Establecer el derecho a la información en los sistemas de IA y mejorar la transparencia algorítmica

Habilitar y realizar evaluaciones obligatorias del impacto sobre los derechos humanos

Desarrollar medidas de rendición de cuentas para los sistemas y procesos algorítmicos del sector público

Puntos de acción

**Mapear políticas y marcos normativos internacionales relevantes sobre derechos humanos, así como compromisos regionales o nacionales aplicables. **

**Mapear y evaluar si los tratados internacionales y leyes nacionales existentes son suficientes para proteger, promover y garantizar los derechos involucrados, revisando análisis legales disponibles para determinar la solidez de dichas protecciones. **

Considerar las características sociales, económicas, demográficas, políticas, históricas y culturales que puedan influir en el proceso, identificando las principales causas de exclusión de grupos marginados y cómo la IA puede amplificarlas.

Reviewer #3 (Public review):

Summary:

It has been previously reported in many high-profile papers, that C. elegans can learn to avoid pathogens. Moreover, this learned pathogen avoidance can be passed on to future generations - up to the F5 generation in some reports. In this paper, Gainey et al. set out to replicate these findings. They successfully replicated pathogen avoidance in the exposed animals, as well as a strong increase in daf-7 expression in ASI neurons in F1 animals, as determined by a daf-7::GFP reporter construct. However, they failed to see strong evidence for pathogen avoidance or daf-7 overexpression in the F2 generation. The failure of replication is the major focus of this work.<br /> Given their failure to replicate these findings, the authors embark on a thorough test of various experimental confounders that may have impacted their results. They also re-analyze the small RNA sequencing and mRNA sequencing data from one of the previously published papers and draw some new conclusions, extending this analysis.

Strengths:

• The authors provide a thorough description of their methods, and a marked-up version of a published protocol that describes how they adapted the protocol to their lab conditions. It should be easy to replicate the experiments.<br /> • The authors test source of bacteria, growth temperature (of both C. elegans and bacteria), and light/dark husbandry conditions. They also supply all their raw data, so that sample size for each testing plate can be easily seen (in the supplementary data). None of these variations appears to have a measurable effect on pathogen avoidance in the F2 generation, with all but one of the experiments failing to exhibit learned pathogen avoidance.<br /> • The small RNA seq and mRNA seq analysis is well performed and extends the results shown in the original paper. The original paper did not give many details of the small RNA analysis, which was an oversight. Although not a major focus of this paper, it is a worthwhile extension on the previous work.<br /> • It is rare that negative results such as these are accessible. Although the authors were unable to determine the reason that their results differ from those previously published, it is important to document these attempts in detail, as has been done here. Behavioral assays are notoriously difficult to perform and public discourse around these attempts may give clarity to the difficulties faced by a controversial field.

Weaknesses:

• Although the "standard" conditions have been tested over multiple biological replicates, many of the potential confounders that may have altered the results have been tested only once or twice. For example, changing the incubation temperature to 25{degree sign}C was tested in only two biological replicates (Exp 5.1 and 5.2) - and one of these experiments actually resulted in apparent pathogen avoidance inheritance in the F2 generation (but not in the F1). An alternative pathogen source was tested in only one biological replicate (Exp 3). Given the variability observed in the F2 generation, increasing biological replicates would have added to the strengths of the report.<br /> • A key difference between the methods used here and those published previously, is an increase in the age of the animals used for training - from mostly L4 to mostly young adults. I was unable to find a clear example of an experiment when these two conditions were compared, although the authors state that it made no difference to their results.<br /> • The original paper reports a transgenerational avoidance effect up to the F5 generation. Although in this work the authors failed to see avoidance in the F2 generation, it would have been prudent to extend their tests for more generations in at least a couple of their experiments to ensure that the F2 generation was not an aberration (although this reviewer acknowledges that this seems unlikely to be the case).

Reviewer #3 (Public review):

In this manuscript, Casas-Tintó et al. explore the role of glial cell in the response to a neurodegenerative injury in the adult brain. They used Drosophila melanogaster as a model organism, and found that glial cells are able to generate new neurons through the mechanism of transdifferentiation in response to injury. This paper provides a new mechanism in regeneration, and gives an understanding to the role of glial cells in the process.

Comments on revisions:

In the previous version of the manuscript, I had suggested several recommendations for the authors. Unfortunately, none of these were addressed in the author's revision.

Reviewer #3 (Public review):

This manuscript describes an investigation of how intracellular calcium stores are regulated and provides evidence that is in line with the role of the SERCA-Ca2+-ATPase in this important homeostasis pathway. Calcium uptake by mitochondria is further investigated and the authors suggest that ER-mitochondria membrane contact sites may be involved in mediating this, as demonstrated in other organisms.

The significance of the findings is in shedding light on key elements within the mechanism of calcium storage and regulation/homeostasis in the medically important parasite Toxoplasma gondii whose ability to infect and cause disease critically relies on calcium signalling. An important strength is that despite its importance, calcium homeostasis in Toxoplasma is understudied and not well understood.

A difficulty in the field, and a weakness of the work, is that following calcium in the cell is technically challenging and thus requires reliance on artificial conditions. In this context, the main weakness of the manuscript is the extrapolation of data. The language used could be more careful, especially considering that the way to measure the ER calcium is highly artificial - for example utilising permeabilization and over-loading the experiment with calcium. Measures are also indirect - for example, when the response to ionomycin treatment was not fully in line with the suggested model the authors hypothesise that the result is likely affected by other storage, but there is no direct support for that.

Below we provide some suggestions to improve controls, however, even with those included, we would still be in favour of revising the language and trying to avoid making strong and definitive conclusions. For example, in the discussion perhaps replace "showed" with "provide evidence that are consistent with..."; replace or remove words like "efficiently" and "impressive"; revise the definitive language used in the last few lines of the abstract (lines 13-17); etc. Importantly we recommend reconsidering whether the data is sufficiently direct and unambiguous to justify the model proposed in Figure 7 (we are in favour of removing this figure at this early point of our understanding of the calcium dynamic between organelles in Toxoplasma).

Another important weakness is poor referencing of previous work in the field. Lines 248-250 read almost as if the authors originally hypothesised the idea that calcium is shuttled between ER and mitochondria via membrane contact sites (MCS) - but there is extensive literature on other eukaryotes which should be first cited and discussed in this context. Likewise, the discussion of MCS in Toxoplasma does not include the body of work already published on this parasite by several groups. It is informative to discuss observations in light of what is already known.

Reviewer #3 (Public review):

Summary:

In this study by Haley et al, the authors investigated explore-exploit foraging using C. elegans as a model system. Through an elegant set of patchy environment assays, the authors built a GLM based on past experience that predicts whether an animal will decide to stay on a patch to feed and exploit that resource, instead of choosing to leave and explore other patches.

Strengths:

I really enjoyed reading this paper. The experiments are simple and elegant, and address fundamental questions of foraging theory in a well-defined system. The experimental design is thoroughly vetted, and the authors provide a considerable volume of data to prove their points. My only criticisms have to do with the data interpretation, which I think is easily addressable.

Weaknesses:

(1) Sensing vs. non-sensing

The authors claim that when animals encounter dilute food patches, they do not sense them, as evidenced by the shallow deceleration that occurs when animals encounter these patches. This seems ethologically inaccurate. There is a critical difference between not sensing a stimulus, and not reacting to it. Animals sense numerous stimuli from their environment, but often only behaviorally respond to a fraction of them, depending on their attention and arousal state. With regard to C. elegans, it is well-established that their amphid chemosensory neurons are capable of detecting very dilute concentrations of odors. In addition, the authors provide evidence that osm-6 animals have altered exploit behaviors, further supporting the importance of amphid chemosensory neurons in this behavior.

(2) Search vs. sample & sensing vs. non-sensing

In Figures 2H and 2I, the authors claim that there are three behavioral states based on quantifying average velocity, encounter duration, and acceleration, but I only see three. Based on density distributions alone, there really only seem to be 2 distributions, not 3. The authors claim there are three, but to come to this conclusion, they used a QDA, which inherently is based on the authors training the model to detect three states based on prior annotations. Did the authors perform a model test, such as the Bayesian Information Criterion, to confirm whether 2 vs. 3 Gaussians is statistically significant? It seems like the authors are trying to impose two states on a phenomenon with a broad distribution. This seems very similar to the results observed for roaming vs. dwelling experiments, which again, are essentially two behavioral states.

(4) History-dependence of the GLM

The logistic GLM seems like a logical way to model a binary choice, and I think the parameters you chose are certainly important. However, the framing of them seems odd to me. I do not doubt the animals are assessing the current state of the patch with an assessment of past experience; that makes perfect logical sense. However, it seems odd to reduce past experience to the categories of recently exploited patch, recently encountered patch, and time since last exploitation. This implies the animals have some way of discriminating these past patch experiences and committing them to memory. Also, it seems logical that the time on these patches, not just their density, should also matter, just as the time without food matters. Time is inherent to memory. This model also imposes a prior categorization in trying to distinguish between sensed vs. not-sensed patches, which I criticized earlier. Only "sensed" patches are used in the model, but it is questionable whether worms genuinely do not "sense" these patches.

(5) osm-6

The osm-6 results are interesting. This seems to indicate that the worms are still sensing the food, but are unable to assess quality, therefore the default response is to exploit. How do you think the worms are sensing the food? Clearly, they sense it, but without the amphid sensory neurons, and not mechanosensation. Perhaps feeding is important? Could you speculate on this?

(7) Impact:

I think this work will have a solid impact on the field, as it provides tangible variables to test how animals assess their environment and decide to exploit resources. I think the strength of this research could be strengthened by a reassessment of their model that would both simplify it and provide testable timescales of satiety/starvation memory.

Reviewer #3 (Public review):

Summary:

The authors tried to characterize the neuronal deficiency in Mid1 knockout mice. They performed behavioral, neuroelectrophysiological, and pathological experiments to show that Mid1 knockout mice have cognitive function, impaired synaptic plasticity, and changes in gene expression.

Strengths:

The evidence provides insight into the mechanisms of cognitive impairments in Opitz syndrome. Overall, the manuscript is well-organized.

Weaknesses:

(1) The major weakness is that the proposed molecular mechanism is not fully supported by the current data. The data presented here only show that changes in gene expression levels, cognitive impairments, and electrophysiological impairments are correlated with each other, but do not support causality.

(2) The main conclusion is that "The main reason is that the deletion of Mid1 gene will increase the accumulation of Pp2ac protein, inhibit the activity of p-Creb, affect the downstream cAMP pathway, lead to the decrease of synaptic density and plasticity, and ultimately affect the learning and memory ability". This should be toned down, since causality is not supported here.

(3) The description of the results should be improved. Only one figure is presented in the manuscript. Some key information in the supplementary figures should be moved to the main figures. This is very strange since four display items are allowed even for a short report.

Reviewer #3 (Public review):

Summary:

Seidenthal et al. investigated the role of the Flower protein, FLWR-1, in C. elegans and confirmed its involvement in endocytosis within both synaptic and non-neuronal cells, possibly by contributing to the fission of bulk endosomes. They also uncovered that FLWR-1 has a novel inhibitory effect on neuronal excitability at GABAergic and cholinergic synapses in neuromuscular junctions.

Strengths:

This study not only reinforces the conserved role of the Flower protein in endocytosis across species but also provides valuable ultrastructural data to support its function in the bulk endosome fission process. Additionally, the discovery of FLWR-1's role in modulating neuronal excitability broadens our understanding of its functions and opens new avenues for research into synaptic regulation.

Weaknesses:

The study does not address the ongoing debate about the Flower protein's proposed Ca2+ channel activity, leaving an important aspect of its function unexplored. Furthermore, the evidence supporting the mechanism by which FLWR-1 inhibits neuronal excitability is limited. The suggested involvement of MCA-3 as a mediator of this inhibition lacks conclusive evidence, and a more detailed exploration of this pathway would strengthen the findings.

Reviewer #3 (Public review):

Summary:

This study by Park and colleagues investigated how the medial prefrontal cortex (mPFC) influences behavior and hippocampal place cell activity during a two-frame active place avoidance task in rats. Rats learned to avoid the location of mild shock within a rotating arena, with the shock zone being defined relative to distal cues in the room. Permanent chemical lesions of the mPFC did not impair the ability to avoid the shock zone by using distal cues and ignoring proximal cues in the arena. In parallel, hippocampal place cells alternated between two spatial tuning patterns, one anchored to the distal cues and the other to the proximal cues, and this alteration was not affected by the mPFC lesion. Based on these findings, the authors argue that the mPFC is not essential for differentiating between task-relevant and irrelevant information.

Strengths:

This study was built on substantial work by the Fenton lab that validated their two-frame active place avoidance task and provided sound theoretical and analytical foundations. Additionally, the effectiveness of mPFC lesions was validated by several measures, enabling the authors to base their argument on the lack of lesion effects on behavior and place cell dynamics.

Weaknesses:

The authors define cognitive control as "the ability to judiciously use task-relevant information while ignoring salient concurrent information that is currently irrelevant for the task." (Lines 77-78). This definition is much simpler than the one by Miller and Cohen: "the ability to orchestrate thought and action in accordance with internal goals (Ref. 1)" and by Robbins: "processes necessary for optimal scheduling of complex sequence of behaviour." (Dalley et al., 2004, PMID: 15555683). Differentiating between task-relevant and irrelevant information is required in various behavioral tasks, such as differential learning, reversal learning, and set-shifting tasks. Previous rodent behavioral studies have shown that the integrity of the mPFC is necessary for set-shifting but not for differential or reversal learning (e.g., Enomoto et al., 2011, PMID: 21146155; Cho et al., 2015, PMID: 25754826). In the present task design, the initial training is a form of differential learning between proximal and distal cues, and the conflict training is akin to reversal learning. Therefore, the lack of lesion effects is somewhat expected. It would be interesting to test whether mPFC lesions impair set-shifting in their paradigm (e.g., the shock zone initially defined by distal cues and later by proximal cues). If the mPFC lesions do not impair this ability and associated hippocampal place dynamics, it will provide strong support for the authors' local-computation hypothesis.

Reviewer #3 (Public review):

Summary:

This manuscript outlines a series of very exciting and game-changing experiments examining the role of peripheral MORs in OIRD. The authors outline experiments that demonstrate a peripherally restricted MOR antagonist (NLX Methiodide) can rescue fentanyl-induced respiratory depression and this effect coincides with a lack of conditioned place aversion. This approach would be a massive boon to the OUD community, as there are a multitude of clinical reports showing that naloxone rescue post fentanyl over-intoxication is more aversive than the potential loss-of-life to the individuals involved. This important study reframes our understanding of successful overdose rescue with potential for reduced aversive withdrawal effects.

Strengths:

Strengths include the plethora of approaches arriving at the same general conclusion, the inclusion of both sexes and the result that a peripheral approach for OIRD rescue may side-step severe negative withdrawal symptoms of traditional NLX rescue.

Weaknesses:

The major weakness of this version relates to the data analysis assessed sex-specific contributors to the results.

Reviewer #3 (Public review):

Summary:

This study aims to address the significant challenge of age-related decline in bone healing by developing a dual therapeutic strategy that rejuvenates osteogenic function in aged calvarial bone tissue. Specifically, the authors investigate the efficacy of combining local Wnt3a-mediated osteoprogenitor stimulation with systemic intermittent fasting (IF) to restore bone repair capacity in aged mice. The highlights are:

(1) Novel Approach with Aged Models:<br /> This pioneering study is among the first to demonstrate the rejuvenation of osteoblasts in significantly aged animals through intermitted fasting, showcasing a new avenue for regenerative therapies.

(2) Rejuvenation Potential in Aged Tissues:<br /> The findings reveal that even aged tissues retain the capacity for rejuvenation, highlighting the potential for targeted interventions to restore youthful cellular function.

(3) Enhanced Vascular Health:<br /> The study also shows that vascular structure and function can be significantly improved in aged tissues, further supporting tissue regeneration and overall health.<br /> Through this innovative approach, the authors seek to overcome intrinsic cellular deficits and environmental changes within aged osteogenic compartments, ultimately achieving bone healing levels comparable to those seen in young mice.

Strengths:

The study is a strong example of translational research, employing robust methodologies across molecular, cellular, and tissue-level analyses. The authors leverage a clinically relevant, immunocompetent mouse model and apply advanced histological, transcriptomic, and functional assays to characterise age-related changes in bone structure and function. Major strengths include the use of single-cell RNA sequencing (scRNA-seq) to profile osteoprogenitor populations within the calvarial periosteum and suture mesenchyme, as well as quantitative assessments of mitochondrial health, vascular density, and osteogenic function. Another important point is the use of very old animals (up to 88 weeks, almost 2 years) modelling the human bone aging that usually starts >65 yo. This comprehensive approach enables the authors to identify critical age-related deficits in osteoprogenitor number, function, and microenvironment, thereby justifying the combined Wnt3a and IF intervention.

Weaknesses:

One limitation is the use of female subjects only and the limited exploration of immune cell involvement in bone healing. Given the known role of the immune system in tissue repair, future studies including a deeper examination of immune cell dynamics within aged osteogenic compartments could provide further insights into the mechanisms of action of IF.

Reviewer #3 (Public review):

Summary:

Kim et al. present a study of the neural dynamics underlying reversal learning in monkey PFC and neural networks. The concept of studying neural dynamics throughout the task (including intervening behaviour) is interesting, and the data provides some insights into the neural dynamics driving reversal learning. The modelling seems to support the analyses, but both the modelling and analyses also leave several open questions.

Strengths:

The paper addresses an interesting topic of the neural dynamics underlying reversal learning in PFC, using a combination of biological and simulated data. Reversal learning has been studied extensively in neuroscience, but this paper takes a step further by analysing neural dynamics throughout the trials instead of focusing on just the evidence integration epoch.

The authors show some close parallels between the experimental data and RNN simulations, both in terms of behaviour and neural dynamics. The analyses of how rewarded and unrewarded trials differentially affect dynamics throughout the trials in RNNs and PFC were particularly interesting. This work has the potential to provide new insights into the neural underpinnings of reversal learning.

Weaknesses:

Conceptual:

A substantial focus of the paper is on the within-trial dynamics associated with "intervening behaviour", but it is not clear whether that is well-modelled by the RNN. In particular, since there is little description of the experimental task, and the RNN does not have to do any explicit computation during the non-feedback parts of the trial, it is unclear whether the RNN 'non-feedback' dynamics can be expected to reasonably model the experimental data.

Data analyses:

While the basic analyses seem mostly sound, it seems like a potential confound that they are all aligned to the inferred reversal trial rather than the true experimental reversal trial. For example, the analyses showing that 'x_rev' decays strongly after the reversal trial, irrespective of the reward outcome, seem like they are true essentially by design. The choice to align to the inferred reversal trial also makes this trial seem 'special' (e.g. in Figure 2, Figure 5A), but it is unclear whether this is a real feature of the data or an artifact of effectively conditioning on a change in behaviour. It would be useful to investigate whether any of these analyses differ when aligned to the true reversal trial. It is also unsurprising that x_rev increases before the reversal and decreases after the reversal (it is hard to imagine a system where this is not the case), yet all of Figure 5 and much of Figure 4 is devoted to this point.

Most of the analyses focus on the dynamics specifically in the x_rev subspace, but a major point of the paper is to say that biological (and artificial) networks may also have to do other things at different times in the trial. If that is the case, it would be interesting to also ask what happens in other subspaces of neural activity, that are not specifically related to evidence integration or choice - are there other subspaces that explain substantial variance? Do they relate to any meaningful features of the experiment?

On a related note, descriptions of the task itself, the behaviour of the animal(s?), and the neural recordings are largely absent, making it difficult to know what we should expect from neural dynamics throughout a trial. In fact, we are not even told how many monkeys were used for the paper or which part of PFC the recordings are from.

Modelling:

There are a number of surprising and non-standard modelling choices made in this paper. For example, the choice to only use inhibitory neurons is non-conventional and not consistent with prior work. The authors cite van Vreeswijk & Sompolinsky's balanced network paper, but this and most other balanced networks use a combination of excitatory and inhibitory neurons.

It also seems like the inputs are provided without any learnable input weights (and the form of the inputs is not described in any detail). This makes it hard to interpret the input-driven dynamics during the different phases of a trial, despite these dynamics being a central topic of the paper.

It is surprising that the RNN is "trained to flip its preferred choice a few trials after the inferred scheduled reversal trial", with the reversal trial inferred by an ideal Bayesian observer. A more natural approach would be to directly train the RNN to solve the task (by predicting the optimal choice) and then investigate the emergent behaviour & dynamics. If the authors prefer their imitation learning approach (which should at least be motivated), it is also surprising that the network is trained to predict the reversal trial inferred using Bayesian smoothing instead of Bayesian filtering.

Reviewer #3 (Public review):

Summary:

This work investigated how uncertainty, repetition bias, and win-stay-lose-shift processes influence reward-based decision-making. Using a modified two-armed bandit task and computational models, the authors provide evidence for individual variation in the integration of uncertainty on choice behaviour that remains somewhat stable across two experiment sessions. The authors also find a number of interesting results due to their ability to disentangle components of this decision-making process using their novel task and models. Specifically, they find that higher total uncertainty leads people to use more heuristic-based strategies like making repetitive choices or engaging in win-stay-lose-shift behaviour. However, they also find that there are individual differences in how people use uncertainty to guide their choices, and that these differences are consistent within individuals across multiple experiment sessions. This finding can help explain prior inconsistencies in the literature, where researchers have found evidence for both uncertainty-seeking and uncertainty-avoidance tendencies. Overall, this research adds to our understanding of the mechanisms of uncertainty-modulated learning and decision-making.

Strengths:

One of the primary strengths of this research is that it helps provide support for the idea that mixed and null results in the prior literature could be due to individual differences in uncertainty preferences and that this individual variation is somewhat stable within subjects across multiple experiment sessions. The authors cleverly disentangle expected reward and uncertainty by interleaving free and forced choice trials in their behavioural task, illuminating the novel impact of reward and uncertainty on this particular decision process. However, it should be noted that this behavioural decorrelation does not persist beyond the first few trials after a forced choice period, so whether or not the decorrelation is truly robust remains unclear.

The authors also use computational modelling to further probe the influence of uncertainty on reward-based choices. Specifically, they compare a Bayesian ideal observer learning model and a variation on a standard Rescorla-Wagner model, finding that a version of the Bayesian model fits the participants' behaviour best. The model descriptions and analyses are clearly explained and methodologically rigorous.

Interestingly, the authors find that both repetition bias and model parameters that capture a win-stay-lose-shift strategy (signed and unsigned previous prediction error) significantly improve their model fits. They also make an important point that if win-stay-lose-shift behaviour is not controlled for, then switch behaviour (for example, switching to a lower expected reward option after receiving a large loss) may appear to be uncertainty-seeking when it is not. This idea speaks to a larger point that future research should be careful to not conflate "exploration" with "uncertainty-seeking."

Weaknesses:

This research has some weaknesses regarding the correlations between the psychopathology measures and the computational model parameters. First, the choice of self-report measures is not well supported by any specific hypotheses. Relatedly, the authors do not include sufficient rationale for their choice to include only results from the anxiety and impulsivity measures in the main text while leaving out significant findings for a number of correlations between other measures and parameter coefficients. It is also not clear how the model parameters are being derived for use in each of these correlational analyses. In sum, the manuscript as-is contains inconsistent and/or confusing reporting of correlation results that require further clarification.

Reviewer #3 (Public review):

Summary:

The authors convincingly demonstrate that a population of CCK+ spinal neurons in the deep dorsal horn express the G protein-coupled estrogen receptor GPR30 to modulate pain sensitivity in the chronic constriction injury (CCI) model of neuropathic pain in mice. Using complementary pharmacological and genetic knockdown experiments they convincingly show that GPR30 inhibition or knockdown reverses mechanical, tactile, and thermal hypersensitivity, conditioned place aversion, and c-fos staining in the spinal dorsal horn after CCI. They propose that GPR30 mediates an increase in postsynaptic AMPA receptors after CCI using slice electrophysiology which may underlie the increased behavioral sensitivity. They then use anterograde tracing approaches to show that CCK and GPR30 positive neurons in the deep dorsal horn may receive direct connections from the primary somatosensory cortex. Chemogenetic activation of these dorsal horn neurons proposed to be connected to S1 increased nociceptive sensitivity in a GPR30-dependent manner. Overall, the data are very convincing and the experiments are well conducted and adequately controlled. However, the proposed model of descending corticospinal facilitation of nociceptive sensitivity through GPR30 in a population of CCK+ neurons in the dorsal horn is not fully supported.

Strengths:

The experiments are very well executed and adequately controlled throughout the manuscript. The data are nicely presented and supportive of a role for GPR30 signaling in the spinal dorsal horn influencing nociceptive sensitivity following CCI. The authors also did an excellent job of using complementary approaches to rigorously test their hypothesis.

Weaknesses:

The primary weakness in this manuscript involves overextending the interpretations of the data to propose a direct link between corticospinal projections signaling through GPR30 on this CCK+ population of spinal dorsal horn neurons. For example, even in the cropped images presented, GPR30 is present in many other CCK-negative neurons. Only about a quarter of the cells labeled by the anterograde viral tracing experiment from S1 are CCK+. Since no direct evidence is provided for S1 signaling through GPR30, this conclusion should be revised.

Reviewer #3 (Public review):

Summary:

The authors have submitted a comprehensive manuscript on the production and central pathways that they propose mediate cough-like behaviors in a TRAP2 transgenic mouse model. While the central pathway data are good, there is significant uncertainty regarding the identity of the presumptive cough-like behavior that has been produced in their model which reduces enthusiasm for the manuscript.

Strengths:

(1) The use of the TRAP2 model in the investigation of coughing is strong.

(2) The implication of SP5 in the production of coughing in response to ammonia inhalation is a novel finding. Further, this area can be activated by AAV-CaMKII to induce coughing in the absence of coincident afferent activation is an important observation.

Weaknesses:

(1) A fundamental aspect of this investigation is the unequivocal identification of the behavior that has been evoked. In this case, the authors have not established that they are actually studying cough. The evidence that they present (especially Figure 1 - Supplement 1) clearly shows that the citric acid (2nd example), capsaicin (2nd example), and ammonia (2nd example) box flows lack a large inspiratory component which is a requirement of cough. The referenced behaviors appear to be expulsion/inspiration which is not cough. The only way these behaviors could be cough is if the conventional polarity of presentation of the flow signals are reversed. However, inspection of the flows during breathing strongly indicates that inspiration is down in your records. Again, this makes these behaviors expulsion/inspiration.

An additional issue is that there are compression phases marked when the flow is occurring. The compression phase is a period of no flow so this is not accurate. There also is no evidence that the mouse has a compression phase at all. In cough flow records in humans, the compression phase can clearly be seen when it happens but not all coughs have one. You must show that a compression phase happens according to the actual description of what this segment of cough actually is.

It may be that you are evoking behaviors that primarily occur in the mouse. As such, they would be novel airway protective behaviors that are worthy of description and study. Ironically, another manuscript in the journal Cell (Jiang et al, 2024, Cell 187:5981-5997) shows similar box flow polarities as your own and clear cough airflows (Fig. 5B). However, they also show other airflow patterns that resemble what you call cough (Figure 5A) but they call them sneeze. Those airflows are expulsion/inspiration and are clearly not sneezing as the expulsion in this behavior also is preceded not followed by inspiration.

The definitive manuscript on cough in the mouse is Zhang et al Am J Physiol Reg Integr Comp 312:R718-R726, 2017. In this manuscript, Figure 2 clearly shows both box pressures and intrapleural pressures during airway protective behaviors in the awake mouse. Note that both cough and a behavior known as expiration reflex were recorded. The key element here is that the cough elicited a tri-phasic box flow. The last excursion was associated with a sound. When compared to the pressure it is clear that this last flow excursion is mechanical chest wall recoil from residual volume. The fact that this segment of the flow record was associated with sound strongly suggests that the vocal folds were adducting at the time to "brake" the chest wall recoil. In other words, the airway resistance went up to slow inspiratory airflow as the chest returned to its resting position. As such, this observation suggests that the chest wall mechanics of cough in the mouse are different than that of larger animals.

(2) Roger Shannon and coworkers have published a number of papers on the detailed brainstem circuits that are responsible for coughing. I recommend that the authors assimilate this knowledge in the context of their results.

Reviewer #3 (Public review):

Summary:

This paper points out an inconsistency of the roles of the striatal spiny neurons projecting to the indirect pathway (iSPN) and the synaptic plasticity rule of those neurons expressing dopamine D2 receptors and proposes a novel, intriguing mechanisms that iSPNs are activated by the efference copy of the chosen action that they are supposed to inhibit.

The proposed model was supported by simulations and analysis of the neural recording data during spontaneous behaviors.

Strengths:

Previous models suggested that the striatal neurons learn action-value functions, but how the information about the chosen action is fed back to the striatum for learning was not clear. The author pointed out that this is a fundamental problem for iSPNs that are supposed to inhibit specific actions and its synaptic inputs are potentiated with dopamine dips.

The authors propose a novel hypothesis that iSPNs are activated by efference copy of the selected action which they are supposed to inhibit during action selection. Even though intriguing and seemingly unnatural, the authors demonstrated that the model based on the hypothesis can circumvent the problem of iSPNs learning to disinhibit the actions associated with negative reward errors. They further showed by analyzing the cell-type specific neural recording data by Markowitz et al. (2018) that iSPN activities tend to be anti-correlated before and after action selection.

Weaknesses:

(1) It is not correct to call the action value learning using the externally-selected action as "off-policy." Both off-policy algorithm Q-learning and on-policy algorithm SARSA update the action value of the chosen action, which can be different from the greedy action implicated by the present action values. In standard reinforcement learning terminology, on-policy or off-policy is regarding the actions in the subsequent state, whether to use the next action value of (to be) chosen action or that of greedy choice as in equation (7).

It is worth noting that this paper suggested that dopamine neurons encode on-policy TD errors:<br /> Morris G, Nevet A, Arkadir D, Vaadia E, Bergman H (2006). Midbrain dopamine neurons encode decisions for future action. Nat Neurosci, 9, 1057-63. https://doi.org/10.1038/nn1743

(2) It is also confusing to contract TD learning and Q-learning, as the latter is considered as one type of TD learning. In the TD error signal by state value function (6) is dependent on the chosen action a_{t-1} implicitly in r_t and s_t based on the reward and state transition function.

(3) It is not clear why interferences of the activities for action selection and learning can be avoided, especially when actions are taken with short intervals or even temporal overlaps. How can the efference copy activation for the previous action be dissociated with the sensory cued activation for the next action selection?

(4) Although it may be difficult to single out the neural pathway that carries the efference copy signal to the striatum, it is desired to consider their requirements and difference possibilities. A major issue is that the time delay from actions to reward feedback can be highly variable.

An interesting candidate is the long-latency neurons in the CM thalamus projecting to striatal cholinergic interneurons, which are activated following low-reward actions:<br /> Minamimoto T, Hori Y, Kimura M (2005). Complementary process to response bias in the centromedian nucleus of the thalamus. Science, 308, 1798-801. https://doi.org/10.1126/science.1109154

(5) In the paragraph before Eq. (3), Eq. (1) should be Eq. (2) for the iSPN.

Reviewer #3 (Public review):

Summary:

In this manuscript the authors begin with the interesting phenotype of sub inhibitory concentrations of the aminoglycoside tobramycin proving toxic to a knockout of the tRNA-guanine transglycosylase (Tgt) of the important human pathogen, Vibrio cholerae. Tgt is important for incorporating queuosine (Q) in place of guanosine at the wobble position of GUN codons. The authors go on to define a mechanism of action where environmental stressors control expression of tgt to control translational decoding of particularly tyrosine codons, skewing the balance from TAC towards TAT decoding in the absence of the enzyme. The authors use advanced proteomics and ribosome profiling to reveal that the loss of tgt results in increased translation of proteins like RsxA and a cohort of DNA repair factors, whose genes harbor an excess of TAT codons in many cases. These findings are bolstered by a series of molecular reporters, mass spectrometry, and tRNA overexpression strains to provide support for a model where Tgt serves as a molecular pivot point to reprogram translational output in response to stress. The manuscript therefore improves our understanding of the phenotype of focus and will prove useful for the field in our understanding of Modification Tunable Transcripts.

Strengths:

The manuscript has many strengths. The authors use a variety of strains, assays, and advanced techniques to discover a mechanism of action for Tgt in mediating tolerance to sub inhibitory concentrations of tobramycin. They observe a clear phenotype for a tRNA modification in facilitating reprogramming of the translational response, and the manuscript certainly has value in defining how microbes tolerate antibiotics.

Weaknesses:

The conclusions of the manuscript are mostly very well-supported by the data, but a few experimental directions remain inconclusive. The finding linking Tgt and UV damage susceptibility is one example where the phenotype is striking, but the mechanism remains somewhat unclear. Future work in this direction will likely be required to fully understand how Tgt influences the repair of DNA after UV.

Reviewer #3 (Public review):

Gatt et al. present a novel take on single-cell RNA-sequencing from complex planktonic samples, introducing an approach they aptly named Ukiyo-e-Seq. This work combines environmental sampling with cell picking, microscopic imaging, and Smart-seq2 single-cell RNA sequencing to profile uncultured eukaryotic plankton. Developing single-cell approaches for such ecosystems is critical, given the poor representation of many planktonic species in cultures and reference databases. This work could help bridge existing technological gaps between morphological and molecular studies of aquatic microeukaryotes

The authors argue that microscopy does not provide information on the biochemistry of species under consideration. At best, it provides taxonomic labeling of species within a sample, yet imaging fails to assess their metabolic state or to disentangle cryptic species. In a standard metatranscriptomic setup, the sequence pool is described by aligning assembled contigs with reference databases to obtain functional and taxonomic information. This complex community-level data is impossible to parse at the single-organism level. Moreover, by relying on reference datasets, a lot of potential information can be missed. The aim of the approach is to combine the strengths of both methods, generating single-cell transcriptomic data linked to individual plankton images.

Strengths:

Ukiyo-e-Seq generated a valuable dataset by combining imaging and transcriptomics for individual planktonic organisms from environmental samples. This multimodal approach has the potential to improve taxonomic predictions and functional insights at the single-organism level. This manuscript demonstrates the technical feasibility of such an approach. Data of this type is rare and thus represents a valuable resource to further advance single-cell sequencing of planktonic species from environmental samples.

Weaknesses:

(1) The merge-split strategy, where single-cell reads are pooled prior to assembly, is counterintuitive. Pooling obscures the single-organism resolution that single-cell methods aim to achieve. The approach might be useful for assembling low-coverage contigs, but risks masking unique expression profiles for transcripts unique to a given well. As an alternative, the authors could assemble each well independently to obtain well-specific transcriptomic bins. Assemblies could then be clustered based on sequence similarity, thereby imposing strict clustering parameters to maintain resolution, to create a common reference for downstream analysis if needed. In my opinion, better results would be obtained by implementing a per-well assembly and read mapping.

(2) The focus on the top five most expressed contigs throughout the manuscripts' data analysis is a limiting choice, as it excludes most contigs. In the preprint, we are presented with a very narrow view of the data. Visualising the entire range of assembled contigs would provide a better picture of the transcriptomic composition and diversity per well. It would be interesting to assess if the full information could be used to preliminary bin transcriptomic sequences from individual wells, for example, by gathering all 'private' contigs with high read coverage in a single well. Does such a set represent a single complete eukaryotic transcriptome?

(3) I missed a verification with (broad-scale) taxonomic assessments based on the associated microscopic images. In their goals, the authors state that a joint approach has the potential to discover new taxonomic biodiversity. I agree, and to me, this is what is exciting about the preprint, yet I miss an example or the right bioinformatic implementation to drive home this claim. Are there organisms in wells where poor taxonomic annotations, based on alignment to a reference database or the LCA approach implemented in Kraken2, would usually result in ignoring the species in classic metatranscriptomics? Can you advance the taxonomic annotation by referring back to the organisms' picture? Can manual assessment of taxonomy advance the results from the LCA approach?

(4) The current use of AlphaFold to predict protein structures does not convincingly add to the study's core objectives.

Overall, Ukiyo-e-Seq presents a promising method for studying single-cell diversity in environmental samples, though the bioinformatic pipeline requires refinement to support some of the claims made by the authors. Additionally, the manuscript would benefit from clarity and additional details in its methods and a more consistent approach to presenting results and summary statistics across all assembled contigs and all sampled wells, rather than focusing on selected wells.

Reviewer #3 (Public review):

This work brings a computational approach to the study of promoters and transcription. The paper is improved but there are still factual errors and implausible explanations. I am not convinced by the response from the authors, concerning the promoter -35 element, in their rebuttal.

Comments on author rebuttal:

- We respectfully but strongly disagree that our analysis has misrepresented the true nature of -35 boxes. First, accounting for more A's at position 5 in the PWM is not going to lead to a "critical error." This is because positions 4-6 of the motif barely have any information content (bits) compared to positions 1-3 (see Fig 1A).

The analysis does misrepresent the consensus -35 element, which is, unequivocally, TTGACA. I agree that positions 4-6 of the element are less well-conserved.

- This assertion is not just based on our own PWM, but based on ample precedent in the literature. In PMID 14529615, TTG is present in 38% of all -35 boxes, but ACA only in 8%.

This does not mean that TTGACA is not the consensus, or that "ACA" is not important at promoters where it's present.

- In PMID 29388765, with the -10 instance TATAAT, the -35 instance TTGCAA yields stronger promoters compared to the -35 instance TTGACA (See their Figure 3B).

This is a known phenomenon and results from "perfect" promoters being limited at the point of RNA polymerase promoter escape (because the RNAP struggles to "let go" of perfect promoters). This does not mean the TTGACA is not the consensus. Indeed, and this is a key point, it is evident in the figure the authors refer to that TTGACA stimulates more transcription than alternative -35 sequences when -10 elements are not perfect.

- In PMID 29745856 (Figure 2), the most information content lies in positions 1-3, with the A and C at position 5 both nearly equally represented, as in our PWM.

The motif shown in this paper suffers from exactly the same issue as the paper under review; the variable spacing between the -35 hexamer and -10 element isn't taken into account by MEME.

- In PMID 33958766 (Figure 1) an experimentally-derived -35 box is even reduced to a "partial" -35 box which only includes positions 1 and 2, with consensus: TTnnnn.

This paper does not show an "experimentally-derived -35 box" in Figure 1 (or anywhere else, as far as I can see).

- In addition, we did not derive the PWMs as the reviewer describes. The PWMs we use are based on computational predictions that are in excellent agreement with experimental results. Specifically, the PWMs we use are from PMID 29728462, which acquired 145 -10 and -35 box sequences from the top 3.3% of computationally predicted boxes from Regulon DB.

The paper mentioned states "for the genomic RNAP logo, sequences were taken from computationally predicted RNAP binding sites on RegulonDB" so these are not experimentally defined promoters? It's not obvious from the paper, or regulon DB, which sequences these are or how they were predicted.

- Thank you for pointing out that our original submission was incomplete in this regard. We address these concerns by new analyses, including some new experiments. First, Rho dependent termination is associated with the RUT motif, which is very rich in Cytosines (PMID: 30845912). Given that our sequences confer between 65%-78% of AT-content, canonical rho dependent termination is unlikely. However, we computationally searched for rho-dependent terminators using the available code from PMID: 30845912, but the algorithm did not identify any putative RUTs. Because this analysis was not informative, we did not include it in the paper.

I don't believe it is the case that Rho absolutely requires a RUT sequence. My understanding is that, if an RNA is not translated, Rho will intervene (e.g. see PMID: 18487194).

- We respectfully disagree that the reviewer's point is pertinent because what the reviewer is referring to is the likelihood that the sequence is a promoter, which indeed increases with AT content, but we are focused on the likelihood that a sequence becomes a promoter through DNA mutation

I disagree that this distinction is relevant. An AT-rich sequence will much more closely resemble a promoter by chance than a GC rich sequence. As an extreme example, the sequence TTTTTT can be converted into a reasonable -10 element by one change (to TATTTT) but the sequence GGGGGG can't.

Reviewer #3 (Public review):

Summary:

m6Am is an abundant mRNA modification present on the TSN. Unlike the structurally similar and abundant internal mRNA modification m6A, m6Am's function has been controversial. One way to resolve controversies surrounding mRNA modification functions has been to develop new ways to better profile said mRNA modification. Here, Liu et al. developed a new method (based on GLORI-seq for m6A-sequencing), for antibody-independent sequencing of m6Am (CROWN-seq). Using appropriate spike-in controls and knockout cell lines, Liu et al. clearly demonstrated CROWN-seq's precision and quantitative accuracy for profiling transcriptome-wide m6Am. Subsequently, the authors used CROWN-seq to greatly expand the number of known m6Am sites in various cell lines and also determine m6Am stoichiometry to generally be high for most genes. CROWN-seq identified gene promoter motifs that correlate best with high stoichiometry m6Am sites, thereby identifying new determinants of m6Am stoichiometry. CROWN-seq also helped reveal that m6Am does not regulate mRNA stability or translation (as opposed to past reported functions). Rather, m6Am stoichiometry correlates well with transcription levels. Finally, Liu et al. reaffirmed that FTO mainly demethylates m6Am, not of mRNA but of snRNAs and snoRNAs.

Strengths:

This is a well-written manuscript that describes and validates a new m6Am-sequencing method: CROWN-seq as the first m6Am-sequencing method that can both quantify m6Am stoichiometry and profile m6Am at single-base resolution. These advantages facilitated Liu et al. to uncover new potential findings related to m6Am regulation and function. I am confident that CROWN-seq will likely be the gold standard for m6Am-sequencing henceforth.

Weaknesses:

Though the authors have uncovered a potentially new function for m6Am, they need to be clear that without identifying a mechanism, their data might only be demonstrating a correlation between the presence of m6Am and transcriptional regulation rather than causality.

Reviewer #3 (Public review):

Summary:

Chen et al. develop and characterize a new approach for screening drugs for epilepsy. The idea is to increase the ability to study seizures in animals with epilepsy because most animal models have rare seizures. Thus, the authors use the existing intrahippocampal kainic acid (IHKA) mouse model, which can have very unpredictable seizures with long periods of time between seizures. The authors employ an additional method to trigger seizures in the IHKA model. This method is closed-loop optogenetic stimulation of area CA1. There are several assumptions: area CA1 is the best location, triggered seizures are the same as spontaneous seizures, and this method will be useful despite requiring a great deal of effort. Regarding the latter, using a mouse model with numerous seizures (such as the pilocarpine model) might be more efficient than using a modified IHKA protocol that requires viral injection for optogenetics, fiber insertion requiring additional surgery, and accurate targeting to reliably trigger seizures on-demand. Aside from these caveats, the authors do succeed in studying seizures more readily in a mouse model of rare seizures. However, the seizures are evoked, not spontaneous. As currently presented, it is not clear how the triggered seizures can be used to investigate if antiseizure medication can reduce seizure burden as measured by seizure severity and seizures per day.

The authors modified the IHKA model to inject KA into CA3 instead of CA1 in order to preserve the CA1 pyramidal cells that they will later stimulate. To express the excitatory opsin channelrhodopsin (ChR2) in area CA1, they use a virus that expresses ChR2 in cells that express the Thy-1 promoter. The authors demonstrate that CA3 delivery of KA can induce a very similar chronic epilepsy phenotype to the injection of KA in CA1 and show that optical excitation of CA1 can reliably induce seizures. These are the strengths of the study.

While the authors show that electrophysiological signatures of induced vs spontaneous seizures are similar in many ways, the authors also show several differences and it is not clear if these differences are meaningful. Notably, the induced seizures are robustly inhibited by the antiseizure medication levetiracetam and variably but significantly inhibited by diazepam, similar to many mouse models with chronic recurrent seizure activity. I agree with the authors that this modified IHKA model will be of most value for higher throughput screening of potential antiseizure therapies, but with the caveat that the data may not generalize to other epilepsy models or humans. The authors evaluate the impact of repeated stimulation on the reliability of seizure induction and show that seizures can be reliably induced by CA1 stimulation for as long as 16 days, but the utility of the model would be better demonstrated if seizures could be shown to be inducible over the range of weeks to months.

Strengths:

(1) The authors show that the IHKA model of chronic epilepsy can be modified to preserve CA1 pyramidal cells (but at a cost of CA3 cells), allowing on-demand optogenetic stimulation of CA1 that appears to lower seizure threshold and thus trigger a seizure event.

(2) The authors show that repeated reactivation of CA1 even in untreated mice can promote kindling and induction of seizure activity, indeed generating two mouse models in total.

(3) Many electrophysiological signatures are similar between the induced and spontaneous seizures, and induced seizures reliably respond to treatment with antiseizure medications.

(4) Given that more seizures can be observed per mouse using on-demand optogenetics, this model enhances the utility of each individual mouse.

Weaknesses:

(1) Evaluation of seizure similarity using the SVM modeling and clustering is not sufficiently explained to show if there are meaningful differences between induced and spontaneous seizures. SVM modeling did not include analysis to assess the overfitting of each classifier since mice were modeled individually for classification.

(2) The difference between seizures and epileptiform discharges or trains of spikes (which are not seizures) is not made clear.

(3) The utility of increasing the number of seizures for enhancing statistical power is limited unless the sample size under evaluation is the number of seizures. However, the standard practice is for the sample size to be the number of mice.

(4) Seizure burden is not easily tested.

(5) It is unlikely that long-term adaptation to CA1-stimulated seizure induction is absent in these mice. A duration of evaluation longer than 16 days is warranted in light of the downward slope at days 13-16 for induced seizures in Figure 4C.

(6) Human epilepsy is extensively heterogeneous in both etiology and individual phenotype, and it may be hard to generalize the approach.

(7) No mention or assessment of mouse sex as a biological variable.

Reviewer #3 (Public review):

Summary:

This study aims to investigate the stoichiometric effect between core factors and partners forming the heterodimeric transcription factor network in living cells at endogenous expression levels. Using state-of-the-art single-molecule analysis techniques, the authors tracked individual RARα and RXRα molecules labeled by HALO-tag knock-in. They discovered an asymmetric response to the overexpression of counter-partners. Specifically, the fact that an increase in RARα did not lead to an increase in RXRα chromatin binding is incompatible with the previous competitive core model. Furthermore, by using a technique that visualizes only molecules proximal to partners, they directly linked transcription factor heterodimerization to chromatin binding.

Strengths:

The carefully designed experiments, from knock-in cell constructions to single-molecule imaging analysis, strengthen the evidence of the stoichiometric perturbation response of endogenous proteins. The novel finding that RXR, previously thought to be a target of competition among partners, is in excess provides new insight into key factors in dimerization network regulation. By combining the cutting-edge single-molecule imaging analysis with the technique for detecting interactions developed by the authors' group, they have directly illustrated the relationship between the physical interactions of dimeric transcription factors and chromatin binding. This has enabled interaction analysis in live cells that was challenging in single-molecule imaging, proving it is a powerful tool for studying endogenous proteins.

Weaknesses:

None noted.

Reviewer #3 (Public review):

Summary:

Yao et al use CHART to identify chromatin associated with Xist in female mouse ESCs, and, as control, male ESCs at various timepoints of differentiation. Besides binding of Xist to X chromosome regions they found significant binding to autosomes, concentrating mostly on promoter regions of around 100 autosomal genes, as elucidated by MACS. The authors went on to show that the RepB repeat is mostly responsible for these autosomal interactions using a female ESC line in which RepB is deleted. Evidence is provided that Xist interacts with active autosomal genes containing lower coverage of repressive marks H3K27me3 and H2AK119ub and that RepB dependent Xist binding leads to dampening of expression, but not silencing of autosomal genes. These results were confirmed by overexpression studies using transgenic ESCs with doxycycline-inducible Xist as well as via a small molecule inhibitor of Xist (X1), inducing/inhibiting the dampening of autosomal genes, respectively. Finally, using MEFs and Xist mutants RepB or RepE the authors provide evidence that Xist is bound to autosomal genes in cells after the XCI process but appears not to affect gene expression. The data presented appear generally clear and consistent and indicate some differences between human and mouse autosomal regulation by Xist. Thus, these results are timely and should be published.

Strengths:

Regulation of autosomal gene expression by Xist is a "big deal" as misregulation of this lncRNA causes developmental defects and human disease. Moreover, this finding may explain sex-specific developmental differences between the sexes. The results in this manuscript identify specific mouse autosomal genes bound by Xist and decipher critical Xist regions that mediate this binding and gene dampening. The methods used in this study are appropriate, and the overall data presented appear convincing and are consistent, indicating some differences between human and mouse autosomal regulation by Xist.

Comments on revisions:

In the revised manuscript, the authors have addressed my previous criticisms satisfactorily. Moreover, the manuscript has been much improved with new confirmatory results and additional control experiments. This, combined with more detailed descriptions/explanations facilitates data interpretation, making the paper more transparent and easier to read.

Reviewer #3 (Public review):

This study begins with a chemogenetic screen to discover previously unrecognized regulators of the cell cycle. Using a CRISPR-Cas9 library in HAP1 cells and an assay that scores cell fitness, the authors identify genes that sensitize or desensitize cells to the presence of palbociclib, colchicine, and camptothecin. The results suggest that these three drugs inhibit proliferation through different mechanisms, and with each treatment, expected and unexpected pathways were found to affect drug sensitivity. The authors focus the rest of the experiments and analysis on the polycomb complex PRC2, as deletion of several of its subunits in the screen conferred palbociclib resistance. The authors find that PRC2, specifically a complex dependent on the MTF2 subunit, methylates histone 3 lysine 27 (H3K27) in promoters of genes associated with various processes including cell-cycle control. Further experiments demonstrate that Cyclin D expression increases upon loss of PRC2 subunits, providing a potential mechanism for palbociclib resistance.

The strengths of the paper are the design and execution of the chemogenetic screen, which provides a wealth of potentially useful information. The data convincingly demonstrate in the HAP1 cell line that the MTF2-PRC2 complex sustains the effects of palbociclib (Fig. 4), methylates H3K27 in CpG-rich promoters (Fig. 5), and represses Cyclin D expression (Fig. 6). The correlation between MTF2-PRC2 inhibition and increased Cyclin D levels is shown in multiple cell lines using both genetic and chemical approaches. These results could be of great interest to those studying cell-cycle control, resistance mechanisms to therapeutic cell-cycle inhibitors, and chromatin regulation and gene expression.

There are a few weaknesses that somewhat temper the overall quality and potential impact of the study. First, the results from the colchicine and camptothecin screens (Fig. 1 and 2) are not experimentally validated, which lessens the rigor of those data and conclusions. Second, some experiments validating and further exploring results from the palbociclib screen (Figs. 4 and 5) are restricted to the Hap1 cell line, so the generality of some conclusions is not established. Third, conclusions drawn from data in Fig. 4D are not fully supported by proper use of biological replicates and analysis of the results.

Comments on revisions:

Proper statistical analysis considering biological replicates is still not applied to determine whether differences in palbociclib IC50 values at different GSK126 concentrations are significant.

Reviewer #3 (Public review):

Summary:

The decrease in estrogen levels is strongly associated with postmenopausal hypertension. Dr. Yao Li and colleagues aimed to investigate the metabolomic mechanisms of underlying postmenopausal hypertension using OVX and OVX+E2 rat models. They successfully established a correlation between reduced estrogen levels and the development of hypertension in rats. They identified L-alpha-aminobutyric acid (AABA) as a potential marker for postmenopausal hypertension. The research explored the metabolic alterations in aortic tissues and proposed several potential mechanisms contributing to postmenopausal hypertension.

Strengths:

The group performed a comprehensive enrichment analysis and various statistical analyses of the metabolomics data.

Weaknesses:

(1) The manuscript is descriptive in nature, although they mentioned their primary objective is to explore the potential mechanisms linking low estrogen levels with postmenopausal hypertension. No mechanism insights have been interrogated in this study, which has been mentioned by the authors in the discussion. The connection between E2, AABA, and macrophage needs to be validated in endothelial cells, vascular smooth muscle cells, and other aortic tissue cells. Without such verification, the manuscript predominantly raises hypotheses only based on metabolomic data.

(2) The serum contains three forms of estrogen: Estradiol, Estrone, and Estriol. The authors used the Rat E2 ELISA kit. Ideally, all three forms of estrogen should be measured.

Reviewer #3 (Public review):

Summary:

The authors have shown that oxydifficidin is a potent inhibitor of Neisseria gonorrhoeae. They were able to identify the target of action to rpsL and showed that resistance could occur via mutation in the DedA flippase and RpsL.

Strengths:

This was a very thorough and clearly argued set of experiments that supported their conclusions.

Weaknesses:

There was no obvious weakness in the experimental design. Although it is promising that the DedA mutations resulted in attenuation of fitness, it remains an open question whether secondary rounds of mutation could overcome this selective disadvantage which was untried in this study.

Comments on revisions:

All of my suggestions were considered and the responses to the other reviewer's appears sound and has improved the manuscript.

Reviewer #3 (Public review):

Summary:

In this paper, Chang and Meliala et al. demonstrate that PEBP1 is a modulator of the ISR, specifically through the induction of mitochondrial stress. The authors utilize thermal proteome profiling (TPP) by which they identify PEPB1 as a thermally stabilized protein upon oligomycin treatment, indicating its role in mitochondrial stress. Moreover, RNA-sequencing analysis indicated that PEBP1 may be specifically modulating the mitochondrial stress-induced ISR, as PEBP1 knock-out reduces phosphorylation of eIF2α. They also show that PEBP1 function is independent of ER stress specifically tunicamycin treatment and loss of PEBP1 does affect mitochondrial ISR but in an OMA1, DELE1 independent manner. Thus, the authors hypothesized that PEBP1 interacts directly with eIF2α, functioning as a scaffolding protein. However, direct co-immunoprecipitation failed to demonstrate PEBP1 and eIF2α potential interaction. The authors then used a NanoBiT luminescence complementation assay to show the PEBP1-eIF2a interaction and its disruption by S51 phosphorylation.

Strengths:

Taken together, this work is novel, and the data presented suggests PEBP1 has a role as a modulator of the mitochondrial ISR, enhancing the signal to elicit the necessary response.

Weaknesses:

The one major issue of this work is the lack of a mechanism showing precisely how PEBP1 amplifies the mitochondrial integrated stress response. The work, as it is described, presents data suggesting PEBP1's role in the ISR but fails to present a more conclusive mechanism.

Reviewer #3 (Public review):

Summary:

The study investigated stable RNA-chromatin interactions by applying RNase treatment before the iMARGI (in situ mapping of RNA-genome interactome) procedure to remove promiscuous, unprotected RNA transcripts and selectively enrich for RNA-inaccessible, potentially functional RNA-chromatin interactions (RNA-Transcription factor and RNA-histone). The researchers found that short-range interactions (<1kb) are RNase resistant, possibly due to the protection from RNA polymerases. They noticed that long-range RNA-chromatin interactions (>2Mbp or interchromosomal) were also enriched after RNase treatment, hypothesizing that these interactions are stabilized by chromatin-binding proteins. They found that genic caRNAs were sensitive, while repeat-derived caRNAs, such as rRNA and satellite repeats, were resistant to RNase. Long non-coding RNAs (lncRNAs), particularly those associated with diseases, were over-represented among RNase-insensitive transcripts, indicating their potential regulatory significance. Additionally, RNase-insensitive caRNAs exhibited higher evolutionary conservation, implying that they are protected by protein complexes, especially in long-range interactions. RNA Attachment Hot Zones (RAHs) enriched post-RNase treatment were found to localize in functional genomic regions such as promoters, transcription factor binding sites (TFBS), and histone modification sites. Importantly, RNase treatment amplified specific RNA-transcription factor interactions, with caRNA signals being preserved at TFBS for factors with RNA-binding capabilities, suggesting that direct RNA-protein binding helps protect caRNAs from degradation. They also found that different TFs are enriched with specific caRNA species, distinguishing them from their genomic footprints. In addition, transcripts with higher abundance tend to enrich at more TFBS. Overall, the study highlights the role of RNase-inaccessible caRNAs in chromatin regulation and provides insight into their functional significance in genome organization.

Strengths:

This study involves rigorous and comprehensive data analysis involving datasets with very high sequencing depth and appropriate statistical tests (e.g., chi-square tests to validate the association between caRNAs and TFBS statistically). This analysis was further strengthened by comparing their results with orthogonal datasets, such as RedChIP and fRIP-seq, providing robust, cross-validated evidence for the caRNA-TFBS associations. In addition to examining broad interactions, the authors identified specific long-range RNA-chromatin interactions and pinpointed specific transcription factors and histone modification markers that are associated with these interactions. The authors explored the evolutionary implications of RNase-insensitive caRNAs and their potential medical relevance, particularly by identifying caRNAs linked to disease-associated genes and long non-coding RNAs (lncRNAs). This combination of detailed analysis, along with functional relevance, broadens the scope of the research, making it a significant contribution to chromatin biology.

Weaknesses:

However, I have the following concerns regarding the studies:<br /> (1) I don't understand the logic behind calling promoters, enhancers, and similar regions "functionally important regions" when describing the enrichment of RNase-insensitive interactions. Genic regions that are RNase-sensitive are also functionally relevant. So, what makes promoters, enhancers, etc, unique in terms of functionality?<br /> (2) First, while the study offers strong evidence for associations between caRNAs, transcription factors, and chromatin markers, it lacks direct functional validation experiments such as RNA knockdown or CRISPR interference, to confirm the specific roles of these RNAs in gene regulation or chromatin structure modifications.<br /> (3) Another limitation is the incomplete investigation of caRNAs with short-range interactions (<1kb). The authors hypothesized that these are protected by RNA polymerases but did not provide supporting experimental evidence or references to previous studies. Offering either experimental validation or a rationale for excluding these short-range interactions would strengthen this hypothesis. The conclusion that authors drew on that "chromatin-associated RNAs (caRNAs) involved in short- to middle-range interactions are more susceptible to RNase treatment" was unclear for the specific "short-range" distance. The data shown in Supplementary Figure 2a contradicted the conclusion in the discussion that "long-distance RNA-chromatin interactions are preferentially preserved after RNase treatment, while short-range interactions are depleted." as well as the suggestion made linking RNase inaccessibility to evolutionarily conserved in the paper.<br /> (4) The study heavily relies on RNase treatment to isolate stable RNA-chromatin interactions, which might neglect important transient or weak interactions and overlook the functional relevance of RNase-sensitive interactions, hence missing the dynamic nature of RNA-chromatin interactions.<br /> (5) Tthe analysis is limited to human embryonic stem cells (H1 cells), which might restrict the generalizability of the findings. Expanding the study to include a cell type that represents a broader range of cell types or tissues will strengthen the conclusions.<br /> (6) The term "RNase A treatment" in the methods section could be clearer if specified as "RNase-treated iMARGI," which encompasses the standard iMARGI protocol.<br /> (7) There is some ambiguity regarding whether the researchers generated new data or reanalyzed existing datasets. While it is mentioned early on that previously published RNase-treated iMARGI datasets were reanalyzed, the text later states that "three biological replicates were generated for the RNase-treated samples." Clarifying whether the data were newly generated in this study or obtained from public datasets would improve the clarity.<br /> (8) The color scheme should be the same for heatmaps for control, and RNase-treated samples in Figure 4.

Reviewer #3 (Public review):

Summary:

Membrane-bound pyrophosphatases (mPPases) are homodimeric proteins that hydrolyze pyrophosphate and pump H+/Na+ across membranes. They are attractive drug targets against protist pathogens. Non-hydrolysable PPi analogue bisphosphonates such as risedronate (RSD) and pamidronate (PMD) serve as primary drugs currently used. Bisphosphonates have a P-C-P bond, with its central carbon can accommodate up to two substituents, allowing a large compound variability. Here the authors solved two TmPPase structures in complex with the bisphosphonates etidronate (ETD) and zoledronate (ZLD) and monitored their conformational ensemble using DEER spectroscopy in solution. These results reveal the inhibition mechanism of these compounds, which is crucial for developing future small molecule inhibitors.

Strengths:

The authors show that seven different bisphosphonates can inhibit TmPPase with IC50 values in the micromolar range. Branched aliphatic and aromatic modifications showed weaker inhibition.

High-resolution structures for TmPPase with ETD (3.2 Å) and ZLD (3.3 Å) are determined. These structures reveal the binding mode and shed light on the inhibition mechanism. The nature of modification on the bisphosphonate alters the conformation of the binding pocket.

The conformational heterogeneity is further investigated using DEER spectroscopy under several conditions.

Weaknesses:

The authors observed asymmetry in the TmPPase-ELD structure above the hydrolytic center. The structural asymmetry arises due to differences in the orientation of ETD within each monomer at the active site. As a result, loop5-6 of the two monomers is oriented differently, resulting in the observed asymmetry. The authors attempt to further establish this asymmetry using DEER spectroscopy experiments. However, the (over)interpretation of these data leads to more confusion than any further understanding. DEER data suggest that the asymmetry observed in the TmPPase-ELD structure in this region might be funneled from the broad conformational space under the crystallization conditions.

DEER data for position T211R1 at the enzyme entrance reveal a highly flexible conformation of loop5-6 (and do not provide any direct evidence for asymmetry, Figure EV8). Similarly, data for position S521R1 near the exit channel do not directly support the proposed asymmetry for ETD. Despite the high quality of the data, they reveal a very similar distance distribution. The reported changes in distances are very small (+/- 0.3 nm), which can be accommodated by a change of spin label rotamer distribution alone. Further, these spin labels are located on a flexible loop, thereby making it difficult to directly relate any distance changes to the global conformation.

The interpretations listed below are not supported by the data presented:

(1) 'In the presence of Ca2+, the distance distribution shifts towards shorter distances, suggesting that the two monomers come closer at the periplasmic side, and consistent with the predicted distances derived from the TmPPase:Ca structure.'

Problem: This is a far-stretched interpretation of a tiny change, which is not reliable for the reasons described in the paragraph above.

(2) 'Based on the DEER data on the IDP-bound TmPPase, we observed significant deviations between the experimental and the in silico distances derived from the TmPPase:IDP X-ray structure for both cytoplasmic- (T211R1) and periplasmic-end (S525R1) sites (Figure 4D and Figure EV8D). This deviation could be explained by the dimer adopting an asymmetric conformation under the physiological conditions used for DEER, with one monomer in a closed state and the other in an open state.'

Problem: The authors are trying to establish asymmetry using the DEER data. Unfortunately, no significant difference is observed (between simulation and experiment) for position 525 as the authors claim (Figure 4D bottom panel). The observed difference for position 112 must be accounted for by the flexibility and the data provide no direct evidence for any asymmetry.

(3) 'Our new structures, together with DEER distance measurements that monitor the conformational ensemble equilibrium of TmPPase in solution, provide further solid experimental evidence of asymmetry in gating and transitional changes upon substrate/inhibitor binding.'

Problem: See above. The DEER data do not support any asymmetry.

(4) Based on these observations, and the DEER data for +IDP, which is consistent with an asymmetric conformation of TmPPase being present in solution, we propose five distinct models of TmPPase (Figure 7).

Problem: Again, the DEER data do not support any asymmetry and the authors may revisit the proposed models.

(5) 'In model 2 (Figure 7), one active site is semi-closed, while the other remains open. This is supported by the distance distributions for S525R1 and T211R1 for +Ca/ETD informed by DEER, which agrees with the in silico distance predictions generated by the asymmetric TmPPase:ETD X-ray structure'

Problem: Neither convincing nor supported by the data

Reviewer #3 (Public review):

Summary:

I really like this topic and study. But I think much can be more focused and tightened up. All the components are here - just some more refining to really make the storyline clear, the journey of discovery, and the impact of such knowledge.

Strengths:

The authors dive directly into the question of genomic ancestry as compared to the breed club's reported ancestry with heavy, quantitative data and critical analytical methods. The questioning line is direct and does not meander. The reader learns about the challenges of breeding associations, and values of understood ancestry, and presents a clear need of re-evaluating the breed standards and expectations of beefalo (if ancestry is indeed the primary goal instead of a phenotype-driven breed mission).

Weaknesses:

Much of the quantitative results are only referred to in the main text with qualitative language. Please incorporate more written quantitative results to highlight evidence that underlines the study narrative because it is quite an interesting study!

Reviewer #3 (Public review):

Summary:

The authors demonstrated MK2i could enhance the therapeutic efficacy of MTAs. With Tumor xenograft and migration assay, the author suggested that the p38-MK2 pathway may serve as a promising therapeutic target in combination with MTAs in cancer treatment.

Strengths:<br /> The authors provided a potential treatment for breast cancer.

Weaknesses:

(1) In Figure 2, the authors used a human retinal pigment epithelial-1 (RPE1) cell line to show that breast cancer cells are more sensitive to CMPD1 treatment. MCF10A cells would be suggested here as a suitable control. Besides, to compare the sensitivity, IC50 indifferent cell lines should be measured.

(2) The data of MDA-MB-231 in Figure 1D is not consistent with CAL-51 and T47D, also not consistent with the data in Figures 2B-C.

(3) To support the authors' conclusion in Figure 5, an additional animal experiment performed by tail vein injection would be helpful.

(4) Page 14, to evaluate the combination result of MK2i and vinblastine, an in vivo animal assay must be performed.

(5) The authors used RNA-seq to show some pathways affected by CMPD1. What are the key/top genes that were affected? How about the mechanism?

(6) Line 127, more experiments should be involved to support the conclusion.

Reviewer #3 (Public review):

Summary:

The authors investigated how non-alcoholic fatty liver disease (NAFLD) influences liver damage during endotoxemia (a condition characterized by elevated endotoxins, like lipopolysaccharide or LPS, in the bloodstream) using a mouse model. Mice with NAFLD were given a moderate dose of LPS, which intensified liver inflammation and mortality compared to controls. The study concludes that targeting neutrophil activity and TNF-α signaling could be a promising approach to reducing excessive inflammation and liver injury in NAFLD patients experiencing endotoxemia. This can have important implications for the treatment but I think the manuscript requires revisions.

Strengths:

(1) The study presents both in vivo and ex vivo assay and results to support their hypothesis.

(2) Several cell types and their interaction with each other have been analyzed.

(3) The authors made use of the publicly available databases.

Weaknesses:

(1) Some figures contradict each other.

(2) Some of the cause-and-effect presentations need additional experiments and different approaches to be proven correct.

(3) Candidate/mechanism selection strategies are not very clear.

Reviewer #3 (Public review):

Summary:

The authors demonstrate that activation of TFEB facilitates cholesterol clearance in cell models of Niemann-Pick type C (NPC). This is done through a variety of approaches including activation of TFEB by sulforaphane (SFN), a naturally occurring small-molecule TFEB agonist. SFN induces TFEB nuclear translocation and promotes lysosomal exocytosis. In an NPC mouse model, SFN dephosphorylates/activates TFEB in the brain and rescues the loss of Purkinje cells.

Strengths:

NPC is a severe disease and there is little in the way of treatment. The manuscript points towards some treatment options. However, the title, the title "Small-molecule activation of TFEB Alleviates Niemann-Pick Disease..." is far too strong and should be changed.

Weaknesses:

(1) The manuscript is extremely hard to read due to the writing; it needs careful editing for grammar and English.

(2) There are a number of important technical issues that need to be addressed.

(3) The TFEB influence on filipin staining in Figure 1A is somewhat subtle. In the mCherry alone panels there is a transfected cell with no filipin staining and the mCherry-TFEBS211A cells still show some filipin staining.

(4) Figure 1C is impressive for the upregulation of filipin with U18666A treatment. However, SFN is used at 15 microM. This must be hitting multiple pathways. Vauzour et al (PMID: 20166144) use SFN at 10 nM to 1microM. Other manuscripts use it in the low microM range. The authors should repeat at least some key experiments using SFN at a range of concentrations from perhaps 100 nM to 5 microM. The use of 15 microM throughout is an overall concern.

Reviewer #3 (Public review):

Summary:

The authors sought to understand social interactions both within and between groups of feral pigs, with the intent of applying their findings to models of disease transmission. The authors analyzed GPS tracking data from across various populations to determine patterns of contact that could support the transmission of a range of zoonotic and livestock diseases. The analysis then focused on the effects of sex, group dynamics, and seasonal changes on contact rates that could be used to base targeted disease control strategies that would prioritize the removal of adult males for reducing intergroup disease transmission.

Strengths:

It utilized GPS tracking data from 146 feral pigs over several years, effectively capturing seasonal and spatial variation in the social behaviors of interest. Using proximity-based social network analysis, this work provides a highly resolved snapshot of contact rates and interactions both within and between groups, substantially improving research in wildlife disease transmission. Results were highly useful and provided practical guidance for disease management, showing that control targeted at adult males could reduce intergroup disease transmission, hence providing an approach for the control of zoonotic and livestock diseases.

Weaknesses:

Despite their reliability, populations can be skewed by small sample sizes and limited generalizability due to specific environmental and demographic characteristics. Further validation is needed to account for additional environmental factors influencing social dynamics and contact rates

Reviewer #3 (Public review):

Summary:

The authors describe an exciting 400-drug screening using a MMV pathogen box to select compounds that effectively affect the medically important Toxoplasma parasite bradyzoite stage. This work utilises a bradyzoites culture technique that was published recently by the same group. They focused on compounds that affected directly the mitochondria electron transport chain (mETC) bc1-complex and compared them with other bc1 inhibitors described in the literature such as atovaquone and HDQs. They further provide metabolomics analysis of inhibited parasites which serves to provide support for the target and to characterise the outcome of the different inhibitors.

Strengths:

This work is important as, until now, there are no effective drugs that clear cysts during T. gondii infection. So, the discovery of new inhibitors that are effective against this parasite stage in culture and thus have the potential to battle chronic infection is needed. The further metabolic characterization provides indirect target validation and highlights different metabolic outcomes for different inhibitors. The latter forms the basis for new studies in the field to understand the mode of inhibition and mechanism of bc1-complex function in detail.

The authors focused on the function of one compound, MMV1028806, that is demonstrated to have a similar metabolic outcome to burvaquone. Furthermore, the authors evaluated the importance of ATP production in tachyzoite and bradyzoites stages and under atovaquone/HDQs drugs.

Weaknesses:

Although the authors did experiments to identify the metabolomic profile of the compounds and suggested bc-1 complex as the main target of MMV1028806, they did not provide experimental validation for that.

Reviewer #3 (Public review):

Summary:

In this study, Wang et al utilized the available GTEx data to compile a comprehensive analysis that attempt to reveal aging-related sex-dimorphic gene expression as well as alternative splicing changes in humans.

The key conclusions based on their analysis are that

(1) extensive sex-dimorphisms during aging with distinct patterns of change in gene expression and alternative splicing (AS), and

(2) the male-biased age-associated AS events have a stronger association with Alzheimer's disease, and

(3) the female-biased events are often regulated by several sex-biased splicing factors that may be controlled by estrogen receptors. They further performed break-point analysis and revealed that in males there are two main breakpoints around ages 35 and 50, while in females, there is only one breakpoint at 45.

Strengths:

This study sets an ambitious goal, leveraging the extensive GTEx dataset to investigate aging-related, sex-dimorphic gene expression and alternative splicing changes in humans. The research addresses a significant question, as our understanding of sex-dimorphic gene expression in the context of human aging is still in its early stages. Advancing our knowledge of these molecular changes is vital for identifying therapeutic targets for age-related diseases and extending the human health span. The study is highly comprehensive, and the authors are commendable for their attempted thorough analysis of both gene expression and alternative splicing - an area often overlooked in similar studies.

Weaknesses:

Due to the inherent noise within the GTEx dataset - which includes numerous variables beyond aging and sex - there are significant technical concerns surrounding this study. Additionally, the lack of cross-validation with independent, existing data raises questions about whether the observed gene expression changes genuinely reflect those associated with human aging. For instance, the break-point analysis in this study identifies two major breakpoints in males around ages 35 and 50, and one breakpoint in females at age 45; however, these findings contradict a recent multi-omics longitudinal study involving 108 participants aged 25 to 75 years, where breakpoint at 44 and 60 years was observed in both male and females (Shen et al, 2024). These issues cast doubt on the robustness of the study's conclusions. Specific concerns are outlined below:

(1) The primary method used in this study is linear regression, incorporating age, sex, and age-by-sex interactions as covariates, alongside other confounding factors (such as ethnicity) as unknown variables. However, the analysis overlooks two critical known variables in the GTEx dataset: time of death (TOD) and postmortem interval (PMI). Both TOD and PMI are recorded for each sample and account for substantial variance in gene expression profiles. A recent study by Wucher et al.(Wucher et al, 2023) demonstrated the powerful impact of TOD on gene expression by using it to reconstruct human circadian and even circannual datasets. Similarly, Ferreira et al. (Ferreira et al, 2018) highlighted PMI's influence on gene expression patterns. Without properly adjusting for these two variables, confidence in the study's conclusions remains limited at best.

(2) To demonstrate that their analysis is robust and that the covariates TOD and PMI are otherwise negligible - the authors should cross-validate their findings with independent datasets to confirm that the identified gene expression changes are reproducible for some tissues. For instance, the recent study by Shen et al. (Shen et al., 2024) in Nature Aging offers an excellent dataset for cross-validation, particularly for blood samples. Comparing the GTEx-derived results with this longitudinal transcriptome dataset would enable verification of gene expression changes at both the individual gene and pathway levels. Without such validation, confidence in the study's conclusions remains limited.

(3) As a demonstration of the lack of such validation, in the Shen et al. study (Shen et al., 2024), breakpoints at 44 and 60 years were observed in both males and females, while this study identifies two major breakpoints in males around ages 35 and 50, and one breakpoint in females at age 45. What caused this discrepancy?

(4) Although the alternative splicing analysis is intriguing, the authors did not differentiate between splicing events that alter the protein-coding sequence and those that do not. Many splicing changes occurring in the 5' UTR and 3' UTR regions do not impact protein coding, so it is essential to filter these out and focus specifically on alternative splicing events that can modify protein-coding sequences.

(5) One of the study's main conclusions - that "male-biased age-associated AS events have a stronger association with Alzheimer's disease" - is not supported by the data presented in Figure 4A, which shows an association with "regulation of amyloid precursor formation" only in female, not male, alternative splicing genes. Additionally, the gene ontology term "Alzheimer's disease" is absent from the unbiased GO analysis in Figure S6. These discrepancies suggest that the focus on Alzheimer's disease may reflect selective data interpretation rather than results driven by an unbiased analysis.

(6) The experimental data presented in Figures 5E - I merely demonstrate that estrogen receptor regulates the expression of two splicing factors, SRSF1 and SRSF7, in an estradiol-dependent manner. However, this finding does not support the notion that this regulation actually contributes to sex-dimorphic alternative splicing changes during human aging. Notably, the authors do not provide evidence that SRSF1 and SRSF7 expression changes actually occur in a sex-dependent manner with human aging (in a manner similar to TIA1). As such, this experimental dataset is disconnected from the main focus of the study and does not substantiate the conclusions on sex-dimorphic splicing during human aging. The authors performed RNA-seq in wild-type and ER mutant cells, and they should perform a comprehensive analysis of ER-dependent alternative splicing and compare the results with the GTEx data. It should be straightforward.

References:

Ferreira PG, Muñoz-Aguirre M, Reverter F, Sá Godinho CP, Sousa A, Amadoz A, Sodaei R, Hidalgo MR, Pervouchine D, Carbonell-Caballero J et al (2018) The effects of death and post-mortem cold ischemia on human tissue transcriptomes. Nature Communications 9: 490.

Shen X, Wang C, Zhou X, Zhou W, Hornburg D, Wu S, Snyder MP (2024) Nonlinear dynamics of multi-omics profiles during human aging. Nature Aging.

Wucher V, Sodaei R, Amador R, Irimia M, Guigó R (2023) Day-night and seasonal variation of human gene expression across tissues. PLOS Biology 21: e3001986.

endomorph body type = good weight gainers = bad weight losers.<br /> thats the whole secret of the "fat acceptance" tragedy / comedy.

Reviewer #3 (Public review):

Summary:

Bos et al study a computational model of cortical circuits with excitatory (E) and two subtypes of inhibition - parvalbumin (PV) and somatostatin (SOM) expressing interneurons. They perform stability and gain analysis of simplified models with nonlinear transfer functions when SOM neurons are perturbed. Their analysis suggests that in a specific setup of connectivity, instability and gain can be untangled, such that SOM modulation leads to both increases in stability and gain, in contrast to the typical direction in neuronal networks where increased gain results in decreased stability.

Strengths:

- Analysis of the canonical circuit in response to SOM perturbations. Through numerical simulations and mathematical analysis, the authors have provided a rather comprehensive picture of how SOM modulation may affect response changes.<br /> - Shedding light on two opposing circuit motifs involved in the canonical E-PV-SOM circuitry - namely, direct inhibition (SOM -> E) vs disinhibition (SOM -> PV -> E). These two pathways can lead to opposing effects, and it is often difficult to predict which one results from modulating SOM neurons. In simplified circuits, the authors show how these two motifs can emerge and depend on parameters like connection weights.<br /> - Suggesting potentially interesting consequences for cortical computation. The authors suggest that certain regimes of connectivity may lead to untangling of stability and gain, such that increases in network gain are not compromised by decreasing stability. They also link SOM modulation in different connectivity regimes to versatile computations in visual processing in simple models.

Weaknesses

Computationally, the analysis is solid, but it's very similar to previous studies (del Molino et al, 2017). Many studies in the past few years have done the perturbation analysis of a similar circuitry with or without nonlinear transfer functions (some of them listed in the references). This study applies the same framework to SOM perturbations, which is a useful computational analysis, in view of the complexity of the high-dimensional parameter space.

Link to biology: the most interesting result of the paper with regard to biology is the suggestion of a regime in which gain and stability can be modulated in an unconventional way - however, it is difficult to link the results to biological networks:<br /> - A general weakness of the paper is a lack of direct comparison to biological parameters or experiments. How different experiments can be reconciled by the results obtained here, and what new circuit mechanisms can be revealed? In its current form, the paper reads as a general suggestion that different combinations of gain modulation and stability can be achieved in a circuit model equipped with many parameters (12 parameters). This is potentially interesting but not surprising, given the high dimensional space of possible dynamical properties. A more interesting result would have been to relate this to biology, by providing reasoning why it might be relevant to certain circuits (and not others), or to provide some predictions or postdictions, which are currently missing in the manuscript.<br /> - For instance, a nice motivation for the paper at the beginning of the Results section is the different results of SOM modulation in different experiments - especially between L23 (inhibition) and L4 (disinhibition). But no further explanation is provided for why such a difference should exist, in view of their results and the insights obtained from their suggested circuit mechanisms. How the parameters identified for the two regimes correspond to different properties of different layers?<br /> - One of the key assumptions of the model is nonlinear transfer functions for all neuron types. In terms of modelling and computational analysis, a thorough analysis of how and when this is necessary is missing (an analysis similar to what has been attempted in Figure 6 for synaptic weights, but for cellular gains). A discussion of this, along with the former analysis to know which nonlinearities would be necessary for the results, is needed, but currently missing from the study. The nonlinearity is assumed for all subtypes because it seems to be needed to obtain the results, but it's not clear how the model would behave in the presence or absence of them, and whether they are relevant to biological networks with inhibitory transfer functions.<br /> - Tuning curves are simulated for an individual orientation (same for all), not considering the heterogeneity of neuronal networks with multiple orientation selectivity (and other visual features) - making the model too simplistic.

Reviewer #3 (Public review):

Summary:

The authors report a novel, direct interaction of Spt6p tSH2 domain to Tom1p. This extends the function of Spt6p from communication with factors associated with RNAPII transcription to processes of ubiquitination. Tom1p is known to ubiquitinate a large variety of substrates, but it is unknown how substrate recognition is done in a specific manner. The team identified a conserved central acidic region of Tom1p which is essential for in vivo functions and binds to histones and nucleosomes, as well as Spt6p. They further describe the Tom1p occupancy pattern on chromatin, assigning it a stabilizing effect on nucleosomes near promotors and a destabilizing effect on nucleosomes within the gene bodies. The authors were able to resolve two different conformational states of Tom1p which are likely connected to its activity, and possibly substrate selectivity.

Overall, the authors show that an intrinsically disordered region in Tom1p is important for substrate interaction and function of Tom1p. The protein is further involved in chromatin architecture and structural transitions control its activity.

Strengths:

By revealing the interaction of Spt6p and Tom1p, the authors discover a novel connection between transcriptional elongation and processes of ubiquitination.<br /> In recent years, disordered regions of MDa protein complexes have become a focus of research projects. The effects of disordered regions on protein localization and specificity of binding interactions have been discussed in great extent, including proteins that are involved in chromatin remodeling and transcription. Adding to these current efforts, the authors assign a function to a highly conserved disordered region of Tom1p in technically clean experiments. Furthermore, with their data, they pin down a specific functional region in Tom1p which is relevant for the previously observed temperature sensitivity caused by Tom1p deletion in yeast.<br /> The team performs a thorough and complete analysis of the cryo-EM structure and they nicely model the hinge motion and details of an open and closed conformation.

Weaknesses:

Despite the high number of interesting findings, there is little connection between the individual sections of the manuscript. For example, many experiments are not related to Spt6p binding although this protein is presented as a major actor in this manuscript during the introduction. Furthermore, the structural analysis is well done, but it is also not quite clear how structural rearrangements are connected to Spt6 binding or chromatin remodeling. Some experimental results lack novelty, as similar data has previously been presented for the human homolog.

To confirm the novel, direct binding interaction of Spt6p and Tom1p, no orthogonal binding assays (SPR, MST, ITC) have been performed to confirm the interaction. To me, this is insufficient, especially since the team has purified both proteins to high quality levels, or could use peptides to test the function of the relevant regions.<br /> Additionally, interaction of Tom1p with Spt6p in the context of transcription elongation is proposed. Yet it is not clear on the mechanistic level how this is regulated if Tom1p and Rpb1p bind in a competitive manner. How is Tom1p tethered to the elongation complex if not through Spt6p? In addition to WT vs. knockout, the authors should further perform the genetic analyses with the intΔ11 mutant. This way they might be able pin down which interactions on chromatin are mediated by Spt6 vs. by other factors and could strengthen the overall model involving Spt6P.

Although the authors try to describe a final model in the discussion, this section is not easy to follow and needs more explanation, ideally drawn as a Figure of the proposed mechanism.

Reviewer #3 (Public review):

This paper discusses how non-sensory and latent, sensory-like attentional templates are represented during attentional preparation. Using multivariate pattern analysis, they found that visual impulses can enhance the decoding generalization from perception to attention tasks in the preparatory stage in the visual cortex. Furthermore, the emergence of the sensory-like template coincided with enhanced information connectivity between V1 and frontoparietal areas and was associated with improved behavioral performance. It is an interesting paper with supporting evidence for the latent, sensory-like attentional template, but several problems still need to be solved.

(1) The title is "Dual-format Attentional Template," yet the supporting evidence for the non-sensory format and its guiding function is quite weak. The author could consider conducting further generalization analysis from stimulus selection to preparation stages to explore whether additional information emerges.

(2) In Figure 2, the author did not find any decodable sensory-like coding in IPS and PFC, even during the impulse-driven session, indicating that these regions do not represent sensory-like information. However, in the final section, the author claimed that the impulse-driven sensory-like template strengthens informational connectivity between sensory and frontoparietal areas. This raises a question: how can we reconcile the lack of decodable coding in these frontoparietal regions with the reported enhancement in network communication? It would be helpful if the author provided a clearer explanation or additional evidence to bridge this gap.

(3) Given that the impulse-driven sensory-like template facilitated behavior, the author proposed that it might also enhance network communication. Indeed, they observed changes in informational connectivity. However, it remains unclear whether these changes in network communication have a direct and robust relationship with behavioral improvements.

(4) I'm uncertain about the definition of the sensory-like template in this paper. Is it referring to the Ping impulse-driven condition or the decodable performance in the early visual cortex? If it is the former, even in working memory, whether pinging identifies an activity-silent mechanism is currently debated. If it's the latter, the authors should consider whether a causal relationship - such as "activating the sensory-like template strengthens the informational connectivity between sensory and frontoparietal areas" - is reasonable.

Reviewer #3 (Public review):

Summary:

Cryptococcus neoformans is a global critical threat pathogen and the manuscript by Mota et al demonstrates that the pathogen's N-glycan-dependent protein quality control system regulates the capacity of the fungus to cause disease. The system makes sure that glycoproteins are folded correctly. The system is involved in the fitness and virulence of the fungus by regulating aspects of cellular robustness and the trafficking of virulence-associated compounds outside of the cell via transport in extracellular vesicles.

Strengths:

The investigators use multiple modalities to demonstrate that the system is involved in cryptococcal pathogenesis. The investigators generated mutant C. neoformans to explore the role of genes involved in the protein folding system. Basic microbiology, genetic analyses, proteomics, fluorescence and transmission microscopy, nanotracking analyses, and murine studies were performed. The validity of the findings are thus very high. Hypotheses are robustly demonstrated.

Weaknesses:

Aspects of the results should be better explained. Some results are extrapolated in their meaning beyond the extent of the data.

Reviewer #3 (Public review):

Summary:

Here Wengert et al., establish a rodent model of KCNC1 (Kv3.1) epilepsy by introducing the A421V mutation. The authors perform video-EEG, slice electrophysiology, and in vivo 2P imaging of calcium activity to establish disease mechanisms involving impairment in the excitability of fast-spiking parvalbumin (PV) interneurons in the cortex and thalamic PV cells.

Outside-out nucleated patch recordings were used to evaluate the biophysical consequence of the A421V mutation on potassium currents and showed a clear reduction in potassium currents. Similarly, action potential generation in cortical PV interneurons was severely reduced. Given that both potassium currents and action potential generation were found to be unaffected in excitatory pyramidal cells in the cortex the authors propose that loss of inhibition leads to hyperexcitability and seizure susceptibility in a mechanism similar to that of Dravet Syndrome.

Strengths:

This manuscript establishes a new rodent model of KCNC1-developmental and epileptic encephalopathy. The manuscript provides strong evidence that parvabumin-type interneurons are impaired by the A421V Kv3.1 mutation and that cortical excitatory neurons are not impaired. Together these findings support the conclusion that seizure phenotypes are caused by reduced cortical inhibition.

Weaknesses:

The manuscript identifies a partial mechanism of disease that leaves several aspects unresolved including the possible role of the observed impairments in thalamic neurons in the seizure mechanism. Similarly, while the authors identify a reduction in potassium currents and a reduction in PV cell surface expression of Kv3.1 it is not clear why these impairments would lead to a more severe disease phenotype than other loss-of-function mutations which have been characterized previously. Lastly, additional analysis of video-EEG data would be helpful for interpreting the extent of the seizure burden and the nature of the seizure types caused by the mutation.

Reviewer #3 (Public review):

Summary:

The obligate intracellular bacterium Chlamydia trachomatis (Ct) divides by binary fission. It lacks FtsZ, but still has many other proteins that regulate the synthesis of septal peptidoglycan, including FtsW and FtsI (PBP3) as well as divisome proteins that recruit and activate them, such as FtsK and FtsQLB. Interestingly, MreB is also required for the division of Ct cells, perhaps by polymerizing to form an FtsZ-like scaffold. Here, Harpring et al. show that MreB does not act early in division and instead is recruited to a protein complex that includes FtsK and PBP2/PBP3. This indicates that Ct cell division is organized by a chimera between conserved divisome and elongasome proteins. Their work also shows convincingly that FtsK is the earliest known step of divisome activity, potentially nucleating the divisome as a single protein complex at the future division site. This is reminiscent of the activity of FtsZ, yet fundamentally different.

Strengths:

The study is very well written and presented, and the data are convincing and rigorous. The data underlying the proposed localization dependency order of the various proteins for cell division is well justified by several different approaches using small molecule inhibitors, knockdowns, and fluorescent protein fusions. The proposed dependency pathway of divisome assembly is consistent with the data and with a novel mechanism for MreB in septum synthesis in Ct.

Weaknesses:

The paper could be improved by including more information about FtsK, the "focus" of this study. For example, if FtsK really is the FtsZ-like nucleator of the Ct divisome, how is the Ct FtsK different sequence-wise or structurally from FtsK of, e.g. E. coli? Is the N-terminal part of FtsK sufficient for cell division in Ct like it is in E. coli, or is the DNA translocase also involved in focus formation or localization? Addressing those questions would put the proposed initiator role of FtsK in Ct in a better context and make the conclusions more attractive to a wider readership.

Another weakness is that the title of the paper implies that FtsK alone initiates divisome assembly. However, the data indicate only that FtsK is important at an early stage of divisome assembly, not that it is THE initiator. I suggest modifying the title to account for this--perhaps "FtsK is required to initiate....".

Reviewer #3 (Public review):

Summary:

This manuscript by Duan and Song interrogates the gating mechanisms and specifically force transmission in mechanosensitive NOMPC channels using steered molecular dynamics simulations. They propose that the ankyrin spring can transmit force to the gate through torsional forces adding molecular detail to the force transduction pathways in this channel.

3. Constant velocity or constant force<br /> For the SMD the authors write "and a constant velocity or constant force". It's unclear from this reviewer's perspective what is used to generate the simulation data.

Strengths:

Detailed, rigorous simulations coupled with a novel model for force transduction.

Weaknesses:

Experimental validation of reduced mechanosensitivity through mutagenesis of proposed ankyrin/TRP domain coupling interactions would greatly enhance the manuscript. I have some additional questions documented below:

(1) The membrane-parallel torsion force can open NOMPC<br /> How does the TRP domain interact with the S4-S5 linker? In the original structural studies, the coordination of lipids in this region seems important for gating. In this manner does the TRP domain and S4-S5 linker combined act like an amphipathic helix as suggested first for MscL (Bavi et al., 2016 Nature Communications) and later identified in many MS channels (Kefauver et al., 2020 Nature).

(2) Torsional forces on shorter ankyrin repeats of mammalian TRP channels<br /> Is it possible torsional forces applied to the shorter ankyrin repeats of mammalian TRPs may also convey force in a similar manner?

(3) Constant velocity or constant force<br /> For the SMD the authors write "and a constant velocity or constant force". It's unclear from this reviewer's perspective which is used to generate the simulation data.

Reviewer #3 (Public review):

Summary:

The paper begins by analyzing the drift in individual behavior over time. Specifically, it quantifies the circling direction of freely walking flies in an arena. The main takeaway from this dataset is that while flies exhibit an individual turning bias (when averaged over time), their preferences fluctuate over slow timescales.

To understand whether genetic or neuromodulatory mechanisms influence the drift in individual preference, the authors test different fly strains concluding that both genetic background and the neuromodulator serotonin contribute to the degree of drift.

Finally, the authors use theoretical approaches to identify the range of environmental conditions under which drift in individual bias supports population growth.

Strengths:

The model provides a clear prediction of the environmental fluctuations under which a drift in bias should be beneficial for population growth.

The approach attempts to identify genetic and neurophysiological mechanisms underlying drift in bias.

Weaknesses:

Different behavioral assays are used and are differently analysed, with little discussion on how these behaviors and analyses compare to each other.

Some of the model assumptions should be made more explicit to better understand which aspects of the behaviors are covered.

Reviewer #3 (Public review):

Summary:

The paper by Zhu et al is on an important topic in visual neuroscience, the emergence in the visual cortex of signals about figures and ground. This topic also goes by the name border ownership. The paper utilizes modern recording techniques very skillfully to extend what is known about border ownership. It offers new evidence about the prevalence of border ownership signals across different cortical layers in V1 cortex. Also, it uses pairwise cross-correlation to study signal flow under different conditions of visual stimulation that include the border ownership paradigm.

Strengths:

The paper's strengths are its use of multi-electrode probes to study border ownership in many neurons simultaneously across the cortical layers in V1, and its innovation of using cross-correlation between cortical neurons -- when they are viewing border-ownership patterns or instead are viewing grating patterns restricted to the classical receptive field (CRF).

Weaknesses:

The paper's weaknesses are its largely incremental approach to the study of border ownership and the lack of a critical analysis of the cross-correlation data. The paper as it is now does not advance our understanding of border ownership; it mainly confirms prior work, and it does not challenge or revise consensus beliefs about mechanisms. However, it is possible that, in the rich dataset the authors have obtained, they do possess data that could be added to the paper to make it much stronger.

Critique:

The border ownership data on V1 offered in the paper replicates experimental results obtained by Zhou and von der Heydt (2000) and confirms the earlier results using the same analysis methods as Zhou. The incremental addition is that the authors found border ownership in all cortical layers extending Zhou's results that were only about layer 2/3.

The cross-correlation results show that the pattern of the cross-correlogram (CCG) is influenced by the visual pattern being presented. However, the results are not analyzed mechanistically, and the interpretation is unclear. For instance, the authors show in Figure 3 (and in Figure S2) that the peak of the CCG can indicate layer 2/3 excites layer 4C when the visual stimulus is the border ownership test pattern, a large square 8 deg on a side. But how can layer 2/3 excite layer 4C? The authors do not raise or offer an answer to this question. Similar questions arise when considering the CCG of layer 4A/B with layer 2/3. What is the proposed pathway for layer 2/3 to excite 4A/B? Other similar questions arise for all the interlaminar CCG data that are presented. What known functional connections would account for the measured CCGs?

The problems in understanding the CCG data are indirectly caused by the lack of a critical analysis of what is happening in the responses that reveal the border ownership signals, as in Figure 2. Let's put it bluntly - are border ownership signals excitatory or inhibitory? The reason I raise this question is that the present authors insightfully place border ownership as examples of the action of the non-classical receptive field (nCRF) of cortical cells. Most previous work on the nCRF (many papers cited by the authors) reveal the nCRF to be inhibitory or suppressive. In order to know whether nCRF signals are excitatory or inhibitory, one needs a baseline response from the CRF, so that when you introduce nCRF signals you can tell whether the change with respect to the CRF is up or down. As far as I know, prior work on border ownership has not addressed this question, and the present paper doesn't either. This is where the rich dataset that the present authors possess might be used to establish a fundamental property of border ownership.

Then we must go back to consider what the consequences of knowing the sign of the border ownership signal would mean for interpreting the CCG data. If the border ownership signals from extrastriate feedback or, alternatively, from horizontal intrinsic connections, are excitatory, they might provide a shared excitatory input to pairs of cells that would show up in the CCG as a peak at 0 delay. However, if the border ownership manuscript signals are inhibitory, they might work by exciting only inhibitory neurons in V1. This could have complicated consequences for the CCG. The interpretation of the CCG data in the present version of the m is unclear (see above). Perhaps a clearer interpretation could be developed once the authors know better what the border ownership signals are.

My critique of the CCG analysis applies to Figure 5 also. I cannot comprehend the point of showing a very weak correlation of CCG asymmetry with Border Ownership Index, especially when what CCG asymmetry means is unclear mechanistically. Figure 5 does not make the paper stronger in my opinion.

In Figure 3, the authors show two CCGs that involve 4C--4C pairs. It would be nice to know more about such pairs. If there are any 6--6 pairs, what they look like also would be interesting. The authors also in Figure 3 show CCG's of two 4C--4A/B pairs and it would be quite interesting to know how such CCGs behave when CRF and nCRF stimuli are compared. In other words, the authors have shown us they have many data but have chosen not to analyze them further or to explain why they chose not to analyze them. It might help the paper if the authors would present all the CCG types they have. This suggestion would be helpful when the authors know more about the sign of border ownership signals, as discussed at length above.

Reviewer #3 (Public review):

Summary:

Fahrenfort et al. investigate how liberal or conservative criterion placement in a detection task affects the construct validity of neural measures of unconscious cognition and conscious processing. Participants identified instances of "seen" or "unseen" in a detection task, a method known as post hoc sorting. Simulation data convincingly demonstrate that, counterintuitively, a conservative criterion inflates effect sizes of neural measures compared to a liberal criterion. While the impact of criterion shifts on effect size is suggested by signal detection theory, this study is the first to address this explicitly within the consciousness literature. Decoding analysis of data from two EEG experiments further shows that different criteria lead to differential effects on classifier performance in post hoc sorting. The findings underscore the pervasive influence of experimental design and participants report on neural measures of consciousness, revealing that criterion placement poses a critical challenge for researchers.

Strengths and Weaknesses:

One of the strengths of this study is the inclusion of the Perceptual Awareness Scale (PAS), which allows participants to provide more nuanced responses regarding their perceptual experiences. This approach ensures that responses at the lowest awareness level (selection 0) are made only when trials are genuinely unseen. This methodological choice is important as it helps prevent the overestimation of unconscious processing, enhancing the validity of the findings.

A potential area for improvement in this study is the use of single time-points from peak decoding accuracy to generate current source density topography maps. While we recognize that the decoding analysis employed here differs from traditional ERP approaches, the robustness of the findings could be enhanced by exploring current source density over relevant time windows. Event-related peaks, both in terms of timing and amplitude, can sometimes be influenced by noise or variability in trial-averaged EEG data, and a time-window analysis might provide a more comprehensive and stable representation of the underlying neural dynamics.

It is helpful that the authors show the standard error of the mean for the classifier performance over time. A similar indication of a measure of variance in other figures could improve clarity and transparency.<br /> That said, the paper appears solid regarding technical issues overall. The authors also do a commendable job in the discussion by addressing alternative paradigms, such as wagering paradigms, as a possible remedy to the criterion problem (Peters & Lau, 2015; Dienes & Seth, 2010). Their consideration of these alternatives provides a balanced view and strengthens the overall discussion.

Impact of the Work:

This study effectively demonstrates a phenomenon that has been largely unexplored within the consciousness literature. Subjective measures may not reliably capture the construct they aim to measure due to criterion confounds. Future research on neural measures of consciousness should account for this issue, and no-report measures may be necessary until the criterion problem is resolved.

These call records do not fully support Adnan's defense, nor do they clearly prove his whereabouts at the time of the crime. In fact, there are some ambiguous parts in these records, which lead her to question whether the investigation may have missed some crucial clues or, whether intentionally or unintentionally, misled the progress of the case.

I thought that in this conversation, Sarah would believe Adnan is more innocent, but instead, she seems to lose more confidence in him. Why is that?

In this segment, Sarah Koenig raises an intriguing question about the nature of investigative work: "Are you only working with cases where you're pretty sure from the get-go that the person is guilty or innocent?" This line touches on the ethical and practical complexities of investigative journalism. It challenges the assumption that journalists or detectives can be certain from the start about someone's guilt or innocence, emphasizing the uncertainty and nuances that often come with real-life cases.

Reviewer #3 (Public review):

Summary:

The paper by Li et al explored the role of Estrogen receptor 1 (Esr1) expressing neurons in the pontine micturition center (PMC), a brainstem region also known as Barrington's nucleus (Hou et al 2016, Keller et al 2018). First, the author conducted bulk Ca2+ imaging/unit recording from PMCESR1 to investigate the correlations of PMCESR1 neural activity to voiding behavior in conscious mice and bladder pressure/external urethral muscle activity in urethane anesthetized mice. Next, the authors conducted optogenetics inactivation/activation of PMCESR1 to confirm the contribution to the voiding behavior also conducted peripheral nerve transection together with optogenetics activation to confirm the independent control of bladder pressure and urethral sphincter muscle.

Weaknesses:

(1) The study demonstrates that pelvic nerve transection reduces urinary volume triggered by PMCESR1+ cell photoactivation in freely moving mice. Could the role of pudendal nerve transection also be examined in awake mice to provide a more comprehensive understanding of neural involvement?

(2) While the paper primarily focuses on PMCESR1+ cells in bladder-sphincter coordination, the analysis of PMCESR1+-DGC/SPN neural circuits - given their distinct anatomical projections in the sacral spinal cord - feels underexplored. How do these circuits influence bladder and sphincter function when activated or inhibited? Also, do you have any tracing data to confirm whether bladder-sphincter innervation comes from distinct spinal nuclei?

(3) Although the paper successfully identifies the physiological role of PMCESR1+ cells in bladder-sphincter coordination, the study falls short in examining the electrophysiological properties of PMCESR1+-DGC/SPN cells. A deeper investigation here would strengthen the findings.

(4) The parameters for photoactivation (blue light pulses delivered at 25 Hz for 15 ms, every 30 s) and photoinhibition (pulses at 50 Hz for 20 ms) vary. What drove the selection of these specific parameters? Moreover, for photoactivation experiments, the change in pressure (ΔP = P5 sec - P0 sec) is calculated differently from photoinhibition (Δpressure = Ppeak - Pmin). Can you clarify the reasoning behind these differing approaches?

(5) The discussion could further emphasize how PMCESR1+ cells coordinate bladder contraction and sphincter relaxation to control urination, highlighting their central role in the initiation and suspension of this process.

(6) In Figure 8, The authors analyze the temporal sequence of bladder pressure and EUS bursting during natural voiding and PMC activation-induced voiding. It would be acceptable to consider the existence of a lower spinal reflex circuit, however, the interpretation of the data contains speculation. Bladder pressure measurement is hard to say reflecting efferent pelvic nerve activity in real time. (As a biological system, bladder contraction is mediated by smooth muscle, and does not reflect real-time efferent pelvic nerve activity. As an experimental set-up, bladder pressure measurement has some delays to reflect bladder pressure because of tubing, but EUS bursting has no delay.) Especially for the inactivation experiment, these factors would contribute to the interpretation of data. This reviewer recommends a rewrite of the section considering these limitations. Most of the section is suitable for the results.

Reviewer #3 (Public Review):

Summary:

The article is an interesting approach to determining the MPOX receptor using "in silico" tools. The results show the presence of two regions of the H3 protein with a high probability of being involved in the interaction with the HS cell receptor. However, the α-helical region seems to be the most probable, since modifications in this region affect the virus binding to the HS receptor.

Strengths:

In my opinion, it is an informative article with interesting results, generated by a combination of "in silico" and wet science to test the theoretical results. This is a strong point of the article.

Comments on revised version:

After a review of the changes to the manuscript and the author's responses, no further changes are needed.

Reviewer #3 (Public review):

Summary:

In this manuscript, Hirai et al investigated the release properties of glutamate/GABA co-transmission at SuM-GC synapses and reported that glutamate/GABA co-transmission exhibits distinct short-term plasticity with segregated postsynaptic targets. Using optogenetics, whole-cell patch-clamp recordings, and immunohistochemistry, the authors reveal distinct transmission modes of glutamate/GABA co-release as frequency-dependent filters of incoming SuM inputs.

Strengths:

Overall, this study is well-designed and executed; conclusions are supported by the results. This study addressed a long-standing question of whether GABA and glutamate are packaged in the same vesicles and co-released in response to the same stimuli in the SuM-GC synapses (Pedersen et al., 2017; Hashimotodani et al., 2018; Billwiller et al., 2020; Chen et al., 2020; Li et al., 2020; Ajibola et al., 2021). Knowledge gained from this study advances our understanding of neurotransmitter co-release mechanisms and their functional roles in the hippocampal circuits.

Comments on revisions:

The authors have addressed my comments, and now the manuscript is in a good form as it currently stands.

Reviewer #3 (Public review):

Summary:

In the present work, Zhang et al investigate involvement of the bacterial DNA damage repair SOS response in the evolution of beta-lactam drug resistance evolution in Escherichia coli. Using a combination of microbiological, bacterial genetics, laboratory evolution, next-generation, and live-cell imaging approaches, the authors propose short-term (transient) drug resistance evolution can take place in RecA-deficient cells in an SOS response-independent manner. They propose the evolvability of drug resistance is alternatively driven by the oxidative stress imposed by accumulation of reactive oxygen species and compromised DNA repair. Overall, this is a nice study that addresses a growing and fundamental global health challenge (antimicrobial resistance).

Strengths:

The authors introduce new concepts to antimicrobial resistance evolution mechanisms. They show short-term exposure to beta-lactams can induce durably fixed antimicrobial resistance mutations. They propose this is due to comprised DNA repair and oxidative stress. Antibiotic resistance evolution under transient stress is poorly studied, so the authors' work is a nice mechanistic contribution to this field.

Weaknesses:

The authors do not show any direct evidence of altered mutation rate or accumulated DNA damage in their model.

for - examples - climate leader hypocrisy - 3 examples - provided by Kevin Anderson

for - climate crisis - IPCC - working group 3 - Oil companies delay action through fake Carbon Dioxide Removal (CDR) technologies - We need to act before 2030 - CDR technologies create inaction now when we most need it - Kevin Anderson

for - climate crisis - IPCC - warning - working group 3 - integrated assessment models - are basically economic models - with a bit of mythical technology - a bit of social science - Kevin Anderson

for - climate crisis - IPCC - warning - working group 3 - integrated assessment models - is just about reducing emissions - inherently political - Kevin Anderson

for - climate crisis - IPCC - warning - working group 3 - Integrated Assessment Models - Some good people here but - It's just Exxon in disguise - Kevin Anderson

Reviewer #3 (Public review):

Summary:

In this manuscript, Costa and colleagues investigate how asymmetry in dorsal root ganglion (DRG) neurons is established. The authors developed an in vitro system that mimics the pseudo-unipolar morphology and asymmetry of DRG neurons during the regeneration of the peripheral and central branch axons. They suggest that central-like DRG axons exhibit a higher density of growing microtubules. By reducing the polymerization of microtubules in these central-like axons, they were able to eliminate the asymmetry in DRG neurons.

Strengths:

The authors point out a distinct microtubule-associated protein signature that differentiates between DRG neurons' central and peripheral axonal branches. Experimental results demonstrate that genetic deletion of spastin eliminated the differences in microtubule dynamics and axon regeneration between the central and peripheral branches.

Weaknesses:

While some of the data are compelling, experimental evidence only partially supports the main claims.

In its current form, the study is primarily descriptive and lacks convincing mechanistic insights. It misses important controls and further validation using 3D in vitro models.

Given the heterogeneity of dorsal root ganglion (DRG) neurons, it is unclear whether the in vitro model described in this study can be applied to all major classes of DRG neurons. Also unclear is the inconsistency with embryonic DRG cultures with embryonic (E)16 from rats and E13 from mice (spastin knockout and wild-type controls). Furthermore, the authors stated (line 393) that only a small subset of cultured DRG neurons exhibited a pseudo-unipolar morphology. The authors should include the percentage of the neurons that exhibit a pseudo-unipolar morphology.

The significance of studying microtubule polymerization to DRG asymmetry in vitro is questionable, especially considering the model's validity. The authors might consider eliminating the in vitro data and instead focus on characterizing DRG asymmetry in vivo both before and after a conditioning lesion. If the authors choose to retain the in vitro data, classifying the central and peripheral-like branches in cultured DRG neurons will require further in-depth characterization. Additional validation should be performed in adult DRG neuron cultures not aged in vitro.

The comparison of asymmetry associated with a regenerative response between in vitro and in vivo paradigms has significant limitations due to the nature of the in vitro culture system. When cultured in isolation, DRG neurons fail to form functional connections with appropriate postsynaptic target neurons (the central branch) or to differentiate the peripheral domains associated with the innervation of target organs. Rather than growing neurons on a flat, hard surface like glass, more physiologically relevant substrates and/or culturing conditions should be considered. This approach could help eliminate potential artifacts caused by plating adult DRG neurons on a flat surface. Additionally, the authors should consider replicating their findings in a 3D culture model or using dorsal root ganglia explants, where both centrally and peripherally projecting axons are present.

Panels 5H-J require additional processing with astrocyte markers to accurately define the lesion borders. Furthermore, including a lower magnification would facilitate a direct comparison of the lesion site. The use of cholera toxin subunit B (CTB) to trace dorsal column sensory axons is prone to misinterpretation, as the tracer accumulates at the axon's tip. This limitation makes it extremely challenging to distinguish between regenerating and degenerating axons.

Reviewer #3 (Public review):

Summary:

A long noncoding RNA, lnc-FANCI-2, was reported to be regulated by HPV E7 oncoprotein and a cell transcription factor, YY1 by this group. The current study focuses on the function of lnc-FANCI-2 in HPV-16 positive cervical cancer is to intrinsically regulate RAS signaling, thereby facilitating our further understanding of additional cellular alterations during HPV oncogenesis. The authors used advanced technical approaches such as KO, transcriptome and (IRPCRP) and LC- MS/MS analyses in the current study and concluded that KO Inc-FANCI-2 significantly increases RAS signaling, especially phosphorylation of Akt and Erk1/2.

Strengths:

(1) HPV E6E7 are required for full immortalization and maintenance of the malignant phenotype of cervical cancer, but they are NOT sufficient for full transformation and tumorigenesis. This study helps further understanding of other cellular alterations in HPV oncogenesis.

(2) lnc-FANCI-2 is upregulated in cervical lesion progression from CIN1, CIN2-3 to cervical cancer, cancer cell lines, and HPV transduced cell lines.

(3) Viral E7 of high-risk HPVs and host transcription factor YY1 are two major factors promoting lnc-FANCI-2 expression.

(4) Proteomic profiling of cytosolic and secreted proteins showed inhibition of MCAM, PODXL2, and ECM1 and increased levels of ADAM8 and TIMP2 in KO cells.

(5) RNA-seq analyses revealed that KO cells exhibited significantly increased RAS signaling but decreased IFN pathways.

(6) Increased phosphorylated Akt and Erk1/2, IGFBP3, MCAM, VIM, and CCND2 (cyclin D2) and decreased RAC3 were observed in KO cells.

Weaknesses:

(1) The authors observed the increased Inc-FANCI-2 in HPV 16 and 18 transduced cells, and other cervical cancer tissues as well, HPV-18 positive HeLa cells exhibited different expressions of Inc-FANCI-2.

(2) Previous studies and data in the current showed a steadily increased Inc-FANCI-2 during cancer progression, however, the authors did not observe significant changes in cell behaviors (both morphology and proliferation) in KO Inc-FANCI-2.

(3) The authors observed the significant changes of RAS signaling (downstream) in KO cells, but they provided limited interpretations of how these results contributed to full transformation or tumorigenesis in HPV-positive cancer.

Reviewer #3 (Public review):

The paper from Liang and Guan details the calculation of the potential mean force for the transition between two key states of the melibiose (Mel) transporter MelB. The authors used the string method along with replica-exchange umbrella sampling to model the transition between the outward and inward-facing Mel-free states, including the binding and subsequent release of Mel. They find a barrier of ~6.8 kcal/mol and an overall free-energy difference of ~6.4 kcal/mol. They also investigate the same process without the co-transported Na+, finding a higher barrier, while in the D59C mutant, the barrier is nearly eliminated.

I found this to be an interesting and technically competent paper. I was disappointed actually to see that the authors didn't try to complete the cycle. I realize this is beyond the scope of the study as presented.

The results are in qualitative agreement with expectations from experiments. Could the authors try to make this comparison more quantitative? For example, by determining the diffusivity along the path, the authors could estimate transition rates.

Relatedly, could the authors comment on how typical concentration gradients of Mel and Na+ would affect these numbers?

Reviewer #3 (Public review):

This study aimed to investigate the impact of seasonality on the malaria parasite population genetic. To achieve this, the researchers conducted a longitudinal study in a region characterized by seasonal malaria transmission. Over a 2.5-year period, blood samples were collected from 1,516 participants residing in four villages in the Upper River Region of The Gambia and tested the samples for malaria parasite positivity. The parasites from the positive samples were genotyped using a genetic barcode and/or whole genome sequencing, followed by a genetic relatedness analysis.

The study identified three key findings:

(1) The parasite population continuously recombines, with no single genotype dominating, in contrast to viral populations;

(2) The relatedness of parasites is influenced by both spatial and temporal distances; and

(3) The lowest genetic relatedness among parasites occurs during the transition from low to high transmission seasons. The authors suggest that this latter finding reflects the increased recombination associated with sexual reproduction in mosquitoes.

The results section is well-structured, and the figures are clear and self-explanatory. The methods are adequately described, providing a solid foundation for the findings. While there are no unexpected results, it is reassuring to see the anticipated outcomes supported by actual data. The conclusions are generally well-supported; however, the discussion on the burden of asymptomatic infections falls outside the scope of the data, as no specific analysis was conducted on this aspect and was not stated as part of the aims of the study. Nonetheless, the recommendation to target asymptomatic infections is logical and relevant.

Reviewer #3 (Public review):

Summary:

The purpose of the current manuscript was to investigate a magnetic cell steering technique for efficiency and tissue-specific targeting, using two types of stem cells, in a mouse model of glaucoma. As the authors point out, trabecular meshwork (TM) cell therapy is an active area of research for treating elevated intraocular pressure as observed in glaucoma. Thus, further studies determining the ideal cell choice for TM cell therapy is warranted. The experimental protocol of the manuscript involved the injection of either human adipose derived mesenchymal stem cells (hAMSCs) or induced pluripotent cell derivatives (iPSC-TM cells) into a previously reported mouse glaucoma model, the transgenic MYOCY437H mice and wild-type littermates followed by the magnetic cell steering. Numerous outcome measures were assessed and quantified including IOP, outflow facility, TM cellularity, retention of stem cells, and the inner wall BM of Schlemm's canal.

Strengths:

All of these analyses were carefully carried out and appropriate statistical methods were employed. The study has clearly shown that the hAMSCs are the cells of choice over the iPSC-TM cells, the latter of which caused tumors in the anterior chamber. The hAMSCs were shown to be retained in the anterior segment over time and this resulted in increased cellular density in the TM region and a reduction in IOP and outflow facility. These are all interesting findings and there is substantial data to support it.

Weaknesses:

However, where the study falls short is in the MYOCY437H mouse model of glaucoma that was employed. The authors clearly state that a major limitation of the study is that this model, in their hands, did not exhibit glaucomatous features as previously reported, such as a significant increase in IOP, which was part of the overall purpose of the study. The authors state that it is possible that "the transgene was silenced in the original breeders". The authors did not show PCR, western blot, or immuno of angle tissue of the tg to determine transgenic expression (increased expression of MYOC was shown in the angle tissue of the transgenics in the original paper by Zode et al, 2011). This should be investigated given that these mice were rederived. Thus, it is clearly possible that these are not transgenic mice. If indeed they are transgenics, the authors may want to consider the fact that in the Zode paper, the most significant IOP elevation in the mutant mice was observed at night and thus this could be examined by the authors. Other glaucomatous features of these mice could also have been investigated such as loss of RGCs, to further determine their transgenic phenotype. Finally, while increased cellular density in the TM region was observed, proliferative markers could be employed to determine if the transplanted cells are proliferating.

Reviewer #3 (Public review):

Summary:

In this study, several novel class IIb microcin biosynthetic gene clusters have been discovered by specific homology searches and manual curation. Using a specific E. coli expression system, the microcins were expressed and conjugated to monoglycosylated enterobactin as siderophore moiety. While this synthetic biology approach cannot account for other siderophores being coupled to the microcin core peptide in the original producing strains, it nonetheless allows for a general screening for the activity of the heterologously produced compounds. Through this approach, the activity of several predicted microcins has been confirmed and three novel class IIb microcin clades were identified.

Strengths:

The experimental design is sound, the results are corroborated by suitable controls, and the findings have a high level of novelty and significance. Furthermore, the comments of the initial round of peer review have been answered satisfactorily by the authors.

Reviewer #3 (Public review):

This study attempted to investigate the relations between processing in the human brain during movie watching and corresponding thought processes. This is a highly interesting question, as movie watching presents a semi-constrained task, combining naturally occurring thoughts and common processing of sensory inputs across participants. This task is inherently difficult because in order to know what participants are thinking at any given moment, one has to interrupt the same thought process which is the object of study.

This study attempts to deal with this issue by aggregating staggered experience sampling data across participants in one behavioral study and using the population level thought patterns to model brain activity in different participants in an open access fMRI dataset.

The behavioral data consist of 120 participants who watched 3 11-minute movie clips. Participants responded to the mDES questionnaire: 16 visual scales characterizing ongoing thought 5 times, two minutes apart, in each clip. The 16 items are first reduced to 4 factors using PCA, and their levels are compared across the different movies. The factors are "episodic knowledge", "intrusive distraction", "verbal detail", and "sensory engagement". The factors differ between the clips, and distraction is negatively correlated with movie comprehension and sensory engagement is positively correlated with comprehension.

The components are aggregated across participants (transforming single subject mDES answers into PCA space and concatenating responses of different participants) and are used as regressors in a GLM analysis. This analysis identifies brain regions corresponding to the components. The resulting brain maps reveal activations that are consistent with the proposed mental processes (e.g. negative loading for intrusion in frontoparietal network, positive loadings for visual and auditory cortices for sensory engagement).

Then, the coordinates for brain regions which were significant for more than one component are entered into a paper search in neurosynth. It is not clear what this analysis demonstrates beyond the fact that sensory engagement contained both visual and auditory components.

The next analysis projected group-averaged brain activation onto gradients (based on previous work) and used gradient timecourses to predict the behavioral report timecourses. This revealed that high activations in gradient 1 (sensory→association) predicted high sensory engagement, and that "episodic knowledge" thought patterns were predicted by increased visual cortex activations. Then, permutation tests were performed to see whether these thought pattern related activations corresponded to well defined regions on a given cluster.

In conclusion, this study tackles a highly interesting subject and does it creatively and expertly.

Reviewer #3 (Public review):

Summary:

Individuals with Down syndrome (DS) have high rates of autoimmunity and can have exaggerated immune responses to infection that can unfortunately cause significant medical complications. Prior studies from these authors and others have convincingly demonstrated that individuals with DS have immune dysregulation including increased Type I IFN activity, elevated production of inflammatory cytokines (hypercytokinemia), increased autoantibodies, and populations of dysregulated adaptive immune cells that pre-dispose to autoimmunity. Prior studies have demonstrated that using JAK inhibitors to treat patient samples in vitro, in small case series of patients, and in mouse models of DS leads to improvement of immune phenotype and/or clinical disease. This manuscript provides two major advances in our understanding of the immune dysregulation and therapy for patients. First, they perform deep immune phenotyping on several hundred individuals with DS and demonstrate that immune dysregulation is present from infancy. Second, they report promising interim analysis of a Phase II clinical trial of a JAK inhibitor in 10 people with DS and moderate to severe skin autoimmunity.

Strengths and weaknesses:

The relatively large cohort and careful clinical annotation here provides new insights into the immune phenotype of patients with DS. For example, it is interesting that regardless of autoimmune disease or autoantibody status, individuals with DS have elevated cytokines and CRP. Analysis of the cohorts by age demonstrated that some cytokines are significant elevated in people with DS starting in infancy (e.g., IL-9 and IL-17C). Nearly all adults with DS in this study had autoantibodies (98%) and most had six or more autoantibodies (63%), which differed significantly from euploid study participants. This implies that all patients with DS might benefit from early intervention with therapy to reduce inflammation. However, it is also worth considering that an alternative interpretation that since hypercytokinemia does not vary based on disease state in individuals with DS, that this may not be a key factor driving autoimmunity (although it may be relevant for other clinical symptoms such as neuroinflammation).

Small case series have suggested the benefit of JAK inhibitors to treat autoimmunity in DS. This is the first report of a prospective clinical trial to test a JAK inhibitor in this setting. The clinical trial entry criteria included moderate to severe autoimmune skin disease in patients aged 12-50 years with DS, and treatment was with the JAK1/3 inhibitor tofacitinib. This clinical trial is a critically important step for the field. The early results support that treatment is well tolerated with improvement of interferon scores in patients and reduction of autoantibodies. Most patients experienced clinical improvement, with alopecia areata having the greatest response. Treatment may not affect all skin disease equally, for example of the 5 patients with hidradenitis suppurativa, only 1 showed clinical improvement based on skin score. While very promising, the clinical trial results reported here are preliminary and based on interim analysis of 10 patients at 16 weeks. Individuals with DS have a lifelong risk of immune dysregulation and thus it is unclear how long therapy, if of benefit, would need to be continued. Results of longer-term therapy will be informative when considering the risks/benefits of this therapy.

Comments on revised version:

The authors have made appropriate revisions to this important contribution to the literature.

Reviewer #3 (Public review):

Summary:

The manuscript by Pooja Popli and co-authors tested the importance of Atg14 in the female reproductive tract by conditionally deleting Atg14 use PrCre and also Foxj1cre. The authors showed that loss of Atg14 leads to infertility due to the retention of embryos within the oviduct. The authors further concluded that the retention of embryos within the oviduct is due to pyroptosis in oviduct cells leading to defective cellular integrity. The revised manuscript has included new experimental data (Figs. S2B, 5B, 5C, and S3) that satisfied the concerns of this reviewer. The manuscript should provide important advancement to the field.

Reviewer #3 (Public review):

Summary:

In this paper, Arimura et al report a new method, termed MagIC-Cryo-EM, which refers to the method of using magnetic beads to capture specific proteins out of a lysate via, followed immunoprecipitation and deposition on EM grids. The so-enriched proteins can be analzyed structurally. Importantly, the nanoparticles are further functionalized with protein-based spacers, to avoid a distorted halo around the particles. This is a very elegant approach and allows the resolution of the stucture of small amounts of native proteins at atomistic resolution.<br /> Here, the authors apply this method to study the chromatosome formation from nucleosomes and the oocyte-specific linker histone H1.8. This allows them to resolve H1.8-containing chromatomosomes from oocyte extract in both interphase and metaphase conditions at 4.3 A resolution, which reveal a common structure with H1 placed right at the dyad and contacting both entry-and exit linker DNA.<br /> They then investigate the origin of H1.8 loss during interphase. They identify a non-nucleosomal H1.8-containing complex from interphase preparations. To resolve its structure, the authors develop a protocol (DuSTER) to exclude particles with ambiguous center, revealing particles with five-fold symmetry, that matches the chaperone NPM2. MS and WB confirms that the protein is present in interphase samples but not metaphase. The authors further separate two isoforms, an open and closed form that coexist. Additional densities in the open form suggest that this might be bound H1.8.

Strengths:

Together this is an important addition to the suite of cryoEM methods, with broad applications. The authors demonstrate the method using interesting applications, showing that the methods work and they can get high resolution structures from nucleosomes in complex with H1 from native environments.

Weaknesses:

The structures of the NPM2 chaperone is less well resolved, and some of the interpretation in this part seems only weakly justified.

Reviewer #3 (Public review):

Summary:

The authors were interested in how Ankle2 regulates nuclear envelope reformation after cell division. Other published manuscripts, including those from the authors, show without a doubt that Ankle2 plays a role in this critical process. However, the mechanism by which Ankle2 functions was unclear. Previous work using worms and humans (Asencio et al., 2012) established that human ANKLE2 could bind endogenous PP2A subunits. The binding was direct and was mediated through a region before and including the first ankyrin repeat in human ANKLE2. In addition to its interaction with PP2A, Asencio et al., 2012 also show that ANKLE2 regulates VRK1 kinase activity. Together PP2A and VRK1 regulate BAF phosphorylation for proper nuclear envelope reformation. Here, the authors provide more evidence for interaction with PP2A by also mapping the domain of interaction to the ankyrin repeat in Drosophila. In addition, the ankyrin repeat is essential for nuclear envelope reformation after division. They show that Ankle2 can bind in a PP2A complex without other known regulatory subunits of PP2A. The authors also identify a novel interaction with ER protein Vap33, but functional relevance for this interaction in nuclear envelope reformation is not provided in the manuscript, which the authors explicitly state. This manuscript does not comment on the activity of Ballchen/VRK1 in relation to Ankle2 loss and BAF phosphorylation or nuclear envelope reformation, even though links were previously shown by multiple studies (Asencio et al., Link et al., Apridita Sebastian et al.,). Nuclear envelope defects were rescued by the reduction of VRK1 in two of these manuscripts. It is possible that BAF phosphorylation phenotypes can be contributed by both PP2A inactivity and VRK1 overactivity due to the loss of Ankle2.

Strengths:

This manuscript is a useful finding linking Ankle2 function during nuclear envelope reformation to the PP2A complex. The authors present solid data showing that Ankle2 can form a complex with PP2A-29B and Mts and generate a phosphoproteomic resource that is fundamentally important to understanding Ankle2 biology.

Weaknesses:

However, the main findings/conclusions about subcellular localization might be incomplete since they are drawn from overexpression experiments. In addition, throughout the text, some conclusions are overstated or are not supported by data.

Reviewer #3 (Public review):

In this very thorough study, the authors characterize the function of a novel Drosophila gene, which they name Sakura. They start with the observation that sakura expression is predicted to be highly enriched in the ovary and they generate an anti-sakura antibody, a line with a GFP-tagged sakura transgene, and a sakura null allele to investigate sakura localization and function directly. They confirm the prediction that it is primarily expressed in the ovary and, specifically, that it is expressed in germ cells, and find that about 2/3 of the mutants lack germ cells completely and the remaining have tumorous ovaries. Further investigation reveals that Sakura is required for piRNA-mediated repression of transposons in germ cells. They also find evidence that sakura is important for germ cell specification during development and germline stem cell maintenance during adulthood. However, despite the role of sakura in maintaining germline stem cells, they find that sakura mutant germ cells also fail to differentiate properly such that mutant germline stem cell clones have an increased number of "GSC-like" cells. They attribute this phenotype to a failure in the repression of Bam by dpp signaling. Lastly, they demonstrate that sakura physically interacts with otu and that sakura and otu mutants have similar germ cell phenotypes. Overall, this study helps to advance the field by providing a characterization of a novel gene that is required for oogenesis. The data are generally high-quality and the new lines and reagents they generated will be useful for the field. However, there are some weaknesses and I would recommend that they address the comments in the Recommendations for the authors section below.

Reviewer #3 (Public review):

Summary:

Recently, the off-target activity of antibiotics on human mitoribosome has been paid more attention in the mitochondrial field. Hafner et al applied mitoribosome profilling to study the effect of antibiotics on protein translation in mitochondria as there are similarities between bacterial ribosome and mitoribosome. The authors conclude that some antibiotics act on mitochondrial translation initiation by the same mechanism as in bacteria. On the other hand, the authors showed that chloramphenicol, linezolid and telithromycin trap mitochondrial translation in a context-dependent manner. More interesting, during deep analysis of 5' end of ORF, the authors reported the alternative start codon for ND1 and ND5 proteins instead of previously known one. This is a novel finding in the field and it also provides another application of the technique to further study on mitochondrial translation.

Strengths:

This is the first study which applied mitoribosome profiling method to analyze mutiple antibiotics treatment cells.<br /> The mitoribosome profiling method had been optimized carefully and has been suggested to be a novel method to study translation events in mitochondria. The manuscript is constructive and written well.

Weaknesses:

This is a novel and interesting study, however, most of the conclusion comes from mitoribosome profiling analysis, as a result, the manuscript lacks the cellular biochemical data to provide more evidence and support the findings.

Reviewer #3 (Public review):

Summary:

Rayshubskiy et al. performed whole-cell recordings from descending neurons (DNs) of fruit flies to characterize their role in steering. Two DNs implicated in "walking control" and "steering control" by previous studies (Namiki et al., 2018, Cande et al., 2018, Chen et al., 2018) were chosen by the authors for further characterization. In-vivo whole-cell recordings from DNa01 and DNa02 showed that their activity predicts spontaneous ipsilateral turning events. The recordings also showed that while DNa02 predicts transient turns DNa01 predicts slow sustained turns. However, optogenetic activation or inactivation showed relatively subtle phenotypes for both neurons (consistent with data in other recent preprints, Yang et al 2023 and Feng et al 2024). The authors also further characterized DNa02 with respect to its inputs and showed a functional connection with olfactory and thermosensory inputs as well as with the head-direction system. DNa01 is not characterized to this extent.

Strengths:

(1) In-vivo recordings and especially dual recordings are extremely challenging in Drosophila and provide a much higher resolution DN characterization than other recent studies that have relied on behavior or calcium imaging. Especially impressive are the simultaneous recordings from bilateral DNs (Figure 3). These bilateral recordings show clearly that DNa02 cells not only fire more during ipsilateral turning events but that they get inhibited during contralateral turns. In line with this observation, the difference between left and right DNa02 neuronal activity is a much better predictor of turning events compared to individual DNa02 activity.

(2) Another technical feat in this work is driving local excitation in the head-direction neuronal ensemble (PEN-1 neurons), while simultaneously imaging its activity and performing whole-cell recordings from DNa02 (Figure 4). This impressive approach provided a way to causally relate changes in the head-direction system to DNa02 activity. Indeed, DNa02 activity could predict the rate at which an artificially triggered bump in the PEN-1 ring attractor returns to its previous stable point.

(3) The authors also support the above observations with connectomics analysis and provide circuit motifs that can explain how the head direction system (as well as external olfactory/thermal stimuli) communicated with DNa02. All these results unequivocally put DNa02 as an essential DN in steering control, both during exploratory navigation as well as stimulus-directed turns.

Weaknesses:

(1) I understand that the first version of this preprint was already on biorxiv in 2020, and some of the "weaknesses" I list are likely a reflection of the fact that I'm tasked to review this manuscript in late 2024 (more than 4 years later). But given this is a 2024 updated version it suffers from laying out the results in contemporary terms. For instance, the manuscript lacks any reference to the DNp09 circuit implicated in object-directed turning and upstream to DNa02 even though the authors cite one of the papers where this was analyzed (Braun et al, 2024). More importantly, these studies (both Braun et al 2024 and Sapkal et al 2024) along with recent work from the authors' lab (Yang et al 2023) and other labs (Feng et al 2024) provide a view that the entire suite of leg kinematics changes required for turning are orchestrated by populations of heterogeneous interconnected DNs. Moreover, these studies also show that this DN-DN network has some degree of hierarchy with some DNs being upstream to other DNs. In this contemporary view of steering control, DNa02 (like DNg13 from Yang et al 2023) is a downstream DN that is recruited by hierarchically upstream DNs like DNa03, DNp09, etc. In this view, DNa02 is likely to be involved in most turning events, but by itself unable to drive all the motor outputs required for the said events. This reasoning could be used while discussing the lack of major phenotypes with DNa02 activation or inactivation observed in the current study, which is in stark contrast to strong phenotypes observed in the case of hierarchically upstream DNs like DNp09 or DNa03. In the section, "Contributions of single descending neuron types to steering behavior": the authors start off by asking if individual DNs can make measurable contributions to steering behavior. Once more, any citations to DNp09 or DNa03 - two DNs that are clearly shown to drive strong turning-on activation (Bidaye et al, 2020, Feng et al 2024) - are lacking. Besides misleading the reader, such statements also digress the results away from contemporary knowledge in the field. I appreciate that the brief discussion in the section titled "Ensemble codes for steering" tries to cover these recent updates. However, I think this would serve a better purpose in the introduction and help guide the results.

(2) The second major weakness is the lack of any immunohistochemistry (IHC) images quantifying the expression of the genetic tools used in these studies. Even though the main split-Gal4 tools for DNa01 and DNa02 were previously reported by Namiki et al, 2018, it is important to document the expression with the effectors used in this work and explicitly mention the expression in any ectopic neurons. Similarly, for any experiments where drivers were combined together (double recordings, functional connectivity) or modified for stochastic expression (Figure 8), IHC images are absolutely necessary. Without this evidence, it is difficult to trust many of the results (especially in the case of behavioral experiments in Figure 8). For example, the DNa01 genetic driver used by the authors is also expressed in some neurons in the nerve cord (as shown on the Flylight webpage of Janelia Research Campus). One wonders if all or part of the results described in Figure 8 are due to DNa01 manipulation or manipulation of the nerve cord neurons. The same applies for optic lobe neurons in the DNa02 driver.

(3) The paper starts off with a comparative analysis of the roles of DNa01 and DNa02 during steering. Unfortunately, after this initial analysis, DNa01 is largely ignored for further characterization (e.g. with respect to inputs, connectomics, etc.), only to return in the final figure for behavioral characterization where DNa01 seems to have a stronger silencing phenotype compared to DNa02. I couldn't find an explanation for this imbalance in the characterization of DNa01 versus DNa02. Is this due to technical reasons? Or was it an informed decision due to some results? In addition to being a biased characterization, this also results in the manuscript lacking a coherent thread, which in turn makes it a bit inaccessible to the non-specialist.

(4) There seems to be a discrepancy with regard to what is emphasized in the main text and what is shown in Figures S3/S4 in relation to the role of these DNs in backward walking. There are only two sentences in the main text where these figures are cited.<br /> a) "DNa01 and DNa02 firing rate increases were not consistently followed by large changes in forward velocity (Figs. 1G and S3)."<br /> b) "We found that rotational velocity was consistently related to the difference in right-left firing rates (Fig. 3B). This relationship was essentially linear through its entire dynamic range, and was consistent across paired recordings (Fig. 3C). It was also consistent during backward walking, as well as forward walking (Fig. S4)."<br /> These main text sentences imply the role of the difference between left and right DNa02 in turning. However, the actual plots in the Figures S3 and S4 and their respective legends seem to imply a role in "backward walking". For instance, see this sentence from the legend of Figure S3 "When (ΔvoltageDNa02>>ΔvoltageDNa01), the fly is typically moving backward. When (firing rateDNa02>>firing rateDNa01), the fly is also often moving backward, but forward movement is still more common overall, and so the net effect is that forward velocity is small but still positive when (firing rateDNa02>>firing rateDNa01). Note that when we condition our analysis on behavior rather than neural activity, we do see that backward walking is associated with a large firing rate differential (Fig. S4)." This sort of discrepancy in what is emphasized in the text, versus what is emphasized in the figures, ends up confusing the reader. More importantly, I do not agree with any of these conclusions regarding the implication of backward walking. Both Figures S3 and S4 are riddled with caveats, misinterpretations, and small sample sizes. As a result, I actually support the authors' decision to not infer too much from these figures in the "main text". In fact, I would recommend going one step further and removing/modifying these figures to focus on the role of "rotational velocity". Please find my concerns about these two figures below:<br /> a) In Figures S3 and S4, every heat map has a different scale for the same parameter: forward velocity. S3A is -10 to +10mm/s. S3B is -6 to +6 S4B (left) is -12 to +12 and S4B (right) is -4 to +4. Since the authors are trying to depict results based on the color-coding this is highly problematic.<br /> b) Figure S3A legend "When (ΔvoltageDNa02>>ΔvoltageDNa01), the fly is typically moving backward." There are also several instances when ΔvoltageDNa02= ΔvoltageDNa01 and both are low (lower left quadrant) when the fly is typically moving backwards. So in my opinion, this figure in fact suggests DNa02 has no role in backward velocity control.<br /> c) Based on the example traces in S4A, every time the fly walks backwards it is also turning. Based on this it is important to show absolute rotational velocity in Figure S4C. It could be that the fly is turning around the backward peak which would change the interpretation from Figure S4C. Also, it is important to note that the backward velocities in S4A are unprecedentedly high. No previous reports show flies walking backwards at such high velocities (for example see Chen et al 2018, Nat Comm. for backward walking velocities on a similar setup).<br /> d) In my opinion, Figure S4D showing that right-left DNa02 correlates with rotational velocity, regardless of whether the fly is in a forward or backward walking state, is the only important and conclusive result in Figures S3/S4. These figures should be rearranged to only emphasize this panel.

(5) Figure 3 shows a really nice analysis of the bilateral DNa02 recordings data. While Figure S5 shows that authors have a similar dataset for DNa01, a similar level analysis (Figures 3D, E) is not done for DNa01 data. Is there a reason why this is not done?

(6) In Figure 4 since the authors have trials where bump-jump led to turning in the opposite direction to the DNa02 being recorded, I wonder if the authors could quantify hyperpolarization in DNa02 as is predicted from connectomics data in Figure 7.

(7) Figure 6 suggests that DNa02 contains information about latent steering drives. This is really interesting. However, in order to unequivocally claim this, a higher-resolution postural analysis might be needed. Especially given that DNa02 activation does not reliably evoke ipsilateral turning, these "latent" steering events could actually contain significant postural changes driven by DNa02 (making them "not latent"). Without this information, at least the authors need to explicitly mention this caveat.

(8) Figure 7 would really benefit from connectome data with synapse numbers (or weighted arrows) and a corresponding analysis of DNa01.

(9) In Figure 8E, the most obvious neuronal silencing phenotype is decreased sideways velocity in the case of DNa01 optogenetic silencing. In Figure S2, the inverse filter for sideways velocity for DNa01 had a higher amplitude than the rotational velocity filter. Taken together, does this point at some role for DNa01 in sideways velocity specifically?

(10) In Figure 8G, the effect on inner hind leg stance prolongation is very weak, and given the huge sample size, hard to interpret. Also, it is not clear how this fits with the role of DNa01 in slow sustained turning based on recordings.

Reviewer #3 (Public review):

Summary:

In this work, the authors investigated the molecular and cellular basis of sour taste perception in Drosophila melanogaster, focusing on identifying receptors that mediate attractive responses to certain carboxylic acids. It builds on previous work from the same group that had identified the IR co-receptors IR25a and IR76b for this sensory process, screening a set of mutants in IRs to identify three, IR51b, IR94a, and IR94h, required for feeding preference responses to some or all of the tested acids.

Strengths:

The work is of interest because it assigns sensory roles to IRs of previously unknown function, in particular IR94a and IR94h, and points to pharyngeal neurons in which these receptors are expressed as the relevant sensory neurons (potentially with different roles for IR94a- and IR94h-expressing neurons). The work combines elegant genetics, simple but effective feeding and taste assays, chemo-/opto-genetic activation, and some calcium imaging. Overall the presented data look solid and well-controlled.

Weaknesses:

The in situ expression analysis relies entirely on transgenic driver lines for IR94a and IR94h (which had been previously described, though not fully cited in this work). Importantly, given that many of the behavioral experiments (genetic rescue, physiology, artificial activation) use the IR94a and IR94h GAL4 driver lines, it would be helpful to validate that these faithfully reflect IR94a and IR94h expression (as far as I can tell, such validation wasn't done in the original papers describing these lines as part of a large collection of IR drivers). For IR51b, pharyngeal expression is concluded indirectly from non-quantitative RT-PCR analysis (genetic reporters did not work). The lack of direct detection of gene/protein expression (for example, through RNA FISH, immunofluorescence, or protein tagging) would have made for a more complete characterization of these receptors (for example, there is no direct evidence that they also express IR25a and IR76b, as one might expect). Finally, the relationship of IR94a and IR94h neurons to other types of pharyngeal neurons remains unclear, as are their projection patterns in the SEZ.

Conceptually, the work is of interest mostly to those in the immediate field; there have been a very large number of studies in the past decade (several from this lab) characterizing the contributions of different IRs to various chemosensory processes. The current work doesn't lend much insight into the nature of the minimal functional unit of gustatory IRs (reconstitution of a functional IR in a heterologous neuron/cell has not been achieved here, but this is a limitation of many other previous studies), nor to how different pharyngeal sensory pathways might collaborate to control behavior. Nevertheless, the findings provide a useful contribution to the literature.

for - paper - Translating Earth system boundaries for cities and businesses - Fig. 3 - Ten principles of translation - Bai et al. 2024 - from - paper - Cross-scale translation of Earth system boundaries should use methods that are more science-based - citation of Fig.3 - Xue & Bakshi

from - paper - citation - Cross-scale translation of Earth system boundaries should use methods that are more science-based - citation of Fig.3 - Xue & Bakshi - https://hyp.is/xf3MxqveEe-pGZeWkHHcLA/jgvw2024.peergos.me/StopResetGo/2024/11/PDFs/MattersArisingBaietal.pdf

for - to - paper - Translating Earth system boundaries for cities and businesses - figure 3 - Bai et al.

to - paper - Translating Earth system boundaries for cities and businesses - figure 3 - Bai et al., 2024 - https://hyp.is/_uwZDKveEe-GOx-pvnYEFg/jgvw2024.peergos.me/StopResetGo/2024/11/PDFs/NS_earth_boundaries_2024.pdf

Reviewer #3 (Public review):

Summary:

Sukhina et al are trying to understand the impacts of malnutrition on immunity. They model malnutrition with a diet switch from ad libitum to 40% caloric restriction (CR) in post-weaned mice. They test impacts on immune function with listeriosis. They then test whether re-feeding corrects these defects and find aspects of emergency myelopoiesis that remain defective after a precedent period of 40% CR. Overall, this is a very interesting observational study on the impacts of sudden prolonged exposure to less caloric intake.

Strengths:

The study is rigorously done. The observation of lasting defects after a bout of 40% CR is quite interesting. Overall, I think the topic and findings are of interest.

Weaknesses:

While the observations are interesting, in this reviewer's opinion, there is both a lack of mechanistic understanding of the phenomena and also some lack of resolution/detail about the phenomena itself. Addressing the following major issues would be helpful towards aspects of both:

(1) Is it calories, per se, or macro/micronutrients that drive these phenotypes observed with 40% CR. At the least, I would want to see isocaloric diets (primarily protein, fat, or carbs) and then some of the same readouts after 40% CR. Ie does low energy with relatively more eg protein prevent immunosuppression (as is commonly suggested)? Micronutrients would be harder to test experimentally and may be out of the scope of this study. However, it is worth noting that many of the malnutrition-associated diseases are micronutrient deficiencies.

(2) Is immunosuppression a function of a certain weight loss threshold? Or something else? Some idea of either the tempo of immunosuppression (happens at 1, in which weight loss is detected; vs 2-3, when body length and condition appear to diverge; or 5 weeks), or grade of CR (40% vs 60% vs 80%) would be helpful since the mechanism of immunosuppression overall is unclear (but nailing it may be beyond the scope of this communication).

(3) Does an obese mouse that gets 40% CR also become immunodeficient? As it stands, this ad libitum --> 40% CR model perhaps best models problems in the industrial world (as opposed to always being 40% CR from weaning, as might be more common in the developing world), and so modeling an obese person losing a lot of weight from CR (like would be achieved with GLP-1 drugs now) would be valuable to understanding generalizability.

(4) Generalizing this phenomenon as "bacterial" with listeriosis, which is more like a virus in many ways (intracellular phase, requires type I IFN, etc.) and cannot be given by the natural route of infection in mice, may not be most accurate. I would want to see an experiment with E.Coli, or some other bacteria, to test the statement of generalizability (ie is it bacteria, or type I IFN-pathway dominant infections, like viruses). If this is unique listeriosis, it doesn't undermine the story as it is at all, but it would just require some word-smithing.

(5) Previous reports (which the authors cite) implicate Leptin, the levels of which scale with fat mass, as "permissive" of a larger immune compartment (immune compartment as "luxury function" idea). Is their phenotype also leptin-mediated (ie leptin AAV)?

(6) The inability of re-feeding to "rescue" the myeloid compartment is really interesting. Can the authors do a bone marrow transplantation (CR-->ad libitum) to test if this effect is intrinsic to the CR-experienced bone marrow?

(7) Is the defect in emergency myelopoiesis a defect in G-CSF? Ie if the authors injected G-CSF in CR animals, do they equivalently mobilize neutrophils? Does G-CSF supplementation (as one does in humans) rescue host defense against Listeria in the CR or re-feeding paradigms?

Reviewer #3 (Public review):

Summary:

This study established a single-cell RNA sequencing atlas of pipefish embryos. The results obtained identified unique gene expression patterns for pipefish-specific characteristics, such as fgf22 in the tip of the palatoquadrate and Meckel's cartilage, broadly informing the genetic mechanisms underlying morphological novelty in teleost fishes. The data obtained are unique and novel, potentially important in understanding fish diversity. Thus, I would enthusiastically support this manuscript if the authors improve it to generate stronger and more convincing conclusions than the current forms.

Weakness:

Regarding the expression of sfrp1a and bmp4 dorsal to the elongating ethmoid plate and surrounding the ceratohyal: Are their expression patterns spatially extended or broader compared to the pipefish ancestor? Is there a much closer species available to compare gene expression patterns with pipefish? Did the authors consider using other species closely related to pipefish for ISH? Sfrp1a and bmp4 may be expressed in the same regions of much more closely related species without face elongation. I understand that embryos of such species are not always accessible, but it is also hard to argue responsible genes for a specific phenotype by only comparing gene expression patterns between distantly related species (e.g., pipefish vs. zebrafish). Due to the same reason, I would not directly compare/argue gene expression patterns between pipefish and mice, although I should admit that mice gene expression patterns are sometimes helpful to make a hypothesis of fish evolution. Alternatively, can the authors conduct ISH in other species of pipefish? If the expression patterns of sfrp1a and bmp4 are common among fishes with face elongation, the conclusion would become more solid. If these embryos are not available, is it possible to reduce the amount of Wnt and BMP signal using Crispr/Cas, MO, or chemical inhibitor? I do think that there are several ways to test the Wnt and/or BMP hypothesis in face elongation.

Reviewer #3 (Public review):

Summary:

Ruan and colleagues consider a branching process model (in their terminology the "Haldane model") and the most basic Wright-Fisher model. They convincingly show that offspring distributions are usually non-Poissonian (as opposed to what's assumed in the Wright-Fisher model), and can depend on short-term ecological dynamics (e.g., variance in offspring number may be smaller during exponential growth). The authors discuss branching processes and the Wright-Fisher model in the context of 3 "paradoxes" --- 1) how Ne depends on N might depend on population dynamics; 2) how Ne is different on the X chromosome, the Y chromosome, and the autosomes, and these differences do match the expectations base on simple counts of the number of chromosomes in the populations; 3) how genetic drift interacts with selection. The authors provide some theoretical explanations for the role of variance in the offspring distribution in each of these three paradoxes. They also perform some experiments to directly measure the variance in offspring number, as well as perform some analyses of published data.

Strengths:

- The theoretical results are well-described and easy to follow.<br /> - The analyses of different variances in offspring number (both experimentally and analyzing public data) are convincing that non-Poissonian offspring distributions are the norm.<br /> - The point that this variance can change as the population size (or population dynamics) change is also very interesting and important to keep in mind.<br /> - I enjoyed the Density-Dependent Haldane model. It was a nice example of the decoupling of census size and effective size.<br /> - Equation (10) is a nice result (but see below)

Weaknesses:

- I am not convinced that these types of effects cannot just be absorbed into some time-varying Ne and still be well-modeled by the Wright-Fisher process. As a concrete example, Mohle and Sagitov 2001 show that a "coalescent Ne" for the WF model should be (N-1)/Var(K). This resolves the exponentially growing bacteria "paradox" raised in the present paper --- when the bacteria are growing Var(K) ~ 0, and hence there should be very little drift. This exactly resolves the "paradox" raised by the authors. Instead, it merely underscores that Ne does not need to be equal to (or even positively correlated!) with N. I absolutely do not see this as a failure of the WF model. Whether one finds branching processes or the WF model more biologically intuitive is a matter of taste, but to say that WF models cannot explain this "paradox" is false, when a well-known paper from more than 20 years ago does just that.<br /> - Along these lines, the result that Ne in the Wright-Fisher process might not be related to N in any straightforward (or even positively correlated) way are well-known (e.g., Neher and Hallatschek 2012; Spence, Kamm, and Song 2016; Matuszewski, Hildebrandt, Achaz, and Jensen 2018; Rice, Novembre, and Desai 2018; the work of Lounès Chikhi on how Ne can be affected by population structure; etc...)<br /> - I was also missing some discussion of the relationship between the branching process and the Wright-Fisher model (or more generally Cannings' Exchangeable Models) when conditioning on the total population size. In particular, if the offspring distribution is Poisson, then conditioned on the total population size, the branching process is identical to the Wright-Fisher model.<br /> - Given that Cannings' exchangeable models decouple N and Ne, it would not surprise me if something like equation (10) could be derived under such a model. I have not seen such a derivation, and the authors' result is nice, but I do not see it as proof that WF-type models (i.e., Cannings' models) are irreparably broken.

Editors' comments (Public review):

Weaknesses:

(1) Figure 7C, 7E, 7I, and "Figure7-figure supplement 1 ": All data in these data panels are based on only n=3, which is insufficient. Sample sizes of n=3 are too low to correctly assess normality of distribution and, as a consequence, do not allow to select the appropriate parametric/non-parametric tests. Accordingly, no statistical comparison can be performed and all p values and symbols currently indicating statistically significant differences between groups must be removed.

(2) Figure 3A, 3B, or 3C: No information about n numbers per group. Should n numbers per group be n=4 or less, no statistical comparison can be performed and all p values and symbols indicating statistically significant differences between groups must be removed.

(3) Figure 4 E and 4F: No information about n numbers per group. Should n numbers per group be n=4 or less, no statistical comparison can be performed and all p values and symbols indicating statistically significant differences between groups must be removed.

(4) Figure 5: No information about n numbers per group is provided. Should n numbers per group be n=4 or less, no statistical comparison can be performed and all p values and symbols indicating statistically significant differences between groups must be removed.

Reviewer #3 (Public review):

Summary:

The study aims to determine whether the endosomal protein SNX4 performs a role in neurotransmitter release and synaptic vesicle recycling. The authors exploited a newly generated conditional knockout mouse to allow them to interrogate SNX4 function. A series of basic parameters were assessed, with an observed impact on neurotransmitter release and active zone morphology. The work is interesting, however as things currently stand, the work is descriptive with little mechanistic insight. There are a number of places where some of the conclusions require further validation.

Strengths:

The strengths of the work are the state-of-the-art methods to monitor presynaptic function.

Weaknesses:

The weaknesses are the fact that the work is largely descriptive, with no mechanistic insight into the role of SNX4.

Comments on revisions:

The authors have addressed a couple of the more major concerns with the manuscript, however many of the original weaknesses remain. The primary weakness being the lack of mechanism. It is disappointing that real-time VAMP2 trafficking was not investigated, and the authors justification as to why the experiment was not performed was not convincing (especially since this is the approach that all other groups employ to examine SV cargo trafficking). In a number of instances "contractual constraints" are referred to as an explanation for not performing additional experiments. It was unclear whether this refers to licencing issues with the mouse line or the lack of personnel to perform the work. Regardless it still leaves this work as somewhat incomplete.

Reviewer #3 (Public review):

Summary of the review process from the Reviewing Editor:

The authors and the reviewers did not agree on several important points made in this paper. The reviewers were critical of the operationalisation of the concept of visual homogeneity (VH), and questioned its validity. For instance, they found it unsatisfying that VH was not calculated on the basis of images themselves, but on the basis of reaction times instead. The authors responded by providing further explanation and argumentation for the importance of this novel concept, but the reviewers were not persuaded. The reviewers also pointed out some data features that did not fit the theory (e.g., overlapping VH between present and absent stimuli), which the authors acknowledge as a point that needs further refining. Finally, the reviewers pointed out that the new so-called visual homogeneity brain region does not overlap very much in the two studies, to which the authors have responded that it is remarkable that there is even partial overlap, given the many confounding differences between the two studies. Altogether, the authors have greatly elaborated their case for VH as an important concept, but the reviewers were not persuaded, and we conclude that the current evidence does not yet meet the high bar for declaring that a novel image property, visual homogeneity, is computed in a localised brain region.

Reviewer #3 (Public review):

Summary:

In this study, the authors set out to investigate whether GPRC6A mediates kokumi taste initiated by the amino acid L-ornithine. They used Wistar rats, a standard laboratory strain, as the primary model and also performed an informative taste test in humans, in which miso soup was supplemented with various concentrations of L-ornithine. The findings are valuable and overall the evidence is solid. L-Ornithine should be considered to be a useful test substance in future studies of kokumi taste and the class C G protein-coupled receptor known as GPRC6A (C6A) along with its homolog, the calcium-sensing receptor (CaSR) should be considered candidate mediators of kokumi taste.

Strengths:

The overall experimental design is solid based on two bottle preference tests in rats. After determining the optimal concentration for L-Ornithine (1 mM) in the presence of MSG, it was added to various tastants, including inosine 5'-monophosphate; monosodium glutamate (MSG); mono-potassium glutamate (MPG); intralipos (a soybean oil emulsion); sucrose; sodium chloride (NaCl); citric acid and quinine hydrochloride. Robust effects of ornithine were observed in the cases of IMP, MSG, MPG, and sucrose, and little or no effects were observed in the cases of sodium chloride, citric acid, and quinine HCl. The researchers then focused on the preference for Ornithine-containing MSG solutions. The inclusion of the C6A inhibitors Calindol (0.3 mM but not 0.06 mM) or the gallate derivative EGCG (0.1 mM but not 0.03 mM) eliminated the preference for solutions that contained Ornithine in addition to MSG. The researchers next performed transections of the chord tympani nerves (with sham operation controls) in anesthetized rats to identify the role of the chorda tympani branches of the facial nerves (cranial nerve VII) in the preference for Ornithine-containing MSG solutions. This finding implicates the anterior half-two thirds of the tongue in ornithine-induced kokumi taste. They then used electrical recordings from intact chorda tympani nerves in anesthetized rats to demonstrate that ornithine enhanced MSG-induced responses following the application of tastants to the anterior surface of the tongue. They went on to show that this enhanced response was insensitive to amiloride, selected to inhibit 'salt tastant' responses mediated by the epithelial Na+ channel, but eliminated by Calindol. Finally, they performed immunohistochemistry on sections of rat tongue demonstrating C6A positive spindle-shaped cells in fungiform papillae that partially overlapped in its distribution with the IP3 type-3 receptor, used as a marker of Type-II cells, but not with (i) gustducin, the G protein partner of Tas1 receptors (T1Rs), used as a marker of a subset of type-II cells; or (ii) 5-HT (serotonin) and Synaptosome-associated protein 25 kDa (SNAP-25) used as markers of Type-III cells.

Weaknesses:

The researchers undertook what turned out to be largely confirmatory studies in rats with respect to their previously published work on Ornithine and C6A in mice (Mizuta et al Nutrients 2021).

The authors point out that animal models pose some difficulties of interpretation in studies of taste and raise the possibility in the Discussion that umami substances may enhance the taste response to ornithine (Line 271, Page 9). They miss an opportunity to outline the experimental results from the study that favor their preferred interpretation that ornithine is a taste enhancer rather than a tastant.

At least two other receptors in addition to C6A might mediate taste responses to ornithine: (i) the CaSR, which binds and responds to multiple L-amino acids (Conigrave et al, PNAS 2000), and which has been previously reported to mediate kokumi taste (Ohsu et al., JBC 2010) as well as responses to Ornithine (Shin et al., Cell Signaling 2020); and (ii) T1R1/T1R3 heterodimers which also respond to L-amino acids and exhibit enhanced responses to IMP (Nelson et al., Nature 2001). While the experimental results as a whole favor the authors' interpretation that C6A mediates the Ornithine responses, they do not make clear either the nature of the 'receptor identification problem' in the Introduction or the way in which they approached that problem in the Results and Discussion sections. It would be helpful to show that a specific inhibitor of the CaSR failed to block the ornithine response. In addition, while they showed that C6A-positive cells were clearly distinct from gustducin-positive, and thus T1R-positive cells, they missed an opportunity to clearly differentiate C6A-expressing taste cells and CaSR-expressing taste cells in the rat tongue sections.

It would have been helpful to include a positive control kokumi substance in the two-bottle preference experiment (e.g., one of the known gamma-glutamyl peptides such as gamma-glu-Val-Gly or glutathione), to compare the relative potencies of the control kokumi compound and Ornithine, and to compare the sensitivities of the two responses to C6A and CaSR inhibitors.

The results demonstrate that enhancement of the chorda tympani nerve response to MSG occurs at substantially greater Ornithine concentrations (10 and 30 mM) than were required to observe differences in the two bottle preference experiments (1.0 mM; Figure 2). The discrepancy requires careful discussion and if necessary further experiments using the two-bottle preference format.

Reviewer #3 (Public review):

Summary:

This study helps to clarify the mixed literature on dopamine responses to aversive stimuli. While it is well accepted that dopamine in the ventral striatum increases in response to various rewarding and appetitive stimuli, aversive stimuli have been shown to evoke phasic increases or decreasing depending on the exact aversive stimuli, behavioral paradigm, and/or dopamine recording method and location examined. Here the authors use a well-designed set of experiments to show differential responses to an appetitive primary reward (sucrose) that later becomes a conditioned aversive stimulus (sucrose previously paired with lithium chloride in a conditioned taste aversion paradigm). The results are interesting and add valuable data to the question of how the mesolimbic dopamine system encodes aversive stimuli, however, the conclusions are strongly stated given that the current data do not necessarily align with prior conflicting data in terms of recording location, and it is not clear exactly how to interpret the generally biphasic dopamine response to the CTA-sucrose which also evolves over exposures within a single session.

Strengths:

• The authors nicely demonstrate that their two aversive stimuli examined, quinine and sucrose following CTA, evoked aversive facial expressions and paw movements that differed from those following rewarding sucrose to support that the stimuli experienced by the rats differ in valence.

• Examined dopamine responses to the exact same sensory stimuli conditioned to have opposing valences, avoiding standard confounds of appetitive and aversive stimuli being sensed by different sensory modalities (i.e., sweet taste vs. electric shock).

• The authors examined multiple measurements of dopamine activity - cell body calcium (GCaMP6f) in midbrain and release in NAc (Grab-DA2h), which is useful as the prior mixed literature on aversive dopamine responses comes from a variety of recording methods.

• Correlations between sucrose preference and dopamine signals demonstrate behavioral relevance of the differential dopamine signals.

• The delayed testing experiment in Figure 7 nicely controls for the effect of time to demonstrate that the "rewarding" dopamine response to sucrose only recovers after multiple extinction sucrose exposures to extinguish the CTA.

Weaknesses for consideration:

• Regional differences in dopamine signaling to aversive stimuli are mentioned in the introduction and discussion. For instance, the idea that dopamine encodes salience is strongly argued against in the discussion, but the paper cited as arguing for that (Kutlu et al. 2021) is recording from the medial core in mice. Given other papers cited in the text about the regional differences in dopamine signaling in the NAc and from different populations of dopamine neurons in midbrain, it's important to mention this distinction wrt to salience signaling. Relatedly, the text says that the lateral NAc shell was targeted for accumbens recordings, but the histology figure looks like the majority of fibers were in the anterior lateral core of NAc. For the current paper to be a convincing last word on the issue, it would be extremely helpful to have similar recordings done in other parts of the NAc to do a more thorough comparison against other studies.

• Dopamine release in the NAc never dips below baseline for the conditioned sucrose. Is it possible to really consider this as a signal for valence per se, as opposed to it being a weaker response relative to the original sucrose response?

• Related to this, the main measure of the dopamine signal here, "mean z-score," obscures the temporal dynamics of the aversive dopamine response across a trial. This measure is used to claim that sucrose after CTA is "suppressing" dopamine neuron activity and release, which is true relative to the positive valence sucrose response. However, both GRAB-DA and cell-body GCaMP measurements show clear increases after onset of sucrose infusion before dipping back to baseline or slightly below in the average of all example experiments displayed. One could point to these data to argue either that aversive stimuli cause phasic increases in dopamine (due to the initial increase) or decreases (due to the delayed dip below baseline) depending on the measurement window. Some discussion of the dynamics of the response and how it relates to the prior literature would be useful.<br /> - Would this delayed below-baseline dip be visible with a shorter infusion time?<br /> - Does the max of the increase or the dip of the decrease better correlate with the behavioral measures of aversion (orofacial, paw movements) or sucrose preference than "mean z-score" measure used here?<br /> - The authors argue strongly in the discussion against the idea that dopamine is encoding "salience." Could this initial peak (also seen in the first few trials of quinine delivery, fig 1c color plot) be a "salience" response?

• Related to this, the color plots showing individual trials show a reduction in the increases to positive valence sucrose across conditioning day trials and a flip from infusion-onset increase to delayed increases across test day trials. This evolution across days makes it appear that the last few conditioning day trials would be impossible to discriminate from the first few test day trials in the CTA-paired. Presumably, from strength of CTA as a paradigm, the sucrose is already aversive to the animals at the first trial of test day. Why do the authors think the response evolves across this session?

• Given that most of the work is using a conditioned aversive stimulus, the comparison to a primary aversive tastant quinine is useful. However, the authors saw basically no dopamine response to a primary aversive tastant quinine (measured only with GRAB-DA) and saw less noticeable decreases following CTA for NAc recordings with GRAB-DA2h than with cell body GCaMP. Given that they are using the high-affinity version of the GRAB sensor, this calls into question whether this is a true difference in release vs. soma activity or issue of high affinity release sensor making decreases in dopamine levels more difficult to observe.

Reviewer #3 (Public review):

Summary:

The authors use high-depth, full-length scRNA-Seq analysis of fetal human retina to identify novel regulators of photoreceptor specification and retinoblastoma progression.

Strengths:

The use of high-depth, full-length scRNA-Seq to identify functionally important alternatively spliced variants of transcription factors controlling photoreceptor subtype specification, and identification of SYK as a potential mediator of RB1-dependent cell cycle reentry in immature cone photoreceptors.

Human developing fetal retinal tissue samples were collected between 13-19 gestational weeks and this provides a substantially higher depth of sequencing coverage, thereby identifying both rare transcripts and alternative splice forms, and thereby representing an important advance over previous droplet-based scRNA-Seq studies of human retinal development.

Weaknesses:

The weaknesses identified are relatively minor. This is a technically strong and thorough study, that is broadly useful to investigators studying retinal development and retinoblastoma.

Reviewer #3 (Public review):

Summary:

At the abandoned replication fork, loading of DnaB helicase requires assistance from PriABC, repA, and other protein partners, but it does not require replication initiator protein, DnaA. In contrast, nucleotide-dependent DnaA binding at the specific functional elements is fundamental for helicase loading, leading to the DUE region's opening. However, the authors questioned in this study that in case of impeding replication at the bacterial chromosomal origins, oriC, a strategy similar to an abandoned replication fork for loading DnaB via bypassing the DnaA interaction step could be functional. The study by Yoshida et al. suggests that PriC could promote DnaB helicase loading on the chromosomal oriC ssDNA without interacting with the DnaA protein. However, the conclusions drawn from the primarily qualitative data presented in the study could be slightly overwhelming and need supportive evidence.

Strengths:

Understanding the mechanism of how DNA replication restarts via reloading the replisomes onto abandoned DNA replication forks is crucial. Notably, this knowledge becomes crucial to understanding how bacterial cells maintain DNA replication from a stalled replication fork when challenging or non-permissive conditions prevail. This critical study combines experiments to address a fundamental question of how DnaB helicase loading could occur when replication initiation impedes at the chromosomal origin, leading to replication restart.

Weaknesses:

The term colony formation used for a spotting assay could be misleading for apparent reasons. Both assess cell viability and growth; while colony formation is quantitative, spotting is qualitative. Particularly in this study, where differences appear minor but draw significant conclusions, the colony formation assays representing growth versus moderate or severe inhibition are a more precise measure of viability.

Figure 2<br /> The reduced number of two oriC copies per cell in the dnaA46priC-deficient strain was considered moderate inhibition. When combined with the data suggested by the dnaAC2priC-deficient strain containing two origins in cells with or without PriC (indicating no inhibition)-the conclusion was drawn that PriC rescue blocked replication via assisting DnaC-dependent DnaB loading step at oriC ssDNA.

The results provided by Saifi B, Ferat JL. PLoS One. 2012;7(3):e33613 suggests the idea that in an asynchronous DnaA46 ts culture, the rate by which dividing cells start accumulating arrested replication forks might differ (indicated by the two subpopulations, one with single oriC and the other with two oriC). DnaA46 protein has significantly reduced ATP binding at 42C, and growing the strain at 42C for 40-80 minutes before releasing them at 30 C for 5 minutes has the probability that the two subpopulations may have differences in the active ATP-DnaA. The above could be why only 50% of cells contain two oriC. Releasing cells for more time before adding rifampicin and cephalexin could increase the number of cells with two oriCs. In contrast, DnaC2 cells have inactive helicase loader at 42 C but intact DnaA-ATP population (WT-DnaA at 42 or 30 C should not differ in ATP-binding). Once released at 30 C, the reduced but active DnaC population could assist in loading DnaB to DnaA, engaged in normal replication initiation, and thus should appear with two oriC in a PriC-independent manner.

Broadly, the evidence provided by the authors may support the primary hypothesis. Still, it could call for an alternative hypothesis: PriC involvement in stabilizing the DnaA-DnaB complex (this possibility could exist here). To prove that the conclusions made from the set of experiments in Figures 2 and 3, which laid the foundations for supporting the primary hypothesis, require insights using on/off rates of DnaB loading onto DnaA and the stability of the complexes in the presence or absence of PriC, I have a few other reasons to consider the latter arguments.

Figure 3<br /> One should consider the fact that dnA46 is present in these cells. Overexpressing pdnaAFH could produce mixed multimers containing subunits of DnaA46 (reduced ATP binding) and DnaAFH (reduced DnaB binding). Both have intact DnaA-DnaA oligomerization ability. The cooperativity between the two functions by a subpopulation of two DnaA variants may compensate for the individual deficiencies, making a population of an active protein, which in the presence of PriC could lead to the promotion of the stable DnaA: DnaBC complexes, able to initiate replication. In the light of results presented in Hayashi et al. and J Biol Chem. 2020 Aug 7;295(32):11131-11143, where mutant DnaBL160A identified was shown to be impaired in DnaA binding but contained an active helicase function and still inhibited for growth; how one could explain the hypothesis presented in this manuscript. If PriC-assisted helicase loading could bypass DnaA interaction, then how growth inhibition in a strain carrying DnaBL160A should be described. However, seeing the results in light of the alternative possibility that PriC assists in stabilizing the DnaA: DnaBC complex is more compatible with the previously published data.

Figure 4<br /> Overexpression of DiaA could contribute to removing a higher number of DnaA populations. This could be more aggravated in the absence of PriC (DiaA could titrate out more DnaA)- the complex formed between DnaA: DnaBC is not stable, therefore reduced DUE opening and replication initiation leading to growth inhibition (Fig. 4A ∆priC-pNA135). Figure 7C: Again, in the absence of PriC, the reduced stability of DnaA: DnaBC complex leaves more DnaA to titrate out by DiaA, and thus less Form I*. However, adding PriC stabilizes the DnaA: DnaBC hetero-complexes, with reduced DnaA titration by DiaA, producing additional Form I*. Adding a panel with DnaBL160A that does not interact with DnaA but contains helicase activity could be helpful. Would the inclusion of PriC increase the ability of mutant helicase to produce additional Form I*?

Figure 5<br /> The interpretation is that colony formation of the Left-oriC ∆priC double mutant was markedly compromised at 37˚C (Figure 5B), and 256 the growth defects of the Left-oriC mutant at 25{degree sign}C and 30{degree sign}C were aggravated. However, prima facia, the relative differences in the growth of cells containing and lacking PriC are similar. Quantitative colony-forming data is required to claim these results. Otherwise, it is slightly confusing.

A minor suggestion is to include cells expressing PriC using plasmid DNA to show that adding PriC should reverse the growth defect of dnaA46 and dnaC2 strains at non-permissive temperatures. The same should be added at other appropriate places.

Reviewer #3 (Public review):

Summary:

Obray et al. investigate the long-lasting effects of adolescent intermittent ethanol (AIE) in rats, a model of alcohol dependence, on a neural circuit within the prefrontal cortex. The studies are focused on inputs from the basolateral amygdala (BLA) onto parvalbumin (PV) interneurons and pyramidal cells that project to the periaqueductal gray (PAG). The authors found that AIE increased BLA excitatory drive onto parvalbumin interneurons and increased BLA feedforward inhibition onto PAG-projecting neurons.

Strengths:

Fully powered cohorts of male and female rodents are used, and the design incorporates both AIE and an acute pain model. The authors used several electrophysiological techniques to assess synaptic strength and excitability from a few complimentary angles. The design and statistical analysis are sound, and the strength of evidence supporting synaptic changes following AIE results is solid.

Weaknesses:

(1) There is incomplete evidence supporting some of the conclusions drawn in this manuscript. The authors claim that the changes in feedforward inhibition onto pyramidal cells are due to the changes in parvalbumin interneurons, but evidence is not provided to support that idea. PV cells do not spontaneously fire action potentials spontaneously in slices (nor do they receive high levels of BLA activity while at rest in slices). It is possible that spontaneous GABA release from PV cells is increased after AIE but the authors did not report sIPSC frequency. Second, the authors did not determine that PV cells mediate the feedforward BLA op-IPSCs and changes following AIE (this would require manipulation to reduce/block PV-IN activity). This limitation in results and interpretation is important because prior work shows BLA-PFC feedforward IPSCs can be driven by somatostatin cells. Cholecystokinin cells are also abundant basket cells in PFC and have been recently shown to mediate feedforward inhibition from the thalamus and ventral hippocampus, so it's also possible that CCK cells are involved in the effects observed here.

(2) The authors conclude that the changes in this circuit likely mediate long-lasting hyperalgesia, but this is not addressed experimentally. In some ways, the focused nature of the study is a benefit in this regard, as there is extensive prior literature linking this circuit with pain behaviors in alternative models (e.g., SNI), but it should be noted that these studies have not assessed hyperalgesia stemming from prior alcohol exposure. While the current studies do not include a causative behavioral manipulation, the strength of the association between BLA-PL-PAG function and hyperalgesia could be bolstered by current data if there were relationships detected between electrophysiological properties and hyperalgesia. Have the authors assessed this? In addition, this study is limited by not addressing the specificity of synaptic adaptations to the BLA-PL-PAG circuit. For instance, PL neurons send reciprocal projections to BLA and send direct projections to the locus coeruleus (which the authors note is an important downstream node of the PAG for regulating pain).

(3) I have some concerns about methodology. First, 5-ms is a long light pulse for optogenetics and might induce action-potential independent release. Does TTX alone block op-EPSCs under these conditions? Second, PV cells express a high degree of calcium-permeable AMPA receptors, which display inward rectification at positive holding potentials due to blockade from intracellular polyamines. Typically, this is controlled/promoted by including spermine in the internal solution, but I do not believe the authors did that. Nonetheless, the relatively low A/N ratios for this cell type suggest that CP-AMPA receptors were not sampled with the +40/+40 design of this experiment, raising concerns that the majority of AMPA receptors in these cells were not sampled during this experiment. Finally, it should be noted that asEPSC frequency can also reflect changes in a number of functional/detectable synapses. This measurement is also fairly susceptible to differences in inter-animal differences in ChR2 expression. There are other techniques for assessing presynaptic release probability (e.g., PPR, MK-801 sensitivity) that would improve the interpretation of these studies if that is intended to be a point of emphasis.

(4) In a few places in the manuscript, results following voluntary drinking experiments (especially Salling et al. and Sicher et al.) are discussed without clear distinction from prior work in vapor models of dependence

(5) Discussion (lines 416-420). The authors describe some differing results with the literature and mention that the maximum current injection might be a factor. To me, this does not seem like the most important factor and potentially undercuts the relevance of the findings. Are the cells undergoing a depolarization block? Did the authors observe any changes in the rheobase or AP threshold? On the other hand, a more likely difference between this and previous work is that the proportion of PAG-projecting cells is relatively low, so previous work in L5 likely sampled many types of pyramidal cells that project to other areas. This is a key example where additional studies by the current group assessing a distinct or parallel set of pyramidal cells would aid in the interpretation of these results and help to place them within the existing literature. Along these lines, PAG-projecting neurons are Type A cells with significant hyperpolarization sag. Previous studies showed that adolescent binge drinking stunts the development of HCN channel function and ensuing hyperpolarization sag. Have the authors observed this in PAG-projecting cells? Another interesting membrane property worth exploring with the existing data set is the afterhyperpolarization / SK channel function.

Reviewer #3 (Public Review):

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

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

Comment from Reviewing Editor: The revised manuscript has addressed the points raised during the review of the original submission.

Reviewer #3 (Public review):

Summary:

The authors are showing evidence that they claim establishes the controversial epigenetic mark, DNA 6mA, as promoting genome stability.

Strengths:

The identification of a poorly understood protein, METTL3, and its subsequent characterization in DDR is of high quality and interesting.

Weaknesses:

(1) The very presence of 6mA (DNA) in mammalian DNA is still highly controversial and numerous studies have been conclusively shown to have reported the presence of 6mA due to technical artifacts and bacterial contamination. Thus, to my knowledge there is no clear evidence for 6mA as an epigenetic mark in mammals, and consequently, no evidence of writers and readers of 6mA. None of this is mentioned in the introduction. Much of the introduction can be reduced, but a paragraph clearly stating the controversy and lack of evidence for 6mA in mammals needs to be added, otherwise, the reader is given an entirely distorted view of the field.

These concerns must also be clearly in the limitations section and even in the results section which fails to nuance the authors' findings.

(2) What is the motivation for using HT-29 cells? Moreover, the materials and methods do not state how the authors controlled for bacterial contamination, which has been the most common cause of erroneous 6mA signals to date. Did the authors routinely check for mycoplasma?

(3) The single cell imaging of 6mA in various cells is nice. The results are confirmed by mass spec as an orthogonal approach. Another orthogonal and quantitative approach to assessing 6mA levels would be PacBio. Similarly, it is unclear why the authors have not performed dot-blots of 6mA for genomic DNA from the given cell lines.

(4) The results of Figure 3 need further investigation and validation. If the results are correct the authors are suggesting that the majority of 6mA in their cell lines is present in the DNA, and not the RNA, which is completely contrary to every other study of 6mA in mammalian cells that I am aware of. This could suggest that the antibody is not, in fact, binding to 6mA, but to unmodified adenine, which would explain why the signal disappears after DNAse treatment. Indeed, binding of 6mA to unmethylated DNA is a commonly known problem with most 6mA antibodies and is well described elsewhere.

(5) Given the lack of orthologous validation of the observed DNA 6mA and the lack of evidence supporting the presence of 6mA in mammalian DNA and consequently any functional role for 6mA in mammalian biology, the manuscript's conclusions need to be toned down significantly, and the inherent difficulty in assessing 6mA accurately in mammals acknowledged throughout.

Reviewer #3 (Public review):

This manuscript reports a detailed model of the rat non-barrel somatosensory cortex, consisting of 4.2 million morphologically and biophysically detailed neuron models, arranged in space and connected according to highly sophisticated rules informed by diverse experimental data. Due to its breadth and sophistication the model will undoubtedly be of interest to the community, and the reporting of anatomical details of modeling in this paper is important for understanding all the assumptions and procedures involved in constructing the model. While a useful contribution to this field, the model and the manuscript could be improved by employing data more directly and comparing simple features of the model's connectivity - in particular, connection probabilities - with relevant experimental data.

The manuscript is overall well-written, but contains a substantial number of confusing or unclear statements, and some important information is not provided.

Comments on revisions:

The authors mostly addressed all my points and improved the paper substantially. I do not have further extensive comments except one general point below.

Regarding section 2.3 and metrics of connectivity like pairwise connection probabilities, it is great that the authors rewrote that section and added comparisons with experimental data in Figs. 4 and S9. Unfortunately, what one finds when direct comparisons are made is that the modeled pairwise connectivity is quite different from the data. Fig. S9 shows that the model's results do not agree with data in about half of the cases (purple and red arrows). Similarly large discrepancies can be seen for some other metrics, like in Fig. S10B and S10C1,C2. (And similar concerns apply to thalamocortical connections in section 2.5, where it looks like little to no data are available to verify the pairwise connectivity between the thalamic and cortical neurons via a direct comparison.)

This is concerning since this model forms the basis for multiple other studies of cortical dynamics and function by the same group and potentially others in the community, with multiple papers relying on it, whereas basic properties of connectivity are apparently not captured well.

On the other hand, this is also a "glass half full" situation, showing that the sophisticated algorithms for establishing connections, developed by the authors, are working well in at least half of the connection types explored. It is therefore imperative that the authors continue refining these algorithms to capture the remaining half in future iterations and producing improved models that the community can better rely on.

Please also note that Fig. S11 does not have a caption.

Reviewer #3 (Public review):

Summary:

The submission from Cronshagen and colleagues describes the application of a previously described method (selection linked integration) to the systematic study of PfEMP1 trafficking in the human malaria parasite Plasmodium falciparum. PfEMP1 is the primary virulence factor and surface antigen of infected red blood cells and is therefore a major focus of research into malaria pathogenesis. Since the discovery of the var gene family that encodes PfEMP1 in the late 1990s, there have been multiple hypotheses for how the protein is trafficked to the infected cell surface, crossing multiple membranes along the way. One difficulty in studying this process is the large size of the var gene family and the propensity of the parasites to switch which var gene is expressed, thus preventing straightforward gene modification-based strategies for tagging the expressed PfEMP1. Here the authors solve this problem by forcing the expression of a targeted var gene by fusing the PfEMP1 coding region with a drug-selectable marker separated by a skip peptide. This enabled them to generate relatively homogenous populations of parasites all expressing tagged (or otherwise modified) forms of PfEMP1 suitable for study. They then applied this method to study various aspects of PfEMP1 trafficking.

Strengths:

The study is very thorough, and the data are well presented. The authors used SLI to target multiple var genes, thus demonstrating the robustness of their strategy. They then perform experiments to investigate possible trafficking through PTEX, they knock out proteins thought to be involved in PfEMP1 trafficking and observe defects in cytoadherence, and they perform proximity labeling to further identify proteins potentially involved in PfEMP1 export. These are independent and complimentary approaches that together tell a very compelling story.

Weaknesses:

(1) When the authors targeted IT4var19, they were successful in transcriptionally activating the gene, however, they did not initially obtain cytoadherent parasites. To observe binding to ICAM-1 and EPCR, they had to perform selection using panning. This is an interesting observation and potentially provides insights into PfEMP1 surface display, folding, etc. However, it also raises questions about other instances in which cytoadherence was not observed. Would panning of these other lines have been successfully selected for cytoadherent infected cells? Did the authors attempt panning of their 3D7 lines? Given that these parasites do export PfEMP1 to the infected cell surface (Figure 1D), it is possible that panning would similarly rescue binding. Likewise, the authors knocked out PTP1, TryThrA, and EMPIC3 and detected a loss of cytoadhesion, but they did not attempt panning to see if this could rescue binding. To ensure that the lack of cytoadhesion in these cases is not serendipitous (as it was when they activated IT4var19), they should demonstrate that panning cannot rescue binding.

(2) The authors perform a series of trafficking experiments to help discern whether PfEMP1 is trafficked through PTEX. While the results were not entirely definitive, they make a strong case for PTEX in PfEMP1 export. The authors then used BioID to obtain a proxiome for PfEMP1 and identified proteins they suggest are involved in PfEMP1 trafficking. However, it seemed that components of PTEX were missing from the list of interacting proteins. Is this surprising and does this observation shed any additional light on the possibility of PfEMP1 trafficking through PTEX? This warrants a comment or discussion.

Reviewer #3 (Public review):

The authors aim to understand how gene pleiotropy affects parallel evolutionary changes among independent replicates of adaptation to a new hot environment of a set of experimental lines of Drosophila simulans using experimental evolution. The flies were RNAsequenced after more than 100 generations of lab adaptation and the changes in average gene expression were obtained relative to ancestral expression levels from reconstructed ancestral lines. Parallelism of gene expression change among lines is evaluated as variance in differential gene expression among lines relative to error variance. Similarly, the authors ask how the standing variation in gene expression estimated from a handful of flies from a reconstructed outbred line affects parallelism. The main findings are that parallelism in gene expression responses is positively associated with pleiotropy and negatively associated with expression variation. Those results are in contradiction with theoretical predictions and empirical findings. To explain those seemingly contradictory results the authors invoke the role of synergistic pleiotropy and correlated selection, although they do not attempt to measure either.

Strengths:

(1) The study uses highly replicated outbred laboratory lines of Drosophila simulans evolved in the lab under a constant hot regime for over 100 generations. This allows for robust comparisons of evolutionary responses among lines.

(2) The manuscript is well written and the hypotheses are clearly delineated at the onset.

(3) The authors have run a causal analysis to understand the causal dependencies between pleiotropy and expression variation on parallelism.

(4) The use of whole-body RNA extraction to study gene expression variation is well justified.

Weaknesses:

(1) It is unclear how well phenotypic variation in gene expression of the evolved lines has been estimated by the sample of 20 males from a reconstructed outbred line not directly linked to the evolved lines under study. I see this as a general weakness of the experimental design.

(2) There are no estimates of standing genetic variation of expression levels of the genes under study, only phenotypic variation. I wished the authors had been clear about that limitation and had discussed the consequences of the analysis. This also constitutes a weakness of the study.

(3) Moreover, since the phenotype studied is gene expression, its genetic basis extends beyond expressed sequences. The phenotypic variation of a gene's expression may thus likely misrepresent the genetic variation available for its evolution. The genetic variation of gene expression phenotypes could be estimated from a cross or pedigree information but since individuals were pool-sequenced (by batches of 50 males), this type of analysis is not possible in this study.

(4) The authors have not attempted to estimate synergistic pleiotropy among genes, nor how selection acts on gene expression modules. It makes any conclusion regarding the role of synergistic pleiotropy highly speculative.

I don't understand the reason why the analysis would be restricted to significantly differentially expressed genes only. It is then unclear whether pleiotropy, parallelism, and expression variation do play a role in adaptation because the two groups of adaptive and non-adaptive genes have not been compared. I recommend performing those comparisons to help us better understand how "adaptive" genes differentially contribute to adaptation relative to "non-adaptive" genes relative to their difference in population and genetic properties.

There is a lack of theoretical groundings on the role of so-called synergistic pleiotropy for parallel genetic evolution. The Discussion does not address this particular prediction. It could be removed from the Introduction.

Reviewer #3 (Public review):

Summary:

Heterochromatin is characterized by low transcription activity and late replication timing, both dependent on the NAD-dependent protein deacetylase Sir2, the founding member of the sirtuins. This manuscript addresses the mechanism by which Sir2 delays replication timing at the rDNA in budding yeast. Previous work from the same laboratory (Foss et al. PLoS Genetics 15, e1008138) showed that Sir2 represses transcription-dependent displacement of the Mcm helicase in the rDNA. In this manuscript, the authors show convincingly that the repositioned Mcms fire earlier and that this early firing partly depends on the ATPase activity of the nucleosome remodeler Fun30. Using read-depth analysis of sorted G1/S cells, fun30 was the only chromatin remodeler mutant that somewhat delayed replication timing in sir2 mutants, while nhp10, chd1, isw1, htl1, swr1, isw2, and irc5 had no effect. The conclusion was corroborated with orthogonal assays including two-dimensional gel electrophoresis and analysis of EdU incorporation at early origins. Using an insightful analysis with an Mcm-MNase fusion (Mcm-ChEC), the authors show that the repositioned Mcms in sir2 mutants fire earlier than the Mcm at the normal position in wild type. This early firing at the repositioned Mcms is partially suppressed by Fun30. In addition, the authors show Fun30 affects nucleosome occupancy at the sites of the repositioned Mcm, providing a plausible mechanism for the effect of Fun30 on Mcm firing at that position. However, the results from the MNAse-seq and ChEC-seq assays are not fully congruent for the fun30 single mutant. Overall, the results support the conclusions providing a much better mechanistic understanding how Sir2 affects replication timing at rDNA,

Strengths:

(1) The data clearly show that the repositioned Mcm helicase fires earlier than the Mcm in the wild type position.

(2) The study identifies a specific role for Fun30 in replication timing and an effect on nucleosome occupancy around the newly positioned Mcm helicase in sir2 cells.

Comments on revisions:

In the previous revision the authors addressed my concerns and improved the manuscript and the presentation of the data. All my recommendations were implemented.

Reviewer #3 (Public review):

Significance:

About 5% of metastatic castration-resistant prostate cancers (mCRPC) display genomic alterations in the transcriptional kinase CDK12. The mechanisms by which CDK12 alterations drive tumorigenesis in this molecularly-defined subset of mCRPC have remained elusive. In particular, some studies have suggested that CDK12 loss confers a homologous recombination deficiency (HRd) phenotype, However, clinical studies have not borne out the benefit to PARP inhibitors in patients with CDK12 alterations, despite the fact that these agents are typically active against tumors with HRd.

In this study, Frank et al. reconcile these findings by showing that: (1) tumors with biallelic CDK12 alterations do not have genomic features of HRd; (2) in vitro, HR gene downregulation occurs with acute depletion of CDK12 but is far less pronounced with chronic CDK12 loss; (3) CDK12-altered cells are uniquely sensitive to genetic or pharmacologic inhibition of CDK13.

Strengths:

Overall, this is an important study that reconciles disparate experimental and clinical observations. The genomic analyses are comprehensive and conducted with a high degree of rigor and represent an important resource to the community regarding the features of this molecular subtype of mCRPC.

Weaknesses:

(1) It is generally assumed that CDK12 alterations are inactivating, but it is noteworthy that homozygous deletions are comparatively uncommon (Figure 1a). Instead many tumors show missense mutations on either one or both alleles, and many of these mutations are outside of the kinase domain (Figure 1b). It remains possible that the CDK12 alterations that occur in some tumors may retain residual CDK12 function, or may confer some other neomorphic function, and therefore may not be accurately modeled by CDK12 knockout or knockdown in vitro. This would also reconcile the observation that knockout of CDK12 is cell-essential while the human genetic data suggest that CDK12 functions as a tumor suppressor gene.

(2) It is not entirely clear whether CDK12 altered tumors may require a co-occurring mutation to prevent loss of fitness, either in vitro or in vivo (e.g. perhaps one or more of the alterations that occur as a result of the TDP may mitigate against the essentiality of CDK12 loss).

Reviewer #4 (Public review):

Summary:

Li and Chouhan et al. follow up on a previous publication describing the role of anterior-posterior (ap) and medial (m) ɑ′/β′ Kenyon cells in mediating sleep-dependent and sleep-independent memory consolidation, respectively, based on feeding state in Drosophila melanogaster. The authors sequenced bulk RNA of ap ɑ′/β′ Kenyon cells 1h after flies were either trained-fed, trained-starved or untrained-fed and find a small number of genes (59) differentially expressed (3 upregulated, 56 downregulated) between trained-fed and trained-starved conditions. Many of these genes encode proteins involved in the regulation of gene expression. The authors then screened these differentially expressed genes for sleep phenotypes by expressing RNAi hairpins constitutively in ap ɑ′/β′ Kenyon cells and measuring sleep patterns. Two hits were selected for further analysis: Polr1F, which promoted sleep, and Regnase-1, which reduced sleep. The pan-neuronal expression of Polr1F and Regnase-1 RNAi constructs was then temporally restricted to adult flies using the GeneSwitch system. Polr1F sleep phenotypes were still observed, while Regnase-1 sleep phenotypes were not, indicating developmental defects. Appetitive memory was then assessed in flies with constitutive knockdown of Polr1F and Regnase-1 in ap ɑ′/β′ Kenyon cells. Polr1F knockdown did not affect sleep-dependent or sleep-independent memory, while Regnase-1 knockdown disrupted sleep-dependent memory, sleep-independent memory, as well as learning. Polr1F knockdown increased pre-ribosomal RNA transcripts in the brain, as measured by qPCR, in line with its predicted role as part of the RNA polymerase I complex. A puromycin incorporation assay to fluorescently label newly synthesized proteins also indicated higher levels of bulk translation upon Polr1F knockdown. Regnase-1 knockdown did not lead to observable changes in measurements of bulk translation.

Strengths:

The proposed involvement of RNA processing genes in regulating sleep and memory processes is interesting, and relatively unexplored. The methods are satisfactory.

Weaknesses:

The main weakness of previous versions of the paper was the over-interpretation of results, particularly relating to the proposed link between sleep and memory consolidation. This has now been appropriately addressed, as reflected in the change of title and incorporation of alternative interpretations of the data in the text.

Reviewer #3 (Public review):

Yan et al. ("When do visual category representations emerge in infant brains?") present an EEG study of category-specific visual responses in infancy from 3 to 15 months of age. In their experiment, infants viewed visually controlled images of faces and several non-face categories in a steady state evoked potential paradigm. The authors find visual responses at all ages, but face responses only at 4-6 months and older, and other category-selective responses at later ages. They find that spatiotemporal patterns of response can discriminate faces from other categories at later ages.

Overall, I found the study well-executed and a useful contribution to the literature. The study advances prior work by using well-controlled stimuli, subgroups at different ages, and new analytic approaches. The data and analyses support their conclusions regarding developmental change in neural responses to high-level visual stimuli.

Reviewer #3 (Public review):

Summary:

Non-opioid analgesics derived from human amniotic membrane (AM) product represents a novel and unique approach to analgesia that may avoid the traditional harms associated with opioids. Here, the study investigators demonstrate that HC-HAPTX3 is the primary bioactive component of the AM product FLO responsible for anti-nociception in mouse-model and in-vitro dorsal root ganglion (DRG) cell culture experiments. The mechanism is demonstrated to be via CD44 with an acute cytoskeleton rearrangement that is induced that inhibits Na+ and Ca++ current through ion channels. Taken together, the studies reported in the manuscript provide supportive evidence clarifying the mechanisms and efficacy of HC-HAPTX3 antinociception and analgesia.

Strengths:

Extensive experiments including murine behavioral paw withdrawal latency and Catwalk test data demonstrating analgesic properties. Breadth and depth of experimental data are clearly supporting mechanisms and antinociceptive properties.

Weaknesses:

None. Only a few minor directed changes to the text of the manuscript.<br /> P4 last sentence - "Our findings highlight the potential of a naturally derived biologic from human birth tissues as an effective non-opioid treatment for post-surgical pain and unravel the underlying mechanisms." - another sentence clause is required before "unravel"<br /> P7 second paragraph - please edit the following sentence for clarity: "Since HC-HA/PTX3 mimics FLO in producing pain inhibition, and it has high-purity and is more water-soluble than FLO, making it suitable for probing cellular mechanisms."

Reviewer #3 (Public review):

Summary:

In the study presented by Burnicka-Turek et al., the authors generated for the first time a mouse model to cause the combined conditional deletion of Tbx3 and Tbx5 genes. This has been impossible to achieve to date due to the proximity of these genes in chromosome 5, preventing the generation of loss of function strategies to delete simultaneously both genes. It is known that both Tbx3 and Tbx5 are required for the development of the cardiac conduction system by transcription factor-specific but also overlapping roles as seen in the common and diverse cardiac defects found in patients with mutations for these genes. After validating the deletion efficiency and specificity of the line, the authors characterised the cardiac phenotype associated with the cardiac conduction system (CCS)-specific combined deletion of Tbx5 and Tbx3 in the adult by inducing the activation of the CCS-specific tamoxifen-inducible Cre recombination (MinK-creERT) at 6 weeks after birth. Their analysis of 8-9-week-old animals did not identify any major morphological cardiac defects. However, the authors found conduction defects including prolonged PR and QTR intervals and ventricular tachycardia causing the death of the double mutants, which do not survive more than 3 months after tamoxifen induction. Molecular and optical mapping analysis of the ventricular conduction system (VCS) of these mutants concluded that, in the absence of Tbx5 and Tbx3 function, the cells forming the ventricular conduction system (VCS) become working myocardium and lose the specific contractile features characterising VCS cells. Altogether, the study identified the critical combined role of Tbx3 and Tbx5 in the maintenance of the VCS in adulthood.

Strengths:

The study generated a new animal model to study the combined deletion of Tbx5 and Tbx3 in the cardiac conduction system. This unique model has provided the authors with the perfect tool to answer their biological questions. The study includes top-class methodologies to assess the functional defects present in the different mutants analysed, and gathered very robust functional data on the conduction defects present in these mutants. They also applied optical action potential (OAP) methods to demonstrate the loss of conduction action potential and the acquisition of working myocardium action potentials in the affected cells because of Tbx5/Tbx3 loss of function. The study used simpler molecular and morphological analysis to demonstrate that there are no major morphological defects in these mutants and that indeed, the conduction defects found are due to the acquisition of working myocardium features by the VCS cells. Altogether, this study identified the critical role of these transcription factors in the maintenance of the VCS in the adult heart.

Weaknesses:

In the opinion of this reviewer, the weakness in the study lies in the morphological and molecular characterization. The morphological analysis simply described the absence of general cardiac defects in the adult heart, however, whether the CCS tissues are present or not was not investigated. Lineage tracing analysis using the reporter lines included in the crosses described in the study will determine if there are changes in CCS tissue composition in the different mutants studied. Similarly, combining this reporter analysis with the molecular markers found to be dysregulated by qPCR and western blot, will demonstrate that indeed the cells that were specified as VCS in the adult heart, become working myocardium in the absence of Tbx3 and Tbx5 function.

Reviewer #3 (Public review):

Summary:

The manuscript by Patel et al investigates the hypothesis that CDHR1a on photoreceptor outer segments is the binding partner for PCDH15 on the calyceal processes, and the absence of either adhesion molecule results in separation between the two structures, eventually leading to degeneration. PCDH15 mutations cause Usher syndrome, a disease of combined hearing and vision loss. In the ear, PCDH15 binds CDH23 to form tip links between stereocilia. The vision loss is less understood. Previous work suggested PCDH15 is localized to the calyceal processes, but the expression of CDH23 is inconsistent between species. Patel et al suggest that CDHR1a (formerly PCDH21) fulfills the role of CDH23 in the retina.

The experiments are mainly performed using the zebrafish model system. Expression of Pcdh15b and Cdhr1a protein is shown in the photoreceptor layer through standard confocal and structured illumination microscopy. The two proteins co-IP and can induce aggregation in vitro. Loss of either Cdhr1a or Pcdh15, or both, results in degeneration of photoreceptor outer segments over time, with cones affected primarily.

The idea of the study is logical given the photoreceptor diseases caused by mutations in either gene, the comparisons to stereocilia tip links, and the protein localization near the outer segments. The work here demonstrates that the two proteins interact in vitro and are both required for ongoing outer segment maintenance. The major novelty of this paper would be the demonstration that Pcdh15 localized to calyceal processes interacts with Cdhr1a on the outer segment, thereby connecting the two structures. Unfortunately, the data presented are inadequate proof of this model.

Strengths:

The in vitro data to support the ability of Pcdh15b and Cdhr1a to bind is well done. The use of pcdh15b and cdhr1a single and double mutants is also a strength of the study, especially being that this would be the first characterization of a zebrafish cdhr1a mutant.

Weaknesses:

(1) The imaging data in Figure 1 is insufficient to show the specific localization of Pcdh15 to calyceal processes or Cdhr1a to the outer segment membrane. The addition of actin co-labelling with Pcdh15/Cdhr1a would be a good start, as would axial sections. The division into rod and cone-specific imaging panels is confusing because the two cell types are in close physical proximity at 5 dpf, but the cone Cdhr1a expression is somehow missing in the rod images. The SIM data appear to be disrupted by chromatic aberration but also have no context. In the zebrafish image, the lines of Pcdh15/Cdhr1a expression would be 40-50 um in length if the scale bar is correct, which is much longer than the outer segments at this stage and therefore hard to explain.

(2) Figure 3E staining of Cdhr1a looks very different from the staining in Figure 1. It is unclear what the authors are proposing as to the localization of Cdhr1a. In the lab's previous paper, they describe Cdhr1a as being associated with the connecting cilium and nascent OS discs, and fail to address how that reconciles with the new model of mediating CP-OS interaction. And whether Cdhr1a localizes to discrete domains on the disc edges, where it interacts with Pcdh15 on individual calyceal processes.

(3) The authors state "In PRCs, Pcdh15 has been unequivocally shown to be localized in the CPs". However, the immunostaining here does not match the pattern seen in the Miles et al 2021 paper, which used a different antibody. Both showed loss of staining in pcdh15b mutants so unclear how to reconcile the two patterns.

(4) The explanation for the CRISPR targets for cdhr1a and the diagram in Figure 3 does not fit with crRNA sequences or the mutation as shown. The mutation spans from the latter part of exon 5 to the initial portion of exon 6, removing intron 5-6. It should nevertheless be a frameshift mutation but requires proper documentation.

(5) There are complications with the quantification of data. First, the number of fish analyzed for each experiment is not provided, nor is the justification for performing statistics on individual cell measurements rather than using averages for individual fish. Second, all cone subtypes are lumped together for analysis despite their variable sizes. Third, t-tests are inappropriately used for post-hoc analysis of ANOVA calculations.

(6) Unclear how calyceal process length is being measured. The cone measurements are shown as starting at the external limiting membrane, which is not equivalent to the origin of calyceal processes, and it is uncertain what defines the apical limit given the multiple subtypes of cones. In Figure 5, the lines demonstrating the measurements seem inconsistently placed.

(7) The number of fish analyzed by TEM and the prevalence of the phenotype across cells are not provided. A lower magnification view would provide context. Also, the authors should explain whether or not overgrowth of basal discs was observed, as seen previously in cdhr1-null frogs (Carr et al., 2021).

(8) The statement describing the separation between calyceal processes and the outer segment in the mutants is not backed up by the data. TEM or co-labelling of the structures in SIM could be done to provide evidence.

(9) "Based on work in the murine model and our own observations of rod CPs, we hypothesize that zebrafish rod CPs only extend along the newly forming OS discs and do not provide structural support to the ROS." Unclear how murine work would support that conclusion given the lack of CPs in mice, or what data in the manuscript supports this conclusion.

(10) The authors state "from the fact that rod CPs are inherently much smaller than cone CPs" without providing a reference. In the manuscript, the measurements do show rod CPs to be shorter, but there are errors in the cone measurements, and it is possible that the RPE pigment is interfering with the rod measurements.

(11) The discussion should include a better comparison of the results with ocular phenotypes in previously generated pcdh15 and cdhr1 mutant animals.

(12) The images in panels B-F of the Supplemental Figure are uncannily similar, possibly even of the same fish at different focal planes.

Reviewer #3 (Public review):

Summary:

Transcriptionally silent HIV-1 genomes integrated into the host`s genome represent the main obstacle to an HIV-1 cure. Therefore, agents aimed at promoting HIV transcription, the so-called latency reactivating agents (LRAs) might represent useful tools to render these hidden proviruses visible to the immune system. The authors successfully identified, through multiple techniques, INTS12, a component of the Integrator complex involved in 3' processing of small nuclear RNAs U1 and U2, as a factor promoting HIV-1 latency and hindering elongation of the HIV RNA transcripts. This factor synergizes with a previously identified combination of LRAs, one of which, AZD5582, has been validated in the macaque model for HIV persistence during therapy (https://pubmed.ncbi.nlm.nih.gov/37783968/). The other compound, I-BET151, is known to synergize with AZD5582, and is a inhibitor of BET, factors counteracting the elongation of RNA transcripts.

Strengths:

The findings were confirmed through multiple screens and multiple techniques. The authors successfully mapped the identified HIV silencing factor at the HIV promoter.

Weaknesses:

(1) Initial bias:<br /> In the choice of the genes comprised in the library, the authors readdress their previous paper (Hsieh et al.) where it is stated: "To specifically investigate host epigenetic regulators involved in the maintenance of HIV-1 latency, we generated a custom human epigenome specific sgRNA CRISPR library (HuEpi). This library contains sgRNAs targeting epigenome factors such as histones, histone binders (e.g., histone readers and chaperones), histone modifiers (e.g., histone writers and erasers), and general chromatin associated factors (e.g., RNA and DNA modifiers) (Fig 1B and 1C)".

From these figure panels, it clearly appears that the genes chosen are all belonging to the indicated pathways. While I have nothing to object to on the pertinence to HIV latency of the pathways selected, the authors should spend some words on the criteria followed to select these pathways. Other pathways involving epigenetic modifications and containing genes not represented in the indicated pathways may have been left apart.

(2) Dereplication:<br /> From Figure 1 it appears that INTS12 alone reactivates HIV -1 from latency alone without any drug intervention as shown by the MACGeCk score of DMSO-alone controls. If INTS12 knockdown alone shows antilatency effects, why, then were they unable to identify it in their previous article (Hsieh et al., 2023)? The authors should include some words on the comparison of the results using DMSO alone with those of the previous screen that they conducted.

(3) Translational potential:<br /> In order to propose a protein as a drug target, it is necessary to adhere to the "primum non nocere" principle in medicine. It is therefore fundamental to show the effects of INTS12 knockdown on cell viability/proliferation (and, advisably, T-cell activation). These data are not reported in the manuscript in its current form, and the authors are strongly encouraged to provide them.

Finally, as many readers may not be very familiar with the general principles behind CRISPR Cas9 screening techniques, I suggest addressing them in this excellent review: https://pmc.ncbi.nlm.nih.gov/articles/PMC7479249/.