analysis of mitotic phosphorylation including the identification of the modified site and its role in spindle recruitment is interesting and useful.

Weakness:<br /> The authors seem to largely ignore previously published work that contrasts with the findings presented in the current study. The previous work found a role of CEP44 in centriole formation and centrosome conversion and observed reduced centriole numbers in depleted cells, whereas the current study claims the opposite, a role in centriole engagement that leads to overduplication and increased centriole number in depleted cells. However, the supporting evidence is not strong enough, especially in light of the previous work. Considering that CEP44 depletion also disrupts mitosis, which could affect centriole numbers by failed segregation/division, a more careful analysis in synchronized cultures would be needed. Also, cell cycle analysis would be required to rule out cell cycle effects in CEP44-depleted cells, which could also explain altered centriole numbers. Moreover, the quality of the imaging is often not sufficient to support the claims.<br /> The second part is largely disconnected from the first and reads as if it was a separate study. There is no attempt to integrate both parts. For example, the second part seems to largely focus on normal bipolar spindles, even though the first part reveals multipolarity as a phenotype after CEP44 knockdown. It remains unclear if the spindle defects are due to centriole defects, defective spindle microtubule stability/organization, or both, and whether the centriole-localized or spindle-localized CEP44 is involved.

Another weak aspect is that neither for RNAi nor for KO cells the authors show that CEP44 is depleted at centrioles and to what extent. This is only shown in cell extract.

Reviewer #3 (Public Review):

Summary:<br /> The DNA damage checkpoint (DDC) inhibits the metaphase-anaphase transition to repair various types of DNA damage, including DNA double strand breaks (DSBs). One irreparable DSB can maintain the DDC for 12-15 hours in yeast, after which the cells resume the cell cycle. If there are two DSBs, the DDC is maintained for at least 24 hours. In this study, the authors take advantage of this tighter DDC to investigate whether the best-known proteins involved in establishing the DDC are also responsible for its long-term maintenance during irreparable DSBs. They do this by cleverly degrading such proteins after DSB formation. They show that most, but not all, DDC proteins maintain the cell cycle block. Interestingly, DDC proteins become dispensable after 15 hours and the block is then maintained by spindle assembly checkpoint (SAC) proteins.

Strengths:<br /> The authors have engineered a tight yeast system to study DDC shutdown after irreparable DSBs and used it to address whether checkpoint proteins (DDC and SAC) contribute to the long-term maintenance of DSB-mediated G2/M block. The different roles of Ddc2, Chk1, and Dun1 are interesting, while the fact that SAC overtakes DDC after 15 hours is intriguing and highlights how DSBs near and far from centromeres can have a profound impact on cell adaptation to DSBs.

Weaknesses:<br /> Some of the results they present essentially confirm their own previous findings, albeit with a tighter strain design for long-term arrest. In addition, some conclusions about the role of specific DDC proteins in cell cycle arrest at G2/M need further experimental support. The results with Bfa1/Bub2 are surprising and somewhat unexpected. There is no clear mechanism for how depletion of Bub2, but not Bfa1, can relieve the G2/M (metaphase) block.

Reviewer #3 (Public Review):

Summary:<br /> Prior research on SCC3, a cohesin subunit protein, in yeast and Arabidopsis has underscored its vital role in cell division. This study investigated into the specific functions of SCC3 in rice mitosis and meiosis. In a weakened SCC3 mutant, sister chromatids separating was observed in anaphase I, resulting in 24 univalents and subsequent sterility. The authors meticulously documented SCC3's loading and degradation dynamics on chromosomes, noting its impact on DNA replication. Despite the loss of homologous chromosome pairing and synapsis in the mutant, chromosomes retained double-strand breaks without fragmenting. Consequently, the authors inferred that in the scc3 mutant, DNA repair more frequently relies on sister chromatids as templates compared to the wild type.

Strengths:<br /> The study presents exceptionally well-executed research in the field of rice cytogenetics.

Weaknesses:<br /> While the paper's conclusions are generally well-supported, further substantiation is needed for the claim that SCC3 inhibits template choice for sister chromatids. To bolster this conclusion, I recommend that the authors perform whole-genome sequencing on parental and F1 individuals from two rice variants, subsequently calculating the allele frequencies at heterozygous sites in the F1 individuals. If SCC3 indeed inhibits inter-sister chromatid repair in the wild type, we would anticipate a higher frequency of inter-homologous chromosome repair (i.e., gene conversion). This should be manifested as a bias away from the Mendelian inheritance ratio (50:50) in the offspring of the wild type compared to the offspring of the scc3+/- mutant.

Reviewer #3 (Public Review):

Summary:<br /> This manuscript reports the novel observation of alterations in the nuclear pore (NUP) components and the function of the nuclear envelope in knock-in models of APP and presenilin mutations. The data show that loss of NUP immunoreactivity (IR) and pore density are observed at times prior to plaque deposition in this model. The loss of NUP IR is correlated with an increase in intraneuronal Abeta IR with two monoclonal antibodies that react with the N-terminus of Abeta. Similar results are observed in cultured neurons from APP-KI and Wt mice where further results with cultured neurons indicate that Abeta "drives" this process: incubation of neurons with oligomeric, but not monomeric or fibrillar Abeta causes loss of NUP IR, incubation with conditioned media from KI cells but not wt cells also causes loss of NUP IR and treatment with the gamma secretase inhibitor, NAPT partially blocks the loss of NUP IR. Further data show that nuclear envelope function is altered in KI cells and KI cells are more sensitive to TNFalpha-induced necroptosis. This is potentially an important and significant report, but how this fits within the larger picture of what is known about amyloid aggregation and accumulation and pathogenesis in neurons needs to be clarified. The results from mouse brains are strong, while the results from cultured cells are in some instances are of a lower magnitude, less convincing, ambiguous, and sometimes over-interpreted.

Strengths:<br /> 1. Loss of NUP expression and activity is a novel observation.<br /> 2. Its association with intraneuronal Abeta immunoreactivity suggests an association with Alzheimer's disease.<br /> 3. The experiments generally appear to be well-controlled.<br /> 4. Multiple approaches are sometimes used to increase the robustness of the data.

Weaknesses:<br /> 1. It does not consider the relationship of the findings here to other published work on the intraneuronal perinuclear and nuclear accumulation of amyloid in other transgenic mouse models and in humans.<br /> 2. It appears to presume that soluble, secreted Abeta is responsible for the effect rather than the insoluble amyloid fibrils.<br /> 3. Most of the critical findings on the association with Abeta and the functional consequences are done in cultured neurons and not in mouse models.<br /> 4. There is no evidence from the human brain that would strengthen the significance.<br /> 5. It is not clear when the alteration in NUP expression begins in the KI mice as there is no time at which there is no difference between NUP expression in KI and Wt and the earliest time shown is 2 months. If NUP expression is decreased from the earliest times at birth, then this makes the significance of the observation of the association with amyloid pathology less clear.

Reviewer #3 (Public Review):

This study presents results arguing that the mammalian acetyltransferase Tip60/KAT5 auto-acetylates itself on one specific lysine residue before the MYST domain, which in turn favors not only nuclear localization but also condensate formation on chromatin through LLPS. The authors further argue that this modification is responsible for the bulk of Tip60 autoacetylation and acetyltransferase activity towards histone H4. Finally, they suggest that it is required for association with txn factors and in vivo function in gene regulation and DNA damage response.

These are very wide and important claims and, while some results are interesting and intriguing, there is not really close to enough work performed/data presented to support them. In addition, some results are redundant between them, lack consistency in the mutants analyzed, and show contradiction between them. The most important shortcoming of the study is the fact that every single experiment in cells was done in over-expressed conditions, from transiently transfected cells. It is well known that these conditions can lead to non-specific mass effects, cellular localization not reflecting native conditions, and disruption of native interactome. On that topic, it is quite striking that the authors completely ignore the fact that Tip60 is exclusively found as part of a stable large multi-subunit complex in vivo, with more than 15 different proteins. Thus, arguing for a single residue acetylation regulating condensate formation and most Tip60 functions while ignoring native conditions (and the fact that Tip60 cannot function outside its native complex) does not allow me to support this study.

Specific points:<br /> -It is known that over-expression after transient transfection can lead to non-specific acetylation of lysines on the proteins, likely in part to protect from proteasome-mediated degradation. It is not clear whether the Kac sites targeted in the experiments are based on published/public data. In that sense, it is surprising that the K327R mutant does not behave like a HAT-dead mutant (which is what exactly?) or the K187R mutant as this site needs to be auto-acetylated to free the catalytic pocket, so essential for acetyltransferase activity like in all MYST-family HATs. In addition, the effect of K187R on the total acetyl-lysine signal of Tip60 is very surprising as this site does not seem to be a dominant one in public databases.

-As the physiological relevance of the results is not clear, the mutants need to be analyzed at the native level of expression to study real functional effects on transcription and localization (ChIP/IF). It is not clear the claim that Tip60 forms nuclear foci/punctate signals at physiological levels is based on what. This is certainly debated because in part of the poor choice of antibodies available for IF analysis. In that sense, it is not clear which Ab is used in the Westerns. Endogenous Tip60 is known to be expressed in multiple isoforms from splice variants, the most dominant one being isoform 2 (PLIP) which lacks a big part (aa96-147) of the so-called IDR domain presented in the study. Does this major isoform behave the same?

-It is extremely strange to show that the K187R mutant fails to get in the nuclei by cell imaging but remains chromatin-bound by fractionation... If K187 is auto-acetylated and required to enter the nucleus, why would a HAT-dead mutant not behave the same?

-If K187 acetylation is key to Tip60 function, it would be most logical (and classical) to test a K187Q acetyl-mimic substitution. In that sense, what happens with the R188Q mutant? That all goes back to the fact that this cluster of basic residues looks quite like an NLS.

-The effect of the mutant on the TIP60 complex itself needs to be analyzed, e.g. for associated subunits like p400, ING3, TRRAP, Brd8...

-The discussion is excessively long without addressing the obvious questions mentioned above.

Reviewer #3 (Public Review):

In this study, the authors demonstrated a new mechanism by which the protein stability of EZH2 is regulated. This mechanism is multifaceted and yet the authors provided evidence for every step of regulation. EZH2 is monomethylated at K20 by SET7, which can be removed by LSD1 and recognized by L3MBTL3. L3MBTL3 recruits the ubiquitin E3 ligase CRLDCAF5 to EZH2 via methylation of K20, which results in polyubiquitylation and proteasomal degradation of the histone methyltransferase. Additionally, they found that AKT-mediated phosphorylation of EZH2 at S21 blocks monomethylation at K20 and vice versa. Finally, they demonstrated in the K20R GEMM model that stabilization of EZH2 protein leads to reactive hyperplasia and hematopoiesis. In general, this study reveals an interesting and novel mechanism underlying the regulation of the epigenetic mark H3K27me3 and the oncogenic function of EZH2. The authors have considered every aspect of the signaling pathway that regulates the protein stability of EZH2. The data was comprehensive, rigorous, and supportive of the conclusions they made. Their results may help explain some of the conflicting results that previous studies have reported.

However, there are still some issues with the significance of the work and the quality of the data. The major issues are:<br /> 1. The converged effect of EZH2 methylation and phosphorylation on H3K27me3 is unclear.<br /> 2. How the methylation-phosphorylation switch of EZH2 determines the biological phenotypes they observed is not addressed.<br /> 3. Some of the data in the manuscript is conflicting.

Reviewer #3 (Public Review):

In their manuscript, Pivirotto et al. make an unexpected observation that a set of candidate beneficial alleles according to the Evolutionary Probability method (EP) have estimated ages thousands of years older than control alleles of similar frequency and outside of functional segments. To explain this unexpectedly older ages, the authors propose a number of interesting evolutionary processes related to balancing selection, including staggered sweeps.

It is important to first mention that the authors do find that as expected, deleterious alleles are younger than controls. This provides evidence that the allele age estimates used by the authors are of sufficient quality to detect age differences between groups of genes. I am also convinced by the fact that EP can be used to focus on a set of alleles substantially enriched in deleterious ones, given the very clear frequency patterns related to EP.

I have a number of concerns about the manuscript, including one rather serious one.

My main concern is that many of the observations made by the authors could be caused by mispolarization of alleles, where either (i) mostly low frequency derived alleles are mischaracterized as ancestral and the other, actually ancestral allele is mischaracterized as a high frequency derived allele, or (ii) mostly low frequency ancestral alleles are mischaracterized as derived. Unfortunately, the authors do not even mention the risk of mispolarization in their manuscript. This is a serious problem for this manuscript because ancestral alleles annotated as derived are by definition going to generate older age estimates than if they were truly derived. It would be very useful to be able to have a look at the full distribution of allele ages rather than just confidence intervals as in Figure 1. I happen to have experience with mispolarization of high frequency ancestral alleles as derived by a maximum likelihood method, different from the one used by the authors (Keightley et al Genetics 2018), where the mispolarization became visible as a very suspicious SFS with a visible excess of high frequency variants, especially those expected to be functional (because of the relatively larger corresponding supply of low frequency deleterious functional variants). Even if the ML method used by the authors is not the same, mispolarization is still a serious risk. Glémin et al. Genome Research 2015 also found that mispolarization is far from being a negligible issue.

Mispolarization of low frequency alleles may be especially prominent in the case of mispolarized deleterious alleles associated with a very negative delta-EP, that then appear as alleles with a very positive delta-EP. Focusing on high delta-EP alleles may then in fact enrich the dataset in mispolarized alleles that then result in older age estimates. Looking at Figure 1B especially, I am worried by the fact that very high delta-EP values seem to go back to the frequencies observed for very negative delta-EP. This is what mispolarization of low frequency alleles might cause as a pattern, in this case especially low frequency ancestral alleles being misidentified as derived?

The authors can address the possible issue of mispolarization in multiple ways. First, they can use simulations of sequences to estimate amounts of mispolarization based on their polarization approach, using substitutions/mutation rates as realistic as possible.<br /> Second, the authors could check if there is suspicious symmetry in the distribution of delta-EP between alleles at frequency f and alleles at frequency 1-f. This pattern could be generated by mispolarization.

My second less serious concern has to do with the use of high delta-EP as evidence that alleles are beneficial. The validation set from the Patel & Kumar 2019 paper is arguably small with 24 known selected variants. It does not follow from the fact that a small set of known selected variants have higher delta-EP, that all variants with high delta-EP tend to be beneficial. This is especially true in the case where beneficial variants tend to be rare, and there are then far more variants expected with high delta-EP than there are beneficial variants. I am willing to change my mind on this if the overall results can be shown to be robust after accounting for allele mispolarization.

Third, I like the idea of staggered sweeps to explain the results, but I am wondering if there is any evidence in the literature of interference between deleterious and advantageous variants that the authors could base their proposed explanation on.

Finally, and I realize that it is a bit of a stretch, I am wondering if the authors could better justify their choices of methods to estimate the age of alleles. What about ARGweaver, Relate or tsdate? How do these methods compare with GEVA? From looking at the literature I could not find a direct comparison of the precision of GEVA compared to these other tools, but it may be worth at least discussing that the results could be further put to the test with other available ARG-based tools to estimate allele ages. Wilder Wohns et al. Science 2022 compare the performance of these different ARG methods with ancient DNA data, and in fact find that GEVA does not perform as well as for example Relate or tsdate.

Reviewer #3 (Public Review):

Summary: In this manuscript, Blaeser et al. explore the link between CSD and headache pain. How does an electrochemical wave in the brain parenchyma, which lacks nociceptors, result in pain and allodynia in the V1-3 distribution? Prior work had established that CSD increased the firing rate of trigeminal neurons, measured electrophysiologically at the level of the peripheral ganglion. Here, Blaeser et al. focus on the fine afferent processes of the trigeminal neurons, resolving Ca2+ activity of individual fibers within the meninges. To accomplish these experiments, the authors injected AAV encoding the Ca2+ sensitive fluorophore GCamp6s into the trigeminal ganglion, and 8 weeks later imaged fluorescence signals from the afferent terminals within the meninges through a closed cranial window. They captured activity patterns at rest, with locomotion, and in response to CSD. They found that mechanical forces due to meningeal deformations during locomotion (shearing, scaling, and Z-shifts) drove non-spreading Ca2+ signals throughout the imaging field, whereas CSD caused propagating Ca2+ signals in the trigeminal afferent fibers, moving at the expected speed of CSD (3.8 mm/min). Following CSD, there were variable changes in basal GCamp6s signals: these signals were unchanged in the majority of fibers, signals increased (after a ~20 min delay) in 10% of fibers, and signals decreased in 20% of fibers. Bouts of locomotion were less frequent following CSD, but when they did occur, they elicited more robust GCamp6s signals than pre-CSD. These findings advance the field, suggesting that headache pain following CSD can be explained on the basis of peripheral cranial nerve activity, without invoking central sensitization at the brain stem/thalamic level. This insight could open new pathways for targeting the parenchymal-meningeal interface to develop novel abortive or preventive migraine treatments.

Strengths: The manuscript is well-written. The studies are broadly relevant to neuroscientists and physiologists, as well as neurologists, pain clinicians, and patients with migraine with aura and acephalgic migraine. The studies are well-conceived and appear to be technically well-executed.

Weaknesses: In the present study, conclusions are based entirely on fluorescence signals from GCamp6s. Fluorescence experiments should be interpreted cautiously in the context of CSD. GCamp6 fluorophores are strongly pH dependent, with decreased signal at acidic pH values (at matched Ca2+ concentration). CSD induces an impressive acidosis transient in the brain parenchyma, so one wonders whether the suppression of activity reported in the wake of CSD (Figure 2) in fact reflects decreased sensitivity of the GCamp6 reporter, rather than decreased activity in the fibers. If intracellular pH in trigeminal afferent fibers acidifies in the wake of CSD, GCamp6s fluorescence may underestimate the actual neuronal activity.

Reviewer #3 (Public Review):

The potential for sexual selection and the extent of sexual dimorphism in gene expression have been studied in great detail in animals, but hardly examined in plants so far. In this context, the study by Zhao, Zhou et al. al represents a welcome addition to the literature.

Relative to the previous studies in Angiosperms, the dataset is interesting in that it focuses on reproductive rather than somatic tissues (which makes sense to investigate sexual selection), and includes more than a single developmental stage (buds + mature flowers).

Reviewer #3 (Public Review):

The potential for sexual selection and the extent of sexual dimorphism in gene expression have been studied in great detail in animals, but hardly examined in plants so far. In this context, the study by Zhao, Zhou et al. al represents a welcome addition to the literature.

Relative to the previous studies in Angiosperms, the dataset is interesting in that it focuses on reproductive rather than somatic tissues (which makes sense to investigate sexual selection), and includes more than a single developmental stage (buds + mature flowers).

DOI: 10.1016/j.xpro.2023.102810

Resource: ZFIN_ZDB-GENO-040723-3

Curator: @scibot

SciCrunch record: RRID:ZFIN_ZDB-GENO-040723-3

What is this?

Reviewer #3 (Public Review):

Summary:<br /> The authors consider various statistical testing frameworks for model selection in the context of neuronal tuning. They consider cross-validation as a baseline scheme, and show various corrections and modifications to existing cross-validation schemes together with the underlying data/sign shuffling procedures for finding null distributions. Through careful simulations, they show that some of these tests are expectedly too conservative or too optimistic, and show that a log-likelihood-based test statistic with a cyclic shift permutation test for obtaining null distribution and Bonferroni correction strikes the right balance between hits and false detection. They further apply these tests to calcium imaging data from the mouse entorhinal cortex to identify grid cells (i.e., cells for which position is selected as a relevant variable).

Strengths:<br /> The paper is very well written, easy to follow, and enjoyable to read. It addresses an important issue in modern neuroscience, which is drawing conclusions based on data with missing or (unaccounted for) auto-correlated covariates.

Weaknesses:<br /> The paper would benefit from including more rigorous theoretical justification on why some of the procedures examined here outperform the others. This could be done in a stylized example with a Gaussian linear model, for which some of the used statistics have well-known distributions.

Comparisons with false discovery rate (FDR) control, as a more appropriate measure of performance when dealing with many comparisons, would benefit the existing comparisons merely based on Bonferroni correction.

Including spiking history in the generalized linear models (GLMs) used in analyzing the mouse data could be beneficial, as existing literature points to the importance of spiking history as a relevant covariate.

Reviewer #3 (Public Review):

Summary: Drosophila larvae exhibit characteristic escape behavior in response to a noxious stimulus. The underlying nociceptive circuit that regulates the temporal dynamics of escape behavior - bending, rolling, and crawling remains unclear. Using behavioral prototypes with optical stimulation and imaging, the authors show the function of descending neurons (SeIN128) in the termination of the rolling and subsequent initiation of the crawling behavior. The study further establishes the functional connectome of SeIN128, Basin-2, and A00c neurons, forming an inhibitory feedback circuit that regulates the rolling-escape sequences.

Strength: The study provides anatomical and functional evidence for temporal dynamics of escape behaviors in Drosophila larvae. Authors convincingly show the function of bilaterally descending neurons (previously identified SeIN128 neurons) in the transition of escape sequences. Based on the previous studies and functional connectome analysis, the study shows that SeIN128 neurons form a GABAergic feedback circuit with Basin-2, a second-order interneuron, and A00c, an ascending neuron downstream of Basin-2. Activation of SeIN128 neurons terminates the rolling by suppressing Basin-2 activity, facilitating subsequent rapid escape crawling. Thus, it establishes the function of feedback inhibition in temporal dynamics of escape behavior and contributes to a mechanistic understanding of the nociceptive circuits.

Weakness: The manuscript is written clearly; however, the presentation of the data needs to be improved for readability. The data and discussion establish the function of SeIN128 and Basin-2 in escape behavior, but the role of A00c neurons needs to be clarified.

Reviewer #3 (Public Review):

Summary<br /> In this paper, the authors model motor adaptation as a Bayesian process that combines visual uncertainty about the error feedback, uncertainty about proprioceptive sense of hand position, and uncertainty of predicted (=planned) hand movement with a learning and retention rate as used in state space models. The model is built with results from several experiments presented in the paper and is compared with the PReMo model (Tsay, Kim, et al., 2022) as well as a cue combination model (Wei & Körding, 2009). The model and experiments demonstrate the role of visual uncertainty about error feedback in implicit adaptation.

In the introduction, the authors notice that implicit adaptation (as measured in error-clamp-based paradigms) does not saturate at larger perturbations, but decreases again (e.g. Moorehead et al., 2017 shows no adaptation at 135{degree sign} and 175{degree sign} perturbations). They hypothesized that visual uncertainty about cursor position increases with larger perturbations since the cursor is further from the fixated target. This could decrease the importance assigned to visual feedback which could explain lower asymptotes.

The authors characterize visual uncertainty for 3 rotation sizes in the first experiment, and while this experiment could be improved, it is probably sufficient for the current purposes. Then the authors present a second experiment where adaptation to 7 clamped errors is tested in different groups of participants. The models' visual uncertainty is set using a linear fit to the results from experiment 1, and the remaining 4 parameters are then fit to this second data set. The 4 parameters are 1) proprioceptive uncertainty, 2) uncertainty about the predicted hand position, 3) a learning rate, and 4) a retention rate. The authors' Perceptual Error Adaptation model ("PEA") predicts asymptotic levels of implicit adaptation much better than both the PReMo model (Tsay, Kim et al., 2022), which predicts saturated asymptotes, or a causal inference model (Wei & Körding, 2007) which predicts no adaptation for larger rotations. In a third experiment, the authors test their model's predictions about proprioceptive recalibration, but unfortunately, compare their data with an unsuitable other data set. Finally, the authors conduct a fourth experiment where they put their model to the test. They measure implicit adaptation with increased visual uncertainty, by adding blur to the cursor, and the results are again better in line with their model (predicting overall lower adaptation) than with the PReMo model (predicting equal saturation but at larger perturbations) or a causal inference model (predicting equal peak adaptation, but shifted to larger rotations). In particular, the model fits experiment 2 and the results from experiment 4 show that the core idea of the model has merit: increased visual uncertainty about errors dampens implicit adaptation.

Strengths<br /> In this study, the authors propose a Perceptual Error Adaptation model ("PEA") and the work combines various ideas from the field of cue combination, Bayesian methods, and new data sets, collected in four experiments using various techniques that test very different components of the model. The central component of visual uncertainty is assessed in the first experiment. The model uses 4 other parameters to explain implicit adaptation. These parameters are 1) learning and 2) retention rate, as used in popular state space models, and the uncertainty (variance) of 3) predicted and 4) proprioceptive hand position. In particular, the authors observe that asymptotes for implicit learning do not saturate, as claimed before, but decrease again when rotations are very large and that this may have to do with visual uncertainty (e.g. Tsay et al., 2021, J Neurophysiol 125, 12-22). The final experiment confirms predictions of the fitted model about what happens when visual uncertainty is increased (overall decrease of adaptation). By incorporating visual uncertainty depending on retinal eccentricity, the predictions of the PEA model for very large perturbations are notably different from and better than, the predictions of the two other models it is compared to. That is, the paper provides strong support for the idea that visual uncertainty of errors matters for implicit adaptation.

Weaknesses<br /> Although the authors don't say this, the "concave" function that shows that adaptation does not saturate for larger rotations has been shown before, including in papers cited in this manuscript.

The first experiment, measuring visual uncertainty for several rotation sizes in error-clamped paradigms has several shortcomings, but these might not be so large as to invalidate the model or the findings in the rest of the manuscript. There are two main issues we highlight here. First, the data is not presented in units that allow comparison with vision science literature. Second, the 1 second delay between the movement endpoint and the disappearance of the cursor, and the presentation of the reference marker, may have led to substantial degradation of the visual memory of the cursor endpoint. That is, the experiment could be overestimating the visual uncertainty during implicit adaptation.

The paper's third experiment relies to a large degree on reproducing patterns found in one particular paper, where the reported hand positions - as a measure of proprioceptive sense of hand position - are given and plotted relative to an ever-present visual target, rather than relative to the actual hand position. That is, 1) since participants actively move to a visual target, the reported hand positions do not reflect proprioception, but mostly the remembered position of the target participants were trying to move to, and 2) if the reports are converted to a difference between the real and reported hand position (rather than the difference between the target and the report), those would be on the order of ~20{degree sign} which is roughly two times larger than any previously reported proprioceptive recalibration, and an order of magnitude larger than what the authors themselves find (1-2{degree sign}) and what their model predicts. Experiment 3 is perhaps not crucial to the paper, but it nicely provides support for the idea that proprioceptive recalibration can occur with error-clamped feedback.

Perhaps the largest caveat to the study is that it assumes that people do not look at the only error feedback available to them (and can explicitly suppress learning from it). This was probably true in the experiments used in the manuscript, but unlikely to be the case in most of the cited literature. Ignoring errors and suppressing adaptation would also be a disastrous strategy to use in the real world, such that our brains may not be very good at this. So the question remains to what degree - if any - the ideas behind the model generalize to experiments without fixation control, and more importantly, to real-life situations.

Specific comments:<br /> A small part of the manuscript relies on replicating or modeling the proprioceptive recalibration in a study we think does NOT measure proprioceptive recalibration (Tsay, Parvin & Ivry, JNP, 2020). In this study, participants reached for a visual target with a clamped cursor, and at the end of the reach were asked to indicate where they thought their hand was. The responses fell very close to the visual target both before and after the perturbation was introduced. This means that the difference between the actual hand position, and the reported/felt hand position gets very large as soon as the perturbation is introduced. That is, proprioceptive recalibration would necessarily have roughly the same magnitude as the adaptation displayed by participants. That would be several times larger than those found in studies where proprioceptive recalibration is measured without a visual anchor. The data is plotted in a way that makes it seem like the proprioceptive recalibration is very small, as they plot the responses relative to the visual target, and not the discrepancy between the actual and reported hand position. It seems to us that this study mostly measures short-term visual memory (of the target location). What is astounding about this study is that the responses change over time to begin with, even if only by a tiny amount. Perhaps this indicates some malleability of the visual system, but it is hard to say for sure.

Regardless, the results of that study do not form a solid basis for the current work and they should be removed. We would recommend making use of the dataset from the same authors, who improved their methods for measuring proprioception shifts just a year later (Tsay, Kim, Parvin, Stover, and Ivry, JNP, 2021). Although here the proprioceptive shifts during error-clamp adaptation (Exp 2) were tiny, and not quite significant (p<0.08), the reports are relative to the actual location of the passively placed unseen hand, measured in trials separate from those with reach adaptation and therefore there is no visual target to anchor their estimates to.

Experiment 1 measures visual uncertainty with increased rotation size. The authors cite relevant work on this topic (Levi & Klein etc) which has found a linear increase in uncertainty of the position of more and more eccentrically displayed stimuli.

First, this is a question where the reported stimuli and effects could greatly benefit from comparisons with the literature in vision science, and the results might even inform it. In order for that to happen, the units for the reported stimuli and effects should (also) be degrees of visual angle (dva).

As far as we know, all previous work has investigated static stimuli, where with moving stimuli, position information from several parts of the visual field are likely integrated over time in a final estimate of position at the end of the trajectory (a Kalman filter type process perhaps). As far as we know, there are no studies in vision science on the uncertainty of the endpoint of moving stimuli. So we think that the experiment is necessary for this study, but there are some areas where it could be improved.

Then, the linear fit is done in the space of the rotation size, but not in the space of eccentricity relative to fixation, and these do not necessarily map onto each other linearly. If we assume that the eye-tracker and the screen were at the closest distance the manufacturer reports it to work accurately at (45 cm), we would get the largest distances the endpoints are away from fixation in dva. Based on that assumed distance between the participant and monitor, we converted the rotation angles to distances between fixation and the cursor endpoint in degrees visual angle: 0.88, 3.5, and 13.25 dva (ignoring screen curvature, or the absence of it). The ratio between the perturbation angle and retinal distance to the endpoint is roughly 0.221, 0.221, and 0.207 if the minimum distance is indeed used - which is probably fine in this case. But still, it would be better to do fit in the relevant perceptual coordinate system.

The first distance (4 deg rotation; 0.88 dva offset between fixation and stimulus) is so close to fixation (even at the assumed shortest distance between eye and screen) that it can be considered foveal and falls within the range of noise of eye-trackers + that of the eye for fixating. There should be no uncertainty on or that close to the fovea. The variability in the data is likely just measurement noise. This also means that a linear fit will almost always go through this point, somewhat skewing the results toward linearity. The advantage is that the estimate of the intercept (measurement noise) is going to be very good. Unfortunately, there are only 2 other points measured, which (if used without the closest point) will always support a linear fit. Therefore, the experiment does not seem suitable to test linearity, only to characterize it, which might be sufficient for the current purposes. We'd understand if the effort to do a test of linearity using many more rotations requires too much effort. But then it should be made much clearer that the experiment assumes linearity and only serves to characterize the assumed linearity.

Final comment after the consultation session:<br /> There were a lot of discussions about the actual interpretation of the behavioral data from this paper with regards to past papers (Tsay et al. 2020 or 2021), and how it matches the different variables of the model. The data from Tsay 2020 combined both proprioceptive information (Xp) and prediction about hand position (Xu) because it involves active movements. On the other hand, Tsay et al. 2021 is based on passive movements and could provide a better measure of Xp alone. We would encourage you to clarify how each of the variables used in the model is mapped onto the outcomes of the cited behavioral experiments.

The reviewers discussed this point extensively during the consultation process. The results reported in the Tsay 2020 study reflect both proprioception and prediction. However, having a visual target contributes more than just prediction, it is likely an anchor in the workspace that draws the response to it. Such that the report is dominated by short-term visual memory of the target (which is not part of the model). However, in the current Exp 3, as in most other work investigating proprioception, this is calculated relative to the actual direction.

The solution is fairly simple. In Experiment 3 in the current study, Xp is measured relative to the hand without any visual anchors drawing responses, and this is also consistent with the reference used in the Tsay et al 2021 study and from many studies in the lab of D. Henriques (none of which also have any visual reach target when measuring proprioceptive estimates). So we suggest using a different data set that also measures Xp without any other influences, such as the data from Tsay et al 2021 instead.

These issues with the data are not superficial and can not be solved within the model. Data with correctly measured biases (relative to the hand) that are not dominated by irrelevant visual attractors would actually be informative about the validity of the PEA model. Dr. Tsay has so much other that we recommend using a more to-the-point data set that could actually validate the PEA model.

Reviewer #3 (Public Review):

Summary:<br /> In this manuscript, Davidsen and collaborators introduce jAspSnFR3, a new version of aspartate biosensor derived from iGluSnFR3, that allows to monitor in real-time aspartate levels in cultured cells. A selective amino acids substitution was applied in a key region of the template to switch its specificity from glutamate to aspartate. The jAspSnFR3 does not respond to other tested metabolites and performs well, is not toxic for cultured cells, and is not affected by temperature ensuring the possibility of using this tool in tissues physiologically more relevant. The high affinity for aspartate (KD=50 uM) allowed the authors to measure fluctuations of this amino acid in the physiological range. Different strategies were used to bring aspartate to the minimal level. Finally, the authors used jAspSnFR3 to estimate the intracellular aspartate concentration.

Strengths:<br /> One of the highlights of the manuscript was a treatment with asparagine during glutamine starvation. Although didn`t corroborate the essentiality of asparagine in glutamine depletion, the measurement of aspartate during this supplementation is a glimpse of how useful this sensor can be.

Weaknesses:<br /> Although this is a well-performed study, I have some comments for the authors to address:<br /> 1-A red tag version of the sensor (jAspSnFR3-mRuby3) was generated for normalization purposes, with this the authors plan to correct GFP signal from expression and movement artifacts. I naturally interpret "movement artifacts" as those generated by variations in cell volume and focal plane during time-lapse experiments. However, it was mentioned that jAspSnFR3-mRuby3 included a histidine tag that may induce a non-specific effect (responses to the treatment with some amino acids). This suggests that a version without the tag needs to be generated and that an alternative design needs to be set for normalization purposes. A nuclear-localized RFP was expressed in a second attempt to incorporate RFP as a normalization signal. Here the cell lines that express both signals (sensor and RFP) were generated by independent lentiviral transductions (insertions). Unless the number of insertions for each construct is known, this approach will not ensure an equimolar expression of both proteins (sensor and RFP). In this scenario is not clear how the nuclear expression of RFP will help the correction by expression or monitor changes in cell volume. The authors may be interested in attempting a bicistronic system to express both the sensor and RFP.<br /> 2-The authors were interested in establishing the temporal dynamics of aspartate depletion by genetics and pharmaceutical means. For the inhibition of mitochondrial complex I rotenone and metformin were used. Although the assays are clearly showing aspartate depletion the report of cell viability is missing. Considering that glutamine deprivation induces arrest in cell proliferation, I think will be important to know the conditions of the cell cultures after 60 hours of treatment with such inhibitors.<br /> 3-The pH sensitivity was checked in vitro with jAspSnFR3-mRuby3 and the sensor reported suitable for measurements at physiological pH. It would be an opportunity to revisit the analysis for pH sensitivity in cultured cells using an untagged version of jAspSnFR3 coupled, for example, to a sensor for pH.<br /> 4-While the authors take an interesting approach to measuring intracellular aspartate concentration, it will be highly desirable if a calibration protocol can be designed for this sensor. Clearly, glutamine depletion grants a minimal ("zero") aspartate concentration. However, having a more dynamic way for calibration will facilitate the introduction of this tool for metabolism studies. This may be achieved by incorporating a cultured cell that already expresses the transporter or by ectopic expression in the cells that have already been used.

Reviewer #3 (Public Review):

The first part of the review was prepared after the first submission of the paper. After this, the authors made several changes in the manuscript. These changes are assessed at the end of the review.

First part:

The paper by Deng, Kumar, Cavalli, Klyachko describe that, unlike in other cell types, loss of Fmr1 decreases the excitability of hippocampal mossy cells due to up-regulation of Kv7 currents. They also show evidence that while muting mossy cells appears to be a compensatory mechanism, it contribute to the higher activity of the dentate gyrus, because the removal of mossy cell output alleviate the inhibition of dentate principal cells. This may be important for the patho-mechanism in Fragile X syndrome caused by the loss of Fmr1.

These experiments were carefully designed, and the results are presented ‎in a very logical, insightful and self-explanatory way. Therefore, this paper represents strong evidences for the claims of the authors. In the current state of the manuscript there are only a few points that need additional explanation.

One of the results, that is shown in the supplementary dataset, does not fit to the main conclusions. Changes in the mEPSC frequency suggest that in addition to the proposed network effects, there are additional changes in the synaptic machinery or synapse number that are independent of the actual activity of the neurons. Since the differences of the mEPSC and sEPSC frequencies are similar and because only the latter can signal network effects, while the former is typically interpreted as a presynaptic change, it cannot be claimed that sEPSC frequency changes are due to the hypo-excitability of mossy cells.

An apparent technical issue may imply a second weak point in the interpretation of the results. Because the IPSCs in the PP stimulation experiments (Fig8) start within a few milliseconds, it is unlikely that its first ‎components originate from the PP-GC-MC-IN feedforward inhibitory circuit. The involvement of this circuit and MCs in the Kv7-dependent excitability changes is the main implication of the results of this paper. But this feedforward inhibition requires three consecutive synaptic steps and EPSP-AP couplings, each of them lasting for at least 1ms + 2-5ms. Therefore, the inhibition via the PP-GC-MC-IN circuit can be only seen from 10-20ms after PP stimulation. The earlier components of the cPSCs should originate from other circuit elements that are not related to the rest of the paper. Therefore, more isolated measurements on the cPSC recordings are needed ‎which consider only the later phase of the IPSCs. This can be either a measurement on the decay phase or a pharmacological manipulation that selectively enhance/inhibit a specific component of the proposed circuit.

I suggest refraining from the conclusions saying "‎MCs provide at least ~51% of the excitatory drive onto interneurons in WT and ~41% in KO mice", because too many factors (eg. IN celll types, slice condition, synaptic reliability) are not accounted for these actual numbers, and these values are not necessary for the general observation of the paper.

There are additional minor issues about the presentation of the results that are explained in the private recommendation for the Authors.

Review after the revision:

The authors accepted my suggestions and made changes in the manuscript to address my point about the interpretation of the mEPSC changes.<br /> The second point was related to the interpretation of the stimulation evoked multisynaptic compound responses. Specifically, the IPSC components in the PP stimulation experiments start within a few milliseconds, and I pointed out that it is unlikely that its first ‎components originate from the PP-GC-MC-IN feedforward inhibitory circuit. The authors provided strong arguments for the interpretation of these compound responses in their reply and the conclusions are consistent with these complex results.

Additional minor issues were fully addressed.

I still think that this is a strong paper that provides new insights into the mechanisms of Fragile X syndrome at the level of single neurons and local network. The extensive series of experiments convincingly support the main findings that in addition to contributing to the underlying mechanisms of this disease also highlight how delicately neuronal activity is balanced even in constrained conditions.

Reviewer #3 (Public Review):

The goal of the current manuscript is to investigate how changes in transporter substrate specificity emerge through experimental evolution. The authors investigate the APC family of amino acid transporters, a large family with many related transporters that together cover the spectrum of amino acid uptake in yeast.

The authors use a clever approach for their experimental evolutions. By deleting 10 amino acid uptake transporters in yeast, they develop a strain that relies on amino acid import by introducing APC transporters under nitrogen-limiting conditions. They can thus evolve transporters towards the transport of new substrates if no other nitrogen source is available. The main takeaway from the paper is that it is relatively easy for the spectrum of substrates in a particular transporter of this family to shift, as a number of single mutants are identified that modulate substrate specificity. In general, transporters evolved towards gain-of-function mutations (better or new activities) and also confer transport promiscuity, expanding the range of amino acids transported.

The data in the paper support the conclusions, in general, and the outcomes (evolution towards promiscuity) agree with the literature available for soluble enzymes. However, it is also a possibility that the design of these experiments selects for promiscuity among amino acids. The selections were designed such that yeast had access to amino acids that were already transported, with a greater abundance of the amino acid that was the target of selection. Under these conditions, it seems probable that the fittest variants will provide the yeast access to all amino acid substrates in the media, and unlikely that a specificity swap would occur, limiting the yeast to only the new amino acid.

The authors also examine the fitness costs of mutants, but only in the narrow context of growth on a single (original) amino acid under conditions of nitrogen limitation. Amino acid uptake is typically tightly controlled because some amino acids (or their carbon degradation products) are toxic in excess. This paper does not address or discuss whether there might be a fitness cost to promiscuous mutants in conditions where nitrogen is not limiting.

Reviewer #3 (Public Review):

Summary:

In this study, Bomba-Warczak et al focused on reproductive aging, and they presented a map for long-lived proteins that were stable during reproductive lifespan. The authors used MIMS to examine and show distinct molecules in different cell types in the ovary and tissue regions in a 6 month mice group, and they also used proteomic analysis to present different LLPs in ovaries between these two timepoints in 6-month and 10-month mice. The authors also examined the LLPs in oocytes in the 6-months mice group and indicated that these were nuclear, cytoskeleton, and mitochondria proteins.

Strengths:

Overall, this study provided basic information or a 'map' of the pattern of long-lived proteins during aging, which will contribute to the understanding of the defects caused by reproductive aging.

Weaknesses:

The 6-month mice were used as an aged model; no validation experiments were performed with proteomics analysis only.

Reviewer #3 (Public Review):

Summary:

The manuscript authored by Lan Guan and colleagues reveals the structure of the cytosol-facing conformation of the MelB sodium/Li coupled permease using the nab-Fab approach and cryoEM for structure determination. The study reveals the conformational transitions in the melB transport cycle and allows understanding the role of sugar and ion specificities within this transporter.

Strengths:

The study employs a very exciting strategy of transferring the CDRS of a conformation specific nano body to the nab-fab system to determine the inward-open structure of MelB. The resolution of the structure is reasonable enough to support the major conclusions of the study. This is overall a well-executed study.

Weaknesses:

The authors seem to have mixed up the exothermic and endothermic aspects of ITC binding in their description. Positive heats correspond to endothermic heat changes in ITC and negative heat changes correspond to exothermic heats. The authors seem to suggest the opposite. This is consistently observed throughout the manuscript.

Reviewer #3 (Public Review):

Hung et al provide a well-written manuscript focused on understanding how Eklf mutation confers anticancer and longevity advantages in vivo.

The authors were responsive to the reviewers comments in some aspects. However, the manuscript continues to suffer from significantly overstated claims that are not mitigated in the revision. While additional data has been added, it is unclear how this new data provides clarity to the overall premise of this observational study. Importantly, the authors have added a second model of hepatocellular carcinoma with findings that are consistent with the melanoma model previously reported. In addition, they make more clear that the previously published manuscript on this subject was use of older donors for BMT while now they use younger donors. This is at best incremental. It remains unclear whether Eklf exerts its effect on resistance to malignant progression / metastasis by modulating Pd1 or Pdl1 vs. increasing NK cells as the authors provide evidence of both and do not resolve which mechanism is primarily involved. Finally, there is no evidence that Eklf mutation confers "an anti-disease and anti-aging" effect as at best the data provides evidence of resistance to malignant progression / metastasis in melanoma and hepatoma models.

The work is impactful as it provides evidence of anticancer effect of a specific hematological mutation but the mechanism by which this occurs is not completely elucidated by this work.

Reviewer #3 (Public Review):

In multiple cancers, the key roles of B cells are emerging in the tumor microenvironment (TME). The authors of this study appropriately introduce that B cells are relatively under-characterised in the TME and argue correctly that it is not known how the B cell receptor (BCR) repertoires across tumors, lymph nodes, and peripheral blood relate. The authors therefore supply a potentially useful study evaluating the tumor, lymph node, and peripheral blood BCR repertoires and site-to-site as well as intra-site relationships. The authors employ sophisticated analysis techniques, although the description of the methods is incomplete. Among other interesting observations, the authors argue that the tumor BCR repertoire is more closely related to that of draining lymph node (dLN) than the peripheral blood in terms of clonal and isotype composition. Furthermore, the author's findings suggest that tumor-infiltrating B cells (TIL-B) exhibit a less mature and less specific BCR repertoire compared with circulating B cells. Overall, this is a potentially useful work that would be of interest to both medical and computational biologists working across cancer. However, there are aspects of the work that would have benefitted from further analysis and areas of the manuscript that could be written more clearly and proofread in further detail.

Major Strengths:

1. The authors provide a unique analysis of BCR repertoires across tumor, dLN, and peripheral blood. The work provides useful insights into inter- and intra-site BCR repertoire heterogeneity. While patient-to-patient variation is expected, the findings with regard to intra-tumor and intra-dLN heterogeneity with the use of fragments from the same tissue are of importance, contribute to the understanding of the TME, and will inform future study design.

2. A particular strength of the study is the detailed CDR3 physicochemical properties analysis which leads the authors to observations that suggest a less-specific BCR repertoire of TIL-B compared to circulating B cells.

Major Weaknesses:

1. The study would have benefitted from a deeper biological interpretation of the data. While given the low number of patients one can plausibly understand a reluctance to speculate about clinical details, there is limited discussion about what may contribute to observed heterogeneity. For example, for the analysis of three lymph nodes taken per patient which were examined for inter-LN heterogeneity, there is a lack of information regarding these lymph nodes. 'LN3' is deemed as exhibiting the most repertoire overlap with the tumor but there is no discussion as to why this may be the case.

2. At times the manuscript is difficult to follow. In particular, the 'Intra-LN heterogeneity' section follows the 'LN-LN heterogeneity in colorectal cancer' section and compares the overlap of LN fragments (LN11, LN21, LN31) with the tumor in two separate patients (Fig 6A). In the previous section (LN-LN), LN11, LN21, LN31 are names given to separate lymph nodes from the same patient. The fragments are referred to as 'LN2' and the nodes in the previous section are referred to similarly. This conflation of naming for nodes and fragments is confusing.

3. There is a duplicated paragraph in 'Short vs long trees' and the following section 'Productive involvement in hypermutation lineages depends on CDR3 characteristics.

Reviewer #3 (Public Review):

Summary:<br /> The manuscript reports new molecular characterization of the Haemophilus influenza tripartite ATP-independent periplasmic (TRAP) transporter of N-acetylneuraminate (Neu5Ac). This membrane transporter is important for the virulence of the pathogen. H. influenza lacks Neu5Ac biosynthetic pathway, and utilizes the TRAP transporter to import it. Neu5Ac is used as a nutrient source but also as a protection from human immune response. The transporter is composed of two fused membrane subunits, HiSiaQM, and one soluble, periplasmic subunit HiSiaP. HiSiaP, by binding to the substrate Neu5Ac, changes its conformation, allowing its binding to HiSiaQM, followed by Neu5Ac and Na+ transport to the cytoplasm. The combination of structural, biophysical and biochemical approaches provides a solid basis for describing the functioning of the Haemophilus influenza Neu5Ac TRAP transporter, which is essential for the pathogen virulence.

Strengths:<br /> The paper describes the electron microscopy structure of HiSiaQM, thanks to its solubilization in L-MNG followed by exchange to amphipol or nanodisc. In these conditions, HiSiaQM consists in a mixture of monomers and dimers, as characterized by analytical ultracentrifugation. The cryo-EM analysis shows two types of dimers: one in an antiparallel configuration, which is artifactual, and a parallel one, which may be physiologically relevant. Cryo-EM on the dimers allows high resolution (≈ 3 Å) structure determination. The structure is the first one of a fused SiaQM, and is the first obtained without megabody. The work highlights structural elements (fusion helix, lipids) that could modulate transport. The authors checked the functionality of the purified HiSiaQM, which, after reconstitution in liposome, displays a significantly larger Neu5Ac transport activity compared to the non-fused PpSiaQM homolog. The work identifies Na+ binding sites, and the putative Neu5Ac binding site. From analytical ultracentrifugation using fluorescently labelled HiSiaP, the authors show that HiSiaP is able to interact with HiSiaQM monomer and dimer, with a low but physiologically relevant affinity. HiSiaP interaction with HiSiaQM was modelled using AlphaFold2, and discussed in view of published activity on mutants, and new transport activity assays using SiaQM and SiaP from different organisms. In conclusion, the combination of structural, biophysical and biochemical approaches provides a solid basis for describing the functioning of this TRAP fused transporter.

Weakness: This work evidences in vitro a HiSiaQM dimer, whose in vivo relevance is not ascertained. However, the authors are very careful, they do not to over-interpret their data, and their conclusions regarding the transporter structure and function are valid irrespective of its state of association.

Reviewer #3 (Public Review):

Summary:

In this manuscript, the authors are trying to delineate the mechanism underlying the osteonecrosis of the femoral head.

Strengths:

The authors provided compelling in vivo and in vitro data to demonstrate Col2+ cells and Osx+ cells were differentially expressed in the<br /> the femoral head. Moreover, inducible knockout of β-catenin in Col2+ cells but not<br /> Osx+ cells lead to a GONFH-like phenotype including fat accumulation, subchondral<br /> bone destruction and femoral head collapse, indicating that imbalance of osteogenic/adipogenic differentiation of Col2+ cells play an important role in GONFH pathogenesis. Therefore, this manuscript provided the mechanistic insights of osteonecrosis as well as potential therapeutic target for disease treatment.

Weaknesses: Additional in depth discussion regarding the phenotype observed in mice is highly encouraged.

Reviewer #3 (Public Review):

Nguyen, Zhao, et al. used bioinformatic analysis of mutational variants of SARS-CoV-2 Nucleocapsid (N) protein from the large genomic database of SARS-CoV-2 sequences to identify domains and regions of N where mutations are more highly represented and computationally determined the effects of these mutations on the physicochemical properties of the protein. They found that the intrinsically disordered regions (IDRs) of N protein are more highly mutated than structured regions and that these mutations can lead to higher variability in the physical properties of these domains. These computational predictions are compared to in vitro biophysical experiments to assess the effects of identified mutations on the thermodynamic stability, oligomeric state, particle formation, and liquid-liquid phase separation of a few exemplary mutants.

The paper is well-written and easy to follow, and the conclusions drawn are supported by the evidence presented. The analyses and conclusions are interesting and will be of value to virologists, cell biologists, and biophysicists studying SARS-CoV-2 function and assembly. It would be nice if some further extrapolation or comments could be made regarding the effects of the observed mutations on the in vivo behavior and properties of the virus, but I appreciate that this is much higher-order than could be addressed with the approaches employed here.

Reviewer #3 (Public Review):

Summary:<br /> In this manuscript, the authors investigated the interaction between the pattern recognition receptor MDA5 and 5'ppp-RNA in a teleost fish called Miiuy croaker. They claimed that MDA5 can replace RIG-I in sensing 5'ppp-RNA of Siniperca cheats rhabdovirus (SCRV) in the absence of RIG-I in Miiuy croaker. The recognition of MDA5 to 5'ppp-RNA was also observed in the chicken (Gallus gallus), a bird species that lacks RIG-I. Additionally, they reported that the function of MDA5 can be impaired through m6A-mediated methylation and degradation of MDA5 mRNA by the METTL3/14-YTHDF2/3 regulatory network in Miiuy croaker under SCRV infection. This impairment weakens the innate antiviral immunity of fish and promotes the immune evasion of SCRV.

Strengths:<br /> These findings provide insights into the adaptation and functional diversity of innate antiviral activity in vertebrates.

Weaknesses:<br /> However, there are some major and minor concerns that need to be further addressed. Addressing these concerns will help the authors improve the quality of their manuscript.

One significant issue with the manuscript is that the authors claim to be investigating the role of MDA5 as a substitute for RIG-I in recognizing 5'ppp-RNA, but their study extends beyond this specific scenario. Based on my understanding, it appears that sections 2.2, 2.3, 2.5, 2.6, and 2.7 do not strictly adhere to this particular scenario. Instead, these sections tend to investigate the functional involvement of Miiuy croaker MDA5 in the innate immune response to viral infection. Furthermore, the majority of the data is focused on Miiuy croaker MDA5, with only a limited and insufficient study on chicken MDA5. Consequently, the authors cannot make broad claims that their research represents events in all RIG-I deficient species, considering the limited scope of the species studied.

The current title of the article does not align well with its actual content. It is recommended that the focus of the research be redirected to the recognition function and molecular mechanism of MDA5 in the absence of RIG-I concerning 5'ppp-RNA. This can be achieved through bolstering experimental analysis in the fields of biochemistry and molecular biology, as well as enhancing theoretical research on the molecular evolution of MDA5. It is advisable to decrease or eliminate content related to m6A modification.

Additionally, the main body of the writing contains several aspects that lack rigor and tend to exaggerate, necessitating significant improvement.

Reviewer #3 (Public Review):

Summary:<br /> This study considers how to model distinct host cell states that correspond to different stages of a viral infection: from naïve and susceptible cells to infected cells and a minority of important interferon-secreting cells that are the first line of defense against viral spread. The study first considers the distinct host cell states by analyzing previously published single-cell RNAseq data. Then an agent-based model on a square lattice is used to probe the dependence of the system on various parameters. Finally, a simplified version of the model is explored, and shown to have some similarity with the more complex model, yet lacks the dependence on the interferon range. By exploring these models one gains an intuitive understanding of the system, and the model may be used to generate hypotheses that could be tested experimentally, telling us "when to be surprised" if the biological system deviates from the model predictions.

Strengths:<br /> - Clear presentation of the experimental findings and a clear logical progression from these experimental findings to the modeling.<br /> - The modeling results are easy to understand, revealing interesting behavior and percolation-like features.<br /> - The scaling results presented span several decades and are therefore compelling.<br /> - The results presented suggest several interesting directions for theoretical follow-up work, as well as possible experiments to probe the system (e.g. by stimulating or blocking IFN secretion).

Weaknesses:<br /> - Since the "range" of IFN is an important parameter, it makes sense to consider lattice geometries other than the square lattice, which is somewhat pathological. Perhaps a hexagonal lattice would generalize better.

- Tissues are typically three-dimensional, not two-dimensional. (Epithelium is an exception). It would be interesting to see how the modeling translates to the three-dimensional case. Percolation transitions are known to be very sensitive to the dimensionality of the system.

- The fixed time-step of the agent-based modeling may introduce biases. I would consider simulating the system with Gillespie dynamics where the reaction rates depend on the ambient system parameters.

- Single-cell RNAseq data typically involves data imputation due to the high sparsity of the measured gene expression. More information could be provided on this crucial data processing step since it may significantly alter the experimental findings.

Justification of claims and conclusions:<br /> The claims and conclusions are well justified.

Reviewer #3 (Public Review):

Summary: Bidirectional transsynaptic signaling via cell adhesion molecules and cell surface receptors contributes to the remarkable specificity of synaptic connectivity in the brain. Zaman et al., investigates how the receptor tyrosine kinase Kit and its trans-cellular kit ligand regulate molecular layer interneuron (MLI)- Purkinje cell (PC) connectivity in the cerebellum. Presynaptic Kit is specific for MLIs, and forms a trans-synaptic complex with Kit ligand in postsynaptic PC cells. The authors begin by generating Kit cKOs via an EUCOMM allele to enable cell-type specific Kit deletion. They cross this Kit cKO to the MLI-specific driver Pax2-Cre and conduct validation via Kit IHC and immunoblotting. Using this system to examine the functional consequences of presynaptic MLI Kit deletion onto postsynaptic PC cells, they record spontaneous and miniature synaptic currents from PC cells and find a selective reduction in IPSC frequency. Deletion of Kit ligand from postsynaptic PC cells also results in reduced IPSC frequency, together supporting that this trans-synaptic complex regulates GABAergic synaptic formation or maturation. The authors then show that sparse Kit ligand overexpression in PCs decreases neighboring uninfected control sIPSCs in a potential competitive manner.

Strengths: Overall, the study addresses an important open question, the data largely supports the authors conclusions, the experiments appear well-performed, and the manuscript is well-written. I just have a few suggestions to help shore up the author's interpretations and improve the study.

Weaknesses:

The strong decrease in sIPSC frequency and amplitude in control uninfected cells in Figure 4 is surprising and puzzling. The competition model proposed is one possibility, and I think the authors need to do additional experiments to help support or refute this model. The authors can conduct similar synaptic staining experiments as in Fig S4 but in their sparse infection paradigm, comparing synapses on infected and uninfected cells. Additional electrophysiological parameters in the sparse injection paradigm, such as mIPSCs or evoked IPSCs, would also help support their conclusions.

The authors should validate KL overexpression and increased cell surface levels using their virus to support their overexpression conclusions.

Reviewer #3 (Public Review):

Summary:

In this manuscript, the authors found that FBXO24, a testis-enriched F-box protein, is indispensable for male fertility. Fbxo24 KO mice exhibited malformed sperm flagellar and compromised sperm motility.

Strengths:

The phenotype of Fbxo24 KO spermatozoa was well analyzed.

Weaknesses:

The authors observed numerous membraneless electron-dense granules in the Fbxo24 KO spermatozoa. They also showed abnormal accumulation of two importins, IPO5 and KPNB1, in the Fbxo24 KO spermatozoa. However, the data presented in the manuscript do not support the conclusion that FBXO24 ensures male fertility by preventing the abnormal accumulation of membraneless granules in sperm flagella, as indicated in the manuscript title.

Reviewer #3 (Public Review):

Overall, this is a strong manuscript that uses multiple current techniques to provide specific mechanistic insight into prior discoveries of the contributions of the Bcl11b transcription factor to mossy fiber synapses of dentate gyrus granule cells. The authors employ an adult deletion of Bcl11b via Tamoxifen-inducible Cre and use immunohistochemical, electron microscopy, and electrophysiological studies of synaptic plasticity, together with viral rescue of C1ql2, a direct transcriptional target of Bcl11b or Nrxn3, to construct a molecular cascade downstream of Bcl11b for DG mossy fiber synapse development. They find that C1ql2 re-expression in Bcl11b cKOs can rescue the synaptic vesicle docking phenotype and the impairments in MF-LTP of these mutants. They also show that C1ql2 knockdown in DG neurons can phenocopy the vesicle docking and plasticity phenotypes of the Bcl11b cKO. They also use artificial synapse formation assays to suggest that C1ql2 functions together with a specific Nrxn3 splice isoform in mediating MF axon development, extending these data with a C1ql2-K262E mutant that purports to specifically disrupt interactions with Nrxn3. All of the molecules involved in this cascade are disease-associated and this study provides an excellent blueprint for uncovering downstream mediators of transcription factor disruption. Together this makes this work of great interest to the field. Strengths are the sophisticated use of viral replacement and multi-level phenotypic analysis while weaknesses include the linkage of C1ql2 with a specific Nrxn3 splice variant in mediating these effects.

Here is an appraisal of the main claims and conclusions:

1. C1ql2 is a downstream target of Bcl11b which mediates the synaptic vesicle recruitment and synaptic plasticity phenotypes seen in these cKOs. This is supported by the clear rescue phenotypes of synapse anatomy (Fig.2) and MF synaptic plasticity (Fig.3). One weakness here is the absence of a control assessing over-expression phenotypes of C1ql2. It's clear from Fig.1D that viral rescue is often greater than WT expression (totally expected). In the case where you are trying to suppress a LoF phenotype, it is important to make sure that enhanced expression of C1ql2 in a WT background does not cause your rescue phenotype. A strong overexpression phenotype in WT would weaken the claim that C1ql2 is the main mediator of the Bcl11b phenotype for MF synapse phenotypes.

2. Knockdown of C1ql2 via 4 shRNAs is sufficient to produce the synaptic vesicle recruitment and MF-LTP phenotypes. This is supported by clear effects in the shRNA-C1ql2 groups as compared to nonsense-EGFP controls. One concern (particularly given the use of 4 distinct shRNAs) is the potential for off-target effects, which is best controlled for by a rescue experiment with RNA-insensitive C1ql2 cDNA as opposed to nonsense sequences, which may not elicit the same off-target effects.

3. C1ql2 interacts with Nrxn3(25b+) to facilitate MF terminal SV clustering. This claim is theoretically supported by the HEK cell artificial synapse formation assay (Fig.5), the inability of the K262-C1ql2 mutation to rescue the Bcl11b phenotype (Fig.6) and the altered localization of C1ql2 in the Nrxn1-3 deletion mice (Fig.7). Each of these lines of experimental evidence has caveats that should be acknowledged and addressed. Given the hypothesis that C1ql2 and Nrxn3b(25b) are expressed in DG neurons and work together, the heterologous co-culture experiment seems weird. Up till now, the authors are looking at pre-synaptic function of C1ql2 since they are re-expressing it in DGNs. The phenotypes they are seeing are also pre-synaptic and/or consistent with pre-synaptic dysfunction. In Fig.5, they are testing whether C1ql2 can induce pre-synaptic differentiation in trans, i.e. theoretically being released from the 293 cells "post-synaptically". But the post-synaptic ligands (Nlgn1 and and GluKs) are not present in the 293 cells, so a heterologous synapse assay doesn't really make sense here. The effect that the authors are seeing likely reflects the fact that C1ql2 and Nrxn3 do bind to each other, so C1ql2 is acting as an artificial post-synaptic ligand, in that it can cluster Nrxn3 which in turn clusters synaptic vesicles. But this does not test the model that the authors propose (i.e. C1ql2 and Nrxn3 are both expressed in MF terminals). Perhaps a heterologous assay where GluK2 is put into HEK cells and the C1ql2 and Nrxn3 are simultaneously or individually manipulated in DG neurons?

4. K262-C1ql2 mutation blocks the normal rescue through a Nrxn3(25b) mechanism (Fig.6). The strength of this experiment rests upon the specificity of this mutation for disrupting Nrxn3b binding (presynaptic) as opposed to any of the known postsynaptic C1ql2 ligands such as GluK2. While this is not relevant for interpreting the heterologous assay (Fig.5), it is relevant for the in vivo phenotypes in Fig.6. Similar approaches as employed in this paper can test whether binding to other known postsynaptic targets is altered by this point mutation.

5. Altered localization of C1ql2 in Nrxn1-3 cKOs. These data are presented to suggest that Nrx3(25b) is important for localizing C1ql2 to the SL of CA3. Weaknesses of this data include both the lack of Nrxn specificity in the triple a/b KOs as well as the profound effects of Nrxn LoF on the total levels of C1ql2 protein. Some measure that isn't biased by this large difference in C1ql2 levels should be attempted (something like in Fig.1F).

Reviewer #3 (Public Review):

Kang, Huang, and colleagues have provided new data to address concerns regarding confirmation of LRRK1 and LRRK2 deletion in their mouse model and the functional impact of the modest loss of TH+ neurons observed in the substantia nigra of their double KO mice. In the revised manuscript, the new data around the characterization of the germline-deleted LRRK1 and LRRK2 mice add confidence that LRRK1 and LRRK2 can be deleted using the genetic approach. They have also added new text to the discussion to try and address some of the comments and questions raised regarding how LRRK1/2 loss may impact cell survival and the implications of this work for PD-linked variants in LRRK2 and therapeutic approaches targeting LRRK2.

The new data provides additional support for the author's claims. I have provided below some suggestions for clarification/additions to the text that can be addressed without additional experiments.

1) The authors added additional text highlighting that more studies are warranted in mice where LRRK1/2 are deleted in other CNS cell types (microglia/astrocytes) to understand cell extrinsic drivers of the autophagy deficits observed in their previous work. It still remains unclear how loss of LRRK1/2 leads to increased apoptosis and gliosis in dopaminergic neurons in a cell-intrinsic manner, and, as suggested in the original review, it would be helpful to add some text to the discussion speculating on potential mechanisms by which this might occur.

2) Revisions have been made to the discussion to clarify their rationale around how variants in LRRK2 associated with PD may be loss-of-function to support the relevance of this mouse model to phenotypes observed in PD. However, as written, the argument that PD-linked variants are loss-of-function is based on the fact that the double KO mice have a mild loss of TH+ neurons while the transgenic mice overexpressing PD-linked LRRK2 variants often do not and that early characterization of kinase activity was done in vitro are relatively weak. Given that the majority of evidence generated by many labs in the field supports a gain-of-function mechanism, the discussion should be further tempered to better highlight the uncertainty around this (rather than strongly arguing for a loss-of-function effect). This could include the mention of increased Rab phosphorylation observed in cellular and animal models and opposing consequences on lysosomal function observed in cellular studies in KO and pathogenic variant expressing cells. Further, a reference to the Whiffen et al. 2020 paper mentioned by another reviewer should be included in the discussion for completeness.

Reviewer #3 (Public Review):

Summary:<br /> Animals can evaluate food quality in many ways. In contrast to the rapid sensory evaluation with smell and taste, the mechanism of slow nutrient sensation and its impact on food choice is unexplored. The authors utilize C. elegans larvae and their bacterial food as an elegant model to tackle this question and reveal the detailed molecular mechanism to avoid nutrient-poor foods.

Strengths:<br /> The strength of this study is that they identified the molecular identities of the critical players in bacterial food and C. elegans using unbiased approaches, namely metabolome analysis, E. coli mutant screening, and RNA sequencing. Furthermore, they strengthen their findings by thorough experiments combining multiple methods such as genetics, fluorescent reporter analysis, and Western blot.

Weaknesses:<br /> The major caveat of this study is the reporter genes. The transcriptional reporters were used to monitor the UPRER and immune responses in the intestine of C. elegans. However, their tissue-specific rescue experiments suggest that the genes in the UPRER and immune response function in the neurons. Thus, we should carefully interpret the results of the reporter genes.

Overall, this work provides convincing data to support their model. In the C. elegans field, the behaviors of larvae are not well studied compared to adults. This work will pose an interesting question about the difference between larvae and adults in nutrition sensing in C. elegans and provide a framework and candidate molecules to be studied in other organisms.

Reviewer #3 (Public Review):

Summary<br /> In their manuscript, Vickers and McCormick have demonstrated the potential of leveraging mesoscale two-photon calcium imaging data to unravel complex behavioural motifs in mice. Particularly commendable is their dedication to providing detailed surgical preparations and corresponding design files, a contribution that will greatly benefit the broader neuroscience community as a whole. The quality of the data is high, but it is not clear whether this is available to the community, some datasets should be deposited. More importantly, the authors have acquired activity-clustered neural ensembles at an unprecedented spatial scale to further correlate with high-level behaviour motifs identified by B-SOiD. Such an advancement marks a significant contribution to the field. While the manuscript is comprehensive and the analytical strategy proposed is promising, some technical aspects warrant further clarification. Overall, the authors have presented an invaluable and innovative approach, effectively laying a solid foundation for future research in correlating large-scale neural ensembles with behavioural. The implementation of a custom sound insulator for the scanner is a great idea and should be something implemented by others.

This is a methods paper, but there is no large diagram that shows how all the parts are connected, communicating, and triggering each other. This is described in the methods, but a visual representation would greatly benefit the readers looking to implement something similar. The authors should cite sources for the claims stated in lines 449-453 and cite the claim of the mouse's hearing threshold mentioned in lines 463. No stats for the results shown in Figure 6e, it would be useful to know which of these neural densities for all areas show a clear statistical significance across all the behaviors. While I understand that this is a methods paper, it seems like the authors are aware of the literature surrounding large neuronal recordings during mouse behavior. Indeed, in lines 178-179, the authors mention how a significant portion of the variance in neural activity can be attributed to changes in "arousal or self-directed movement even during spontaneous behavior.". Why then did the authors not make an attempt at a simple linear model that tries to predict the activity of their many thousands of neurons by employing the multitude of regressors at their disposal (pupil, saccades, stimuli, movements, facial changes, etc). These models are straightforward to implement, and indeed it would benefit this work if the model extracts information on par with what is known from the literature.

Specific strengths and weaknesses with areas to improve:

The paper should include an overall cartoon diagram that indicates how the various modules are linked together for the sampling of both behaviour and mesoscale GCAMP. This is a methods paper, but there is no large diagram that shows how all the parts are connected, communicating, and triggering each other.

The paper contains many important results regarding correlations between behaviour and activity motifs on both the cellular and regional scales. There is a lot of data and it is difficult to draw out new concepts. It might be useful for readers to have an overall figure discussing various results and how they are linked to pupil movement and brain activity. A simple linear model that tries to predict the activity of their many thousands of neurons by employing the multitude of regressors at their disposal (pupil, saccades, stimuli, movements, facial changes, etc) may help in this regard.

Previously, widefield imaging methods have been employed to describe regional activity motifs that correlate with known intracortical projections. Within the authors' data it would be interesting to perhaps describe how these two different methods are interrelated, they do collect both datasets. Surprisingly, such macroscale patterns are not immediately obvious from the authors' data. Some of this may be related to the scaling of correlation patterns or other factors. Perhaps there still isn't enough data to readily see these and it is too sparse.

In lines 71-71, the authors described some disadvantages of one-photon widefield imaging including the inability to achieve single-cell resolution. However, this is not true. In recent years, the combination of better surgical preparations, camera sensors, and genetically encoded calcium indicators has enabled the acquisition of single-cell data even using one-photon widefield imaging methods. These methods include miniscopes (Cai et al., 2016), multi-camera arrays (Hope et al., 2023), and spinning disks (Xie et al., 2023).

Cai, Denise J., et al. "A shared neural ensemble links distinct contextual memories encoded close in time." Nature 534.7605 (2016): 115-118.<br /> Hope, James, et al. "Brain-wide neural recordings in mice navigating physical spaces enabled by a cranial exoskeleton." bioRxiv (2023).<br /> Xie, Hao, et al. "Multifocal fluorescence video-rate imaging of centimetre-wide arbitrarily shaped brain surfaces at micrometric resolution." Nature Biomedical Engineering (2023): 1-14.

The authors' claim of achieving optical clarity for up to 150 days post-surgery with their modified crystal skull approach is significantly longer than the 8 weeks (approximately 56 days) reported in the original study by Kim et al. (2016). Since surgical preparations are an integral part of the manuscript, it may be helpful to provide more details to address the feasibility and reliability of the preparation in chronic studies. A series of images documenting the progression optical quality of the window would offer valuable insight.

Reviewer #3 (Public Review):

Wu and colleagues are characterising the function of Styxl2 during muscle development, a pseudo-phosphatase that was already described to have some function in sarcomere morphogenesis or maintenance (Fero et al. 2014). The authors verify a role for Styxl2 in sarcomere assembly/maintenance using zebrafish embryonic muscles by morpholino knock-down and by a conditional Styxl2 allele in mice (knocked-out in satellite cells with Pax7 Cre).

Experiments using a tamoxifen inducible Cre suggest that Styxl2 is dispensable for sarcomere maintenance and only needed for sarcomere assembly.

BioID experiments with Styxl2 in C2C 12 myoblasts suggest binding of nonmuscle myosins (NMs) to Styxl2. Interestingly, both NMs are downregulated when muscles differentiate after birth or during regeneration in mice. This down-regulation is reduced in the Styxl2 mutant mice, demonstrating that Styxl2 is required for the degradation of these NMs.

Impressively, reducing one NM (zMyh10) by double morpholino injection in a Styxl2 morphant zebrafish, does improve zebrafish mobility and sarcomere structure. Degradation of Mhy9 is also stimulated in cell culture if Styxl2 is co-expressed. Surprisingly, the phosphatase domain is not needed for these degradation and sarcomere structure rescue effects. Inhibitor experiments suggest that Styxl2 does promote the degradation of NMs by promoting the selective autophagy pathway.

Strengths:<br /> A major strength of the paper is the combination of various systems, mouse and fish muscles in vivo to test Styxl2 function, and cell culture including a C2C12 muscle cell line to assay protein binding or protein degradation as well as inhibitor studies that can suggest biochemical pathways.<br /> A second strength is that this manuscript sheds new light on the still ill-characterised mechanism of sarcomere assembly in skeletal muscles.

Weakness:<br /> The weaknesses of this manuscript have been largely eliminated during revision.

Reviewer #3 (Public Review):

Perhaps not unexpectedly, the proposed revisions consist of textual revisions only. Yewdell added a touch of levity with his H.G. Wells foundation as a source of $$ for a time machine. The paper does not establish striking new facts, in my opinion, but will stimulate discussion.

One point to consider: the relevance of the human T cell activation experiments is now downplayed even further, by the authors themselves, no less. I would suggest leaving the actual data out altogether and conclude with a statement: "Similar experiments conducted on activated human T cells showed significantly worse activation and may therefore not allow a head-to-head comparison with the results of our experimentst performed on mouse T cells. Not only might one consider the mode of activation (PMA/ionomycin) non-physiological, the activation status achievedwas less than that seen for the OT-1 model. " or something similar to that effect. In the present weakened form, I do not believe that the human data add anything of substance to the paper and are more of a distraction. The authors would increase the impact and readability of their paper if they omitted the human data.

Reviewer #3 (Public Review):

In this manuscript, the authors investigated the effects of deletion of the ER-plasma membrane/Golgi tethering proteins tricalbins (Tcb1-3) on vacuolar morphology to demonstrate the role of membrane contact sites (MCSs) in regulating vacuolar morphology in Saccharomyces cerevisiae. Their data show that tricalbin deletion causes vacuolar fragmentation possibly in parallel with TORC1 pathway. In addition, their data reveal that levels of various lipids including ceramides, long-chain base (LCB)-1P, and phytosphingosine (PHS) are increased in tricalbin-deleted cells. The authors find that exogenously added PHS can induce vacuole fragmentation and by performing analyses of genes involved in sphingolipid metabolism, they conclude that vacuolar fragmentation in tricalbin-deleted cells is due to the accumulated PHS in these cells. Importantly, exogenous PHS- or tricalbin deletion-induced vacuole fragmentation was suppressed by loss of the nucleus vacuole junction (NVJ), suggesting the possibility that PHS transported from the ER to vacuoles via the NVJ triggers vacuole fission. Of note, the authors find that hyperosmotic shock increases intracellular PHS levels, suggesting a general role of PHS in vacuole fission in response to physiological vacuolar division-inducing stimuli.

This work provides valuable insights into the relationship between MCS-mediated sphingolipid metabolism and vacuole morphology. The conclusions of this paper are mostly supported by their results, but inclusion of direct evidence indicating increased transport of PHS from the ER to vacuoles via NVJ in response to vacuolar division-inducing stimuli would have strengthened this study.

There is another weakness in their claim that the transmembrane domain of Tcb3 contributes to the formation of the tricalbin complex which is sufficient for tethering ER to the plasma membrane and the Golgi complex. Their claim is based only on the structural simulation, but not on by biochemical experiments such as co-immunoprecipitation and pull-down.

Reviewer #3 (Public Review):

Summary:<br /> Studying evolutionary trajectories provides important insight into the genetic architecture of adaptation and provides a potential contribution to evaluating the predictability (or unpredictability) of biological processes involving adaptation. While many papers in the field address adaptation to environmental challenges, the number of studies on how genomic contexts, such as large-scale variation, can impact evolutionary outcomes adaptation is relatively low. This research experimentally evolved a genome-reduced strain for ~1000 generations with 9 replicates and dissected their evolutionary changes. Using the fitness assay of OD measurement, the authors claimed that there is a general trend of increasing growth rate and decreasing carrying capacity, despite a positive correlation among all replicates. The authors also performed genomic and transcriptomic research at the end of experimental evolution, claiming the dissimilarity in the evolution at the molecular level.

Strengths:<br /> The experimental evolution approach with a high number of replicates provides a good way to reveal the generality/diversity of the evolutionary routes.

The assay of fitness, genome, and transcriptome all together allows a more thorough understanding of the evolutionary scenarios and genetic mechanisms.

Weaknesses:<br /> My major concern is the current form of statistical analysis leads to the conclusion that the dissimilarity is not very strong. Adding some more statistical analysis should substantially improve the strength of the manuscript. As mentioned in the Discussion, I understand that there are more available methods to test for generality in experimental evolution but less for diversity. When it is improper to use a canonical statistical test, a test with some simulation and resampling can be useful. For example, I particularly appreciate the analysis done in Figure 2B. An analysis like that should be done more throughout the entire manuscript.

Reviewer #3 (Public Review):

Summary:

SMN expression in non-neuronal cells, particularly in limb mesenchymal progenitors is essential for the proper growth of chondrocytes and the formation of adult NMJ junctions.

Strengths:

The authors show copy numbers of smndelta7 in MPC influence NMJ structure.

Weaknesses:

Functional recovery by FAP transplantation is not complete. Mesenchymal progenitors are heterogeneous, and how heterogeneity influences this study is not clear. Part of the main findings to show the importance of SMN expression in non-neuronal cells is partly published by the same group (Kim et al., JCI Insight 2022). In the study, the authors used Dpp4(+) cells. The difference between the current study and the previous study is not so clear.

What to listen for in jazz Kernfeld 1995