Reviewer #1 (Public Review):

This work seeks to understand how behaviour-related information is represented in the neural activity of the primate motor cortex. To this end, a statistical model of neural activity is presented that enables a non-linear separation of behaviour-related from unrelated activity. As a generative model, it enables the separate analysis of these two activity modes, here primarily done by assessing the decoding performance of hand movements the monkeys perform in the experiments. Several lines of analysis are presented to show that while the neurons with significant tuning to movements strongly contribute to the behaviourally-relevant activity subspace, less or un-tuned neurons also carry decodable information. It is further shown that the discovered subspaces enable linear decoding, leading the authors to conclude that motor cortex read-out can be linear.

Strengths:

In my opinion, using an expressive generative model to analyse neural state spaces is an interesting approach to understand neural population coding. While potentially sacrificing interpretability, this approach allows capturing both redundancies and synergies in the code as done in this paper. The model presented here is a natural non-linear extension of a previous linear model PSID) and uses weak supervision in a manner similar to a previous non-linear model (TNDM).

Weaknesses:

This revised version provides additional evidence to support the author's claims regarding model performance and interpretation of the structure of the resulting latent spaces, in particular the distributed neural code over the whole recorded population, not just the well-tuned neurons. The improved ability to linearly decode behaviour from the relevant subspace and the analysis of the linear subspace projections in my opinion convincingly demonstrates that the model picks up behaviour-relevant dynamics, and that these are distributed widely across the population. As reviewer 3 also points out, I would, however, caution to interpret this as evidence for linear read-out of the motor system - your model performs a non-linear transformation, and while this is indeed linearly decodable, the motor system would need to do something similar first to achieve the same. In fact to me it seems to show the opposite, that behaviour-related information may not be generally accessible to linear decoders (including to down-stream brain areas).

As in my initial review, I would also caution against making strong claims about identifiability although this work and TNDM seem to show that in practise such methods work quite well. CEBRA, in contrast, offers some theoretical guarantees, but it is not a generative model, so would not allow the type of analysis done in this paper. In your model there is a para,eter \alpha to balance between neural and behaviour reconstruction. This seems very similar to TNDM and has to be optimised - if this is correct, then there is manual intervention required to identify a good model.

Somewhat related, I also found that the now comprehensive comparison with related models shows that the using decoding performance (R2) as a metric for model comparison may be problematic: the R2 values reported in Figure 2 (e.g. the MC_RTT dataset) should be compared to the values reported in the neural latent benchmark, which represent well-tuned models (e.g. AutoLFADS). The numbers (difficult to see, a table with numbers in the appendix would be useful, see: https://eval.ai/web/challenges/challenge-page/1256/leaderboard) seem lower than what can be obtained with models without latent space disentanglement. While this does not necessarily invalidate the conclusions drawn here, it shows that decoding performance can depend on a variety of model choices, and may not be ideal to discriminate between models. I'm also surprised by the low neural R2 for LFADS I assume this is condition-averaged) - LFADS tends to perform very well on this metric.

One statement I still cannot follow is how the prior of the variational distribution is modelled. You say you depart from the usual Gaussian prior, but equation 7 seems to suggest there is a normal prior. Are the parameters of this distribution learned? As I pointed out earlier, I however suspect this may not matter much as you give the prior a very low weight. I also still am not sure how you generate a sample from the variational distribution, do you just draw one for each pass?

Summary:

This paper presents a very interesting analysis, but some concerns remain that mainly stem from the complexity of deep learning models. It would be good to acknowledge these as readers without relevant background need to understand where the possible caveats are.

Reviewer #1 (Public Review):

Summary:<br /> The paper investigates visual processing in primates and deep neural networks (DNNs), focusing on factorization in the encoding of scene parameters. It challenges the conventional view that object classification is the primary function of the ventral visual stream, suggesting instead that the visual system employs a nuanced strategy involving both factorization and invariance. The study also presents empirical findings suggesting a correlation between high factorization scores and good neural predictivity.

Strengths:

1. Novel Perspective: The paper introduces a fresh viewpoint on visual processing by emphasizing the factorization of non-class information.

2. Methodology: The use of diverse datasets from primates and humans, alongside various computational models, strengthens the validity of the findings.

3. Detailed Analysis: The paper suggests metrics for factorization and invariance, contributing to a future understanding & measurements of these concepts.

Weaknesses:

1. Vagueness (Perceptual or Neural Invariance?): The paper uses the term 'invariance', typically referring to perceptual stability despite stimulus variability [1], as the complete discarding of nuisance information in neural activity. This oversimplification overlooks the nuanced distinction between perceptual invariance (e.g., invariant object recognition) and neural invariance (e.g., no change in neural activity). It seems that by 'invariance' the authors mean 'neural' invariance (rather than 'perceptual' invariance) in this paper, which is vague. The paper could benefit from changing what is called 'invariance' in the paper to 'neural invariance' and distinguish it from 'perceptual invariance,' to avoid potential confusion for future readers. The assignment of 'compact' representation to 'invariance' in Figure 1A is misleading (although it can be addressed by the clarification on the term invariance). [1] DiCarlo JJ, Cox DD. Untangling invariant object recognition. Trends in cognitive sciences. 2007 Aug 1;11(8):333-41.

2. Details on Metrics: The paper's explanation of factorization as encoding variance independently or uncorrelatedly needs more justification and elaboration. The definition of 'factorization' in Figure 1B seems to be potentially misleading, as the metric for factorization in the paper seems to be defined regardless of class information (can be defined within a single class). Does the factorization metric as defined in the paper (orthogonality of different sources of variation) warrant that responses for different object classes are aligned/parallel like in 1B (middle)? More clarification around this point could make the paper much richer and more interesting.

3. Factorization vs. Invariance: Is it fair to present invariance vs. factorization as mutually exclusive options in representational hypothesis space? Perhaps a more fair comparison would be factorization vs. object recognition, as it is possible to have different levels of neural variability (or neural invariance) underlying both factorization and object recognition tasks.

4. Potential Confounding Factors in Empirical Findings: The correlation observed in Figure 3 between factorization and neural predictivity might be influenced by data dimensionality, rather than factorization per se [2]. Incorporating discussions around this recent finding could strengthen the paper.

[2] Elmoznino E, Bonner MF. High-performing neural network models of the visual cortex benefit from high latent dimensionality. bioRxiv. 2022 Jul 13:2022-07.

Conclusion:<br /> The paper offers insightful empirical research with useful implications for understanding visual processing in primates and DNNs. The paper would benefit from a more nuanced discussion of perceptual and neural invariance, as well as a deeper discussion of the coexistence of factorization, recognition, and invariance in neural representation geometry. Additionally, addressing the potential confounding factors in the empirical findings on the correlation between factorization and neural predictivity would strengthen the paper's conclusions.

Reviewer #1 (Public Review):

Summary:<br /> This study examines the cortical modular functional organization of visual texture in comparison with that of color and disparity. While color, disparity, and orientation have been shown to exhibit clear functional organizations within the thin, thick, and thick/pale stripes of V2, whether the feature of texture is also organized within V2 is unknown. Using ultrahigh field 7T fMRI in humans viewing color-, disparity-, and texture-specific visual stimuli, the authors find that, unlike color and disparity, texture does not exhibit stripe-specific organization in V2. Moreover, using laminar imaging methods and calculations of informational connectivity, they find V2 color and disparity stripes exhibit the expected feedforward and feedback relationships with V1 & V4, and with V1 & V3ab, respectively. In contrast, texture activation, found predominantly in the deep layers of V2, is driven preferentially by feedback from V4. Based on these findings, the authors suggest that texture is a visual feature computed in higher-order areas and not generated by local intra-V2 computation.

Strengths:<br /> This study poses an interesting and fundamental question regarding the relationship between functional modularity and the hierarchical origin of computed properties. This question is thus highly significant and deserves study. The methodology is appropriate for the question and the areal and laminar resolution achieved across 10 subjects is commendable. The combination of high-resolution functional imaging and informational connectivity analysis introduces a useful way for examining feedforward and feedback relationships in mesoscale imaging data.

Weaknesses:<br /> While the data are suggestive, further controls are needed.

To support the finding that texture is not represented in a modular fashion, additional possibilities must be considered. These include the effectiveness and specificity of the texture stimulus and control stimuli, (b) further analysis of possible structure in images that may have been missed, and (c) limitations of imaging resolution.

More in-depth analysis of subject data is needed. The apparent structure in the texture images in peripheral fields of some subjects calls for more detailed analysis. e.g Relationship to eccentricity and the need for a 'modularity index' to quantify the degree of modularity. A possible relationship to eccentricity should also be considered.

Given what is known as a modular organization in V4 and V3 (e.g. for color, orientation, curvature), did images reveal these organizations? If so, connectivity analysis would be improved based on such ROIs. This would further strengthen the hierarchical scheme.

Reviewer #1 (Public Review):

Summary:<br /> The authors aim to test the sensory recruitment theory of visual memory, which assumes that visual sensory areas are recruited for working memory, and that these sensory areas represent visual memories in a similar fashion to how perceptual inputs are represented. To test the overlap between working memory (WM) and perception, the authors use coarse stimulus (aperture) biases that are known to account for (some) orientation decoding in the visual cortex (i.e., stimulus energy is higher for parts of an image where a grating orientation is perpendicular to an aperture edge, and stimulus energy drives decoding). Specifically, the authors show gratings (with a given "carrier" orientation) behind two different apertures: one is a radial modulator (with maximal energy aligned with the carrier orientation) and the other an angular modulator (with maximal energy orthogonal to the carrier orientation). When the subject detects contrast changes in these stimuli (the perceptual task), orientation decoding only works when training and testing within each modulator, but not across modulators, showing the impact of stimulus energy on decoding performance. Instead, when subjects remember the orientation over a 12s delay, orientation decoding works irrespective of the modulator used. The authors conclude that representations during WM are therefore not "sensory-like", given that they are immune to aperture biases. This invalidates the sensory recruitment hypothesis, or at least the part assuming that when sensory areas are recruited during WM, they are recruited in a manner that resembles how these areas are used during perception.

Strengths:<br /> Duan and Curtis very convincingly show that aperture effects that are present during perception, do not appear to be present during the working memory delay. Especially when the debate about "why can we decode orientations from human visual cortex" was in full swing, many may have quietly assumed this to be true (e.g., "the memory delay has no stimuli, and ergo no stimulus aperture effects"), but it is definitely not self-evident and nobody ever thought to test it directly until now. In addition to the clear absence of aperture effects during the delay, Duan and Curtis also show that when stimulus energy aligns with the carrier orientation, cross-generalization between perception and memory does work (which could explain why perception-to-memory cross-decoding also works). All in all, this is a clever manipulation, and I'm glad someone did it, and did it well.

Weaknesses:<br /> There seems to be a major possible confound that prohibits strong conclusions about "abstractions" into "line-like" representation, which is spatial attention. What if subjects simply attend the endpoints of the carrier grating, or attend to the edge of the screen where the carrier orientation "intersects" in order to do the task? This may also result in reconstructions that have higher bold at areas close to the stimulus/screen edges along the carrier orientation. The question then would be if this is truly an "abstracted representation", or if subjects are merely using spatial attention to do the task.

Alternatively (and this reaches back to the "fine vs coarse" debate), another argument could be that during memory, what we are decoding is indeed fine-scale inhomogenous sampling of orientation preferences across many voxels. This is clearly not the most convincing argument, as the spatial reconstructions (e.g., Figure 3A and C) show higher BOLD for voxels with receptive fields that are aligned to the remembered orientation (which is in itself a form of coarse-scale bias), but could still play a role.

To conclude that the spatial reconstruction from the data indeed comes from a line-like representation, you'd need to generate modeled reconstructions of all possible stimuli and representations. Yes, Figure 4 shows that line results in a modeled spatial map that resembles the WM data, but many other stimuli might too, and some may better match the data. For example, the alternative hypothesis (attention to grating endpoints) may very well lead to a very comparable model output to the one from a line. However testing this would not suffice, as there may be an inherent inverse problem (with multiple stimuli that can lead to the same visual field model).

The main conclusion, and title of the paper, that visual working memories are abstractions of percepts, is therefore not supported. Subjects could be using spatial attention, for example. Furthermore, even if it is true that gratings are abstracted into lines, this form of abstraction would not generalize to any non-spatial feature (e.g., color cannot become a line, contrast cannot become a line, etc.), which means it has limited explanatory power.

Additional context:<br /> The working memory and perception tasks are rather different. In this case, the perception task does not require the subject to process the carrier orientation (which is largely occluded, and possibly not that obvious without paying attention to it), but attention is paid to contrast. In this scenario, stimulus energy may dominate the signal. In the WM task, subjects have to work out what orientation is shown to do the task. Given that the sensory stimulus in both tasks is brief (1.5s during memory encoding, and 2.5s total in the perceptual task), it would be interesting to look at decoding (and reconstructions) for the WM stimulus epoch. If abstraction (into a line) happens in working memory, then this perceptual part of the task should still be susceptible to aperture biases. It allows the authors to show that it is indeed during memory (and not merely the task or attentional state of the subject) that abstraction occurs.

What's also interesting is what happens in the passive perceptual condition, and the fact that spatial reconstructions for areas beyond V1 and V2 (i.e., V3, V3AB, and IPS0-1) align with (implied) grating endpoints, even when an angular modulator is used (Figure 3C). Are these areas also "abstracting" the stimulus (in a line-like format)?

Reviewer #1 (Public Review):

The main focus of the current study is to identify the anatomical core of an expiratory oscillator in the medulla using pharmacological disinhibition. Although expiration is passive in normal eupneic conditions, activation of the parafacial (pFL) region is believed to evoke active expiration in conditions of elevated ventilatory demands. The authors and others in the field have previously attempted to map this region using pharmacological, optogenetic, and chemogenetic approaches, which present their own challenges.

In the present study, the authors take a systematic approach to determine the precise anatomical location within the ventral medulla's rostrocaudal axis where the expiratory oscillator is located. The authors used a bicuculline (a GABA-A receptor antagonist) and fluorobeads solution at 5 distinct anatomical locations to study the effects on neuronal excitability and functional circuitry in the pFL. The effects of bicuculline on different phases of the respiratory cycle were characterized using a multidimensional cycle-by-cycle analysis. This analysis involved measuring the differences in airflow, diaphragm electromyography (EMG), and abdominal EMG signals, as well as using a phase-plane analysis to analyze the combined differences of these respiratory signals. Anatomical immunostaining techniques were also used to complement the functional mapping of the pFL.

Major strengths of this work include a robust study design, complementary neurophysiological and immunohistochemical methods, and the use of a novel phase-plane analysis. The authors construct a comprehensive functional map revealing functional nuances in respiratory responses to bicuculline along the rostrocaudal axis of the parafacial region. They convincingly show that although bicuculline injections at all coordinates of the pFL generated an expiratory response, the most rostral locations in the lateral parafacial region play the strongest role in generating active expiration. These were characterized by a strong impact on the duration and strength of ABD activation and a robust change in tidal volume and minute ventilation. The authors also confirmed histologically that none of the injection sites overlapped grossly with PHOX2B+ neurons, thus confirming the specificity of the injections in the pFL and not the neighboring RTN.

Collectively, these findings advance our understanding of the presumed expiratory oscillator, the pFL, and highlight the functional heterogeneity in the functional response of this anatomical structure.

Reviewer #1 (Public Review)

Cav1.4 calcium channels control voltage-dependent calcium influx at photoreceptor synapses, and congenital loss of Cav1.4 function causes stationary night blindness CSNB2. Based on a broad portfolio of methodological approaches - genetic mouse models, immunolabeling and microscopic imaging, serial block-face-SEM, ERGs, and electrophysiology - the authors show that cone photoreceptor synapse development is strongly perturbed in the absence of Cav1.4 protein, and that expression of a nonconducting Cav1.4 channel mitigates these perturbations. Further data indicate that Cav3 channels are present, which, according to the authors, may compensate for the loss of Cav1.4 calcium currents and thus maintain cone synaptic transmission. These data, which are in agreement with a similar study by the same authors on rod photoreceptor synapses, help to explain what functional defects exactly cause CSNB2 and why it is accompanied by only mild visual impairment.

The strengths of the present study are its conceptual and experimental soundness, the broad spectrum of cutting-edge methodological approaches pursued, and the convincing differential analysis of mutant phenotypes.

Weaknesses mainly concern the experiments and arguments leading to the authors' notion that Cav3 channels may partially compensate for the loss of Cav1.4 calcium currents in cone synapses. It is possible that the non-conducting Cav1.4 variant supports synapse development and the Cav3 channel then provides the calcium influx. However, in its current state, the study does not unequivocally assess Cav3 expression in wild-type cones, it lacks direct evidence of Cav3 expression and upregulation, e.g. via single cell transcriptomics, immunolabeling, or an elaboration on electrophysiology, and it does not test the authors' earlier idea that Cav1.4 might couple to intracellular calcium stores at photoreceptor synapses

Reviewer #1 (Public Review):

Summary:<br /> This manuscript aims at a quantitative model of how visual stimuli, given as time-dependent light intensity signals, are transduced into electrical currents in photoreceptors of macaque and mouse retina. Based on prior knowledge of the fundamental biophysical steps of the transduction cascade and a relatively small number of free parameters, the resulting model is found to fairly accurately capture measured photoreceptor currents under a range of diverse visual stimuli and with parameters that are (mostly) identical for photoreceptors of the same type.

Furthermore, as the model is invertible, the authors show that it can be used to derive visual stimuli that result in a desired, predetermined photoreceptor response. As demonstrated with several examples, this can be used to probe how the dynamics of phototransduction affect downstream signals in retinal ganglion cells, for example, by manipulating the visual stimuli in such a way that photoreceptor signals are linear or have reduced or altered adaptation. This innovative approach had already previously been used by the same lab to probe the contribution of photoreceptor adaptation to differences between On and Off parasol cells (Yu et al, eLife 2022), but the present paper extends this by describing and testing the photoreceptor model more generally and in both macaque and mouse as well as for both rods and cones.

Strengths:<br /> The presentation of the model is thorough and convincing, and the ability to capture responses to stimuli as different as white noise with varying mean intensity and flashes with a common set of model parameters across cells is impressive. Also, the suggested approach of applying the model to modify visual stimuli that effectively alter photoreceptor signal processing is thought-provoking and should be a powerful tool for future investigations of retinal circuit function. The examples of how this approach can be applied are convincing and corroborate, for example, previous findings that adaptation to ambient light in the primate retina, as measured by responses to light flashes, mostly originates in photoreceptors.

Weaknesses:<br /> In the current form of the presentation, it doesn't become fully clear how easily the approach is applicable at different mean light levels and where exactly the limits for the model inversion are at high frequency. Also, accessibility and applicability by others could be strengthened by including more details about how parameters are fixed and what consensus values are selected.

Joint Public Review:

Summary:

In this interesting work, the authors investigated an important topical question: when we see travelling waves in cortical activity, is this due to true wave-like spread, or due to sequentially activated sources? In simulations, it is shown that sequential brain module activation can show up as a travelling wave - even in improved methods such as phase delay maps - and a variety of parameters is investigated. Then, in ex-vivo turtle eye-brain preparations, the authors show that visual cortex waves observable in local field potentials are in fact often better explained as areas D1 and D2 being sequentially activated. This has implications for how we think about travelling wave methodology and relevant analytical tools.

Strengths:

I enjoyed reading the discussion. The authors are careful in their claims, and point out that some phenomena may still indeed be genuine travelling waves, but we should have a higher evidence bar to claim this for a particular process in light of this paper and Zhigalov & Jensen (2023) (ref 44). Given this careful discussion, the claims made are well-supported by the experimental results. The discussion also gives a nice overview of potential options in light of this and future directions.

The illustration of different gaussian covariances leading to very different latency maps was interesting to see.

Furthermore, the methods are detailed and clearly structured and the Supplementary Figures, particularly single trial results, are useful and convincing.

Reviewer #1 (Public Review):

Summary:<br /> The authors explore mechanisms through which T-regs attenuate acute pain using a heat sensitivity paradigm. Analysis of available transcriptomic data revealed expression on the proenkephalin (Penk) gene in T-regs. The authors explore the contribution of T-reg Penk in the resolution of heat sensitivity.

Strengths:<br /> Investigating the potential role of T-reg Penk in the resolution of acute pain is a strength.

Weaknesses:<br /> The overall experimental design is superficial and lacks sufficient rigor to draw any meaningful conclusions.

For instance:<br /> 1) The were no TAM controls. What is the evidence that TAM does not alter heat-sensitive receptors.<br /> 2) There are no controls demonstrating that recombination actually occurred. How do the authors know a single dose of TAM is sufficient?<br /> 3) Why was only heat sensitivity assessed? The behavioral tests are inadequate to derive any meaningful conclusions. Further, why wasn't the behavioral data plotted longitudinally

Reviewer #1 (Public Review):

In this paper by Lui and colleagues, the authors examine the role of locus coeruleus (LC)-noradrenaline (NA) neurons in the extinction of appetitive instrumental conditioning. They report that optogenetic activation of global LC-NA neurons during the conditioned stimulus (CS) period of extinction enhances long-term extinction memory without affecting within-session extinction. In contrast, LC-NA activation during the intertrial interval doesn't affect extinction and long-term memory. They then show that optogenetic activation of LC-NA neurons doesn't induce conditioned place preference/avoidance. Finally, they assess the necessity of LC-NA neurons in appetitive extinction and find that optogenetic inactivation of LC-NA neurons during CS period results in enhancement of within-session extinction. The experiments are well-designed, including offset control in the optogenetic activation study. I think this study adds new insight into the LC-NA system in the context of appetitive extinction.

Strength:<br /> ・These studies identify the artificial activation of LC-NA neurons enhances long-term memory of appetitive extinction while this activation can't induce long-term conditioned place aversion. Thus, optogenetic activation of LC-NA neurons can inhibit spontaneous recovery of appetitive extinction without causing long-term aversive memory.<br /> ・Optoinhibition study demonstrates the reduction of conditioned response of within-session extinction. Therefore, LC-NA neuronal activity at the CS period of extinction could act as anti-extinction or be important for the expression of conditioned response.

Weakness:<br /> ・It is unclear how LC-NA neurons behave during the CS period of appetitive extinction from this study. This weakens the importance of the optogenetic inactivation result.<br /> ・While authors manipulate global LC-NA neurons, many people find functionally heterogeneous populations in the LC. It remains unsolved if there is specific LC-NA subpopulation responsible for appetitive extinction.

Reviewer #1 (Public Review):

Summary:<br /> The Roco proteins are a family of GTPases characterized by the conserved presence of an ROC-COR tandem domain. How GTP binding alters the structure and activity of Roco proteins remains unclear. In this study, Galicia C et al. took advantage of conformation-specific nanobodies to trap CtRoco, a bacterial Roco, in an active monomeric state and determined its high-resolution structure by cryo-EM. This study, in combination with the previous inactive dimeric CtRoco, revealed the molecular basis of CtRoco activation through GTP-binding and dimer-to-monomer transition.

Strengths:<br /> The reviewer is impressed by the authors' deep understanding of the CtRoco protein. Capturing Roco proteins in a GTP-bound state is a major breakthrough in the mechanistic understanding of the activation mechanism of Roco proteins and shows similarity with the activation mechanism of LRRK2, a key molecule in Parkinson's disease. Furthermore, the methodology the authors used in this manuscript - using conformation-specific nanobodies to trap the active conformation, which is otherwise flexible and resistant to single-particle average - is highly valuable and inspiring.

Weakness:<br /> Though written with good clarity, the paper will benefit from some clarifications.

1. The angular distribution of particles for the 3D reconstructions should be provided (Figure 1 - Sup. 1 & Sup. 2).

2. The B-factors for protein and ligand of the model, Map sharpening factor, and molprobity score should be provided (Table 1).

3. A supplemental Figure to Figure 2B, illustrating how a0-helix interacts with COR-A&LRR before and after GTP binding in atomic details, will be helpful for the readers to understand the critical role of a0-helix during CtRoco activation.

4. For the following statement, "On the other hand, only relatively small changes are observed in the orientation of the Roc a3 helix. This helix, which was previously suggested to be an important element in the activation of LRRK2 (Kalogeropulou et al., 2022), is located at the interface of the Roc and CORB domains and harbors the residues H554 and Y558, orthologous to the LRRK2 PD mutation sites N1337 and R1441, respectively."<br /> It is not surprising the a3-helix of the ROC domain only has small changes when the ROC domain is aligned (Figure 2E). However, in the study by Zhu et al (DOI: 10.1126/science.adi9926), it was shown that a3-helix has a "see-saw" motion when the COR-B domain is aligned. Is this motion conserved in CtRoco from inactive to active state?

5. A supplemental figure showing the positions of and distances between NbRoco1 K91 and Roc K443, K583, and K611 would help the following statement. "Also multiple crosslinks between the Nbs and CtRoco, as well as between both nanobodies were found. ... NbRoco1-K69 also forms crosslinks with two lysines within the Roc domain (K583 and K611), and NbRoco1-K91 is crosslinked to K583".

6. It would be informative to show the position of CtRoco-L487 in the NF and GTP-bound state and comment on why this mutation favors GTP hydrolysis.

Reviewer #1 (Public Review):

Summary:<br /> Medina et al, 2023 investigated the peripheral blood transcriptional responses in patients with diversifying disease outcomes. The authors characterized the blood transcriptome of four non-hospitalized individuals presenting mild disease and four patients hospitalized with severe disease. These individuals were observed longitudinally at three time points (0-, 7-, and 28-days post recruitment), and distinct transcriptional responses were observed between severe hospitalized patients and mild non-hospitalized individuals, especially during 0- and 7-day collection time points. Particularly, the authors found that increased expression of genes associated with NK cell cytotoxicity is associated with mild outcomes. Additional co-regulated gene network analyses positively correlate T cell activity with mild disease and neutrophil degranulation with severe disease.

Strengths:<br /> The longitudinal measurements in individual participants at consistent collection intervals can offer an added dimension to the dataset that involves temporal trajectories of genes associated with disease outcomes and is a key strength of the study. The use of co-expressed gene networks specific to the cohort to complement enrichment results obtained from pre-determined genesets can offer valuable insights into new associations/networks associated with disease progression and warrants further analyses on the biological functions enriched within these co-expressed network modules.

Weaknesses:<br /> There is a large difference in terms of infection timeline (onset of symptom to recruitment) between mild and severe patient cohorts. As immune responses during early infection can be highly dynamic, the differences in infection timeline may contribute to differences in transcriptional signatures. The study is also limited by a small cohort size.

Reviewer #1 (Public Review):

Summary:<br /> The study entitled "Rifampicin tolerance and growth fitness among isoniazid-resistant clinical Mycobacterium tuberculosis isolates: an in-vitro longitudinal study" by Vijay et al. provides valuable insights into the association of rifampicin tolerance and growth fitness with isoniazid resistance among clinical isolates of M. tuberculosis. Antibiotic tolerance in M. tuberculosis is an important topic since it contributes to the lengthy and complicated treatment required to cure tuberculosis disease and may portend the emergence of antibiotic resistance. The authors found that rifampicin tolerance was correlated with bacterial growth, rifampicin minimum inhibitory concentrations, and isoniazid-resistance mutations.

Strengths:<br /> The large number of clinical isolates evaluated and their longitudinal nature during treatment for TB (including exposure to rifampin) are strengths of the study.

Weaknesses:<br /> Some of the methodologies are not well explained or justified and the association of antibiotic tolerance with growth rate is not a novel finding. In addition, the molecular mechanisms underlying rifampicin tolerance only in rapidly growing isoniazid-resistant isolates have not been elucidated and the potential implications of these findings for clinical management are not immediately apparent.

Reviewer #1 (Public Review):

The manuscript by Chen et al. investigated the interaction between CHI3L1, a chitinase-like protein in the 18 glycosyl hydrolase family, and gut bacteria in the mucosal layers. The authors provided evidence to document the direct interaction between CHI3L1 and peptidoglycan, a major component of bacterial cell walls. In doing so, Chi3l1 produced by gut epithelial cells regulates the balance of the gut microbiome and diminishes DSS-induced colitis, potentially through the colonization of protective gram-positive bacteria such as lactobacillus.

The study is the first to systemically document the interactions between Chi3L1 and microbiome. Convincing data were shown to characterize the imbalance of gram-positive bacteria in the newly generated gut epithelial-specific Chi3L1 deficient mice. Comprehensive FMT experiments were performed to demonstrate the contributions of gut microbiome using the mouse colitis model. However, the manuscript could've been strengthened by additional mechanistic studies concerning the binding between Chi3l1 and peptidoglycan, and how this interaction could facilitate the colonization of gram-positive bacteria. Additionally, the conclusion by the authors that disordered intestinal bacteria in gut epithelial-specific Chi3L1 deficient mice, rather than an effect by host cells, contributes to exacerbated colitis, needs further validation. In fact, the fact that FMT did not completely rescue the phenotype may point to the role of host cells in the processes. On the contrary, there is an existing body of literature demonstrating the detrimental roles of Chi3l1 in the mouse IBD model, conflicting with the current study. The differences in study design and approaches in these studies that lead to controversial findings will need to be discussed.

Specifically,<br /> 1) In Figure 1, it is curious that the authors only chose E.coli and staphytlococcus sciuri to test the induction of Chi3l1. What about other bacteria? Why does only E.coli but not staphytlococcus sciuri induce chi3l1 production? It does not prove that the gut microbiome induces the expression of Chi3l1. If it is the effect of LPS, does it trigger a cell death response or inflammatory responses that are known to induce chi3l1 production? What is the role of peptidoglycan in this experiment? Also, it is recommended to change WT to SPF in the figure and text, as no genetic manipulation was involved in this figure.

2) In Figure 2, the binding between Chi3l1 and PGN needs better characterization, regarding the affinity and how it compares with the binding between Chi3l1 and chitin. More importantly, it is unclear how this interaction could facilitate the colonization of gram-positive bacteria.

3) In Figure 3, the abundance of furmicutes and other gram-positive species is lower in the knockout mice. What is the rationale for choosing lactobacillus in the following transfer experiments?

4) FDAA-labeled E. faecalis colonization is decreased in the knockouts. Is it specific for E. faecalis, or it is generally true for all gram-positive bacteria? What about the colonization of gram-negative bacteria?

5) In Figure 5, the fact that FMT did not completely rescue the phenotype may point to the role of host cells in the processes. The reason that lactobacillus transfer did completely rescue the phenotypes could be due to the overwhelming protective role of lactobacillus itself, as the experiments were missing villin-cre mice transferred with lactobacillus.

6) Conflicting literature demonstrating the detrimental roles of Chi3l1 in mouse IBD model needs to be acknowledged and discussed.

Joint Public Review:

Summary:<br /> Identifying dietary biomarkers, in particular, has become a main focus of nutrition research in the drive to develop personalized nutrition.

The aim of this study was to determine the accuracy of using food composition databases to assess the association between dietary intake and health outcomes. The authors found that using food composition data to assess dietary intake of specific bioactives and the impact consumption has on systolic blood pressure provided vastly different outcomes depending on the method used. These findings demonstrate the difficulty in elucidating the relationship between diet and health outcomes and the need for more stringent research in the development of dietary biomarkers.

Strengths:<br /> The primary strength of the study is the use of a large cohort in which dietary data and the measurement of three specific bioactives and blood pressure were collected on the same day. The bioactives selected have been extensively researched for their health effects. Another strength is that the authors controlled for as many variables as possible when running the simulations to get a more accurate account of how the variability in food composition can impact research findings that associate the intake of certain food components with health outcomes.

Weaknesses:<br /> The authors address the large variability when using food composition data, e.g. the range of tea and apple intake needed to meet recommendations depending on using the mean food composition data or using the lowest reported food content, however, there is no discussion on the intake needed if the biomarker is used. So how many cups of tea are needed to reach the suggested 200 mg/day of flavan-3-ols when using biomarker data instead of the food composition data? More information should be added on the effect of using biomarker data on dietary recommendations and risk assessment.

Reviewer #1 (Public Review):

Summary:<br /> Ye et al. identified a novel tumour microenvironment (TME) signature that can help to prognosticate DLBCL. They first interrogated a publicly available dataset to identify tumour purity-related genes (TPGs) and found these TPGs were associated with extracellular matrix organisation and immune response. Protein-protein interaction analysis identified hub genes that were associated with prognosis, and 3 genes (VCAN, CD3G, C1QB) were selected to construct a prognosis model. The authors attempted to validate the findings on immunohistochemistry (IHC) and showed prognostication using an IHC assay. Finally, they showed a possible prediction of drug sensitivity using the novel signature in DLBCL.

Strengths:<br /> This study investigated both immune and non-immune TME related to tumour purity. Tumour purity has not been thoroughly investigated in DLBCL. Hence, the prognostic significance of tumour purity demonstrated in this paper brought into light another potential area of research in DLBCL. Similarly, the investigation into non-immune TME was novel and thought-provoking, as most research in DLBCL TME has mostly been in the immune microenvironment.

The bioinformatics approach in identifying the key TPGs was well conducted, such as the GO and KEGG enrichment analysis which supported the role of these TPGs in the modulation of the microenvironment. The findings were also validated in another dataset, which increased the confidence in this model. However, it was not clear to me why the authors chose VCAN, CD3G, and C1QB out of the 9 intersection genes that they found. It would perhaps be useful to show the statistical justification in the Supplementary Results section.

The possible translation of these findings into clinical practice by immunohistochemistry (IHC) was a useful tool to make the findings applicable in the clinical setting. However, as stated by the authors, the real-life clinical application of these findings may be more challenging as these antigens seemed to be expressed in a continuum, rather than in a discrete manner. For example, in Figure 5A, even the low VCAN status still demonstrated strong cytoplasmic staining. Similarly, in Figure 5C, it seemed to be difficult to differentiate strong from background staining. This means pre-analytical variables may affect the staining and standardisation among different laboratories may be difficult to achieve without external controls.

Weaknesses:<br /> Though the rationale behind choosing the TPG genes and its correlation with non-immune TME was clear, the justification for investigating CD68+ macrophages, CD4+ T cells, and CD8+ T cells was not as strong. This was done in a subsection that was supposed to investigate the prognostic values of IHC staining in VCAN, CD3G, and C1QB. Hence, the analysis of the immune compartment of the TME was rather superficial. For example, it would be insufficient to correlate CD4+ and CD8T+ T cells without understanding their deeper phenotypes such as regulatory vs memory or exhausted vs activated. An attempt was made to subtype the macrophages by bioinformatics approach but it was not further investigated with IHC.

Similarly, the investigation into drug sensitivity was only done in-silico. This investigation was adequate for hypothesis generation. However, it was not enough to substantiate the claim that TPGs can be used to predict drug sensitivity. This claim requires functional in-vitro experiments to validate the bioinformatics approach, or even correlation with clinical data when the identified drugs were used in DLBCL, for example in the ReMODL-B cohort that used bortezomib.

Reviewer #1 (Public Review):

The present study provides a phylogenetic analysis of the size prefrontal areas in primates, aiming to investigate whether relative size of the rostral prefrontal cortex (frontal pole) and dorsolateral prefrontal cortex volume vary according to known ecological or social variables.

I am very much in favor of the general approach taken in this study. Neuroimaging now allows us to obtain more detailed anatomical data in a much larger range of species than ever before and this study shows the questions that can be asked using these types of data. In general, the study is conducted with care, focusing on anatomical precision in definition of the cortical areas and using appropriate statistical techniques, such as PGLS.

I have read the revised version of the manuscript with interest. I agree with the authors that a focus on ecological vs laboratory variables is a good one, although it might have been useful to reflect that in the title.

I am happy to see that the authors included additional analyses using different definitions of FP and DLPFC in the supplementary material. As I said in my earlier review, the precise delineation of the areas will always be an issue of debate in studies like this, so showing the effects of different decisions in vital.

I am sorry the authors are so dismissive of the idea of looking the models where brain size and area size are directly compared in the model, rather preferring to run separate models on brain size and area size. This seems to me a sensible suggestion.

Similarly, the debate about whether area volume and number of neurons can be equated across the regions is an important one, of which they are a bit dismissive.

Nevertheless, I think this is an important study. I am happy that we are using imaging data to answer more wider phylogenetic questions. Combining detailed anatomy, big data, and phylogenetic statistical frameworks is a important approach.

Joint Public Review:

Summary:<br /> Desiderio and colleagues investigated the role of the TALE (three amino acid loop extension) homeodomain transcription factor Meis2 during maturation and target innervation of mechanoreceptors and their sensation to touch. They start with a series of careful in situ hybridizations and immunohistochemical analyses to examine Meis2 transcript expression and protein distribution in mouse and chick DRGs of different embryonic stages. By this approach, they identify Meis2+ neurons as slowly- and rapidly adapting A-beta LTMRs, respectively. Retrograde tracing experiments in newborn mice confirmed that Meis2-expressing sensory neurons project to the skin, while unilateral limb bud ablations in chick embryos in ovo showed that these neurons require target-derived signals for survival. The authors further generated a conditional knock-out (cKO) mouse model in which Meis2 is selectively lost in Islet1-expressing, postmitotic neurons in the DRG (IsletCre/+::Meis2flox/flox, abbreviated below as cKO). WT and Islet1Cre/+ littermates served as controls. cKO mice did not exhibit any obvious alteration in volume or cellular composition of the DRGs but showed significantly reduced sensitivity to touch stimuli and various innervation defects to different end-organ targets. RNA-sequencing experiments of E18.5 DRGs taken from WT, Islet1Cre/+ and cKO mice reveals extensive gene expression differences between cKO cells and the two controls, including synaptic proteins and components of GABAergic- and glutamatergic transmission. Histological analysis and electrophysiological recordings shed light on the physiological defects resulting from the loss of Meis2. By immunohistochemical approaches, the authors describe distinct innervation defects in glabrous and hairy skin (reduced innervation of Merkel cells by SA1-LTMRs in glabrous but not hairy skin, reduced complexity of A-beta RA1-LTMs innervating Meissner's corpuscles in glabrous skin, reduced branching and innervation of A-betA RA1-LTMRs in hairy skin). Electrophysiological recordings from ex vivo skin nerve preparations found that several, but not all of these histological defects are matched by altered responses to external stimuli, indicating that compensation may play a considerable role in this system. This study will be of interest to developmental biologists and neuroscientists, in particular those interested in the sensation of touch.

Strengths:<br /> This is a well-conducted study that combines different experimental approaches to convincingly show that the transcription factor Meis2 plays an important role in the perception of light touch. The authors describe a new mouse model for compromised touch sensation, characterize it by histology and electrophysiological recordings, and identify several genes whose expression depends on Meis2 in mouse DRGs.

Weaknesses:<br /> The authors use different experimental approaches to investigate the role of Meis2 in touch sensation, but the results obtained by these techniques could be better connected. For instance, the authors identify several genes involved in synapse formation, synaptic transmission, neuronal projections, or axon and dendrite maturation that are up- or downregulated upon targeted Meis2 deletion, but it remains to be resolved whether these chances explain the histological, electrophysiological, or behavioral deficits observed in cKO animals.

Reviewer #1 (Public Review):

Summary:<br /> In the current study, Papandreou et al. developed an iPSC-based midbrain dopaminergic neuronal cell model of Beta-Propeller Protein-Associated Neurodegeneration (BPAN), which is caused by mutations in the WDR45 gene and is known to impair autophagy. They also noted defective autophagy and abnormal BPAN-related gene expression signatures. Further, they performed a drug screening and identified five cardiac glycosides. Treatment with these drugs effectively in improved autophagy defects and restored gene expression.

Strengths:<br /> Seeing the autophagy defects and impaired expression of BPAN-related genes adds strength to this study. Importantly, this work shows the value of iPSC-based modeling in studying disease and finding therapeutic strategies for genetic disorders, including BPAN.

Weaknesses:<br /> It is unclear whether these cells show iron metabolism defects and whether treatment with these drugs can ameliorate the iron metabolism phenotypes.

Reviewer #1 (Public Review):

Summary:<br /> Zhang et al., investigated the relationship between monocular and binocular responses of V1 superficial-layer neurons using two-photon calcium imaging. They found a strong relationship in their data: neurons that exhibited a greater preference for one eye or the other (high ocular dominance) were more likely to be suppressed under binocular stimulation, whereas neurons that are more equivalently driven by each other (low ocular dominance) were more likely to be enhanced by binocular stimulation. This result chiefly demonstrates the relationship between ocular dominance and binocular responses in V1, corroborating what has been shown previously using electrophysiological techniques but now with greater spatial resolution (albeit less temporal resolution). The binocular responses were well-fitted by a model that institutes divisive normalization between the eyes that accounts for both the suppression and enhancement phenomena observed in the subpopulation of binocular neurons. In so doing, the authors reify the importance of incorporating ocular dominance in computational models of binocular combination.

The conclusions of this paper are mostly well supported by the data, but there are some limitations of the methodology that need to be clarified, and an expansion of how the results relate to previous work would better contextualize these important findings in the literature.

Strengths:<br /> The two-photon imaging technique used to resolve the activity of individual neurons within intact brain tissue grants a host of advantages. Foremost, two-photon imaging confers considerably high spatial resolution. As a result, the authors were able to sample and analyze the activity from thousands of verified superficial-layer V1 neurons. The animal model used, awake macaques, is also highly relevant for the study of binocular combination. Macaques, like humans, are binocular animals, meaning they have forward-facing eyes that confer overlapping visual fields. Importantly, macaque V1 is organized into cortical columns that process specific visual features from the separate eyes just like in humans. In combination with a powerful imaging technique, this allowed the authors to evaluate the monocular and binocular response profiles of V1 neurons that are situated within neighboring ocular dominance columns, a novel feat. To this aim, the approach was well-executed and should instill further confidence in the notion that V1 neurons combine monocular information in a manner that is dependent on the strength of their ocular dominance.

Weaknesses:<br /> While two-photon imaging provides excellent spatial resolution, its temporal resolution is often lower compared to some other techniques, such as electrophysiology. This limits the ability to study the fast dynamics of neuronal activity, a well-understood trade-off of the method. The issue is more so that the authors draw comparisons to electrophysiological studies without explicit appreciation of the temporal difference between these techniques. In a similar vein, two-photon imaging is limited spatially in terms of cortical depth, preferentially sampling from neurons in layers 2/3. This limitation does not invalidate any of the interpretations but should be considered by readers, especially when making comparisons to previous electrophysiological reports using microelectrode linear arrays that sample from all cortical layers. Indeed, it is likely that a complete picture of early cortical binocular processing will require high spatial resolution (i.e., sampling from neurons in neighboring ocular dominance columns, from pia mater to white matter) at the biophysically relevant timescales (1ms resolution, capturing response dynamics over the full duration of the stimulus presentation, including the transient onset and steady-state periods).

Reviewer #1 (Public Review):

Summary:<br /> The authors ran an explorative analysis in order to describe how a "tri-partite" brain network model could describe the combination of resting fMRI data and individual characteristics. They utilized previously obtained fMRI data across four scanning runs in 144 individuals. At the end of each run, participants rated their patterns of thinking on 12 statements (short multi-dimensional experience sampling-MDES) using a 0-100% visual analog scale. Also, 71 personality traits were obtained on 21 questionnaires. The authors ran two separate principal component analyses (PCA) to obtain low dimensional summaries of the two individual characteristics (personality traits from questionnaires, and thought patterns from MDES). The dimensionality reduction of the fMRI data was done by means of gradient analysis, which was combined with Neurosynth decoding to visualize the functional axis of the gradients. To test the reliability of thought components across scanning time, intra-class correlation coefficients (ICC) were calculated for the thought patterns, and discriminability indices were calculated for whole gradients. The relationship between individual differences in traits, thoughts, and macro-scale gradients was tested with multivariate regression.

The authors found: a) reliability of thought components across the one hour of scanning, b) Gradient 1 differentiated between visual regions and DMN, Gradient 2 dissociated somatomotor from visual cortices, Gradient 3 differentiated the DMN from the fronto-parietal system, c) the associations between traits/thought patterns and brain gradients revealed significant effects of "introversion" and "specific internal" thought: "Introversion" was associated with variant parcels on the three gradients, with most of parcels belonging to the VAN and then to the DMN; and "Specific internal thought" was associated with variant parcels on the three gradients with most of parcels belonging to the DAN and then the visual. The authors conclude that interactions between attention systems and the DMN are important influences on ongoing thought at rest.

Strengths:<br /> The study's strength lies in its attempt to combine brain activity with individual characteristics using state-of-the-art methodologies.

Weaknesses:<br /> The study protocol in its current form restricts replicability. This is largely due to missing information on the MRI protocol and data preprocessing. The article refers the reader to the work of Mendes et al 2019 which is said to provide this information, but the paper should rather stand alone with all this crucial material mentioned here, as well. Also, effect sizes are provided only for the multiple multivariate regression of the inter-class correlations, which makes it difficult to appreciate the power of the other obtained results.

Reviewer #1 (Public Review):

Summary:<br /> This paper describes a reanalysis of data collected by Gagne et al. (2020), who investigated how human choice behaviour differs in response to changes in environmental volatility. Several studies to date have demonstrated that individuals appear to increase their learning rate in response to greater volatility and that this adjustment is reduced amongst individuals with anxiety and depression. The present authors challenge this view and instead describe a novel Mixture of Strategies (MOS) model, that attributes individual differences in choice behaviour to different weightings of three distinct decision-making strategies. They demonstrate that the MOS model provides a superior fit to the data and that the previously observed differences between patients and healthy controls may be explained by patients opting for a less cognitively demanding, but suboptimal, strategy.

Strengths:<br /> The authors compare several models (including the original winning model in Gagne et al., 2020) that could feasibly fit the data. These are clearly described and are evaluated using a range of model diagnostics. The proposed MOS model appears to provide a superior fit across several tests.

The MOS model output is easy to interpret and has good face validity. This allows for the generation of clear, testable, hypotheses, and the authors have suggested several lines of potential research based on this.

Weaknesses:<br /> The authors justify this reanalysis by arguing that learning rate adjustment (which has previously been used to explain choice behaviour on volatility tasks) is likely to be too computationally expensive and therefore unfeasible. It is unclear how to determine how "expensive" learning rate adjustment is, and how this compares to the proposed MOS model (which also includes learning rate parameters), which combines estimates across three distinct decision-making strategies.

As highlighted by the authors, the model is limited in its explanation of previously observed learning differences based on outcome value. It's currently unclear why there would be a change in learning across positive/negative outcome contexts, based on strategy choice alone.

Reviewer #1 (Public Review):

Nitrogen metabolism is of fundamental importance to biology. However, the metabolism and biochemistry of guanidine and guanidine containing compounds, including arginine and homoarginine, have been understudied over the last few decades. Very few guanidine forming enzymes have been identified. Funck et al define a new type of guanidine forming enzyme. It was previously known that 2-oxogluturate oxygenase catalysis in bacteria can produce guanidine via oxidation of arginine. Interestingly, the same reported enzyme that produces guanidine from arginine also oxidises 2-oxogluturate to give the plant signalling molecule ethylene. Funck et al show that a mechanistically related oxygenase enzyme from plants can also produce guanidine, but instead of using arginine as a substrate, it uses homoarginine and does not produce ethylene. The work will stimulate interest in the cellular roles of homoarginine, a metabolite present in plants and other organisms including humans and, more generally, in the biochemistry and metabolism of guanidine derivatives.

1. Significance<br /> Studies on the metabolism and biochemistry of the small nitrogen rich molecule guanidine and related compounds including arginine have been largely ignored over the last few decades. Very few guanidine forming enzymes have been identified. Funck et al define a new guanidine forming enzyme that works by oxidation of homoarginine, a metabolite present in organisms ranging from plants to humans. The new enzyme requires oxygen and 2-oxogluturate as cosubstrates and is related, but distinct from a known enzyme that oxidises arginine to produce guanidine, but which can also oxidise 2-oxogluturate to produce the plant signalling molecule ethylene.

I thought this was an exceptionally well-written and interesting manuscript. Although a 2-oxogluturate dependent guanidine forming enzyme is known (EFE), the discovery that a related enzyme oxidises homoarginine is really interesting, especially given the presence of homoarginine in plant seeds. There is more work to be done in terms of functional assignment, but this can be the subject of future studies. I also fully endorse the authors' view that guanidine and related compounds have been massively understudied in recent times. Congratulations to the authors on a very nice study.

Overall, I thought this was a very interesting study, comprising biochemical, cellular, and in vivo studies. Of course, more could be done on each of these, and likely will be, but I think the assignment of biochemical function is very strong, across all three approaches. The one new experiment I requested was a demonstration of whether ethylene is produced by the new enzymes - this was clearly shown not to be the case.

Reviewer #1 (Public Review):

Summary:<br /> "Phosphorylation, disorder, and phase separation govern the behavior of Frequency in the fungal circadian clock" is a convincing manuscript that delves into the structural and biochemical aspects of FRQ and the FFC under both LLPS and non-LLPS conditions. Circadian clocks serve as adaptations to the daily rhythms of sunlight, providing a reliable internal representation of local time.

All circadian clocks are composed of positive and negative components. The FFC contributes negative feedback to the Neurospora circadian oscillator. It consists of FRQ, CK1, and FRH. The FFC facilitates close interaction between CK1 and the WCC, with CK1-mediated phosphorylation disrupting WCC:c-box interactions necessary for restarting the circadian cycle.

Despite the significance of FRQ and the FFC, challenges associated with purifying and stabilizing FRQ have hindered in vitro studies. Here, researchers successfully developed a protocol for purifying recombinant FRQ expressed in E. coli.

Armed with full-length FRQ, they utilized spin-labeled FRQ, CK1, and FRH to gain structural insights into FRQ and the FFC using ESR. These studies revealed a somewhat ordered core and a disordered periphery in FRQ, consistent with prior investigations using limited proteolysis assays. Additionally, p-FRQ exhibited greater conformational flexibility than np-FRQ, and CK1 and FRH were found in close proximity within the FFC. The study further demonstrated that under LLPS conditions in vitro, FRQ undergoes phase separation, encapsulating FRH and CK1 within LLPS droplets, ultimately diminishing CK1 activity within the FFC. Intriguingly, higher temperatures enhanced LLPS formation, suggesting a potential role of LLPS in the fungal clock's temperature compensation mechanism.

Biological significance was supported by live imaging of Neurospora, revealing FRQ foci at the periphery of nuclei consistent with LLPS. The amino acid sequence of FRQ conferred LLPS properties, and a comparison of clock repressor protein sequences in other eukaryotes indicated that LLPS formation might be a conserved process within the negative arms of these circadian clocks.

In summary, this manuscript represents a valuable advancement with solid evidence in the understanding of a circadian clock system that has proven challenging to characterize structurally due to obstacles linked to FRQ purification and stability. The implications of LLPS formation in the negative arm of other eukaryotic clocks and its role in temperature compensation are highly intriguing.

Joint Public Review:

The authors previously showed that expressing formate dehydrogenase, rubisco, carbonic anhydrase, and phosphoribulokinase in Escherichia coli, followed by experimental evolution, led to the generation of strains that can metabolise CO2. Using two rounds of experimental evolution, the authors identify mutations in three genes - pgi, rpoB, and crp - that allow cells to metabolise CO2 in their engineered strain background. The authors make a strong case that mutations in pgi are loss-of-function mutations that prevent metabolic efflux from the reductive pentose phosphate autocatalytic cycle. The authors also use proteomic analysis to probe the role of the mutations in crp and rpoB. While they do not reach strong conclusions about how these mutations promote autotrophic growth, they provide some clues, leading to valuable speculation.

Comments on revised version:<br /> The authors have thoroughly addressed the reviewers' comments. The major addition to the paper is the proteomic analysis of single and double mutants of crp and rpoB. These new data provide clues as to the role of the crp and rpoB mutations in promoting autotrophic growth, which the authors discuss. The authors acknowledge that it will require additional experiments to determine whether the speculated mechanisms are correct. Nonetheless, the new data provide valuable new insight into the role of the crp and rpoB mutations. The authors have also expanded their description of the crp and rpoB mutations, making it clearer that the effects of these mutations are likely to be distinct, albeit with potential for overlap in function.

Joint Public Review:

The manuscript highlights a mechanistic insight into meiotic initiation in budding yeast. In this study, the authors analyzed the genetic link between the mitotic cell cycle regulator SBF (the Swi4-Swi6 complex) and a meiosis inducing regulator Ime1 in the context of meiotic initiation. The authors' comprehensive analyses with cytology, imaging, RNA-seq using mutant strains lead to the conclusion that Swi4 levels regulates Ime1-Ume6 interaction to activate expression of early meiosis genes for meiotic initiation.

The authors first show a down regulation of Swi4 at the protein level upon meiosis entry and then investigate downstream consequences. This study reveals several regulations: 1) Mutations in CLN1 and 2, which are targets of Swi4, allow rescuing the delay in meiotic entry observed when Swi4 is overexpressed; 2) Ime1 activity is antigonized by Swi4, and more specifically its interaction with Ume6. 3) Expression of SWI4 is regulated by LUTI-based transcription at the SWI4 locus that impedes expression of canonical SWI4 transcripts 4) The expression of SWI4 LUTI is likely negatively regulated by the Ime1-Ume6 complex 5) Whi5 restrict SBF activity during meiotic entry, thereby ensuring Cyclin repression.

The important implication in this paper is that meiotic initiation is regulated by the balance of mitotic cell cycle regulator and meiosis-specific transcription factor.

Joint Public Review:

Summary<br /> Sender et al describe a model to estimate what fraction of DNA becomes cell-free DNA in plasma. This is of great interest to the community, as the amount of DNA from a certain tissue (for example, a tumor) that becomes available for detection in the blood has important implications for disease detection.

Strengths<br /> The question asked by the authors has potentially important implications for disease diagnosis. Understanding how genomic DNA degrades in the human circulation can guide towards ways to enrich for DNA of interest or may lead to unexpected methods of conserving cell-free DNA. Thus, the question "how much genomic DNA becomes cfDNA" is of great interest to the scientific and medical community. I believe this manuscript has the potential to be a widely used resource. As more data is collected on cell-free DNA yields and cellular turnover in the body, this work will only increase in importance.

Appraisal<br /> At this stage of the manuscript (second submission), I think the authors provide important evidence and analysis that aim to answer their research question. Previous concerns about methodology have been addressed.

Impact<br /> This manuscript will be highly impactful on the community. The field of liquid biopsies (non-invasive diagnostics) has the potential to revolutionize the medical field (and has already in certain areas, such as prenatal diagnostics). Yet, there is a lack of basic science questions in the field. This manuscript is an important step forward in asking more "basic science" questions that seek to answer a fundamental biological question.

No feedback! This is great! I love how you touched on so many abiotic factors in one post! If you wanted to expand on any of them, you certainly could, but you have plenty of information already!

This introduction is so much fun! I love the unique way you chose to format this post.

The images are great too, but it might be helpful to add captions with a brief explanation.

Awesome explanation, very clear and informational!

Even though I'm already excited to read this, it may be good to add a little hook/intro paragraph to illuminate why this is so interesting!

Reviewer #1 (Public Review):

Summary:<br /> In the first half of this study, Pham et al. investigate the regulation of TEAD via ubiquitination and PARylation, identifying an E3 ubiquitin ligase, RNF146, as a negative regulator of TEAD activity through an siRNA screen of ubiquitin-related genes in MCF7 cells. The study also finds that depletion of PARP1 reduced TEAD4 ubiquitination levels, suggesting a certain relationship between TEAD4 PARylation and ubiquitination which was also explored through an interesting D70A mutation. Pham et al. subsequently tested this regulation in D. melanogaster by introducing Hpo loss-of-function mutations and rescuing the overgrowth phenotype through RNF146 overexpression.

In the second half of this study, Pham et al. designed and assayed several potential TEAD degraders with a heterobifunctional design, which they term TEAD-CIDE. Compounds D and E were found to effectively degrade pan-TEAD, an effect which could be disrupted by treatment with TEAD lipid pocket binders, proteasome inhibitors, or E1 inhibitors, demonstrating that the TEAD-CIDEs operate in a proteasome-dependent manner. These TEAD-CIDEs could reduce cell proliferation in OVCAR-8, a YAP-deficient cell line, but not SK-N-FI, a Hippo pathway independent cell line. Finally, this study also utilizes ATAC-seq on Compound D to identify reductions in chromatin accessibility at the regions enriched for TEAD DNA binding motifs.

Strengths:<br /> The study provides compelling evidence that the E3 ubiquitin ligase RNF146 is a novel negative regulator of TEAD activity. The authors convincingly delineate the mechanism through multiple techniques and approaches. The authors also describe the development of heterobifunctional pan-degraders of TEAD, which could serve as valuable reagents to more deeply study TEAD biology.

Weaknesses:<br /> The scope of this study is extremely broad. The first half of the paper highlights the mechanisms underlying TEAD degradation; however, the connection to the second half of the paper on small molecule degraders of TEAD is jarring, and it seems as though two separate stories were combined into this single massive study. In my opinion, the study would be stronger if it chose to focus on only one of these topics and instead went deeper.

Additionally, the figure clarity needs to be substantially improved, as readability and interpretation were difficult in many panels. Lastly, there are numerous typos and poor grammar throughout the text that need to be addressed.

Reviewer #1 (Public Review):

Summary:<br /> The current manuscript provides an extensive in vivo analysis of two guidance pathways identifying multiple mechanisms that shape the bifurcation of DRG axons when forming the dorsal funiculus in the DREZ.

Strengths:<br /> Multiple mouse mutant lines were used, together with complementary techniques; the results are very clear and compelling.<br /> The findings are very significant and clearly move forward our understanding of the regulation of axonal development at the DREZ.

Weaknesses:<br /> No major weaknesses were found. As it is I have no recommendations that would increase the clarity or quality of the manuscript.

Reviewer #1 (Public Review):

Yu et al. investigated Fusarium oxysporum f. sp. lycopersici SIX effectors structure using experimental and computational approaches, and while doing so, the authors identified several SIX effectors as member of the FOLD family, and expanded the known diversity of the SIX effectors. A very interesting and novel finding is the presence of FOLD putative effectors in other Ascomycetes secretome, sharing structural similarities with SIX effectors Avr1, Avr3 and SIX6.

By performing technically sound predictions and experimental confirmation, the authors also confirmed co-operative interactions between Fol effectors, something that was previously known for different pairs of proteins, expanding this observation for new SIX effectors. In addition, the authors contributed to the understanding of the interaction Fol effectors, specifically FOLD and LARS effectors, - I receptors to suppress immunity by structurally similar effectors.

The conclusions of this paper are supported by data and I think it is a pioneer study analyzing the correspondence between AlphaFold predictions and experimentally derived structures, highlighting that models can answer the scientific questions in some cases but could not be enough in others.

Reviewer #1 (Public Review):

The author found the nifuroxazide has the potential to augment the efficacy of radiotherapy in HCC by reducing PD-L1 expression. This effect may be attributed to increased degradation of PD-L1 through the ubiquitination-proteasome pathway. These evidences support the future application of nifuroxazide in the treatment of HCC.

Reviewer #1 (Public Review):

Summary:

The authors developed a deep learning method called H3-OPT, which combines the strength of AF2 and PLM to reach better prediction accuracy of antibody CDR-H3 loops than AF2 and IgFold. These improvements will have an impact on antibody structure prediction and design.

Strengths:

The training data are carefully selected and clustered, the network design is simple and effective.

The improvements include smaller average Ca RMSD, backbone RMSD, side chain RMSD, more accurate surface residues and/or SASA, and more accurate H3 loop-antigen contacts.

The performance is validated from multiple angles.

The revised manuscript has cleared my previous concerns.

Reviewer #1 (Public Review):

Summary:<br /> This study presents a valuable finding on the increased activity of two well-studied signal transduction pathways - STAT-3 and TGF-Beta in a specific subtype of pancreatic cancer. Specifically, SMAD4 deficient tumors (commonly observed in pancreatic cancer) are well differentiated in the presence of STAT3. Yet surprisingly, in the presence of SMAD4 in a STAT-3 deficient pancreatic cancer, the phenotype is poorly differentiated in the background of KRASGD12D. The evidence in the animal models supporting the authors' claims is solid, although including TCGA data and/or a larger number of patients would have strengthened the study. The work will be of interest to medical biologists working on pancreatic cancer and potentially the broader field.

Strengths:<br /> Strengths are the animal models and the lead author's expertise in STAT3 signaling.

Weaknesses:<br /> Weaknesses are the absence of correlation between the results from the animal studies and human pancreatic cancers.

Reviewer #1 (Public Review):

Summary:<br /> The manuscript by Chen et al. presents a detailed metabolic characterization of male and female WT and CTRP10 knockout mice. The main finding is that female KO mice become obese on both low-fat and high-fat diets but without evidence of marked insulin resistance, hepatic steatosis, dyslipidemia, or increased inflammatory markers. The authors performed a detailed transcriptomic analysis and identified differentially expressed genes that distinguish high-fat diet-fed CTRP10 KO from WT control mice. They further show that this set of genes exhibits cross-correlation in human tissues, and that this is greater in females than in males. The data indicate that the CTRP10 KO model may be useful to understand how obesity and metabolic dysfunction are coupled to each other, and how this occurs by a sex-biased mechanism.

Strengths:<br /> The work presents a large amount of data, which has been carefully acquired and is convincing. The transcriptomic analysis will further help to define what pathways are associated with obesity, but not necessarily with metabolic dysfunction. The manuscript will be of interest to investigators studying metabolic diseases, and to those studying sex-specific differences in metabolic physiology. The limitations of the study are acknowledged, including that a whole-body knockout was used. The cause of the increased body weight is not entirely clear, despite the careful and detailed analysis that was performed. Notwithstanding these limitations, the phenotype is interesting, and this work will establish a basis for further work to understand the mechanisms that are involved.

Weaknesses:<br /> Genes identified as DEGs in the mouse RNAseq data set were used to identify a set of human orthologous transcripts and the abundances of these transcripts were correlated with each other in Figure 10. This identified a greater correlation ("connectivity") in subQ adipose compared to other tissues, and in females compared to males. The description of how this analysis was done could be clearer. In some cases, the text refers to the software that was used without describing the goal of the analysis. In other instances, specialized terminology was used (e.g. "biweight midcorrelation") without defining what this means.

Reviewer #1 (Public Review):

Summary:

Bartolome et al. report adaptation of proximity labeling using BirA and TurboID fusions to proteasome subunits to identify the proteasome-proximal proteome both in cultured cells and also in a newly developed mouse model. Using this approach, the authors demonstrate identification of many known proteasome-interacting proteins, as well as several new proteins, some of which are validated directly. The authors further evaluate the proteasome-proximal proteome in most mouse organs, and find substantial agreement with the proteome identified from cultured cells, as well as between tissues. This represents one of the first studies of the "proteasome-ome" in vivo, and sets the stage for addressing numerous important future questions regarding how the proteasome's environment changes over time, in response to different stimuli, and in distinct disease conditions.

Strengths:

Generally speaking, the approach provided is rigorous and supported by several complementary lines of evidence, such as demonstration that the interactome is enriched for known proteasome-binding proteins and co-purification or co-elution experiments. Similarly, the high agreement between the outcomes in cultured cells and in the mouse model developed by the authors provides further confidence in the results.

Weaknesses:

The major weakness of the work is arguably the choice of proteasome subunits for tagging with biotinylating enzymes. In most cases, the subunits and termini chosen for tagging are known to either protrude toward functionally important regions (such as the substrate-processing pore of the ATPase component), to have important functional roles likely to be disrupted via tagging, or are subunits known to be substituted by others in some conditions. Thus, the interactome reported may conflate those of normal proteasomes with those harboring tag-induced functional or structural defects. Although the authors made a commendable attempt to demonstrate minimal impacts of tagging, the conclusions would be greatly further strengthened by contrasting the impacts of tagging subunits less likely to cause perturbations and by more rigorously demonstrating normal proteolysis of a broader array of known proteasome substrates.

Reviewer #1 (Public Review):

Summary:<br /> Zhang et al. describe novel roles for the centriolar protein CEP44, namely that it is required for centriole engagement (and thus inhibition of centriole reduplication) and that it promotes microtubule stability. While a function of CEP44 in centriole engagement is somehow convincingly shown, the data do not support a role for CEP44 in microtubule stabilization.

Strengths:<br /> The finding that centriole engagement relies on CEP44 is novel and of great interest to the centriole field. Interestingly, the authors correlate reduced CEP44 expression levels with the occurrence of breast carcinoma, which makes this study also very interesting for a broad audience.

Weaknesses:<br /> The paper has important findings, but unfortunately, the main claims are only partially supported.

1) The role of CEP44 in microtubule stability is not clear from the presented data:<br /> - Fig. 7A and S6 A, there is no visible difference in microtubule density/intensity between the different groups of cells. In Fig. 7C, the CEP44 S324A spindle looks even brighter than the WT spindle. The authors need to indicate how many cells were analyzed. This information is actually lacking in all the experiments.

2) Several figure parts are not properly labelled.

3) Several of the experiments (WBs) likely miss proper controls: How did the authors detect proteins that run at very similar sizes: 55 kDa (alpha-tubulin), 44 kDa (Cep44), and 57 kDa (Cep57 and Cep57L)? The loading control needs to be detected in the same lane as the protein of interest. Did the authors strip and reprobe membranes? If so, this needs to be indicated and included in the methods section.

4) It is not clear how such a low CEP44-FLAG expression (Fig. 5A) can rescue a CEP44 KO.

Reviewer #1 (Public Review):

Summary:<br /> In their manuscript, Zhou et al. analyze the factors controlling the activation and maintenance of a sustained cell cycle block in response to persistent DNA DSBs. By conditionally depleting components of the DDC using auxin-inducible degrons, the authors verified that some DDC proteins are only required for the activation (e.g., Dun1) or the maintenance (e.g., Chk1) of the DSB-dependent cell cycle arrest, while others such as Ddc2, Rad24, Rad9 or Rad53 are required for both processes. Notably, they further demonstrate that after a prolonged arrest (>24 h) in a strain carrying two DSBs, the DDC becomes dispensable and the mitotic block is then maintained by SAC proteins such as Mad1, Mad2, or the mitotic exit network (MEN) component Bub2.

Strengths:<br /> The manuscript dissects the specific role that different components of the DDC and the SAC have during the induction of a cell cycle arrest induced by DNA damage, as well as their contribution to the short-term and long-term maintenance of a DNA DSB-induced mitotic block. Overall, the experiments are well described and properly executed, and the data in the manuscript are clearly presented. The conclusions drawn are also generally well supported by the experimental data. The observations contribute to drawing a clearer picture of the relative contribution of these factors to the maintenance of genome stability in cells exposed to permanent DNA damage.

Weaknesses:<br /> The main weakness of the study is that it is fundamentally based only on the use of the auxin-inducible degron (AID) strategy to deplete proteins. This is a widely used method that allows a very efficient depletion of proteins. However, the drawback is that a tag is added to the protein, which can affect the functionality of the targeted protein or modify its capacity to interact with others. In fact, three of the proteins that are depleted using the AID systems are shown to be clearly hypomorphic. Verification of at least some of the results using an alternative manner to eliminate the proteins would help to strengthen the conclusions of the manuscript.

Reviewer #1 (Public Review):

Summary:<br /> The manuscript describes the identification and characterization of rice SCC3, including the generation and characterization of plants containing apparently lethal null mutations in SCC3 as well as mutant plants containing a c-terminal frame-shift mutation. The weak scc3 mutants showed both vegetative and reproductive defects. Specifically, mitotic chromosomes appeared to partially separate during prometaphase, while meiotic chromosomes were diffuse during early meiosis and showed alterations in sister chromatid cohesion, homologous chromosome pairing, and recombination. The authors suggest that SCC3 acts as a cohesin subunit in mitosis and meiosis, but also plays more functions other than just cohesion.

Strengths:<br /> The manuscript contains a large amount of generally high-quality data.

Weaknesses:<br /> Several of the conclusions drawn in the manuscript are not supported by the data. There are many examples where the authors either draw conclusions or make statements that are just not justified based on the data presented or present a conclusion as a new finding, which has already been demonstrated in the past by others. For example, they claim that SCC3 functions in the maintenance of replication. From my reading of the manuscript, nowhere did the authors examine DNA replication. Likewise, several of the conclusions drawn are in direct contrast with what is known about SCC3 in other organisms. Therefore, the conclusions are either groundbreaking or incorrect.

Reviewer #1 (Public Review):

Summary:<br /> This manuscript describes a deficiency in nuclear pore complexes (NPCs) to maintain proper compartmentalization between the nucleus and cytoplasm in a mouse model of AD-related Aβ pathology. Experiments demonstrate NPC dysfunction in cultured neurons and mouse tissue as a result of intracellular Aβ, which may cause reduced levels of certain nucleoporins, leading to a reduced number of NPCs, and their dysfunction in nuclear protein import and maintaining nucleocytoplasmic compartmentalization. In addition, the authors also report a potential mechanism for how NPC dysfunction may result in increased vulnerability to inflammation-induced necroptosis, where core components are reportedly activated via phosphorylation through nucleocytoplasmic shutting. Overall, the study is interesting and well conducted and reveals striking NCT defects in a Aβ pathology disease model that may have important implications for our understanding of AD pathology.

Strengths:<br /> Previous studies have found nucleocytoplasmic transport (NCT) defects in other models of age-related neurodegenerative diseases, including Huntington's disease, tauopathy, C9orf72-linked frontotemporal dementia / amyotrophic lateral sclerosis (FTD/ALS), and TDP-43 proteinopathy in FTD/ALS. Typically, NCT defects have been linked mechanistically to aberrant co-aggregation of nucleoporins with e.g. TDP-43 and tau found in disease models and sometimes also human autopsy tissue. This study is novel, in that it describes NCT defects that are caused by Alzheimer's disease (AD) related Aβ pathology, using a human APP knock-in mouse model (AppNL-G-F/NL-G-F) that exhibits robust Aβ pathology in the CNS. The main focus of this study is on the barrier dysfunction of the NPCs leading to compartmentalization defects, while previous publications in the field have focused more on active protein import and RNA export defects. This is of considerable interest since an age-dependent decline in NPC barrier function has been observed in transdifferentiated neurons derived from normal-aged fibroblasts (Mertens et al., 2015). The potential link of NPC dysfunction to an increased vulnerability to inflammation-induced necroptosis may also be relevant to other neurodegenerative disorders with NCT dysfunction. Experiments are largely focused on either dissociated neuronal cultures, or studies using mouse tissue at different stages of disease progression. Experiments are mostly based on immunocytochemistry (ICC) and histochemistry (IHC) of nucleoporins to show morphological NPC defects and fluorescent reporter constructs and dyes of defined MW to show NPC dysfunction. The experiments using an anti-nuclear pore O-linked glycoprotein antibody [RL1], which recognizes multiple metazoan nucleoporins that are modified via post-translational O-GlcNAcylation, show a very striking reduction in staining intensity that is also replicated with antibodies specific for the FG-motif rich Nup98 and the very stable and essential NPC component Nup107. Taken together, the fluorescence microscopy studies convincingly support the claim of NPC dysfunction leading to defective compartmentalization between the nucleus and cytoplasm.

Weaknesses:<br /> However, the molecular mechanisms leading to NPC dysfunction and the cellular consequences of resulting compartmentalization defects are not as thoroughly explored. Results from complementary key experiments using western blot analysis are less impressive than microscopy data and do not show the same level of reduction. The antibodies recognizing multiple nucleoporins (RL1 and Mab414) could have been used to identify specific nucleoporins that are most affected, while the selection of Nup98 and Nup107 is not well explained. There is also no clear hypothesis on how Aβ pathology may affect nucleoporin levels and NPC function. All functional NCT experiments are based on reporters or dyes, although one would expect widespread mislocalization of endogenous proteins, likely affecting many cellular pathways. The second part of this manuscript reports that in App KI neurons, disruption in the permeability barrier and nucleocytoplasmic transport may enhance activation of key components of the necrosome complex that include receptor-interacting kinase 3 (RIPK3) and mixed lineage kinase domain1 like (MLKL) protein, resulting in an increase in TNFα-induced necroptosis. While this is of potential interest, it is not well integrated in the study. This potential disease pathway is not shown in the very simple schematic (Fig. 8) and is barely mentioned in the Discussion section, although it would deserve a more thorough examination.

Reviewer #1 (Public Review):

Summary:<br /> The manuscript aimed at elucidating the substrate specificity of two M23 endopeptidase Lysostaphin (LSS) and LytM in S. aureus. Endopeptidases are known to cleave the glycine-bridges of staphylococcal cell wall peptidoglycan (PG). To address this question, various glycine-bridge peptides were synthesized as substrates, the catalytic domain of LSS and LytM were recombinantly expressed and purified, and the reactions were analyzed using solution-state NMR. The major finding is that LytM is not only a Gly-Gly endopeptidase, but also cleaves D-Ala-Gly. Technically, the advantage of using real-time NMR was emphasized in the manuscript. The study explores an interesting aspect of cell wall hydrolases in terms of substrate-level regulation. It potentially identified new enzymatic activity of LytM. However, the biological significance and relevance of the conclusions remain clear, as the results are mostly from synthetic substrates.

Strengths:<br /> The study explores an interesting aspect of cell wall hydrolases in terms of substrate-level regulation. It potentially identified new enzymatic activity of LytM.

Weaknesses:<br /> 1. Significance: while the current study provided a detailed analysis of various substrates, the conclusions are mainly based on synthesized peptides. One experiment used purified muropeptides (Fig. 3H); however, the results were unclear from this figure. The results from synthesized peptides may not necessarily correlate with their biological functions in vivo. Secondly, the study used only the catalytic domain of both proteins. It is known that the substrate specificity of these enzymes is regulated by their substrate-binding domains. There is no mention of other domains in the manuscript and no justification of why only the catalytic domain was studied. In short, the relevance of the results from the current study to the enzymes' actual physiological functions remains to be addressed, which attenuated the significance of the study.

2. Impact and novelty: (1) the current study provided evidence suggesting the novel function of LytM in cleaving D-Ala-Gly. The impact of this finding is unclear. The manuscript discussed Enterococcus faecalis EnpA. But how about other M23 endopeptidases? What is biological relevance? (2) A very similar study published recently showed that the activity of LSS and LytM is regulated by PG cross-linking: LSS cleaves more cross-linked PG and LytM cleaves less cross-linked PG (Razew, A., Laguri, C., Vallet, A., et al. Staphylococcus aureus sacculus mediates activities of M23 hydrolases. Nat Commun 14, 6706 (2023). The results of this paper are different from the current study whereby both LSS and LytM prefer cross-linked substrates (Fig, 2JKL). Moreover, no D-Ala-Gly cleavage was observed by LytM using purified PG substrate from Razew A et al. An explanation of inconsistent results is needed here. In my opinion, the knowledge generated from the current study has not been fully settled. If the results can be validated, the contribution to the field is incremental, but not substantial. (3) The authors emphasized a few times in the text that it is superior to use NMR technology. In my opinion, NMR has certain advantages, such as measuring the efficacy of cleavage, but it is not that superior. It should be complementary to other methods such as mass spectrometry. In addition, more relevant solid-state NMR using intact PG or bacterial cells was not discussed in the study. I am of the opinion that the corresponding text should be revised.

3. The conclusions are not fully supported by the data<br /> As mentioned above, the conclusions from synthesized peptide substrates may not necessarily reveal physiological functions. The conclusions need to be validated by more physiological substrates.

4. There are some issues with the presentation of the figures, text, and formatting.

Reviewer #1 (Public Review):

Summary:<br /> The manuscript by Dubey et al. examines the function of the acetyltransferase Tip60. The authors show that (auto)acetylation of a lysine residue in Tip60 is important for its nuclear localization and liquid-liquid-phase-separation (LLPS).

The main observations are: (i) Tip60 is localized to the nucleus, where it typically forms punctate foci. (ii) An intrinsically disordered region (IDR) within Tip60 is critical for the normal distribution of Tip60. (iii) Within the IDR the authors show that a lysine residue (K187), that is auto-acetylated, is critical. Mutation of that lysine residue to a non-acetylable arginine abolishes the behavior. (iv) biochemical experiments show that the formation of the punctate foci may be consistent with LLPS.

Strengths:<br /> The experiments are largely convincing and appear to be well executed.

Weaknesses:<br /> The main concern I have is that all in vivo (i.e. in cells) experiments are done with overexpression in Cos-1 cells, in the presence of the endogenous protein. No attempt is made to use e.g. cells that would be KO for Tip60 in order to have a cleaner system or to look at the endogenous protein. It would be reassuring to know that what the authors observe with highly overexpressed proteins also takes place with endogenous proteins.

Also, it is not clear how often the experiments have been repeated and additional quantifications (e.g. of western blots) would be useful.

In addition, regarding the LLPS description (Figure 1), it would be important to show the wetting behavior and the temperature-dependent reversibility of the droplet formation.

On balance, this is an interesting study that describes the role of acetylation of Tip60 in controlling its biochemical behavior as well as its localization and function in cells. The authors mention in their Discussion section other examples showing that acetylation can change the behavior of proteins with respect to LLPS; depending on the specific context, acetylation can promote (as here for Tip60) or impair LLPS.

Reviewer #1 (Public Review):

This study shows that SET7 and LSD1 regulate the dynamic methylation of EZH2 at K20, which is recognized by L3MBTL3 promoting protein degradation via the DCAF5-CRL4 E3 ubiquitin ligase. K20 methylation negatively regulates S21 phosphorylation and vice versa, modulating EZH2 functions. Mice harboring the K20 methylation-deficient mutant (K20R) exhibit hematopoietic defects. Overall, this is an interesting study elucidating a novel mechanism of EZH2 regulation. The methodologies are sound and the conclusions are largely supported by the data provided. However, there are some questions regarding the overall model and some contradictory results.

Reviewer #1 (Public Review):

The study by Schmehl and colleagues asks an important question, i.e. how are multiple objects/stimuli represented in the visual system despite broad tuning properties of neurons along multiple different dimensions (e.g. space, features). This is a continuation of an impactful and highly significant line of work from the Groh lab and their collaborators. In previous work, they showed that fluctuations in firing patterns may be critical in representing multiple objects and parse them in time. In this particular study, the authors ask three specific questions to extend these observations: (i) Are such fluctuations widespread in the visual system?; (ii) Are they related to the perceptual distinction of objects?; (iii) And how are they related to the functional specialization of neuronal populations along feature dimensions (e.g. faces, motion).

It seems to me that there is ample evidence for the first two questions from previous work by these authors. For (i), fluctuations in firing patterns related to multiple stimuli have been shown in the auditory (e.g. inferior colliculus, Caruso et al., 2018) and multiple areas of the visual system (i.e. V1, V4, and the face patch system; Caruso et al., 2018; Jun et al., 2022). The present study adds data from MT to this increasing evidence. For (ii), Jun et al., 2022 already showed that fluctuations are not related to stimuli perceived as merged, or not distinct. Thus, the main contribution appears to be related to functional specialization. I suggest clarifying the major novelty of the present report and to focus the introduction on it.

The present work analyzed three different data sets acquired in different areas (V1, V4, MT, IT face network), using different feature stimuli (motion, faces), obtained under various attention conditions/states (passive fixation, actively ignored). Many of the results are nice confirmations and minor extensions of previous work. The conceptual advance and novelty of the findings are therefore limited.

There is a growing literature on fluctuating neural firing patterns that is not considered in this report. The scholarship appears a bit impoverished with only 19 references, many of which point to work from this group of collaborators. I suggest that the authors consider the present work in the context of the wider literature more scholarly, even if not all the relations of these different lines of work can be conclusively connected at this point. For a few examples, there is work by Kienitz and colleagues on fluctuating neural patterns in V4 evoked by competing grating stimuli. Also, the work by Engel, Moore, and colleagues on 'on' and 'off' states in the context of selective attention seems relevant, or the work by Fiebelkorn and Kastner on rhythmic perception and attention.

I love this introduction! You do a marvelous job of drawing in the reader, especially with these photographs!

Reviewer #1 (Public Review):

Summary:<br /> In this study, the authors attempt to reinvestigate an old question in population genetics regarding the age of alleles that have experienced different strengths (and directions) of natural selection. Under simple population genetic models, alleles that are positively selected are expected to change frequency in populations faster than neutral alleles. So the naïve expectation is that if you look at alleles that are the same population frequency, those that have been evolving neutrally should have been segregating in the population longer than those that have been experiencing natural selection. While this is exactly what the authors find for alleles inferred to be experiencing negative selection (i.e. they tend to be younger than alleles inferred to be neutral that are at the same frequency), the authors find the opposite for alleles inferred to be under positive selection: they tend to be older than alleles inferred to be neutral. The authors argue that this pattern can be explained by a model where positively selected mutations experience a phase of balancing selection that can dramatically extend the period of time that these alleles segregate in the population.

Strengths:<br /> The question that the authors address is very interesting and thought provoking. When confronted with a counter-intuitive finding, the authors describe an interesting hypothesis to explain it. The authors investigate a number of interesting sub analyses to corroborate their findings.

Weaknesses:<br /> While there are some intriguing hypotheses in this manuscript, I struggle to be convinced. The main point that the authors argue is that positively selected alleles are older than their neutral counterparts at the same frequency. They argue that this may be because the positively selected alleles are stuck in some form of balancing selection for a long time before they switch to a more classical form of directional selection. The form of balancing selection they argue is one caused by linkage to deleterious alleles, which takes time for the beneficial alleles to recombine onto a more neutral background. I would really like to see some simulations that demonstrate this can actually occur on average. Reading this paper brought back memories of the classic Birky and Walsh (1988; PMCID: PMC281982) paper that argued that linkage amongst selected alleles does not impact the substitution rate of linked neutral alleles, but does reduce the substitution rate among beneficial alleles. Their simple simulations in 1988 illuminated how this works, and they developed a simple mathematical model that helped us understand how it works. In the current paper, it seems the authors are arguing for a similar effect, but rather than focus on beneficial alleles that fix, they are focusing on beneficial alleles that are still segregating. These seem like similar stories, but without simulations or a mathematical model, I struggle to gain any insight into why the observation is the way it is (and not simply due to a number of possible confounding effects noted below).<br /> There are a number of elements to the methods and interpretation that could use clarification.<br /> • Genetic data. One of the biggest weaknesses of this analysis is the choice of genetic data. The authors use the UK10k dataset, and reference the 2015 paper. Looking at that paper, it seems that the data may be composed of low coverage whole genome sequencing data (7x) and high coverage exome sequence data (80x). It appears that these data were integrated into a single VCF file, similar to the 1000 Genomes Project Phase 3 data. If these are the data that was used, then there are substantial differences between the coding and non-coding variants that are compared. However, it is possible that the authors chose to restrict the analysis to the low coverage WGS data and neglected to indicate it in the methods section. I will assume that this is the case for the rest of the review, but the authors should clarify.<br /> • Recombination rates. I believe the authors use an LD-based recombination map. While these maps are correlated at the longer physical distances with pedigree maps, there are substantial differences at shorter physical scales. These differences have been argued to be due to the action of natural selection skewing patterns of LD. If that is the case, then some of the observations in this paper are circular. Please confirm similar findings with a pedigree-based recombination map.<br /> • Recombination rates, pt 2. The authors compare patterns of non-synonymous coding variants to a set of non-coding, non-regulatory SNPs. They argue "these will necessarily have experienced similar mutational and recombinational processes". I don't know that this is true. There are both distinct recombination patterns and mutational patterns in genes vs non-coding regions of the genome. It would be important to more carefully match coding and non-coding variants based on both recombination as well as the type of nucleotide change. There are substantial differences in CpG composition in coding vs non-coding regions for example. While the authors say "Analyses thought to be sensitive to CpG high mutability were limited to SNPs that did not occur as part of a CpG", it is quite unclear what where CpGs were included vs excluded.<br /> • Identifying ancestral vs derived alleles. It is unclear how the authors identified ancestral vs derived alleles (they say "inferred ancestral sequence from Ensembl (1) and a maximum likelihood estimator". Several studies have shown that ancestral misidentification can cause skews in the site frequency spectrum. If the ancestral state of some fraction of alleles were misidentified, then the estimated allele age would be incorrect. Figure 1B shows that the mean frequency of the alleles with the largest delta-EP tend to be very low. This makes me think that ancestral misidentification may have impacted the results.<br /> • Figure 2B and C. I do not understand how the median can be so far outside the mean and error bars. The legend does not specify what the error bars are, but I feel the distribution must be shown if it is so skewed that the mean and any definition of error does not include the median.<br /> • Inferring allele ages. The authors use two methods for estimating allele ages, but focus on GEVA. They use the default parameter of effective population size 10,000. How sensitive is the model to this assumption? It has been shown that different regions of the genome (particularly coding vs neutral non-coding) experience different rates of deleterious mutations, and therefore different rates of background selection. Simple models of background selection would suggest that these regions will therefore have different effective population sizes.<br /> • Fst analysis. The authors look at Fst among 3 populations as a function of delta-EP compared to frequency-matched control SNPs. They find there is no statistical support for different levels of Fst in any pairwise comparison for any delta-EP bin. It seems strange that alleles with large delta-EP would not show increased Fst compared to control SNPs... If they are indeed positively selected, the assumption must be that they are then positively selected in all populations, which seems unlikely. Alternatively, by considering only narrow allele frequency bins, it is possible that Fst is also being controlled, and therefore this analysis is non-informative. A simulation would help understand what the expected pattern is here.<br /> • It would be great to show more figures like 2A. You can place the x-axis on a log-scale so that it is easier to view the lower allele frequencies. This plot clearly shows differences among the 3 categories. I am very surprised at the much shorter error bars for negative delta-EP at high frequency compared to positive delta-EP variants... Shouldn't there be very few negative delta-EP alleles at such high frequency?

Reviewer #1 (Public Review):

Summary:<br /> Herein, Blaeser et al. explored the impact of migraine-related cortical spreading depression (CSD) on the calcium dynamics of meningeal afferents that are considered the putative source of migraine-related pain. Critically previous studies have identified widespread activation of these meningeal afferents following CSD; however, most studies of this kind have been performed in anesthetized rodents. By conducting a series of technically challenging and compelling calcium imaging experiments in conscious head fixed mice they find in contrast that a much smaller proportion of meningeal afferents are persistently activated following CSD. Instead, they identify that post-CSD responses are differentially altered across a wide array of afferents, including increased and decreased responses to mechanical meningeal deformations and activation of previously non-responsive afferents following CSD. Given that migraine is characterized by worsening head pain in response to movement, the findings offer a potential mechanism that may explain this clinical phenomenon.

Strengths:<br /> Using head fixed conscious mice overcomes the limitations of anesthetized preps and the potential impact of anaesthesia on meningeal afferent function which facilitated novel results when compared to previous anesthetized studies. Further, the authors used a closed cranial window preparation to maximize normal physiological states during recording, although the introduction of a needle prick to induce CSD will have generated a small opening in the cranial preparation, rendering it not fully closed as suggested. However, technical issues with available AAV's and alternate less invasive triggering methodologies necessitate the current approach.

Weaknesses:<br /> Although this is a well conducted technically challenging study that has added valuable knowledge on the response of meningeal afferents the study would have benefited from the inclusion of more female mice. Migraine is a female dominant condition and an attempt to compare potential sex-differences in afferent responses would undoubtedly have improved the outcome. The authors report potential sex-specific effects on AAV transfection rates between males and females which have contributed to this imbalance.

The authors imply that the current method shows clear differences when compared to older anaesthetized studies; however, many of these were conducted in rats and relied on recording from the trigeminal ganglion. Attempts to address this point have proven difficult due to limited GCaMP signalling in anaesthetised mice, meaning that technical differences cannot be ruled out.

Reviewer #1 (Public Review):

The evolution of dioecy in angiosperms has significant implications for plant reproductive efficiency, adaptation, evolutionary potential, and resilience to environmental changes. Dioecy allows for the specialization and division of labor between male and female plants, where each sex can focus on specific aspects of reproduction and allocate resources accordingly. This division of labor creates an opportunity for sexual selection to act and can drive the evolution of sexual dimorphism.

In the present study, the authors investigate sex-biased gene expression patterns in juvenile and mature dioecious flowers to gain insights into the molecular basis of sexual dimorphism. They find that a large proportion of the plant transcriptome is differentially regulated between males and females with the number of sex-biased genes in floral buds being approximately 15 times higher than in mature flowers. The functional analysis of sex-biased genes reveals that chemical defense pathways against herbivores are up-regulated in the female buds along with genes involved in the acquisition of resources such as carbon for fruit and seed production, whereas male buds are enriched in genes related to signaling, inflorescence development and senescence of male flowers. Furthermore, the authors implement sophisticated maximum likelihood methods to understand the forces driving the evolution of sex-biased genes. They highlight the influence of positive and relaxed purifying selection on the evolution of male-biased genes, which show significantly higher rates of non-synonymous to synonymous substitutions than female or unbiased genes. This is the first report (to my knowledge) highlighting the occurrence of this pattern in plants. Overall, this study provides important insights into the genetic basis of sexual dimorphism and the evolution of reproductive genes in Cucurbitaceae.

Reviewer #1 (Public Review):

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

Main comments:

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

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

Reviewer #1 (Public Review):

The manuscript addresses a fundamental question about how different types of communication signals differentially affect brain state and neurochemistry. In addition, their manuscript highlights the various processes that modulate brain responses to communication signals, including prior experience, sex, and hormonal status. Overall, the manuscript is well-written and the research is appropriately contextualized.

That being said, it remains important for the authors to think more about their analytical approaches. In particular, the effect of normalization and the explicit outlining and interpretations of statistical models. As mentioned in the original review, the normalization of neurochemical data seems unnecessary given the repeated-measures design of their analysis and by normalizing all data to the baseline data and including this baseline data in the repeated measures analysis, one artificially creates a baseline period with minimal variation that dramatically differs in variance from other periods (akin to heteroscedasticity). If the authors want to analyze how a stimulus changes neurochemical concentrations, they could analyze the raw data but depict normalized data in their figures (similar to other papers). Or they could analyze group differences in the normalized data of the two stimulus periods (i.e., excluding the baseline period used for normalization).

It would also be useful for the authors to provide further discussion of the potential contributions of different types of experiences (mating vs. restraint) to the change in behavior and neurochemical responses to the vocalization playbacks and to try to disentangle sensory and motor contributions to neurochemical changes.

Reviewer #1 (Public Review):

Summary:<br /> This paper describes a comparison of different statistical methods for model comparison and covariate selection in neural encoding models. It shows in particular that issues arising from temporal autocorrelation and missing variables can lead to statistical tests with substantially higher false positive rates than expected from theory. The paper proposes methods for overcoming these problems, in particular cross-validation with cyclical shift permutation tests. The results are timely, important, and likely to have a broad impact. In particular, the paper shows that cell tuning classification can vary dramatically with the testing procedure, which is an important lesson for the field as a whole.

Strengths:<br /> - Novel and important comparison of different methods for variable selection in nested models.

Weaknesses:<br /> - Does not (yet) examine effect sizes<br /> - Does not motivate/explain key methods clearly enough in the main text.

General Comments:<br /> 1. My first general comment is that the paper in its current form focuses on the "null hypothesis significance testing" (NHST) paradigm. That is, it is focused on binary tests about what variables to include (or not include) in a regression model, and the false-positive rates of such tests. However, the broader statistics community has recently seen a shift away from NHST and towards a statistical reporting paradigm focused on effect sizes. See for example:<br /> - "Scientists rise up against statistical significance". Nature, March 2019.<br /> - Moving to a World Beyond "p < 0.05". RL Wasserstein, AL Schirm, NA Lazar. The American Statistician, 2019.

In light of this shift, I think the paper would be substantially strengthened if the authors could add a description of effect sizes for the statistical procedures they consider. Thus, for example, in cases where a procedure selects the wrong model (e.g., by selecting a variable that should not be included), how large is the inferred regression weight, and/or how large is the improvement in prediction performance (e.g. test log-likelihood) from including the erroneous regressor? How strong is the position tuning ascribed to a MEC cell that is inappropriately classified as having position tuning under one of the sub-optimal procedures? (Figure 7 shows some example place maps, but it would be nice to see a more thorough and rigorous analysis).

My suspicion would be that even when the hypothesis test gives a false positive, the effect sizes tend to remain small... but it is certainly possible that I'm mistaken, or that inferred effect sizes are more accurate for some procedures than others.

2. My only other major criticism relates to clarity and readability: in particular, the various procedures discussed in the paper ("forward selection", "maxT correction", "permutation test with cyclic shifts") are not clearly explained in the main paper, but are relegated to the Methods. Although I think it is useful to keep many of the mathematical details in the methods section, it would benefit the reader to have a general and intuitive explanation of the key methods within the flow of the main paper. The first paragraph of the Results section is particularly underdeveloped and hard to read and could benefit from a substantial revision to introduce and motivate the terms and procedures more clearly. I would recommend moving much of the text from the Methods into the Results section, or at the very least adding a paragraph describing the general idea/motivation for each method in Results.

Reviewer #1 (Public Review):

Summary:<br /> Zhu et al. set out to better understand the neural mechanisms underlying Drosophila larval escape behavior. The escape behavior is comprised of several sequenced movements, including a lateral roll motion followed by fast crawling. The authors specifically were looking to identify neurons important for the roll-to-crawl transition.

Strengths:<br /> This paper is clearly written. The experiments are logical and complementary. They support the author's main claim that SeIN128 is a type of descending neuron that is both necessary and sufficient to modulate the termination of rolling.

Weaknesses:<br /> -This manuscript is narrowly focused on Drosophila larval escape behavior. It would be more accessible to a broader audience if this work was put into a larger context of descending control.<br /> -In general, the rigor is high. However, a few control experiments are missing.

Reviewer #1 (Public Review):

This valuable study demonstrates a novel mechanism by which implicit motor adaptation saturates for large visual errors in a principled normative Bayesian manner. Additionally, the study revealed two notable empirical findings: visual uncertainty increases for larger visual errors in the periphery, and proprioceptive shifts/implicit motor adaptation are non-monotonic, rather than ramp-like. This study is highly relevant for researchers in sensory cue integration and motor learning. However, I find some areas where statistical quantification is incomplete, and the contextualization of previous studies to be puzzling.

Issue #1: Contextualization of past studies.

While I agree that previous studies have focused on how sensory errors drive motor adaptation (e.g., Burge et al., 2008; Wei and Kording, 2009), I don't think the PReMo model was contextualized properly. Indeed, while PReMo should have adopted clearer language - given that proprioception (sensory) and kinaesthesia (perception) have been used interchangeably, something we now make clear in our new study (Tsay, Chandy, et al. 2023) - PReMo's central contribution is that a perceptual error drives implicit adaptation (see Abstract): the mismatch between the felt (perceived) and desired hand position. The current paper overlooks this contribution. I encourage the authors to contextualize PReMo's contribution more clearly throughout. Not mentioned in the current study, for example, PReMo accounts for the continuous changes in perceived hand position in Figure 4 (Figure 7 in the PReMo study).

There is no doubt that the current study provides important additional constraints on what determines perceived hand position: Firstly, it offers a normative Bayesian perspective in determining perceived hand position. PReMo suggests that perceived hand position is determined by integrating motor predictions with proprioception, then adding a proprioceptive shift; PEA formulates this as the optimal integration of these three inputs. Secondly, PReMo assumed visual uncertainty to remain constant for different visual errors; PEA suggests that visual uncertainty ought to increase (but see Issue #2).

Issue #2: Failed replication of previous results on the effect of visual uncertainty.

2a. A key finding of this paper is that visual uncertainty linearly increases in the periphery; a constraint crucial for explaining the non-monotonicity in implicit adaptation. One notable methodological deviation from previous studies is the requirement to fixate on the target: Notably, in the current experiments, participants were asked to fixate on the target, a constraint not imposed in previous studies. In a free-viewing environment, visual uncertainty may not attenuate as fast, and hence, implicit adaptation does not attenuate as quickly as that revealed in the current design with larger visual errors. Seems like this current fixation design, while important, needs to be properly contextualized considering how it may not represent most implicit adaptation experiments.

2b. Moreover, the current results - visual uncertainty attenuates implicit adaptation in response to large, but not small, visual errors - deviates from several past studies that have shown that visual uncertainty attenuates implicit adaptation to small, but not large, visual errors (Tsay, Avraham, et al. 2021; Makino, Hayashi, and Nozaki, n.d.; Shyr and Joshi 2023). What do the authors attribute this empirical difference to? Would this free-viewing environment also result in the opposite pattern in the effect of visual uncertainty on implicit adaptation for small and large visual errors?

2c. In the current study, the measure of visual uncertainty might be inflated by brief presentation times of comparison and referent visual stimuli (only 150 ms; our previous study allowed for a 500 ms viewing time to make sure participants see the comparison stimuli). Relatedly, there are some individuals whose visual uncertainty is greater than 20 degrees standard deviation. This seems very large, and less likely in a free-viewing environment.

2d. One important confound between clear and uncertain (blurred) visual conditions is the number of cursors on the screen. The number of cursors may have an attenuating effect on implicit adaptation simply due to task-irrelevant attentional demands (Parvin et al. 2022), rather than that of visual uncertainty. Could the authors provide a figure showing these blurred stimuli (gaussian clouds) in the context of the experimental paradigm? Note that we addressed this confound in the past by comparing participants with and without low vision, where only one visual cursor is provided for both groups (Tsay, Tan, et al. 2023).

Issue #3: More methodological details are needed.

3a. It's unclear why, in Figure 4, PEA predicts an overshoot in terms of perceived hand position from the target. In PReMo, we specified a visual shift in the perceived target position, shifted towards the adapted hand position, which may result in overshooting of the perceived hand position with this target position. This visual shift phenomenon has been discovered in previous studies (e.g., (Simani, McGuire, and Sabes 2007)).

3b. The extent of implicit adaptation in Experiment 2, especially with smaller errors, is unclear. The implicit adaptation function seems to be still increasing, at least by visual inspection. Can the authors comment on this trend, and relatedly, show individual data points that help the reader appreciate the variability inherent to these data?

3c. The same participants were asked to return for multiple days/experiments. Given that the authors acknowledge potential session effects, with attenuation upon re-exposure to the same rotation (Avraham et al. 2021), how does re-exposure affect the current results? Could the authors provide clarity, perhaps a table, to show shared participants between experiments and provide evidence showing how session order may not be impacting results?

3d. The number of trials per experiment should be detailed more clearly in the Methods section (e.g., Exp 4). Moreover, could the authors please provide relevant code on how they implemented their computational models? This would aid in future implementation of these models in future work. I, for one, am enthusiastic to build on PEA.

3f. In addition to predicting a correlation between proprioceptive shift and implicit adaptation on a group level, both PReMo and PEA (but not causal inference) predict a correlation between individual differences in proprioceptive shift and proprioceptive uncertainty with the extent of implicit adaptation (Tsay, Kim, et al. 2021). Interestingly, shift and uncertainty are independent (see Figures 4F and 6C in Tsay et al, 2021). Does PEA also predict independence between shift and uncertainty? It seems like PEA does predict a correlation.

References:

Avraham, Guy, Ryan Morehead, Hyosub E. Kim, and Richard B. Ivry. 2021. "Reexposure to a Sensorimotor Perturbation Produces Opposite Effects on Explicit and Implicit Learning Processes." PLoS Biology 19 (3): e3001147.<br /> Makino, Yuto, Takuji Hayashi, and Daichi Nozaki. n.d. "Divisively Normalized Neuronal Processing of Uncertain Visual Feedback for Visuomotor Learning."<br /> Parvin, Darius E., Kristy V. Dang, Alissa R. Stover, Richard B. Ivry, and J. Ryan Morehead. 2022. "Implicit Adaptation Is Modulated by the Relevance of Feedback." BioRxiv. https://doi.org/10.1101/2022.01.19.476924.<br /> Shyr, Megan C., and Sanjay S. Joshi. 2023. "A Case Study of the Validity of Web-Based Visuomotor Rotation Experiments." Journal of Cognitive Neuroscience, October, 1-24.<br /> Simani, M. C., L. M. M. McGuire, and P. N. Sabes. 2007. "Visual-Shift Adaptation Is Composed of Separable Sensory and Task-Dependent Effects." Journal of Neurophysiology 98 (5): 2827-41.<br /> Tsay, Jonathan S., Guy Avraham, Hyosub E. Kim, Darius E. Parvin, Zixuan Wang, and Richard B. Ivry. 2021. "The Effect of Visual Uncertainty on Implicit Motor Adaptation." Journal of Neurophysiology 125 (1): 12-22.<br /> Tsay, Jonathan S., Anisha M. Chandy, Romeo Chua, R. Chris Miall, Jonathan Cole, Alessandro Farnè, Richard B. Ivry, and Fabrice R. Sarlegna. 2023. "Implicit Motor Adaptation and Perceived Hand Position without Proprioception: A Kinesthetic Error May Be Derived from Efferent Signals." BioRxiv. https://doi.org/10.1101/2023.01.19.524726.<br /> Tsay, Jonathan S., Hyosub E. Kim, Darius E. Parvin, Alissa R. Stover, and Richard B. Ivry. 2021. "Individual Differences in Proprioception Predict the Extent of Implicit Sensorimotor Adaptation." Journal of Neurophysiology, March. https://doi.org/10.1152/jn.00585.2020.<br /> Tsay, Jonathan S., Steven Tan, Marlena Chu, Richard B. Ivry, and Emily A. Cooper. 2023. "Low Vision Impairs Implicit Sensorimotor Adaptation in Response to Small Errors, but Not Large Errors." Journal of Cognitive Neuroscience, January, 1-13.

Reviewer #2 (Public Review):

Summary:<br /> This study seeks to understand the connection between protein sequence and function in disordered regions enriched in polar amino acids (specifically Q, N, S and T). While the authors suggest that specific motifs facilitate protein-enhancing activities, their findings are correlative, and the evidence is incomplete. Similarly, the authors propose that the re-assignment of stop codons to glutamine-encoding codons underlies the greater user of glutamine in a subset of ciliates, but again, the conclusions here are, at best, correlative. The authors perform extensive bioinformatic analysis, with detailed (albeit somewhat ad hoc) discussion on a number of proteins. Overall, the results presented here are interesting but are unable to exclude competing hypotheses.

Strengths:<br /> Following up on previous work, the authors wish to uncover a mechanism associated with poly-Q and SCD motifs explaining proposed protein expression-enhancing activities. They note that these motifs often occur IDRs and hypothesize that structural plasticity could be capitalized upon as a mechanism of diversification in evolution. To investigate this further, they employ bioinformatics to investigate the sequence features of proteomes of 27 eukaryotes. They deepen their sequence space exploration uncovering sub-phylum-specific features associated with species in which a stop-codon substitution has occurred. The authors propose this stop-codon substitution underlies an expansion of ploy-Q repeats and increased glutamine distribution.

Weaknesses:<br /> The authors were provided with a series of suggested changes to improve clarity, and a series of concerns raised. Some of these have been addressed but many have not. At this point, I do not see my role as telling the authors how to re-write their manuscript, but many of the concerns raised in my original review remain, and the authors have done little to allay those concerns in their revisions.

Reviewer #2 (Public Review):

The authors have greatly expanded their helpful hippocampome.org resource for the community regarding hippocampal cell types and their interactions from many perspectives. The many updates from v1.0 to v1.12 are nicely summarized in Table 1.

With v2.0, they now achieve the original vision of their project - to enable data-driven spiking neural network simulations of rodent hippocampal circuits. This work thus moves hippocampome.org from not only being a useful resource but also being able to launch simulations in which the models have direct links to the experimental literature. This will not only be of interest to the vast hippocampal community, but also to the diverse computational neuroscience community as theoretical models can potentially be "experimentally tested" with v2.0 to allow theoretical insights to be more biologically applicable.

Reviewer #1 (Public Review):

The authors investigate the function of the PTB domain containing adaptor protein Numb in skeletal muscle structure and function. In particular, the effects of reduced Numb expression in aging muscle is proposed as a mechanism for reduced contractile function associated with sarcopenia. Using ex-vivo analysis of conditional Numb and Numblike knockout muscle the authors demonstrate that loss of Numb but not the related Numblike gene expression perturbs muscle force generation. In order to explore the molecular mechanisms involved, Numb interacting proteins were identified in C2C12 cell cultured myotubes by immunoprecipitation and LC-MS/MS. The authors identify Septin 7 as well as Septin 2, 9 and 10 as a Numb binding proteins and demonstrate that loss of Numb/Numblike in myofibers causes changes in Septin 7 subcellular localization. Of note, whether additional septins form a complex or are also disrupted by Numb/Numblike loss remains an interesting area for further investigation. Additional investigation of the specificity and mapping of the Numb-Septin 7 (or another Septin) interaction would be of interest and provide an approach for future studies to demonstrate the biological relevance and specificity of the Numb-Septin 7 interaction in skeletal muscle

Reviewer #1 (Public Review):

In this manuscript, Davidsen and coworkers describe the development of a novel aspartate biosensor jAspSNFR3. This collaborative work supports and complements what was reported in a recent preprint by Hellweg et al., (bioRxiv.; doi: 10.1101/2023.05.04.537313). In both studies, the newly engineered aspartate sensor was developed from the same glutamate biosensor previously developed by the authors of this manuscript. This coincidence is not casual but is the result of the need to find tools capable of measuring aspartate levels in vivo. Therefore, it is undoubtedly a relevant and timely work carried out by groups experienced in aspartate metabolism and in the generation of metabolite biosensors.

Reviewer #1 (Public Review):

Summary:

In this work the authors provide evidence to show that an increase in Kv7 channels in hilar mossy cells of Fmr1 knock out mice results in a marked decrease in their excitability. The reduction in excitatory drive onto local hilar interneurons produces an increased excitation/inhibition ratio in granule cells. Inhibiting Kv7 channels can help normalize the excitatory drive in this circuit, suggesting that they may represent a viable target for targeted therapeutics for fragile-x syndrome.

Strengths:

The work is supported by a compelling and thorough set of electrophysiological studies. The authors do an excellent job of analysing their data and present a very complete data set.

Weaknesses:

There are no significant weaknesses in the experimental work, however the complexity of the data presentation and the lack of a schematic showing the organizational framework of this circuit make the data less accessible to non-experts in the field. I highly encourage a graphical abstract and network diagram to help individuals understand the implications of this work.

The work is important as it identifies a unique regional and cell specific abnormality in Fmr1 KO mice, showing how the loss of one gene can result in region specific changes in brain circuits.

Reviewer #1 (Public Review):

Summary:<br /> The authors measured the oxygen stable isotope ratios in six orangutan teeth using a state of the art micro-sampling technique (SHRIMP SI) to gather substantial multi-year isotopic data for six modern and five fossil orangutan individuals from Borneo and Sumatra. This fine-scale sampling technique allowed to address the fundamental question if breastfeeding affects the oxygen isotope ratios in teeth forming in the first one to two years of life, during which orangutans can be assumed to largely depend on breastmilk. The authors provide compelling evidence that the consumption of milk does not appear to affect the overall isotopic profile in early forming teeth. They conclude that this allows us to use these teeth as terrestrial/arboreal isotopic proxies in paleoenvironmental research, which would provide an invaluable addition to otherwise largely marine climate records in this regions.

Strengths:<br /> The overall large sample size of orangutan dental isotope records as well as the rigorous dating of the fossil specimens provide a strong dataset for addressing the outlined questions. The direct comparison of modern and fossil orangutan specimens provides a valuable evaluation of the use of these modern and past environmental proxies, with some discussion of the implications for the environmental conditions during the expansion of early modern humans into this region of the world.

Weakness:<br /> The authors illustrate that all orangutan individuals sampled, modern and fossil, show a considerable amount of isotopic variation between and within their teeth. Some of this variation is clearly associated with isotopic shifts in precipitation, but some will also be linked to the variation in oxygen isotopes within the forest itself and the many plant foods it produces for the orangutan. In the future, the systematic measurement of oxygen isotopes across orangutan food items, forest canopies and precipitation could help differentiate how much of the observed isotopic variation in teeth is indeed related to climatic shifts alone.

Reviewer #1 (Public Review):

The article offers a comparative study between various methodologies to evaluate the abundance, richness, and diversity of insects from data obtained in a large-scale field experiment. The experiment is impressive in view of the number of locations, its spatial coverage, the number of instruments or methods used, and the data collected appears rich and worthy of multiple publications. The paper focuses on the validation of a novel approach based on optical sensors. These sensors collect the backscattered light from flying insects in their field of view and can retrieve the wingbeat frequency and the body-to-wing backscattering ratios.<br /> Unfortunately, the paper is poorly written and hard to read, with a lack of clear sections, and an overall confusing structure. The methods, metrics, and data analysis are not properly and thoroughly described, making it sometimes difficult to evaluate the validity of the approach.<br /> Most importantly, the methodology to retrieve the richness and diversity from optical sensors seems flawed. While the scope and scale of the experiment is valuable, I do not believe that this article supports the authors' claim. The main criticisms are described in more detail below.

1) The Material and Method section is poorly structured. The article focuses on a series of metrics to evaluate biodiversity from three independent methods: optical sensors, malaise traps, and net sweeping. The authors need to provide a clear and thorough description of what the metrics to be studied are, and how those metrics are evaluated for each method. While it is the main focus of the paper, the term "biodiversity metrics" is never properly defined, it is used in the singular form in both the title and abstract, then in its plural form in the rest of the paper, making the reader further doubt what exactly it means. It is then discussed using the correlation value retrieved when studying richness, so is the biodiversity metric the same as richness? Studying biodiversity remains a complex and sometimes contentious subject and this term, especially when measured by three different methods, is far from obvious. The term "community metrics" is defined as abundance, richness, and diversity; is that the same as biodiversity metrics? In any case, the method section should thoroughly describe how each of those metrics is calculated from the raw data collected by each method. This information is somewhat there, but in a very unorganized way, making it difficult to read. I would recommend organizing this section with multiple and clear sections: 1) describing the metrics that are meant to be studied, 2) the location, dates and time, type of crops, and other general information about the experiment, 3) description and methods around optical sensors, 4) description and methods around malaise traps, 5) description and methods around the sweeping. The last 3 sections should describe how it retrieves the previously defined metrics, potentially using equations.

2) Regarding the calculation of the body-to-wing ratio, sigma is described as a "signal" line 195, then is described as intensity counts in Figure 2; isn't it really the backscattering optical cross-section? It changes significantly over time during the signal, so how is one value of sigma calculated? Is it the average of the whole insect event? The maximum?

3) The "ecosystem services" paragraph is really confusing and needs to be rewritten.

4) Like for the method section, the result section should be structured around the comparison of each metric, abundance, richness, and diversity, or any other properly defined metrics described in the method, so that the result section is consistent with the method section.

5) The abundance is not correlated; interestingly, malaise traps and sweeping are even less correlated which further supports the claims by the authors that new and improved methods are needed. This part of the results could be further developed. A linear fit could be added to Figure 4.

6) Richness and diversity are the most problematic. Again, the method is poorly described, with pieces of explanation spread out throughout the paper, but my understanding is the following: the optical sensor retrieves two features from each insect signal, wbf, and BWR. Clustering is made using DBSCAN which has 2 parameters: minimum number of signals, and merge distance. It is important to note that these two parameters will greatly influence the number of clusters found by DBSCAN. The richness obtained by optical sensors is defined as the number of clusters and the diversity is evaluated from it as well. Hence, both diversity and richness are greatly dependent on the chosen parameters. The DBSCAN parameters are chosen by maximizing the Spearman correlation between richness obtained by the optical sensors and richness by the capture methods. I see a major problem here: if you optimize the parameters, that directly impact the retrieved diversity and richness by optical sensors, to have the best correlation with either the richness or diversity of the other methods, you will automatically create a correlation between the richness and diversity retrieved by the optical sensors and alternative methods. The p-value in Figure 6 does not represent the probability of the correlation hypothesis being false anymore, since the whole process is based on artificially forcing the correlation from the start.

7) In addition, the clustering method provides values higher than 80, which is quite unrealistic with just 2 features, wbf and BWR. It is clear from many studies using optical sensors that the features from optical sensors are subject to variability. Wbf has naturally some variances within the same species, not to mention temperature dependency. Backscattering cross sections will also heavily function on the insect's orientation (facing or sideways) while crossing the cone of light, and, even though it is a ratio, the collection efficiency of the instrument telescope and scattering efficiency of the target will be impacted by the position of the insects within the cone of light, which will also impact the variability on the BWR. While those features can still be used, obtaining 80 clusters from two variables with such statistical fluctuations is simply not credible. Additional features could help, such as the two wavelengths mentioned in the description of the optical sensor but are never mentioned again.

The conclusion then states that the study serves as the first field validation. I disagree; the abundance doesn't correlate, and the richness and diversity evaluations are flawed. While I do think there is great value in the work done by the authors through this impressive field experiment, and in general in their work toward the development of entomological optical sensors, I believe the data analysis and communication of the results do not support the conclusions drawn.

Reviewer #1 (Public Review):

Summary:<br /> The evolution of transporter specificity is currently unclear. Did solute carrier systems evolve independently in response to a cellular need to transport a specific metabolite in combination with a specific ion or counter metabolite, or did they evolve specificity from an ancestral protein that could transport and counter-transport most metabolites? The present study addresses this question by applying selective pressure to Saccharomyces cerevisiae and studying the mutational landscape of two well-characterised amino acid transporters. The data suggest that AA transporters likely evolved from an ancestral transporter and then specific sub-families evolved specificity depending on specific evolutionary pressure.

Strengths:<br /> The work is based on sound logic and the experimental methodology is well thought through. The data appear accurate, and where ambiguity is observed (as in the case of citruline uptake by AGP1), in vitro transport assays are carried out to verify transport function.

Weaknesses:<br /> Although the data and findings are well described, the study lacked additional contextual information that would support a clear take-home message.

Reviewer #1 (Public Review):

Summary:<br /> This manuscript presents a method to infer causality between two genes (and potentially proteins or other molecules) based on the non-genetic fluctuations among cells using a version of the dual-reporter assay as a causal control, where one half of the dual-reporter pair is causally decoupled, as it is inactive. The authors propose a statistical invariant identity to formalize this idea.

Strengths:<br /> The paper outlines a theoretical formalism, which, if experimentally used, can be useful in causal network inference, which is a great need in the study of biological systems.

Weaknesses:<br /> The practical utility of this method may not be straightforward and potentially be quite difficult to execute. Additionally, further investigations are needed to provide evidence of the broad applicability of the method to naturally occurring systems and its scalability beyond the simple circuit in which it is experimentally demonstrated.

Reviewer #1 (Public Review):

Summary:

This manuscript by Bomba-Warczak describes a comprehensive evaluation of long-lived proteins in the ovary using transgenerational radioactive labelled 15N pulse-chase in mice. The transgenerational labeling of proteins (and nucleic acids) with 15N allowed the authors to identify regions enriched in long-lived macromolecules at the 6 and 10-month chase time points. The authors also identify the retained proteins in the ovary and oocyte using MS. Key findings include the relative enrichment in long-lived macromolecules in oocytes, pregranulosa cells, CL, stroma, and surprisingly OSE. Gene ontology analysis of these proteins revealed enrichment for nucleosome, myosin complex, mitochondria, and other matrix-type protein functions. Interestingly, compared to other post-mitotic tissues where such analyses have been previously performed such as the brain and heart, they find a higher fractional abundance of labeled proteins related to the mitochondria and myosin respectively.

Strengths:

A major strength of the study is the combined spatial analyses of LLPs using histological sections with MS analysis to identify retained proteins.

Another major strength is the use of two chase time points allowing assessment of temporal changes in LLPs associated with aging.

The major claims such as an enrichment of LLPs in pregranulosa cells, GCs of primary follicles, CL, stroma, and OSE are soundly supported by the analyses, and the caveat that nucleic acids might differentially contribute to this signal is well presented.

The claims that nucleosomes, myosin complex, and mitochondrial proteins are enriched for LLPs are well supported by GO enrichment analysis and well described within the known body of evidence that these proteins are generally long-lived in other tissues.

Weaknesses:

One small potential weakness is the lack of a mechanistic explanation of if/why turnover may be accelerating at the 6-10 month interval compared to 1-6.

A mild weakness is the open-ended explanation of OSE label retention. This is a very interesting finding, and the claims in the paper are nuanced and perfectly reflect the current understanding of OSE repair. However, if the sections are available and one could look at the spatial distribution of OSE signal across the ovarian surface it would interesting to note if label retention varied by regions such as the CLs or hilum where more/less OSE division may be expected.

Reviewer #1 (Public Review):

Summary: This study addressed an alternative hypothesis to temporal binding phenomena. In temporal binding, two events that are separated in time are "pulled" towards one another, such that they appear more coincidental. Previous research has shown evidence of temporal binding events in the context of actions and multisensory events. In this context, the author revisits the well-known Libet clock paradigm, in which subjects view a moving clock face, press a button at a time of their choosing to stop the clock, a tone is played (after some delay), and then subjects move the clock dial to the point where the one occurred (or when the action occurred). Classically, the reported clock time is a combination of the action and sound times. The author here suggests that attention can explain this by a mechanism in which the clock dial leads to a roving window of spatiotemporal attention (that is, it extends in both space and time around the dial). To test this, the author conducted a number of experiments where subjects performed the Libet clock experiment, but with a variety of different stimulus combinations. Crucially, a visual detection task was introduced by flashing a disc at different positions along the clock face. The results showed that detection performance was also "pulled" towards the action event or sensory event, depending on the condition. A model of roving spatiotemporal attention replicated these effects, providing further evidence of the attentional window.

The study provides a novel explanation for temporal binding phenomena, with clear and cleverly designed experiments. The results provide a nice fit to the proposed model, and the model itself is able to recapitulate the observed effects.

Reviewer #1 (Public Review):

Summary:

The current study reports a cryo-EM structure of MFS transporter MelB trapped in an inward-facing state by a conformationally selective nanobody. The authors compare this structure to previously-resolved crystal structures of outward-facing MelB. Additionally, the authors report H/D exchange/ mass spec experiments that identify accessible residues in the protein.

Strengths:

The authors overcame very significant technical challenges to solve the first inward-facing structure of the small, model MFS transporter MelB by cryo-EM. The use of conformation-trapping nanobodies (which had been reported previously by this group) is particularly nice.

Weaknesses:

The authors highlight the use of HDX experiments as a measurement of protein conformational dynamics. However, the experiment instead measures the accessibility of different residues. An ideal experiment would trap the transporter in inward- and outward states, but only the inward conformation is trapped here. The outward-facing conformation is instead an ensemble of outward and occluded conformations. It seems obvious that this will be more dynamic than the nanobody-trapped inward state.

Reviewer #1 (Public Review):

Summary: The current study reports a cryo-EM structure of MFS transporter MelB trapped in an inward-facing state by a conformationally selective nanobody. The authors compare this structure to previously-resolved crystal structures of outward-facing MelB. Additionally, the authors report H/D exchange/ mass spec experiments that identify accessible residues in the protein.

Strengths:

The authors overcame very significant technical challenges to solve the first inward-facing structure of the small, model MFS transporter MelB by cryo-EM. The use of conformation-trapping nanobodies (which had been reported previously by this group) is particularly nice.

Weaknesses:

Maps and coordinates were not provided by the authors, which presents a gap in this assessment.

The authors highlight the use of HDX experiments as a measurement of protein conformational dynamics. However, this experiment does not measure the conformational dynamics of the transporter, since in these experiments exchange is not initiated by ligand addition or another trigger. The experiment instead measures the accessibility of different residues, and of course, a freely-exchanging sodium bound transporter would have more exchangeable positions than when a conformation-trapping nanobody is bound. It is not clear what new mechanistic information this provides, since this property of the nanobody has already been established.

Based on the evidence presented, it is somewhat speculative that the structure represents the EIIa-bound regulatory state.

Reviewer #2 (Public Review):

Yanagihara and colleagues investigated the immune cell composition of bronchoalveolar lavage fluid (BALF) samples in a cohort of patients with malignancy undergoing chemotherapy and with with lung adverse reactions including Pneumocystis jirovecii pneumonia (PCP) and immune-checkpoint inhibitors (ICIs) or cytotoxic drug induced interstitial lung diseases (ILDs). Using mass cytometry, their aim was to characterize the cellular and molecular changes in BAL to improve our understanding of their pathogenesis and identify potential biomarkers and therapeutic targets. In this regard, the authors identify a correlation between CD16 expression in T cells and the severity of PCP and an increased infiltration of CD57+ CD8+ T cells expressing immune checkpoints and FCLR5+ B cells in ICI-ILD patients.

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

1) The authors should elaborate on why different set of markers were selected for each analysis step. E.g., Different set of markers were used for UMAP, CITRUS and viSNE in the T cell and myeloid analysis.

2) The authors should state if a normality test for the distribution of the data was performed. If not, non-parametric tests should be used.

3) The authors should explore the correlation between CD16 intensity and the CTCAE grade in T cell subsets such as EMRA CD8 T cells, effector memory CD4, etc as identified in Figure 1B.

4) The authors could use CITRUS to better assess the B cell compartment.

Reviewer #1 (Public Review):

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

The authors have now adapted their manuscript reflecting the novelties of these studies. Overall, the study is a continuation and also corroboration of previous work, without clinical data yet. The authors have now expanded their work to a second mouse model, which strengthens their data.

Reviewer #1 (Public Review):

Summary:

The authors attempt to fully characterise the immunoglobulin (Ig) heavy (H) chain repertoire of tumor-infiltrating B cells from three different cancer types by identifying the IgH repertoire overlap between these, their corresponding draining lymph nodes (DLNs), and peripheral B cells. The authors claim that B cells from tumors and DLNs have a closer IgH profile than those in peripheral blood and that DLNs are differentially involved with tumor B cells. The claim that tumor-resident B cells are more immature and less specific is made based on the characteristics of the CDR-H3 they express.

Strengths:

The authors show great expertise in developing in-house bioinformatics pipelines, as well as using tools developed by others, to explore the IgH repertoire expressed by B cells as a means of better characterising tumour-associated B cells for the future generation of tumour-reactive antibodies as a therapy.

Weaknesses:

This paper needs major editing, both of the text and the figures, because as it stands it is convoluted and extremely difficult to follow. The conclusions reached are often not obvious from the figures themselves. Sufficient a priori details describing the framework for their analyses are not provided, making the outcome of their results questionable and leaving the reader wondering whether the findings are on solid ground. The authors are encouraged to explain in more detail the premises used in their algorithms, as well as the criteria they follow to define clonotypes, clonal groups, and clonal lineages, which are currently poorly defined and are crucial elements that may influence their results and conclusions. Having excluded the IGHD gene segment from some of their analyses (at least those related to clonal lineage inference and phylogenetic trees), it is not well explained which region of CDR-H3 is responsible for the charge, interaction strength, and Kidera factors, since in some cases the authors mention that the central part of CDR-H3 consists of five amino acids and in others of seven amino acids. How can the authors justify that the threshold for CDR-H3 identity varies according to individual patient data?

Throughout the analyses, the reasons for choosing one type of cancer over another sometimes seem subjective and are not well justified in the text.

Overall, the narrative is fragmented. There is a lack of well-defined conclusions at the end of the results subheadings. The exact same paragraph is repeated twice in the results section. The authors have also failed to synchronise the actual number of main figures with the text, and some panels are included in the main figures that are neither described nor mentioned in the text (Venn diagram Fig. 2A and phylogenetic tree Fig. 5D). Overall, the manuscript appears to have been rushed and not thoroughly read before submission.

Reviewers are forced to wade through, unravel, and validate poorly explained algorithms in order to understand the authors' often bold conclusions.

Reviewer #1 (Public Review):

Summary:<br /> This work explored intra and interspecific niche partitioning along spatial, temporal, and dietary niche partitioning between apex carnivores and mesocarnivores in the Qilian Mountain National Park of China, using camera trapping data and DNA metabarcoding sequencing data. They conclude that spatial niche partitioning plays a key role in facilitating the coexistence of apex carnivore species, spatial and temporal niche partitioning facilitate the coexistence of mesocarnivore species, and spatial and dietary niche partitioning facilitate the coexistence between apex and mesocarnivore species. The information presented in this study is important for wildlife conservation and will contribute substantially to the current understanding of carnivore guilds and effective conservation management in fragile alpine ecosystems.

Strengths:<br /> Extensive fieldwork is evident in the study. Aiming to cover a large percentage of the Qilian Mountain National Park, the study area was subdivided into squares, as a geographical reference to distribute the sampling points where the camera traps were placed and the excreta samples were collected.

They were able to obtain many records in their camera traps and collected many samples of excreta. This diversity of data allowed them to conduct robust analyses. The data analyses carried out were adequate to obtain clear and meaningful results that enabled them to answer the research questions posed. The conclusions of this paper are mostly well supported by data.

The study has demonstrated the coexistence of carnivore species in the landscapes of the Qilian Mountains National Park, complementing the findings of previous studies. The information presented in this study is important for wildlife conservation and will contribute substantially to the current understanding of carnivore guilds and effective conservation management in fragile alpine ecosystems.

Weaknesses:<br /> It is necessary to better explain the methodology because it is not clear what is the total sampling effort. In methodology, they only claim to have used 280 camera traps, and in the results, they mention that there are 319 sampling sites. However, the total sampling effort (e.g. total time of active camera traps) carried out in the study and at each site is not specified.

Reviewer #1 (Public Review):

Summary: This papers performs fine-mapping of the silkworm mutants bd and its fertile allelic version, bdf, narrowing down the causal intervals to a small interval of a handful of genes. In this region, the gene orthologous to mamo is impaired by a large indel, and its function is later confirmed using expression profiling, RNAi, and CRISPR KO. All these experiments are convincingly showing that mamo is necessary for the suppression of melanic pigmentation in the silkworm larval integument.

The authors also use in silico and in vitro assays to probe the potential effector genes that mamo may regulate.

Strengths: The genotype-to-phenotype workflow, combining forward (mapping) and reverse genetics (RNAi and CRISPR loss-of-function assays) linking mamo to pigmentation are extremely convincing.

This revision is a much improved manuscript and I command the authors for many of their edits.

I find the last part of the discussion, starting at "It is generally believed that changes in gene expression patterns are the result of the evolution of CREs", to be confusing.<br /> In this section, I believe the authors sequentially:<br /> - emphasize the role of CRE in morphological evolution (I agree)<br /> - emphasize that TF, and in particular their own CRE, are themselves important mutational targets of evolution (I agree, but the phrasing need to insist the authors are here talking about the CRE found at the TF locus, not the CRE bound by the TF).<br /> - use the stickleback Pel enhancer as an example, which I think is a good case study, but the authors also then make an argument about DNA fragility sites, which is hard to connect with the present study.<br /> - then continue on "DNA fragility" using the peppered moth and butterfly cortex locus. There is no evidence of DNA fragility at these loci, so the connection does not work. "The cortex gene locus is frequently mutated in Lepidoptera", the authors say. But a more accurate picture would be that the cortex locus is repeatedly involved in the generation of color pattern variants. Unlike for Pel fragile enhancer, we don't know if the causal mutations at this locus are repeatedly the same, and the haplotypes that have been described could be collateral rather than causal. Overall, it is important to clarify the idea that mutation bias is a possible factor explaining "genetic hotspots of evolution" (or genetic parallelism sensu 10.1038/nrg3483), but it is also possible that many genetic hotspots are repeated mutational targets because of their "optimal pleiotropy" (e.g. hub position in GRNs, such as mamo might be), or because of particularly modular CRE region that allow fine-tuning. Thus, I find the "fragility" argument misleading here. In fact the finding that "bd" and "bdf" alleles are different in nature is against the idea of a fragility bias (unless the authors can show increased mutation rates at this locus in a wild silkmoth species?). These alleles are also artificially-selected ie. they increased in frequency by breeding rather than natural selection in the wild, so while interesting for our understand of the genotype-phenotype map, they are not necessarily representative of the mutations that may underlie evolution in the wild.<br /> - Curiously, the last paragraph ("Some research suggests that common fragile sites...") elaborate on the idea that some sites of the genome are prone to mutation. The connection with mamo and the current article are extremely thin. There is here an attempt to connect meiotic and mitotic breaks to Bm-mamo, but this is confusing : it seems to propose Bm-mamo as a recruiter of epigenetic modulators that may drive higher mutation rates elsewhere. Not only I am not convinced by this argument without actual data, but this would not explain how the mutations at the Bm-mamo itself evolved.

On a more positive note, I find it fascinating that the authors identified a TF that clearly articulates or orchestrate larval pattern development, and that when it is deleted, can generate healthy individuals. In other words, while it is a TF with many targets, it is not too pleiotropic. This idea, that the genetically causal modulators of developmental evolution are regulatory genes, has been described elsewhere (e.g. Fig 4c in 10.1038/s41576-020-0234-z, and associated refs). To me, the beautiful findings about Bm-mamo make sense in the general, existing framework that developmental processes and regulatory networks "shape" the evolutionary potential and trajectories of organisms. There is a degree of "programmability" in the genomes, because some loci are particularly prone to modulate a given type of trait. Here, Bm-mamo, as a potentially regulator of both CPs and melanin pathway genes, appear to be a potent modulator of epithelial traits. Claiming that there are inherent mutational biases behind this is unwarranted.

Reviewer #1 (Public Review):

In 2019, Wilkinson and colleagues (PMID: 31142833) managed to break the veil on a 20-year open question on how to properly culture and expand Hematopoietic Stem Cells (HSCs). Although this study is revolutionizing the HSC biology field, several questions regarding the mechanisms of expansion remain open. Leveraging on this gap, Zhang et al.; embarked on a much-needed investigation regarding HSC self-renewal in this particular culturing setting.

The authors firstly tacked the known caveat that some HSC membrane markers are altered during in vitro cultures by functionally establishing EPCR (CD201) as a reliable and stable HSC marker (Figure 1), demonstrating that this compartment is also responsible for long-term hematopoietic reconstitution (Figure 3). Next in Figure 2, the authors performed single-cell omics to shed light into the potential mechanisms involved into HSC maintenance, and interestingly it was shown that several hematopoietic populations like monocytes and neutrophils are also present in this culture conditions, which has not been reported. The study goes on to functionally characterize these cultured HSCs (cHSC). The authors elegantly demonstrate using state-of-the-art barcoding strategies that these culturing conditions provoke heterogeneity in the expanding HSC pool (Figure 4). In the last experiment (Figure 5), it was demonstrated that cHSC not only retain their high EPCR expression levels but upon transplantation these cells remain more quiescent than freshly-isolated controls.

Taken together, this study independently validates that the proposed culturing system works and provide new insights into the mechanisms whereby HSC expansion takes place.

Following a first round of comments, the authors provided a comprehensive point-by-point response to the different points raised by reviewers, which significantly helps on better understanding some of the decisions taken by the authors. However, it is surprising that the current manuscript is practically unchanged compared to the previous version. Effectively, all major comments raised by reviewers are address in the response letter rather than incorporated into a truly updated version, which would be of great benefit for readers.

Further comments:<br /> 1. It is highly appreciated that the authors provide a comprehensive and cohesive explanations on i) the rationale for employing SAILERX for single-cell RNA and ATAC-seq, ii) data on HSC signature projected on independent scRNA-seq datasets and iii) further context on the Fgd5 expression limitations. These are important snippets of information which do not only further validate this manuscript's data but also provide context within the HSC biology field.<br /> However, I do not fully agree with the author statement "our primary objective in this study was to highlight the relatively low content of HSCs in cultures" (page 1, response to Reviewers) justifying why single-cell genome-wise approaches were used. As the authors are aware HSCs are defined by functional characterization rather than transcriptional/chromatin accessibility profiles, so it seems odd that this was the rationale to perform omics for this purpose. More importantly, the authors had gone through the lengths of already performing this costly and time-consuming experiment, but miss out on the opportunity to take a deeper dive into the molecular characteristics that could explain divergent behavior between freshly-isolated and cultured HSCs. It would be extremely relevant to the HSC biology community to understand, for example, if these two HSC populations have differences in enhancer accessibility (if the data quality allows), which could provide an upstream explanation for differences in transcription (is also not explored in this manuscript version).

2. It intriguing that the authors acknowledge that there are already more recent versions of this expansion protocol (page 2, response to Reviewers) and provided a convoluted explanation on why these were not included in the original manuscript. Both papers (PMID: 36809781 and PMID: 37385251) have now been published in respected peer-reviewed journals and provide insights which are pertinent for this work. Yet, the authors decided not to discuss these findings. It is understandable that repeating experiments with these updated conditions is outside of the scope of this manuscript, but it would be relevant to discuss how these recent advances in the protocol impact the work presented in this manuscript.

3. Regarding the previous comment on how cultured HSC are related to HSC aging, I highly appreciate both data on serial transplantation and also on scRNA-seq.

Reviewer #1 (Public Review):

Summary:<br /> TRAP transporters are an unusual class of secondary active transporters that utilize periplasmic binding proteins to deliver their substrates. This paper contributes a new 3 Å structure of the Haemophilus influenzae TRAP transporter. The structure joins two other recent cryo-EM structures of TRAP transporters, including a lower resolution structure of the same H. influenzae protein (overall 4.7 Å), and a ~3 Å structure of a homologue from P. profundum. In addition to reporting a higher resolution cryo-EM structure, the authors also recapitulate protein activity in a reconstituted system, investigate protein oligomerization using analytic ultracentrifugation, and evaluate interactions and function in "mix and match" configurations with periplasmic subunits from other homologues.

Strengths:<br /> The strength of the paper is that the better resolution cryo-EM data permits sidechain assignment, the identification of bound lipids, and the identification of sodium ions. It is important to get this structure out there, since the resolution passes an important threshold for model building accuracy. The current structure nicely explains a lot of prior mutagenesis data on the H. influenzae TRAP. This is also the first structure of a TRAP protein to be solved without a fiducial, although the overall structure is not very different than those solved with fiducials.

Weaknesses:<br /> The experiments examining the monomer/dimer equilibrium appear somewhat preliminary. The biological or mechanistic importance of oligomerization is not established, so these experiments are inherently of limited scope. Moreover, cryo-EM datasets exhibit both parallel and antiparallel dimers, the latter of which are clearly not biologically relevant. It is probably impossible to distinguish these in the AUC experiments, which makes interpretation of these experiments more difficult.

Similarly, the importance of the lipid binding sites observed in cryo-EM aren't experimentally established (for example by mutating the binding site) and it is thus unknown whether they are important for function (as the authors acknowledge).

Reviewer #1 (Public Review):

Summary:

The manuscript by Xia et al. investigated the mechanisms underlying Glucocorticoid-induced osteonecrosis of the femoral head (GONFH). The authors observed that abnormal osteogenesis and adipogenesis is associated with decreased β-catenin in the necrotic femoral head of GONFH patients and inhibition of β-catenin signaling leads to abnormal osteogenesis and adipogenesis in GONFH rats. Of interest, deletion of β-catenin in Col2-expressing cells rather than in osx-expressing cells leads to a GONFH-like phenotype in femoral head of mice.

Strengths:

A strength of the study is that it sets up a Col2-expressing cell-specific β-catenin knockout mouse model that mimics full spectrum of osteonecrosis phenotype of GONFH. This is interesting and provides new insights into the understanding of GONFH. Overall, the data are solid and support their conclusions.

Reviewer #1 (Public Review):

Summary:<br /> This paper investigates the neural mechanisms underlying the change in perception when viewing ambiguous figures. Each possible percept is related to an attractor-like brain state and a perceptual switch corresponds to a transition between these states. The hypothesis is that these switches are promoted by bursts of noradrenaline that change the gain of neural circuits. The authors present several lines of evidence consistent with this view: pupil diameter changes during the time point of the perceptual change; a gain change in neural network models promotes a state transition; and large-scale fMRI dynamics in a different experiment suggests a lower barrier between brain states at the change point. However, some assumptions of the computational model seem not well justified and the theoretical analysis is incomplete. The paper would also benefit from a more in-depth analysis of the experimental data.

Strengths:<br /> The main strength of the paper is that it attempts to combine experimental measurements - from psychophysics, pupil measurements, and fMRI dynamics - and computational modeling to provide an emerging picture of how a perceptual switch emerges. This integrative approach is highly useful because the model has the potential to make the underlying mechanisms explicit and to make concrete predictions.

Weaknesses:<br /> A general weakness is that the link between the three parts of the paper is not very strong. Pupil and fMRI measurements come from different experiments and additional analysis showing that the two experiments are comparable should be included. Crucially, the assumptions underlying the RNN modeling are unclear and the conclusions drawn from the simulation may depend on those assumptions.

Main points:<br /> Perceptual tasks in pupil and fMRI experiments: how comparable are these two tasks? It seems that the timing is very different, with long stimulus presentations and breaks in the fMRI task and a rapid sequence in the pupil task. Detailed information about the task timing in the pupil task is missing. What evidence is there that the same mechanisms underlie perceptual switches at these different timescales? Quantification of the distributions of switching times/switching points in both tasks is missing. Do the subjects in the fMRI task show the same overall behavior as in the pupil task? More information is needed to clarify these points.

Computational model:<br /> 1. Modeling noradrenalin effects in the RNN: The pupil data suggests phasic bursts of NA would promote perceptual switches. But as I understand, in the RNN neuromodulation is modeled as different levels of gain throughout the trial. Making the neural gain time-dependent would allow investigation of whether a phasic gain change can explain the experimentally observed distribution of switching times.

2. Modeling perceptual switches: in the results, it is described that the networks were trained to output a categorical response, but the firing rates in Fig 2B do not seem categorical but rather seem to follow the input stimulus. The output signals of the network are not shown. If I understand correctly, a trivial network that would just represent the two input signals without any internal computation and relay them to the output would do the task correctly (because "the network's choice at each time point was the maximum of the two-dimensional output", p. 22). This seems like cheating: the very operation that the model should perform is to signal the change, in a categorical manner, not to represent the gradually changing input signals.

3. The mechanism of how increased gain leads to faster switches remains unclear to me. My first intuition was that increasing the gain of excitatory populations (the situation shown in Fig. 2E) in discrete attractor models would lead to deeper attractor wells and this would make it more difficult to switch. That is, a higher gain should lead to slower decisions in this case. However, here the switching time remains constant for a gain between 1 and 1.5. Lowering the gain, on the other hand, leads to slower switching. It is, of course, possible that the RNN behaves differently than classical point attractor models or that my intuition is incorrect (though I believe it is consistent with previous literature, e.g. Niyogi & Wong-Lin 2013 (doi:10.1371/journal.pcbi.1003099) who show higher firing rates - more stable attractors - for increased excitatory gain).

4. From the RNN model it is not clear how changes in excitatory and inhibitory gain lead to slower/faster switching. In order to better understand the role of inhibitory and excitatory gain on switching, I would suggest studying a simple discrete attractor model (a rate model, for example as in Wong and Wang 2006 or Roxin and Ledberg, Plos Comp. Bio 2008) which will allow to study these effects in terms of a very few model parameters. The Roxin paper also shows how to map rate models onto simplified one-dimensional systems such as the one in Fig S3. Setting up the model using this framework would allow for making much stronger, principled statements about how gain changes affect the energy landscape, and under which conditions increased inhibitory gain leads to faster switching.

One possibility is that increasing the excitatory gain in the RNN leads to saturated firing rates. If this is the reason for the different effects of excitatory and inhibitory gain changes, it should be properly explained. Moreover, the biological relevance of this effect should be discussed (assuming that saturation is indeed the explanation).

Alternative mechanisms:<br /> It is mentioned in the introduction that changes in attention could drive perceptual switches. A priori, attention signals originating in the frontal cortex may be plausible mechanisms for perceptual switches, as an alternative to LC-controlled gain modulation. Does the observed fMRI dynamics allow us to distinguish these two hypotheses? In any case, I would suggest including alternative scenarios that may be compatible with the observed findings in the discussion.

Reviewer #1 (Public Review):

In this manuscript, the authors explore the effects of DNA methylation on the strength of regulatory activity using massively parallel reporter assays in cell lines on a genome-wide level. This is a follow-up of their first paper from 2018 that describes this method for the first time. In addition to adding more in depth information on sequences that are explored by many researchers using two main methods, reduced bisulfite sequencing and sites represented on the Illumina EPIC array, they now show also that DNA methylation can influence changes in regulatory activity following a specific stimulation, even in absence of baseline effects of DNA methylation on activity. In this manuscript, the authors explore the effects of DNA methylation on the response to Interferon alpha (INFA) and a glucocorticoid receptor agonist (dexamethasone). The author validate their baseline findings using additional datasets, including RNAseq data and show convergences across two cell lines. The authors then map the methylation x environmental challenge (IFNA and dex) sequences identified in vitro to explore whether their methylation status is also predictive of regulatory activity in vivo. This is very convincingly shown for INFA response sequences, where baseline methylation is predictive of the transcriptional response to flu infection in human macrophages, an infection that triggers the INF pathways. The extension of the functional validity of the dex-response altering sequences is less convincing. Sequences altering the response to glucocorticoids, however, were not enriched in DNA methylation sites associated with exposure to early adversity which the authors interpret that "they are not links on the causal pathway between early life disadvantage and later life health outcomes, but rather passive biomarkers. However, this approach does not seem an optimal model to explore this relationship in vivo. This is because exposure to early adversity and its consequences is not directly correlated with glucocorticoid release and changes in DNA methylation levels following early adversity could be related to many physiological mechanisms, and overall, large datasets and meta-analyses do not show robust associations of exposure to early adversity and DNA methylation changes. Here other datasets, such as from Cushing patients maybe of more interest.<br /> ***<br /> After revision, the authors have now discussed this issue carefully, so that this point is addressed.<br /> ***<br /> Overall, the authors provide a great resource of DNA methylation sensitive enhancers that can now be used for functional interpretation of large scale datasets (that are widely generated in the research community), given the focus on sites included in RBSS and the Illumina EPIC array. In addition, their data lends support that difference in DNA methylation can alter responses to environmental stimuli and thus of the possibility that environmental exposures that alter DNS methylation can also alter subsequent response to this exposure, in line with the theory of epigenetic embedding of prior stimuli/experiences. The conclusions related to the early adversity data should be reconsidered in light of the comments above.

Reviewer #1 (Public Review):

Summary:<br /> Alonso-Calleja and colleagues explore the role of TGR5 in adult hematopoiesis at both steady state and post-transplantation. The authors utilize two different mouse models including a TGR5-GFP reporter mouse to analyze the expression of TGR5 in various hematopoietic cell subsets. Using germline Tgr5-/- mice it's reported that loss of Tgr5 has no significant impact on steady-state hematopoiesis, with a small decrease in trabecular bone fraction, associated with a reduction in proximal tibia adipose tissue, and an increase in marrow phenotypic adipocytic precursors. The authors further explored the role of stroma TGR5 expression in the hematopoietic recovery upon bone marrow transplantation of wild-type cells, although the studies supporting this claim are weak. Overall, while most of the hematopoietic phenotypes have negative results or small effects, the role of TGR5 in adipose tissue regulation is interesting to the field.

Strengths:<br /> • This is the first time the role of TGR5 has been examined in the bone marrow.<br /> • This paper supports further exploration of the role of bile acids in bone marrow transplantation and possible therapeutic strategies.

Weaknesses:<br /> • The authors fail to describe whether niche stroma cells or adipocyte progenitor cells (APCs) express TGR5.<br /> • Although the authors note a significant reduction in bone marrow adipose tissue in Tgr5-/- mice, they do not address whether this is white or brown adipose tissue especially since BA-TGR5 signaling has been shown to play a role in beiging.<br /> • In Figure 1, the authors explore different progenitor subsets but stop short of describing whether TGR5 is expressed in hematopoietic stem cells (HSCs).<br /> • Are there more CD45+ cells in the BM because hematopoietic cells are proliferating more due to a direct effect of the loss of Tgr5 or is it because there is just more space due to less trabecular bone?<br /> • In Figure 4 no absolute cell counts are provided to support the increase in immunophenotypic APCs (CD45-Ter119-CD31-Sca1+CD24-) in the stroma of Tgr5-/- mice. Accordingly, the absolute number of total stromal cells and other stroma niche cells such as MSCs, ECs are missing.<br /> • There are issues with the reciprocal transplantation design in Fig 4. Why did the authors choose such a low dose (250 000) of BM cells to transplant? If the effect is true and relevant, the early recovery would be observed independently of the setup and a more robust engraftment dataset would be observed without having lethality post-transplant. On the same note, it's surprising that the authors report ~70% lethality post-transplant from wild-type control mice (Fig 4E), according to the literature 200 000 BM cells should ensure the survival of the recipient post-TBI. Overall, the results even in such a stringent setup still show minimal differences and the study lacks further in-depth analyses to support the main claim.<br /> • Mechanistically, how does the loss of Tgr5 impact hematopoietic regeneration following sublethal irradiation?<br /> • Only male mice were used throughout this study. It would be beneficial to know whether female mice show similar results.

Reviewer #1 (Public Review):

Here, Muronova et al., demonstrate the physiological importance of a centriole and microtubule-associated protein, CCDC146, in sperm flagellar formation and male reproduction. This study identifies novel causal variants to cause male infertility and resolves the pathogenicity by the mutation with characterizing mouse models. Furthermore, the authors' claims are well supported by the biochemical and imaging approaches used in this study.

Reviewer #1 (Public Review):

Summary:<br /> The study by He et al. investigates the relationship of an increased susceptibility of diabetes patients to COVID-19. The paper raises the possibility that hyperglycemia-induced cathepsin L maturation could be one of the driving forces in this pathology, suggesting that an increased activity of CTSL leads to accelerated virus infection rates due to an elevated processing of the SARS-CoV-2 spike protein.

In a clinical case-control study, the team found that the severity of corona infections was higher in diabetic patients, and their CTSL levels correlated well with the progression of the disease. They further showed an increase in CTSL activity in the long term as well as acute hyperglycemia. SARS-CoV-2 increasingly infected cells that were cultured in serum from diabetic patients, the same was observed using high glucose medium. No effect was observed in the medium with increased concentrations of insulin. CTSL knockout abolished the glucose-dependent increase in infection.

Increased glucose levels did not correlate with an increase in CTSL transcription. Rather He et al. could show that high glucose levels led to CTSL translocation from the ER into the lysosome. It was the glucose-dependent processing of the protease to its active form which promoted infection.

Strengths:<br /> It is a complete study starting from a clinical observation and ending on the molecular mechanism. A strength is certainly the wide selection of experiments. The clinical study to investigate the effect of glucose on CTSL concentrations in healthy individuals sets the stage for experiments in cell culture, animal models, and human tissue. The effect of CTSL knockout cell lines on glucose-induced SARS-CoV2 infection rates is convincing. Finally, the team used a combination of Western blots and confocal microscopy to identify the underlying molecular mechanisms. The authors manage to keep the diabetic condition at the center of their study and therefore extend on previous knowledge of glucose-induced CTSL activation and their consequences for COVID-19 infections. By doing so, they create a novel connection between CTSL involvement in SARS-CoV2 infections and diabetes.

Weaknesses:<br /> The authors suggest that hyperglycemia as a symptom of diabetes leads to an increased infection rate in those patients. Throughout their study, the team focuses on two select symptoms of a diabetic condition, hyperglycemia and hyperinsulinemia. The team acknowledges in the discussion that there could be various other reasons. Hyperglycemia can lead to metabolic acidosis and a shift in blood pH. As CTSL activity is highly dependent on pH, it would have been crucial to include this parameter in the study.

The study rarely differentiates between cellular and extracellular CTSL activity. A more detailed explanation for the connection between the intracellular CTSL and serum CTSL in diabetic individuals, presumably via lysosomal exocytosis, could be helpful with regard to the final model to give a more complete picture.

In the early result section, an effect of hyperglycemia on total CTSL concentrations is described, but the data is not very convincing. Over the course of the manuscript, the hypothesis shifts increasingly towards an increase in protease trans-localization and processing to the active form rather than a change in total protease amounts. The overall importance of CTSL concentrations remains questionable.

Reviewer #1 (Public Review):

Summary:<br /> This important study provides a comprehensive evaluation of skeletal muscle mitochondrial function and remodeling in a genetically engineered mouse model of pancreatic cancer cachexia. The study builds upon and extends previous findings that implicate mitochondrial defects in the pathophysiology of cancer cachexia. The authors demonstrate that while the total quantity of mitochondria from skeletal muscles of mice with pancreatic cancer cachexia is similar to controls, mitochondria were elongated with disorganized cristae, and had reduced oxidative capacity. The mitochondrial dysfunction was not associated with exercise-induced metabolic stress (insufficient ATP production), suggesting compensation by glycolysis or other metabolic pathways. However, mitochondrial dysfunction can lead to increased production of ROS/oxidative stress and would be expected to interfere with carbohydrate and lipid metabolism, events that are linked to cancer-induced muscle loss. The data are convincing and were collected and analyzed using state-of-the-art techniques, with unbiased proteomics and transcriptomics analyses supporting most of their conclusions.

Additional Strengths:<br /> The authors utilize a genetically engineered mouse model of pancreatic cancer which recapitulates key aspects of human PDAC including the development of cachexia, making the model highly appropriate and translational.

The authors perform transcriptomic and proteomics analyses on the same tissue, providing a comprehensive analysis of the transcriptional networks and protein networks changed in the context of PDAC cachexia.

Weaknesses:<br /> The authors refer to skeletal muscle wasting induced by PDAC as sarcopenia. However, the term sarcopenia is typically reserved for the loss of skeletal muscle mass associated with aging.

In Figure 2, the MuRF1 IHC staining appears localized to the extracellular space surrounding blood vessels and myofibers-which causes concern as to the specificity of the antibody staining. MuRF1, as a muscle-specific E3 ubiquitin ligase that degrades myofibrillar proteins, would be expected to be expressed in the cytosol of muscle fibers.

Disruptions to skeletal muscle metabolism in PDAC mice are predicted based on mitochondrial dysfunction and the transcriptomic and proteomics data. The manuscript could therefore be strengthened by additional measures looking at skeletal muscle metabolites, or linking the findings to previous work that has looked at the skeletal muscle metabolome in related models of PDAC cachexia (Neyroud et al., 2023).

Reviewer #1 (Public Review):

The study is highly interesting and the applied methods are target-oriented. The biophysical characterization of viable N-protein species and several representative N-protein mutants is supported by the data, including polarity, hydrophobicity, thermodynamic stability, CD spectra, particle size, and especially protein self-association. The physicochemical parameters for viable N-protein and related coronavirus are described for comparison in detail. However, the conclusion becomes less convincing that the interaction of peptides or motifs was judged by different biophysical results, with no more direct data about peptide interaction. Additionally, the manuscript could benefit from more results involving peptide interaction to support the author's opinions or make expression more accurate when concerning the interaction of motifs. Although the authors put a lot of effort into the study, there are still some questions to answer.

Reviewer #1 (Public Review):

This study offers valuable insights into host-virus interactions, emphasizing the adaptability of the immune system. Readers should recognize the significance of MDA5 in potentially replacing RIG-I and the adversarial strategy employed by 5'ppp-RNA SCRV in degrading MDA5 mediated by m6A modification in different species, further indicating that m6A is a conservational process in the antiviral immune response.

However, caution is warranted in extrapolating these findings universally, given the dynamic nature of host-virus dynamics. The study provides a snapshot into the complexity of these interactions, but further research is needed to validate and extend these insights, considering potential variations across viral species and environmental contexts.

Joint Public Review

The present study focuses on the structure and function of human PURA, a regulator of gene transcription and mRNA transport and translation whose mutation causes the neurodevelopmental PURA syndrome, characterized by developmental delay, intellectual disability, hypotonia, epileptic seizures, a.o. deficits. The authors combined structural biology, molecular dynamics simulation, and various cell biological assays to study the effects of disease-causing PURA mutations on protein structure and function. The corresponding data reveal a highly dynamic PURA structure and show that disease-related mutations in PURA cause complex defects in folding, DNA-unwinding activity, RNA binding, dimerization, and partitioning into processing bodies. These findings provide first insights into how very diverse PURA mutations can cause penetrant molecular, cellular, and clinical defects. This will be of substantial interest to cell biologists, neurogeneticists, and neurologists alike.

A particular strength of the present study is the structural characterization of human PURA, which is a challenging target for structural biology approaches. The molecular dynamics simulations are state-of-the-art, allowing a statistically meaningful assessment of the differences between wild-type and mutant proteins. The functional consequences of PURA mutations at the cellular level are fascinating, particularly the differential compartmentalization of wild-type and mutant PURA variants into certain subcellular condensates.

Weaknesses that warrant rectification relate to (i) the interpretation of statistically non-significant effects seen in the molecular dynamics simulations, (ii) the statistical analysis of the differential compartmentalization of PURA variants into processing bodies vs. stress granules, and (iii) the documentation of protein expression levels and knock-down efficiencies.

Reviewer #1 (Public Review):

Summary:<br /> Tian et al. describe how TIPE regulates melanoma progression, stemness, and glycolysis. The authors link high TIPE expression to increased melanoma cell proliferation and tumor growth. TIPE causes dimerization of PKM2, as well as translocation of PKM2 to the nucleus, thereby activating HIF-1alpha. TIPE promotes the phosphorylation of S37 on PKM2 in an ERK-dependent manner. TIPE is shown to increase stem-like phenotype markers. The expression of TIPE is positively correlated with the levels of PKM2 Ser37 phosphorylation in murine and clinical tissue samples. Taken together, the authors demonstrate how TIPE impacts melanoma progression, stemness, and glycolysis through dimeric PKM2 and HIF-1alpha crosstalk.

Strengths:<br /> The authors manipulated TIPE expression using both shRNA and overexpression approaches throughout the manuscript. Using these models, they provide strong evidence of the involvement of TIPE in mediating PKM2 Ser37 phosphorylation and dimerization. The authors also used mutants of PKM2 at S37A to block its interaction with TIPE and HIF-1alpha. In addition, an ERK inhibitor (U0126) was used to block the phosphorylation of Ser37 on PKM2. The authors show how dimerization of PKM2 by TIPE causes nuclear import of PKM2 and activation of HIF-1alpha and target genes. Pyridoxine was used to induce PKM2 dimer formation, while TEPP-46 was used to suppress PKM2 dimer formation. TIPE maintains stem cell phenotypes by increasing the expression of stem-like markers. Furthermore, the relationship between TIPE and Ser37 PKM2 was demonstrated in murine and clinical tissue samples.

Weaknesses:<br /> The evaluation of how TIPE causes metabolic reprogramming can be better assessed using isotope tracing experiments and improved bioenergetic analysis.

Reviewer #1 (Public Review):

Summary:<br /> The manuscript ``Self-inhibiting percolation and viral spreading in epithelial tissue' describes a model based on 5-state cellular automata of development of an infection. The model is motivated and qualitatively justified by time-resolved measurements of expression levels of viral, interferon-producing, and antiviral genes. The model is set up in such a way that the crucial difference in outcomes (infection spreading vs. confinement) depends on the initial fraction of special virus-sensing cells. Those cells (denoted as 'type a') cannot be infected and do not support the propagation of infection, but rather inhibit it in a somewhat autocatalytic way. Presumably, such feedback makes the transition between two outcomes very sharp: a minor variation in concentration of ``a' cells results in qualitative change from one outcome to another. As in any percolation-like system, the transition between propagation and inhibition of infection goes through a critical state with all its attributes. A power-law distribution of the cluster size (corresponding to the fraction of infected cells) with a fairly universal exponent and a cutoff at the upper limit of this distribution.

Strengths:<br /> The proposed model suggests an explanation for the apparent diversity of outcomes of viral infections such as COVID.

Weaknesses:<br /> Those are not real points of weakness, though I think addressing them would substantially improve the manuscript.

The key point in the manuscript is the reduction of actual biochemical processes to the NOVAa rules. I think more could be said about it, be it referring to a set of well-known connections between expression states of cells and their reaction to infection or justifying it as an educated guess.

Another aspect where the manuscript could be improved would be to look a little beyond the strange and 'not-so-relevant for a biomedical audience' focus on the percolation critical state. While the presented calculation of the precise percolation threshold and the critical exponent confirm the numerical skills of the authors, the probability that an actual infected tissue is right at the threshold is negligible. So in addition to the critical properties, it would be interesting to learn about the system not exactly at the threshold: For example, how the speed of propagation of infection depends on subcritical p_a and what is the cluster size distribution for supercritical p_a.

Reviewer #1 (Public Review):

This manuscript from Zaman et al., investigates the role of cKit and Kit ligand in inhibitory synapse function at molecular layer interneuron (MLI) synapses onto cerebellar Purkinje cells (PC). cKit is a receptor tyrosine kinase expressed in multiple tissues, including select populations of neurons in the CNS. cKIt is activated by Kit ligand, a transmembrane protein typically expressed at the membrane of connected cells. A strength of this paper is the use of cell-specific knockouts of cKit and Kit ligand, in MLIs and PCs, respectively. In both cases, the frequency of spontaneous or miniature (in the presence of TTX) IPSCs was reduced. This suggests either a reduction in the number of functional inhibitory release sites or reduced release probability. IPSCs evoked by electrical stimulation in the molecular layer showed no change in paired-pulse ratio, indicating release probability is not changed in the cKit KO, and favoring a reduction in the number of release sites. Changes in IPSC amplitude were more subtle, with some analyses showing a decrease and others not. These data suggest that disruption of the cKit-Kit ligand complex reduces the number of functional synapses with only minor changes in synapse strength.

Reviewer #1 (Public Review):

Summary:

The main goal of the authors was to study the testis-specific role of the protein FBXO24 in the formation and function of the ribonucleoprotein granules (membraneless electron-dense structures rich in RNAs and proteins).

Strengths:

The wide variety of methods used to support their conclusions (including transgenic models)

Weaknesses:

The lack of specific antibodies against FBXO24. Some of the experiments showing a specific phenotype are descriptive and lack of logical explanation about the possible mechanism (i.e. AR or the tail structure).

Questions:

The paper is excellent and employs a wide variety of methods to substantiate the conclusions. I have very few questions to ask:

1) KO mice cannot undergo acrosome reaction (AR) even spontaneously. How do you account for this, given that no visible defects were observed in the acrosome?

2) KO sperm are unable to migrate in the female tract, and, more intriguingly, they do not pass through the utero-tubal junction (UTJ). The levels of ADAM3 are normal, suggesting that the phenotype is influenced by other factors. The authors should investigate the levels of Ly6K since mice also exhibit the same phenotype but with normal levels of ADAM3.

3) In Figure 4A, the authors assert that "RBGS Tg mice revealed that mitochondria were abnormally segmented in Fbxo24 KO spermatozoa." I am unable to discern this from the picture shown in that panel. Could you please provide a more detailed explanation or display the information more explicitly?

Reviewer #1 (Public Review):

Summary:<br /> This study used a unique acute HIV-1 infection cohort, RV217, to study the evolution of transmitted founder viral Envelope sequences under nascent immune pressure. The striking feature of the RV217 cohort is the ability to detect viremia in the first week of infection, which can be followed at discrete Fiebig stages over long time intervals. This study evaluated Env sequences at 1 week, 4 weeks, and 24 weeks to provide a picture of viral and immunological co-evolution from Fiebig Stage I (1 week), Fiebig Stages IV (4 weeks), and Fiebig Stage VI (>24 weeks). This study design enabled lineage tracing of viral variants from a single transmitted founder (T/F) over the Fiebig Stages I, IV, and VI under nascent immune pressure generated in response to the T/F virus and its subsequent mutants.

Strengths:<br /> As expected, there were temporal differences in the appearance of virus quasispecies among the individuals, which were located predominantly in solvent-exposed residues of Env, which is consistent with prior literature. Interestingly, two waves of antibody reactivity were observed for variants with mutations in the V2 region that harbors V2i and V2p epitopes correlated with protection in the RV144 clinical trial. Two waves of antibody response, detected by SPR, were observed, with the first wave being predominated by antibodies specific for the T/F07 V2 epitope associated with H173 located on the C β-strand in the V2 region. The second wave was dominated by antibodies specific for an H to Y mutation at 173 that emerged due to antibody-mediated pressure to the original H173 virus. This is a remarkable finding in three ways.

First, the mutation is in the C β-strand, an unlikely paratope contact residue, as this region of the V2 loop is shielded by glycans in Env trimer structures with full glycan representation (see PDB:5t3x). The structure used for modeling in the current study was an earlier structure, PDB:4TVP, that had many truncated glycans. This does not detract from the finding that the H173Y mutation likely causes a conformational shift from a more rigid helical/coil conformation to a more dynamic conformation with a β-stranded and β-sheet core preference as indicated by the literature and by the MD simulations carried out by the authors. This observation suggests that using V2 scaffolds with both the H173 and H173Y variants will increase the breadth of potentially protective antibody responses to HIV-1, as indicated in reference 42, cited by the authors. Interestingly, the H173Y mutation abrogates reactivity to mAb CH58 and attenuates reactivity to mAb CH59 isolated from RV144 volunteers. These mAbs recognize conformationally distinct V2 epitopes, adding further credence to the conclusion that the H173Y mutation results in a conformational switch of the V2 region.

Second, the H173Y mutation affects the conformation of V2 epitopes recognized by mAbs that do not neutralize T/F HIV-1 while mediating potent ADCC. The ADCC data in the manuscript provides strong support for this hypothesis and augers for further exploration of the V2 epitopes as HIV-1 vaccine targets.<br /> Third, it is striking that immunogens based on the H173Y mutation successfully recapitulated the observed human antibody responses in wild-type Balb/c mice. The investigators used their extensive knowledge of V2 structures and scaffold-immunogens to create the library of constructs used for this study. In this case, the ΔDSV mutation increased the breadth and magnitude of the murine antibody responses.

Weaknesses:<br /> 1. V2 epitopes exhibit properties of CD4i epitopes in that they are largely absent from the native Env surface, probably by glycan-occlusion, but become more exposed upon CD4 binding. Although the V2-scaffolds were produced in GnTi- cells to produce high-mannose proteins, it appears that no systematic analysis of glycan content or structure was carried out save for enzymatic deglycosylation of the constructs to sharpen bands on SDS-PAGE gels. It would be helpful if the authors could comment on how the lack of this information might impact their conclusions.

2. Similarly, the MD simulations appear to be performed without taking glycan structure/occupancy.

Reviewer #1 (Public Review):

Mignerot et al. performed a Herculean effort to measure and describe natural variation in C. elegans egg-laying behavior and egg retention. The paper is well written and organized. The authors show wild strains vary in egg retention with some extremes that appear phenotypically similar to species with viviparity (or live birth / internal hatching of offspring). They previously published a rare variant in the gene kcnl-1 that plays a role in egg retention but identify common variants in this study. They classify wild strains based on egg-retention to separate out the extremely different isolates. Egg laying has been extensively studied in the laboratory strain N2, but rarely addressed in natural strains. The authors investigate egg-laying behaviors using standard assays and find that their classified egg-laying groups have differences in sub-behaviors suggesting diverse roles in the ultimate egg-laying output. Then, they turn to the egg-laying circuit using both exogenous serotonin (5-HT), 5-HT modulatory drugs (e.g. SSRIs), and even genome editing to test epistasis with the mod-5 5-HT reuptake. The effects of 5-HT modulation and mutants are not predictive based on the basal behaviors and egg-retention phenotypes with the most extreme egg-retention strains differing in their responses. Interestingly, strains with more egg retention have decreased fitness (in their laboratory) measures but also provide a protective environment for offspring when exposed to common "natural" stressors. Their final conclusion that egg retention could be a trade-off between antagonistic effects of maternal vs. offspring fitness is supported well and sets the stage for future mechanistic studies across Caenorhabditis.

Reviewer #1 (Public Review):

Koumoundourou et al., identify a pathway downstream of Bcl11b that controls synapse morphology and plasticity of hippocampal mossy fiber synapses. Using an elegant combination of in vivo, ex vivo, and in vitro approaches, the authors build on their previous work that indicated C1ql2 as a functional target of Bcl11b (De Bruyckere et al., 2018). Here, they examine the functional implications of C1ql2 at MF synapses in Bcl11b cKO mice and following C1ql2 shRNA. The authors find that Bcl11b KO and shRNA against C1ql2 significantly reduces the recruitment of synaptic vesicles and impairs LTP at MF synapses. Importantly, the authors test a role for the previously identified C1ql2 binding partner, exon 25b-containing Nrxn3 (Matsuda et al., 2016), as relevant at MF synapses to maintain synaptic vesicle recruitment. To test this, the authors developed a K262E C1ql2 mutant that disrupts binding to Nrxn3. Curiously, while Bcl11b KO and C1ql2 KD largely phenocopy (reduced vesicle recruitment and impaired LTP), only vesicle recruitment is dependent on C1ql2-Nrxn3 interactions. These findings provide new insight into the functional role of C1ql2 at MF synapses. The authors utilize a multidisciplinary approach to convincingly demonstrate a role for C1ql2-Nrxn3(25b+) interactions for vesicle recruitment and a Nrxn3(25b+)-independent role for C1ql2 in LTP, The authors establish an important signaling pathway that offers insight into how disruptions of Bcl11b contribute to synapse dysfunction and provide a much needed advance toward understanding the functional consequences of neurexin alternative splicing.

Reviewer #1 (Public Review):

Summary:<br /> This is an important work showing that loss of LRRK function causes late-onset dopaminergic neurodegeneration in a cell-autonomous manner. One of the LRRK members, LRRK2, is of significant translational importance as mutations in LRRK2 cause late-onset autosomal dominant Parkinson's disease (PD). While many in the field assume that LRRK2 mutant causes PD via increased LRRK2 activity (i.e., kinase activity), it is not a settled issue as not all disease-causing mutant LRRK2 exhibits increased activity. Further, while LRRK2 inhibitors are under clinical trials for PD, the consequence of chronic, long-term LRRK2 inhibition is unknown. Thus, studies evaluating the long-term impact of LRRK deficit have important translational implications. Moreover, because LRRK proteins, particularly LRRK2, are known to modulate immune response and intracellular membrane trafficking, the study's results and the reagents will be valuable for others interested in LRRK function.

Strengths:<br /> This report describes a mouse model where LRRK1 and LRRK2 genes are conditionally deleted in dopaminergic neurons. Previously, this group showed that while loss of LRRK2 expression does not cause brain phenotype, loss of both LRRK1 and LRRK2 causes a later onset, progressive degeneration of catecholaminergic neurons, Dopaminergic (DAergic) neurons in substantia niga (SN) and Noradrenergic neurons in Locus Coeruleus (LC). However, because LRRK genes are widely expressed with some peripheral phenotypes, it was unknown if the neurodegeneration in LRRK double Knock Out (DKO) was cell autonomous. To rigorously test this question, the authors have generated a double conditional KO allele where both LRRK1 and LRRK2 genes were targeted to contain loxP sites. In my view, this was beyond what is normally required as most investigators might just combine one KO allele with another floxed allele. The authors provide a rigorous validation showing that the Driver (DAT-Cre) is expressed in the majority of DAergic neurons in SN and that LRRK levels are decreased selectively in the ventral midbrain. Using these mice, the authors show that the number of DA neurons is average at 15 but significantly decreased at 20 months of age. Moreover, the authors show that the number of apoptotic neurons is increased by ~2X in aged SN, demonstrating increased ongoing cell death, as well as an increase in activated microglia. The degeneration is limited to DA neurons as LC neurons are not lost as this population does not express DAT. Overall, the mouse genetics and experimental analysis were performed in a rigorous manner and the results were statistically sound and compelling.

Weakness: I only have a few minor comments. First, in PD and other degenerative conditions, axons and terminals loss occurs prior to cell bodies. It might be beneficial to show the status of DAergic markers in the striatum. Second, previous studies indicate that very little, if any, LRRK1 is expressed in SN DAergic neurons. This also seems to be the case with the Allen Brain Atlas profile. Thus, it is preferable that authors discuss the discrepancy as authors seem to imply significant LRRK1 expression in DA neurons.

Revision: I appreciate the authors revising the manuscript with additional data that have clarified my prior comments. They now show that TH levels in the striatum decrease with SNpc neurons. Further, while there is some disagreement regarding the expression LRRK1 in SNpc, the authors provide a convincing case that LRRK1 function is important in SNpc DA neurons.

Reviewer #1 (Public Review):

Summary:<br /> This manuscript by Liu et al explores the role of the UPR and immune regulators in the evaluation of nutritional quality in C. elegans. They identify neuronal UPR activation and the MAPK PMK-1 as key responders to low food quality. In particular, the data suggest that these pathways are activated by low levels of vitamin C synthesis that result from the low sugar levels present in heat-killed E. coli.

Strengths:<br /> The results are intriguing and expand our understanding both of physiological food evaluation systems, and of the known roles of stress response pathways in organismal physiology. The authors use a range of techniques, encompassing imaging, metabolomic analysis, gene expression analysis, and behavioural assays, to support their claims.

Weaknesses:<br /> There is limited mechanistic analysis in the study. In particular, how does low vitamin C trigger UPR activation? This is an intriguing finding that, if followed up, could potentially reveal a novel mechanism of UPR activation. In addition, how is the activation of the PMK-1 pathway driven by/coordinated with UPR activation? The data in some figures is not as convincing as it could be: the magnitude of the effect size is small in the supplementation experiments, and the statistical tests used are not always appropriate to enable multiple comparisons.

Reviewer #1 (Public Review):

Summary:<br /> The authors introduce two preparations for observing large-scale cortical activity in mice during behavior. Alongside this, they present intriguing preliminary findings utilizing these methods. This paper is poised to be an invaluable resource for researchers engaged in extensive cortical recording in behaving mice.

Strengths:<br /> -Comprehensive methodological detailing:<br /> The paper excels in providing an exceptionally detailed description of the methods used. This meticulous documentation includes a step-by-step workflow, complemented by thorough workflow, protocols, and a list of materials in the supplementary materials.

-Minimal movement artifacts:<br /> A notable strength of this study is the remarkably low movement artifacts. To further underscore this achievement, a more robust quantification across all subjects, coupled with benchmarking against established tools (such as those from suite2p), would be beneficial.

Insightful preliminary data and analysis:<br /> The preliminary data unveiled in the study reveal interesting heterogeneity in the relationships between neural activity and detailed behavioral features, particularly notable in the lateral cortex. This aspect of the findings is intriguing and suggests avenues for further exploration.

Weaknesses:<br /> -Clarification about the extent of the method in the title and text:<br /> The title of the paper, using the term "pan-cortical," along with certain phrases in the text, may inadvertently suggest that both the top and lateral view preparations are utilized in the same set of mice. To avoid confusion, it should be explicitly stated that the authors employ either the dorsal view (which offers limited access to the lateral ventral regions) or the lateral view (which restricts access to the opposite side of the cortex). For instance, in line 545, the phrase "lateral cortex with our dorsal and side mount preparations" should be revised to "lateral cortex with our dorsal or side mount preparations" for greater clarity.

-Comparison with existing methods:<br /> A more detailed contrast between this method and other published techniques would add value to the paper. Specifically, the lateral view appears somewhat narrower than that described in Esmaeili et al., 2021; a discussion of this comparison would be useful. Furthermore, the number of neurons analyzed seems modest compared to recent papers (50k) - elaborating on this aspect could provide important context for the readers.

-Discussion of methodological limitations:<br /> The limitations inherent to the method, such as the potential behavioral effects of tilting the mouse's head, are not thoroughly examined. A more comprehensive discussion of these limitations would enhance the paper's balance and depth.

-Preliminary nature of results:<br /> The results are at a preliminary stage; for example, the B-soid analysis is based on a single mouse, and the validation data are derived from the training data set. The discrepancy between the maps in Figures 5e and 6e might indicate that a significant portion of the map represents noise. An analysis of variability across mice and a method to assign significance to these maps would be beneficial.

-Analysis details:<br /> More comprehensive details on the analysis would be beneficial for replicability and deeper understanding. For instance, the statement "Rigid and non-rigid motion correction were performed in Suite2p" could be expanded with a brief explanation of the underlying principles, such as phase correlation, to provide readers with a better grasp of the methodologies employed.

Reviewer #1 (Public Review):

Granados-Aparici et al., investigate somatic-germline interactions in female mice. Mammalian oocytes are nurtured in multi-cellular ovarian follicles and communication with surrounding somatic cells is critical for oocyte development. This study focused on transzonal projections (TZP) extending from granulosa cells to the surface of oocytes and documented the importance of SMAD4, a TGF- β mediator, in regulating the TZPs. They propose a model in which individual TZPs contact the surface of the oocyte and stably attach if there is sufficient N-cadherin. In SMAD4-depleted cells, there is insufficient N-cadherin to stabilize the attachment. The TZP continues to elongate but eventually retracts. Their model is well supported by their experimental evidence and the manuscript is both well-formulated and written.

Reviewer #1 (Public Review):

Summary:<br /> The manuscript describes the crystal structures of Streptococcus pneumoniae NOXs. Crystals were obtained for the wild-type and mutant dehydrogenase domain, as well as for the full-length protein comprising the membrane domain. The manuscript further carefully studies the enzyme's kinetics and substrate-specificity properties. Streptococcus pneumoniae NOX is a non-regulated enzyme, and therefore, its structure should provide a view of the NOX active conformation. The structural and biochemical data are discussed on this ground.

Strengths:<br /> This is very solid work. The protein chemistry and biochemical analysis are well executed and carefully described. Similarly, the crystallography must be appreciated given the difficulty of obtaining good enzyme preparations and the flexibility of the protein. Even if solved at medium resolution, the crystal structure of the full-length protein conveys relevant information. The manuscript nicely shows that the domain rotations are unlikely to be the main mechanistic element of NOX regulation. It rather appears that the NADPH-binding conformation is pivotal to enzyme activation. The paper extensively refers to the previous literature and analyses the structures comprehensively with a comparison to previously reported structures of eukaryotic and prokaryotic NOXs.

Weaknesses:<br /> The manuscript is not always very clear with regard to the analysis of NADPH binding. The last section describes a "crevice" featured by the NADPH-binding sites in NOXs. It remains unclear whether this element corresponds to the different conformations of the protein C-terminal residues or more extensive structural differences. This point must be clarified.<br /> A second less convincing point concerns the nature of the electron acceptor. The manuscript states that this NOX might not physiologically act as a ROS producer. A question then immediately arises: Is this protein an iron reductase? Can the authors better discuss or provide more data about this point?

Reviewer #1 (Public Review):

This paper performed a functional analysis of the poorly characterized pseudo-phosphatase Styxl2, one of the targets of the Jak/Stat pathway in muscle cells. The authors propose that Styxl2 is essential for de novo sarcomere assembly by regulating autophagic degradation of non-muscle myosin IIs (NM IIs). Although a previous study by Fero et al. (2014) has already reported that Styxl2 is essential for the integrity of sarcomeres, this study provides new mechanistic insights into the phenomenon. In vivo studies in this manuscript are compelling; however, I feel the contribution of autophagy in the degradation of NM IIs is still unclear.

Reviewer #1 (Public Review):

Summary:<br /> This paper presents evidence that a relatively common genetic variant tied to several disease phenotypes affects the interaction between the mRNA of CCL2 and the RNA binding protein HuR. CCL2 is an immune cell chemoattractant protein.

Strengths:<br /> The study is well conducted with relevant controls. The techniques are appropriate, and several approaches provided concordant results that were generally supportive of the conclusions reached. The impact of this work, identifying a genetic variant that works by altering the binding of an RNA-regulatory protein, has important implications given that the HuR protein could be a drug target to improve its function and override this genetic change. This could have important implications for a number of diseases where this genetic variant contributes to disease risk.

Weaknesses:<br /> The authors need to do a better job of citing prior work. Certain details of the experimental protocols need to be further elaborated or clarified to contextualize the significance of the findings, Some of the findings need to be better described.

Reviewer #1 (Public Review):

The authors examine the fascinating question of how T lymphocytes regulate proteome expression during the dramatic cell state change that accompanies the transition from the resting quiescent state to the activated, dividing state. Orthogonal, complementary assays for translation (RPM/RTA, metabolic labeling) are combined with polyribosome profiling and quantitative, biochemical determinations of protein and ribosome content to explore this question, primarily in the OT-I T lymphocyte model system. The authors conclude that the ratio of protein levels to ribosomes/protein synthesis capacity is insufficient to support activation-coupled T cell division and cell size expansion. The authors hint at cellular mechanisms to explain this apparent paradox, focusing on protein acquisition strategies, including emperipolesis and entosis, though these remain topic areas for future study.

The strengths of the paper include the focus on a fundamental biological question - the transcriptional/translational control mechanisms that support the rapid, dramatic cell state change that accompanies lymphocyte activation from the quiescent to activated state, the use of orthogonal approaches to validate the primary findings, and the creative proposal for how this state change is achieved.

The weakness of the work is that several cellular regulatory processes that could explain the apparent paradox are not explored, though they are accessible to experimental analysis. In the accounting narrative that the authors highlight, a thorough accounting of the cellular process inventory that could support the cell state change should be further explored before committing to the proposal, provocative as it is, that protein acquisition provides a principal mechanism for supporting lymphocyte activation cell state change.

Appraisal and Discussion:

1) Relating to the points raised above, two recent review articles explore this topic area and highlight important areas of study in RNA biology and translational control that likely contribute to the paradox noted by the authors: Choi et al. 2022,<br /> doi.org/10.4110/in.2022.22.e39 ("RNA metabolism in T lymphocytes") and Turner 2023, DOI: 10.1002/bies.202200236 ("Regulation and function of poised mRNAs in lymphocytes"). These should be cited, and the broader areas of RNA biology discussed by these authors integrated into the current manuscript.

2) The authors cite the Wolf et al. study from the Geiger lab (doi.org/10.1038/s41590-020-0714-5, ref. 41) though largely to compare determined values for ribosome number. Many other elements of the Wolf paper seem quite relevant, for example, the very high abundance of glycolytic enzymes (and whose mRNAs are quite abundant as well), where (and as others have reported) there is a dramatic activation of glycolytic flux upon T cell activation that is largely independent of transcription and translation, the evidence for "pre-existing, idle ribosomes", the changes in mRNA copy number and protein synthesis rate Spearman correlation that accompanies activation, and that the efficiencies of mRNA translation are heterogeneous. These data suggest that more accounting needs to be done to establish that there is a paradox.

As one example, what if glycolytic enzyme protein levels in the resting cell are in substantial excess of what's need to support glycolysis (likely true) and so translational upregulation can be directed to other mRNAs whose products are necessary for function of the activated cell? In this scenario the dilution of glycolytic enzyme concentration that would come with cell division would not necessarily have a functional consequence. And the idle ribosomes could be recruited to key subsets of mRNAs (transcriptionally or post-transcriptionally upregulated) and with that a substantial remodeling of the proteome (authors ref. 44). The study of Ricciardi et al. 2018 (The translational machinery of human CD4+ T cells is poised for activation and controls the switch from quiescence to metabolic remodeling (doi.org/10.1016/j.cmet.2018.08.009) is consistent with this possibility. That study, and the short reviews noted above, are useful in highlighting the contributions of selective translational remodeling and the signaling pathways that contribute to the cell state change of T cell activation. From this perspective an alternative view can be posited, where the quiescent state is biologically poised to support activation, where subsets of proteins and mRNAs are present in far higher levels than that necessary to support basal function of the quiescent lymphocyte. In such a model, the early stages of lymphocyte activation and cell division are supported by this surplus inventory, with transcriptional activation, including ribosomal genes, primarily contributing at later stages of the activation process. An obvious analogy is the developing Drosophila embryo where maternal inheritance supports early-stage development and zygotic transcriptional contributions subsequently assuming primary control (e.g. DOI 10.1002/1873-3468.13183 , DOI: 10.1126/science.abq4835). To pursue that biological logic would require quantifying individual mRNAs and their ribosome loading states, mRNA-specific elongation rates, existing individual protein levels, turnover rates of both mRNAs and proteins, ribosome levels, mean ribosome occupancy state, and how each of these parameters are altered in response to activation. Such accounting could go far to unveil the paradox. This is a considerable undertaking, though, and outside the scope of the current paper.

Regarding the revised manuscript:

I am largely satisfied with the authors responses to the review and have but a few remaining thoughts, some mirrored in the comments from the other reviewers and some that came to mind upon reading the revision.

1) In the Introduction, it would be (have been) helpful if in paragraph two, it was stated that the current study was designed to test that assumption made in prior reports that the fold-increase in protein synthesis in response to mitogen activation was sufficient to endow the daughter cells with "the same protein content as their progenitor".

2) The primary conclusion, that "...protein synthesis activity or capacity of in vivo activated T cells does not support their doubling times" remains, to my eye, insufficiently supported by the data, though I agree it is a rational interpretation. My concern is that the devil is deeper in the details and without knowing the mRNA transcriptome composition pre- and post-activation, mean CDS length, 5' UTR structural features, perhaps codon optimality, etc., etc., the broader conclusion could be premature. As a first check, it would be useful to determine poly(A) mRNA and ribosome concentrations/cell, pre- and post-activation, and subsequently to compare mRNA transcriptome compositions in greater detail. Do mRNA:ribosome levels and ratios diverge as a consequence of activation? Poly(A) mRNA compositions? Does protein half-life change pre- and post-activation? mRNA half-life? My view is that additional molecular accounting is likely necessary to be confident in the primary conclusion.

3) I did not provide a clear description of the alternative interpretation I was imagining, which is that in the resting, unstimulated state, mRNA:ribosome and/or protein levels may be much higher than that necessary for lymphocyte viability. As in early development, this could be a mechanism to then provide sufficient protein synthesis capacity and/or proteins to daughter cells following activation of cell division and cell growth. In other words, it's a dynamic range question; the daughter cells exploit "unused" protein synthesis capacity to sustain their growth and division. Quantification and analysis of the additional variables noted in point 2) could reconcile the different interpretations.

Reviewer #1 (Public Review):

The association of vitamin D supplementation in reducing Asthma risk is well studied, although the mechanistic basis for this remains unanswered. In the presented study, Kilic and co-authors aim to dissect the pathway of Vitamin D-mediated amelioration of allergic airway inflammation. They use initial leads from bioinformatic approaches, which they then associate with results from a clinical trial (VDAART) and then validate them using experimental approaches in murine models. The authors identify a role of VDR in inducing the expression of the key regulator Ikzf3, which possibly suppresses the IL-2/STAT5 axis, consequently blunting the Th2 response and mitigating allergic airway inflammation.

The major strength of the paper lies in its interdisciplinary approach, right from hypothesis generation, and linkage with clinical data, as well as in the use of extensive ex vivo experiments and in vivo approaches using knock-out mice. The study presents some interesting findings including an inducible baseline absence/minimal expression of VDR in lymphocytes, which could have physiological implications and needs to be explored in future studies.<br /> The study presents a potential for further dissection of relevant pathophysiological pathways to explain certain seemingly associative results, and allow for a more effective translation.

Several results in the study suggest multiple factors and pathways influencing the phenotype seen, which could be explored in the future. The inferences of this study also need to be read in the context of the different sub-phenotypes and endotypes of Asthma, where the Th2 response may not be predominant. While this does not undermine the importance of this elegant study, it is essential to emphasise a holistic picture while interpreting the results.

Reviewer #1 (Public Review):

The manuscript investigates the role of membrane contact sites (MCSs) and sphingolipid metabolism in regulating vacuolar morphology in the yeast Saccharomyces cerevisiae. The authors show that tricalbin (1-3) deletion leads to vacuolar fragmentation and the accumulation of the sphingolipid phytosphingosine (PHS). They propose that PHS triggers vacuole division through MCSs and the nuclear-vacuolar junction (NVJ). The study presents some solid data and proposes potential mechanisms underlying vacuolar fragmentation driven by this pathway. Although the manuscript is clear in what the data indicates and what is more hypothetical, the story would benefit from providing more conclusive evidence to support these hypothesis. Overall, the study provides valuable insights into the connection between MCSs, lipid metabolism, and vacuole dynamics.

Reviewer #1 (Public Review):

Summary:<br /> This paper addresses the mechanisms positioning microtubule asters in Drosophila explants. Taking advantage of a genetic mutant, blocking the cell cycle in early embryos, the authors generate embryos with centrosomes detached from nuclei and then study the positioning mechanisms of such asters in explants. They conclude that asters interact via pushing forces. While this is an artificial system, understanding the mechanics of asters positioning, in particular, whether forces are pushing or pulling is an important one.

Strengths:<br /> The major strength of this paper is the series of laser cutting experiments supporting that asters position via pushing forces acting both on the boundary (see below for a relevant comment) and between asters. The combination of imaging, data analysis and mathematical modeling is also powerful.

Weaknesses:<br /> This paper has overlap in the conclusions with a previous paper from the same authors, so its impact is reduced. In Figure 2, the tracking of fluid flows is hard to see and better quantifications/analyses would lead to stronger conclusions. In Figure 4, it is not clear that the acceleration is significant and no statistical test is provided or described, as far as I can tell.

Reviewer #1 (Public Review):

In this study, the authors explored how the reduced growth fitness, resulting from genome reduction, can be compensated through evolution. They conducted an evolution experiment with a strain of Escherichia coli that carried a reduced genome, over approximately 1,000 generations. The authors carried out sequencing and found no clear genetic signatures of evolution across replicate populations. They carry out transcriptomics and a series of analyses that lead them to conclude that there are divergent mechanisms at play in individual evolutionary lineages. The authors used gene network reconstruction to identify three gene modules functionally differentiated, correlating with changes in growth fitness, genome mutation, and gene expression, respectively, due to evolutionary changes in the reduced genome.

I think that this study addresses an interesting question. Many microbial evolution experiments evolve by loss of function mutations, but presumably, a cell that has already lost so much of its genome needs to find other mechanisms to adapt. Experiments like this have the potential to study "constructive" rather than "destructive" evolution.

At the top of the results, the authors should say what species they're working with and give some background about the nature of the reduced genome. It is important to know what the changes were and especially how much of the genome was deleted. Some insights into the genes that were deleted would also be useful context for understanding the evolution experiment. This could be included in the introduction or results.

Reviewer #1 (Public Review):

Summary:

The manuscript by Hann et. al examines the role of survival motor neuron protein (SMN) in lateral plate mesoderm-derived cells using the Prrx1Cre to elucidate how changing cell-specific SMN levels coordinate aspects of the spinal muscular atrophy (SMA) pathology. SMN has generally been studied in neuronal cells, and this is one of the first insights into non-neuronal cells that may contribute to SMA disease. The authors generated 3 mouse lines: a Prrx1;Smnf/f conditional null mouse, as well as, single and double copy Prrx1;Smnf/f;SMN2 mice carrying either one or two copies of a human SMN2 transgene. First, the bone development and growth of all three were assessed; the conditional null Smn mutation was lethal shortly after birth, while the SMN2 2-copy mutant did not exhibit bone growth phenotypes. Meanwhile, single-copy SMN2 mutant mice showed reduced size and shorter limbs with shorter proliferative and hypertrophic chondrocyte zones. The authors suggested that this was cell autonomous by assessing the expression of extrinsic factors known to modulate proliferation/differentiation of growth plate chondrocytes. After assessing bone phenotypes, the authors transitioned to the assessments of neuromuscular junction (NMJ) phenotypes, since there are documented neuromuscular impairments in SMA and the Prrx1Cre transgene is expressed in muscle-associated fibro-adipogenic progenitors (FAPs). Neonatal NMJ development was unchanged in mutant mice with two copies of SMN2 , but adult single-copy SMN2 mutant mice had abnormal NMJ morphology, altered presynaptic neurotransmission, and problematic nerve terminal structure. Finally, the authors sought to assess the ability to rescue NMJ phenotypes via FAP cell transplantation and showed wild-type FAPs were able to reduce pre/postsynaptic fragmentation and neurofilament varicosities.

Strengths:

The conditional genetic approaches are novel and interestingly demonstrate the potential for chondrocyte and fibro-adipogenic progenitor-specific contributions to the SMA pathology.

The characterizations of the neuromuscular and NMJ phenotypes are relatively strong.

The data strongly suggest a non-neuronal contribution to SMA, which indicates a need for further mechanistic (cellular and molecular) studies to better understand SMA.

Weaknesses:

The skeletal analyses are not rigorous and likely do not get to the core of how SMN regulates bone development.

The overall work is descriptive and lacks convincing mechanisms.

Additional experimentation is likely needed to fully justify the conclusions.

Reviewer #1 (Public Review):

People can perform a wide variety of different tasks, and a long-standing question in cognitive neuroscience is how the properties of different tasks are represented in the brain. The authors develop an interesting task that mixes two different sources of difficulty, and find that the brain appears to represent this mixture on a continuum, in the prefrontal areas involved in resolving task difficulty. While these results are interesting and in several ways compelling, they overlap with previous findings and rely on novel statistical analyses that may require further validation.

Strengths<br /> 1. The authors present an interesting and novel task for combining the contributions of stimulus-stimulus and stimulus-response conflict. While this mixture has been measured in the multi-source interference task (MSIT), this task provides a more graded mixture between these two sources of difficulty.

2. The authors do a good job triangulating regions that encoding conflict similarity, looking for the conjunction across several different measures of conflict encoding. These conflict measures use several best-practice approaches towards estimating representational similarity.

3. The authors quantify several salient alternative hypothesis, and systematically distinguish their core results from these alternatives.

4. The question that the authors tackle is important to cognitive control, and they make a solid contribution.

Concerns<br /> 1. The framing of 'infinite possible types of conflict' feels like a strawman. While they might be true across stimuli (which may motivate a feature-based account of control), the authors explore the interpolation between two stimuli. Instead, this work provides confirmatory evidence that task difficulty is represented parametrically (e.g., consistent with literatures like n-back, multiple object tracking, and random dot motion). This parametric encoding is standard in feature-based attention, and it's not clear what the cognitive map framing is contributing.

2. The representations within DLPFC appear to treat 100% Stoop and (to a lesser extent) 100% Simon differently than mixed trials. Within mixed trials, the RDM within this region don't strongly match the predictions of the conflict similarity model. It appears that there may be a more complex relationship encoded in this region.

3. To orthogonalized their variables, the authors need to employ a complex linear mixed effects analysis, with a potential influence of implementation details (e.g., high-level interactions and inflated degrees of freedom).

Reviewer #1 (Public Review):

Summary:

Kinase inhibitors represent a highly valuable class of drugs as evidenced by their continued clinical success. The target landscape of kinase targeting small molecules can be leveraged to alter multiple phenotypes with increasing complexity that broadly aligns with increasing target promiscuity. This 'tools and resources' contribution provides a starting point for researchers interested in aligning kinase inhibitor activity with cytokine/chemokine stimulated signal transduction networks.

Strengths:

KinCytE is a forward-thinking database that yields hypothesis-generating options for researchers interested in pharmacologically modulating cytokine/chemokine signaling.

Weaknesses:

As a 'tools and resources' contribution, the primary (potential) weakness will be the authors' willingness to update and improve the tool. KinCytE will require frequent updating to better inform users in terms of contextual cytokine/chemokine stimulated signaling and the target landscape of those agents that are included as options.

Reviewer #1 (Public Review):

Summary:<br /> Tuberculous meningitis (TBM) is one of the most severe forms of extrapulmonary TB. TBM is especially prevalent in people who are immunocompromised (e.g. HIV-positive). Delays in diagnosis and treatment could lead to severe disease or mortality. In this study, the authors performed the largest-ever host whole blood transcriptomics analysis on a cohort of 606 Vietnamese participants. The results indicated that TBM mortality is associated with increased neutrophil activation and decreased T and B cell activation pathways. Furthermore, increased angiogenesis was also observed in HIV-positive patients who died from TBM, whereas activated TNF signaling and down-regulated extracellular matrix organisation were seen in the HIV-negative group. Despite similarities in transcriptional profiles between PTB and TBM compared to healthy controls, inflammatory genes were more active in HIV-positive TBM. Finally, 4 hub genes (MCEMP1, NELL2, ZNF354C, and CD4) were identified as strong predictors of death from TBM.

Strengths:<br /> This is a really impressive piece of work, both in terms of the size of the cohort which took years of effort to recruit, sample, and analyse, and also the meticulous bioinformatics performed. The biggest advantage of obtaining a whole blood signature is that it allows an easier translational development into a test that can be used in the clinical with a minimally invasive sample. Furthermore, the data from this study has also revealed important insights into the mechanisms associated with mortality and the differences in pathogenesis between HIV-positive and HIV-negative patients, which would have diagnostic and therapeutic implications.

Weaknesses:<br /> The data on blood neutrophil count is really intriguing and seems to provide a very powerful yet easy-to-measure method to differentiate survival vs. death in TBM patients. It would be quite useful in this case to perform predictive analysis to see if neutrophil count alone, or in combination with gene signature, can predict (or better predict) mortality, as it would be far easier for clinical implementation than the RNA-based method. Moreover, genes associated with increased neutrophil activation and decreased T cell activation both have significantly higher enrichment scores in TBM (Figure 9) and in morality (Figure 8). While I understand the basis of selecting hub genes in the significant modules, they often do not represent these biological pathways (at least not directly associated in most cases). If genes were selected based on these biologically relevant pathways, would they have better predictive values?

Reviewer #1 (Public Review):

Summary:<br /> The investigators have performed a state-of-the art systematic review and meta-analysis of studies that may help to answer the research question: if administration of multiple antibiotics simultaneously prevents antibiotic resistance development in individuals. The amount of studies eligible for analysis is very low, and within that low number, there is huge variability in bug-drug combinations studied and most studies had a high risk of bias, further limiting the capability of meta-analysis to answer the research question. In addition, based on I2 values there is also huge statistical heterogeneity between outcomes of studies compared, further limiting the predictive value of meta-analysis. In fact, the only 2 studies meeting all eligibility criteria addressed the treatment of mycobacterium tuberculosis, for which the research question is hardly applicable. The authors, therefore, conclude that "our analysis could not identify any benefit or harm of using a higher or a lower number of antibiotics regarding within-patient resistance development." Apart from articulating this knowledge gap, the findings will not have consequences for patient care, but may stimulate the scientific community to better address this research question in future studies.

Strengths:<br /> The systematic and rigorous approach for the review and meta-analysis.

Weaknesses:<br /> None identified.

Reviewer #1 (Public Review):

Summary:

This manuscript by Vuong and colleagues reports a study that pooled data from 3 separate longitudinal studies that collectively spanned an observation period of over 15 years. The authors examined for correlation between viraemia measured at various days from illness onset with thrombocytopaenia and severe dengue, according to the WHO 2009 classification scheme. The motivation for this study is both to support the use of viraemia measurement as a prognostic indicator of dengue and also when an antiviral drug becomes licensed for use, to guide the selection of patients for antiviral therapy. They found that the four DENVs show differences in peak and duration of viraemia and that viraemia levels before day 5 but not those after from illness onset correlated with platelet count and plasma leakage at day 7 onwards. They concluded that the viraemia kinetics call for early measurement of viraemia levels in the early febrile phase of illness.

Strengths:

This is a unique study due to the large sample size and longitudinal viraemia measurements in the study subjects. The data addresses a gap in information in the literature, where although it has been widely indicated that viraemia levels are useful when collected early in the course of illness, this is the first time anyone has systematically examined this notion.

Weaknesses:

The study only analysed data from dengue patients in Vietnam. Moreover, the majority of these patients had DENV-1 infection; few had DENV-4 infection. The data could thus be skewed by the imbalance in the prevalence of the different types of DENV during the period of observation. The use of patient-reported time of symptom onset as a reference point for viraemia measurement is pragmatic although there is subjectivity and thus noise in the data.

Reviewer #1 (Public Review):

Summary:

The authors presented here a novel 3D fibroblast culture and transdifferentiation approach for potential meat production with GelMA hydrogel.

Strengths:

1. Reduced serum concentration for 3D chicken fibroblast culture and transdifferentiation is optimized.<br /> 2. Efficient myogenic transdifferentiation and lipogenesis as well as controlled fat deposition are achieved in the 3D GelMA.

Weaknesses:

1. While the authors stated the rationale of using fibroblasts instead of myogenic/adipogenic stem cells for meat production, the authors did not comment on the drawbacks/disadvantages of genetic engineering (e.g., forced expression of MyoD) in meat production.<br /> 2. While the authors cited one paper to state the properties and applications of GelMA hydrogel in tissue engineering and food processing, concerns/examples of the food safety with GelMA hydrogel are not discussed thoroughly.<br /> 3. In Fig. 4C, there seems no significant difference in the Vimentin expression between Fibroblast_MyoD and Myofibroblast. The conclusion of "greatly reduced in the myogenic transdifferentiated cells" is overstated.<br /> 4. The presented cell culture platform is only applied to chicken fibroblasts and should be tested in other species such as pigs and fish.