Reviewer #2 (Public Review):

The new work from Lemcke et al suggests that the infection with Influenza A virus causes such flu symptoms as sleepiness and loss of appetite through the direct action on the responsible brain region, the hypothalamus. To test this idea, the authors performed single-nucleus RNA sequencing of the mouse hypothalamus in controlled experimental conditions (0, 3, 7, and 23 days after intranasal infection) and analyzed changes in the gene expression in the specific cell populations. The key results are promising.

However, the analysis (cell type annotation, integration, group comparison) is not optimal and incomplete and, therefore should be significantly improved.

More specifically:

1) The current annotation of cell types (especially neuronal but also applicable to the group of heterogeneous "Unassigned cells") did not make a good link to existing cell heterogeneity in the hypothalamus identified with scRNA seq in about 20 recently published works. All information about different peptidergic groups can not be extracted from the current version (except for a few). There are also some mistakes or wrong interpretations (eg, authors assigned hypothalamic dopamine cells to the glutamatergic group, which is not true). This state is feasible to improve (and should be improved) with already existing data.

2) I am confused with the results shown in the label transfer (suppl fig 3 and 4; note, they do not have the references in the text) applied to some published datasets (authors used the Seurat functions 'FindTransferAnchors' and 'TransferData'). The final results don't make sense: while the dataset for the arcuate nucleus (Campbel et al) well covered the GABAergic neurons it is not the case for the whole hypothalamus datasets (Chen et al; Zeisel et al). Similarly, for glutamatergic neurons. Additionally, I could not see that the label transfer works well for PMCH cells which should be present in the dataset for the lateral hypothalamus (Mickelsen et al,2019).

3) There are newly developed approaches to check the shifts in the cell compositions and specific differential gene expression in the cell groups (e.g. Cacoa from Kharchenko lab, scCoda from Büttner et al; etc). Therefore, I did not fully understand why here the authors used the pseudo-bulk approaches for the data analysis (having such a valuable dataset with multiple hashed samples for each timepoint). Therefore it would be great to use at least one of those approaches, which were developed specifically for the scRNAseq data analysis. Or, if there are some reasons - the authors should argue why their approach is optimal

4) When the authors describe the DGE changes upon experimental conditions (Figures 5 and 6), my first comment is again relevant: it is difficult to use the current annotation and cell type description as the reference for testing virus effects and shifts in the DGE in distinct neuronal subtypes.

I have to note that the experimental design is well done and logical. Therefore I believe that to strengthen the conclusions, the already obtained datasets can be used for improved analysis.

Reviewer #2 (Public Review):

In this work, the authors use computational modeling and human neurophysiology (MEG) to uncover behavioral and neural signatures of choice history biases during sequential perceptual decision-making. In line with previous work, they see neural signatures reflecting choice planning during perceptual evidence accumulation in motor-related regions, and further show that the rate of accumulation responds to structured, predictable environments suggesting that statistical learning of environment structure in decision-making can adaptively bias the rate of perceptual evidence accumulation via neural signatures of action planning. The data and evidence show subtle but clear effects, and are consistent with a large body of work on decision-making and action planning.

Overall, the authors achieved what they set out to do in this nice study, and the results, while somewhat subtle in places, support the main conclusions. This work will have impact within the fields of decision-making and motor planning, linking statistical learning of structured sequential effects in sense data to evidence accumulation and action planning.

Strengths:<br /> - The study is elegantly designed, and the methods are clear and generally state-of-the-art<br /> - The background leading up to the study is well described, and the study itself conjoins two bodies of work - the dynamics of action-planning processes during perceptual evidence accumulation, and the statistical learning of sequential structure in incoming sense data<br /> - Careful analyses effectively deal with potential confounds (e.g., baseline beta biases)

Weaknesses:<br /> - Much of the study is primarily a verification of what was expected based on previous behavioral work, with the main difference (if I'm not mistaken) being that subjects learn actual latent structure rather than expressing sequential biases in uniform random environments. Whether this difference - between learning true structure or superstitiously applying it when it's not there - is significant at the behavioral or neural level is unclear. Did the authors have a hypothesis about this distinction? If the distinction is not relevant, is the main contribution here the neural effect?<br /> - The key effects (Figure 4) are among the more statistically on-the-cusp effects in the paper, and the Alternating group in 4C did not reliably go in the expected direction. This is not a huge problem per se, but does make the key result seem less reliable given the clear reliability of the behavioral results<br /> - The treatment of "awareness" of task structure in the study (via informal interviews in only a sub-sample of subjects) is wanting

Reviewer #2 (Public Review):

In this study, Yan et al. report that a cleaved form of METTL3 (termed METTL3a) plays an essential role in regulating the assembly of the METTL3-METTL14-WTAP complex. Depletion of METTL3a leads to reduced m6A level on TMEM127, an mTOR repressor, and subsequently decreased breast cancer cell proliferation. Mechanistically, METTL3a is generated via 26S proteasome in an mTOR-dependent manner.

The manuscript follows a smooth, logical flow from one result to the next, and most of the results are clearly presented. Specifically, the molecular interaction assays are well-designed. If true, this model represents a significant addition to the current understanding of m6A-methyltransferase complex formation.

A few minor issues detailed below should be addressed to make the paper even more robust. The specific comments are contained below.

1. The existence of METTL3a and METTL3b.<br /> In this study, the author found the cleaved form of METTL3 in breast cancer patient tissues and breast cancer cell lines. Is it a specific event that only occurs in breast cancer? The author may examine the METTL3a in other cell lines if it is a common rule.<br /> 2. Generation of METTL3a and METTL3b.<br /> 1) Figure 1 shows that METTL3a and METTL3b were generated from the C-terminal of full-length METTL3. Because the sequence of METTL3a is involved in the sequences of METTL3b, can METTL3b be further cleaved to produce METTL3a?<br /> 2) Based on current data, the generation of METTL3a and METTL3b are separated. Are there any factors that affect the cleavage ratio between METTL3a and METTL3b?<br /> 3. In Figure 2G, the author shows the result that incubation of the Δ198+Δ238 METTL3 protein with T47D cell lysates cannot produce the METTL3a and METTL3b variants. The author may also show the results that Δ198 METTL3 protein or Δ238 METTL3 protein incubates with T47D cell lysates, respectively.<br /> 4. As well as many results published in previous studies, the in vitro methylation assay shows that WT METTL3 is capable of methylating RNA probe (figure 2H). The main point of this study is that METTL3a is required for the METTL3-METTL14 assembly. However, the absence of METTL3a in the in vitro system did not inhibit METTL3-METTL14 methylation activity. Moreover, the presence of METTL3a even resulted in a weak m6A level.<br /> 5. In Figure 4A, the author suggests that WTAP cannot be immunoprecipitated with METTL3a and 3b because WTAP interacted with the N-terminal of METTL3. If this assay is performed in WT cells, the endogenous full-length METTL3 may help to form the complex. In this case, WTAP is supposed to be co-immunoprecipitated.

Reviewer #2 (Public Review):

In the present study, Masson et al. provide an elegant and profound demonstration of utilization of systems genetics data to fuel discovery of actionable therapeutics. The strengths of the study are many: generation of a novel skeletal muscle genetics proteomic dataset which is paired with measures of glucose metabolism in mice, systematic utilization of these data to yield potential therapeutic molecules which target insulin resistance, cross-referencing library screens from connectivity map with an independent validation platform for muscle glucose uptake and preclinical data supporting a new mechanism for thiostrepton in alleviating muscle insulin resistance. Future studies evaluating similar integrations of omics data from genetic diversity with compound screens, as well as detailed characterization of mechanisms such as thiostrepton on muscle fibers will further inform some remaining questions. In general, the thorough nature of this study not only provides strong support for the conclusions made, but additionally offers a new framework for analysis of systems-based data. As a result, my questions/comments below are mostly derived from interest and curiosity.

Line 105: The observation that variance in respiratory proteins is stable while lipid pathways is variable is quite interesting. Is this due to lower overall levels of lipid metabolism enzymes (ex. do these differ substantially from similar pathways ranked from high-low abundance?).

Line 154: the 664 associations are impressive and potentially informative. It would be valuable to know which of these co-map to the same locus - either to distinguish linkage in a 2mb window or identify any cis-proteins which directly exert effects in trans-

Line 194: Cross-platform validation of the CMAP fingerprint results is an admirable set of validations. It might be good to know general parameters like how many compounds were shared/unique for each platform. Also the concordance between ranking scores for significant and shared compounds.

Line 319: Another consideration in the molecular fingerprint is how unique these are for muscle. While studies evaluating gene expression have shown that many cis-eQTLs are shred across tissues, to my knowledge, this hasn't been performed systematically for pQTLs. Therefore, consider adding a point to the discussion pointing out that some of the proteins might be conserved pQTLs whereas others which would be more relevant here present unique druggable targets in muscle.

Line 332: These are fascinating observations. 1, that in general insulin signaling and ampk were not themselves shown as top-ranked enrichments with matsuda and that this was sufficient to alter glucose metabolism without changes in these pathways. While further characterization of this signaling emchanism is beyond the scope of this study, it would be good to speculate as to additional signaling pathways that are relevant beyond ROS (ex. CNYP2 and others)

Line: 314: Remove the statement: "While this approach is less powerful than QTL co-localisation for identifying causal drivers,", as I don't believe that this has been demonstrated. Clearly, the authors provide a sufficient framework to pinpoint causality and produce an actionable set of proteins.

Line 346: I would highlight one more appeal of the approach adopted by the authors. Given that these compound libraries were prioritized from patterns of diverse genetics, these observations are inherently more-likely to operate robustly across target backgrounds.

Line 434: I might have missed but can't seem to find where the muscle data are available to researchers. Given the importance and novelty of these studies, it will be important to provide some way to access the proteomic data.

Reviewer #2 (Public Review):

The authors Yang et al., examine the role of NR2F1/COUPTFI and NR2F2/COUPTF2 genes in hippocampus (HP) development, using two Cre lines, RxCre, and Emx1Cre. They report that loss of COUPTFI leads to a defective specification of dorsal CA1; loss of COUPTF2 leads to defects in the morphogenesis of the ventral HP with some ectopic CA field domains; loss of both results in a greatly shrunken hippocampus.

While the phenotypes are indeed interesting and important to examine carefully, there are major lacunae in (A) the authors' interpretation of the literature that sets up the problem (B) the data itself and the experimental design (C) the interpretation of the data. These are detailed below.

[A] Interpretation of the literature<br /> A1: The author's interpretation of the Lhx5 mutant phenotype (line 74-76) missed the fact that the hem appears to be missing or greatly reduced (Zhao et al., 1999; Figure 4D,I; Miquelajáuregui et al., 2010 Figure 5). If the hem is deficient, shrinkage/ agenesis of hippocampus is not surprising. It is incorrect to conclude that Lhx5 has a role in the hippocampal primordium, not only because of the above, but also because Lhx5 expression has been well characterized to be limited to the early hem and CR cells, but is not known to be expressed in the hippocampal primordium. The immunohistochemistry data in Figure 5B showing Lhx5 presence in the vz of the hippocampal and neocortical primordium is perplexing and not what other studies in the literature show for this gene. This is a major point because "regulation of the Lhx2-Lhx5 axis" is one of the main conclusions of the study.

A2: The Lhx2<->Lhx5 inhibition is pitched as a mechanism, but there's no evidence in the literature for this nor in this study. Lines 78-79 "Intriguingly, deficiency of either Lhx5 or Lhx2 results in agenesis of the hippocampus, and more particularly, these genes inhibit each other" are an incorrect interpretation of the literature. The "agenesis" of the hippocampus in the Lhx5 mutant (Zhao et al., 1999) is likely to be because the hem is deficient (point A1 above). The Lhx2 mutant lacks a hippocampus (and neocortex) because the entire dorsal telencephalon has transformed into hem and antihem (Mangale et al., 2008). To cite this as "agenesis of the hippocampus" as originally described by Porter et al (1997) misinterprets a complex stepwise process that was elucidated subsequently in the literature.

Finally, it has not been shown that Lhx2 and Lhx5 inhibit each other- the literature cited does not contain this information. The phenotype reported by the authors may actually have a basis in the effect of loss of COUPTFI/ II on the hem, and a rostro-caudal variation in this effect (or in the timing of action of the Cre lines used) may explain the phenotype.

Problems in the experimental design:<br /> B1: What is the expression domain and timing of RxCre? If it has a dorso-ventral bias in the early embryo, it could explain the regional difference in the COUPTF phenotypes. The authors must show the domain of Cre activation using an Ai9 reporter at E10.5-E11.5 and also at later embryonic stages to be able to interpret whether the shrunken hippocampal phenotype in the single and double mutants is a due to a defect in induction (from the hem), specification (in the early hippocampal primordium), or growth and maintenance (at later embryonic/ postnatal stages). A related point is whether COUPTFI expressed in the hem at E10.5-E11.5, since the earliest age shown is E14.5 which does show expression in the hem; likewise COUPTFII is shown to be expressed in the hem at E12.5. Emx1Cre acts in the hem and therefore the phenotypes could be partially explained by a deficit in the hem itself. Where RxCre acts is not shown and nor is it cited and the logic of shifting between RxCre and Emx1Cre is not clear. A comparison of the expression domains of these lines at relevant early and late embryonic ages is important.

B2:<br /> Line 187: "We would like to investigate the correlation of the CH and/or amygdala anlage with the duplicated ventral hippocampal domains in the COUP-TFII mutant in detail in our future study."<br /> This is inadequate, the effect of the mutation on the cortical hem may be central to the hippocampal phenotype and therefore is central to this study. Ectopic CA fields arising in unexpected places is a finding that needs an explanation, this is not a mere morphogenesis issue as implied in line 190.

B3: Questionable immunofluoresence data: Figure 5B panel h shows that Lhx2 expression extends into the region of the hem at E14.5, suggesting that the hem may in fact not have been specified in the first place. However, the choroid plexus appears to be LHX2 positive in the same image, which it isn't supposed to be, and this calls into question the quality and specificity of the immunofluoresence data. LHX5 staining in Figure 5B panel has been mentioned in point A1- it does not reflect the known expression pattern of this gene (Allen Brain atlas, Zhao et al., 2009). SOX2 also shouldn't be seen in the choroid plexus.

[C] Interpretation of the data<br /> C1: In the COUPTFII mutant, the ectopic presence of HuB+ve cells is intriguing, however it is a stretch to conclude that these cells are born at the expense of CTIP2+ve cells (line 179) without experiments that examine this point.

C2: Line 251: "Unexpectedly, an ectopic nucleus was observed in the region of the prospected temporal hippocampus, indicated by the arrowhead, in the double-mutant mice (Figure 3Ag, h)"<br /> These data are unclear and difficult to appreciate.

C3: The hippocampus is shrunken in the double mutants but the underlying cause has not been examined from the perspective of early cell cycle exit or cell death. How does the reduction of Tbr2+ and NeuroD1+ cells speak to the hippocampal defect? (Figure 5)

Reviewer #2 (Public Review):

Previous studies have extensively explored the rules by which patterned inputs from the two eyes are combined in the visual cortex. Here the authors explore these rules for un-patterned inputs (luminance flicker) at both the level of the cortex, using Steady-State Visual Evoked Potentials (SSVEPs) and at the sub-cortical level using pupillary responses. They find that the pattern of binocular combination differs between cortical and sub-cortical levels with the cortex showing less dichoptic masking and somewhat more binocular facilitation.

Importantly, the present results with flicker differ markedly from those with gratings (Hou et al., 2020, J Neurosci, Baker and Wade 2017 cerebral cortex, Norcia et al, 2000 Nuroreport, Brown et al., 1999, IOVS). When SSVEP responses are measured under dichoptic conditions where each eye is driven with a unique temporal frequency, in the case of grating stimuli, the magnitude of the response in the fixed contrast eye decreases as a function of contrast in the variable contrast eye. Here the response increases by varying (small) magnitudes. The authors favor a view that cortex and perception pool binocular flicker inputs approximately linearly using cells that are largely monocular. The lack of a decrease below the monocular level when modulation strength increase is taken to indicate that previously observed normalization mechanism in pattern vision does not play a substantial role in the processing of flicker. The authors present a computational model of binocular combination that captures features of the data when fit separately to each data set. Because the model has no frequency dependence and is based on scalar quantities, it cannot make joint predictions for the multiple experimental conditions which is one of its limitations.

A strength of the current work is the use of frequency-tagging of both pupil and EEG responses to measure responses for flicker stimuli at two anatomical levels of processing. Flicker responses are interesting but have been relatively neglected. The tagging approach allows one to access responses driven by each eye, even when the other eye is stimulated which is a great strength. The tagging approach can be applied at both levels of processing at the same time when stimulus frequencies are low, which is an advantage as they can be directly compared. The authors demonstrate the versatility of frequency tagging in a novel experimental design which may inspire other uses, both within the present context and others. A disadvantage of the tagging approach for studying sub-cortical dynamics via pupil responses is that it is restricted to low temporal frequencies given the temporal bandwidth of the pupil. The inclusion of a behavioral measure and a model is also a strength, but there are some limitations in the modeling (see below).

The authors suggest in the discussion that luminance flicker may preferentially drive cortical mechanisms that are largely monocular and in the results that they are approximately linear in the dichoptic cross condition (no effect of the fixed contrast stimulus in the other eye). By contrast, prior research using dichoptic dual frequency flickering stimuli has found robust intermodulation (IM) components in the VEP response spectrum (Baitch and Levi, 1988, Vision Res; Stevens et al., 1994 J Ped Ophthal Strab; France and Ver Hoeve, 1994, J Ped Ophthal Strab; Suter et al., 1996 Vis Neurosci). The presence of IM is a direct signature of binocular interaction and suggests that at least under some measurement conditions, binocular luminance combination is "essentially" non-linear, where essential implies a point-like non-linearity such as squaring of excitatory inputs. The two views are in striking contrast. It would thus be useful for the authors could show spectra for the dichoptic, two-frequency conditions to see if non-linear binocular IM components are present.

If the IM components are indeed absent, then there is a question of the generality of the conclusions, given that several previous studies have found them with dichoptic flicker. The previous studies differ from the authors' in terms of larger stimuli and in their use of higher temporal frequencies (e.g. 18/20 Hz, 17/21 Hz, 6/8 Hz). Either retinal area stimulated (periphery vs central field) or stimulus frequency (high vs low) could affect the results and thus the conclusions about the nature of dichoptic flicker processing in cortex. It would be interesting to sort this out as it may point the research in new directions.

Whether these components are present or absent is of interest in terms of the authors' computational model of binocular combination. It appears that the present model is based on scalar magnitudes, rather than vectors as in Baker and Wade (2017), so it would be silent on this point. The final summation of the separate eye inputs is linear in the model. In the first stage of the model, each eye's input is divided by a weighted input from the other eye. If we take this input as inhibitory, then IM would not emerge from this stage either.

Related to the model: One of the more striking results is the substantial difference between the dichoptic and dichoptic-cross conditions. They differ in that the latter has two different frequencies in the two eyes while the former has the same frequency in each eye. As it stands, if fit jointly on the two conditions, the model would make the same prediction for the dichoptic and dichoptic-cross conditions. It would also make the same prediction whether the two eyes were in-phase temporally or in anti-phase temporally. There is no frequency/phase-dependence in the model to explain differences in these cases or to potentially explain different patterns at the different VEP response harmonics. The model also fits independently to each data set which weakens its generality. An interpretation outside of the model framework would thus be helpful for the specific case of differences between the dichoptic and dichoptic-cross conditions.

Prior work has defined several regimes of binocular summation in the VEP (Apkarian et al.,1981 EEG Journal). It would be useful for the authors to relate the use of their terms "facilitation" and "suppression" to these regimes and to justify/clarify differences in usage, when present. Experiment 1, Fig. 3 shows cases where the binocular response is more than twice the monocular response. Here the interpretation is clear: the responses are super-additive and would be classed as involving facilitation in the Apkarian et al framework.

In the Apkarian et al framework, a ratio of 2 indicates independence/linearity. Ratios between 1 and 2 indicate sub-additivity and are diagnostic of the presence of binocular interaction but are noted by them to be difficult to interpret mechanistically. This should be discussed. A ratio of <1 indicates frank suppression which is not observed here with flicker.

Can the model explore the full range of binocular/monocular ratios in the Apkarian et al framework? I believe much of the data lies in the "partial summation" regime of Apkarian et al and that the model is mainly exploring this regime and is a way of quantifying varying degrees of partial summation.

Reviewer #2 (Public Review):

One of the major strengths in the current study is the implementation of the fully data-driven, gradient-based method for mapping connectopies of the LC. This approach is especially suited for brain structures that are difficult to localise because the resulted connectopic mapping is relatively robust to ROI definition (Fig. 7 in Haak et al., 2018). However, as a very inclusive definition of the LC (the "meta atlas") was adopted in the study, to what extent the gradient approach can tolerate changes of accuracy and specificity for LC ROI definition is unknown. Some comparative analyses would be helpful to provide assessments on the specificity and stability of the reported gradient pattern.

Haak et al. showed distinct reproducibility within and between subjects when comparing connectopic mappings between M1 and V1. M1 connectopic mapping showed very high consistency across subjects (ICCs > 0.9) compared with V1. This is very reasonable because the functional organisation within M1 is relatively homogeneous. Regarding the reliability of the LC rostro-caudal gradient, the authors only stated that "individual gradient estimation is often not consistent", but direct measurement on the consistency across subjects for the LC gradient was missing. This is important for future LC fMRI studies as more consistent pattern might warrant the application of an atlas-based method otherwise a more individualised pipeline is needed for investigating functional dissociation in LC subregions.

It puzzles me that why a dichotomous rostral vs caudal comparison was used to demonstrate the difference in connectivity patterns along the rostro-caudal gradient which might be an oversimplistic approach as described by the authors themselves? In fact, it might be more interesting to include the central "core" LC which is structurally organized in high density (Fernandes et al., 2012) and functionally distinguishable to the peri-LC "shell" region (Totah et al., 2018; Poe et al., 2022).

The composition of rostral vs caudal connectivity pattern changes over ageing, where the loss of rostral-like connectivity was consistent in bilateral LC whereas the gain of caudal-like connectivity in older subjects was only evident in the left LC. Do authors have any explanations on this left-lateralised ageing effect which is interestingly coincided with a lot of observations such as increased left LC contrast ratios was found during ageing (Betts et al., 2017) and in PD patients (Ye et al., 2022), reduced left LC-parahippocampal gyrus connectivity was reported in aMCI patients (Jacobs et al., 2015).

Reviewer #2 (Public Review):

Summary, general appraisal

This study examines the construct of "cognitive spaces" as they relate to neural coding schemes present in response conflict tasks. The authors utilize a novel paradigm, in which subjects must map the direction of a vertically oriented arrow to either a left or right response. Different types of conflict (spatial Stroop, Simon) are parametrically manipulated by varying the spatial location of the arrow (a task-irrelevant feature). The vertical eccentricity of the arrow either agrees or conflicts with the arrow's direction (spatial Stroop), while the horizontal eccentricity of the arrow agrees or conflicts with the side of the response (Simon). A neural coding model is postulated in which the stimuli are embedded in a cognitive space, organized by distances that depend only on the similarity of congruency types (i.e., where conditions with similar relative proportions of spatial-Stroop versus Simon congruency are represented with similar activity patterns). The authors conduct a behavioral and fMRI study to provide evidence for such a representational coding scheme. The behavioral findings replicate the authors' prior work in demonstrating that conflict-related cognitive control adjustments (the congruency sequence effect) shows strong modulation as a function of the similarity between conflict types. With the fMRI neural activity data, the authors report univariate analyses that identified activation in left prefrontal and dorsomedial frontal cortex modulated by the amount of Stroop or Simon conflict present, and multivariate representational similarity analyses (RSA) that identified right lateral prefrontal activity encoding conflict similarity and correlated with the behavioral effects of conflict similarity.<br /> This study tackles an important question regarding how distinct types of conflict, which have been previously shown to elicit independent forms of cognitive control adjustments, might be encoded in the brain within a computationally efficient representational format. The ideas postulated by the authors are interesting ones and the utilized methods are rigorous. However, the study has critical limitations that are due to a lack of clarity regarding theoretical hypotheses, serious confounds in the experimental design, and a highly non-standard (and problematic) approach to RSA. Without addressing these issues it is hard to evaluate the contribution of the authors findings to the computational cognitive neuroscience literature.

The primary theoretical question and its implications are unclear.

The paper would greatly benefit from more clearly specifying potential alternative hypotheses and discussing their implications. Consider, for example, the case of parallel conflict monitors. Say that these conflict monitors are separately tuned for Stroop and Simon conflict, and are located within adjacent patches of cortex that are both contained within a single cortical parcel (e.g., as defined by the Glasser atlas used by the authors for analyses). If RSA was conducted on the responses of such a parcel to this task, it seems highly likely that an activation similarity matrix would be observed that is quite similar (if not identical) to the hypothesized one displayed in Figure 1. Yet it would seem like the authors are arguing that the "cognitive space" representation is qualitatively and conceptually distinct from the "parallel monitor" coding scheme. Thus, it seems that the task and analytic approach is not sufficient to disambiguate these different types of coding schemes or neural architectures.

The authors also discuss a fully domain-general conflict monitor, in which different forms of conflict are encoded within a single dimension. Yet this alternative hypothesis is also not explicitly tested nor discussed in detail. It seems that the experiment was designed to orthogonalize the "domain-general" model from the "cognitive space" model, by attempting to keep the overall conflict uniform across the different stimuli (i.e., in the design, the level of Stroop congruency parametrically trades off with the level of Simon congruency). But in the behavioral results (Fig. S1), the interference effects were found to peak when both Stroop and Simon congruency are present (i.e., Conf 3 and 4), suggesting that the "domain-general" model may not be orthogonal to the "cognitive space" model. One of the key advantages of RSA is that it provides the ability to explicitly formulate, test and compare different coding models to determine which best accounts for the pattern of data. Thus, it would seem critical for the authors to set up the design and analyses so that an explicit model comparison analysis could be conducted, contrasting the domain-general, domain-specific, and cognitive space accounts.<br /> Relatedly, the reasoning for the use of the term "cognitive space" is unclear. The mere presence of graded coding for two types of conflict seems to be a low bar for referring to neural activity patterns as encoding a "cognitive space". It is discussed that cognitive spaces/maps allow for flexibility through inference and generalization. But no links were made between these cognitive abilities and the observed representational structure. Additionally, no explicit tests of generality (e.g., via cross-condition generalization) were provided. Finally, although the design elicits strong CSE effects, it seems somewhat awkward to consider CSE behavioral patterns as a reflection of the kind of abilities supported by a cognitive map (if this is indeed the implication that was intended). In fact, CSE effects are well-modeled by simpler "model-free" associative learning processes, that do not require elaborate representations of abstract structures.

More generally, it seems problematic that Stroop and Simon conflict in the paradigm parametrically trade-off against each other. A more powerful design would have de-confounded Stroop and Simon conflict so that each could be separately estimation via (potentially orthogonal) conflict axes. Additionally, incorporating more varied stimulus sets, locations, or responses might have enabled various tests of generality, as implied by a cognitive space account.

Serious confounds in the design render the results difficult to interpret.

As much prior neuroimaging and behavioral work has established, "conflict" per se is perniciously correlated with many conceptually different variables. Consequently, it is very difficult to distinguish these confounding variables within aggregate measures of neural activity like fMRI. For example, conflict is confounded with increased time-on-task with longer RT, as well as conflict-driven increases in coding of other task variables (e.g., task-set related coding; e.g., Ebitz et al. 2020 bioRxiv). Even when using much higher resolution invasive measures than fMRI (i.e., eCoG), researchers have rightly been wary of making strong conclusions about explicit encoding of conflict (Tang et al, 2019; eLife). As such, the researchers would do well to be quite cautious and conservative in their analytic approach and interpretation of results.

This issue is most critical in the interpretation of the fMRI results as reflecting encoding of conflict types. A key limitation of the design, that is acknowledged by the authors is that conflict is fully confounded within-subject by spatial orientation. Indeed, the limited set of stimulus-response mappings also cast doubt on the underlying factors that give rise to the CSE modulations observed by the authors in their behavioral results. The CSE modulations are so strong - going from a complete absence of current x previous trial-type interaction in the cos(90) case all the way to a complete elimination of any current trial conflict when the prior trial was incongruent in the cos(0) case - that they cause suspicion that they are actually driven by conflict-related control adjustments rather than sequential dependencies in the stimulus-response mappings that can be associatively learned.

To their credit, the authors recognize this confound, and attempt to address it analytically through the use of a between-subject RSA approach. Yet the solution is itself problematic, because it doesn't actually deconfound conflict from orientation. In particular, the RSA model assumes that whatever components of neural activity encode orientation produce this encoding within the same voxel-level patterns of activity in each subject. If they are not (which is of course likely), then orthogonalization of these variables will be incomplete. Similar issues underlie the interpretation target/response and distractor coding. Given these issues, perhaps zooming out to a larger spatial scale for the between-subject RSA might be warranted. Perhaps whole-brain at the voxel level with a high degree of smoothing, or even whole-brain at the parcel level (averaging per parcel). For this purpose, Schaefer atlas parcels might be more useful than Glasser, as they more strongly reflect functional divisions (e.g., motor strip is split into mouth/hand divisions; visual cortex is split into central/peripheral visual field divisions). Similarly, given the lateralization of stimuli, if a within-parcel RSA is going to be used, it seems quite sensible to pool voxels across hemispheres (so effectively using 180 parcels instead of 360).

The strength of the results is difficult to interpret due to the non-standard analysis method.

The use of a mixed-level modeling approach to summarize the empirical similarity matrix is an interesting idea, but nevertheless is highly non-standard within RSA neuroimaging methods. More importantly, the way in which it was implemented makes it potentially vulnerable to a high degree of inaccuracy or bias. In this case, this bias is likely to be overly optimistic (high false positive rate).

A key source of potential bias comes from the fact that the off-diagonal cells are not independent (e.g., the correlation between subject A and B is strongly dependent on the correlation between subject A and C). For appropriate degrees of freedom calculation, the model must take this into account somehow. As fitted, the current models do not seem to handle this appropriately. That being said, it may be possible to devise an appropriate test via mixed-level models. In fact, Chen et al. have a series of three recent Neuroimage articles that extensively explore this question (all entitled "Untangling the relatedness among correlations") - adopting one of the methods described in the papers, seems much safer, if possible.

Another potential source of bias is in treating the subject-level random effect coefficients (as predicted by the mixed-level model) as independent samples from a random variable (in the t-tests). The more standard method for inference would be to use test statistics derived from the mixed-model fixed effects, as those have degrees of freedom calculations that are calibrated based on statistical theory.

No numerical or formal defense was provided for this mixed-level model approach. As a result, the use of this method seems quite problematic, as it renders the strength of the observed results difficult to interpret. Instead, the authors are encouraged using a previously published method of conducting inference with between-subject RSA, such as the bootstrapping methods illustrated in Kragel et al. (2018; Nat Neurosci), or in potentially adopting one of the Chen et al. methods mentioned above, that have been extensively explored in terms of statistical properties.

Reviewer #2 (Public Review):

The manuscript describes an approach to monitor microglial structural dynamics and correlate it to ongoing changes in brain state during sleep-wake cycles. The main novelty here is the use of miniaturized 2p microscopy, which allows tracking microglia surveillance over long periods of hours, while the mice are allowed to freely behave. Accordingly, this experimental setup would permit to explore long-lasting changes in microglia in a more naturalistic environment, which were previously not possible to identify otherwise. The findings could provide key advances to the research of microglia during natural sleep and wakefulness, as opposed to anesthesia. The main findings of the paper are that microglia increase their process motility and surveillance during REM and NREM sleep as compared to the awake state. The authors further show that sleep deprivation induces opposite changes in microglia dynamics- limiting their surveillance and size. The authors then demonstrate potential causal role for norepinephrine secretion from the locus coeruleus (LC) which is driven by beta 2 adrenergic receptors (b2AR) on microglia. However, there are several methodological and experimental concerns which should be addressed.

The major comments are summarized below:

1. The main technological advantage of the 2p miniaturized microscope is the ability to track single cells over sleep cycles. A main question that is unclear from the analysis and the way the data is presented is: are the structural changes in microglia reversible? Meaning, could the authors provide evidence that the same cell can dynamically change in sleep state and then return to similar size in wakefulness? The same question arises again with the data which is presented for anesthesia, is this change reversible?<br /> 2. The binary comparison between brain states is misleading, shouldn't the changes in structural dynamics compared to the baseline of the state onset? The authors method describes analysis of the last 5 minutes in each sleep/wake state. However, these transitions are directional- for instance, REM usually follows NREM, so the description of a decrease in length during REM sleep could be inaccurate.<br /> 3. Sleep deprivation- again, it is unclear whether these structural changes are reversible. This point is straightforward to address using this methodology by measuring sleep following SD. In addition, the authors chose a method to induce sleep deprivation that is rather harsh. It is unclear if the effect shown is the result of stress or perhaps an excess of motor activity.<br /> 4. The authors perform measurements of norepinephrine with a recently developed GRAB sensor. These experiments are performed to causally link microglia surveillance during sleep to norepinephrine secretion. They perform 2p imaging and collect data points which are single neurons, and it is unclear why the normalization and analysis is performed for bulk fluorescence similar to data obtained with photometry.<br /> 5. The experiments involving b2AR KO mice are difficult to interpret and do not provide substantial mechanistic insight. Since b2AR are expressed throughout numerous cell types in the brain and in the periphery, it is entirely not clear whether the effects on microglia dynamics are direct. The conclusion and the statement regarding the expression of b2AR in microglia is not supported by the references the authors present, which simply demonstrate the existence and function of b2AR in microglia. In addition, these mice show significant changes in sleep pattern and increased REM sleep. This could account for reasons for the changes in microglia structure rather than the interpretation that these are direct effects.<br /> To summarize, the main conclusions of the paper require further support with analysis of existing data and experimental validation.

Reviewer #2 (Public Review):

The authors decipher the signaling between vitamin D and proteins that are downstream of SIRT1. The importance of vitamin D in physiology is clear. However, the link between vitamin D and cancer is less clear. This study provides very interesting and solid information on the link between vitamin D and colorectal cancer. It is likely that this study will provide insight into the importance of vitamin D in other types of cancer. It may also lead to new therapeutic strategies for specific cases.

The authors focus on vitamin D-mediated signaling through VDR, SIRT1 and Ace H3K9. They highlight the importance of K610 in SIRT1 in this process. This article is convincing, although the authors can improve their study as outlined below:

* The authors should specify which cell line was used to perform the experiment in Figure 1E,F. What would be the result in the presence/absence of 1,25(OH)2D3? In Figure 1G, what is the meaning of # and ###?

* Figure 2C, it would have been ideal to show the VDR-SIRT1 interaction after a Sirt1 IP.

* I understand the authors' overall message for this figure, but it is far from clear. This section needs to be improved. For example, in Figure 3G, does this mean that the level of AceH3K9 is independent of the level of SIRT1? Is there a contradiction? The authors should indicate the color of the different stainings for Figure 3D. Do the authors mean that the secondary antibody marks in brown/red? If so, these results are inconsistent with the text considering that hematoxylin was used for non-tumor tissue. This part needs to be clarified. What about the level of FOXO3A in these tissues/tumors? What is the level of 1,25(OH)2D3 in these patients? In Figure 3D, the following information is missing: "A detailed amplification is shown in the lower left of each micrograph." In Figure 3E, it says p=0.325, in the legend p<0.01, and in the text there is a trend. Which is the correct version?

* Figure 4F. The quality of the presented blots is not optimal. It needs to be improved. In addition, the number of independent biological experiments is not indicated. In general, the authors should better indicate the number of independent biological experiments performed, at least for some of them. For example, see Figure 1G. Regarding Figure 2C, we understand that the WB was performed 3 times. Is this the case for the PI? etc...

Reviewer #2 (Public Review):

In this study, the authors validated a positive feedback loop between ZEB2 and ACSL4 in breast cancer, which regulates lipid metabolism to promote metastasis.

Overall, the study is original, well structured, and easy to read. Despite the reliability of the data discussed in this article, there are still some deficiencies that need to be addressed through further explanation.

Major issues:

1. The authors demonstrated that ACSL4 regulates ZEB2 not only via a post-transcriptional mechanism but also via a transcriptional mechanism. The authors have not provided a comprehensive explanation of the specific mechanism in this paper. Therefore, it is recommended that the author delve into the potential mechanisms in the discussion section. For example, related mechanisms affecting ZEB2 ubiquitination degradation, as well as factors affecting ZEB2 upstream transcriptional regulation, etc.

2. To further clarify the interaction of ZEB2 and ACSL4, it is best to perform in vitro glutathione-S-transferase (GST) pulldown assay and immunofluorescence assay.

3. In Figure 7B, the protein level of ZEB2 seems not to be altered in BT549 BCSC cell line after the depletion of ACSL4.

4. EMT is characterized by changes in cell morphology, so the staining of cytoskeletons with Phalloidin is needed.

5. Additional breast cancer cases or cohorts (such as TMA) should be used to validate the positive correlation between ACSL4 and ZEB2 expression through IHC analysis.

Reviewer #2 (Public Review):

Summary:

This work investigates the effects of various antipsychotic drugs on cortical responses during visuomotor integration. Using wide-field calcium imaging in a virtual reality setup, the researchers compare neuronal responses to self-generated movement during locomotion-congruent (closed loop) or locomotion-incongruent (open loop) visual stimulation. Moreover, they probe responses to unexpected visual events (halt of visual flow, sudden-onset drifting grating). The researchers find that, in contrast to a variety of excitatory and inhibitory cell types, genetically defined layer 5 excitatory neurons distinguish between the closed and the open loop condition and exhibit activity patterns in visual cortex in response to unexpected events, consistent with unsigned prediction error coding. Motivated by the idea that prediction error coding is aberrant in psychosis, the authors then inject the antipsychotic drug clozapine, and observe that this intervention specifically affects closed loop responses of layer 5 excitatory neurons, blunting the distinction between the open and closed loop conditions. Clozapine also leads to a decrease in long-range correlations between L5 activity in different brain regions, and similar effects are observed for two other antipsychotics, aripripazole and haloperidol, but not for the stimulant amphetamine. The authors suggest that altered prediction error coding in layer 5 excitatory neurons due to reduced long-range correlations in L5 neurons might be a major effect of antipsychotic drugs and speculate that this might serve as a new biomarker for drug development.

Strengths:

- Relevant and interesting research question:

The distinction between expected and unexpected stimuli is blunted in psychosis but the neural mechanisms remain unclear. Therefore, it is critical to understand whether and how antipsychotic drugs used to treat psychosis affect cortical responses to expected and unexpected stimuli. This study provides important insights into this question by identifying a specific cortical cell type and long-range interactions as potential targets. The authors identify layer 5 excitatory neurons as a site where functional effects of antipsychotic drugs manifest. This is particularly interesting as these deep layer neurons have been proposed to play a crucial role in computing the integration of predictions, which is thought to be disrupted in psychosis. This work therefore has the potential to guide future investigations on psychosis and predictive coding towards these layer 5 neurons, and ultimately improve our understanding of the neural basis of psychotic symptoms.

- Broad investigation of different cell types and cortical regions:

One of the major strengths of this study is quasi-systematic approach towards cell types and cortical regions. By analysing a wide range of genetically defined excitatory and inhibitory cell types, the authors were able to identify layer 5 excitatory neurons as exhibiting the strongest responses to unexpected vs. expected stimuli and being the most affected by antipsychotic drugs. Hence, this quasi-systematic approach provides valuable insights into the functional effects of antipsychotic drugs on the brain, and can guide future investigations towards the mechanisms by which these medications affect cortical neurons.

- Bridging theory with experiments:

Another strength of this study is its theoretical framework, which is grounded in the predictive coding theory. The authors use this theory as a guiding principle to motivate their experimental approach connecting visual responses in different layers with psychosis and antipsychotic drugs. This integration of theory and experimentation is a powerful approach to tie together the various findings the authors present and to contribute to the development of a coherent model of how the brain processes visual information both in health and in disease.

Weaknesses:

- Unclear relevance for psychosis research:

From the study, it remains unclear whether the findings might indeed be able to normalise altered predictive coding in psychosis. Psychosis is characterised by a blunted distinction between predicted and unpredicted stimuli. The results of this study indicate that antipsychotic drugs further blunt the distinction between predicted and unpredicted stimuli, which would suggest that antipsychotic drugs would deteriorate rather than ameliorate the predictive coding deficit found in psychosis. However, these findings were based on observations in wild-type mice at baseline. Given that antipsychotics are thought to have little effects in health but potent antipsychotic effects in psychosis, it seems possible that the presented results might be different in a condition modelling a psychotic state, for example after a dopamine-agonistic or a NMDA-antagonistic challenge. Therefore, future work in models of psychotic states is needed to further investigate the translational relevance of these findings.

- Incomplete testing of predictive coding interpretation:

While the investigation of neuronal responses to different visual flow stimuli Is interesting, it remains open whether these responses indeed reflect internal representations in the framework of predictive coding. While the responses are consistent with internal representation as defined by the researchers, i.e., unsigned prediction error signals, an alternative interpretation might be that responses simply reflect sensory bottom-up signals that are more related to some low-level stimulus characteristics than to prediction errors. Moreover, This interpretational uncertainty is compounded by the fact that the used experimental paradigms were not suited to test whether behaviour is impacted as a function of the visual stimulation which makes it difficult to assess what the internal representation of the animal actual was. For these reasons, the observed effects might reflect simple bottom-up sensory processing alterations and not necessarily have any functional consequences. While this potential alternative explanation does not detract from the value of the study, future work would be needed to explain the effect of antipsychotic drugs on responses to visual flow. For example, experimental designs that systematically vary the predictive strength of coupled events or that include a behavioural readout might be more suited to draw from conclusions about whether antipsychotic drugs indeed alter internal representations.

- Methodological constraints of experimental design:

While the study findings provide valuable insights into the potential effects of antipsychotic drugs, it is important to acknowledge that there may be some methodological constraints that could impact the interpretation of the results. More specifically, the experimental design does not include a negative control condition or different doses. These conditions would help to ensure that the observed effects are not due to unspecific effects related to injection-induced stress or time, and not confined to a narrow dose range that might or might not reflect therapeutic doses used in humans. Hence, future work is needed to confirm that the observed effects indeed represent specific drug effects that are relevant to antipsychotic action.

Conclusion:

Overall, the results support the idea that antipsychotic drugs affect neural responses to predicted and unpredicted stimuli in deep layers of cortex. Although some future work is required to establish whether this observation can indeed be explained by a drug-specific effect on predictive coding, the study provides important insights into the neural underpinnings of visual processing and antipsychotic drugs, which is expected to guide future investigations on the predictive coding hypothesis of psychosis. This will be of broad interest to neuroscientists working on predictive coding in health and in disease.

Reviewer #2 (Public Review):

The authors of the current study set out to improve the purity of extracellular vesicles obtained from plasma. A well-described problem is that various means of separating extracellular vesicles from other plasma constituents tend to leave residual impurities such as lipoproteins and free proteins in the final extracellular vesicles preparation. Van Deun and colleagues had previously improved on the size exclusion chromatography approach by adding a second form of chromatography using separation based on charge. The current authors have evaluated that method and another gold standard approach, iodixanol gradient ultracentrifugation, and they have extended the work with the addition of a third form of chromatography. They are building on their prior work on separating albumin from plasma extracellular vesicles.

A major strength of the paper is that the authors have used complementary methods including a digital immunoassay method and transmission electron microscopy to demonstrate the purity of their sample preparation method. In addition, they have used mass spectrometry to show that they are able to profile hundreds of proteins in their plasma extracellular vesicle sample preparations.

Another major strength of the work is that the authors have taken pains to aid others in reproducing and extending the work. The authors used commercially available human pooled plasma, which is a good decision in terms of reproducibility, compared with a single person's plasma. The authors have explained exactly how to make their new chromatography columns, and they've also explained how to make a manual or an automated apparatus to improve the parallel processing of samples. They explained exactly how to fabricate each apparatus, with computer-aided design files and Raspberry Pi software. I anticipate many others will be able to implement what the authors have done because they shared these resources.

Moreover, the authors have shared the essential data needed to understand and vet their work.

Meanwhile, they shared simple and practical information about the preparation of Sepharose columns to improve the yield of chromatography. They showed that in-column washing with PBS yielded more extracellular vesicles compared with washing Sepharose prior to making the column. This finding should help anyone using size-exclusion chromatography or the more sophisticated combinations of chromatography studied herein.

The major weakness of the method is that it remains unclear to what extent the results of proteomic profiling of these purer plasma extracellular vesicles continue to be confounded by free proteins. This is a problem that will take sustained efforts to resolve, but the authors have built the next piece of the road heading in that direction.

The authors have succeeded in their main aims, albeit without being able to completely rid the sample preparations of lipoproteins, which may or may not be possible.

The results support the authors' conclusions.

This work is going to be useful to the increasing number of researchers who find that circulating extracellular vesicles hold promise for the diagnosis of diseases. In order to find the "signal" within the noise of the complex admixture constituting human plasma, a suitable process for separating vesicles from what I would call impurities is essential. The ability to automate that process while also scaling it up are additional essential components for the extracellular vesicle biomarker field to develop into a clinically useful source of biomarkers. The authors have made progress in each of these areas.

Reviewer #2 (Public Review):

In this manuscript, authors had to circumvent some challenges in protein design that included the generation of peptide-receptive MHCI and a defined Man9GlcNAc2 glycan tree on the MHC I recognizable by UGGT1. Production of peptide-receptive MHCI was achieved by forming a fos/jun dimerized single-chain MHC1-fos with TAPBPR-jun in the presence of the α-mannosidase I inhibitor kifunensine. Glucozylation of MHCI by UGGT1 was monitored on protease-cleaved MHCI/TAPBPR, and liquid chromatography-mass spectrometry was used to monitor reglucosylation. Authors have provided convincing evidence that TAPBPR is sufficient and necessary for glucosylation of MHC 1, hence TAPBPR in addition to serving as an accessory protein in regulating peptide selection has a second function in quality control and fitness of newly synthesized MHC I during maturation.

The strength of the study lies in the generation of a complete in vitro system where different steps and direct interactions between different components of MHCI maturation can be monitored, hence leading to a better mechanistic understanding of MHC I maturation. However, some potential weakness might be that the major finding of the manuscript describing the critical role of TAPBPR as a chaperon in optimizing peptide selection and regulation of MHC I glucosylation and reglucosylation has been previously reported. Nonetheless, the current study further establishes and better defines some prior findings, thus quite valuable.

Reviewer #2 (Public Review):

This paper purports to unveil a mechanism controlling telomere length through SUMO modifications controlling interactions between PCNA unloader Elg1 and the CST complex that functions at telomeres. This is an extremely interesting mechanism to understand, and this paper indeed reveals some interesting genetic results, leading to a compelling model, with potential impact on the field. The conclusions are largely supported by experiments examining protein-protein interactions at low resolution and ambiguous regarding directness of interactions like co-IP and yeast two-hybrid (Y2H) combined with genetics. However, some results appear contradictory and there's a lack of rigor in the experimental data needed to support claims. There is significant room for improvement and this work could certainly attain the quality needed to support the claims. The current version needs substantial revision and lacks the necessary experimental detail. Stronger support for the claims would add detail to help distinguish competing models.

Reviewer #2 (Public Review):

The manuscript "Detecting and validating influential organisms for rice growth: An ecological network approach" explores the influence of biotic and abiotic entities that are often neglected on rice growth. The study has a straightforward experimental design, and well thought hypothesis for explorations. Monitoring data is collected to infer relationships between species and the environment empirically. It is analyzed with an up-to-date statistical method. This allowed the manuscript to hypothesize and test the effects most influential entities in a controlled experiment.

The manuscript is interesting and sets up a nice framework for future studies. In general, the manuscript can be improved significantly, when this workflow is smoothly connected and communicated how they follow each other more than the sequence and dates provided. It is valuable philosophical thinking, and the research community can benefit from this framework.

I understand the length and format of the manuscript make it difficult to add more details, but I am sure it can refer to/clear some concepts/methods that might be new for the audience. How/why variables are selected as important parts of the system, a tiny bit of information about the nonlinear time series analysis in the early manuscript, and the biological reasoning behind these statistically driven decisions are some examples.

Reviewer #2 (Public Review):

This is a well-written manuscript about a strong comparative study of diversity of facial movements in three macaque species to test arguments about social complexity influencing communicative complexity. My major criticism has to do with the lack of any reporting of inter-observer reliability statistics - see comment below. Reporting high levels of inter-observer reliability is crucial for making clear the authors have minimized chances of possible observer biases in a study like this, where it is not possible to code the data blind with regard to comparison group. My other comments and questions follow by line number:

38-40. Whereas I am an advocate of this hypothesis and have tested it myself, the authors should probably comment here, or later in the discussion, about the reverse argument - greater communicative complexity (driven by other selection pressures) could make more complicated social structures possible. This latter view was the one advocated by McComb & Semple in their foundational 2005 Biology Letters comparative study of relationships between vocal repertoire size and typical group size in non-human primate species.

72-84 and 95-96. In the paragraph here, the authors outline an argument about increasing uncertainty / entropy mapping on to increasing complexity in a system (social or communicative). In lines 95-96, though, they fall back on the standard argument about complex systems having intermediate levels of uncertainty (complete uncertainty roughly = random and complete certainty roughly = simple). Various authors have put forward what I think are useful ways of thinking about complexity in groups - from the perspective of an insider (i.e., a group member, where greater randomness is, in fact, greater complexity) vs from the perspective of an outside (i.e., a researcher trying to quantify the complexity of the system where is it relatively easy to explain a completely predictable or completely random system but harder to do so for an intermediately ordered or random system). This sort of argument (Andrew Whiten had an early paper that made this argument) might be worth raising here or later in the discussion? (I'm also curious where the authors sentiments lie for this question - they seem to touch on it in lines 285-287, but I think it's worth unpacking a little more here!)

115-129. See also:<br /> Maestripieri, D. (2005). "Gestural communication in three species of macaques (Macaca mulatta, M. nemestrina, M. arctoides): use of signals in relation to dominance and social context." Gesture 5: 57-73.<br /> Maestripieri, D. and K. Wallen (1997). "Affiliative and submissive communication in rhesus macaques." Primates 38(2): 127-138.<br /> On that note, it is probably worth discussing in this paragraph and probably later in the discussion exactly how this study differs from these earlier studies of Maestripieri. I think the fact that machine learning approaches had the most difficulty assigning crested data to context is an important methodological advance for addressing these sorts of questions - there are probably other important differences between the authors' study here and these older publications that are worth bringing up.

220-222. What is known about visual perception in these species? Recent arguments suggest that more socially complex species should have more sensitive perceptual processing abilities for other individuals' signals and cues (see Freeberg et al. 2019 Animal Behaviour). Are there any published empirical data to this effect, ideally from the visual domain but perhaps from any domain?

274-277. I am not sure I follow this - could not different social and non-social contexts produce variation in different affective states such that "emotion"-based signals could be as flexible / uncertain as seemingly volitional / information-based / referential-like signals? This issue is probably too far away from the main points of this paper, but I suspect the authors' argument in this sentence is too simplified or overstated with regard to more affect-based signals.

288 on. Given there are only three species in this study, the chances of one of the species being the 'most complex' in any measure is 0.33. Although I do not believe this argument I am making here, can the authors rule out the possibility that their findings related to crested macaques are all related to chance, statistically speaking?

329-330. The fact that only one male rhesus macaque was assessed here seems problematic, given the balance of sexes in the other two species. Can the authors comment more on this - are the gestures they are studying here identical across the sexes?

354-371. Inter-observer reliability statistics are required here - one of the authors who did not code the original data set, or a trained observer who is not an author, could easily code a subset of the video files to obtain inter-observer reliability data. This is important for ruling out potential unconscious observer biases in coding the data.

Reviewer #2 (Public Review):

This study presents a useful investigation of eccDNAs in spermatogenesis of mouse. It provides evidence about the biogenesis of eccDNAs and suggests that eccDNAs are derived from oligonucleosmal DNA fragmentation during apoptosis by MMEJ and may not be the direct products of germline deletions. However, the method of data analyses were not fully described and data analysis is incomplete. It provides additional observations about the eccDNA biogenesis and can be used as a starting point for functional studies of eccDNA in sperms. However, many aspects about data analyses and data interpretations need to be improved.

• Most of the conclusions made by the work are only based on the bioinformatics analyses, the validation of these foundlings using other method (biochemistry/molecular biology method) are missing. For example, no QC results presented for the eccDNA purification, which may show whether contaminates such as linear DNA or mitochondria DNA have been fully removed. Additionally, it is also helpful to use simple PCR to test the existence of identified eccDNAs in sperm or other samples to validate the specificity of the Circle-seq method.

• The reliability of the data analysis methods is uncertain, as the authors constructed and utilized their own pipeline to identify eccDNAs, despite the availability of established bioinformatics tools such as ECCsplorer, eccFinder, and Amplicon Architect. Moreover, the lack of validation of the pipeline using either ground truth datasets or simulation data raises concerns about its accuracy. Additionally, the methodology employed for identifying eccDNA that encompasses multiple gene loci remains unclear.

• Although the author stated that previous studies utilizing short-read sequencing technologies may have incorrectly annotated eccDNA breakpoints, this claim requires careful scrutiny and supporting evidence, which was not provided in the manuscript.

• The similarity between the eccDNA profiles of human and mouse sperm remains uncertain, and therefore, analyses of human eccDNA data and comparisons between the two are necessary if the authors claim that their findings of widespread eccDNA formation in mouse spermatogenesis extend to human sperms.

Time Shooter 2 is an enjoyable first-person shooter in which time only moves when the player moves. Slow-motion combat forces the player to plan their movements.

The game features impressive graphics and sound effects that enhance the overall gaming experience. The high-quality visuals and sound effects immerse players in the game, making it more enjoyable and entertaining.

Let's play and have fun with the game in shooting mode.

Reviewer #2 (Public Review):

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

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

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

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

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

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

Reviewer #2 (Public Review):

This important work presents an example of a contextual computation in a navigation task through a comparison of task driven RNNs and mouse neuronal data. Authors perform convincing state of the art analyses demonstrating compositional computation with valuable properties for shared and distinct readouts. This work will be of interest to those studying contextual computation and navigation in biological and artificial systems.

This work advances intuitions about recent remapping results. Authors trained RNNs to output spatial position and context given velocity and 1-bit flip-flops. Both of these tasks have been trained separately, but this is the first time to my knowledge that one network was trained to output both context and spatial position. This work is also somewhat similar to previous work where RNNs were trained to perform a contextual variation on the Ready-Set-Go with various input configurations (Remington et al. 2018). Additionally findings in the context of recent motor and brain machine interface tasks are consistent with these findings (Marino et al in prep). In all cases contextual input shifts neural dynamics linearly in state space. This shift results in a compositional organization where spatial position can be consistently decoded across contexts. This organization allows for generalization in new contexts. These findings in conjunction with the present study make a consistent argument that remapping events are the result of some input (contextual or otherwise) that moves the neural state along the remapping dimension.

The strength of this paper is that it tightly links theoretical insights with experimental data, demonstrating the value of running simulations in artificial systems for interpreting emergent properties of biological neuronal networks. For those familiar with RNNs and previous work in this area, these findings may not significantly advance intuitions beyond those developed in previous work. It's still valuable to see this implementation and satisfying demonstration of state of the art methods. The analysis of fixed points in these networks should provide a model for how to reverse engineer and mechanistically understand computation in RNNs.

I'm curious how the results might change or look the same if the network doesn't need to output context information. One prediction might be that the two rings would collapse resulting in completely overlapping maps in either context. I think this has interesting implications about the outputs of the biological system. What information should be maintained for potential readout and what information should be discarded? This is relevant for considering the number of maps in the network. Additionally, I could imagine the authors might reproduce their current findings in another interesting scenario: Train a network on the spatial navigation task without a context output. Fix the weights. Then provide a new contextual input for the network. I'm curious whether the geometric organization would be similar in this case. This would be an interesting scenario because it would show that any random input could translate the ring attractor that maintains spatial position information without degradation. It might not work, but it could be interesting to try!

I was curious and interested in the authors choice to not use activity or weight regularization in their networks. My expectation is that regularization might smooth the ring attractor to remove coding irrelevant fluctuations in neural activity. This might make Supplementary Figure 1 look more similar across model and biological remapping events (Line 74). I think this might also change the way authors describe potential complex and high dimensional remapping events described in Figure 2A.

Overall this is a nice demonstration of state-of-the-art methods to reverse engineer artificial systems to develop insights about biological systems. This work brings together concepts for various tasks and model organisms to provide a satisfying analysis of this remapping data.

Reviewer #2 (Public Review):

Starting from the observation that difficulty estimation lies at the core of human cognition, the authors acknowledge that despite extensive work focusing on the computational mechanisms of decision-making, little is known about how subjective judgments of task difficulty are made. Instantiating the question with a perceptual decision-making task, the authors found that how humans pick the easiest of two stimuli, and how quickly these difficulty judgments are made, are best described by a simple evidence accumulation model. In this model, perceptual evidence of concurrent stimuli is accumulated and difficulty is determined by the difference between the absolute values of decision variables corresponding to each stimulus, combined with a threshold crossing mechanism. Altogether, these results strengthen the success of evidence accumulation models, and more broadly sequential sampling models, in describing human decision-making, now extending it to judgments of difficulty.

The manuscript addresses a timely question and is very well written, with its goals, methods and findings clearly explained and directly relating to each other. The authors are specialists in evidence accumulation tasks and models. Their modelling of human behaviour within this framework is state-of-the-art. In particular, their model comparison is guided by qualitative signatures which are diagnostic to tease apart the different models (e.g., the RT criss-cross pattern). Human behaviour is then inspected for these signatures, instead of relying exclusively on quantitative comparison of goodness-of-fit metrics. This work will likely have a wide impact in the field of decision-making, and this across species. It will echo in particular with many other studies relying on the similar theoretical account of behaviour (evidence accumulation).

A few points nevertheless came to my attention while reading the manuscript, which the authors might find useful to answer or address in a new version of their manuscript.

1. The authors acknowledge that difficulty estimation occurs notably before exploration (e.g., attempting a new recipe) or learning (e.g., learning a new musical piece) situations. Motivated by the fact that naturalistic tasks make difficult the identification of the inference process underlying difficulty judgments, the authors instead chose a simple perceptual decision-making task to address their question. While I generally agree with the authors's general diagnostic, I am nevertheless concerned so as to whether the task really captures the cognitive process of interest as described in the introduction. As coined by the authors themselves, the main function of prospective difficulty judgment is to select a task which will then ultimately be performed, or reject one which won't. However, in the task presented here, participants are asked to produce difficulty judgments without those judgements actually impacting the future in the task. A feature thus key to difficulty judgments thus seems lacking from the task. Furthermore, the trial-by-trial feedback provided to participants also likely differ from difficulty judgments made in real world. This comment is probably difficult to address but it might generally be useful to discuss the limitations of the task, in particular in probing the desired cognitive process as described in introduction. Currently, no limitations are discussed.

2. The authors take their findings as the general indication that humans rely on accumulation evidence mechanisms to probe the difficulty of perceptual decisions. I would probably have been slightly more cautious in excluding alternative explanations. First, only accumulation models are compared. It is thus simply not possible to reach a different conclusion. Second, even though it is particularly compelling to see untested predictions from the winning model in experiment #1 to be directly tested, and validated in a second experiment, that second experiment presents data from only 3 participants (1 of which has slightly different behaviour than the 2 others), thereby limiting the generality of the findings. Third, the winning model in experiment #1 (difference model) is the preferred model on 12 participants, out of the 20 tested ones. Fourth, the raw BIC values are compared against each other in absolute terms without relying on significance testing of the differences in model frequency within the sample of participants (e.g., using exceedance probabilities; see Stephan et al., 2009 and Rigoux et al., 2014). Based on these different observations, I would thus have interpreted the results of the study with a bit more caution and avoided concluding too widely about the generality of the findings.

3. Deriving and describing the optimal model of the task was particularly appreciated. It was however a bit disappointing not to see how well the optimal model explains participants behaviour and whether it does so better than the other considered models. Also, it would have been helpful to see how close each of the 4 models compared in Figures 2 & 3 get to the optimal solution. Note however that neither of these comments are needed to support the authors' claims.

4. The authors compared the difficulty vs. color judgment conditions to conclude that the accumulation process subtending difficulty judgements is partly distinct from the accumulation process leading to perceptual decisions themselves. To do so, they directly compared reaction times obtained in these two conditions (e.g. "in other cases, the two perceptual decisions are almost certainly completed before the difficulty decision"). However, I find it difficult to directly compare the 'color' and 'difficulty' conditions as the latter entails a single stimulus while the former comprises two stimuli. Any reaction-time difference between conditions could thus I believe only follow from asymmetric perceptual/cognitive load between conditions (at least in the sense RT_color < RT_difficulty). One alternative could have been to present two stimuli in the 'color' condition as well, and asking participants to judge both (or probe which to judge later in the trial). Implementing this now would however require to run a whole new experiment which is likely too demanding. Perhaps the authors could instead also acknowledge that this a critical difference between their conditions, which makes direct comparison difficult.

Reviewer #2 (Public Review):

The authors investigated the role of the Jak1-Stat1 signaling pathway in myogenic differentiation by screening the transcriptional targets of Jak1-Stat1 and identified Styxl2, a pseudophosphatase, as one of them. Styxl2 expression was induced in differentiating muscles. The authors used a zebrafish knockdown model and conditional knockout mouse models to show that Styxl2 is required for de novo sarcomere assembly but is dispensable for the maintenance of existing sarcomeres. Styxl2 interacts with the non-muscle myosin IIs, Myh9 and Myh10, and promotes the replacement of these non-muscle myosin IIs by muscle myosin IIs through inducing autophagic degradation of Myh9 and Myh10. This function is independent of its phosphatase domain.

A previous study using zebrafish found that Styxl2 (previously known as DUSP27) is expressed during embryonic muscle development and is crucial for sarcomere assembly, but its mechanism remains unknown. This paper provides important information on how Styxl2 mediates the replacement of non-muscle myosin with muscle myosin during differentiation. This study may also explain why autophagy deficiency in muscles and the heart causes sarcomere assembly defects in previous mouse models.

Reviewer #2 (Public Review):

The C. elegans embryo has been model system of study for more than 30 years because of the ease of doing forward and reverse genetics, coupled with its nearly invariant lineage which allows a description of development at high resolution. 4D time lapse imaging coupled with spatially resolved gene expression has enabled identification of transcriptional signatures of cells in space and time, and in the past decade this has been advanced with single-cell transcriptomics methods, using individually isolated embryonic cells (which can retain their identity) or by deconvolving complex mixtures of early cells. Recent work using these methods has resolved spatiotemporal expression patterns for many genes, defining cells up to gastrulation stage, but then changing to more tissue-specific patterns during morphogenesis. A key paradigm of specification in C. elegans and other systems is that early maternal factors initiate or restrict patterns of transcription factor expression from the zygotic genome. Combinatorial expression patterns and some symmetries broken by autonomous or extrinsic cell inductions ultimately program lineages towards their fates. To date, only simple networks have been elucidated, as the increasing complexity of these networks and the high level of redundancy has made functional dissection of such pathways difficult. Hence, almost all of the work in recent years has been descriptive.

In this work the authors fill a knowledge gap from the early embryo (~16 cells) to the ~100-cell stage and describe new patterns of gene expression. They reconcile their findings with that of others who have defined expression patterns using other methods, such as scRNA-Seq from complex mixtures of cells, and from transcription factor expression analyses. The resulting description of embryonic develop is the most precise to date, and offers a potentially useful resource for other researchers.<br /> The authors attempt to use their results to find patterns of gene expression that could hint at phylogenetic conservation of specification mechanisms. They find some supporting evidence that expression of homeobox genes occurs in anterior-posterior stripes, which recalls the elaborate A/P patterning system elucidated in the Drosophila embryo, which belongs to the sister phylum Arthropoda in the Ecdysozoan clade of molting animals. It felt as if the authors chose the Hox genes they need to support this conclusion.

Some caveats exist to the work. The expression patterns seem to be well-validated, and following prior work from the Yanai group are likely to be strongly correlated with expression in living embryos. When cells are separated, they could lose some expression patterns that require cell-cell interactions, so it is expected that there might be a small minority of expression patterns that are more complex than what has been documented here.

A major caveat is the idea of the stripes of Hox expression. I just did not find these arguments to be compelling. Seeing these 'stripes' requires organizing the data in a way that maximizes their appearance, for one. Since there is not a lot of movement of cells away from their birth in the early embryo, the AB descendants are anterior to those of MS, anterior to those of E, anterior to those of C, D, and P4. Lineage-specific expression will just naturally fall into 'stripes'. Second, the conservation of Hox expression patterns typically comes with collinearity of the genes along the length of a chromosome (i.e. the so-called Hox clusters) with expression along the body axis, as well as posterior-to-anterior fate transformations when Hox specification is disrupted.

A minor note is the detection of an enrichment of GATA factors in the early E lineage. This has now been found to be a derived condition even within the genus (see Broitman-Maduro et al. Development 149 (21): dev200984, as other species like C. angaria show only a simpler network of elt-3 -> elt-2. This suggests that many of the other patterns of gene expression, particularly in the early embryo, could be highly derived as well; some caution is warranted in generalizing the results as being conserved with arthropods as some of this could be convergent.

Given what the authors are proposing about Hox stripes, some omissions of prior work were surprising (or maybe I missed them). For example, a comprehensive study of Hox genes in C. elegans by Hench et al. (2015) (PLoS One 10(5): e0126947) evaluated all the homeobox genes and examined their genomic locations and expression patterns in the embryo at high spatiotemporal resolution. Work from the Hobert lab (Nature 2020, 584(7822):595-601) showed how homeobox codes specify classes of C. elegans neurons, and the Murray lab (PLoS Genet. 18(5):e1010187) examined Hox control of posterior lineage specification at high resolution, with functional assays.

The Discussion section of the paper is brief, consistent with the descriptive nature of the work overall, but it would have been nice to see the findings related to other published studies as indicated above.

Reviewer #2 (Public Review):

The study by Acosta et al. is very interesting as it presents a simple and easy method for identifying live and dead bacteria DNA in the skin - PMA labeling, verified by FISH. This study provides several meaningful conclusions that could inform future skin microbiome studies:

Firstly, the 16s rRNA gene sequencing of skin microbial samples collected by cotton swabs may include DNA from a large number of dead bacteria, leading to an over-representation of skin bacteria in the analysis.

Secondly, the study found that there were fewer live bacteria on the skin surface than the detected bacterial DNA predicted, with most skin bacteria harboring in the hair follicles. This conclusion aligns with the physiological properties of the skin, as the hair follicle epithelium creates a moist, nutrient-rich, low-UV, and immune-privileged environment, which is conducive to the growth, colonization, and development of microorganisms.

Finally, the authors propose that the bacteria on the skin surface originate from the proliferation and replenishment of hair follicle resident bacteria, which could be one reason for the short-term instability and long-term stability of the skin microbiome.

Overall, this study provides valuable insights into the composition and distribution of skin bacteria and highlights the importance of using appropriate methods to identify live bacteria in skin microbiome studies.

Reviewer #2 (Public Review):

The findings reported by Diaz-Vegas et al. extend those described in a previous paper from the same group establishing a role for mitochondrial CoQ depletion in the development of insulin resistance in muscle and adipose tissue (Fazakerley, 2018). In this new report, investigators sought to determine whether CoQ depletion contributes to insulin resistance caused by palmitate exposure and/or intracellular ceramide accumulation. To this end, researchers employed a widely used in vitro model of insulin resistance wherein L6 myocytes develop impaired Glut4 translocation upon exposure to palmitate (in this case, 150 uM for 16 hours). This model was combined with a variety of pharmacologic and genetic manipulations aimed at augmenting or inhibiting CoQ biosynthesis and/or ceramide biosynthesis, specifically in mitochondria. This series of experiments produced a valuable and provocative body of evidence positioning CoQ depletion downstream of mitochondrial ceramide accumulation and necessary for both palmitate- and ceramide-induced insulin resistance in L6 myocytes. Investigators concluded that mitochondrial ceramides, CoQ depletion and respiratory dysfunction are part of a core pathway leading to insulin resistance.

Strengths:

The study provides exciting, first-time evidence linking palmitate-induced insulin resistance to ceramide accumulation within the mitochondria and subsequent depletion of CoQ. Ceramide accumulation specifically in mitochondria was found to be necessary and sufficient to cause insulin resistance in cultured L6 myocytes.

The in vitro experiments featured a set of mitochondrial-targeted genetic manipulations that permitted up/down-regulation of ceramide levels specifically in the mitochondrial compartment. Genetically induced mitochondrial ceramide accumulation led to CoQ depletion, which was accompanied by increased ROS production and diminution of ETC proteins and OXPHOS capacity and impaired insulin action, thereby establishing cause/effect.

Analysis of mitochondria isolated from human muscle biopsies obtained from individuals with disparate metabolic phenotypes revealed a positive correlation between complex I proteins and insulin sensitivity and a negative correlation with mitochondrial ceramide content. While it is likely that many factors contribute to these correlations, the results support the possibility that the ceramide/CoQ mechanism might be relevant to glucose control in humans.

These important findings offer valuable new insights into mechanisms that connect ceramides to insulin resistance and mitochondrial dysfunction, and could inform new therapeutic approaches towards improved glucose control.

Weaknesses:

The mechanistic aspect of the work and conclusions put forth rely heavily on studies performed in cultured myocytes, which are highly glycolytic and generally viewed as a poor model for studying muscle metabolism and insulin action. Nonetheless, the findings provide a strong rationale for moving this line of investigation into mouse gain/loss of function models.

One caveat of the approach taken is that exposure of cells to palmitate alone is not reflective of in vivo physiology. It would be interesting to know if similar effects on CoQ are observed when cells are exposed to a more physiological mixture of fatty acids that includes a high ratio of palmitate, but better mimics in vivo nutrition.

While the utility of targeting SMPD5 to the mitochondria is appreciated, the results in Figure 5 suggest that this manoeuvre caused a rather severe form of mitochondrial dysfunction. This could be more representative of toxicity rather than pathophysiology. It would be helpful to know if these same effects are observed with other manipulations that lower CoQ to a similar degree. If not, the discrepancies should be discussed.

The conclusions could be strengthened by more extensive studies in mice to assess the interplay between mitochondrial ceramides, CoQ depletion and ETC/mitochondrial dysfunction in the context of a standard diet versus HF diet-induced insulin resistance. Does P053 affect mitochondrial ceramide, ETC protein abundance, mitochondrial function, and muscle insulin sensitivity in the predicted directions?

Reviewer #2 (Public Review):

This report describes a large-scale analysis of cell counts in mouse brains. The authors found that the Allen Mouse Connectivity project has a rich dataset for cell counting that is yet to be analyzed, and they developed methods to quantify cells in different nuclei. They go on to compare males vs females and two different strains. From this analysis, they found specific differences between male versus female brains, left versus right hemispheres, and C57BL/6 versus FVB.CD1 mice, especially with regard to cell counts and density.

Overall, the methodology is sound and the quality of the data seems high. In fact, this study uses >100 brains for the statistics, and this is one of the major strengths of this study. For researchers who are interested in interrogating the differences at the macroscopic level in brain structures, this study will be a great resource. For example, the manuscript contains an interesting finding that for most brain areas, females have larger volumes but fewer cell numbers.

Reviewer #2 (Public Review):

In this manuscript, the authors use generative adversarial networks (GANs) to manipulate neural data recorded from intracortical arrays in the context of intracortical BCIs so that these decoders are robust. Specifically, the authors deal with the hard problem where signals from an intracortical array change over time and decoders that are trained on day 0 do not work on day K. Either the decoder or the neural data needs to be updated to achieve the same performance as initially. GANs try to alter the neural data from day K to make it indistinguishable to day 0 and thus in principle the decoder should perform better. The authors compare their GAN approach to an older GAN approach (by an overlapping group of authors) and suggest that this new GAN approach is somewhat better.

Major Strengths are multiple datasets from behaving monkeys performing various tasks that involve motor function. Comparison between two different GAN approaches and a classical approach that uses factor analysis. The weakness is insufficient comparison to another state-of-the-art approach that has been applied on the same dataset (NoMAD, Karpowicz et al. BioRxiv 2022).

The results are very reasonable and they show their approach, Cycle GANs, does slightly better than the traditional GAN approach. However, the Cycle GANs have many more modules and also as I understand it performs a forward backward mapping of the day - 0 and day - k and thus theoretically better. But, it seems quite slow.

I think the results are interesting but as such, I am not sure this is such a fundamental advance compared to the Farashcian et al. paper, which introduced GANs to improve decoding in the face of changing neural data. There are other approaches that also use GANs and I think they all need to be compared against each other. Finally, these are all offline results and what happens online is anyone's real guess. Of course, this is not just a weakness of this study but many such studies of its ilk.

Reviewer #2 (Public Review):

In this interesting study, Wang et al. demonstrated a critical role of the key focal adhesion protein vinculin in the control of bone mass in mice. Specifically, the authors deleted vinculin expression by using the mouse 10-kb Dmp1-Cre transgenic mice that were reported to primarily target osteocytes and mature osteoblasts. The authors found that vinculin loss in these cells caused severe osteopenia in mice due to impairment of osteoblast and bone formation with minimal impact on osteoclast formation and bone resorption. Interestingly, the vinculin loss also reduced the mechanical loading induction of bone formation in mice. Mechanically, the authors found that vinculin knockdown increased, while vinculin overexpression decreased, sclerostin expression in osteocytes without affecting that of Mef2c, a major transcriptional regulator of the Sost gene, which encodes sclerostin. Mechanistically, the authors found that vinculin protein bound to Mef2c and vinculin loss increased Mef2c nuclear translocation and binding to the Sost enhancer ECR5. Deleting Sost expression largely reversed the osteopenic phenotypes caused by vinculin deletion. Finally, the authors demonstrated that estrogen promoted vinculin expression in osteocytes and that vinculin loss abolished the estrogen deficiency induction of bone loss in mice. In this study, with a tremendous amount of convincing in vitro and in vivo data, the authors have established a critical role of vinculin in bone and defined a novel mechanism that regulates bone mass. The findings from this study are important and interesting.

Reviewer #2 (Public Review):

This study used direct recording from the soma, the terminal and the postsynaptic cell in cerebellar inter-neuron- Purkinje cell synapses. The authors nicely showed that action potentials travel reliably from the soma to the axon. In addition, they showed that the postsynaptic responses elicited at the dendrites reliably traveled along the axon. Such sub-threshold potential could potentiate transmitter release in short-term (for tens of ms at most), by "priming" Ca channels and accelerating activation kinetics of Ca channels. Results are based on the technically demanding electrophysiological technique and are in general. The study directly solves the mechanism of short-term facilitation induced by sub-threshold depolarization.

Reviewer #2 (Public Review):

In this study, the authors aim to uncover the neuroanatomical and metabolite underpinnings of an intriguing phenomenon observed in some insects due to the infection of fungal pathogens. They very cleverly develop a high-throughput assay to examine and quantify this behaviour in a tractable model organism - Drosophila melanogaster which the authors have previously shown to also exhibit this phenomenon. They characterize the details of this behaviour and clearly show the temporal gating of this summiting-followed-by-death behavior to occur shortly before the dusk transition. They go on to examine using a candidate (over 200) screen approach potential neuronal circuits and genes based on the hypothesis that they may be related to 'arousal and gravitaxis'. They narrow down to a line that is restricted to the PI based on the fact that it has a significant effect on the summiting behaviour and that it is known to affect locomotion. They can demonstrate that flies when a subset of PI neurons (R19G10) are transiently activated, they will show summiting even without exposure to the pathogen. Based on Syt-eGFP staining they conclude that PI communicates with the carpora cardiaca (CA). They also show that CA itself is necessary for this behavior, but cannot demonstrate the role of Juvenile hormones using their pharmacological methods.

The authors then describe an automated classifier to identify an upcoming summiting behaviour. Further, they use this real-time classifier to stage different steps of the summiting and match it to the extent of pathology observed by microscopy. They also ask whether the constituents of the hemolymph differ between the summiting and not-yet summiting flies for which they conduct metabolome analysis of the hemolymphs. They are also able to show that cross-injection of uninfected or infected but not summiting flies can be induced to show summiting-like behaviour upon injection with the hemolymph.<br /> Finally, they propose the sequence by which the fungal pathogen may modulate the behaviours of the host fly so as to execute this highly gated act of increased locomotion prior to death.

Strengths<br /> • The detailed characterization of the behaviour in D melanogaster and development of the high-throughput behavioural arena.<br /> • Development of the automated classifier which appears to accurately predict this behaviour.<br /> • Narrowing down to a small group of PI neurons having a strong impact on this behaviour although sufficiency is not clearly demonstrated.

Weaknesses<br /> • The evidence of temporal (circadian) gating is weak despite the proposed DN1p - PI - CA connections.<br /> • The eventual modification of the behavior to enable enhanced locomotion and negative geotaxis to occur appears to be mediated by yet unknown factors<br /> • The metabolite analysis did not help to narrow down to candidates that can be speculated to cause this behaviour.

Reviewer #2 (Public Review):

In this manuscript, Gochman et al. studied the molecular mechanism by which cannabidiol (CBD) sensitizes the TRPV2 channel to activation by 2-APB. While CBD itself can activate TRPV2 with low efficacy, it can sensitize TRPV2 current activated by 2-APB by two orders of magnitude. The authors showed, via single-channel recording, that the CBD-dependent sensitization arises from an increase in Po when the channel binds to both CBD and 2-APB. The authors then used cryo-EM to investigate how CBD binds to TRPV2 and identified two CBD binding sites in each subunit, with one site being previously reported and the other being newly discovered.

TRPV1 and TRPV2 are two channels closely related to TRPV2. All three channels can be activated by CBD and 2-APB, but only TRPV2 and 3 are strongly sensitized by CBD. To understand the molecular basis of the different sensitivity to CBD, the authors compared the residues within the CBD binding sites and generated mutants by swapping non-conserved residues between TRPV1 and TRPV2. They then performed patch-clamp recordings on these mutants and found that mutations on non-conserved residues indeed influenced the CBD-dependent sensitization, thereby supporting the observed CBD binding sites.

Unexpectedly, the authors did not identify the binding site of 2-APB, despite its robust effect in electrophysiology recordings, especially when combined with CBD. Although previous structural studies of TRPV2 have reported 2-APB binding sites, the associated densities in these studies were not well-resolved. Therefore, the authors called on the field to re-examine published structural data with regard to the 2-APB binding sites.

Overall, this is an important study with well-designed and well-conducted experiments.

Reviewer #2 (Public Review):

The manuscript is well written, the data are based on well-performed experiments, and the conclusions are supported by the data. The authors study thoroughly the global phenotype of T and NK cells and also analyze antigen-specific T cell frequencies. The data confirm that individuals who had severe COVID-19 disease (required ventilation and/or ITU admission) have slightly more activated CD4 and CD8 T cells at 3 months post-infection and report more frequently long COVID symptoms, yet the novelty of this manuscript is to show that these two are not linked to each other. Moreover, the manuscript confirms that patients across all disease severities mount and maintain memory T cell and antibody responses to SARS-CoV-2.

In the introduction, the authors want to highlight the extent of patients who suffer from long COVID symptoms, yet it should be noted that these high frequencies (8-21%) are coming from unvaccinated and hospitalized patients (like those included in this study), while a large group of individuals experience asymptomatic SARS-CoV-2 infection, and these individuals are not integrated into these studies.

The authors find that patients who recovered from severe COVID-19 3 months ago have more activated CD4+ and CD8+ T cells than patients who recovered from the mild disease. Although the difference is significant, the frequency of CD4+ T cells with an activated phenotype is increased only by about 2-fold (~2% vs ~1%), while the frequency of activated CD8+ T cells is about 6% vs 4%, which should be added to the results to better describe the extent of the activation.

As the authors mention in the discussion, it cannot be excluded that the more activated T cell phenotype in patients who recovered from severe COVID-19 is not rather a consequence of the increased comorbidities associated with this group. However, their Luminex analysis of the serum shows that the levels of cytokines TNF-a, IL-4, IL-12, IL-15, and IL-17A decline by 8 and 12 months, suggesting that the immune activation by 3 months is most likely a consequence of the previous severe viral infection.<br /> To strengthen this point, PBMC is probably not available at a later time point, to see if the increased T cell activation decreases in line with the serum cytokines. Yet, the authors should at least try to repeat the experiments of coculturing CD3+ T cells from healthy volunteers with the serum of mild/severe patients at 8-12 months post-recovery (Fig. 3 D-E).

The authors tried to find if the activated T cell phenotype or increased serum cytokines at 3 months post-infection is linked with increased long COVID symptoms. The study does not find any direct association when the data are adjusted for age, sex, and severity. This is the only novelty of this study, yet it is an important piece of information in the attempt to broaden our understanding of the underlying causes of long COVID symptoms.

Overall, it would be important to understand if increased frequencies of T cell activation (~2-fold) and increased levels of serum cytokines at 3 months following severe COVID-19 that resulted in ventilation and/or ITU admission is specific to severe SARS-CoV-2 infection, or if similar consequences are resulting also from other severe acute viral infections. Addressing this question is beyond the scope of the manuscript, yet it should be discussed.

Reviewer #2 (Public Review):

This report uses massively parallel reporter assays to examine the impact on gene expression of >2000 uORFs found in yeast mRNAs with 5'UTR lengths <181nt, by comparing expression of two YFP reporters for each uORF, one containing the WT 5'UTR and the other with the uORF AUG codon mutated to a near-cognate AAG triplet. All of the mRNAs were expressed from the same promoter from the ENO2 gene, which is expected to produce the predicted 5' ends for all of the mRNAs being sampled. The results indicated that most AUG uORFs are repressive, while most nonAUG (near-cognate) uORFs have little effect on reporter expression; and a small fraction of AUG uORFs are stimulatory to YFP expression. They corroborated these results by sequencing the reporter library mRNAs in polysome vs monosome fractions and showing a good correlation (R=0.78) between the effects of the uORF AUG mutations on YFP expression versus fraction of the mRNA in polysomes. The reporter library was assayed in in both WT and upf1 mutants to evaluate the impact of NMD on uORF regulation of reporter expression and polysome association, which allowed them to determine that, on average, NMD accounts for ~35% of the uORF-mediated repression of reporter expression, ie. the magnitude of the repression is blunted in the upf1 mutant. Consistent with this, the reductions in YFP expression are frequently associated with reductions in reporter mRNA levels, measured by RNA-seq. Moreover, the repressive effects of the uORFs calculated from YFP expression versus polysome association of reporter mRNAs are more congruent in the upf1 mutant where NMD effects are absent versus the WT. Their bioinformatic analyses provide some evidence that NMD control is lessened by inefficient termination at uORFs with UGAC stop codons, for long vs. short uORFs, and by decreasing the distance of the uORF stop codon from the mRNA cap. Their large dataset allowed them to conduct machine learning to identify features of uORFs that are associated with their effects on YFP expression, finding that repression by the uORF is associated about equally with a good Kozak context for the start codon, a shorter distance of the uORF from the cap, and shorter distance of the uORF stop codon to the downstream CDS, with a somewhat weaker association with a longer uORF CDS. These findings for Kozak context were predictable from prior work, as were the associations with uORF length and distance to the YFP AUG in the context of known effects of these parameters on reinitiation. However, the association with distance of the uORF from the cap is more novel. They provide some additional support for the latter by analyzing the influence of different TSSs/5'UTR lengths on uORF repressive function for a subset of 333 uORFs, finding that the repressive effect can vary depending on the TSS, with several instances in which the uORF was less inhibitory when the TSS is located further upstream from the uORF AUG. Finally, they provide some evidence that uORFs conserved between closely related yeast species are generally less repressive and have poorer AUG contexts, leading to the conclusion that they are under purifying selection to make them less inhibitory.

This study is valuable in providing an unprecedented, comprehensive analysis of the regulatory effects of naturally occurring AUG and near-cognate uORFs on gene expression in a manner that distinguishes between repression of translation versus repression of mRNA stability via NMD. Owing to the large number of uORFs analyzed in a system that eliminates variations in transcription rate, it was possible to identify certain statistically significant associations between uORF features and the extent to which they repress translation or evoke NMD.

There are several areas in which the authors' claims or conclusions are not fully justified and require either additional statistical analysis or new experimentation to support the claims. In particular, additional experiments are needed to confirm that the reporter mRNAs initiate at the predicted TSS; to bolster the novel conclusion that moving a uORF farther from the cap reduces its inhibitory effect on translation initiation downstream, independently of the inclusion of other uORFs in the intervening interval; and to test their interpretations concerning the differences in uORF function between S. cerevisiae and S. paradoxus for particular mRNAs.

Reviewer #2 (Public Review):

Despite the fact that CTLA-4 is a critical molecule for inhibiting the immune response, surprisingly individuals with heterozygous CTLA-4 mutations exhibit immunodeficiency, presenting with antibody deficiency secondary to B cell loss. Why the loss of a molecule that regulates T cell activation should lead to B cell loss has remained unclear. In this study, Muthana and colleagues use an anti-CTLA-4 antibody drug conjugate (aCTLA-4 ADC) to delete cells expressing high levels of CTLA-4, and show that this leads to a reduction in B cells. The aCTLA-4 ADC is found to delete a subset of Tregs, leading to hyperactivation of T cells that is associated with B cell depletion. Using blocking antibodies, the authors implicate TNFa in the observed B cell loss.

The reciprocal regulation of T and B cell homeostasis is an important research area. While it has been shown that Treg defects are associated with B cell loss, the mechanisms at play are incompletely understood. CTLA-4 is not normally expressed in B cells so an indirect mechanism of action is assumed. The authors show that the decrease in Treg following aCTLA-4 ADC treatment is associated with activation of T cells, and that B cell loss is blunted if T cells are depleted. A role for both CD4 and CD8 T cells is identified by selective CD4/CD8 depletion. T cells appear to require CD28 costimulation in order to mediate B cell loss, since the response is partially inhibited in the presence of the costimulation blockade drug belatacept (CTLA-4-Ig). Finally, experiments using the anti-TNFa antibody adalimumab suggest a potential role for TNFa in the depletion of B cells.

While the manuscript makes a useful contribution, a number of questions remain. Perhaps most important is the extent to which this model mimics the natural situation in individuals with CTLA-4 mutations (or following CTLA-4-based clinical interventions). aCTLA-4 ADC treatment permits acute deletion of Treg expressing high levels of CTLA-4, whereas in patients the Treg population remains but is specifically impaired in CTLA-4 function. Secondly, although the requirement for T cells to mediate B cell loss is convincingly demonstrated, the incomplete reversal by TNFa blockade suggests additional unidentified factors contribute to this effect. Finally, although the manuscript favours peripheral killing of mature B cells over alterations to B cell lymphopoiesis, one concern is that this may simply reflect the model employed: the short-term (6 day) treatment used here may be too acute to alter B cell development, but this may nevertheless be a feature of prolonged immune dysregulation in humans.

Reviewer #2 (Public Review):

The manuscript by Kaneko set out to understand the mechanisms underlying cell proliferation in hepatocytes lacking Shp2 signals. To do this, the authors focused on CD133 as the proliferating clusters of cells in the Shp2 knockout (SKO) livers are CD133 expressing. After excluding the contribution of progenitors that are CD133 to this cell population, the authors focused on the intrinsic regulation of CD133 by Met/Shp2 regulated Ras/Erk parthway and showed upregulation of CD133 to be a compensatory signal to overcome loss of Ras/Erk signal and suggested Wnt10a in the regulation of CD133 signal. The study then focused on the observed filament localization of CD133 in the CD133+ cluster of cells. The study went on to identify the CD133+ vesicles that contain primarily mRNA vs. microRNA like other EVs. Specifically, the authors identified several mRNA species that encode IEGs, indicating a potential role for these CD133+ vesicles in cell proliferation signal transmission to neighboring cells via delivery of the IEG mRNAs as cargos. Finally, they showed that the induction of CD133 (and by derivative, the CD133+ vesicles) are necessary for maintaining cell proliferation in the cell cluster with high proliferation capacities in the SKO livers; and in intestinal crypt organoids treated with Met inhibitors to block Ras/ERk signal.

1) The identification of CD133+ vesicles is largely based on staining and costainings. Though the experiments are very well done with many controls and approaches, the authors may want to perform one or two key experiments with EM to definitively demonstrate the colocalization. For example, the mCherry experiment in Fig6H and the colocalization experiments for CD133 and HuR in Fig 7.

2) Since CD133+ marks the 50nM intracellsome defined by the authors, it is unclear what the CD133- vesicles used as controls are. Are they regular EVs that are larger in size? This needs better clarification as they are used as a control for many experiments such as Fig 7A.

Reviewer #2 (Public Review):

I agree that minor genetic variation could potentially be used to more accurately infer who-infected- whom in an outbreak scenario. Indeed, the use of minor genetic variation has proven very useful in reconstructing transmission chains for chronic infections such as HIV (e.g., see applications using Phyloscanner). To me, it seems that considering the full spectrum of viral genetic diversity within infected hosts would necessarily do the same if not better than considering only consensus-level viral sequence data. This is because there is a necessarily a loss of data and potentially a loss of information when going from considering the genetic composition of viral populations within a host to only considering the consensus sequences of those viral populations. As such, Ortiz et al.'s hypothesis stated on lines 66-70 is a reasonable one, and I was looking forward to seeing this hypothesis evaluated in detail in this manuscript.<br /> There are several parts of this manuscript I really like. In particular, encoding within-sample diversity as character states and using that alternative representation of sequence data for phylogenetic inference (as shown in Figure 3) is a very interesting idea, I think. There are some limitations that are not explicitly mentioned, however. For example, when using this 16-character state representation for phylogenetic inference, they assume independence between nucleotide sites. This is a major assumption that can be violated when considering longitudinal intrahost data and transmission dynamics in an outbreak setting, given genetic linkage between sites.

I have several major concerns about the work as it stands, particularly in the context of the SARS-CoV-2 application.

Concerns not related to the SARS-CoV-2 application:<br /> Concern #1: Figure 4 shows that a model using within-sample diversity can more accurately reconstruct evolutionary histories than a model that uses only consensus-level genetic data. This is really interesting. The Materials and Methods section (particularly lines 351-354) indicates that the sequence data were generated using certain specified substitution rates. The rates specified seem to be chosen in such a way to facilitate finding an improvement when using within-sample diversity. I don't know whether the relative rates of these 'substitutions' at all mirror "real-life". It would be very useful to have a broader set of analyses here to examine the effect of these 'substitution' rates on the utility of incorporating within-sample diversity into phylogenetic inference. (Also, 1, 100, 200 (line 353) inconsistent with 1, 20, 200 in Supp Table 3)

Concern #2: Figure 5 is very interesting, particularly the results at bottleneck sizes of 1-10. What are the 'substitution' rates that are inferred here from using this simulated dataset? The Material and Methods section also does not mention the within-host viral generation time anywhere, as far as I can see (~line 384 states the mutation rate per base per generation cycle but not the length of the generation cycle anywhere).

Concerns related to the SARS-CoV-2 application:<br /> Concern #3: I am very concerned about the testing of this hypothesis on the SARS-CoV-2 data presented. First, 1% is a very low variant calling threshold. Second, analysis of the 17 samples that were resequenced (out of 454) indicated that on average, 39% of iSNVS (intrahost single nucleotide variants) called between duplicate runs were only observed in one of the two runs (line 117). Their analysis in Figure 1 indicates that these discrepant (and seemingly spurious) variants occur at higher levels in high Ct samples (which makes sense; Figure 1b). They therefore decide to limit their analyses to samples with Ct values <= 30. This results in 249 samples. However, if we look at Figure 1b, only ~10% of iSNVs called across duplicate runs with Ct = 30 are shared! That means that 90% of iSNVs in the set appear to be spurious. If we assume that each duplicate run of a sample has approximately the same number of spurious iSNVs, then approximately 82% of iSNVs called in a sample with a Ct of 30 would be spurious. This fraction decreases with samples that have lower Ct values, but even at a Ct of 27, only ~60% of iSNVs called across duplicate runs are shared. All the downstream SARS-CoV-2 analyses based on within-host sample diversity therefore are based on samples where the large majority of considered sample diversity is not real. This leads to me necessarily discounting all of those downstream SARS-CoV-2 results.

Concern #4: Lines 153-167: I can't figure out how to square the quantitative results given in this paragraph with what is shown in Figure 2. To me, Figure 2 shows only that Technical Replicates have higher probabilities of sharing a variant than with 'No' relationship. What would also be helpful here so that the reader can get a better feel for the data would be to see the iSNV frequencies plotted over time for the longitudinal replicate samples in the supplement and, for the 'epidemiological' samples to show 'TV plots' in the supplement (as in Fig 3c in McCrone et al. eLife)

Concern #5: Figure 6 and associated text: (a) root-to-tip distance: what units is this distance in? (b) That the authors find a temporal signal in these transmission clusters (where all consensus sequences within a cluster are the same) is interesting but also a bit baffling to me. Given the inference of very small transmission bottlenecks in previous studies (e.g., Martin & Koelle - reanalysis of Popa et al.; Lythgoe et al.; Braun et al.), I don't understand where the temporal signal comes in. Do the samples become more genetically diverse over the outbreak (this seems to be indicated in lines 260-262 but never shown and unlikely given bottleneck sizes)? Additional analyses to help the reader understand WHY within-sample diversity allows for the identification of temporal signal is important. This could involve plotting genetic diversity of the samples by collection date or some other, similar analyses.

Concern #6: Paragraph consisting of lines 229-238 and Figure 7: This analysis stops abruptly. What are the conclusions here? Figure 7a (right) seems inconsistent to me with Figure 7b and 7C results. Also, the main hypothesis put forward in this paper is that within-sample sequence data can better resolve who-infected-whom in an outbreak setting. Figure 7b and 7c however are never compared against analogous panels that use just consensus sequences. (Even though the consensus sequences are the same, according to Figure 7a, the inferences shown in Figures 7b and 7c could use additional data such as collection times, etc. that would provide information even when using exclusively consensus-level data). Also, do the analyses in Figures 7b and 7c use the 16-character state model at all? I think Supp Figure 9 is relevant here but not sure how?)

Additional concerns:<br /> Concern #7: Some of the stated conclusions, particularly in the Discussion section and in the Abstract, do not seem to be supported by the presented results. For example, line 27: 'within-sample diversity is stable among repeated serial samples from the same host': Figure 2 does not show this conclusively. Line 28: 'within-sample diversity... is transmitted between those cases with known epidemiological links': Figure 2 also does not show this conclusively. Line 29: 'within-sample diversity... improves phylogenetic inference and our understanding of who infected whom': Figure 7b/c results using within-sample diversity is never compared against results that use only consensus, so improvement not demonstrated. Line 272-273: 'samples with shorter distance in the consensus phylogeny were more likely to share low frequency variants'. Line 287: 'We demonstrated that phylogenies... were heavily biased'.

Concern #8: The manuscript at times does not cite previous work that is highly relevant and thus overstates the novelty of the current work. For example: lines 21-23: '..conventional whole-genome sequencing phylogenetic approaches to reconstruct outbreaks exclusively use consensus sequences...' Phyloscanner uses within-sample diversity, for example, as does SCOTTI. These are finally cited in the discussion section (~line 310), but because this previous work is not acknowledged earlier in the manuscript, the novelty of the work presented here is somewhat overstated.

In sum, I think that the 16 character-state model is a very interesting model. More analyses on simulated data would be helpful to expand on when below-the-consensus level genetic data would truly be informative of phylogenetic relationships and who-infected-whom in outbreak settings. The SARS-CoV-2 analyses are very worrisome to me, given the inclusion of samples where the majority of considered within-sample genetic diversity is very likely not real. Some of the stated conclusions appear to either be at odds with the results presented or not directly evaluated.

Reviewer #2 (Public Review):

Targeted genetic engineering with programmable nucleases and other targetable enzymes (aka "genome editing") has emerged as a technology with curative potential in hemoglobinopathies, sickle cell disease, and beta-thalassemia. Multiple ongoing clinical trials are evaluating such editing using distinct approaches: elevation of fetal hemoglobin (HbF), direct repair of the mutation causing SCD, and engineering of a Hb variant. The present work explores a different strategy: the targeted engineering of the promoter of a paralog of adult beta-globin known as HBD. This is a timely effort because there has emerged, over the past decade, a clear and charted path for advancing any such approach to human clinical trials. The study identifies three transcription factor binding sites as divergent in the HBD promoter vs the HBB one. A homology-directed repair (HDR)-based scheme using oligonucleotide repair templates in combination with a CRISPR-Cas9-induced double-strand break (DSB) is designed and used to generate pools of human immortalized cells bearing one, two, or all three such de novo introduced TF binding sites at the HBD promoter. Only the latter scheme is shown to measurably increase HBD (following erythroid differentiation) in pools of cells and single-cell-derived clones as gauged by qPCR and HPLC. A similar analysis is performed on pools of erythroid-like cells generated from genome-edited human hematopoietic stem and progenitor cells (HSPCs), as well as genetically clonal erythroid colonies bearing the edits of interest; trends in these data support the observations made on the immortalized cells. Overall the data support the notion that HBD promoter genome editing has the potential as a strategy to normalize hemoglobin synthesis in hemoglobinopathies. Further, the data support an advance of this approach down a well-established path of preclinical development in such cases: increasing the efficiency of genome editing in HSPCs to what would be deemed therapeutically useful, assessing the genotoxic burden from the editing, evaluating the potential negative impact on stemness, and determining whether this approach would normalize hemoglobin synthesis in the erythroid progeny of patient HSPCs.

The genome editing scheme for the "KDT" strategy in Fig 1B involves the introduction of three binding sites for transcription factors at progressively increasing distances from the site of the DSB induced by Cas9. It would be of interest to determine from the next-generation-sequencing data whether partial gene conversion tracks are observed at the edited locus (Elliott and Jasin MCB 18: 93), and if yes, whether these affect in some way the pool-level measurement by qPCR on HBD mRNA levels (Fig 1D).

The data in Fig 2A show an analysis of transcription factor and RNA pol II occupancy following genome editing at HBD. The figure legend refers to these data as having been obtained on single-cell-derived clones bearing the edits in homozygous or heterozygous form, but it is unclear from fig 2A, which clones were used for which analysis.

The data in Fig 3C present an analysis of HBD levels in erythroid colonies derived from genome-edited HSPCs. It would be helpful to clarify whether an individual dot represents a single such colony (this would seem to be the case from the cognate figure legend). If so, what number of such colonies would one need to obtain to gain a clearer sense of the effect on HBD levels from the various genome editing strategies used?

It would be helpful to comment, in the Discussion, on potential genome editing strategies to obtain high-efficiency pool-level uniform long-track gene conversion that is necessary to obtain high HBD levels in the progeny of edited CD34 cells. Would this be a good application of the AAV6 strategy developed by the Sangamo and Porteus groups? Would prime editing as developed by Liu be an option here?

It would be equally helpful, in the Discussion, to place the level of HbA2 obtained via the strategy shown in the manuscript in the context of other genome-editing-based approaches for normalizing Hb synthesis in the hemoglobinopathies (ie HbF elevation by editing the BCL11A enhancer, or the gamma-globin promoter; or direct repair of the SCD mutation; or engineering of Hb Makassar).

Reviewer #2 (Public Review):

This paper set out to understand the impact of early life stress on the behavior and individuality of animals, and how that impact might be amplified or masked by neuromodulation. To do so, the authors built on a previously established assay (Stern et al 2017) to measure the roaming fraction and speed of individuals. This technique allowed the authors to assess the effects of early life starvation on behavior across the entire developmental trajectory of the individual. By combining this with strains with mutant neuromodulatory systems, this enabled the authors to produce a rich dataset ripe for analysis to analyze the complicated interactions between behavior, starvation intensity, developmental time, individuality, and neuromodulatory systems.

The richness of this dataset - 2 behavioral measures continuous across 5 developmental stages, 3 different neuromodulatory conditions (with the dopamine system subject to decomposition by receptor types) and 4 different levels of starvation, with ~50-500 individuals in each condition-underlies the strength of this paper. This dataset enabled the authors to convincingly demonstrate that starvation triggers a behavioral effect in L1 and adult animals that is largely masked in intermediate stages, and that this effect becomes larger with increased severity of starvation. Furthermore, they convincingly show that the masking of the effect of starvation in L2-L4 animals depends on dopaminergic systems. The richness of the dataset also allowed a careful analysis of individuality, though only neuromodulatory mutants convincingly manipulated individuality, recapitulating earlier research. Nonetheless, a few caveats exist on some of their findings and conclusions:

1. Lack of quantitative analysis for effects within developmental stages. In making the argument for buffered effects of starvation on behavior during periods of larval development, the authors make claims regarding the temporal structure of behavior within specific stages. However, no formal analysis is performed and and the traces are provided without confidence intervals, making it difficult to judge the significance of potential deviations between starvation conditions.

2. Incorrect inferences from differences in significance demonstrating significant differences. The authors claim that there is an increase in PC1 inter-individual variation in tph-1 individuals, however the difference in significance is not evidence of a significant difference between conditions (see Nieuwenhuis et al. 2011). This undermines claims about an interaction of starvation, neuromodulators, and individuality.

3. Sensitivity of analysis to baseline effects and assumptions of additive/proportional effects. The neuromodulatory and stress conditions in this paper have a mixture of effects on baseline activity and differences from baseline. The authors normalize to the roaming fraction without starvation, making the reasonable assumption that the effect due to starvation is proportional to baseline, rather than an additive effect. This confound is most visible in the adult subpanel of figure 5d, where an ~2-3 fold difference in relative roaming due to starvation is clearly noted, however, this is from a baseline roaming fraction in tph-1 animals that are ~2 fold higher, suggesting that the effect could plausibly be comparable in absolute terms.

Unavoidably, any such assumptions on the expected interaction between multiple effects will be a gross simplification in complicated nonlinear systems, and the data are largely shown with sufficient clarity to allow the reader to make their own conclusions. However, some of the interpretations in the paper lean heavily on an assumption that the data support a direct interpretation (e.g. "neuronal mechanisms actively buffer behavioral alterations at specific development times") rather than an indirect interpretation (e.g. that serotonin reduces baseline roaming fraction which makes a fixed sized effect more noticeable). Parsing the differences requires either more detailed mechanistic study or careful characterization of the effect of different baselines on the sensitivity of behavior to perturbation-barring that it's worth noting that many of these interactions may be due to differences in biological and experimental sensitivity to change under different conditions, rather than a direct interaction of stress and neuromodulatory processes or evidence of differing neuromodulatory activity at different stages of development.

Reviewer #2 (Public Review):

Using an approach that combines synthetic genetic array (SGA) analysis with high-throughput microscopic analysis of the GFP-tagged yeast ORF collection in the budding yeast, Saccharomyces cerevisiae, this study has examined the contribution of the critical checkpoint kinases Mec1 and Rad53 to the subcellular relocalization of 322 candidate proteins in response to HU- and MMS-induced replication stress. Previous studies have established that Mec1 is required for Rad53 activation during replication stress and that Mec1 also serves checkpoint functions independent of Rad53. Unexpectedly, this study identifies groups of proteins whose stress-induced relocalization is dependent on Rad53 but not Mec1. This data indicates that Rad53 mediates some replication stress responses in a non-canonical manner that is independent of Mec1.

The authors confirm their initial observations from the screening approach by focusing on the Rad53-dependent and Mec1-independent focus formation of GFP-Rad54. Moreover, using mass-spec analysis the authors demonstrate that some Rad53 phosphorylation sites known to be critical for Rad53 activation, including a consensus Mec1 phosphorylation site, are phosphorylated after replication stress even in the absence of Mec1. Motivated by this finding the authors screen for potential kinase and phosphatase pathways that may regulate Rad53 function during MMS-induced replication stress. Top hits identified include members of the retrograde signaling pathway, which is confirmed by conventional genetic assays while mass spec analysis supports the involvement of Rtg3 in mediating Rad53 phosphorylation during replication stress in the absence of Mec1.

Overall this is a solid study reporting unexpected new findings that significantly advance our view of the global replication checkpoint response. The data are generally of high quality, well presented and quantified, and overall support the authors' claims. The mass spec approach used here to identify Rad53 phosphorylation sites offers an unbiased alternative to the simpler and more widely employed gel-shift method to monitor Rad53 activation. The hits identified in the various screens presented here provide a platform for potential follow-up studies by the community. The main drawback is that it remains unclear how Rtg3 promotes Rad53 activtation. However, this could be considered to be beyond the scope of this study.

Reviewer #2 (Public Review):

The relationship between measures of brain state, behavioral state, and performance has long been speculated to be relatively simple - with arousal and engagement reflecting EEG desynchronization and improved performance associated with increases in engagement and attention. The present study demonstrates that the outcome of the previous trial, specifically a miss, allows these associations to be seen - while a correct response appears less likely to do so. This is an interesting advance in our understanding of the relationship between brain state, behavioral state, and performance.

While the study is well done, the results are likely to be specific to their trial structure and states exhibited by the mice. To examine the full range of arousal states, it needs to be demonstrated that animals are varying between near-sleep (e.g. drowsiness) and high-alertness such as in rapid running. The fact that the trials occurred rapidly means that the physiological and neural variables associated with each trial will overlap with upcoming trials - it takes a mouse more than a few seconds to relax from a previous miss or hit, for example. Spreading the rapidity of the trials out would allow for a broader range of states to be examined, and perhaps less cross-talk between adjacent trials. The interpretation of the results, therefore, must be taken in light of the trial structure and the states exhibited by the mice.

Reviewer #2 (Public Review):

The manuscript by Mohebi et al. examines a critical open question regarding the interaction of cholinergic interneurons of the striatum and transmitter release from dopaminergic axons in behaving animals. Activation of cholinergic interneurons in the striatum can evoke dopamine release in brain slices and in vivo as measured with voltammetry. However, it remains an open question in what context and to what extent this acetylcholine-mediated dopamine occurs in behaving animals. Here, the authors argue that CIN activity triggers dopamine release in the nucleus accumbens which encodes the motivation to obtain a reward through increasing "ramps" of dopamine release. Their data suggest that the ramps are not reflected in the firing of dopaminergic neurons. Rather, they provide compelling evidence that the ramps of dopamine release correlate with ramps in cholinergic interneuron activity as measured with GCaMP6. What's more, the authors show that ACh-mediated dopamine release has no paired-pulse depression, a striking result that differs from all prior ex vivo brain slice data. The manuscript is extremely well written and the data are of very high quality. Overall, this study represents an important step forward in our understanding of how ACh-mediated dopamine release regulates behavior, and more broadly how axons can generate behaviors independently from somatic activity.

Major comments<br /> 1. The complete absence of any short-term plasticity in CIN-mediated dopamine release is a striking result that is important for the field. The authors should strengthen this result with additional quantitative analysis demonstrating the lack of STP. They have analyzed paired-pulse ratios, but they should analyze this for stimuli at the higher frequencies (4 Hz, etc) that are more physiologically relevant. For example, Fig 1e shows a CIN-evoked DA release at many optically-stimulated frequencies. The authors should quantify short-term plasticity by generating fits of the single stimulus signal and comparing the mathematical sum predicted from 4 stim DA signals at different frequencies to the recorded data. A similar analysis has been done with Ca signals (Koester and Sakmann, 2000).

2. The authors show that optical activation of CINs results in DA release as measured by dLight. To clearly establish that these signals are generated by DA release driven by nicotinic receptors (and not a partial effect of some unknown artifact), it would be useful to show that the optical CIN-evoked dLight signals shown in Fig. 1 are inhibited by nicotinic receptor antagonists such as DHbE. This control experiment would significantly strengthen the result shown here.

3. Similarly, the authors show clear correlations between CIN activity and DA release during behavior. The authors should consider determining whether CINs play a causal role in triggering DA release during behavior. For example, does infusion of DHbE in the NAc prevent the light-mediated DA release during behavior? As an alternative hypothesis, some groups have been suggesting that CIN activity has almost no direct influence over DA. Therefore, testing whether a causal relationship exists between CINs and DA release would be an important experiment in addressing these two opposing viewpoints.

4. The ramps that are described in this manuscript are an order of magnitude faster (increasing over 100s of milliseconds) than ramps described in other studies that occur over seconds. In fact, the two signals may be completely different functionally. Discussion of this topic would be helpful.

Early library card catalogs used cards that were 2 x 5" cards, the Harvard College size, before the standardization of 3 x 5" index cards.

Reviewer #2 (Public Review):

In this study the authors investigate functional associations made by transcription factor ZMYM2 with chromatin regulators, and the impact of perturbing these complexes on the transcriptome of the U2OS cell line. They focus on validating two novel chromatin-templated interactions: with TRIM28/KAP1 and with ADNP, concluding that via these distinct chromatin regulators, ZMYM2 contributes to transcriptional control of LTR and SINE retrotransposons, respectively.

Strengths and weakness of the study:

- The co-localization of ZMYM2 with ADNP and TRIM28 is validated through RIME, ChIP-seq and co-IP. (Notably, since both RIME and ChIP-seq rely on crosslinking, and the co-IP with TRIM28 required crosslinking due to being SUMO-dependent, only the ZMYM2-ADNP co-IP experiment demonstrates an interaction in the absence of crosslinking).

- It is good that uniquely-mapped reads are used in the ChIP-seq analysis given the interest in repetitive elements. Likewise, though the RT-qPCR data in Fig5 should be complemented by analysis of the RNA-seq data that the authors already have, it seems that the primers are carefully designed such that a single retrotransposon copy is amplified.

- The top-scoring interactors are highly-abundant nuclear proteins: for example, data from the contaminant repository for affinity purification mass-spec data (https://reprint-apms.org/) show that TRIM28 is identified in 466 / 716 AP-MS experiments with a mean spectral count of 16. While this does not indicate that the ZMYM2-TRIM28 interaction is not 'true', it would have been helpful to further dissect the interaction to strengthen this conclusion. For example, it would be nice to see the co-IP (fig 3A) repeated from the cells expressing the ZMYM2 mutant that is no longer competent to bind SUMO (used in the ChIP-seq data of Fig 2). Alternatively - if the model is that ZMYM2 recruits SUMOylated TRIM28 - with well-characterized TRIM28 mutants that lack SUMOylation.

- The transcriptional response using bulk RNA-seq in ZMYM2-depleted cells is rather gene-centric despite the title of the paper being about TE transcription. In fact the only panels about TE transcription are the RT-qPCR data in Fig 5D,F. I may be missing something (and there aren't many details given about the RNA-seq experiments) but why not look at TE transcription in an unbiased way with the transcriptomic data at hand? I appreciate potential hazards of multi-mapping etc but it would be interesting to see at least some subfamily analysis (e.g. using the TEtranscripts tool). On a similar point, why not show some RNA-seq in the genome browser snapshots of the epigenomics - together with a RepeatMasker annotation track of TEs...

While the results broadly support the authors' conclusions, I have the overall impression that the central claim of TE transcriptional regulation by ZMYM2 could be strengthened a lot with some fairly straightforward additional experiments and analyses.

Reviewer #2 (Public Review):

In this manuscript, Chen et al. determined the structural basis for pre-RNA processing by Las1-Grc3 endoribonuclease and polynucleotide kinase complexes from S. cerevisiae (Sc) and C. jadinii (Cj). Using a robust set of biochemical assays, the authors identify that the sc- and CjLas1-Grc3 complexes can cleave the ITS2 sequence in two specific locations, including a novel C2' location. The authors then determined X-ray crystallography and cryo-EM structures of the ScLas1-Grc3 and CjLas1-Grc3 complexes, providing structural insight that is complimentary to previously reported Las1-Grc3 structures from C. thermophilum (Pillon et al., 2019, NSMB). The authors further explore the importance of multiple Las1 and Grc3 domains and interaction interfaces for RNA binding, RNA cleavage activity, and Las1-Grc3 complex formation. Finally, evidence is presented that suggests Las1 undergoes a conformational change upon Grc3 binding that stabilizes the Las1 HEPN active site, providing a possible rationale for the stimulation of Las1 cleavage by Grc3.

Several of the conclusions in this manuscript are supported by the data provided, particularly the identification and validation of the second cleavage site in the ITS2. However, several aspects of the structural analysis and complimentary biochemical assays would need to be addressed to fully support the conclusions drawn by the authors.

• There is a lack of clarity regarding the number of replicates performed for the biochemical experiments throughout the manuscript. This information is critical for establishing the rigor of these biochemical experiments.

• The authors conclude that Rat1-Rai1 can degrade the phosphorylated P1 and P2 products of ITS2 (lines 160-162, Figure 1H). However, the data in Fig. 1H shows complete degradation of 5'Phos-P2 and 5'Phos-P4 of ITS2, while the P1 and 5'Phos-P3 fragments remain in-tact. Additional clarification for this discrepancy should be provided.

• The authors determined X-ray crystal structures of the ScLas1-Grc3 (PDB:7Y18) and CjLas1-Grc3 (PDB:7Y17) complexes, which represents the bulk of the manuscript. However, there are major concerns with the structural models for ScLas1-Grc3 (PDB:7Y18) and CjLas1-Grc3 (PDB:7Y17). These structures have extremely high clashscores (>100) as well as a significant number of RSRZ outliers, sidechain rotamer outliers, bond angle outliers, and bond length outliers. Moreover, both structures have extensive regions that have been modeled without corresponding electron density, and other regions where the model clearly does not fit the experimental density. These concerns make it difficult to determine whether the structural data fully support several of the conclusions in the manuscript. A more careful and thorough reevaluation of the models is important for providing confidence in these structural conclusions.

• The presentation of the cryo-EM datasets is underdeveloped in the results section drawing and the contribution of these structures towards supporting the main conclusions of the manuscript are unclear. An in-depth comparison of the structures generated from X-ray crystallography and cryo-EM would have greatly strengthened the structural conclusions made for the ScLas1-Grc3 and CjLas1-Grc3 complexes.

• The authors conclude that truncation of the CC-domain contributes to Las1 IRS2 binding and cleavage (lines 220-222, Fig. 4C). However, these assays show that internal deletion of the CC-domain alone has minimal effect on cleavage (Fig 4C, sample 3). The loss in ITS2 cleavage activity is only seen when truncating the LCT and LCT+CC-domain (Fig 4C, sample 2 and 4, respectively). Consistently, the authors later show that Las1 is unable to interact with Grc3 when the LCT domain is deleted (Fig. 6 and Fig. 6-figure supplement 2). These data indicate the LCT plays a critical role in Las1-Grc3 complex formation and subsequent Las1 cleavage activity. However, it is unclear how this data supports the stated conclusion that the CC-domain is important for LasI cleavage.

• The authors conclude that the HEPN domains undergo a conformational change upon Grc3 binding, which is important for stabilization of the Las1 active site and Grc3-mediated activation of Las1. This conclusion is based on structural comparison of the HEPN domains from the CjLas1-Grc3 complex (PDB:7Y17) and the structure of the isolated HEPN domain dimer (PDB:7Y16). However, it is also possible that the conformational changes observed in the HEPN domain are due to truncation of the Las1 CC and CGT domains. A rationale for excluding this possibility would have strengthened this section of the manuscript.

Reviewer #2 (Public Review):

Caveney et al have overexpressed an engineered construct of the human membrane receptor guanyl cyclase GC-C in hamster cells and co-purified it with the endogenous HSP90 and CDC37. They have then determined the structure of the resultant complex by single particle cryoEM reconstruction at sufficient resolution to dock existing structures of HSP90 and CDC37, plus an AlphaFold model of the pseudo-kinase domain of the guanylyl cyclase. The novelty of the work stems from the observation that the pseudo-kinase domain of GC-C associates with CDC37 and HSP90 similarly to how the bona fide protein kinases CDK4, CRAF and BRAF have been previously shown to interact.

The experimentation is limited to the cryoEM analysis, and is lacking additional studies that would give deeper insight into the oligomeric nature - if any - of the GC-C when bound to HSP90-CDC37 as compared to the free protein. This is relevant, as the dimerization domain downstream of the pseudokinase, is evident in the maps - albeit not well resolved - and it is not clear whether it is still able to mediate dimerization with a second free or HSP90-CDC37-bound GC-C. It would also be good to see some experimentation that asks whether association with HSP90-CDC37 inhibits the guanyl cyclase activity. It is clear from previous work that HSP90-CDC37 silence the kinase activity of their bound client kinases, but in this case the catalytic guanyl cyclase is not directly associated with the chaperone complex and may still be able to function.

Although the sequence alignment presented in SuppFig 2 shows that GC-C conserves the classic DFG motif that plays a critical role in the regulation of most kinases, the numbering of the sequence is absent, making it very difficult to relate this to the structural detail shown in Fig 2B. This needs to be clarified, as the interaction of CDC37-Trp31 with the DFG motifs and downstream activation loops in CRAF and BRAF have been proposed as important features of the selectivity of these kinases for the HSP90-CDC37 system, and it would be good to be able to see clearly how much of this is also conserved in the GC-C pseudokinase domain interaction. For example, is the much shorter activation segment (DFG -> APE) ordered in the complex or disordered?

It was not easy to follow what was in the sample used for cryoEM. The cloning of the guanylyl cyclase (GC) component is described in the methods and they have shown some illustrations in fig 1 but a proper numbered figure of the domain organisation clearly showing domain boundaries and linker segments is really needed for a reader not familiar with the structure of GCs, especially since they have replaced the ECD with a leucine zipper in their construct. It is important to show a domain figure of what this construct looks like as well, as from the illustrations in fig 1 for examples its hard to see what's PK, DD, GC domains. It would also be helpful to see in the supplementary a gel of complex they put on the grids, to make it clearer what exactly the sample is and to reassure that the GC-C domains that are not resolved in the cryoEM are nonetheless present in the sample.

Overall there is only minimal proposal of mechanism or biological function based on the structure. The speculation in the Discussion of two fates - PP5 dephosphorylation or E3 ligase recruitment, is not supported by any experimentation, which is reasonable for speculation, but is also not underpinned by reference to any previously published work suggesting that these additional processes may be important. In the absence of any work by the authors can they put these speculations more in context with previously published work that supports the importance of these processes specifically for GC regulation?

Reviewer #2 (Public Review):

The study focuses on a mechanism of pest/pathogen resistance identified in Solanum commersonii, which appears to offer dominant resistance to Alternaria solani through the activity of specific glycosyltransferases which facilitate the production of tetraose glycoalkaloids in leaf tissue. The authors demonstrated that these glycoalkaloids are suppressive to the growth of multiple pathogenic ascomycetes and furthermore, that transgenic plants expressing these glycosyltransferases in susceptibleS. commersonii clones demonstrate improved resistance to a specific strain of A. solani and a genotype of Colorado Potato Beetle. The study design is straightforward, yet thorough, and does a good job demonstrating the importance of these genes in resistance. While the research findings are significant there are statements throughout the manuscript that overstate both the novelty and utility of the findings.

Title: While the protection is impressive, the title suggests that these glycoalkaloids provide protection against all fungi and insects, which is both unlikely and essentially impossible to prove. This should be changed to something more measured. This is especially true given that only a single fungus and insect were tested against transgenic plants, but would be an overstatement even with more robust evaluation.

Throughout the paper: A single isolate of A. solani and a single genotype of CPB were used in this study. While this is in line with the typical limitations of such a study, the authors need to be careful about claiming broad resistance to either of the species. Variability in fungicide tolerance and detoxification activity have been noted in both fungi and CPB, so more specific language should be used throughout (such as L213 and L221).

Reviewer #2 (Public Review):

Cacioppo et al describe a mechanism of translation regulation of Aurora A, which is dependent on alternative polyadenylation. They suggest that altered expression of the resulting isoforms in cancers is at least partly responsible for elevated Aurora A levels, which in turn is known to indicate poor prognosis.

The authors exploit publicly available databases and patient data to highlight the correlation of increased abundance of the SHORT isoform (relative to the LONG one) and poor patient survival in TNBC, as well as breast and lung cancer.

In their thorough mechanistic study they use a number of reporters to assess the impact of alternative polyadenylation on mRNA stability and translation efficiency and explore whether this process accounts for cell-cycle-regulated expression of Aurora A. These reporters are carefully controlled and well explained. I particularly commend the authors for the clear graphical presentations of the reporters (eg fig 2A, fig 3D, fig 4A). Rigorous control experiments are performed to make sure that the reporters work and "report" what they are meant to do, and to show that previous findings can be reproduced in experiments based on the reporters (eg higher protein expression from the short 3' UTR APA isoform of CDC6 mRNA, targeting of MZF1 3'UTR by hsa-let-7a).

They show that translation of the longer isoform is subject to suppression by hsa-let-7a, while the shorter isoform is not. They attribute cell-cycle regulated expression of Aurora A at least in part to the suppression of translation of the LONG isoform in G1 and S.<br /> In Figure 6 they address whether the APA-based regulatory mechanism alters Aurora A levels sufficiently to confer features associated with oncogenic transformation and overexpression of Aurora A. These data nicely tie together the observations in databases and the mechanistic part of the study.

The logic is clear and the conclusions are well supported by the data.

The authors state themselves that the impact of translation regulation on Aurora A levels in the cell cycle is an important but unanswered question. The evidence that suppression of translation of the LONG transcript contributes to the cell-cycle regulation of Aurora A is convincing, but the extent could be explored further. I wonder whether published genome-wide studies (eg PMCID 4548207, PMC3959127) have relevant data on the translation rate of Aurora A in the cell cycle.

In the paper this question is addressed in cells enriched in G1/S (Fig 6) and using the reporters (Fig 5). Having generated the ΔdPAS mutants, Aurora A levels could be easily assessed in each cell-cycle phase. The best way to do this would be sorting followed by immunoblotting.

The fact that Aurora A levels are reduced by a 6h treatment with 0.1 mg/ml CHX (Fig 6D) is interpreted as "AURKA expression in G1/S was reduced in the mutated cell lines when treated with CHX, indicating that translation of the short isoform is active in this phase" It is rather expected that using a translation inhibitor will stop the accumulation of a protein and so this experiment does not add much. A better approach to address the effect of the mutations on translation would be to add a proteasome inhibitor and follow accumulation of Aurora A, preferably not only in G1/S but also in other cell-cycle phases. Accumulation of the protein in this experiment would better reflect translation rates.

Reviewer #2 (Public Review):

Preserving and restoring the fertility of prepubertal patients undergoing gonadotoxic treatments involves freezing testicular fragments and waking them up in a culture in the context of medically assisted procreation. This implies that spermatogenesis must be fully reproduced ex vivo. The parameters of this type of culture must be validated using non-human models. In this article, the authors make an extensive study of the quality of the organotypic culture of neonatal mouse testes, paying particular attention to the differentiation and endocrine function of Leydig cells. They show that fetal Leydig cells present at the start of culture fail to complete the differentiation process into adult Leydig cells, which has an impact on the nature of the steroids produced and even on the signaling of these hormones.

The authors make an extensive study of the different populations of Leydig cells which are supposed to succeed each other during the first month of life of the mouse to end up with a population of adult and fully functional cells. The authors combine quantitative in situ studies with more global analyzes (RT-QtPCR Western blot, hormonal assays), which range from gene to hormone. This study is well written and illustrated, the description of the methods is honest, the analyses systematic, and are accompanied by multiple relevant control conditions.

Since the aim of the study was to study Leydig cell differentiation in neonatal mouse testis cultures, the study is well conceived, the results answer the initial question and are not over-interpreted.

My main concern is to understand why the authors have undertaken so much work when they mention RNA extractions and western blot, that the necrotic central part had to be carefully removed. There is no information on how this parameter was considered for immunohistochemistry and steroid measurements. The authors describe the initial material as a quarter testis, but they don't mention the resulting size of the fragment. A brief review of the literature shows that if often the culture medium is crucial for the quality of the culture (and in particular the supplementations as discussed by the authors here), the size of the fragments is also a determining factor, especially for long cultures. The main limitation of the study is therefore that the authors cannot exclude that central necrosis can have harmful effects on the survival and/or the growth and/or the differentiation of the testis in culture. In this sense, the general interpretation that the authors make of their work is correct, the culture conditions are not optimized.

Organotypic culture is currently trying to cross the doors of academic research laboratories to become a clinical tool, but it requires many adjustments and many quality controls. This study shows a perfect example of the pitfall often associated with this approach. The road is still long, but every piece of information is useful.

Reviewer #2 (Public Review):

This manuscript is an impressive "resurrection" of physiology regarding an enigmatic though unfortunately extinct species, and their potential adaptation to cold-water environments. I am largely convinced of their findings, which I feel are very straightforward and thorough.

One place where the authors perhaps fell a bit short was regarding some conclusions associated with maternal/fetal oxygen delivery. The sirenian versions of fetal & embryonic hemoglobin genes have been identified and assessed to some degree in previously published work the same research group. I feel the manuscript would have benefited from actual analysis of the fetal & embryonic hemoglobin (epsilon, gamma, zeta) to strengthen their assertions.

Reviewer #2 (Public Review):

Parasitic African trypanosomes are agents of devastating diseases in humans and animals. Currently, no vaccines exist, with control of human disease being realized thru vector suppression and elimination of infected hosts while animal diseases remain rampant on the continent. The molecular aspects of the multiple developmental stages the parasite undergoes thru its mammal and tsetse hosts, and the unique aspects of parasite gene expression regulation and host evasion mechanisms have been extensively investigated. Recent applications of single-cell transcriptomics (scRNA) to these approaches have expanded knowledge gained from total RNA and revealed new insights.

In this paper, Briggs et al., set out to determine the cell cycle-related genes (CCR) of T. brucei, which follows the typical eukaryotic progression through G1, S, G2, and M phases followed by cytokinesis, although trypanosomes are unusual in that both nuclear and mitochondrial genome replication and segregation are orchestrated during cell division. while many regulators remain unidentified, are absent, or have been replaced by trypanosomatid-specific factors. For these studies, they apply scRNA methodology using asynchronous mixed populations of cultured 'monomorphic' slender mammalian (BSF) and insect stage (PCF) cells and then determine their cell cycle phases computationally. Of interest, performing similar analysis with fresh and cryopreserved cells made minimal difference to the outcome, thus enabling future investigations with preserved cells.

The study identified 1,550 genes with dynamic transcript level changes reflective of the cell cycle, 1,151 of which had not been previously identified by bulk analysis. These revealed a common set of highly conserved CCR genes as well as unique gene transcript levels expressed thru the cell cycle for BSF and PCF cells. Expression patterns of the G1 and S phase genes are highly comparable between BSF and PCF forms, whereas, after the S phase, the timing of gene expression for the S-G2 transition is far less synchronized. Comparison between transcript expression patterns and previously published protein abundance changes identified a relative delay in peak levels for transcript and protein for at least 50% of the genes that could be compared. Collectively, this foundational analysis generates transcript atlases for BSF and PCF cell cycles, which can be further mined for downstream functional investigations.

Reviewer #2 (Public Review):

Nurr1 is a nuclear receptor and is important for mammalian brain development and homeostasis. Dysfunctional Nurr1 transcriptional activities are implicated in neurodegenerative diseases like Parkinson's. This exquisite ligand-dependent and specific transcriptional reprogramming make nuclear receptors ideal drug targets. However, the design of Nurr1-selective ligands has been confounded by the fact that Nurr1's ligand binding pocket appears to collapse in x-ray crystal structures. Interestingly, RXRalpha-targeted ligands, Nurr1's obligate heterodimer binding partner, show differential effects on Nurr1's transcriptional activities. In this study, the authors aimed to address how RXRalpha ligands lead to Nurr1 transcriptional activation. By combining biochemical approaches, NMR spectroscopy, and transcriptional reporter gene assays in neuronal cells, the authors convincingly show that these select RXRalpha ligands elicit an allosteric effect that reduces Nurr1 binding affinity. They further show that monomeric Nurr1 is a highly effective enhancer of the promoter that is repressed in the presence of RXRalpha. Overall, this is a well-presented and robust study as presented and the conclusions are supported by their evidence. This study should have a profound impact on the field as it provides a clear structural mechanism for ligand-dependent Nurr1 activation in neuronal cells.

Reviewer #2 (Public Review):

The authors identified a novel TNFAIP3 variation Leu236Pro located in the A20 OUT domain and demonstrated its pathogenicity. Proinflammatory cytokines were substantially elevated in the patients. In vitro study showed decreased stability of the Leu236Pro A20 protein and Leu236Pro mutant failed to suppress TNF induced NF-κB activity. Review of previously reported TNFAIP3 missense variations revealed that only 3/7 are pathogenic. Truncating A20 mutations are easy to determine the pathogenicity, while missense TNFAIP3 variants require more functional studies to determine the pathogenicity. The results of this study can help interpretation of TNFAIP3 missense variations.

Reviewer #2 (Public Review):

Sachiko et al. study presents strong evidence that implicates environmental volatile odorants, particularly diacetyl, in an alternate role as an inhibitors HDAC proteins and gene expression. HDACs are histone deacetylases that generally have repressive role in gene expression. In this paper the authors test the hypothesis that diacetyl, which is a compound emitted by rotting food sources, can diffuse through blood-brain-barrier and cell membranes to directly modulate HDAC activity to alter gene expression in a neural activity independent manner. This work is significant because the authors also link modulation of HDAC activity by diacetyl exposure to transcriptional and cellular responses to present it as a potential therapeutic agent for neurological diseases, such as inhibition of neuroblastoma and neurodegeneration.

The authors first demonstrate that exposure to diacetyl, and some other odorants, inhibits deacetylation activity of specific HDAC proteins using in vitro assays, and increases acetylation of specific histones in cultured cells. Consistent with a role for diacetyl in HDAC inhibition, the authors find dose dependent alterations in gene expression in different fly and mice tissues in response to diacetyl exposure. In flies they first identify a decrease in the expression of chemosensory receptors in olfactory neurons after exposure to diacetyl. Subsequently, they also observe large gene expression changes in the lungs, brain, and airways in mice. In flies, some of the gene expression changes in response to diacetyl are partially reversable and show an overlap with genes that alter expression in response to treatment with other HDAC inhibitors. Given the use of HDAC inhibitors as chemotherapy agents and treatment methods for cancers and neurodegenerative diseases, the authors hypothesize that diacetyl as an HDAC inhibitor can also serve similar functions. Indeed, they find that exposure of mice to diacetyl leads to a decrease in the brain expression of many genes normally upregulated in neuroblastomas, and selectively inhibited proliferation of cell lines which are driven from neuroblastomas. To test the potential for diacetyl in treatment of neurodegenerative diseases, the authors use the fly Huntington's disease model, utilizing the overexpression of Huntingtin protein with expanded poly-Q repeats in the photoreceptor rhabdomeres which leads to their degeneration. Exposing these flies to diacetyl significantly decreases the loss of rhabdomeres, suggesting a potential for diacetyl as a therapeutic agent for neurodegeneration.

The findings are very intriguing and highlight environmental chemicals as potent agents which can alter gene expression independent of their action through chemosensory receptors.

Reviewer #2 (Public Review):

Granell et al. investigated genetic factors underlying wheezing from birth to young adulthood using a robust data-driven approach with the aim of understanding the genetic architecture of different wheezing phenotypes. The association of 8.1 million single nucleotide polymorphisms (SNPs) with wheeze phenotypes derived from birth to 18 years of age was evaluated in 9,568 subjects from five independent cohorts from the United Kingdom. This meta-genome-wide association study (GWAS) revealed the suggestive association of 134 independent SNPs with at least one wheezing subtype. Among these, 85 genetic variants were found to be potentially causative. Indeed, some of these were located nearby well-known asthma loci (e.g., the 17q21 chromosome band), although ANXA1 was revealed for the first time to play an important role in early-onset persistent wheezing. This was strongly supported by functional evidence. One of the top ANXA1 SNPs associated with wheezing was found to be potentially involved in the regulation of the transcription of this gene due to its location at the promoter region. This polymorphism (rs75260654) had been previously evidenced to regulate the ANXA1 expression in immune cells, as well as in pulmonary cells through its association as an eQTL. Protein-protein network analyses revealed the interaction of ANXA1 with proteins involved in asthma pathophysiology and regulation of the inflammatory response. Additionally, the authors conducted a murine model, finding increased anxa1 levels after a challenge with house dust mite allergens. Mice deficient in anxa1 showed decreased lung function, increased eosinophilia, and Th2 cell levels after allergen stimulation. These results suggest the dysregulation of the immune response in the lungs, eosinophilia, and Th2-driven exacerbations in response to allergens as a result of decreased levels of anxa1. This coincides with evidence of lower plasmatic ANXA1 levels in patients with uncontrolled asthma, suggesting this locus is a very promising candidate as a target of novel therapeutic strategies.

Limitations of this piece of work that need to be acknowledged: (1) the manual and visual inspection of Locus Zoom plots for the refinement of association signals and identification of functional elements does not seem to be objective enough; (2) the sample size is limited, although the statistical power was improved by the assessment of very accurate disease sub-phenotype; (3) association signals with moderate significance levels but with strong functional evidence were found; (4) no direct replication of the findings in independent populations including diverse ancestry groups was described. Nonetheless, the robustness and consistency of the findings supported by different analytical and experimental layers is the major strength of this study.

The authors successfully achieved the aims of the study, strongly supported by the results presented. This study not only provides an exciting novel locus for wheezing with potential implications in the development of alternative therapeutic strategies but also opens the path for better-powered research of asthma genetics, focused on accurate disease phenotypes derived by innovative data-driven approaches that might speed up the process to disentangle the missing heritability of asthma, making use of still useful GWAS approaches.

Reviewer #2 (Public Review):

This is an excellent study. It starts with the identification of two bactofilins in H. neptunium, a demonstration of their important role for the determination of cell shape and discovery of an associated endopeptidase to provide a convincing model for how these two classes of proteins interact to control cell shape. This model is backed up by a quantitative characterisation of their properties using high-resolution imaging and image analysis methods.

Overall, all evidence is very convincing and I do not have many recommendations on how to improve the manuscript.

In my opinion, there are only two issues that I have with the paper:

1. The single particle dynamics of BacA is presented as analysed and I would like to give some suggestions how to maybe extract even more information from the already acquired data:

1.1. Presentation: Figure 5A is only showing projections of single particle time-lapse movies. To convince the reader that it was indeed possible to detect single molecules it would be helpful if the authors present individual snapshots and intensity traces. In case of single molecules these will show step wise bleaching.

1.2. Analysis: Figure 5B and Supplement Figure 1 are showing the single particle tracking results, revealing that there are two populations of BacA-YFP in the cell. However, this data does not show if individual BacA particles transition between these two populations or not. A more detailed analysis of the existing data, where one can try to identify confinement events in single particle trajectories could be very revealing and help to understand the behaviour of BacA in more detail.

2. The title of Fig. 3 says that BacA and BacD copolymerise, however, the data presented to confirm this conclusion is actually rather weak. First, the Alphafold prediction does not show the co-polymer, and second, the in vitro polymerisation experiments were only done with BacA in the absence of BacD. Accordingly, the only evidence that supports this is their colocalization in fluorescence microscopy. I suggest either weakening the statement or changing the title adds more evidence.

Finally, did the authors think about biochemical experiments to study the interaction between the cytoplasmic part of LmdC and the bactofilins? These could further support their model.

Reviewer #2 (Public Review):

This work sheds new light on the growth trajectory of Bonobo and contributes heavily to the discussion of the exclusivity of certain aspects of growth in modern humans. These results are also interesting as long as they are based on the study of the largest sample ever considered in the study of the growth of this species by including morphometric measurements as well as endocrinological factors.

The authors approach the study of the presence of growth spurs (GS) in Bonobo on the basis that GS are exclusive to the growth in modern humans. This idea is fairly widespread, however studies on non-human primates have shown an acceleration of growth during adolescence in several species, these works are recalled, presented and discussed by the authors. The originality of this work lies in highlighting the importance of scaling in studies of growth trajectories. The absence of GS in Bonobo but also in other primate species may result from not considering the conjunction of weight and height in the analysis of growth, because the pronounced changes in the speed of the height are in relation to the speed of changes in weight and this is modified according to the size/age. The authors apply scaling corrections to their results and the GS become evident (or more obvious) in Bonobo. Thus, the exclusivity of GS in growth in modern humans may in fact result only by the application of analytical approach not very appropriate in non-human primates.

Reviewer #2 (Public Review):

In this manuscript, Castanera et al. investigated how transposable elements (TEs) altered gene expression in rice and how these changes were selected during the domestication of rice. Using GWAS, the authors found many TE polymorphisms in the proximity of genes to be correlated to distinct gene expression patterns between O. sativa ssp. japonica and O. sativa ssp. indica and between two different growing conditions (wet and drought). Thereby, the authors found some evidence of positive selection on some TE polymorphisms that could have contributed to the evolution of the different rice subspecies. These findings are underlined by some examples, which illustrate how changes in the expression of some specific genes could have been advantageous under different conditions. In this work, the authors manage to show that TEs should not be ignored when investigating the domestication of rise as they could have played an important role in contributing to the genetic diversity that was selected. However, this study stops short of identifying causations as the used method, GWAS, can only identify promising correlations. Nevertheless, this study contributes interesting insights into the role TEs played during the evolution of rice and will be of interest to a broader audience interested in the role TEs played during the evolution of plants in general.

Reviewer #2 (Public Review):

This paper extends prior work demonstrating the importance of K145 acetylation of TDP-43 as a post-translational modification that impacts its RNA-binding capacity and may contribute to pathology in FTLD-ALS. The main strengths of this paper are the generation of a novel mouse model, using CRISPR gene editing, in which an acetylation-mimetic mutation (K to Q) is introduced at position 145. Behavioral, biochemical, and genetic analyses indicate that these mice display phenotypes relevant to TDP-43-associated disease and will be a valuable contribution to the field. While most of the data are rigorous and clearly presented, several weaknesses should be addressed to strengthen the manuscript and further characterize the phenotype of mutant mice.

Reviewer #2 (Public Review):

This study reports a novel role of the natriuretic receptors Npr3 and Npr1 in the formation of neural crest (NC) and cranial placode (CP) progenitor populations in frog embryos. The authors discovered this receptor family in a screen for genes activated during NC development. They show the relevant expression of these receptors and the corresponding ligands in the NC and CP populations. Knockdown and rescue experiments combined with pharmacological drug treatment demonstrated that Npr3 clearance activity is required for NC progenitor formation. Surprisingly, adenylyl cyclase inhibition was required for cGMP production and the effect on CP development. The authors conclude that the two second messengers downstream participate in the segregation of the NC and CP progenitors in embryonic development.

The significance of this study is in the demonstration of two distinct developmental programs that are separately controlled by different activities of the same receptor. The study is well designed and executed with proper controls. Nevertheless, the data suggesting that Npr3 regulates NC and CP fates via different mechanisms are limited and need further support, such as the analysis of additional markers for CP progenitors, to be unambiguously interpreted. The work is likely to impact two different areas: early embryonic development and natriuretic peptide signaling.

Reviewer #2 (Public Review):

Rapan and colleagues did perform an impressive multi-modal parcellation of the macaque frontal cortex. In addition to qualitative cytoarchitectonic and resting-state functional fMRI data analyses, the authors based their parcellation on quantitative receptor density analysis of 14 receptors. Compared with the classic Walker map of the macaque frontal cortex, the authors produced a more refined map. Those results should be discussed in light of previous work on the same topic (Petrides et al. 2012 Cortex; Reveley et al. 2017 Cerebral Cortex; Saleem and Logothetis 2012).

Reviewer #2 (Public Review):

Immature lattice assembly remains an arcane topic, and these simulations provide high resolution data such as assembly kinetics and large-scale lattice rearrangement. Further, the authors extend their model to compare directly with experiments, e.g. SNAP-HALO dimerization, which provides a basis to interpret their conclusions. The manuscript is difficult to read, as it is a technical manuscript that overuses jargon; overall, it seems written for a specialized audience. Additionally, there are several aspects of the model design that remain opaque, such as the implicit lipid method and the suppression of multi-site nucleation. Further, analyses such as time auto-correlation and mean first passage time are not given much context by the authors. Altogether, it is the opinion of this reviewer that several revisions to the manuscript should be incorporated to improve clarity and strengthen the significance of the authors' efforts.

Reviewer #2 (Public Review):

This study by Yamaguchi and Peltier provides a detailed investigation of the brainstem CPG functional organization that rules vocal behaviors in several Xenopus species, from an evolutionary perspective. The main conclusion of the paper reveals that vocal CPGs, located in the brainstem, generating fast and slow clicks in Xenopus male courtship calls are conserved across various Xenopus species. But the development of the fast CPG depends on testosterone only in species producing fast-click courtship calls.

Reviewer #2 (Public Review):

ATM and Rad3-related (ATR) interact with ATRIP and plays a central role in DNA damage response. Previous studies have established the idea that ATR is recruited to RPA-covered ssDNA via ATRIP-RPA interaction. In this paper, the authors propose a new RPA-independent mechanism for ATR recruitment.

Reviewer #2 (Public Review):

The authors used single cell transcriptome analysis of zebrafish skin cells and characterized various types of cells that are involved in scale formation and stripe patterning. The methods employed in this study is highly powerful to provide mechanistic explanation of these fundamental biological issues and will be a good example for many researchers studying other biological issues. Furthermore, the results characterizing differences in gene expression patterns among various types of cells will be informative for other researchers in the field.

For scale formation, it is known that mineralized tissues may significantly differ in rayfins and lobefins since sox9, col2a1, and col10a1 are all expressed in osteoblasts, in addition to chondrocytes, in zebrafish and gar (Eames et al., 2012, BMC Evol. Biol.). Furthermore, in mammals, Col10 is expressed in chondrocytes in mature cartilage that undergoes ossification. Thus, unlike the authors argue, col10a1 expression is not apparently relevant to the elasticity of scales.

The authors also state that the expression of dlx4a, msx2a, and runx2b characterize cells homologous to mammalian ameloblasts. However, dlx4, runx2, and msx2 are all duplicated in zebrafish, and the function of duplicated genes in teleost fishes may differ from that of single ancestral gene. Moreover, none of Dlx4, Msx2, and Runx2 is expressed specifically by ameloblasts in mammals. Indeed, both Msx2 and Runx2 are expressed in osteoblasts, while the expression of Dlx4 in ameloblasts is not reported. These results, together with the expression of an enamel gene, enam, in dermal cells (SFC), do not appear to support the homology of the surface tissue of mammalian teeth and zebrafish scales.

Reviewer #2 (Public Review):

In a neonatal model of bacterial meningitis induced by s.c. injection of E. coli, transcriptional changes were found across all major cell types including endothelial cells, fibroblasts and macrophages. Among macrophages, they describe 2 resident subsets and 2 inflammatory subsets. By immunohistochemistry of arachnoid and dura flatmounts, they show vascular changes upon infection, including clustering of CLDN5 and PECAM1, and disorganized capillary morphology, which was dependent on Tlr4 signaling but independent of arachnoid macrophages.

The manuscript would benefit from rewriting, it is not written in a concise manner and the rationale for experiments, time points for analyses and their conclusions are not clear. The model of s.c. bacterial infection is not well introduced and overall changes in the periphery, survival curves or bacterial counts (in the KO models) in the meninges/brain are not mentioned.

Reviewer #2 (Public Review):

This study aimed to classify colorectal cancer (CRC) samples based on the expression of genes in selected gene lists, where the gene lists were chosen to represent aspects of the tumour microenvironment, tumour-associated immune cells, and tumour cells. The resulting clusters were then used to define a classifier, followed by a detailed description of molecular features of the tumours and tumour microenvironments assigned to each cluster. The authors claim this study is more "holistic" than previous work on CRC clustering/classifiers because they aimed to explicitly include additional components of the tumour microenvironment in both the clustering/classifier definition and in the subsequent description of molecular characteristics.

The CCCRC clustering and the resulting classifier presented in this paper are derived from published RNAseq studies. The multi-omics aspect of the work is restricted to smaller sample numbers for which both transcriptomic and another omics dataset were available in public resources and comprises a description or correlative analysis of each omics data type within each of the assigned CCCRC subtypes.

By applying solid computational methods to a compendium of published RNAseq datasets (n~1500 tumours), they found that tumour samples from colorectal cancers clustered into 4 subtypes ("CCCRC" subtypes) on the basis of 61 pre-defined gene expression signatures. These subtypes correlated with but did not correspond to, the previously described Consensus Molecular Subtypes (CMS) of colorectal tumours.

Other types of molecular data were available for some tumours, obtained from the same published resources: whole-slide images, mutations, tumour proteomics, and/or scRNAseq. The authors reanalysed these datasets using standard methods and drew correlations with the CCCRC subtypes they had assigned in this work. To (semi-)quantify immune infiltration characteristics from whole-slide images (WSI), they additionally performed automated segmentation in addition to review by pathologists, which in combination produced a convincing WSI-derived dataset.

In combination with existing CRC classifications, this study could facilitate future biomarker discoveries. This appears to be the authors' main claim, and the data and methods broadly support this claim.

Some aspects of the work need to be clarified:

This work relies on the definition of 4 clusters of CRC tumours based on consensus clustering of the 61 gene lists, which in turn depends on the choice of clustering method and the choice of gene lists. Sufficient detail is provided about the gene lists and resulting clusters, but this paper does not show how robust the 4 clusters are to these choices; for example, the "Energy" gene list appears to be a relatively strong component of clusters C2 and C3.

The authors examined whether their CCCRC classification showed differential disease progression in available retrospective cohorts of people treated with anti-PDL1 therapy. The authors presented this work as "significance of CCCRC in guiding the clinical treatment of colorectal cancer", but the data presented in this section cannot support clinical treatment decisions, which would require prospective studies and clinical trial designs. However, this section is potentially useful for generating hypotheses about potential biomarkers related to the CCCRC subtypes, and might, in the future with additional evidence, contribute to the design of a trial. The authors point out that additional experimental evidence would be required.

Other prognostic or predictive clinicopathological variables for colorectal cancer are not discussed in detail in the present work but are important for further work on the prognostic and predictive value of CRC molecular subtypes and biomarker derivation. Discrepancies in treatment response have previously been observed in separate CRC trials of biologically targeted agents with different chemotherapy backbones and other authors have suggested that treatment interactions with the tumour microenvironment might in part explain these discrepancies (e.g. Aderka (2019) PMID:31044725, and others).

Reviewer #2 (Public Review):

This paper provides an important and insightful investigation into patterns of activations that emerge in external task states. The authors use state-of-the-art methods and novel analytic approaches to establish that deactivations in the default mode network during external tasks are driven by activity in brain regions that are important in the current tasks (such as the visual or dorsal attention networks). It will be important in the future to understand whether this is a symmetrical phenomenon by studying this behaviour in states that maximize activity within the default mode network and also drive reductions in networks that are not relevant to these situations.

Reviewer #2 (Public Review):

Since this study is a long-term cohort study in children and adolescents, it is advisable to decide whether to highlight differences by age group or to show consistent effect after exposure. In particular, obesity and related diseases are closely related to socio-economic environmental factors, and its impact might be different according to age (group) at exposure.

The part described in comparison with previous studies is a good attempt. However, some results are consistent with those of previous studies and some are not. This may be related to the time difference in socio-economic environmental factors rather than simply the difference between the West and China (Hong Kong). According to modernization/urbanization, changes in living environment, changes in family relationships, and changes in the care environment can also be factors especially in children.

In studying the effect of environment on gene expression, it can be thought that the influence of genes and the degree of expression might be different depending on the age of the subject (newborn, infant, infant, adolescent, adult) duration of exposure and these still need to be elucidated.

Reviewer #2 (Public Review):

In the manuscript, Chen and colleagues reconstituted the minimal system that indicates the coupling of PSD condensates with actin polymerization. While the functional connection between the assembly and dynamics of PSD and actin was known, the molecular mechanism remained elusive. Using a series of elegant biochemical reconstitutions and in-vitro assays complemented with analysis in living cells and primary neurons, the authors characterized whether PSD condensates of Homer-1, Shank-3 and SAPAP/GKAP are sufficient to induce F-actin bundling. Furthermore, they dissected the positively-charged Arg patch within EVH1 domain of Homer to be crucial for the F-actin bundling. Postsynaptic CaMKII and a short isoform of Homer, Homer1a, can both attenuate this process, suggesting various mechanisms neurons can regulate this process. Overall, the topic is timely, the study is well-designed, and the assays are clearly executed. However, several aspects need to be experimentally addressed, including some important controls:

1. It is well established that molecular crowding plays a crucial role in F-actin bundling. For example, in the reconstitution assays in Fig.1, the authors use 10 µM of each component of PSD (total of 60 µM), to which 5 µM actin is added. Yet, in their control assays (Supp. Fig. 1), only 10 µM of each protein was checked with the same amount of actin. A control is missing where the total protein crowding would be preserved, for example, by adding BSA or protein to mimic non-specific protein crowding.<br /> 2. Is the F-bunding observed under these physiological ratios of PSD proteins and actin? For instance, a recent quantitative study (PMID: 34168338) suggests actin:Homer-1 is 200:1 or 100:1, which is in stark difference from the 1:2 molar ratio used in the study. The protein concentrations (molar ratios) need to match the physiological.<br /> 3. In the cell migration assays, it is somewhat unclear to what extent the interaction is direct. For instance, co-sedimentation at ultra-speed (100,000 g) was used to suggest a direct binding of EVH1-GNC4 fusions (Homer1, Enah) with F-actin. The control that needs to be included is a protein known not to bind to F-actin incubated under the same conditions (salt concentration, duration of incubation) and spun down at 100,000xg. This is important to exclude that the tested proteins non-specifically entangle into F-actin without specifically binding to it, particularly at such high speed.<br /> 4. The imaging assay in hippocampal neurons uses an increased spine head size as a proxy for F-actin bundling. However, one needs to be careful as the baseline includes soluble mCherry, which is both much smaller in size and does not specifically enrich the spines. The image of Homer 1 R3E shows overall lower localization at the spines. Thus, one cannot exclude that the spine enlargement upon overexpression of Homer 1 wt and R3E+EN is not primarily driven by their overall enrichment in the PSD phase. A suitable control for this assay would be mCherry-tagged PSD95, which would localize to the spines yet is not directly involved in F-actin bundling.

Reviewer #2 (Public Review):

This work is a cross-validation of an x-ray tomography technique (SAXS) and an optical microscopy technique (SLI) for imaging axonal orientations ex vivo. These innovative methods were introduced in recent papers by the authors, who have teamed up here to compare them side-by-side on the same tissue samples for the first time. The two methods are both label-free (do not require staining) and they are quite complementary. SAXS can provide full 3D orientation measurements on intact tissue, but it operates at a mesoscopic resolution and requires access to a synchrotron. SLI can measure the orientations of multiple fascicles per voxel at a microscopic resolution and relies on more widely accessible equipment, but its accuracy suffers for fiber orientations perpendicular to the imaging plane and it requires tissue to be sectioned before it is imaged. Therefore it makes a lot of sense to explore the complementary strengths of these two techniques, and to use one to "fill in the blanks" of the other. The paper also compares the orientation measurements obtained with SAXS and SLI to those obtained with diffusion MRI. The latter provides only indirect measurements based on water diffusion, at a mesoscopic resolution somewhat lower than that of SAXS, but has the benefit of being feasible in vivo.

A limitation of this study is that conclusions on the comparison between SAXS and SLI are drawn from only 2 sections of a partial monkey brain sample and 2 sections of a partial human brain sample. Conclusions on diffusion MRI are drawn only on the 2 human sample sections. This is particularly an issue for the comparison to diffusion MRI, as the diffusion MRI voxels are wider than the section thickness, hence one cannot preclude that any orientations detected with diffusion MRI but not with SAXS and SLI come from the portion of the voxel that is missing from the corresponding SAXS/SLI section.

The stated aim of the paper is to provide a framework for combining the complementary benefits of SAXS and SLI, rather than simply presenting the results of a cross-validation study. This is a significant and ambitious aim. However, in order for this to serve as a framework, there would have to be clear prescriptions for how researchers interested in obtaining ground-truth measurements of axonal orientations would do so by using these two methods in tandem. This is not adequately developed in the paper in its present form. For example, the results show reasonable agreement between SAXS and SLI orientations when fibers lie within the SLI imaging plane and decreasing agreement for fibers with increasing through-plane inclination. How would the two methods be combined in voxels where they disagree? Would one use SLI orientations in voxels with fewer through-plane fibers and SAXS orientations in voxels with more through-plane fibers? How would voxels be assigned to each category? How would the orientation vectors from the two modalities be composed and how would the resolution difference between the two be handled? When the through-plane measurement of SLI is unreliable, is its in-plane measurement still reliable? That is if there were one mainly in-plane and one mainly through-plane fiber population, would the orientation of the former still be measured correctly by SLI? There is also considerable agreement reported here between through-plane orientations obtained with SAXS and diffusion MRI. Would this mean that diffusion MRI itself could be used to supplement SLI with through-plane orientations? Any clear set of prescriptions along these lines would represent a framework for imaging orientations by combining modalities. This, however, would require detailed steps for how to perform the combination and use the multi- vs. uni-modal framework to reconstruct connectional anatomy.

A key advantage of SAXS is that it can be performed on intact samples, i.e., before any nonlinear distortions of the tissue are introduced by sectioning. Thus it can provide an undistorted reference, with contrast on axonal orientations that would be absent in, say, a structural MRI of comparable resolution. This contrast could be used to drive registration of the distorted SLI sections to an undistorted SAXS volume, and therefore is a key way in which the two techniques can complement each other. Here, however, this is not explored, as SAXS is performed after sectioning. It is not clear if this is the authors' prescription for how a combined SAXS/SLI framework would be implemented, or if it was done specifically for this study. First, it would seem that SAXS on the intact sample would be lower maintenance, requiring less setup time and hence potentially less overall beamtime than performing SAXS on each section separately. This would make it more practical for routine deployment beyond a few sections. Second, because the SAXS data are now nonlinearly distorted, they cannot be affinely aligned to the MRI volumes. While, in principle, performing both SAXS and SLI on the sections may facilitate the comparison between the two, having to unmount, rehydrate, and remount the sections in between may negate this advantage, as now there is no guarantee that SAXS and SLI can be affinely registered to each other. Here all these registration steps are performed affinely, so it is unclear to which extent the computed errors between modalities are characterizing the inherent limitations of the respective contrasts, or limitations of the registration technique. Some of the alignment is performed manually, for example, specific regions of the images are realigned by hand, and the slice of the diffusion MRI volume that is aligned to the SAXS/SLI sections is chosen by hand. Again, for this to serve as a framework that can be deployed on whole samples, there would have to be clear prescriptions for how to perform these steps robustly, how to ensure that the MRI can be acquired in a coordinate frame parallel to the sections, etc.

Finally, the paper puts forth a general conclusion that diffusion MRI overestimates the number of fiber populations per voxel, on the basis of small ODF peaks appearing perpendicular to the main ODF peaks. Of all conclusions in the paper, this is the least convincingly supported by evidence. First, these small perpendicular peaks are a known artifact, which would be typically eliminated by ignoring ODF peaks below a certain amplitude, a common practice in diffusion tractography algorithms. The authors refrain from using an amplitude threshold, with the rationale that it may also remove true diffusion orientations. However, they apply a threshold when they detect SLI peaks (a rather stringent 8% of the maximum). Second, the explanation that these artifactual peaks may appear due to vessel walls is not convincing. Vasculature is sparse. A single vessel wall will not impact the diffusion signal in the same way as a bundle of parallel axons. In an axon bundle, water molecule displacements are restricted in all directions except parallel to the axons. A single vessel wall in a voxel will not have the same effect on displacements (which are much smaller than the size of the voxel). From Figure 5, it looks like there would be at most 1-2 of these vessels in a diffusion MRI voxel, and they would not be in all voxels. This cannot explain the widespread appearance of these small artifactual peaks. Third, many ODF reconstruction methods have parameters that can be adjusted to make these artifactual peaks more or less prominent. The default parameters may be optimal for in vivo but not ex vivo data, due to the effects of fixation. In light of these concerns, I would caution against making such a general statement about all diffusion MRI in the human brain, especially on the basis of a single diffusion reconstruction method applied to a single location in one brain.

Reviewer #2 (Public Review):

Londoño-Nieto et al. investigated the influence of temperature on the form and intensity of sexual conflict in Drosophila melanogaster. They aimed to test the effect of naturally occurring temperature fluctuations on a wild population of Drosophila while disentangling pre- and post-copulatory episodes of sexual conflict. To this end, they exposed females to males under monogamy or polyandry, hence manipulating the degree of male harm experienced by females. The effect of temperature was explored by exposing these groups to 20, 24, or 28{degree sign}C. They found that female fitness suffered from male harm most at 24{degree sign}C and less at the other two temperatures. Interestingly, pre- and postcopulatory episodes of sexual conflict were affected differently by temperature. Overall, these data suggest that the relationship between sexual conflict and temperature can be strong and complex. Hence, these results can have important implications for the impact of sexual conflict on population viability, especially in light of the climate crisis.

This paper tackles a highly relevant question using an established model organism for sexual conflict and contains a rich dataset obtained using a series of carefully planned experiments and analysed in an appropriate way. Importantly, the authors used biologically meaningful temperatures and mating treatments, which increases the relevance of the data. The main conclusions are well supported by the data. Nevertheless, the devil is in the detail, and given the way the authors frame their study (i.e. testing a natural population under naturally occurring temperature fluctuations) and their results (i.e. sexual conflict is buffered by temperature effects in the wild) there are some limitations to be considered:

1) The authors frame their study as addressing the question of how sexual conflict reacts to naturally occurring temperature fluctuations in the wild. Nevertheless, the population used in this experiment had been kept for nearly 3 years in the laboratory prior to the experiment. Importantly, the authors ensured that the laboratory population maintained genetic diversity, by regularly crossing wild lines into it. Nevertheless, this population remained for some time in the laboratory under standardized conditions. The applied temperature fluctuations are in a biologically meaningful range (though only during the reproductive season), but it remains unclear if the applied fluctuations were in a standardized way (i.e. pre-programmed) or included random fluctuations (i.e. a more natural setting). This laboratory setup has certainly clear advantages, for example, it enables the exclusion of any effects other than the temperature on sexual conflict. Nevertheless, how these will then ultimately play out in the wild could be a different story.

2) The authors highlight clearly that temperature fluctuations in the wild might play an important part in how sexual conflict plays out in natural populations. This very interesting and highly relevant point might lead the reader to assume that this is what was actually tested in the experiment. Nevertheless, in the experiments, different constant temperatures were applied to the flies, while only the stock population was kept at a fluctuating temperature regime. Hence, the influence of fluctuations during episodes of sexual conflict remains untested. While the present data show that sexual conflict can be modulated by temperature, the effect of naturally occurring fluctuations on the net cost of sexual conflict to a population remains unclear.

3) The authors conclude that the effect of sexual conflict can be buffered by temperature in the wild. In general, I agree with this, although a more conservative way of framing this would be to say that temperature modulates or moderates sexual conflict instead of buffers it. If there really is a buffering effect of temperature in the wild remains to be tested, I believe. This will depend on how actual changes in temperature affect this dynamic (see point 2). In addition, I think another interesting open question is what the mechanism behind the observed differences might be. Are male and female interests really more aligned at different temperatures (i.e. males plastically reduce harm)? This would really buffer the harm of sexual conflict at those temperatures. Nevertheless, alternatively, males might not be perfectly adapted to manipulate the female optimally at lower or higher temperatures. This would mean that if the temperatures change, males might evolve to increase the manipulation of females, and hence the scope for sexual conflict might not change in the end under this scenario. Nevertheless, as the authors themselves state: 'An intriguing possibility is thus that SFPs are more effective at lowering female re-mating rates at warm temperatures, thereby buffering these costs.' Therefore, a temperature-dependent increase in the effectiveness of male manipulation might counterintuitively reduce sexual conflict in this species.

4) In the end the authors argue that the climate crisis might have 'unexpected positive consequences via its effect on male harm'. Sexual conflict is indeed widespread, but it takes many different forms (as has been nicely described in the introduction of this paper). Because the studied system seems to be quite a specific example, it is questionable how far spread this phenomenon is in nature. In addition, it remains unclear how male harm will evolve in response to the climate crisis (see point 3). Finally, the relative fitness of females increased in the present experiment, as the tested range was within the reproductive optimum of the species. Nevertheless, the relative importance of the positive effect of sexual conflict on fitness outside of optimal temperatures seems questionable.

Nonetheless, I believe these results to be of exceeding interest to the scientific community and of importance to the field. It opens up many potential research directions and adds further data to the fascinating field of sexual conflict, SFPs, and male harm in Drosophila.

Reviewer #2 (Public Review):

The paper starts with the premise that given the broad immature connectivity between the retina and thalamus during development, locally homogeneous synaptic currents should generate precise spike correlations (on a millisecond timescale) which are not seen in development and could be bad for developmental refinements and "network diversity". Rather, the correlations during development are over much longer timescales. The authors propose that two main factors, the dominance of NMDA (over AMPA) currents and the absence of recurrent connections prevents these precise correlations and preserves diversity.

The paper consists of three parts: (I) develop a biophysical model for a thalamic neuron, (II) use the model to determine which factors govern precise correlations, and then (III) simulate a cortical network and demonstrate loss of network diversity when precise correlations are used. While all parts are interesting, there are several claims in each (and the links between them) that are not fully justified.

What is commending about the paper is that it is one of few theoretical/modeling papers that focuses on neural circuit development and it manages to link experimental results to principles of circuit function. The authors apply quite a few modeling approaches ranging from single-neuron models (including building a database of thalamic neurons based on experimental data) and network models. Some of the claims regarding the timescales of correlations (long in development) are unjustified because the authors use a fixed-timescales kernel to compute these correlations and mainly investigate their amplitude or level, not their timescales. It is also not clear how important is the heterogeneity among thalamic neurons. Are the effects on the correlations the result of NMDA currents or the neuronal diversity from their database? Are precise correlations generated because of the diverse/heterogeneous neurons, or because of the levels of convergence? What happens to homogeneous neurons? The authors also propose that precise correlations impair network diversity but never show this impairment directly beyond a diversity of excitatory-to-excitatory connection strengths. If the authors were to clarify these issues then the paper could be a valuable contribution to the field of developmental systems and computational neuroscience.

Reviewer #2 (Public Review):

The overall objective of this paper is to characterize the cells that are responsible for producing the secretions of the parasitic larvae, Brugia malayi. This parasite is a human pathogen that is one of three responsible for lymphatic filariasis/elephantiasis a disease that threatens half of the world's population. The specific focus of this work is protein secretions made by the parasites. In general, it is well-known that parasitic worms can manipulate and evade host immune immunity via secreted products. Studies have focused on the activities of these secretions and specific molecules. What is lacking is a detailed description of the identity and anatomical location of the cells that produce them. This is especially important as these cells are the target of different classes of anthelminthic drugs. This knowledge could allow new strategies to target these pathogens and to better understand the mechanism of actions.

To better understand this important topic, this manuscript describes a method to dissociate cells of the pre-larval stage (microfilariae) of the human parasitic filarial nematode, Brugia malayi. This method is then used to create an atlas of cells based on the expression profiles of individual dissociated cells. Cells are grouped into clusters with similar patterns of expression using single-cell mRNA sequencing analysis pipelines and the clusters are defined by using a combination of known functionalities based on the well-established, free living, soil nematode, C. elegans, and different functional classifications based on genes of interest. These include known antigens as well as targets of 3 classes of anthelmintic molecules. Using the scRNA-seq data, clear hypotheses can be made about ion channel and structural protein composition, the putative targets of the anthelminthics. Finally, it is proposed that the dissociated cells can be cultured which can facilitate future studies since cell lines or primary cultures of cells from filarial worms are not available.

This paper represents a huge undertaking on an important and understudied area. The authors have taken on a major challenge to gain novel insights, and to provide data and protocols for the field to use. The data are well-presented and support the conclusions of the work. The authors have broadly achieved their goals and the data generated and methodology will be important for the community.

Reviewer #2 (Public Review):

The manuscript is rigorous and clearly written; the experiments are well described, and the conclusions are consistent with the experimental results. Particularly interesting are the data demonstrating the role of cytoneme-like structures. The microscopy images supporting the experimental data are clear and fascinating.<br /> The work is, in my opinion, well conducted.

Reviewer #2 (Public Review):

In this manuscripts, the authors investigated differential role of two closely related proteins, S27 and S27L , which are one of the subunits of ribosome. Ribosomes containing each protein associate with a distinct set of mRNAs, suggesting that ribosomes in the cells play distinct roles depending on which subtype of S27 subunits they contain. The authors also performed functional analyses using mutant mice, and demonstrated that functions of S27-containing ribosome can be rescued by S27L-containing ribosome and vice versa. These findings provide new experimental insights into the origin of family genes fixed during the course of evolution.

Reviewer #2 (Public Review):

In this manuscript, the authors characterize the extent of RNA transfer between cells in culture, with an emphasis on trying to identify RNAs that are transferred through tunneling nanotubes (TNTs). They use an in vitro human-mouse cell co-culture model, consisting of mouse embryonic fibroblasts and human MCF7 breast cancer cells. They take advantage of the CD326 cell surface molecule, which is specifically expressed on MCF7 cells, to separate the two cell populations using magnetic beads conjugated to anti-CD326 antibodies, followed by deep sequencing to identify human RNAs present in mouse cells. They identify many 'transferred' RNAs. Further analysis of sequencing data together with experiments using synthetic reporters indicate that RNA transfer is non-selective, that the amount of transfer strongly correlates with the level of expression in donor cells, and does not appear to require specific RNA motifs. The authors also note that co-culture with MCF7 cells leads to significant changes in the MEF transcriptome.

The experiments are overall carefully designed, and the data are clearly and quite carefully presented to point out limitations in interpretation and to distinguish speculations from experimental conclusions. It should however be kept in mind that it is unclear to what extent these limitations influence the conclusions reached. For example, the identification of transferred RNAs relies on the purity of the isolated cell populations and, while the authors provide some supporting evidence for this, nevertheless potential caveats remain. For instance, the isolated MEF samples used for analysis appear to lack single MCF7 cells, but still contain components, labeled as 'double stained' and 'unstained' cells, which are uncharacterized. The authors present some arguments as to why these would not contribute to 'transferred' reads, but given the low level of detectable transferred RNAs, and the unclear origin of these components, whether they influence the results could be debatable. Furthermore, the small number of replicates (2 replicates for the genome-wide studies and 1 replicate for most of the subsequent experiments) minimizes the confidence in the conclusions. In this context, it is also notable that the profile of transferred RNAs between the two replicates of co-cultured samples appears quite different by PCA analysis. It is thus conceivable that there might be specificity in the RNA 'transferome', influenced by unknown experimental variables, which is though masked when averaging those samples in subsequent analyses.

While the manuscript emphasizes the role of TNTs in RNA transfer, the actual involvement of TNTs relies solely on the observation that potential TNTs form between co-cultured cells. Other means of transfer, such as through engulfment or phagocytosis of cell fragments, could still possibly contribute. Furthermore, the dependence of mRNA transfer on direct cell-to-cell contact is demonstrated for 5 RNAs and extrapolated to transcriptome-wide RNA transfer, an assumption which might, or might not, be valid.

Finally, the results on gene expression changes induced by co-culture (Figures 7, 8) are of unclear relevance. As the authors point out, it is uncertain whether RNA transfer or other paracrine or adhesion-mediated signaling events, underlie these changes. It is therefore not easy to see how these results relate to the rest of the presented work. Furthermore, while the authors expand on the potential significance of changes observed in genes related to cancer-associated fibroblasts or to immunity-related genes, these remain speculative and untested.

Overall, the manuscript presents evidence indicating that RNA is transferred non-selectively in co-cultured cells, under specific conditions and between the cell types tested. The impact of the work is reduced by the lack of mechanistic understanding underlying this transfer and the uncertainty of whether this phenomenon has any subsequent physiological relevance.

Reviewer #2 (Public Review):

This manuscript by Laturney et al. has found a previously uncharacterized neural link between female mating status and upregulation of sugar intake in the common fruit fly, Drosophila melanogaster. Although mated female flies have been known to increase both yeast and salt intake compared to virgin females, changes in sugar intake have not been previously described. Using quantitative monitoring of food intake, functional calcium imaging, connectome tracing, and neuronal manipulations, authors convincingly demonstrated that the Sex Peptide sensory neurons (SPSN) and their downstream neural circuit control the activity of female-specific Lgr3 neurons in a mating-dependent manner. In virgin females, the SPSN circuit (including its output pCd-2 neurons) is active, which is predicted to inhibit hunger-promoting Lgr3 neurons. After mating, the SPSN circuit becomes silent, which should disinhibit Lgr3 neurons. Indeed, they found that optogenetic silencing of pC2-d neurons promoted sucrose consumption. The newly characterized pCd-2 neurons are sexually dimorphic, consistent with their role in female-specific post-mating modulation of sucrose consumption.

Aside from the novelty of the mating-dependent changes in sugar intake, an exciting discovery of the current study is that separate circuits control different aspects of post-mating behavioral changes (increased egg-laying, mating rejection, increased sugar consumption). This finding illustrates a general neural mechanism by which a single "internal state" exerts its influences on multiple behaviors via branches of circuits from a hub for the given state (pC1 for the female mating status), which is a powerful mechanistic model for other internal states.

The high-quality data based on elegant yet rigorous experiments deserve praise as a textbook example. They presented multiple independent lines of evidence to demonstrate the function of each component of the SPSN circuit over the sucrose consumption Lgr3 neurons, which convincingly proves that the pCd-2a/b neurons transmit information of mating status to a hunger-controlling hub. Experiments have been exceptionally rigorous. Genetic manipulations were performed with multiple controls. They used multiple split GAL4 lines to target specific classes of neurons to eliminate the neuronal off-target effect. They also used multiple types of feeding assays to clarify the feeding phenotype induced by mating. Overall, the scientific rigor of this work sets a standard for researchers in the field to follow.

That the activity levels of pCd-2 neurons and their downstream Lgr3 neurons are indeed influenced by mating has not been directly tested. Since multiple previous publications consistently demonstrated that the SPSN-SAG-pC1 axis is suppressed by the Sex Peptide, the authors' conclusion that pCd-2 neurons are suppressed after mating (for example, see line 319) is very likely correct. However, what the authors showed was that silencing of the SPSN circuit "can" increase sucrose consumption in virgin females. To what extent mating suppresses pCd-2 neurons (and disinhibits Lgr3 neurons) remains uncharacterized. The inhibition exerted by the Sex Peptide is likely partial, which might not be precisely recapitulated by the optogenetic silencing. Mated female flies show an increased preference for protein and salt. The authors' finding that they also increase sugar consumption after mating indicates that mating causes a substantial change in female feeding patterns. The current work elevates the value of Drosophila as a neurogenetic model to understand how the nervous system achieves the complex tasks of nutritional homeostasis after mating, which dramatically alters the energy allocation in many species (including mammals). Data presented in this work will advance our understanding of how females coordinate feeding priorities in a face of changing nutritional demands after mating, which is one of the fundamental questions in neuroscience.

Reviewer #2 (Public Review):

The large genetic association studies conducted in East Africa have shown that the Dantu blood group provides substantial protection against severe malaria. The proposed mechanism of protection is reduced red cell invasion resulting in reduced parasite multiplication. This hypothesis was tested in adult Kenyan volunteers infected with P. falciparum under careful monitoring. The strength of the study is that the CHMI model using a single parasite strain has few confounders and it provides a very clear answer. The data reported on the other "protective" genetic polymorphisms is also fascinating. The hypothesis that Dantu reduces merozoite invasion has some support from previous laboratory studies, but it would be useful to confirm, once invasion is successful, that intraerythrocytic growth is unimpaired (e.g. count merozoites per schizont, measure asexual cycle length etc).

Reviewer #2 (Public Review):

In this manuscript, Seelbinder et al, introduce a novel, elegant approach to study the organization of cell nuclei, which complements currently existing technology. The authors employ localized temperature gradients to move chromatin inside the nucleus noninvasively, and they study flow fields and deformations of different nuclear compartments in different experimental settings. The study is timely and should be of broad interest to a wide readership, in particular since the method can also be applied to study mechanical relationships of subcellular compartments in other cellular and extracellular systems.

The non-invasive manipulation of cell organelles in intact cells has been a challenge for decades. The new technique introduced in this study contributes to closing this important gap, enabling experiments to better understand spatial and mechanical relationships between different cell compartments. This study is a very nice example of how concepts and approaches from physics can be exploited to better understand biology.

Reviewer #2 (Public Review):

Ramesh, Liu et al. investigated the dynamics of the histone H3 lysine 27 trimethyl mark (H3K27me3) in the cerebellum during postnatal development. They profile the mark and measure gene expression at three time points (P7, P14, P60) to show that there is a global increase in the amounts of H3K27me3 genome-wide, but a generalized loss of the mark at promoters. This loss is associated with neuronal genes that become expressed in the mature cerebellum. Through conditional knockout and transcription factor analysis, they implicate the autism-associated lysine demethylase gene KDM6B in the removal of H3K27me3 at genes that become active postnatally and show that the ZIC transcription factors are candidates to mediate some of these effects. They then use pharmacologic inhibition of KDM6B and the PRC2 component, EZH2, in a granule neuron culture system to further dissect the function of these enzymes in H3K27me3 dynamics.

The authors employ multiple genomic methods to carry out rigorously controlled experiments and their conclusions are well supported by the data. The study provides fundamental insights into the dynamics of H3K27me3 during the postnatal development of circuits in the brain. In particular, the findings that substantial changes in the H3K27me3 mark continue through the later steps of cerebellar maturation (P14 to P60) and that the autism-associated gene KDM6B is involved in this process, will be of significant interest to the field.

The study has some limitations with regard to scope and mechanism. For example, given the importance of enhancers in the regulation of gene expression, the omission of any analysis of H3K27me3 at defined enhancer elements is a limitation of the study. In addition, while the observations supporting the role of ZIC proteins in the removal of H3K27me3 during gene activation are interesting, the lack of direct mechanistic analysis investigating this biology limits the strength of the conclusions that can be made about the direct function of these factors in H3K27me3 dynamics.

Reviewer #2 (Public Review):

In this work Ushio et al. combine environmental DNA metabarcoding with novel statistical approaches to demonstrate how fish communities respond to changing sea temperatures over a seasonal cycle. These findings are important due to the need for new techniques that can better measure community stability under climate change. The eDNA metabarcoding dataset of 550 water samples over two years is, I feel, of sufficient scale to provide power to detect fine-scale ecological interactions, the experiments are well controlled, and the statistical analysis is thorough.

The major strengths of the manuscript are: (1) the magnitude of the dataset, which provides densely replicated sampling that can overcome some of the noise associated with eDNA metabarcoding data and scale up the number of data points to make unique inferences; (2) the novel method of transforming the metabarcode reads using endogenous qPCR "spike-in" data from a common reference species to obtain estimates of DNA concentration across other species; and (3) the statistical analysis of time-series and network data and translating it into interaction strengths between species provides a cross-disciplinary dimension to the work.

I feel like this kind of study showcases the power of eDNA metabarcoding to answer some really interesting questions that were previously unobtainable due to the complexities and cost of such an exercise. Notwithstanding the problems associated with PCR primer bias and PCR stochasticity, the qPCR "spike-in" method is easy to implement and will likely become a standardised technique in the field. Further studies will examine and improve on it.

Overall I found the manuscript to be clear and easy to follow for the most part. I did not identify any serious weaknesses or concerns with the study, although I am not able to comment on the more complex statistical procedures such as the "unified information-theoretic causality" method devised by the authors. The section on limitations of the study is important and acknowledges some issues with interpretation that need to be explained. The methods, while brief in parts, are clear. The code used to generate the results has been made available via a GitHub repository. The figures are clear and attractive.

Reviewer #2 (Public Review):

The authors convincingly show multiple inner and outer leaflet non-protein (lipid) densities in a cryo-EM closed state structure of GLIC, a prokaryotic homologue of canonical pentameric ligand-gated ion channels, and observe lipids in similar sites during extensive simulations at both resting and activating pH. The simulations not only corroborate structural observations, but also suggest the existence of a state-dependent lipid intersubunit site only occupied in the open state. These important findings will be of considerable interest to the ion channel community and provide new hypotheses about lipid interactions in conjunction with channel gating.

Reviewer #2 (Public Review):

This is an interesting study from Admin Peng's laboratory that builds on previous work by the PI implicating Greatwall Kinase (the mammalian gene is called MASTL) in checkpoint recovery.

The main claims of this study are:

1) Greatwall stability is regulated by the E6-AP ubiquitin ligase and this is inhibited following DNA damage in an ATM dependent manner.<br /> 2) Greatwall directly interacts with E6-AP and this interaction is suppressed by ATM dependent phosphorylation of E6-AP on S218<br /> 3) E6-AP mediates Greatwall stability directly via ubiqitylation<br /> 4) E6-AP knock out cells show reduced ATM/ATR activation and quicker checkpoint recovery following ETO and HU treatment<br /> 5) Greatwall mediated checkpoint recovery via increased phosphorylation of Cdk substrates

In my opinion, there are several interesting findings presented here but the overall model for a role of the E6-AP -Greatwall axis is not fully supported by the current data and will require further work. Moreover, there are a number of technical issues making it difficult to assess and interpret the presented data.

Major points:

1) The notion that Greatwall is indeed required for checkpoint recovery hinges on two experiments shown in Figures 5A and B where Greatwall depletion blocks the accumulation of HELA cells in mitosis following recovery from ETO treatment and in G2/M following release from HU. An alternative possibility to the direct involvement of Greatwall in checkpoint recovery could be that Greatwall in HeLA cells is required for S-phase progression (as for example Charrasse et al. suggested). A simple control would be to monitor the accumulation of mitotic cells by microscopy or FACS following Greatwall depletion without any further checkpoint activation.

2) The changes in protein levels of Greatwall and the effects of E6-AP on Greatwall stability are rather subtle and depend mostly on a qualitative assessment of western blots. Where quantifications have been made (Figures 2D and 4F) the loading control and the starting conditions for Greatwall (0 timepoints in the right panel) appear saturated making precise quantification impossible. I would argue that the authors should at least quantify the immuno-blots that led them to conclude on changes in Greatwall levels and make sure that the exposure times used are in the dynamic range of the camera (or film). A more precise experiment would be to use the exogenously expressed CFP-Greatwall that is described in Figure 6 and measure the acute changes in protein levels using quantitative fluorescence microscopy in live cells. This is, in my opinion, a lot more trustworthy than quantitative immuno-blots.<br /> I also note here that most experiments linking Greatwall levels to E6-AP were done using siRNA, while the E6-AP ko cells would be a more reliable background for these experiments, especially with reconstituted controls.

3) This study has no data linking the effects of Greatwall to its canonical target PP2A:B55. The model shown in Figure 9 is therefore highly speculative. The possibility that Greatwall could act independently of PP2A:B55 should at least be considered in the discussion given the lack of experimental evidence.

4) The major effect of E6-AP depletion on the checkpoint appears to be a striking reduction in ATM/ATR activation, suggesting that this ubiquitin ligase is involved in checkpoint activation rather than recovery. It is not clear if this phenotype is dependent on Greatwall. If so it would be hard to reconcile with the default model that E6-AP acts via the destabilisation of Greatwall. In the permanent absence of E6-AP, increased Greatwall levels should inactivate B55:PP2A. How would this lead to a decrease in ATM/ATR activation? This is unlikely, and indeed Figure 5E shows that the reduction of MASTL in parallel to E6-AP does not result in elevated levels of ATR/ATM activation. Conversely, the S215A E6-AP mutant does have a strong rescue impact on ATR/ATM (Figure 8D).

5) In summary, I do not think that the presented experiments clearly dissect the involvement of E6-AP and Greatwall in checkpoint activation and recovery. E6-AP depletion has a strong effect on checkpoint activation while Greatwall depletion is likely to have various checkpoint-independent effects on cell cycle progression.

Reviewer #2 (Public Review):

This paper describes a novel synthetic lethal interaction between BRCA2 loss and the cytokinesis regulators, including ROCK. The SL effects are restricted to short-term in vitro assays, and the underlying mechanisms remain largely elusive. The impact of the work in its current form is limited.

Strengths:<br /> - A novel synthetic lethal (SL) interaction, which appears independent from the -BRCA2 SL interaction that depends on replication fork stalling and DNA damage induction.<br /> - The SL interaction is validated in a panel of genetic models of BRCA2 deficiency.<br /> - The SL interaction can be induced using clinically approved agents.

Weaknesses<br /> - The evidence that this SL interaction is independent of replication defects is not solid.<br /> - The SL interaction is based on chemical inhibitors only, with 6 out of 9 ROCK inhibitors not demonstrating the SL interaction.<br /> - The mechanisms by which ROCKi specifically affects BRCA2-defective cells are elusive.<br /> - It remains unclear what the cause of the multiple mitotic defects is.

Combined, it remains unclear if the identified SL interaction is therapeutically meaningful. Clinical stage inhibitors are available, and various BRCA2-deficient cancer models have been described, allowing the authors to address this in long-term in vitro assays and in vivo assays. Also, the authors describe multiple phenotypic consequences, but the order of events and the reason why the effects are specific to BRCA2 remain largely unclear. Furthermore, the notion that the observed effects are independent of replication defects requires further substantiation.

Reviewer #2 (Public Review):

Zheng et al., investigated the molecular and functional mechanisms of two homeodomain missense mutations causing human retinal photoreceptor degeneration diseases in photoreceptor development regulated by the CRX transcription factor. They analyzed the E80A mutation associated with dominant cone-rod dystrophy (CRD) and the K88N mutation associated with dominant Leber Congenital Amaurosis (LCA). The authors found that E80A CRX binds to the same target DNA sites as WT CRX, but the binding specificity of K88N CRX is altered from that of WT in an in vitro assay. They generated Crx(E80A) and Crx(K88N) KI mice and performed ChIP assay and observed that K88N CRX binds to novel genomic regions from the WT-binding sites, while E80A binds to the WT sites. In addition, using the KI mice, they found that E80A and K88N differently affect the expression of Crx target genes. This study is well executed with proper and solid methodologies, and the manuscript is clearly written. This study gives us the insights how single missense CRX mutations lead to different types of human retinal photoreceptor degeneration diseases.

While the study has strengths in principle, it has a couple of weaknesses. One is how well E80A KI mice function as a pathological model of dominant CRD, in which cones are mainly first affected, is not clearly shown in this study. More data investigating how cones are affected by performing histological, molecular, and physiological analyses will be helpful and useful. For example, in the Discussion, the authors describe that E80A associates with S-cone opsin promoter results is "data now shown". This data must be presented for the readers. In addition, more molecular insights as to how E80A affects cones will strengthen this study. Another point is that it will be very valuable if the authors could show how E80A and K88N differently affect the 3D structure of the CRX homeodomain. Even a simulation model would be valuable.

Reviewer #2 (Public Review):

In this work from Zhou et al., the authors address mechanisms of mitotic chromosome size scaling during development. Their approach, which employs complementary use of in vivo (Xenopus embryos) and in vitro systems (Xenopus extracts), rendered investigation of this relationship experimentally tractable and allowed the authors to convincingly demonstrate that mitotic chromosome scaling is mediated by differential loading of maternal chromatin remodeling factors during interphase. The authors show that this scaling is dependent on an increasing nucleo-cytoplasmic (N/C) and that condensin I is titrated away from chromosomes as the N/C ratio is increased. Interestingly, the authors found that spindle and nuclei did not scale with changes in N/C ratio, suggesting that although mitotic chromosome scaling correlates with spindle and nuclei scaling, it is mechanistically distinct. Complementary Hi-C analyses of chromatin architectures of both larger condensin I-rich chromosomes and smaller condensin I-poor chromosomes support a condensin-based looping model to explain the inverse relationship between chromosome-associated condensin and chromosome length, however, this model seems somewhat contrived due to inherent limitations of the approach. A characterization of an independent importin-α-dependent mitotic chromosome scaling mechanism, though potentially interesting, is too premature to be included and a bit of a non sequitur in terms of the overarching narrative and major findings of the work. Though there is some room for improvement in terms of image analysis and measurements, the work is well-written, comprehensive in scope, and addresses a fundamental biological question. Furthermore, the authors' major conclusions and substantive claims are well-supported by the experimental results.

Reviewer #2 (Public Review):

The Covid-19 pandemic has had major adverse impacts on cancer screening globally. Despite this, most prior reports have not included observations from LMICs. This paper aims to address this important gap.

Because comparable data were not available across the countries reported here, comparisons would not be appropriate, so the authors chose a case study design, which was a prudent decision and a strength of the work.

The authors make use of data from IARC's CanScreen5 reporting system, which is completely appropriate. In addition, this aspect serves to demonstrate the usefulness of the CanScreen5 system, as it can be used to support this type of study. National data were not available in all countries.

The main findings in the paper describe the early impact of the Covid-19 pandemic on cancer screening participation for the screening programs reported on in the 6 countries that were selected.

I would anticipate that, having demonstrated that this type of case study focusing on cancer screening in LMICs is feasible, this would encourage others to conduct further studies among LMICs, which would be welcomed by the field.

Reviewer #2 (Public Review):

The abstract and introduction framework asserts that ketamine's enhancement of excitatory synaptic drive in the hippocampus is presumed to underlie its rapid antidepressant effects. This is not the only, and perhaps not the primary effect mechanism suggested by prior experiments, also strongly implicating disinhibitory effects in the prefrontal cortex as necessary and sufficient to mediate antidepressant effects. Nevertheless, it is valuable to seek mechanistic motifs that provide multiple paths for explaining the seemingly counterintuitive effects where NMDAR blocker enhances excitatory transmission. These need not be conserved across brain regions and cell classes. The primary result of this study demonstrates that 1 hr-long ketamine application to cultured cells reduces calcineurin and GCaMP activity to elevate AMPA receptor subunit GluA1 phosphorylation and enhance the expression of Ca2+-permeable, GluA2-lacking (CP-)AMPARs. These observations are then evaluated in vivo, where calcineurin shows a similar response to ketamine and CP-AMPAR antagonist-abolished ketamine effects on behavior in the open field and tail suspension tests. One significant uncertainty this study helps resolve is whether GluA2-containing AMPARs are removed from synapses or whether GluA2-lacking AMPARs are inserted following ketamine administration. GCaMP imaging, FRET and glutamate uncaging assays provide a strong complement to biochemistry and in vivo data. There are several significant technical and conceptual limitations in this work, which substantially limit the extent of conclusions that can be drawn at this point.

1. The age of neurons in cell culture experiments was 14 days in vitro (DIV), representing developing cultures that are just starting to form synapses. How these effects carry over to more mature cultures or adult animals is unclear.

2. Phosphorylation analyses, forming the foundation of this work, are carried out 1 hr after ketamine treatment. This is prior to the observed clinical effects of ketamine and this point should be acknowledged. Whether and how long this effect lasts remains to be examined. If the goal is to highlight the earliest likely effects of ketamine that should precede potential clinical effects, this should be acknowledged, and in that case, the onset of effects should be clarified. At this point, the temporal features remain undersampled, with a single time-point.

3. A lower dose (50%) treatment was used to evaluate potential sex differences in ketamine effects, which is not sufficiently justified, except post hoc based on behavioral data. The discussion section does consider potential factors that can account for observed differences.

4. The 1-hr timeline to behavioral testing is fast, relative to clinical effects on behavior as well as behavioral effects measured in most studies using mouse models.

5. Tail suspension test is broadly acknowledged as an inadequate model of antidepressant effects.

6. There is no evidence from the in vivo experiments that effects in the hippocampus are due to direct actions of ketamine, as those reported for the cell culture studies. Intraperitoneal injections cannot be used to localize primary effects in vivo to the hippocampus, which would require local delivery.

7. If (MNI)-caged L-glutamate was used at 1 μM concentration, as stated in methods, this is considerably below typical concentrations reported in the literature.

Reviewer #2 (Public Review):

In this paper by Banerjee et al., the authors described the potential role of two universal stress proteins in M. smegmatis and M. tuberculosis in regulating intracellular free cAMP concentration, which was a unique observation. The experiments were logically designed to prove the expression and interactions; it would have been worthwhile to explore beyond to gain an insight into how the changing levels of free cAMP could modulate any key phenotypes in the bacteria such as virulence, antibiotic resistance, etc. in the content of knockout/knockdown and overexpression of MSMEG_3811 and Rv1636 in individual organisms. The preliminary data of natural inhibitor STOCKIN43384 impacting the survival of M. smegmatis was interesting, but authors need to prove the MOA by using knockdown and overexpression strains of Rv1636.

Reviewer #2 (Public Review):

The evolution and control of the three-part life history of holometabolous insects have been controversial issues for over a century. While the functioning of broad as a master gene controlling the pupal stage and of E93 as a master gene for the adult stage has been known for about a decade or more, chinmo has only recently been proposed as being the master gene responsible for maintaining the larval stage (Truman & Riddiford, 2022). While the former paper focused on the embryonic and early larval function of Chinmo, this paper explores its metamorphic effects and defines the roles of Broad and E93 in the phenotypes produced by manipulations of Chinmo expression.

Overall, the paper is well presented but in places, readers would be helped if the authors were more explicit about the logic and details of their manipulations. There are a couple of conceptual issues that the authors should address.

The role of Broad in larval tissues:<br /> One intriguing issue relates to the relationship of Chinmo to Broad and E93 in larval versus imaginal tissues prior to metamorphosis. The knock-down of chinmo in imaginal discs results in severe suppression of growth and the lack of metamorphic patterning genes such as cut and wingless. Normal growth and patterning are reestablished though, if broad is also knocked-down, supporting the notion that the effects of the lack of Chinmo are mediated through the premature expression of Broad.<br /> In the salivary glands, by contrast, chinmo knock-down suppresses growth, and this growth suppression is not reversed by simultaneous broad knockdown. They properly conclude that the role of Chinmo in supporting the growth of larval tissues does not involve Broad, but their data on the expression of salivary gland proteins suggest that Broad still plays some role in Chinmo function in salivary glands. Fig. 5E shows the levels of various salivary glue proteins in the glands of Chinmo knock-down larvae. The levels are reduced, as expected by the lack of salivary gland growth, but a significant finding is that they are there at all! The Costantino et al. (2008) paper shows that these genes are only induced in the mid-L3. Ecdysone, acting through Broad isoforms, is necessary for their appearance and these SGS genes can be induced in the L1 and L2 stages by ectopic expression of some Broad isoforms. Their low levels in Fig 5, would be due to the small size of the gland, but the gland's premature expression of Broad likely causes their induction. In larval cells, then, Chinmo may feed into two parallel pathways, one that does not involve broad and regulates growth and the other, utilizing Broad, regulating premetamorphic changes.<br /> It would be useful to look at early larval salivary gland proteins such as ng-1 to -3 that are expressed in salivary glands before the critical weight. Also, it would be interesting if the appearance of the SGS proteins after chinmo knock-down (Fig 5E) is abolished by simultaneous knock-down of broad.

Role of Chinmo and Broad in Hemimetabolous insects:<br /> In the conclusion of their comparative studies on the cockroach (line 342), the authors state that Broad exerts no role in the development of hemimetabolous insects. However, this conclusion is not consistent with the literature. The first study of broad knockdown in a hemimetabolous insect was in the milkweed bug Oncopeltus fasciatus by Erezyilmaz et al. (2006). Surprisingly to Erezyilmaz et al., broad knock-down in early-stage nymphs did not cause premature metamorphosis. However, Broad expression was essential for tissues of the wing pads and dorsal thorax to undergo morphogenetic growth (rather than simple isomorphic growth), and for stage-specific changes in coloration through the nymphal series (but not for the nymph to adult color change). A similar function for Broad on wing growth during the later nymphal stages was later shown in Blattella (Fernandez-Nicolas et al., 2022; Huang et al., 2013). The wing- and genital pads represent "imaginal" tissues in the nymph and the need for Broad in these tissues are the same as seen in imaginal discs as the latter shift from isomorphic growth to morphogenesis at the critical weight checkpoint in the L3.<br /> This would suggest that important roles for Broad and E93 are already established in the hemimetabolous insects with E93 controlling the shift from immature (nymphal) to adult phenotypes and Broad controlling the premetamorphic growth of imaginal tissues in early-stage nymphs. Chinmo might then be needed to keep both in check.

Reviewer #2 (Public Review):

Like humans, Bengalese finches rely on auditory feedback to maintain the acoustic stability of their learned vocalizations, and deafening causes acoustic degradation of their songs. How disruptions to sensory input alter gene expression in brain regions important for singing and song learning remains relatively unexplored. The authors develop an innovative serial laser capture RNA-sequencing method, which allows them to conduct large-scale analyses of gene expression in spatially defined singing-related regions, as well as in surrounding non-singing-related regions. These methods are used to demonstrate that deafening preferentially alters gene expression in song-related regions relative to surrounding song-related areas, and that deafening reduces correlations in gene expression between connected song-related regions. The authors then compare their findings to a previous single-cell RNA sequencing dataset to determine the cell types whose gene expression is likely to be most strongly affected by deafening and song degradation. Finally, the authors repeat their measurements of gene expression changes in RA following unilateral lesion of LMAN and find that LMAN lesions have the largest effect on groups of genes whose expression was also strongly affected by deafening. The study is elegant and rigorous, and its conclusions are well-supported. This work reveals candidate genes that may play a role in stable vocal performance and whose changes in expression may contribute to the acoustic degradation of vocal performance following deafening.

Reviewer #2 (Public Review):

Neurons of the inferior olive exhibit strong subthreshold oscillations, and drive complex spiking through climbing fiber synapses onto Purkinje cells in the cerebellar cortex. This activity plays an essential role in coordinating motor control and the induction of cerebellar plasticity. In this study, the authors make use of optogenetic and electrophysiological approaches to examine the interplay between intrinsic oscillations and two important excitatory and inhibitory input populations to the inferior olive. The authors show that excitation is enhanced when it occurs in the rebound phase of the preceding inhibition. Using a computer model, the authors also show that enhanced excitation can effectively recruit larger populations of neurons, presumably through gap junctional coupling. The strengths of the study include the authors' ability to independently control both excitatory and inhibitory pathways, as well as the rigorous and systematic examination of input timing and amplitude and their effects on spike output. There were some weaknesses; high variability in cell resting potentials raised questions about how cell health impacted the findings, and there needed to be better documentation of recording conditions and parameters. There also needed to be a more extensive discussion about the nature of input timing and frequency under behaviorally relevant conditions. Given these relatively minor issues, the study provides new insight and depth into synaptic integration in the inferior olive and adds to our understanding of how input timing is translated into climbing fiber signals.

Reviewer #2 (Public Review):

The authors report a study comparing self-reported stable and unstable knees with total knee arthroplasty. Advanced imaging methods (dynamic fluoroscopy with model-image registration) and analysis of muscle activities were used to characterize the study subjects during three ambulatory activities (level gait, downhill walking, and stair descent).

The subject cohort all received one design of TKA in a similar time period. The unstable subgroup was >60% female, while the stable knee cohort was 70% male, which is a notable limitation. The measurement methods are state-of-the-art and expertly applied.

The results suggest there may be measurable differences in knee kinematics in subjects with unstable knees, but this was not strongly supported across groups. Rather it was highlighted in 3 individuals who self-reported instability during the test session. The muscle activity analysis supports there being differences between the stable and unstable knee groups.

Despite the limitations of a small subject cohort with only a single TKA design, the study highlights important methods that appear suitable to further study the factors contributing to clinical dissatisfaction with TKA as it relates to joint stability and function during ambulatory tasks.

Reviewer #2 (Public Review):

In this manuscript, Villalobos-Cantor et. al. described a new technique for cell-type specific in vivo labeling of nascent peptides, which they call POPPi. POPPi is based on sequence-independent incorporation of the puromycin analog OPP into an elongating peptide, which also simultaneously terminates the growing peptide. To achieve cell-type-specific labeling, the authors used an OPP derivative, PhAc-OPP, as the labeling substrate. PhAc-OPP contains a blocking group that prevents it from incorporating into the growing peptide, and the blocking group can be cleaved off by the enzyme PGA, which is expressed in the cell type of interest.

The authors validated POPPi in different cell types in the Drosophila brain and showed that this method could be used to image general translation or to biochemically enrich nascent peptides in a cell-type-specific manner. They also showed that with an optimized labeling protocol, it is possible to achieve efficient labeling with minimum effect on animal viability and health. The authors further used POPPi to provide independent support for a previously known phenomenon: age-dependent decline in general translation in the neurons. The results of this work are solid, and the main conclusions are well supported by the data presented. The manuscript is very well written with a clear logic flow and is very easy to read.

What is less clear is how generally useful POPPi will be to the community. The authors pointed out two major cell-type specific applications of POPPi, 1) imaging general translation and 2) biochemically purifying nascent peptides. For application #1, although POPPi might be a more desirable method in some cases, a combination of non-cell-type specific labeling using OPP, and marking the cell type of interest by a fluorescent protein might be simpler. Because labeling with OPP eliminates the enzymatic step that converts non-reactive PhAc-OPP to reactive OPP, the labeling kinetics can be improved, and the toxicity associated with PGA expression can be avoided. For application #2, a currently widely used strategy for a similar purpose is various types of ribosome profiling techniques. Ribosome profiling may be easier to perform than POPPi, and because proteins cannot be amplified, a very large quantity of starting materials will be needed if one wants to use POPPi to characterize cell-type specific nascent proteome. In fact, in this manuscript, the authors used western blots to detect candidate proteins and did not use mass spec to characterize the nascent proteome.

Reviewer #2 (Public Review):

The work from Nakajima-Takagi et al describes the phenotypes and study of a PCGF1 mutant mouse model. PCGF1 is a core component of the non-canonical PRC1.1 complex and specific functions of this complex in hematopoiesis. Using somatic inactivation models, the authors demonstrate that the acute deletion of PCGF1 from adult hematopoiesis leads to a progressive myeloid bias in the bone marrow and peripheral blood. This occurs at the expense of the HSPC compartment, with a reduction in all populations and of the lymphoid committed populations. The myeloid bias is cell intrinsic, as competitive transplant of the PGCF1 deficient bone marrow recapitulates the phenotype. The effect is not due to exhaustion or loss of self-renewal of the HSCs.<br /> To understand the basis for the myeloid bias, the authors first assessed transcriptome signatures and see a shift in gene expression programs related to myeloid development and targets of the key myeloid transcription factor C/EBPa. Further analysis demonstrated an increased expression of Cebp1 in the PCGF1-deficient LSK cells. Reducing the expression of Cebpa could modify the myeloid skewing of Pcgf1 deficient cells in culture. This de-repression of Cebpa correlates with changed local H2AK119ub1 levels in the HPSC populations.

Additional studies assessed how the loss of Pcgf1 changed the response to hemoablation, in this instance with a single dose of 5-FU. This study coupled with scRNA-seq suggested that PRC1.1 was important in regulating the GMP populations, potentially through a self-renewal program. This led to a focussed analysis of the GMPs, with evidence for altered Hoxa9 and b-catenin levels contributing to the altered GMP behaviours. Both have been implicated and demonstrated to have functional roles in these programs in other studies.

Finally, ageing of Pcgf1 deficient mice demonstrated that these mice were predisposed to developing T-ALL and MPN. The authors provide a characterisation of these moribund states and their phenotypes are consistent with the diagnosis.

Overall the work demonstrates a specific requirement for Pcgf1, and therefore PRC1.1, in the regulation of hematopoiesis. I think the authors largely achieved the aims and the results are supportive of the conclusions. The work shows myeloid bias, experimental evidence that this is due to a derepression of a myeloid lineage program in the HPSC and associated chromatin changes, and functions for Pcgf1 in both hematopoietic regeneration and malignancy. This suggests a unique role for non-canonical PRC1.1 compared to canonical PRC 1.

Strengths:<br /> - in vivo experiments and evidence;<br /> - multiple lines of evidence supporting the conclusion;<br /> - mechanistic studies provide direct evidence of the proposed mechanism.

Weaknesses:<br /> - can the authors demonstrate normal maturation of the myeloid lineages as this would be important to differentiate between myeloid bias and a block in myeloid differentiation? This is important to distinguish between.<br /> - include analysis of mature myeloid cells and FACS plots to allow assessment of maturation.

Reviewer #2 (Public Review):

In this manuscript, Niethamer et al. investigate the role of the transcription factor ATF3 in lung regeneration after H1N1 influenza. They focus on endothelial ATF3 which is present in a subset of lung capillaries in the adult mouse lung. Interestingly, they found that influenza infection upregulates endothelial ATF3 and that endothelial deletion of Atf3 results in impaired regeneration, leading to enlarged airspaces after viral infection. They further show that this effect may be due to an increase in apoptosis and a decrease in proliferation, suggesting that endothelial ATF3 is necessary for pulmonary vascular regeneration, as well as recovery of the alveolar architecture.<br /> Given the recent publications in the field describing lung endothelial heterogeneity, as well as its possible role in injury repair, this work is relevant to the community. It also supports the idea that epithelial-endothelial crosstalk is important for lung regeneration and proposes a potential candidate for this process.

Strengths:<br /> The authors identified and tested the role of endothelial Atf3 in lung regeneration using well-established techniques. They identified this transcription factor as a candidate using state-of-the-art scRNA-seq. They also carefully lineage traced ATF3 expressing cells using an inducible reporter before and after infection and then used a pan-endothelial driver Cdh5 to delete Atf3 specifically in the endothelium. Thus, the authors successfully show significant changes in the alveolar structure after infection in their mutant model.

Weaknesses:<br /> Although there is evidence that the author's claims have biological relevance, this paper would benefit from strengthening and/or clarifying some things:

• The scRNA-seq analysis is performed in two separate objects ("control lung" and "H1N1 infected lung 14dpi"). For these two sets of data to be comparable, the authors should have integrated the objects and analyzed them together. This is not only important for deciding the clusters' identities and making sure that the same clusters are compared between control and infected, but also necessary to compare gene expression.<br /> • ATF3 is not only present in Cap1_B, in the infected lung there seems like Cap1_A express less ATF3. The authors should comment on this difference.<br /> • It is unclear how the clusters Cap1_A and Cap1_B were decided. The manuscript would benefit from clarification.<br /> • It would be beneficial to see via immunofluorescence the morphological and spatial differences between ATF3-expressing and non-expressing endothelial cells since this transcription factor is expressed in multiple endothelial cell types.<br /> • The authors mention ATF3 is not endothelial-specific. Expression of ATF3 in other cell types should be evaluated via immunofluorescence.<br /> • The authors should have shown evidence of the deletion in their Atf3EC-KO mouse and addressed whether they had residual ATF3. If there is no antibody available, RNAscope could be used, or Western Blot or RT-PCR on sorted endothelial cells.<br /> • The authors only show the epithelium as evidence that the alveolar region is altered in their mutant after infection. The endothelium should have also been investigated, especially since their mutant is an endothelial-specific deletion. Within this, the different endothelial cells should have been assessed by a method other than RNAscope such as immunofluorescence, given that this method is unable to show morphology and there are antibodies available.<br /> • Bulk RNA-seq from endothelial cells is used in the manuscript. However, because ATF3 is not specific to Cap1_B cells or even capillaries alone, the downstream gene expression analysis of bulk RNA should be placed into the context of lung endothelial heterogeneity.<br /> • Although the authors mentioned that the infection with H1N1 influenza can have regional differences, they do not show how they picked regions for their analysis and quantification, and whether ATF3 upregulation was found in more severely affected regions. Furthermore, since they quantified via FACS, this heterogeneity in the infection itself could have affected their observations.

Reviewer #2 (Public Review):

1) A detailed step-by-step approach to validation of some previously known outcomes.<br /> 2) Useful for more focus to be placed on data from the second half of the paper.<br /> 3) Some reflection on the media used to study paracrine effects is needed - more experiments here would be beneficial.<br /> 4) Path clamp experiments - how does bath solution alter the effect of any limited paracrine effect - we are removing cells from the treatment media and putting them in physiological solutions - an opportunity to recover?

Reviewer #2 (Public Review):

This study assessed the inflammatory and metabolic profiles of a healthy sub-Saharan Africa (Tanzania) population versus a healthy population outside Africa (Dutch). Using plasma samples from these cohorts, an O-Link proteomics inflammatory panel and targeted metabolomics platforms were utilised. The study shows that 'healthy' Tanzanians display an enhanced pro-inflammatory phenotype versus Dutch volunteers. Specific pathways and metabolites identified included - increase activation of the Wnt/Beta catenin pathway, and the metabolites 4E-BP1 and FGF21. The study highlights some interesting findings regarding the impact of diet on inflammatory pathway activation.

Major Strengths & Weaknesses - This is an interesting study and approach that aims to address some challenging questions in underrepresented populations. The findings demonstrate the importance of diet and dietary interventions on metabolic health, as well as key inflammatory proteins. It does raise the question whether anti-inflammatory therapies need to be targeted to specific at-risk populations, more so than other populations.

Impact - The study demonstrates the importance of considering differences between populations and the inclusion of underrepresented populations in such studies. The data suggests that lifestyle changes in sub-Saharan Africa are potentially contributing to altered inflammatory and metabolic profiles. Thus, health initiatives advocating traditional diets may alleviate the NCD epidemic in sub-Saharan Africa.

Reviewer #2 (Public Review):

The authors combine NMR experiments, cell experiments, and molecular simulations to address the question of how lipid interactions of the prolactin receptor contribute to signalling. They assess the interactions of the disordered cytoplasmic tail of the receptor with phosphoinositides among others by chemical shift perturbations from NMR for different PIP2-containing membranes, by coarse-grained simulations, as well as site-directed mutagenesis and subsequent cell signalling experiments to monitor the activation of the mutants. A major result is that PIP2 interactions are functionally important, which so far has not been known for this receptor. Their results are likely relevant for other non-receptor tyrosine kinases.

The hypothesis that the protein complex is regulated by IDR-membrane interactions is very novel. A major strength is the close connection of and feedback between state-of-the-art experiments and simulations.

This is where I see weaknesses:<br /> 1. The motivation of focusing on LID1 is limited.<br /> 2. The data and analysis for the JAK2-PRLR complex appear somewhat superficial, and a connection between conformational states to their functional relevance is lacking. In fact, the majority of the simulation part of the paper is about suggesting different states of the PRLR-JAK2 complex but the states and their hypothesized functional relevance are not further taken up, e.g. by experiments, and yet presented as major results, e.g. in the abstract.<br /> 3. The connection between simulations and mutational study is not very direct.<br /> An open question is if the mutants can distinguish between the effects of PRLR-PIP2 interaction or PRLR-JAK2 interaction, even though this conclusion is still drawn from the data.<br /> 4. The conclusions drawn from the mutagenesis study (lines 547-555) are not directly supported by data. Only a partial correlation between PRLR membrane localisation and STAT5 activation is no reason to attribute the unexplained part of the STAT5 activation to PRLR-JAK2 interactions without further studies.<br /> 5. PIP2 is identified as an important regulator, with very solid support from the presented data. PIP3 is part of the model but not discussed before or as part of the results. The analysis could be similarly applied or the data directly relevant to the understanding of PIP3 plays a similar role, as interactions are likely primarily electrostatically driven.

Reviewer #2 (Public Review):

In A. Ruppel, et al, the authors study the mechanics of one cell, two cells, and cell monolayers upon a transient local activation of contractility. First, the authors characterize the tractions and stress maps (measured via Traction Force Microscopy and Monolayer Stress Microscopy, resp.) for one and two cells in the absence of contractility activation, and found a correlation between the principal stress direction and actin fiber orientation. Next, the authors use the theory of foams to infer, combining traction force data and cell geometry data, the mechanical parameters of cells like the line tension or the force of adherent fibers. Next, the authors activate contractility by means of optogenetic tools on one half of the system and quantify the response on both halves, concluding that the receiver half response is driven by active processes, increasing contractility for two cells, while fluidizing for one cell. Next, the authors estimate the level of active response in cell doublets by comparing the stress maps to numerical simulations of a thin elastic medium with anisotropic contractility. By varying aspect ratios of the H pattern, the authors find a correlation between the principal stress direction and the orientation of stress fibers and find that the previous active response is in general enhanced when the principal stress direction is perpendicular to the orientation of the fibers. Finally, these features are also found in a cell monolayer for a fixed confinement aspect ratio.

Overall, the manuscript contains a broad characterization of the steady state mechanics and the dynamical response to the activation of contractility for one cell, two cells, and cell monolayers.

Reviewer #2 (Public Review):

The authors hypothesized that PTH1R and ZFP467 could constitute a feedback loop that facilitates PTH-induced osteogenesis and that conditional deletion of Zfp467 in osteogenic precursors would lead to high bone mass. Using a number of methods, they have established a regulatory feedback mechanism of this transcription factor and the PTH receptor in osteoblastic precursors as well as showing that PrrxCre deletion of Zfp467 causes an increase in trabecular bone mass, while AdipoCre does not. Nevertheless, they have not established the actual mechanism of action of the transcription factor nor which gene it acts on in the osteoblast. They have mostly achieved their aims and the results partially support their conclusions. However, the work is descriptive and does not address the central issue of how ZFP467 acts. At present, its impact on the field is limited.

Reviewer #2 (Public Review):

This manuscript introduces a novel assay in a 'phenomics' approach to address an important aspect of S. aureus pathogenesis. The authors set out to identify mutations that arise during clinical S. aureus infections that cause a decrease in intracellular host-cell toxicity and increase intracellular persistence. To do this, they use a 'phenomics' approach. For phenotype, they quantify HeLa cell toxicity for each strain in a panel of 387 clinical S. aureus isolates. This is done by measuring HeLa cell death induced by intracellular S. aureus via propidium-iodide uptake. The whole genomes of each of these 387 isolates had previously been sequences. They use the genomic data and phenotype data to carry out a genome-wide association study (GWAS) looking for genetic signatures that correlate with reduced HeLa cell cytotoxicity. As expected, mutations in agr were the strongest locus-level signal, but the study did identify one agr-independent mutation in ausA, which was able to be independently validated, showing that the assay is robust enough to find causal mutations. The analysis is thoughtful, the assay appears robust, and I think the discussion of conclusions and limitations is mostly valid. Thus, my concerns are focused on further understanding the practical utility of the approach and whether or not the HeLa cell model recapitulates what happens in professional phagocytes. For example, it is not clear to me that this system has the statistical power to find novel, biologically relevant rare mutations without first being very mindful in selecting strains that are extremely genetically similar. It is also not clear to me that the toxicity assay captures the important features of the intracellular persistence that occurs in vivo within professional phagocytic cells. Thus, given these practical limitations and a somewhat artificial model system, the impact on the field is likely to be moderate in nature. However, the analysis and approach taken could be re-purposed to any robust quantitative phenotype, and this will certainly be of great interest to others that study bacterial evolution in clinical contexts.

Reviewer #2 (Public Review):

Krishnan, et al describe a unique and powerful approach to assessing the role of genetic variation on mitochondrial and cardiac function and health. Utilizing a panel of inbred mouse strains, on which they performed proteomics on heart samples, they measured 840 mitochondrial proteins and correlated these data to heart function using two heart stress models. This resulted in a number of correlative observations, three of which were explored in more detail to connect three specific genes to cardiac hypertrophy. This is an interesting dataset and there is clearly value in what is presented. The data were largely correlative, however, and there are only a couple of causation-oriented experiments. It's hard to adjudicate between these strengths and weaknesses in determining the overall impact of the manuscript.

Reviewer #2 (Public Review):

This manuscript reports on mermithid nematode fossils from amber which dates from the Cretaceous period. The specimens described in the manuscript consist of insects and associated nematodes which have been trapped in amber and fossilised. The nematodes have been identified as belonging to the Mermithidae family, a family of nematode worm that infect insects.

The findings of this manuscript provide an insight into the evolution history of nematodes and parasitism. Despite the ubiquity of both nematodes and parasites in extant ecosystems, fossil records of both are very rare. This is because nematodes and many parasites are soft bodied, and many are located inside their hosts' bodies, thus they rarely become fossilised. Thus, most of what is known about the evolutionary history of nematodes, and evolution of parasitism are based on what could be inferred from extant examples.

The specimens described in this manuscript provides a valuable contribution to our understanding of parasitism in the geological past. These amber specimens are a snapshot of parasite-host interactions - interactions which are commonly found in nature but are rarely captured in fossils. The identification of the specimens as mermithid nematodes are based on sound scientific reasoning. The worms' morphology and position in relation to the insects are consistent with what have been observed with extant mermithid nematodes.

Additionally, one of the values of such parasite fossils is that they provide us with insight into parasite-host combinations or interactions which may have existed throughout the geological past, but no longer exist today or cannot be inferred from extant taxa. It helps fill in major gaps in our understanding of parasitism. This was the case with the amber fossil that contained a bristletail with its nematode parasite.

Reviewer #2 (Public Review):

This very interesting study uses a combination of high channel count neural recordings and machine learning to characterize neural representations of complex natural and synthetic sounds in the inferior colliculus. The authors use deep neural networks to model sound evoked activity in a large number of IC multiunits with high accuracy in gerbils with normal hearing and hearing loss. They then use the DNNs to simulate activity evoked by a wide range of stimuli and demonstrate systematic differences in latent population representations between normal hearing and hearing-impaired animals. Models for hearing impaired animals show activity consistent with impaired representations of speech in noise. These results lay the groundwork for a potentially valuable approach to improving signal processing in hearing aids and prosthetics.

The large speech dataset and clean hearing loss effects are particularly impressive. While the approach and associated data are novel and likely to be of broad interest, there are some substantial concerns about the study. First, the authors fail to acknowledge substantial previous work on super-threshold activity in cortex of animals with hearing loss, making it appear that they overstate the novelty of the current results. There are also many cases where they fail to clearly report the details of statistics used to support their claims. Finally, while the accuracy of the DNN models is compelling for the speech stimuli in the data set, it is not clear that the comparisons of simulated activity reflect actual neural activity in the stimulus conditions tested.

Reviewer #2 (Public Review):

In the study by Li et al., the authors hypothesize that RELMa, a macrophage-derived protein, plays a sex-dimorphic role as a protective factor in obesity in females vs males. The authors perform largely in vivo studies utilizing male and female WT and RELMa KO mice on a high-fat diet and perform an in-depth analysis of immune cell composition, gene expression, and single-cell RNA Sequencing. The authors find that WT females are protected from obesity and inflammation vs males, and this protection is lost in female RELMa KO mice. Further analysis by the authors including flow cytometry of the visceral fat SVF in female WT mice showed reduced macrophage infiltration, higher levels of eosinophils, and Th2 cytokine expression compared to WT male mice and female KO mice. The authors show that protection from obesity and inflammation in female RELMa KO mice can be rescued with an injection of eosinophils and recombinant RELMa. Lastly, the authors use single-cell RNA-Sequencing to further analyze SVF cells in WT and KO male and female mice on a high-fat diet.

Overall, we find that the study represents an important finding in the immunometabolism field showing that RELMa is a key myeloid-derived factor that helps influence the macrophage-eosinophil function in female mice and protects from diet-induced obesity and inflammation in a sexually dimorphic manner. Overall, the study provides strong and convincing data supporting the authors' hypothesis and conclusion.

Reviewer #2 (Public Review):

Yamaguchi et al. studied the roles of two proteins, Calaxin and Armc4, in the assembly of the outer arm dynein (OAD) docking complex (DC). By combination of the improved cryo-ET analysis and gene knockout zebrafish lacking each of these proteins, they found that Armc4 plays a critical role in the docking of OAD and that Calaxin stabilizes the molecular interaction in the docking.They further showed an evidence that Calaxin changes the conformation of another compartment of DC comprising CCDC151/114. This new information provides an important basis for understanding how the DC is assembled and regulates docking of OAD. The authors' conclusion is well supported by the data but some data presentation and discussion need to be completed.

Gui et al. (2021) already reported on a cryo-EM observation in bovine tracheal cilia, with the conclusion similar to this paper in the structure of OAD/DC on DMT. Using knockout zebrafish strain, the authors present detailed interaction of calaxin with other DC components. They show that the binding of calaxin induces the changes of conformation in N-terminal region of CCDC151/114. The conformation further changes in the presence of Ca2+; specific conformation of N-terminal region of CCDC151/114 becomes undetectable, instead additional structure appears in the vicinity of calaxin.

1) The authors conclude that the Ca2+-dependent conformational change of DC is subtle and not dynamic. This result is eventually valuable information but may be somewhat unexpected from the point of view that calaxin plays an important role in the regulation of flagellar motility in Ciona sperm. The authors found that calaxin changes the conformation of N-terminal CCDC151/114 region but the core dynein structure shows no dynamic change. What about the changes in the interaction between calaxin, core dynein, and DMT? Is this beyond the resolution of cryo-ET analysis?

2) It would be very helpful if the authors could add the cryo-ET images of calaxin-/- axoneme in the presence of 1 mM EGTA in Figure 7. Although these images are thought to be similar or identical to Figure 4F, it would help to confirm that the conformational changes in CCDC151/114 and additional part of DC are induced in a Ca2+-dependent manner.

3) To clarify the molecular interaction of calaxin with other components, it would also be helpful if the authors add the images rotated 80 degree to Figure 4F and G, in similar way in Figure 7,

4) Despite the molecular phylogenetic difference, there are several similarities between calaxin and Chlamydomonas DC3, not only in the in situ structure and configuration but in the phenotype of mutants; Chlamydomonas mutant lacking DC3 shows OAD loss in the distal part of a flagellum (Casey et al, MBC, 2003). It may be a good reference if the authors add the position of DC3 in Figure 4. A', B', and C.

5) There is a significant difference in sperm motility between WT and calaxin-/- or WT and armc4-/- (Figure 2E). However, it is not clear whether immotile sperm were included in the data for VAP (Figure 2F) or BCF (Figure 2G). For example, WT and calaxin-/- show similar VAP, although both are significantly different in the percent of motile sperm.

6) In calaxin-/- mouse, OAD was clearly detected from the base to two-thirds of a flagellum with unclear border (Figure 2A). Typical distribution of OAD+class and OAD-class are shown in Figure 5 in the ~3 micrometer tomograms. Were these taken from around this unclear border? Are proximal most region of a flagellum occupied with OAD+class only? The authors should clearly indicate the region of a flagellum where the tomograms in Figure 5C and D were selected.

7) Line 229~: It is not clear what the authors meant by "probably reflecting the different distance from the sperm head". In relation to this and the comment 6, does the "proximal" in the sentence "OAD loss occurred even in the proximal part of the flagella" (line 232) indicate the region near the base of a flagellum?

8) In conjugation with comment 7, it would be appreciated to show an authors' idea on why distal region of flagella tends to lack calaxin, if they do not discuss anywhere in the text,

9) Immunofluorescence in twister-/- epithelial cilia showed that the localization of calaxin is independent of OAD (line 271-274). Based on the authors' finding, the localization of calaxin requires Armc4, which is preassembled with calaxin in the cytoplasm. If this is true and the localization of calaxin is NOT resulting from diffusion, Armc4 must be localized with calaxin along the entire length of cilia in twister-/- epithelial cilia (Figure 6D). Although Armc4 is shown localized in cryo-ET images (e.g. Figure 1, Figure 7), authors may provide the immunofluorescence of Armc4 along the entire length of sperm flagella and epithelial cilia.

Reviewer #2 (Public Review):

In this study, Rmus and colleagues contribute to the important open question of whether reinforcement learning deficits observed in older adults are due to impairments in basic learning processes, or can be attributed to a decline in working memory function. The authors present cross-sectional behavioral data from a task designed to assess the role of working memory in reinforcement learning. And they use computational modeling in conjunction with MR spectroscopy to demonstrate a relationship between prefrontal glutamate and age-related impairments in learning specific to working memory decay. I found the overall story compelling, the data novel, and the analysis carefully executed. Below I outline some areas in which the claims of the manuscript could be strengthened.

1. I may have missed this, but does glutamate correlate with other model parameters? Or did the authors only focus on the WM parameters because of the age difference? In support of the specificity argument, it would be important to show that glutamate only predicts WM related parameters regardless of whether there was an age difference or not.<br /> 2. As it is somewhat common with these tasks, it seems like the model does not fully capture the performance deficit in OA (Fig. 2B), even when all the individual difference parameters in WM are allowed to vary. Can the authors say more about the discrepancy? This is an interesting datapoint which may give clues to mechanism.<br /> 3. Relatedly, it may not be possible with these data alone, but can authors discuss what the WM decay parameter captures? In particular for OA, the distinction between generating and maintaining a "task set" has been extensively written about. Older adults tend to have difficulty internally generating and flexibly deploying task sets, but somewhat paradoxically can perform better than YA in certain decision situations (e.g. when reward is dependent on previous choices, see Worthy et. Al. 2011). The task in this study necessarily pushes OA in a regime in which relying on familiar decision strategies is sub-optimal, and task sets must be continuously generated. Is there a type of intervention do authors expect would reverse the observed deficit in WM?<br /> 4. There is a wealth of evidence suggesting striatal DA loss in older adults, which served as the basis for many of the original investigations and hypotheses regarding a simple RL deficit in OA (e.g. work by Shu-Chen Li and others). While the authors do not directly measure DA in this study, it would be helpful to place the results in the context of that literature.<br /> 5. Finally, the main argument of the paper as I read it is that PFC glutamate mediates the performance deficits observed in RL because it reflects a compromised WM system. Sample size permitting, it would be helpful to see a formal test of this mediation relationship.

Reviewer #2 (Public Review):

The manuscript by Aderounmu presents an interesting attempt to reconstruct evolution of the function of the helicase domain in ancestral Dicers, RNase III enzymes producing siRNAs from long double-stranded RNA and microRNAs from small hairpin precursors. The helicase has a role in long dsRNA recognition and processing and this function could have an antiviral role. Authors show on reconstructed ancestral Dicer variants that the helicase was losing dsRNA binding affinity and ATPase activity during evolution of the lineage leading to vertebrates while an early divergent Dicer-2 variant in Arthropods retained high activity and seemed better adapted for blunt ended long dsRNA, which would be consistent with antiviral function.

The work is consistent with apparent adaptation of vertebrate Dicers for miRNA biogenesis and two known modes of substrate loading: "bottom up" dsRNA threading through the helicase domain where the helicase domain recognizes the end of dsRNA and feeds it into the enzyme and "top-down" where the substrate is first anchored in the PAZ domain before it locks into the enzyme. Some extant Dicer variants are known to be adapted for just one of these two modes while Dicer in C. elegans exemplifies an "ambidextrous" variant. The reconstruction of the helicase domain complex enabled authors to test how well would be ancestral helicases supporting the "bottom up" feeding of long dsRNA and whether the helicase would be distinguishing blunt-end dsRNA and 3' 2 nucleotide overhang. Although the reconstruction of an ancestral protein from highly divergent extant sequences yields just a hypothetical ancestor, which cannot be validated, the work provides remarkable data for interpreting evolutionary history of the helicase domain and RNA silencing in more general. While it is not surprising that the ancestral helicase was a functional ATPase stimulated by dsRNA, particularly new and interesting are data that the decline of the helicase function started already at the level of the common deuterostome ancestor and the helicase was essentially dead in the vertebrate ancestor. It has been reported two decades ago that human Dicer carries a helicase, which has highly conserved critical residues in the ATPase domain but it is non-functional (10.1093/emboj/cdf582). Recently published mouse mutants showed that these highly conserved residues are not important in vivo (10.1016/j.molcel.2022.10.010). Aderounmu et al. now suggest that Dicer carried this dead ATPase with conserved residues for over 500 million years of vertebrate evolution.

I do not have any major comments to the biochemical analyses and while I think that the ancestral protein reconstruction could yield hypothetical sequences, which did not exist, I think they represent reasonable reconstructions, which yielded data worth of interpretations. My major criticism of the work concerns clarity for the readership and interpretations of some results where I wish authors would clarify/revise the text. The following three examples are particularly significant:

1) It should be explained to which common ancestor during metazoan evolution belongs the ancestral helicase AncD1D2 or at least what that sequence might represent in terms of common ancestry during metazoan evolution.

2) This is linked to the first point - authors work with phylogenetic trees reconstructed from a single protein sequence, which are not well aligned with predicted early metazoan divergence (https://doi.org/10.1098/rstb.2015.0036). While their sequence-based trees show early branching of Dicer-2 as if the two Dicers existed in the common ancestor of almost all animals (except of Placozoa), I do not think there is sufficient support for such a statement, especially since antiviral RNAi-dedicated Dicers evolve faster and Dicer-2 is restricted to a few distant taxonomic group, which might be better explained by independent duplications of ambidextrous ancestral Dicers. I would appreciate if authors would discuss this issue in more detail and make readers more aware of the complexity of the problem.

3) Authors should take more into the account existing literature and data when hypothesizing about sequences of events. Some decline of the helicase activity is apparent in AncD1DEUT suggesting that it initiated between AncD1D2 and AncD1DEUT. This implies that a) antiviral role of Dicer was becoming redundant with other cellular protein sensors by then and b) Dicer was already becoming adapted for miRNA biogenesis, which further progressed in the lineage leading to vertebrates to the unique top-down loading with the distinct pre-dicing state where the helicase forms a rigid arm. Authors even cite Qiao et al. (https://doi.org/10.1016/j.dci.2021.103997) who report primitive interferon-like system in molluscs - this places the ancestry of the interferon response upstream of AncD1DEUT and suggests that this ancestral protein-based system was taking over antiviral role of Dicer much earlier. In fact, a bit weaker performance of AncD1LOPH/DEUT combined with the aforementioned interferon-like system and massive miRNA expansion in extant molluscs (10.1126/sciadv.add9938) suggests that molluscs possibly followed a convergent path like mammals. While I am missing this kind of discussion in the manuscript, I think that the model where "interferon appears ..." in AncD1VERT (Fig. 6) is incorrect and misleading.

Reviewer #2 (Public Review):

In this work, Cunha et al provide an insightful and exhaustive analysis of the role of hypoxia and HIF-1a for T cell activation and function. The work contributes to the field by showing that transient hypoxia occurring simultaneously with T cell stimulation (antigen recognition) induces an effector program in T cells that results in increased cytotoxicity in vivo and in mouse models. Importantly, the induction of this effector phenotype is not necessarily linked with an increase in proliferation in vitro, and in vitro differences are mostly observed upon antigen re-challenging.

The major strengths of the work are the use of different complementary methods to modulate HIF-1a (low oxygen conditions, inhibition of PDH by FG-4592, and deletion of VHL) and the combination of mouse and human models, especially addressing how to implement the findings to the production of CAR-T cells. Besides, the authors not only evaluate T cell function but also dive into the pathways driving the responses observed, which provides mechanistic insight.

While activation of HIF-1a through the different means mentioned before results in similar signatures in terms of T cell effector phenotype and animal response, there are some aspects that differ between the models. This is probably indicating that low levels of oxygen have other effects beyond the regulation of HIF, and that pharmacological modulation of HIF-1a might not be exactly equivalent to HIF-1a stabilization by real hypoxia.

The work is useful to better understand the discrepancies in the field, where it has been previously shown that hypoxia can have both a pro-inflammatory effect and an immunosuppressive effect on T cells. The answer proposed by the authors is that it´s a matter of timing, and not so much the magnitude of the HIF-1a response. Despite this being relatively easy to control ex vivo, the challenge occurs when considering the role of hypoxia in vivo, which probably lasts longer than the transient hypoxia needed for beneficial effects on T cells, causing T cell exhaustion.

From the translational perspective, the study suggests strategies to improve CAR-T cell therapy but also has some limitations. Despite an improvement of cytotoxicity and survival observed in mouse models upon adoptive cell transfer or injection of CAR-T cells with previously increased HIF1a levels, these approaches do not result in curation and survival is still quite low in all groups. Interestingly, improved survival with HER2 CARs exposed ex vivo to low oxygen conditions for 1 day is clear and more promising.