Reviewer #2 (Public review):

Summary:

Chlamydial cell division is a peculiar event, whose mechanism was mysterious for many years. C. trachomatis division was shown to be polar and involve a minimal divisome machinery composed of both homologues of divisome and elongasome components, in the absence of an homologue of the classical division organizer FtsZ. In this paper, Harpring et al., show that FtsK is required at an early stage of the chlamydial divisome formation.

Strengths:

The manuscript is well-written and the results are convincing. Quantification of divisome component localization is well performed, number of replicas and number of cells assessed are sufficient to get convincing data. The use of a CRISPRi approach to knock down some divisome components is an asset and allows a mechanistic understanding of the hierarchy of divisome components.

Weaknesses:

The authors did not analyse the role of all potential chlamydial divisome components and did not show how FtsK may initiate the positioning of the divisome. Their conclusion that FtsK initiates the assembly of the divisome is an overinterpretation and is not backed by the data. However, data show convincingly that FtsK, if perhaps not the initiator of chlamydial division, is definitely an early and essential component of the chlamydial divisome.

Reviewer #2 (Public review):

This study uses all-atom MD simulation to explore the mechanics of channel opening for the NOMPC mechanosensitive channel. Previously the authors used MD to show that external forces directed along the long axis of the protein (normal to the membrane) result in AR domain compression and channel opening. This force causes two changes to the key TRP domains adjacent to the channel gate: 1) a compressive force pushes the TRP domain along the membrane normal, while 2) a twisting torque induces a clock-wise rotation on the TRP domain helix when viewing the bottom of the channel from the cytoplasm. Here, the authors wanted to understand which of those two changes is responsible for increasing the inner pore radius, and they show that it is the torque. The simulations in Figure 2 probe this question with different forces, and we can see the pore open with parallel forces in the membrane, but not with the membrane-normal forces. I believe this result as it is reproducible, the timescales are reaching 1 microsecond, and the gate is clearly increasing diameter to about 4 Å. This seems to be the most important finding in the paper, but the impact is limited since the authors already show how forces lead to channel opening, and this is further teasing apart the forces and motions that are actually the ones that cause the opening.

Reviewer #2 (Public review):

In this study, the authors sought to elucidate the neural mechanisms underlying the role of Naa10 in neurodevelopmental disruptions with a focus on its role in the hippocampus. The authors use an impressive array of techniques to identify a chain of events that occurs in the signaling pathway starting from Naa10 acetylating Btbd3 to regulation of F-actin dynamics that are fundamental to neurite outgrowth. They provide convincing evidence that Naa10 acetylates Btbd3, that Btbd3 facilitates CapZb binding to F-actin in a Naa10 acetylation-dependent manner, and that this CapZb binding to F-actin is key to neurite outgrowth. Besides establishing this signaling pathway, the authors contribute novel lists of Naa10 and Btbd3 interacting partners, which will be useful for future investigations into other mechanisms of action of Naa10 or Btbd3 through alternative cell signaling pathways. The evidence presented for an anxiety-like behavioral phenotype as a result of Naa10 dysfunction is mixed and tenuous, and assays for the primary behaviors known to be altered by Naa10 mutations in humans were not tested. As such, behavioral findings and their translational implications should be interpreted with caution. Finally, while not central to the main cell signaling pathway delineated, the characterization of brain region-specific and cell maturity of Naa10 expression patterns was presented in few to single animals and not quantified, and as such should also be interpreted with caution. On a broader level, these findings have implications for neurodevelopment and potentially, although not tested here, synaptic plasticity in adulthood, which means this novel pathway may be fundamental for brain health.

Summarized list of minor concerns

(1) The early claims of the manuscript are supported by very small sample sizes (often 1-3) and/or lack of quantification, particularly in Figures S1 and 1.

(2) Evidence is insufficient for CA1-specific knockdown of Naa10.

(3) The relationship between the behaviors measured, which centered around mood, and Ogden syndrome, was not clear, and likely other behavioral measures would be more translationally relevant for this study. Furthermore, the evidence for an anxiety-like phenotype was mixed.

(4) Btbd3 is characterized by the authors as an OCD risk gene, but its status as such is not well supported by the most recent, better-powered genome-wide association studies than the one that originally implicated Btbd3. However, there is evidence that Btbd3 expression, including selectively in the hippocampus, is implicated in OCD-relevant behaviors in mice.

(5) The reporting of the statistics lacks sufficient detail for the reader to deduce how experimental replicates were defined.

Reviewer #2 (Public review):

Summary:

This is an inspired study that merges the concept of individuality with evolutionary processes to uncover a new strategy that diversifies individual behavior that is also potentially evolutionarily adaptive.

The authors use a time-resolved measurement of spontaneous, innate behavior, namely handedness or turn bias in individual, isogenic flies, across several genetic backgrounds.

They find that an individual's behavior changes over time, or drifts. This has been observed before, but what is interesting here is that by looking at multiple genotypes, the authors find the amount of drift is consistent within genotype i.e., genetically regulated, and thus not entirely stochastic. This is not in line with what is known about innate, spontaneous behaviors. Normally, fluctuations in behavior would be ascribed to a response to environmental noise. However, here, the authors go on to find what is the pattern or rule that determines the rate of change of the behavior over time within individuals. Using modeling of behavior and environment in the context of evolutionarily important timeframes such as lifespan or reproductive age, they could show when drift is favored over bet-hedging and that there is an evolutionary purpose to behavioral drift. Namely, drift diversifies behaviors across individuals of the same genotype within the timescale of lifespan, so that the genotype's chance for expressing beneficial behavior is optimally matched with potential variation of environment experienced prior to reproduction. This ultimately increases the fitness of the genotype. Because they find that behavioral drift is genetically variable, they argue it can also evolve.

Strengths:

Unlike most studies of individuality, in this study, the authors consider the impact of individuality on evolution. This is enabled by the use of multiple natural genetic backgrounds and an appropriately large number of individuals to come to the conclusions presented in the study. I thought it was really creative to study how individual behavior evolves over multiple timescales. And indeed this approach yielded interesting and important insight into individuality. Unlike most studies so far, this one highlights that behavioral individuality is not a static property of an individual, but it dynamically changes. Also, placing these findings in the evolutionary context was beneficial. The conclusion that individual drift and bet-hedging are differently favored over different timescales is, I think, a significant and exciting finding.

Overall, I think this study highlights how little we know about the fundamental, general concepts behind individuality and why behavioral individuality is an important trait. They also show that with simple but elegant behavioral experiments and appropriate modeling, we could uncover fundamental rules underlying the emergence of individual behavior. These rules may not at all be apparent using classical approaches to studying individuality, using individual variation within a single genotype or within a single timeframe.

Weaknesses:

I am unconvinced by the claim that serotonin neuron circuits regulate behavioral drift, especially because of its bidirectional effect and lack of relative results for other neuromodulators. Without testing other neuromodulators, it will remain unclear if serotonin intervention increases behavioral noise within individuals, or if any other pharmacological or genetic intervention would do the same. Another issue is that the amount of drugs that the individuals ingested was not tracked. Variable amounts can result in variable changes in behavior that are more consistent with the interpretation of environmental plasticity, rather than behavioral drift. With the current evidence presented, individual behavior may change upon serotonin perturbation, but this does not necessarily mean that it changes or regulates drift.

However, I think for the scope of this study, finding out whether serotonin regulates drift or not is less important. I understand that today there is a strong push to find molecular and circuit mechanisms of any behavior, and other peers may have asked for such experiments, perhaps even simply out of habit. Fortunately, the main conclusions derived from behavioral data across multiple genetic backgrounds and the modeling are anyway novel, interesting, and in fact more fundamental than showing if it is serotonin that does it or not.

To this point, one thing that was unclear from the methods section is whether genotypes that were tested were raised in replicate vials and how was replication accounted for in the analyses. This is a crucial point - the conclusion that genotypes have different amounts of behavioral drift cannot be drawn without showing that the difference in behavioral drift does not stem from differences in developmental environment.

Reviewer #2 (Public review):

Summary:

The authors present a study of how modulatory activity from outside the classical receptive field (cRF) differs from cRF stimulation. They study neural activity across the different layers of V1 in two anesthetized monkeys using Neuropixels probes. The monkeys are presented with drifting gratings and border-ownership tuning stimuli. They find that border-ownership tuning is organized into columns within V1, which is unexpected and exciting, and that the flow of activity from cell-to-cell (as judged by cross-correlograms between single units) is influenced by the type of visual stimulus: border-ownership tuning stimuli vs. drifting-grating stimuli.

Strengths:

The questions addressed by the study are of high interest, and the use of Neuropixels probes yields extremely high numbers of single-units and cross-correlation histograms (CCHs) which makes the results robust. The study is well-described.

Weaknesses:

The weaknesses of the study are (a) the use of anesthetized animals, which raises questions about the nature of the modulatory signal being measured and the underlying logic of why a change in visual stimulus would produce a reversal in information flow through the cortical microcircuit and (b) the choice of visual stimuli, which do not uniquely isolate feedforward from feedback influences.

(1) The modulation latency seems quite short in Figure 2C. Have the authors measured the latency of the effect in the manuscript and how it compares to the onset of the visually driven response? It would be surprising if the latency was much shorter than 70ms given previous measurements of BO and figure-ground modulation latency in V2 and V1. On the same note, it might be revealing to make laminar profiles of the modulation (i.e. preferred - non-preferred border orientation) as it develops over time. Does the modulation start in feedback recipient layers?

(2) Can the authors show the average time course of the response elicited by preferred and non-preferred border ownership stimuli across all significant neurons?

(3) The logic of assuming that cRF stimulation should produce the opposite signal flow to border-ownership tuning stimuli is worth discussing. I suspect the key difference between stimuli is that they used drifting gratings as the cRF stimulus, the movement of the stimulus continually refreshes the retinal image, leading to continuous feedforward dominance of the signals in V1. Had they used a static grating, the spiking during the sustained portion of the response might also show more influence of feedback/horizontal connections. Do the initial spikes fired in response to the border-ownership tuning stimuli show the feedforward pattern of responses? The authors state that they did not look at cross-correlations during the initial response, but if they do, do they see the feedforward-dominated pattern? The jitter CCH analysis might suffice in correcting for the response transient.

(4) The term "nCRF stimulation" is not appropriate because the CRF is stimulated by the light/dark edge.

Reviewer #2 (Public review):

Summary:

This manuscript presents an important exploration of how intrinsic and synaptic conductances affect the robustness of neural circuits. This is a well-deserved question, and overall, the manuscript is written well and has a logical progression.

The focus on intrinsic plasticity as a potentially overlooked factor in network dynamics is valuable. However, while the stomatogastric ganglion (STG) serves as a well-characterized and valuable model for studying network dynamics, its simplified structure and specific dynamics limit the generalizability of these findings to more complex systems, such as mammalian cortical microcircuits.

Strengths:<br /> Clean and simple model. Simulations are carefully carried out and parameter space is searched exhaustively.

Weaknesses:

(1) Scope and Generalizability:<br /> The study's emphasis on intrinsic conductance is timely, but with its minimalistic and unique dynamics, the STG model poses challenges when attempting to generalize findings to other neural systems. This raises questions regarding the applicability of the results to more complex circuits, especially those found in mammalian brains and those where the dynamics are not necessarily oscillating. This is even more so (as the authors mention) because synaptic conductances in this study are inhibitory, and changes to their synaptic conductances are limited (as the driving force for the current is relatively low).

(2) Challenges in Comparison:<br /> A significant challenge in the study is the comparison method used to evaluate the robustness of intrinsic versus synaptic perturbations. Perturbations to intrinsic conductances often drastically affect individual neurons' dynamics, as seen in Figure 1, where such changes result in single spikes or even the absence of spikes instead of the expected bursting behavior. This affects the input to downstream neurons, leading to circuit breakdowns. For a fair comparison, it would be essential to constrain the intrinsic perturbations so that each neuron remains within a particular functional range (e.g., maintaining a set number of spikes). This could be done by setting minimal behavioral criteria for neurons and testing how different perturbation limits impact circuit function.

(3) Comparative Metrics for Perturbation:<br /> Another notable issue lies in the evaluation metrics for intrinsic and synaptic perturbations. Synaptic perturbations are straightforward to quantify in terms of conductance, but intrinsic perturbations involve more complexity, as changes in maximal conductance result in variable, nonlinear effects depending on the gating states of ion channels. Furthermore, synaptic perturbations focus on individual conductances, while intrinsic perturbations involve multiple conductance changes simultaneously. To improve fairness in comparison, the authors could, for example, adjust the x-axis to reflect actual changes in conductance or scale the data post hoc based on the real impact of each perturbation on conductance. For example, in Figure 6, the scale of the panels of the intrinsic (e.g., g_na-bar) is x500 larger than the synaptic conductance (a row below), but the maximal conductance for sodium hits maybe for a brief moment during every spike and than most of the time it is close to null. Moreover, changing the sodium conductance over the range of 0-250 for such a nonlinear current is, in many ways, unthinkable, did you ever measure two neurons with such a difference in the sodium conductance? So, how can we tell that the ranges of the perturbations make a meaningful comparison?

Reviewer #2 (Public review):

Summary:

The study investigates the potential influence of the response criterion on neural decoding accuracy in consciousness and unconsciousness, utilizing either simulated data or reanalyzing experimental data with post-hoc sorting data.

Strengths:

When comparing the neural decoding performance of Target versus NonTarget with or without post-hoc sorting based on subject reports, it is evident that response criterion can influence the results. This was observed in simulated data as well as in two experiments that manipulated the subject response criterion to be either more liberal or more conservative. One experiment involved a two-level response (seen vs unseen), while the other included a more detailed four-level response (ranging from 0 for no experience to 3 for a clear experience). The findings consistently indicated that adopting a more conservative response criterion could enhance neural decoding performance, whether in conscious or unconscious states, depending on the sensitivity or overall response threshold.

Weaknesses:

(1) The response criterion plays a crucial role in influencing neural decoding because a subject's report may not always align with the actual stimulus presented. This discrepancy can occur in cases of false alarms, where a subject reports seeing a target that was not actually there, or in cases where a target is present but not reported. Some may argue that only using data from consistent trials (those with correct responses) would not be affected by the response criterion. However, the authors' analysis suggests that a conservative response criterion not only reduces false alarms but also impacts hit rates. It is important for the authors to further investigate how the response criterion affects neural decoding even when considering only correct trials.

(2) The author has utilized decoding target vs. nontarget as the neural measures of unconscious and/or conscious processing. However, it is important to note that this is just one of the many neural measures used in the field. There are an increasing number of studies that focus on decoding the conscious content, such as target location or target category. If the author were to include results on decoding target orientation and how it may be influenced by response criterion, the field would greatly benefit from this paper.

for - climate crisis - planetary tipping points - irreversible? - Hansen disagrees - part 2 - climate crisis - comparison - planetary tipping points - Hansen vs Rockstrom

climate crisis - comparison - planetary tipping points - Hansen vs Rockstrom - Hansen makes a valid point. What Rockstrom might consider irreversible, although he doesn't explicitly say, but implies, Hansen speaks instead in more precise terms - The perspectives may be dependent on the knowledge that informs each scientist - Hansen's research into the unknown area of climate change, aeresols and cloud cover, is not considered in conventional knowledge that IPCC bases its conclusions on since it is unknown - Hansen's research uncovers that aeresols play a very large role, to such an extent that humans may be able to mitigate exceeding dangerous temperature thresholds pragmatically through aeresol interventions that impact cloud behavior -

This is a customer review (Amazon) of Spinfit Superfine tips (SS) for Airpods Pro 2, whose smallest tips (XS) still hurt my ears esp. the right ear. Note: Comply tips.

Reviewer #2 (Public review):

Summary:

The authors have performed a rigorous study to assess the role of ESR1+ neurons in the PMC to control the coordination of bladder and sphincter muscles during urination. This is an important extension of previous work defining the role of these brainstem neurons, and convincingly adds to the understanding of their role as master regulators of urination. This is a thorough, well-done study that clarifies how the Pontine micturition center coordinates different muscle groups for efficient urination, but there are some questions and considerations that remain.

Strengths:

These data are thorough and convincing in showing that ESR1+ PMC neurons exert coordinated control over both the bladder and sphincter activity, which is essential for efficient urination. The anatomical distinctions in pelvic versus pudendal control are clear, and it's an advance to understand how this coordination occurs. This work offers a clearer picture of how micturition is driven.

Weaknesses:

The dynamics of how this population of ESR1+ neurons is engaged in natural urination events remains unclear. Not all ESR1+neurons are always engaged, and it is not measured whether this is simply variation in population activity, or if more neurons are engaged during more intense starting bladder pressures, for instance. In particular, the response dynamics of single and doubly-projecting neurons are not defined. Additionally, the model for how these neurons coordinate with CRH+ neuron activity in the PMC is not addressed, although these cell types seem to be engaged at the same time. Lastly, it would be interesting to know how sensory input can likely modulate the activity of these neurons, but this is perhaps a future direction.

Reviewer #2 (Public Review):

Summary:

The manuscript presenting the discovery of a heparan-sulfate (HS) binding domain in monkeypox virus (MPXV) H3 protein as a new anti-poxviral drug target, presented by Bin Zhen and co-workers, is of interest, given that it offers a potentially broad antiviral substance to be used against poxviruses. Using new computational biology techniques, the authors identified a new alpha-helical domain in the H3 protein, which interacts with cell surface HS, and this domain seems to be crucial for H3-HS interaction. Given that this domain is conserved across orthopoxviruses, authors designed protein inhibitors. One of these inhibitors, AI-PoxBlock723, effectively disrupted the H3-HS interaction and inhibited infection with Monkeypox virus and Vaccinia virus. The presented data should be of interest to a diverse audience, given the possibility of an effective anti-poxviral drug.

Strengths:

In my opinion, the experiments done in this work were well-planned and executed. The authors put together several computational methods, to design poxvirus inhibitor molecules, and then they test these molecules for infection inhibition.

Comments on revised version:

The authors have addressed the comments I made in my review.

Reviewer #2 (Public review):

Summary:

In this study, the authors investigated the release properties of glutamate/GABA co-transmission at the supramammillary nucleus (SuM)-dentate granule cell (DGC) synapses using state -of-the-arts in vitro electrophysiology and anatomical approaches at the light and electron microscopy level. They found that SuM to dentate granule cell synapses, which co-release glutamate and GABA, exhibit distinct differences in paired-pulse ratio, Ca2+ sensitivity, presynaptic receptor modulation, and Ca2+ channel-vesicle coupling configuration for each neurotransmitter. The study shows that glutamate/GABA co-release produces independent glutamatergic and GABAergic synaptic responses, with postsynaptic targets segregated. They show that most SuM boutons form distinct glutamatergic and GABAergic synapses at proximity, characterized by GluN1 and GABAAα1 receptor labeling respectively. Furthermore, they demonstrate that glutamate/GABA co-transmission exhibits distinct short-term plasticity, with glutamate showing frequency-dependent depression and GABA showing frequency-independent stable depression. The authors provide compelling evidence at the anatomical and physiological levels that glutamate and GABA are co-release by different synaptic vesicles within the same synaptic terminal at the SuM-DGC synapses and that the distinct transmission modes of the glutamate and GABA release serve as a frequency-dependent filters of SuM inputs on GC outputs.<br /> This is a fundamental work, that significantly advances our understanding of the mechanism by which the two fast-acting and functionally opposing neurotransmitters glutamate and GABA are co-transmitted at the SuM-DGC synapses and the functional role of this type of Glutamate/GABA co-transmission.

Strengths:

The conclusions of this paper are provided by a large number of compelling data

Reviewer #2 (Public review):

Summary:

The authors are trying to test the hypothesis that ATP bursts are the predominant driver of antibiotic lethality of Mycobacteria

Strengths:

No significant strengths in the current state as it is written.

Weaknesses:

A major weakness is that M. smegmatis has a doubling time of three hours and the authors are trying to conclude that their data would reflect the physiology of M. tuberculossi that has a doubling time of 24 hours. Moreover, the authors try to compare OD measurements with CFU counts and thus observe great variabilities.

Comments on revisions:

I am surprised that the authors simply did not repeat the study in figure one with CFU counts and repeated in triplicate. Since this is M. smegmatis, it would take no longer than two weeks to repeat this experiment and replace the figure. I understand that obtaining CFU counts is much more laborious than OD measurements but it is necessary. Your graph still says that there is 0 bacteria at time 0, yet in your legend it says you started with 600,000 CFU/ml. I don't understand why this experiment was not repeated with CFU counts measured throughout. This is not a big ask since this is M. smegmatis but it appears that the authors do not want to repeat this experiment. Minimally, fix the graph to represent the CFU.

Reviewer #2 (Public review):

Summary:

The authors show convincing data that increasing NK cell function/frequency can reduce development and progression of metastatic disease after primary tumor resection.

Strengths:

The inclusion of a first-in-human trial highlighting some partial responses of metastatic patients treated with in vitro expanded NK cells is tantalising. It is difficult to perform trials in preventing further metastasis since the timelines are very protracted but more data like these highlighting a role for NK cells in improving local cDC1/T cells anti-tumor immunity will encourage deeper thinking around therapeutic approaches to target endogenous NK cells to achieve the same.

Weaknesses:

As always, more patient data would help increase confidence around the human relevance of the approach.

Comments on revisions:

The authors have addressed all my queries

Reviewer #2 (Public review):

This study from Dr. Emura and colleagues addresses the relevance of AGS3 mutations in the execution of asymmetric cell divisions promoting the formation of the micromere during sea-searching development. To this aim, the authors use quantitative imaging approaches to evaluate the localisation of AGS3 mutants truncated at the N-terminal region or at the C-terminal region, and correlate these distributions with the formation of micromere and correct development of embryos to the pluteus stage. The authors also analyse the capacity of these mutated proteins to rescue developmental defects observed upon AGS3 depletion by morpholino antisense nucleotides (MO). Collectively these experiments revealed that the C-terminus of AGS3, coding for four GoLoco motifs binding to cortical Gaphai proteins, is the molecular determinant for cortical localisation of AGS3 at the micromeres and correct pluteus development. Further genetic dissections and expression of chimeric AGS3 mutants carrying shuffled copies of the GoLoco motifs or four copies of the same motifs revealed that the position of GoLoco1 is essential for AGS3 functioning. To understand whether the AGS3-GoLoco1 evolved specifically to promote asymmetric cell divisions, the author analyse chimeric AGS3 variants in which they replaced the sea urchin GoLoco region with orthologs from other echinoids that do not form micromeres, or from Drosophila Pins or human LGN. These analyses corroborate the notion that the GoLoco1 position is crucial for asymmetric AGS3 functions. In the last part of the manuscript, the authors explore whether SpAGS3 interacts with the molecular machinery described to promote asymmetric cell division in eukaryotes, including Insc, NuMA, Par3 and Galphai, and show that all these proteins colocalize at the nascent micromere, together with the fate determinant Vasa. Collectively this evidence highlighted how evolutionarily selected AGS3 modifications are essential to sustain asymmetric divisions and specific developmental programs associated with them.

The manuscript addresses an interesting question and uses elegant genetic approaches associated with imaging analyses to elucidate the molecular mechanisms whereby AGS3 and spindle orientation proteins promote asymmetric divisions and specific developmental programs. This considered, it might be worth clarifying a few aspects of the reported findings.

(1) In some experimental settings, the presence of AGS3 mutants exacerbates the AGS3 deletion by MO (Fig. 4F). Can the author speculate on what can be the molecular explanation?

(2) Imaging analyses of Figure 4B-C suggest that the mutant AGS1111 does not localise at the vegetal cortex while AGS2222 does (Fig. 4C). However these mutants induce similar developmental defects (Fig. 4F) . What could be the reason?

(3) Figure 7 shows the crosstalk between AGS3 and other asymmetry players including NuMA. Vertebrate and Drosophila NuMA are ubiquitously present in tissues and localises to the spindle poles in mitosi. However in Figure 7A and 7E NuMA seems expressed only in a subset of sea urchin embryonic cells. Is this the case?

Reviewer #2 (Public review):

Summary:

Overall, this is a well-executed and insightful study. With some refinement to the presentation and a deeper exploration of the implications, the manuscript will make a significant contribution to the field of cancer genomics and personalized medicine.

Strengths:

The manuscript integrates multi-omics data with machine learning to address the significant heterogeneity of hepatocellular carcinoma (HCC). The use of multiple clustering algorithms and a consensus method strengthens the robustness of the findings. The study successfully develops a prognostic model with excellent predictive accuracy, validated across independent datasets. This adds considerable value to the field, particularly in providing individualized treatment strategies. The identification of two distinct liver cancer subtypes with different biological and metabolic characteristics is well-supported by the data, offering a promising direction for personalized medicine.

Weaknesses:

(1) Consider streamlining the presentation of methods, especially regarding the clustering algorithms and machine learning models. Readers may find it difficult to follow the exact process unless more clearly outlined.

(2) Some figures, such as the signaling pathways and heatmaps, are critical to understanding the study's findings. Ensure that all figures are high quality, easy to interpret, and adequately labeled. You may also want to highlight the key findings within the figure captions more explicitly.

(3) While the manuscript does compare its prognostic model to those previously published, the novelty of the findings could be emphasized more clearly. Discussing the potential limitations of the study (e.g., the reliance on computational models and small sample sizes for scRNA-seq) could strengthen the manuscript.

(4) The manuscript mentions that the data was split into training and validation datasets in a 1:1 ratio. How was the performance verified? Is there an independent test set?

(5) The role of the MIF signaling pathway in subtype differentiation is intriguing, but further mechanistic insights into how this pathway drives the differences between CS1 and CS2 could be discussed in more detail. If experimental evidence for this pathway exists in the literature, it should be mentioned.

(6) Some sentences are quite long and complex, which can affect readability. Breaking them down into shorter, clearer sentences would improve the flow.

Reviewer #2 (Public review):

Summary:

The authors seek to use single-cell sequencing approaches to identify TCRs specific for the SARS CoV2 spike protein, select a candidate TCR for cloning and use it to construct a TCR transgenic mouse. The argument is that this process is less cumbersome than the classical approach, which involves the identification of antigen-reactive T cells in vitro and the construction of T cell hybridomas prior to TCR cloning. TCRs identified by single-cell sequencing that is already paired to transcriptomic data would more rapidly identify TCRs that are likely to contribute to a functional response. The authors successfully identify TCRs that have expanded in response to SARS CoV2 spike protein immunization, bind to MHC tetramers and express genes associated with functional response. They then select a TCR for cloning and construction of a transgenic mouse in order to test the response of resulting T cells in vivo following immunization with spike protein of coronavirus infection.

Strengths:

(1) The study provides proof of principle for the identification and characterization of TCRs based on single-cell sequencing data.

(2) The authors employ a recently developed software tool (DALI) that assists in linking transcriptomic data to individual clones.

(3) The authors successfully generate a TCR transgenic animal derived from the most promising T cell clone (CORSET8) using the TCR sequencing approach.

(4) The authors provide initial evidence that CORSET8 T cells undergo activation and proliferation in vivo in response to immunization or infection.

(5) Procedures are well-described and readily reproducible.

Weaknesses:

(1) The purpose of presenting a failed attempt to generate TCR transgenic mice using a traditional TCR hybridoma method is unclear. The reasons for the failure are uncertain, and the inclusion of this data does not really provide information on the likely success rate of the hybridoma vs single cell approach for TCR identification, as only a single example is provided for either.

(2) There is little information provided regarding the functional differentiation of the CORSET8 T cells following challenge in vivo, including expression of molecules associated with effector function, cytokine production, killing activity and formation of memory. The study would be strengthened by some evidence that CORSET8 T cells are successfully recapitulating the functional features of the endogenous immune response (beyond simply proliferating and expressing CD44). This information is important to evaluate whether the presented sequencing-based identification and selection of TCRs is likely to result in T-cell responses that replicate the criteria for selecting the TCR in the first place.

(3) While I find the argument reasonable that the approach presented here has a lot of likely advantages over traditional approaches for generating TCR transgenic animals, the use of TCR sequencing data to identify TCRs for study in variety of areas, including cancer immunotherapy and autoimmunity, is in broad use. While much of this work opts for alternative methods of TCR expression in primary T cells (i.e. CRISPR or retroviral approaches), the process of generating a TCR transgenic mouse from a cloned TCR is not in itself novel. It would be helpful if the authors could provide a more extensive discussion explaining the novelty of their approach for TCR identification in comparison to other more modern approaches, rather than only hybridoma generation.

Comments on revisions:

The authors have provided additional clarification on the comparisons between the presented method for TCR transgenic generation and the hybridoma method that is more commonly used and added additional verification of the functional response in vivo of T cells expressing the selected TCR. Overall, these additions enhance the evidence that the proposed methods are likely to identify TCRs with a strong immune activation profile and are a reasonable response to the first round of review.

Reviewer #2 (Public review):

Summary:

This study aims to demonstrate that E. coli can acquire rapid antibiotic resistance mutations in the absence of a DNA damage response. The authors employed a modified Adaptive Laboratory Evolution (ALE) workflow to investigate this, initiating the process by diluting an overnight culture 50-fold into an ampicillin selection medium. They present evidence that a recA- strain develops ampicillin resistance mutations more rapidly than the wild-type, as indicated by the Minimum Inhibitory Concentration (MIC) and mutation frequency. Whole-genome sequencing of recA- colonies resistant to ampicillin showed predominant inactivation of genes involved in the multi-drug efflux pump system, contrasting with wild-type mutations that seem to activate the chromosomal ampC cryptic promoter. Further analysis of mutants, including a lexA3 mutant incapable of inducing the SOS response, led the authors to conclude that the rapid evolution of antibiotic resistance occurs via an SOS-independent mechanism in the absence of recA. RNA sequencing suggests that antioxidative response genes drive the rapid evolution of antibiotic resistance in the recA- strain. They assert that rapid evolution is facilitated by compromised DNA repair, transcriptional repression of antioxidative stress genes, and excessive ROS accumulation.

Strengths:

The experiments are well-executed and the data appear reliable. It is evident that the inactivation of recA promotes faster evolutionary responses, although the exact mechanisms driving this acceleration remain elusive and deserve further investigation.

Weaknesses:

Some conclusions are overstated. For instance, the conclusion regarding the LexA3 allele, indicating that rapid evolution occurs in an SOS-independent manner (line 217), contradicts the introductory statement that attributes evolution to compromised DNA repair. The claim made in the discussion of Figure 3 that the hindrance of DNA repair in recA- is crucial for rapid evolution is at best suggestive, not demonstrative. Additionally, the interpretation of the PolI data implies its role, yet it remains speculative. In Figure 2A table, mutations in amp promoters are leading to amino acid changes! The authors' assertion that ampicillin significantly influences persistence pathways in the wild-type strain, affecting quorum sensing, flagellar assembly, biofilm formation, and bacterial chemotaxis, lacks empirical validation. Figure 1G suggests that recA cells treated with ampicillin exhibit a strong mutator phenotype; however, it remains unclear if this can be linked to the mutations identified in Figure 2's sequencing analysis.

Reviewer #2 (Public review):

In the manuscript, the authors combine SARS-CoV-2 sequence data from a state in Germany and mobility data to help in understanding the movement of virus and the potential to help decide where to focus sequencing. The global expansion in sequencing capability is a key outcome of the public health response. However, there remains uncertainty how to maximise the insights the sequence data can give. Improved ability to predict the movement of emergent variants would be a useful public health outcome.

However, I remain unconvinced that changing surveillance strategies is necessarily sensible as it remains unclear what the ultimate benefit of variant hunting is. Decisions to adapt surveillance strategies should not be taken lightly as there are substantial benefits of maintaining a stable and as representative as possible, system over time. It's unclear what public health action would result of detecting a few more sequences of a variant. Once a variant has been identified (arguably anywhere in the world/region), we already have the necessary information to motivate the development of updated vaccines/monoclonals.

Reviewer #2 (Public review):

Summary:

The authors set out to develop a tissue culture method in which to study the different regenerative abilities of the central and peripheral branch of sensory axons. Neurons developed a small and large branch, which have different regenerative abilities, different transport rates, and different microtubule properties. The study provides convincing evidence that the two axonal branches differ in a way to correspond to in vivo. The different regenerative abilities of the two branches are an important observation because until now it has not been clear whether this difference is intrinsic to the neuron and axons or due to differences in the environment surrounding the axons. The authors have then looked for molecular explanations of the differences between the branches. They find different transport rates and different microtubule dynamics. The different microtubule dynamics are explained by differing levels of spastin, an enzyme that severs microtubules encouraging dynamics.

Strengths:

The differences between the two branches are clearly shown, together with differences in transport, microtubule dynamics, and regeneration. The in vitro model is novel and could be widely used. The methods used are robust and generally accepted.

Weaknesses:

In order for the method to be used it needs to be better described. For instance what proportion of neurons develop just two axonal branches, one of which is different? How selective are the researchers in finding appropriate neurons?

Reviewer #2 (Public review):

Summary:

The authors provide four new annotated genomes for an important taxon within Mollusca known as Polyplacophora (chitons). They provide an impressive analysis showing syntenic relationships between the chromosomes of these four genomes but also other available chiton genome sequences and analysis of 20 molluscan linkage groups to expand this analysis across Mollusca.

Strengths:

The authors have selected particular chiton species for genome sequencing and annotation that expand what is known about genomes across portions of chiton phylogenetic diversity lacking genome sequences. The manuscript is well-written and illustrated in a concise manner. The figures are mostly clear, allowing a reader to visually compare the syntenic relationships of chromosomes, especially within chitons. Their phylogenetic analysis provides a simple manner to map important events in molluscan genome evolution. This study greatly expands what is known about molluscan and chiton comparative genomics.

Weaknesses:

I am not especially convinced that chitons have experienced more substantial genomic rearrangements or other genomic events than other molluscan classes, and for this reason, I did not personally find the title compelling: "Still waters run deep: Large scale genome rearrangements in the evolution of morphologically conservative Polyplacophora." Are the documented events "large scale genomic rearrangements"? It seems that mostly they found two cases of chromosome fusion, plus one apparent case of whole genome duplication. What do they mean by "Still waters run deep"? I have no idea. I guess they consider chitons to be morphologically conservative in their appearance and lifestyle so they are calling attention to this apparent paradox. However, most chiton genomes seem to be relatively conserved, but there are unexpected chromosome fusion events within a particular genus, Acanthochitona. Likewise, they found a large-scale gene duplication event in Acanthopleurinae, a different subfamily of chitons, which is quite interesting but these seem to be geologically recent events that do not especially represent the general pattern of genome evolution across this ancient molluscan taxon.

Reviewer #2 (Public review):

The study by Liu et al provides a functional analysis of lnc-FANCI-2 in cervical carcinogenesis, building on their previous discovery of FANCI-2 being upregulated in cervical cancer by HPV E7.

The authors conducted a comprehensive investigation by knocking out (KO) FANCI-2 in CaSki cells and assessing viral gene expression, cellular morphology, altered protein expression and secretion, altered RNA expression through RNA sequencing (verification of which by RT-PCR is well appreciated), protein binding, etc. Verification experiments by RT-PCR, Western blot, etc are notable strengths of the study.

The KO and KD were related to increased Ras signaling and EMT and reduced IFN-y/a responses.

Although the large amount of data is well acknowledged, it is a limitation that most data come from CaSki cells, in which FANCI-2 localization is different from SiHa cells and cancer tissues (Figure 1). The cytoplasmic versus nuclear localization is somewhat puzzling.

Reviewer #2 (Public review):

Summary:

In this manuscript, Ruhling et al propose a rapid uptake pathway that is dependent on lysosomal exocytosis, lysosomal Ca2+ and acid sphingomyelinase, and further suggest that the intracellular trafficking and fate of the pathogen is dictated by the mode of entry.

The evidence provided is solid, methods used are appropriate and results largely support their conclusions, but can be substantiated further as detailed below. The weakness is a reliance on chemical inhibitors that can be non-specific to delineate critical steps.

Specific comments:

A large number of experiments rely on treatment with chemical inhibitors. While this approach is reasonable, many of the inhibitors employed such as amitriptyline and vacuolin1 have other or non-defined cellular targets and pleiotropic effects cannot be ruled out. Given the centrality of ASM for the manuscript, it will be important to replicate some key results with ASM KO cells.

Most experiments are done in HeLa cells. Given the pathway is projected as generic, it will be important to further characterize cell type specificity for the process. Some evidence for a similar mechanism in other cell types S. aureus infects, perhaps phagocytic cell type, might be good.

I'm a little confused about the role of ASM on the surface. Presumably, it converts SM to ceramide, as the final model suggests. Overexpression of b-toxin results in the near complete absence of SM on phagosomes (having representative images will help appreciate this), but why is phagosomal SM detected at high levels in untreated conditions? If bacteria are engulfed by SM-containing membrane compartments, what role does ASM play on the surface? If surface SM is necessary for phagosomal escape within the cell, do the authors imply that ASM is tuning the surface SM levels to a certain optimal range? Alternatively, can there be additional roles for ASM on the cell surface? Can surface SM levels be visualized (for example, in Figure 4 E, F)?

Related to that, why is ASM activity on the cell surface important? Its role in non-infectious or other contexts can be discussed.

If SM removal is so crucial for uptake, can exocytosis of lysosomes alone provide sufficient ASM for SM removal? How much or to what extent is lysosomal exocytosis enhanced by initial signaling events? Do the authors envisage the early events in their model happening in localized confines of the PM, this can be discussed.

How are inhibitor doses determined? How efficient is the removal of extracellular bacteria at 10 min? It will be good to substantiate the cfu experiments for infectivity with imaging-based methods. Are the roles of TPC1 and TPC2 redundant? If so, why does silencing TPC1 alone result in a decrease in infectivity? For these and other assays, it would be better to show raw values for infectivity. Please show alterations in lysosomal Ca2+ at the doses of inhibitors indicated. Is lysosomal Ca2+ released upon S. aureus binding to the cell surface? Will be good to directly visualize this.

The precise identification of cytosolic vs phagosomal bacteria is not very easy to appreciate. The methods section indicates how this distinction is made, but how do the authors deal with partial overlaps and ambiguities generally associated with such analyses? Please show respective images. The number of events (individual bacteria) for the live cell imaging data should be clearly mentioned.

In the phagosome maturation experiments, what is the proportion of bacteria in Rab5 or Rab7 compartments at each time point? Will the decreased Rab7 association be accompanied by increased Rab5? Showing raw values and images will help appreciate such differences. Given the expertise and tools available in live cell imaging, can the authors trace Rab5 and Rab7 positive compartment times for the same bacteria?

The results with longer-term infection are interesting. Live cell imaging suggests that ASM-inhibited cells show accelerated phagosomal escape that reduces by 6 hpi. Where are the bacteria at this time point ? Presumably, they should have reached lysosomes. The relationship between cytosolic escape, replication, and host cell death is interesting, but the evidence, as presented is correlative for the populations. Given the use of live cell imaging, can the authors show these events in the same cell?

Given the inherent heterogeneity in uptake processes and the use of inhibitors in most experiments, the distinction between ASM-dependent and independent pathways might not be as clear-cut as the authors suggest. Some caution here will be good. Can the authors estimate what fraction of intracellular bacteria are taken up ASM-dependent?

Reviewer #2 (Public review):

Summary:

The manuscript by Liang and Guan provides an impressive attempt to characterize the conformational free energy landscape of a melibiose permease (MelB), a symporter member of major facilitator superfamily (MFS) of transporters. Although similar studies have been conducted previously for other members of MFS, each member or subfamily has its own unique features that make the employment of such methods quite challenging. While the methodology is indeed impressive, characterizing the coupling between large-scale conformational changes and substrate binding in membrane transporters is quite challenging and requires a sophisticated methodology. The conclusions obtained from the three sets of path-optimization and free energy calculations done by the authors are generally supported by the provided data and certainly add to our understanding of how sodium binding facilitates the transport of melibiose in MelB. However, the data is not generated reliably which questions the relevance of the conclusions as well. I particularly have some concerns regarding the implementation of the methodology that I will discuss below.

(1) In enhanced sampling techniques, often much attention is given to the sampling algorithm. Although the sampling algorithm is quite important and this manuscript has chosen an excellent pair: string method with swarms of trajectories (SMwST) and replica-exchange umbrella sampling (REUS) for this task, there are other important factors that must be taken into account. More specifically, the collective variables used and the preparation of initial conformations for sampling. I have objectives for both of these (particularly the latter) that I detail below. Overall, I am not confident that the free energy profiles generated (summarized in Figure 5) are reliable, and unfortunately, much of the data presented in this manuscript heavily relies on these free energy profiles.

(2) The authors state that they have had an advantage over other similar studies in that they had two endpoints of the string to work from experimental data. I agree that this is an advantage. However, this could lead to some dangerous flaws in the methodology if not appropriately taken into account. Proteins such as membrane transporters have many slow degrees of freedom that can be fully captured within tens of nanoseconds (90 ns was the simulation time used here for the REUS). Biased sampling allows us to overcome this challenge to some extent, but it is virtually impossible to take into account all slow degrees of freedom in the enhanced sampling protocol (e.g., the collective variables used here do not represent anything related to sidechain dynamics). Therefore, if one mixes initial conformations that form different initial structures (e.g., an OF state and an IF state from two different PDB files), it is very likely that despite all equilibration and relaxation during SMwST and REUS simulations, the conformations that come from different sources never truly mix. This is dangerous in that it is quite difficult to detect such inconsistencies and from a theoretical point of view it makes the free energy calculations impossible. Methods such as WHAM and its various offshoots all rely on overlap between neighboring windows to calculate the free energy difference between two windows and the overlap should be in all dimensions and not just the ones that we use for biasing. This is related to well-known issues such as hidden barriers and metastability. If one uses two different structures to generate the initial conformations, then the authors need to show their sampling has been long enough to allow the two sets of conformations to mix and overlap in all dimensions, which is a difficult task to do.

(3) I also have concerns regarding the choice of collective variables. The authors have split the residues in each transmembrane helix into the cyto- and periplasmic sides. Then they have calculated the mass center distance between the cytoplasmic sides of certain pairs of helices and have also done the same for the periplasmic side. Given the shape of a helix, this does not seem to be an ideal choice since rather than the rotational motion of the helix, this captures more the translational motion of the helix. However, the transmembrane helices are more likely to undergo rotational motion than the translational one.

(4) Convergence: String method convergence data does not show strong evidence for convergence (Figure S2) in my opinion. REUS convergence is also not discussed. No information is provided on the exchange rate or overlap between the windows.

Reviewer #2 (Public review):

Summary:

This manuscript builds upon the work of a previous study published by the group (Dennison, 2021) to further elucidate the coregulatory axis of Srsf3 and PDGFRa on craniofacial development. The authors in this study investigated the molecular mechanisms by which PDGFRa signaling activates the RNA-binding protein Srsf3 to regulate alternative splicing (AS) and gene expression (GE) necessary for craniofacial development. PDGFRa signaling-mediated Srsf3 phosphorylation drives its translocation into the nucleus and affect binding affinity to different proteins and RNA, but the exact molecular mechanisms were not known. The authors performed RNA sequencing on immortalized mouse embryonic mesenchyme (MEPM) cells treated with shRNA targeting 3' UTR of Srsf3 or scramble shRNA (to probe AS and DE events that are Srsf3 dependent) and with and without PDGF-AA ligand treatment (to probe AS and DE events that are PDGFRa signaling dependent). They found that PDGFRa signaling has more effect on AS than on DE. A matching eCLIP-seq experiment was performed to investigate how Srsf3 binding sites change with and without PDGFRa signaling.

Strengths:

(1) The work builds well upon the previous data and the authors employ a variety of appropriate techniques to answer their research questions.

(2) The authors show that Srsf3 binding pattern within the transcript as well as binding motifs change significantly upon PDGFRa signaling, providing a mechanistic explanation for the significant changes in AS.

(3) By combining RNA-seq and eCLIP datasets together, the authors identified a list of genes that are directly bound by Srsf3 and undergo changes in GE and/or AS. Two examples are Becn1 and Wdr81, which are involved in early endosomal trafficking.

Weaknesses:

(1) The authors identify two genes whose AS are directly regulated by Srsf3 and involved in endosomal trafficking; however, they do not validate the differential AS results and whether changes in these genes can affect endosomal trafficking. In Figure 6, they show that PDGFRa signaling is involved in endosome size and Rab5 colocalization, but do not show how Srsf3 and the two genes are involved.

(2) The proposed model does not account for other proteins mediating the activation of Srsf3 after Akt phosphorylation. How do we know this is a direct effect (and not secondary or tertiary effect)?

This is a thoroughly revised manuscript. I would like to congratulate the authors to have invested a lot of time, resources, new data, and a more refined discussion to make this a compelling piece of work. I have no further concerns.

Reviewer #2 (Public review):

Summary:

Malaria transmission in the Gambia is highly seasonal, whereby periods of intense transmission at the beginning of the rainy season are interspersed by long periods of low to no transmission. This raises several questions about how this transmission pattern impacts the spatiotemporal distribution of circulating parasite strains. Knowledge of these dynamics may allow the identification of key units for targeted control strategies, the evaluation of the effect of selection/drift on parasite phenotypes (e.g., the emergence or loss of drug resistance genotypes), and analyze, through the parasites' genetic nature, the duration of chronic infections persisting during the dry season. Using a combination of barcodes and whole genome analysis, the authors try to answer these questions by making clever use of the different recombination rates, as measured through the proportion of genomes with identity-by-descent (IBD), to investigate the spatiotemporal relatedness of parasite strains at different spatial (i.e., individual, household, village, and region) and temporal (i.e., high, low, and the corresponding the transitions) levels. The authors show that a large fraction of infections are polygenomic and stable over time, resulting in high recombinational diversity (Figure 2). Since the number of recombination events is expected to increase with time or with the number of mosquito bites, IBD allows them to investigate the connectivity between spatial levels and to measure the fraction of effective recombinational events over time. The authors demonstrate the epidemiological connectivity between villages by showing the presence of related genotypes, a higher probability of finding similar genotypes within the same household, and how parasite-relatedness gradually disappears over time (Figure 3). Moreover, they show that transmission intensity increases during the transition from dry to wet seasons (Figure 4). If there is no drug selection during the dry season and if resistance incurs a fitness cost it is possible that alleles associated with drug resistance may change in frequency. The authors looked at the frequencies of six drug-resistance haplotypes (aat1, crt, dhfr, dhps, kelch13, and mdr1), and found no evidence of changes in allele frequencies associated with seasonality. They also find chronic infections lasting from one month to one and a half years with no dependence on age or gender.

The use of genomic information and IBD analytic tools provides the Control Program with important metrics for malaria control policies, for example, identifying target populations for malaria control and evaluation of malaria control programs.

Strength:

The authors use a combination of high-quality barcodes (425 barcodes representing 101 bi-allelic SNPs) and 199 high-quality genome sequences to infer the fraction of the genome with shared Identity by Descent (IBD) (i.e. a metric of recombination rate) over several time points covering two years. The barcode and whole genome sequence combination allows full use of a large dataset, and to confidently infer the relatedness of parasite isolates at various spatiotemporal scales.

Reviewer #2 (Public review):

Summary:

This observational study investigates the efficacy of intracameral injected human stem cells as a means to re-functionalize the trabecular meshwork for the restoration of intraocular pressure homeostasis. Using a murine model of glaucoma, human adipose-derived mesenchymal stem cells are shown to be biologically safer and functionally superior at eliciting a sustained reduction in intraocular pressure (IOP). The authors conclude that the use of human adipose-derived mesenchymal stem cells has the potential for long-term treatment of ocular hypertension in glaucoma.

Strengths:

A noted strength is the use of a magnetic steering technique to direct injected stem cells to the iridocorneal angle. An additional strength is the comparison of efficacy between two distinct sources of stem cells: human adipose-derived mesenchymal vs. induced pluripotent cell derivatives. Utilizing both in vivo and ex vivo methodology coupled with histological evidence of introduced stem cell localization provides a consistent and compelling argument for a sustainable impact exogenous stem cells may have on the re-functionalization of a pathologically compromised TM.

Weaknesses:

A noted weakness of the study, as pointed out by the authors, includes the unanticipated failure of the genetic model to develop glaucoma-related pathology (elevated IOP, TM cell changes). While this is most unfortunate, it does temper the conclusion that exogenous human adipose derived mesenchymal stem cells may restore TM cell function. Given that TM cell function was not altered in their genetic model, it is difficult to say with any certainty that the introduced stem cells would be capable of restoring pathologically altered TM function. A restoration effect remains to be seen. Another noted complication to these findings is the observation that sham intracameral-injected saline control animals all showed elevated IOP and reduced outflow facility, compared to WT or Tg untreated animals, which allowed for more robust statistically significant outcomes. Additional comments/concerns that the authors may wish to address are elaborated in the Private Review section.

Reviewer #2 (Public review):

Summary:

The authors used a chemical linker to induce phase separation in U2OS cell nuclei with two different proteins, a coiled-coil protein (Mad1) and a disordered domain (from LAF-1), whose condensates were purported to have different material properties. First, they performed Fluorescence Recovery After Photobleaching (FRAP) and estimated the viscosity via the Stokes-Einstein equation. Combined with droplet fusion assays, this yielded an estimate of the surface tension, wherein the disordered condensates were found to have 130 times higher surface tension than the coiled-coil condensates. Confocal fluorescence microscopy was used to follow condensates over time, enabling classification of growth events as either fusion-, ripening-, or diffusion-based, and subsequent comparison of the relative abundances of these growth events between the two condensate types. Coiled-coil condensates grew primarily by diffusive processes, whereas disordered condensates grew primarily by ripening processes. The coarsening rates were described by growth exponents extracted from power-law fits of average normalized condensate radius over time. In both cases, these growth exponents were smaller than those predicted by theory, leading the authors to propose that nuclear condensate growth is generally suppressed by chromatin mechanics, as found in previous studies albeit with different exponents. The authors developed a theory to understand how the extent of this effect may depend on condensate material properties like surface tension. Treating chromatin as a neo-Hookean elastic solid, the authors assume a form of mechanical pressure that plateaus with increasing condensate size, and the resulting theory is used to analyze the observed condensate growth dynamics. A linearized extension of the theory is used to distinguish between suppressed, elastic, and Ostwald ripening. Finally, the authors consider the impact of different chromatin environments on condensate growth patterns and dynamics, which is achieved experimentally with another cell type (HeLa) and with a drug that decondenses chromatin (TSA). They find that condensate growth patterns are not significantly changed in either condensate type, but that the number of condensates nucleated and their related growth exponent are more sensitive to variations in chromatin stiffness in the coiled-coil system due to its low surface tension.

Strengths:

This work provides evidence that nuclear condensates can coarsen not only by fusion but also by continuous diffusive growth processes, predominant in coiled-coil condensates, and ripening, predominant in disordered condensates. Across these different condensate types and coarsening mechanisms, the authors find growth exponents lower than theoretical expectations, reinforcing the notion that elastic media can suppress condensate growth in the nucleus. Combined with theory, these observed differences in growth patterns and rates are argued to originate from differences in material properties, namely, surface tension relative to local chromatin stiffness. The authors further suggest that the few ripening events that are seen in coiled-coil condensates may be elastic in nature due to gradients in chromatin stiffness as opposed to Ostwald ripening. If this assertion proves to be robust, it would mark an early observation of elastic ripening in living cells.

Weaknesses:

(1) The assertion that nuclear condensates experience an external pressure from the chromatin network implies that chromatin should be excluded from the condensates (Nott et al., Molecular Cell (2015); Shin et al., Cell (2018)). This has not been shown or discussed here. While Movie 1 suggests the coiled-coil condensates may exclude chromatin, Movie 2 suggests the disordered condensates do not. LAF-1, as an RNA helicase, interacts with RNA, and RNA can be associated with chromatin in the nucleus. RNA can also modulate droplet viscosity. The authors' analysis of the disordered condensate data only makes sense if these condensates exclude chromatin, which they have not demonstrated, and which appears not to be the case.

(2) Critical physical parameters like viscosity and surface tension have not been directly measured but rather are estimated indirectly using FRAP and the Stokes-Einstein equation. While not uncommon in the field, this approach is flawed as droplet viscosity is not simply determined by the size of the composing particles. Rather, in polymeric systems, viscosity strongly depends on the local protein concentration and intermolecular interactions (Rubinstein & Semenov Macromolecules (2001)). This unjustified approach propagates to the surface tension estimate since only the ratio of viscosity to surface tension is explicitly measured. Since the paper's conclusions strongly hinge on the magnitude of the surface tension, a more accurate estimate or direct measurement of this salient material property is called for.

(3) The phase diagram of growth modes very much depends on the assumption of neo-Hookean elasticity of the chromatin network. This assumption is poorly justified and calls into question the general conclusions about possible growth phases. The authors need to either provide evidence for neo-Hookean elasticity, or, alternatively, consider a model in which strain stiffening or thinning continues as droplets grow, which would likely lead to very different conclusions, and acknowledge this uncertainty.

(4) There is limited data for the elastic ripening claim. In Figure 3E, only one data point resides in the elastic ripening (δ < 0) range, with a few data points very close to zero.

(5) The authors claim that "our work shows that the elastic chromatin network can stabilize condensates against Ostwald ripening but only when condensate surface tension is low." This claim also depends on the details of the chosen neo-Hookean model of chromatic elasticity, and it is not studied here whether these results are robust to other models.

(6) It is also not clear how the total number of Mad1 proteins and LAF-1 disordered regions change while the condensates evolve with time. As the experiments span longer than 6 hours, continued protein production could lead to altered condensate coarsening dynamics. For example, continued production of Mad1 can lead to the growth of all Mad1 condensates, mimicking the diffusive growth process.

Reviewer #2 (Public review):

Mortzfeld et al. describe their study of class IIb microcins. Furthering our awareness of the presence and action of microcins is an important line of research. However, several issues related to the premise, sequence analysis, and validation require attention to support the claims.

(1) Previous studies have been published on the broader distribution of microcins across bacteria. The software has been published for their identification. Comparison to this software and/or discussion of previous work should be included to place this work in the context of the field.

(2) It is not clear how immunity proteins were identified and there does not appear to be functional confirmation to show these predicted immunity proteins are real. Thus, it is premature to state that immunity genes have been found. This may also confound some of the validation studies below if proper immunity proteins have not been included.

(3) Please show the nt alignment used to generate the tree. Without seeing it, one would guess that the sequences are either quite similar (making the results from this study less novel) or there would be concerns that the phylogenetic relationship derived from the nt alignment is spurious.

(4) Figure 1 B-C: There are numerous branches that do not have phylogenetic support (values <50%). These are not statistically valid phylogenetic relationships and should be collapsed. The resulting tree should be used in the description of clades.

(5) The discovered microcins are not being directly tested since they are expressed heterologous and reliant on non-native modification systems. The results present the statement that novel microcins have been validated. This should be described accordingly.

(6) The key finding of this paper is the claim that 12 novel class IIb microcins have been validated. To substantiate this claim, original images showing evidence of antibacterial activity must be made available rather than a presence/absence chart. The negative controls for this table are unclear and should be included with the original images.

(7) Further data for the purified microcin is needed. The purification method described is standard practice and should allow for product quantification, which should be included. Standard practice includes an SDS page showing the purity of the microcin, or at least the TEV digest to show microcin has been produced, and importantly a control sample (scrambled sequence, empty vector purification, etc) to show that observed activity (Figure 2B) is not from a purification carry over. This data should be included to support that microcin has been purified and is active.

Reviewer #2 (Public review):

Summary:

This study investigates the potential role of CD131, a cytokine receptor subunit shared by GM-CSF and IL-3, in intestinal inflammation. Using heterozygous mice with an inactivating mutation on this gene, the study demonstrates ameliorated inflammation, associated with less infiltration of macrophages. Moreover, the depletion of macrophages prevented many of the inflammatory effects of DSS and made both WT and mutant mice equivalent in terms of inflammation severity. Correlative data showing increased CD131+ cells in tissues of patients with ulcerative colitis is also demonstrating, evidence for plausibility for these pathways in human disease.

Strengths:

The phenotype of mutant mice seems quite robust and the pathways proposed, GM-CSF signaling in macrophages with CCL4 as a downstream pathway, are all plausible and concordant with existing models. Many of the experiments included meaningful endpoints and were overall well performed.

Weaknesses:

(1) Experimental rigor was lacking in this manuscript, which provided limited or no details on the number of independent iterations that each experiment was done, the number of animals per group, the number of technical or biological replicates in each graph, etc.

(2) Details of animal model validation showing that this particular mutant allele results in a lack of CD131 protein expression were not shown. Moreover, since the paper uses heterozygous mice, it is critical to show that at the protein level, there is indeed reduced expression of CD131 in het mice compared to controls (many heterozygous states do not lead to appreciable protein depletion).

(3) Another major weakness is that the paper asserts a causal relationship between CD131 signaling and CCL4 production: the data shown indicates that the phenotypes of CCL4 deficiency (through Ab blockade) and CD131 partial deficiency (in het mice) are similar. However, this does not establish that CD131 signaling acts through CCL4.

(4) Lastly, while the paper claims that CD131 acts through macrophage recruitment, the evidence is circumstantial and not direct. DSS-induced acute colitis is largely mediated by macrophages, so any manipulation associated with less severe inflammation is accompanied by lesser macrophage infiltration in this model: this does not directly establish that CD131 acts directly on macrophages, which would require cell-specific knockout or complex cell reconstitution experiments.

Reviewer #2 (Public review):

In this study, Kavaklıoğlu et al. investigated and presented evidence for a role for domesticated transposon protein L1TD1 in enabling its ancestral relative, L1 ORF1p, to retrotranspose in HAP1 human tumor cells. The authors provided insight into the molecular function of L1TD1 and shed some clarifying light on previous studies that showed somewhat contradictory outcomes surrounding L1TD1 expression. Here, L1TD1 expression was correlated with L1 activation in a hypomethylation dependent manner, due to DNMT1 deletion in HAP1 cell line. The authors then identified L1TD1 associated RNAs using RIP-Seq, which display a disconnect between transcript and protein abundance (via Tandem Mass Tag multiplex mass spectrometry analysis). The one exception was for L1TD1 itself, is consistent with a model in which the RNA transcripts associated with L1TD1 are not directly regulated at the translation level. Instead, the authors found L1TD1 protein associated with L1-RNPs and this interaction is associated with increased L1 retrotransposition, at least in the contexts of HAP1 cells. Overall, these results support a model in which L1TD1 is restrained by DNA methylation, but in the absence of this repressive mark, L1TD1 is expression, and collaborates with L1 ORF1p (either directly or through interaction with L1 RNA, which remains unclear based on current results), leads to enhances L1 retrotransposition. These results establish feasibility of this relationship existing in vivo in either development or disease, or both.

Comments on revised version:

In general, the authors did an acceptable job addressing the major concerns throughout the manuscript. This revision is much clearer and has improved in terms of logical progression.

Reviewer #2 (Public review):

The present study explores how thoughts map onto brain activity, a notoriously challenging question because of the dynamic, subjective, and abstract nature of thoughts. To tackle this question, the authors collected continuous thought ratings from participants watching a movie, and additionally made use of an open-source fMRI dataset recorded during movie watching as well as five established gradients of brain variation as identified in resting state data. Using a voxel-space approach, the results show that episodic knowledge, verbal detail, and sensory engagement of thoughts commonly modulate visual and auditory cortex, while intrusive distraction modulates the frontoparietal network. Additionally, sensory engagement mapped onto a gradient from primary to association cortex, while episodic knowledge mapped onto a gradient from the dorsal attention network to visual cortex. Building on the association between behavioral performance and neural activation, the authors conclude that sensory coupling to external input and frontoparietal executive control are key to comprehension in naturalistic settings.

The manuscript stands out for its methodological advancements in quantifying thoughts over time and its aim to study the implementation of thoughts in the brain during naturalistic movie watching.

Strengths:

(1) The study raises a question that has been difficult to study in naturalistic settings so far but is key to understanding human cognition, namely how thoughts map onto brain activation.

(2) The thought ratings introduce a novel method for continuously tracking thoughts, promising utility beyond this study.

(3) The authors used diverse data types, metrics, and analyses to substantiate the effects of thinking from multiple perspectives.

Reviewer #2 (Public review):

In this manuscript, Rachubinski and colleagues provide a comprehensive clinical, immunological, and autoantibody assessment of autoimmune/inflammatory manifestations of patients with Down syndrome (DS) in a large number of patients with this disorder. These analyses confirm prior results of excess interferon and cytokine signals in DS patients and extend these observations to highlight early-onset immunological aberrancies, far before symptoms occur, as well as characterizing novel autoantibody reactivities in this patient population. Then, the authors report the interim analysis of an open label, Phase II, clinical trial of the JAK1/3 inhibitor, tofacitinib, that aims to define the safety, clinical efficacy, and immunological outcomes of DS patients who suffer from inflammatory conditions of the skin. The clinical trial analysis indicates that the treatment is tolerated without serious adverse effects and that the majority of patients have experienced clinical improvement or remission in their corresponding clinical cutaneous manifestations as well as improvement or normalization of aberrant immunological signals such as cytokines.

The major strength of the study is the recruitment and uniform, systematic evaluation of an impressive number of DS patients. Moreover, the promising early results from the tofacitinib clinical trial pave the way for analysis of a larger number of patients within the Phase II trial and otherwise, which may lead to improved clinical outcomes of affected patients. An inherent weakness of such studies is the descriptive nature of several parameters and the relatively small size of tofacitinib-treated DS patients. However, the descriptive nature of some of the correlative research analyses are of scientific interest and are useful to generate hypotheses for future additional (including mechanistic) work and treatment of 10 DS patients in a formal clinical trial at interim analysis is not a trivial task for a disease like this. The manuscript achieves the aims of the authors and the results support their conclusions. The authors appropriately acknowledge areas that require more research and areas that are not well understood. The results are represented in a useful manner and statistical methods and analyses appear sound.

Comments on revised version:

The authors have satisfactorily addressed my comments in the revised manuscript.

Reviewer #2 (Public review):

In this manuscript, Popli et al investigated the roles of autophagy related gene, Atg14, in the female reproductive tract (FRT) using conditional knockout mouse models. By ablation of Atg14 in both oviduct and uterus with PR-Cre (Atg14 cKO), authors discovered that such females are completely infertile. They went on to show that Atg14 cKO females have impaired embryo implantation as well as embryo transport from oviduct to uterus. Further analysis showed that Atg14 cKO leads to increased pyroptosis in oviduct, which disrupts oviduct epithelial integrity and leads to obstructive oviduct lumen and impaired embryo transport. The authors concluded that Atg14 is critical for maintaining the oviduct homeostasis and keeping the inflammation under check to enable proper embryo transport.

The authors have barely addressed most of my concerns in this revised version with a few minor issues remaining to be addressed:<br /> (1) The authors tried to address my first concern regarding the statement that "autophagy is critical for maintaining the oviduct homeostasis". The revised statement in Line 53-54 "we report that Atg14-dependent autophagy plays a crucial role in maintaining..." is still not correct. It should be corrected as " we report that autophagy-related protein Atg14 plays a crucial role in maintaining...".<br /> (2) Line 349-351 described 80-90% of blastocysts retrieved from oviducts of cKO mice, which is in consistent with Figure 3B (showing more than 98%).<br /> (3) Line 447, "Fig. 5E" should be Fig. 6A. In addition, grammar error in the next sentence.<br /> (4) In Figure 6D, why the composition of blastocysts in chemical treated group do not add up to 100%.

Reviewer #2 (Public review):

Summary:

Yamawaki et al., conducted a series of neuroanatomical tracing and whole cell recording experiments to elucidate and characterise a relatively unknown pathway between the endopiriform (EN) and CA1 of the ventral hippocampus (vCA1) and to assess its functional role in social and object recognition using fibre photometry and dual vector chemogenetics. The main findings were that the EN sends robust projections to the vCA1 that collateralise to the prefrontal cortex, lateral entorhinal cortex and piriform cortex, and these EN projection neurons terminate in the stratum lacunosum-moleculare (SLM) layer of distal vCA1, synapsing onto GABAergic neurons that span across the Pyramidal-Stratum Radiatum (SR) and SR-SML borders. It was also demonstrated that EN input disynaptically inhibits vCA1 pyramidal neurons. vCA1 projecting EN neurons receive afferent input from piriform cortex, and from within EN. Finally, fibre photometry experiments revealed that vCA1 projecting EN neurons are most active when mice explore novel objects or conspecifics, and pathway-specific chemogenetic inhibition led to an impairment in the ability to discriminate between novel vs. familiar objects and conspecifics.

Revision 1:<br /> The authors have addressed most of my concerns, but a few weaknesses remain :

(1) I expected to see the addition of raw interaction times with objects and conspecifics for each phase of social testing (pre-test, sociability test, social discrimination), as per my comment on including raw data. However, the authors only provided total distance traveled and velocity, and total interaction time in Figure S9, which is less informative.

(2) The authors observed increased activity in vCA1-projecting EN neurons tracking with the preferred object during the pre-test (object-object exploration) phase of the social tests, and the summary schematic (Figure 9A) depicts animals as showing a preference for one object over the other (although they are identical) in both the social and object recognition tests. However, in the chemogenetic experiment, the data (Fig S9B) indicate that animals did not show this preference for one object over another, making the expected baseline for this task unclear. This also raises an important question of whether the lack of effect from chemogenetic inhibition of vCA1-projecting EN neurons could be attributed to the absence of this baseline preference.<br /> Additionally, the finding that vCA1-projecting EN activity is associated with the preferred object exploration appears to counter the authors' argument that novelty engages this circuit (since both objects are novel in this instance). This discrepancy warrants further discussion.

Reviewer #2 (Public review):

Summary:

This study describes a deep mutational scan across CDKN2A using suppression of cell proliferation in pancreatic adenocarcinoma cells as a readout for CDKN2A function. The results are also compared to in silico variant predictors currently utilized by the current diagnostic frameworks to gauge these predictors' performance. The authors also functionally classify CDKN2A somatic mutations in cancers across different tissues

Review:

The goal of this paper was to perform functional classification of missense mutations in CDKN2A in order to generate a resource to aid in clinical interpretation of CDKN2A genetic variants identified in clinical sequencing. In our initial review, we concluded that this paper was difficult to review because there was a lack of primary data and experimental detail. The authors have significantly improved the clarity, methodological detail and data exposition in this revision, facilitating a fuller scientific review. Based on the data provided we do not think the functional characterization of CDKN2A variants is robust or complete enough to meet the stated goal of aiding clinical variant interpretation. We think the underlying assay could be used for this purpose but different experimental design choices and more replication would be required for these data to be useful. Alternatively, the authors could also focus on novel CDKN2A variants as there seems to be potential gain of function mutations that are simply lumped into "neutral" that may have important biological implications.

Major concerns:

Low experimental concordance. The p-value scatter plot (Figure 2 Figure Supplement 3A) across 560 variants shows low collinearity indicating poor replicability. These data should be shown in log2fold changes, but even after model fitting with the gamma GLM still show low concordance which casts strong doubt on the function scores.<br /> The more detailed methods provided indicate that the growth suppression experiment is done in 156 pools with each pool consisting of the 20 variants corresponding to one of the 156 aa positions in CKDN2A. There are several serious problems with this design.

Batch effects in each of the pools preventing comparison across different residues. We think this is a serious design flaw and not standard for how these deep mutational scans are done. The standard would be to combine all 156 pools in a single experiment. Given the sequencing strategy of dividing up CDKN2A into 3 segments, the 156 pools could easily have been collapsed into 3 (1 to 53, 54 to 110, 111 to 156). This would significantly minimize variation in handling between variants at each residue and would be more manageable for performance of further replicates of the screen for reproducibility purposes. The huge variation in confluency time 16-40 days for each pool suggest that this batch effect is a strong source of variation in the experiment

Lack of experimental/biological replication: The functional assay was only performed once on all 156 CDKN2A residues and was repeated for only 28 out of 156 residues, with only ~80% concordance in functional classification between the first and second screens. This is not sufficiently robust for variant interpretation. Why was the experiment not performed more than once for most aa sites?

For the screen, the methods section states that PANC-1 cells were infected at MOI=1 while the standard is an MOI of 0.3-0.5 to minimize multiple variants integrating into a single cell. At an MOI =1 under a Poisson process which captures viral integration, ~25% of cells would have more than 1 lentiviral integrant. So in 25% of the cells the effect of a variant would be confounded by one or more other variants adding noise to the assay.

While the authors provide more explanation of the gamma GLM, we strongly advise that the heatmap and replicate correlations be shown with the log2 fold changes rather than the fit output of the p-values.

In this study, the authors only classify variants into the categories "neutral", "indeterminate", or "deleterious" but they do not address CDKN2A gain-of-function variants that may lead to decreased proliferation. For example, there is no discussion on variants at residue 104, whose proliferation values mostly consist of higher magnitude negative log2fold change values. These variants are defined as neutral but from the one replicate of the experiment performed, they appear to be potential gain-of-function variants.

Reviewer #2 (Public review):

In this paper, the authors aim to explore whether an AI model trained on natural protein data can aid in designing proteins that are resistant to extreme environments. While this is an interesting attempt, the study's computational contributions are weak, and the design of the computational experiments appears arbitrary.

(1) The writing throughout the paper is poor. This leaves the reader confused.

(2) The main technical issue the authors address is whether AI can identify protein mutations that adapt to extreme environments based solely on natural protein data. However, the introduction could be more concise and focused on the key points to better clarify the significance of this question.

(3) The authors did not develop a new model but instead used their previously developed Pro-PRIME model. This significantly weakens the novelty and contribution of this work.

(4) The computational experiments are not well-justified. For instance, the authors used a zero-shot setting for single-point mutation experiments but opted for fine-tuning in multiple-point mutation experiments. There is no clear explanation for this discrepancy. How does the model perform in zero-shot settings for multiple-point mutations? How would fine-tuning affect single-point mutation results? The choice of these strategies seems arbitrary and lacks sufficient discussion.

Reviewer #2 (Public review):

Summary:

The authors present a straightforward and convincing demonstration of a reagent and workflow that they collectively term "MagIC-cryo-EM", in which magnetic nanobeads combined with affinity linkers are used to specifically immobilize and locally concentrate complexes that contain a protein-of-interest. As a proof of concept, they localize, image, and reconstruct H1.8-bound nucleosomes reconstructed from frog egg extracts. The authors additionally devised an image-processing workflow termed "DuSTER", which increases the true positive detections of the partially ordered NPM2 complex. The analysis of the NPM2 complex {plus minus} H1.8 was challenging because only ~60 kDa of protein mass was ordered. Overall, single-particle cryo-EM practitioners should find this study useful.

Strengths:

The rationale is very logical and the data are convincing.

Weaknesses: I have seen an earlier version of this study at a conference. The conference presentation was much easier to follow than the current manuscript. It is as if this manuscript had undergone review at another journal and includes additional experiments to satisfy previous reviewers. Specifically, the NPM2 results don't seem to add much to the main story (MagIC-cryo-EM), and read more like an addendum. The authors could probably publish the NPM2 results separately, which would make the core MagIC results (sans DusTER) easier to read.

Reviewer #2 (Public review):

Summary:

This work uses genomic and biochemical approaches for HCMV infection in human fibroblasts and retinal epithelial cell lines, followed by comparisons and some validations using strategies such as immunoblots. Based on these analyses, they propose several mechanisms that could contribute to the HCMV-induced diseases, including closing of TEAD1-occupying domains and reduced TEAD1 transcript and protein levels, decreased YAP1 and phospho-YAP1 levels, and exclusion of TEAD1 exon 6.

Strengths:

The genomics experiments were done in duplicates and data analyses show good technical reproducibility. Data analyses are performed to show changes at the transcript and chromatin level changes, followed by some Western blot validations.

Weaknesses:

This work, at the current stage, is quite correlative since no functional studies are done to show any causal links. For readers who are outside the field, some clarifications of the system and design need to be stated.

Reviewer #2 (Public review):

Summary:<br /> CTCF is one of the most well-characterized regulators of chromatin architecture in mammals. Given that CTCF is an essential protein, understanding how its binding is regulated is a very active area of research. It has been known for decades that CTCF is sensitive to 5-cystosine DNA methylation (5meC) in certain contexts. Moreover, at genomic imprints and in certain oncogenes, 5meC-mediated CTCF antagonism has very important gene regulatory implications. A number of labs (eg, Schubeler and Stamatoyannopoulos) have assessed the impact of DNA methylation on CTCF binding, but it is important to also interrogate the effect on chromatin organization (ie, looping). Here, Roseman and colleagues used a DNMT1 inhibitor in two established human cancer lines (HCT116 [colon] and K562 [leukemia]), and performed CTCF ChIPseq and HiChIP. They showed that "reactivated" CTCF sites-that is, bound in the absence of 5meC-are enriched in gene bodies, participate in many looping events, and intriguingly, appear associated with nuclear speckles. This last aspect suggests that these reactivated loops might play an important role in increased gene transcription. They showed a number of genes that are upregulated in the DNA hypomethylated state actually require CTCF binding, which is an important result.

Strengths:<br /> Overall, I found the paper to be succinctly written and the data presented clearly. The relationship between CTCF binding in gene bodies and association with nuclear speckles is an interesting result. Another strong point of the paper was combining DNMT1 inhibition with CTCF degradation.

Weaknesses:<br /> The most problematic aspect of this paper in my view is the insufficient evidence for the association of "reactivated" CTCF binding sites with nuclear speckles needs to be more diligently demonstrated (see Major Comment). One unfortunate aspect was that this paper neglected to discuss findings from our recent paper, wherein we also performed CTCF HiChIP in a DNA methylation mutant (Monteagudo-Sanchez et al., 2024 PMID: 39180406). It is true, this is a relatively recent publication, although the BioRxiv version has been available since fall 2023. I do not wish to accuse the authors of actively disregarding our study, but I do insist that they refer to it in a revised version. Moreover, there are a number of differences between the studies such that I find them more complementary rather than overlapping. To wit, the species (mouse vs human), the cell type (pluripotent vs human cancer), the use of a CTCF degron, and the conclusions of the paper (we did not make a link with nuclear speckles). Furthermore, we used a constitutive DNMT knockout which is not viable in most cell types (HCT116 cells being an exception), and in the discussion mentioned the advantage of using degron technology:

"With high-resolution techniques, such as HiChIP or Micro-C (119-121), a degron system can be coupled with an assessment of the cis-regulatory interactome (118). Such techniques could be adapted for DNA methylation degrons (eg, DNMT1) in differentiated cell types in order to gauge the impact of 5meC on the 3D genome."

The authors here used a DNMT1 inhibitor, which for intents and purposes, is akin to a DNMT1 degron, thus I was happy to see a study employ such a technique. A comparison between the findings from the two studies would strengthen the current manuscript, in addition to being more ethically responsible.

Reviewer #2 (Public review):

The authors first identify Ankle2 as a regulatory subunit and direct interactor of PP2A, showing they interact both in vitro and in vivo to promote BAF dephosphorylation. The Ankyrin domain of Ankle2 is important for the interaction with PP2A. They then show Ankle2 also interacts with the ER protein Vap33 through FFAT motifs and they particularly co-localize during mitosis. The recruitment of Ankle2 to Vap33 is essential to ER and nuclear envelop membrane in telophase while earlier in mitosis, it relies on the C terminus but not the FFAT motifs for recruitments to the nuclear membrane and spindle envelop in early mitosis. The molecular determinants and receptors are currently not known. The authors check the function of the PP2A recruitment to Ankle2/Vap33 in the context of embryos and show this recruitment pathway is functionally important. While the Ankle2/Vap33 interaction is dispensable in adult flies -looking at wing development, the PP2A/Ankle2 interaction is essential for correct wing and fly development. Overall, this is a very complete paper that reveals the molecular mechanism of PP2A recruitment to Ankle2 and studies both the cellular and the physiological effect of this interaction in the context of fly development.

Strengths:

The paper is well written and the narrative is well-developed. The figures are of high quality, well-controlled, clearly labelled, and easy to understand. They support the claims made by the authors.

Weaknesses:

The study would benefit from being discussed in the context of what is already known on Ankle2 biology in C.elegans and human cells. It is important to highlight the structures shown in the paper are alphafold models, rather than validated structures.

Reviewer #2 (Public review):

In this study, the authors identified CG14545 (and named it Sakura), as a key gene essential for Drosophila oogenesis. Genetic analyses revealed that Sakura is vital for both oogenesis progression and ultimate female fertility, playing a central role in the renewal and differentiation of germ stem cells (GSC).

The absence of Sakura disrupts the Dpp/BMP signaling pathway, resulting in abnormal bam gene expression, which impairs GSC differentiation and leads to GSC loss. Additionally, Sakura is critical for maintaining normal levels of piRNAs. Also, the authors convincingly demonstrate that Sakura physically interacts with Otu, identifying the specific domains necessary for this interaction, suggesting a cooperative role in germline regulation. Importantly, the loss of otu produces similar defects to those observed in Sakura mutants, highlighting their functional collaboration.

The authors provide compelling evidence that Sakura is a critical regulator of germ cell fate, maintenance, and differentiation in Drosophila. This regulatory role is mediated through the modulation of pMad and Bam expression. However, the phenotypes observed in the germarium appear to stem from reduced pMad levels, which subsequently trigger premature and ectopic expression of Bam. This aberrant Bam expression could lead to increased CycA levels and altered transcriptional regulation, impacting piRNA expression. Given Sakura's role in pMad expression, it would be insightful to investigate whether overexpression of Mad or pMad could mitigate these phenotypic defects (UAS-Mad line is available at Bloomington Drosophila Stock Center).

A major concern is the overstated role of Sakura in regulating Orb. The data does not reveal mislocalized Orb; rather, a mislocalized oocyte and cytoskeletal breakdown, which may be secondary consequences of defects in oocyte polarity and structure rather than direct misregulation of Orb. The conclusion that Sakura is necessary for Orb localization is not supported by the data. Orb still localizes to the oocyte until about stage 6. In the later stage, it looks like the cytoskeleton is broken down and the oocyte is not positioned properly, however, there is still Orb localization in the ~8-stage egg chamber in the oocyte. This phenotype points towards a defect in the transport of Orb and possibly all other factors that need to localize to the oocyte due to cytoskeletal breakdown, not Orb regulation directly. While this result is very interesting it needs further evaluation on the underlying mechanism. For example, the decrease in E-cadherin levels leads to a similar phenotype and Bam is known to regulate E-cadherin expression. Is Bam expressed in these later knockdowns?

The manuscript would benefit from a more balanced interpretation of the data concerning Sakura's role in Orb regulation. Furthermore, a more expanded discussion on Sakura's potential role in pMad regulation is needed. For example, since Otu and Bam are involved in translational regulation, do the authors think that Mad is not translated and therefore it is the reason for less pMad? Currently the discussion presents just a summary of the results and not an extension of possible interpretation discussed in context of present literature.

Reviewer #2 (Public review):

In this study, the authors set out to explore how antibiotics known to inhibit bacterial protein synthesis also affect mitoribosomes in HEK cells. They achieved this through mitoribosome profiling, where RNase I and Mnase were used to generate mitoribosome-protected fragments, followed by sequencing to map the regions where translation arrest occurs. This profiling identified the codon-specific impact of antibiotics on mitochondrial translation.

The study finds that most antibiotics tested inhibit mitochondrial translation similarly to their bacterial counterparts, except telithromycin, which exhibited distinct stalling patterns. Specifically, chloramphenicol and linezolid selectively inhibited translation when certain amino acids were in the penultimate position of the nascent peptide, which aligns with their known bacterial mechanism. Telithromycin stalls translation at an R/K-X-R/K motif in bacteria, and the study demonstrated a preference for arresting at an R/K/A-X-K motif in mitochondria. Additionally, alternative translation initiation sites were identified in MT-ND1 and MT-ND5, with non-canonical start codons. Overall, the paper presents a comprehensive analysis of antibiotics in the context of mitochondrial translation toxicity, and the identification of alternative translation initiation sites will provide valuable insights for researchers in the mitochondrial translation field.

From my perspective as a structural biologist working on the human mitoribosome, I appreciate the use of mitoribosome profiling to explore off-target antibiotic effects and the discovery of alternative mitochondrial translation initiation sites. However, the description is somewhat limited by a focus on this single methodology. The authors could strengthen their discussion by incorporating structural approaches, which have contributed significantly to the field. For example, antibiotics such as paromomycin and linezolid have been modeled in the human mitoribosome (PMID: 25838379), while streptomycin has been resolved (10.7554/eLife.77460), and erythromycin was previously discussed (PMID: 24675956). The reason we can now describe off-target effects more meaningfully is due to the availability of fully modified human mitoribosome structures, including mitochondria-specific modifications and their roles in stabilizing the decoding center and binding ligands, mRNA, and tRNAs (10.1038/s41467-024-48163-x).<br /> These and other relevant studies should be acknowledged throughout the paper to provide additional context.

Reviewer #2 (Public review):

Summary:

The study investigates the molecular mechanisms underlying chronic pain-related memory impairment by focusing on S1P/S1PR1 signaling in the dentate gyrus (DG) of the hippocampus. Through behavioural tests (Y-maze and Morris water maze) and RNA-seq analysis, the researchers segregated chronic pain mice into memory impairment-susceptible and -unsusceptible subpopulations. They discovered that S1P/S1PR1 signaling is crucial for determining susceptibility to memory impairment, with decreased S1PR1 expression linked to structural plasticity changes and memory deficits.

Knockdown of S1PR1 in the DG induced a susceptible phenotype, while overexpression or pharmacological activation of S1PR1 promoted resistance to memory impairment and restored normal synaptic structure. The study identifies actin cytoskeleton-related pathways, including ITGA2 and its downstream Rac1/Cdc42 signaling, as key mediators of S1PR1's effects, offering new insights and potential therapeutic targets for chronic pain-related cognitive dysfunction.

This manuscript consists of a comprehensive investigation and significant findings. The study provides novel insights into the molecular mechanisms of chronic pain-related memory impairment, highlighting the critical role of S1P/S1PR1 signaling in the hippocampal dentate gyrus. The clear identification of S1P/S1PR1 as a potential therapeutic target offers promising avenues for future research and treatment strategies. The manuscript is well-structured, methodologically sound, and presents valuable contributions to the field.

Strengths:

(1) The manuscript is well-structured and written in clear, concise language. The flow of information is logical and easy to follow.

(2) The segregation of mice into memory impairment-susceptible and -unsusceptible subpopulations is innovative and well-justified. The statistical analyses are robust and appropriate for the data.

(3) The detailed examination of S1PR1 expression and its impact on synaptic plasticity and actin cytoskeleton reorganization is impressive. The findings are significant and contribute to the understanding of chronic pain-related memory impairment.

Comments on revisions:

The authors have satisfactorily addressed all the issues raised.

Reviewer #2 (Public review):

The data is largely electrophysiological recordings coupled with behavioral measurements (technically impressive) and some gain-of-function experiments in freely walking flies. Loss-of-function was tested but had minimal effect, which is not surprising in a system with partially redundant control mechanisms. The data is also consistent with/complementary to subsequent manuscripts (Yang 2023, Feng 2024, and Ros 2024) showing additional descending neurons with contributions to steering in walking and flying.

The experiments are well executed, the results interesting, and the description clear. Some hypotheses based on connectome anatomy are tested: the insights on the pre-synaptic side - how sensory and central complex heading circuits converge onto these DNs are stronger than the suggestions about biomechanical mechanisms for how turning happens on the motor side.

Of particular interest is the idea that different sensory cues can converge on a common motor program. The turn-toward or turn-away mechanism is initiated by valence rather than whether the stimulus was odor or temperature or memory of heading. The idea that animals choose a direction based on external sensory information and then maintain that direction as a heading through a more internal, goal-based memory mechanism, is interesting but it is hard to separate conclusively.

The "see-saw", where left-right symmetry is broken to allow a turn, presumably by excitation on one side and inhibition of the other leg motor modules, is interesting but not well explained here. How hyperpolarization affects motor outputs is not clear.

The statement near Figure 5B that "DNa02 activity was higher on the side ipsilateral to the attractive stimulus, but contralateral to the aversive stimulus" is really important - and only possible to see because of the dual recordings.

Reviewer #2 (Public review):

Summary:

In the present study, the authors select the coiled-coil protein CCDC113 and revealed its expression in the stages of spermatogenesis in the testis as well as in the different steps of spermiogenesis with expression also mapped in the different parts of the epididymis. Gene deletion led to male infertility in CRISPR-Cas9 KO mice and PAS staining showed defects mapped in the different stages of the seminiferous cycle and through the different steps of spermiogenesis. EM and IF with several markers of testis germ cells and spermatozoa in the epididymis indicated defects in flagella and head-to-tail coupling for flagella as well as acephaly. The authors' co-IP experiments of expressed CCDC113 in HEK293T cells indicated an association with CFAP91 and DRC2 as well as SUN5 and CENTLEIN.

The authors propose that CCDC113 connects CFAP91 and DRC2 to doublet microtubules of the axoneme and CCDC113's association with SUN5 and CENTLEIN to stabilize the sperm flagellum head-to-tail coupling apparatus. Extensive experiments mapping CCDC13 during postnatal development are reported as well as negative co-IP experiments and studies with SUN5 KO mice as well as CENTLEIN KO mice.

Strengths:

The authors provide compelling observations to indicate the relevance of CCDC113 to flagellum formation with potential protein partners. The data are relevant to sperm flagella formation and its coupling to the sperm head.

Weaknesses:

The authors' observations are consistent with the model proposed but the authors' conclusions for the mechanism may require direct demonstration in sperm flagella. The Walton et al paper shows human CCDC96/113 in cilia of human respiratory epithelia. An application of such methodology to the proteins indicated by Wu et al for the sperm axoneme and head-tail coupling apparatus is eagerly awaited as a follow-up study.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors report GCaMP fiber-photometry recordings from the GnRH neuron distal projections in the ventral arcuate nucleus. The recording are taken from intact, male and female, freely behaving mice. The report three patterns of neuronal activity:

1) abrupt increases in the Ca2+ signals that are perfectly correlated with LH pulses.

2) a gradual, yet fluctuating (with a slow ultradian frequency), increase in activity, which is associated with the onset of the LH surge in female animals.

3) clustered (high frequency) baseline activity in both female and male animals.

Strengths:

The GCaMP fiber-photometry recordings reported here are the first direct recordings from GnRH neurones in free behaving mice. These recordings suggest a rich repertoire of activity, including the integration of distinct "surge" and "pulse" generation signals, and an ultradian rhythm during the onset of the surge.

Weaknesses:

The data analysis methods used for the characterisation of the oscillatory behaviour could be complemented with more advanced wavelet methods to quantify and analyse how the frequency content of the observed Ca2+ signal changes over the cycle.

Reviewer #2 (Public review):

Summary:

In this study, the authors identified nuclear genome-encoded transcription factors that regulate mtDNA maintenance and mitochondrial biogenesis. They started with an RNAi screening in developing Drosophila eyes with reduced mtDNA content and identified several putative candidate genes. Subsequently, using ChIP-seq data, they built a potential regulatory network that seems to govern mitochondrial biogenesis. Next, they focused on a candidate gene, CG1603 /clifford, for further characterization. Based on the expression of different markers, such as TFAM and SDHA, in RNAi and overexpression clones in the midgut, they argued that CG1603 promotes mitochondrial biogenesis and the expression of ETC complex genes. They used a CG1603 mutant to show reduced mtDNA and mitochondrial protein levels. Clonal analyses showed a reduction in mitochondrial biogenesis and membrane potential upon loss of CG1603. They further showed that the protein is localized to the mitochondria, and binds to polytene chromosomes in the salivary gland. Based on the RNA-seq results from the mutants and the ChIP data, the authors argued that the nucleus-encoded mitochondrial genes are downregulated >2 folds in the CG1603 mutants and that the regulatory elements bound by CG1603 are related to ETC biogenesis. Finally, they showed that YL-1, another candidate in the network, is an upstream regulator of CG1603. The screening strategy was well-designed, and the follow-up experiments were nicely executed.

Comments on revisions:

The authors have addressed my previous comments satisfactorily.

Reviewer #2 (Public review):

Summary:

Sphingosine-1-phosphate (S1P) metabolic and signaling genes are expressed highly in retinal Müller glia (MG) cells. This study tested how S1P signaling regulates glial phenotype, dedifferentiation of, reprogramming into proliferating MG-derived progenitor cells (MGPCs), and neuronal differentiation of the progeny of MGPCs using in vivo chick retina. Major techniques used are Sc-RNASeq and immunohistochemistry to determine the gene expression and proliferation of MG cells that co-label with signaling antibodies or mRNA FISH following treating the in vivo eyes with various S1P signaling antagonists, agonists, and signal modulators. The major conclusions drawn are supported by the results presented. However, the methodology they have used to modulate the S1P pathway using various chemical drugs raises questions about the outcomes and whether those are the real effects of S1P receptor modulation or S1P synthesis inhibition.

Strengths:

- Use of elaborated single-cell RNAseq expression data.<br /> - Use of FISH for S1P receptors and kinase as a good quality antibody is not available.<br /> - Use of EdU assay in combination with IHC<br /> - Comparison with human and Zebrafish Sc-RNA data

Weaknesses:

The methodology is not very clean. A number of drugs (inhibitors/ antagonists/agonists signal modulators) are used to modulate S1P expression or signaling in the retina without evidence that these drugs are reaching the target cells. No alternative evaluation if the drugs, in fact, are effective. The drug solubility in the vehicle and in the vitreous is not provided, and how did they decide on using a single dose of each drug to have the optimal expected effect on the S1P pathway?

Reviewer #2 (Public review):

Shrestha et al investigated the role of IR receptors in the detection of 3 carboxylic acids in adult Drosophila. A low concentration of either of these carboxylic acids added to 2 mM sucrose (1% lactic acid (LA), citric acid (CA), or glycolic acid (GA)) stimulates the consumption of adult flies in choice conditions. The authors use this behavioral test to screen the impact of mutations within 33 receptors belonging to the IR family, a large family of receptors derived from glutamate receptors and expressed both in the olfactory and gustatory sensilla of insects. Within the panel of mutants tested, they observed that 3 receptors (IR25a, IR51b, and IR76b) impaired the detection of LA, CA, and GA, and that 2 others impacted the detection of CA and GA (IR94a and IR94h). Interestingly, impairing IR51b, IR94a, and IR94h did not affect the electrophysiological responses of external gustatory sensilla to LA, CA, and GA. Thanks to the use of GAL4 strains associated with these receptors and thanks to the use of poxn mutants (which do not develop external gustatory sensilla but still have functional internal receptors), they show evidence that IR94a and IR94h are only expressed in two clusters of gustatory neurons of the pharynx, respectively in the VCSO (ventral cibarial sense organ) and in the VCSO + LSO (labral sense organ). As for IR51b, the GAL4 approach was not successful but RT-PCR made on different parts of the insect showed an expression both in the pharyngeal organs and in peripheral receptors. These main findings are then complemented by a host of additional experiments meant to better understand the respective roles of IR94a and IR94h, by using optogenetics and brain calcium imaging using GCamp6. They also report a failed attempt to co-express IR51b, IR94a, and IR94h into external receptors, a co-expression which did not confer the capability of bitter-sensitive cells (expressing GR33a-GAL4) to detect either of the carboxylic acids. These data complete and expand previous observations made on this group and others, and dot to 2 new IR receptors which show an unsuspected specific expression, into organs that still remain difficult to study.

The conclusions of this paper are supported by the data presented, but it remains difficult to make general conclusions as concerns the mechanisms by which carboxylic acids are detected.

(1) All experiments were done with 1% of carboxylic acids. What is the dose dependency of the behavioral responses to these acids, and is it conceivable that other receptors are involved at other concentrations?

(2) One result needs to be better discussed and hypotheses proposed - which is why the mutations of most receptors lead to a loss of detection (mutant flies become incapable of detecting the acid) while mutations in IR94a and IR94h make CA and GA potent deterrents. Does it mean that CA and GA are detected by another set of receptors that, when activated, make flies actively avoid CA and GA? In that case, do the authors think that testing receptors one by one is enough to uncover all the receptors participating in the detection of these substances?

(3) The paper needs to be updated with a recent paper published by Guillemin et al (2024), indicating that LA is detected externally by a combination of IR94e, IR76b and IR25a. IR25a might help to form a fully functional receptor in GR33a neurons (a former study from Chen et al (2017) indicate that IR25a is expressed in all gustatory neurons of the pharynx).

(4) Although it was not the main focus of the paper, it would have been most interesting if the cells expressing IR94a and IR94h were identified, and placed on the functional map proposed by the group of Dahanukar (Chen et al 2017 Cell Reports, Chen et al 2019 Cell Reports).

Reviewer #2 (Public review):

Summary:

Salt stress is a significant and growing concern for agriculture in some parts of the world. While the effects of sodium excess have been studied in Arabidopsis and (many) crop species, most studies have focused on Na uptake, toxicity and overall effects on yield, rather than on developmental responses to excess Na, per se. The work by Ishka and colleagues aims to fill this gap.

Working from an existing dataset that exposed a diverse panel of A. thaliana accessions to control, moderate, and severe salt stress, the authors identify candidate loci associated with altering the root:shoot ratio under salt stress. Following a series of molecular assays, they characterize a DUF247 protein which they dub SR3G, which appears to be a negative regulator of root growth under salt stress.

Overall, this is a well-executed study which demonstrates the functional role played by a single gene in plant response to salt stress in Arabidopsis.

Review of revised manuscript:

The authors have addressed my point-by-point comments to my satisfaction. In the cases where they have changed their manuscript language, clarified figures, or added analyses I have no further comment. In some cases, there is a fruitful back-and-forth discussion of methodology which I think will be of interest to readers.

I have nothing to add during this round of review. I think that the paper and associated discussion will make a nice contribution to the field

Reviewer #2 (Public review):

Summary:

Sukhina et al. use a chronic murine dietary restriction model to investigate the cellular mechanisms underlying nutritionally acquired immunodeficiency as well as the consequences of a refeeding intervention. The authors report a substantial impact of undernutrition on the myeloid compartment, which is not rescued by refeeding despite rescue of other phenotypes including lymphocyte levels, and which is associated with maintained partial susceptibility to bacterial infection.

Strengths:

Overall, this is a nicely executed study with appropriate numbers of mice, robust phenotypes, and interesting conclusions, and the text is very well-written. The authors' conclusions are generally well-supported by their data.

Weaknesses:

There is little evaluation of known critical drivers of myelopoiesis (e.g. PMID 20535209, 26072330, 29218601) over the course of the 40% diet, which would be of interest with regard to comparing this chronic model to other more short-term models of undernutrition.

Further, the microbiota, which is well-established to be regulated by undernutrition (e.g. PMID 22674549, 27339978, etc.), and also well-established to be a critical regulator of hematopoiesis/myelopoiesis (e.g. PMID 27879260, 27799160, etc.), is completely ignored here.

Reviewer #2 (Public review):

Summary:

The authors present the first single-cell atlas for syngathid fishes, providing a resource for future evolution & development studies in this group.

Strengths:

The concept here is simple and I find the manuscript to be well written. I like the in situ hybridization of marker genes >> this is really nice. I also appreciate the gene co-expression analysis to identify modules of expression. There are no explicit hypotheses tested in the manuscript, but the discovery of these cell types should have value in this organism and in the determination of morphological novelties in seahorses and their relatives.

Weaknesses:

I think there are a few computational analyses that might improve the generality of the results.

(1) The cell types: The authors use marker gene analysis and KEGG pathways to identify cell types. I'd suggest a tool like SAMap (https://elifesciences.org/articles/66747) which compares single cell data sets from distinct organisms to identify 'homologous' cell types -- I imagine the zebrafish developmental atlases could serve as a reasonable comparative reference.

(2) Trajectory analyses: Authors suggest that their analyses might identify progenitor cell states and perhaps related differentiated states. They might explore cytoTRACE and/or pseudotime-based trajectory analyses to more fully delineate these ideas.

(3) Cell-cell communication: I think it's very difficult to identify 'tooth primordium' cell types, because cell types won't be defined by organ in this way. for instance dental glia will cluster with other glia, dental mesenchyme will likely cluster with other mesenchymal cell types. so the histology and ISH in most convincing in this regard. having said this, given the known signaling interactions in the developing tooth (and in development generally) the authors might explore cell-cell communication analysis (e.g., CellChat) to identify cell types that may be interacting.

Comments on revisions:

I feel essentially the same about this manuscript. it's a useful resource for future experimental forays into this unique system. The team made improvements to deal with comments from other reviewers related to quality of confirmatory in situ hybridization. This is good.

Regarding their response that one can't use CellChat if you're not working in mice or human, this is inaccurate. the assumption one makes is that ligand-receptor pairs and signaling pathways have conserved functions across animals (vertebrates). It's the same assumption the authors make when using the KEGG pathway to score enrichment of pathways in clusters. CellChat used in fishes in Johnson et al 2023 Nature Communications | ( 2023) 14:4891.

Reviewer #2 (Public review):

Summary:

Li et al. explored which stage of Stroop conflict processing was influenced by working memory loads. Participants completed a single task (Stroop task) and a dual task (the Sternberg working memory task combined with the Stroop task) while their EEG data was recorded. They adopted the event-related potential (ERP), and multivariate pattern analyses (MVPA) to investigate the interaction effect of task (single/dual) and congruency (congruent/incongruent). The results showed that the interaction effect was significant on the sustained potential (SP; 650-950 ms), the late theta (740-820 ms), and beta (920-1040 ms) power but not significant on the early P1 potential (110-150 ms). They used the representational similarity analyses (RSA) method to explore the correlation between behavioral and neural data, and the results revealed a significant contribution of late theta activity.

Strengths:

(1) The experiment is well-designed.

(2) The data were analyzed in depth from both time and frequency domain perspectives by combining several methods.

Weaknesses:

(1) As the researchers mentioned, a previous study reported a diminished Stroop effect with concurrent working memory tasks to memorize meaningless visual shapes rather than memorize Chinese characters as in the study. My main concern is that lower-level graphic processing when memorizing visual shapes also influences the Stroop effect. The stage of Stroop conflict processing affected by the working memory load may depend on the specific content of the concurrent working memory task. If that's the case, I sense that the generalization of this finding may be limited.

(2) The P1 and N450 components are sensitive to congruency in previous studies as mentioned by the researchers, but the results in the present study did not replicate them. This raised concerns about data quality and needs to be explained.

Reviewer #2 (Public review):

Summary:

This study integrated single-cell sequencing and spatial transcriptome data from mouse heart tissue at different time points post-MI. They identified four transcriptionally distinct subtypes of lymphatic endothelial cells and localized them in space. They observed that LECs subgroups are localized in different zones of infarcted heart with functions. Specifically, they demonstrated that LEC ca III may be involved in directly regulating myocardial injuries in the infarcted zone concerning metabolic stress, while LEC ca II may be related to the rapid immune inflammatory responses of the border zone in the early stage of MI. LEC ca I and LEC collection mainly participate in regulating myocardial tissue edema resolution in the middle and late stages post-MI. Finally, cell trajectory and Cell-Chat analyses further identified that LECs may regulate myocardial edema through Aqp1, and likely affect macrophage infiltration through the galectin9-CD44 pathway. The authors concluded that their study revealed the dynamic transcriptional heterogeneity distribution of LECs in different regions of the infarcted heart and that LECs formed different functional subgroups that may exert different bioeffects in myocardial tissue post-MI.

Strengths:

The study addresses a significant clinical challenge, and the results are of great translational value. All experiments were carefully performed, and their data support the conclusion.

Reviewer #2 (Public review):

Summary:

SNX4 is thought to mediate recycling from endosomes back to the plasma membrane in cells. In this study, the authors demonstrate the increases in the amounts of transmitter release and the number of docked vesicles by combining genetics, electrophysiology and EM. They failed to find evidence for its role in synaptic vesicle cycling and endocytosis, which may be intuitively closer to the endosome function.

Strengths:

The electrophysiological data and EM data are in principle, convincing, though there are several issues in the study.

Weaknesses:

It is unclear why the increase in the amounts of transmitter release and docked vesicles happened in the SNX4 KO mice. In other words, it is unclear how the endosomal sorting proteins in the end regulate or are connected to presynaptic, particularly the active zone function.

Comments on revisions:

I am fine with revision in principle. the authors have addressed my concerns.

Reviewer #2 (Public review):

Summary:

The work by Arafi et al. shows the effect of Familial Alzheimer's Disease presenilin-1 mutants on endoproteinase and carboxylase activity. They have elegantly demonstrated how some mutants alter each step of processing. Together with FLIM experiments, this study provides additional evidence to support their 'stalled complex hypotheses'.

Strengths:

This is a beautiful biochemical work. The approach is comprehensive.

Weaknesses:

(1) It appears that the purified g-secretase complex generates the same amount of Ab40 and Ab42, which is quite different in cellular and biochemical studies. Is there any explanation for this?

(2) It has been reported the Ab production lines from Ab49 and Ab48 can be crossed with various combinations (PMID: 23291095 and PMID: 38843321). How does the production line crossing impact the interpretation of this work?

(3) In Figure 5, did the authors look at the protein levels of PS1 mutations and C99-720, as well as secreted Ab species? Do the different amounts of PS1 full-length and PS1-NTF/CTF influence FILM results?

(4) It is interesting that both Ab40 and Ab42 Elisa kits detect Ab43. Have the authors tested other kits in the market? It might change the interpretation of some published work.

Reviewer #2 (Public review):

Summary:

This study focused on using strictly the slope of the power spectral density (PSD) to perform automated sleep scoring and evaluation of the durations of sleep cycles. The method appears to work well because the slope of the PSD is highest during slow-wave sleep, and lowest during waking and REM sleep. Therefore, when smoothed and analyzed across time, there are cyclical variations in the slope of the PSD, fit using an IRASA (Irregularly resampled auto-spectral analysis) algorithm proposed by Wen & Liu (2016).

Strengths:

The main novelty of the study is that the non-fractal (oscillatory) components of the PSD that are more typically used during sleep scoring can be essentially ignored because the key information is already contained within the fractal (slope) component. The authors show that for the most part, results are fairly consistent between this and conventional sleep scoring, but in some cases show disagreements that may be scientifically interesting.

Weaknesses:

The previous weaknesses were well-addressed by the authors in the revised manuscript. I will note that from the fractal cycle perspective, waking and REM sleep are not very dissimilar. Combining these states underlies some of the key results of this study.

Reviewer #2 (Public review):

Summary

Li et al. explored which stage of Stroop conflict processing was influenced by working memory loads. Participants completed a single task (Stroop task) and a dual task (the Sternberg working memory task combined with the Stroop task) while their EEG data was recorded. They adopted the event-related potential (ERP), and multivariate pattern analyses (MVPA) to investigate the interaction effect of task (single/dual) and congruency (congruent/incongruent). The results showed that the interaction effect was significant on the sustained potential (SP; 650-950 ms), the late theta (740-820 ms), and beta (920-1040 ms) power but not significant on the early P1 potential (110-150 ms). They used the representational similarity analyses (RSA) method to explore the correlation between behavioral and neural data, and the results revealed a significant contribution of late theta activity.

Strength

The experiment is well designed.<br /> The data were analyzed in depth from both time and frequency domain perspectives by combining several methods.

Comments on revisions:

All my concerns have been properly addressed, no further comments.

Reviewer #2 (Public review):

Summary:

This study proposes visual homogeneity as a novel visual property that enables observers perform to several seemingly disparate visual tasks, such as finding an odd item, deciding if two items are same, or judging if an object is symmetric. In Exp 1, the reaction times on several objects were measured in human subjects. In Exp 2, visual homogeneity of each object was calculated based on the reaction time data. The visual homogeneity scores predicted reaction times. This value was also correlated with the BOLD signals in a specific region anterior to LO. Similar methods were used to analyze reaction time and fMRI data in a symmetry detection task. It is concluded that visual homogeneity is an important feature that enables observers to solve these two tasks.

Strengths:

(1) The writing is very clear. The presentation of the study is informative.

(2) This study includes several behavioral and fMRI experiments. I appreciate the scientific rigor of the authors.

Weaknesses:

Before addressing the manuscript itself, I would like to comment the review process first. Having read the lasted revised manuscript, I shared many of the concerns raised by the two reviewers in the last two rounds of review. It appears that the authors have disagreed with the majority of comments made by the two reviewers. If so, I strongly recommend that the authors proceed to make this revision as a Version of Record and conclude this review process. According to eLife's policy that the authors have the right to make a Version of Record at any time during the review process, and I fully respect that right. However, I also ask that the authors respect the reviewer's right to retain the comments regarding this paper.

Beside that, I still have several further questions about this study.

(1) My main concern with this paper is the way visual homogeneity is computed. On page 10, lines 188-192, it says: "we then asked if there is any point in this multidimensional representation such that distances from this point to the target-present and target-absent response vectors can accurately predict the target-present and target-absent response times with a positive and negative correlation respectively (see Methods)". This is also true for the symmetry detection task. If I understand correctly, the reference point in this perceptual space was found by deliberating satisfying the negative and positive correlations in response times. And then on page 10, lines 200-205, it shows that the positive and negative correlations actually exist. This logic is confusing. The positive and negative correlations emerge only because this method is optimized to do so. It seems more reasonable to identify the reference point of this perceptual space independently, without using the reaction time data. Otherwise, the inference process sounds circular. A simple way is to just use the mean point of all objects in Exp 1, without any optimization towards reaction time data.<br /> I raised this question in my initial review. However, the authors did not address whether the positive and negative correlations still hold if the mean point is defined as the reference point without any optimization. The authors also argue that it is similar to a case of fitting a straight line. It is fine that the authors insist on the straight line (e.g., correlation). However, I would not call "straight line correlations" a "quantitative model" as a high-profile journals like eLife. Please remove all related arguments of a novel quantitative model.

(2) Visual homogeneity (at least given the current form) is an unnecessary term. It is similar to distractor heterogeneity/distractor variability/distractor saliency in literature. However, the authors attempt to claim it as a novel concept. Both R1 and me raised this question in the very first review. However, the authors refused to revise the manuscript. In the last review, I mentioned this and provided some example sentences claiming novelty. The authors only revised the last sentence of the abstract, and even did not bother to revise the last sentence of significance: "we show that these tasks can be solved using a simple property WE DEFINE as visual homogeneity". Also, lines 851 still shows "we have defined a NOVEL image property, visual homogeneity...". I am confused about whether the authors agree or disagree that "visual homogeneity is an unnecessary term". If the authors agree, they should completely remove the related phrase throughout the paper. If not, they should keep all these and state the reasons. I don't think this is a correct approach to revising a manuscript.

(3) If the authors agree that visual homogeneity is not new, I suggest a complete rewrite of the title, abstract, significance, and introduction. Let me ask a simple question, can we remove "visual homogeneity" and use some more well-established term like "image feature similarity"? If yes, visual homogeneity is unnecessary.

(4) If I understand it correctly, one of the key findings of this paper is "the response times for target-present searches were positively correlated with visual homogeneity. By contrast, the response times for target-absent searches were negatively correlated with visual homogeneity" (lines 204-207). I think the authors have already acknowledged that this positive correlation is not surprising at all because it reflects the classic target-distractor similarity effect. If this is the case, please completely remove the positive correlation as a novel prediction and finding.

(5) In my last review, I mentioned the seminal paper by Duncan and Humphreys (1989) has clearly stated that "difficulty increases with increased similarity of targets to nontargets and decreased similarity between nontargets" (the sentence in their abstract). Here, "similarity between nontargets" is the same as the visual homogeneity defined here. Similar effects have been shown in Duncan (1989) and Nagy, Neriani, and Young (2005). See also the inconsistent results in Nagy& Thomas, 2003, Vicent, Baddeley, Troscianko&Gilchrist, 2009. More recently, Wei Ji Ma has systematically investigated the effects of heterogeneous distractors in visual search. I think the introduction part of Wei Ji Ma's paper (2020) provides a nice summary of this line of research.

Thanks to the authors' revision, I now better understand the negative correlation. The between-distrator similarity mentioned above describes the heterogeneity of distractors WITHIN an image. However, if I understand it correctly, this study aims to address the negative correlation of reaction time and target-absent stimuli ACROSS images. In other words, why do humans show a shorter reaction time to an image of four pigeons than to an image of four dogs (as shown in Figure 2C), simply because the later image is closer to the reference point of the image space. In this sense, this negative correlation is indeed not the same as distractor heterogeneity. However, this is known as the saliency effect or oddball effects. For example, it seems quite natural to me that humans respond faster to a fish image if the image set contains many images of four-leg dogs that look very different from fish. If this is indeed a saliency effect, why should we define a new term "visual homogeneity"?

(6) The section "key predictions" is quite straightforward. I understand the logic of positive and negative correlations. However, what is the physical meaning of "decision boundary" (Fig. 1G) here? How does the "decision boundary" map on the image space?

(7) In my opinion, one of the advantages of this study is the fMRI dataset, which is valuable because previous studies did not collect fMRI data. The key contribution may be the novel brain region associated with display heterogeneity. If this is the case, I would suggest using a more parametric way to measure this region. For example, one can use Gabor stimuli and systematically manipulate the variations of multiple Gabor stimuli, the same logic also applies to motion direction. If this study uses static Gabor, random dot motion, object images that span from low-level to high-level visual stimuli, and consistently shows that the stimulus heterogeneity is encoded in one brain region, I would say this finding is valuable. But this sounds another experiment. In other words, it is insufficient to claim a new brain region given the current form of the manuscript.

References:

* Duncan, J., & Humphreys, G. W. (1989). Visual search and stimulus similarity. Psychological Review, 96(3), 433-458. doi: 10.1037/0033-295x.96.3.433<br /> * Duncan, J. (1989). Boundary conditions on parallel processing in human vision. Perception, 18(4), 457-469. doi: 10.1068/p180457<br /> * Nagy, A. L., Neriani, K. E., & Young, T. L. (2005). Effects of target and distractor heterogeneity on search for a color target. Vision Research, 45(14), 1885-1899. doi: 10.1016/j.visres.2005.01.007<br /> * Nagy, A. L., & Thomas, G. (2003). Distractor heterogeneity, attention, and color in visual search. Vision Research, 43(14), 1541-1552. doi: 10.1016/s0042-6989(03)00234-7<br /> * Vincent, B., Baddeley, R., Troscianko, T., & Gilchrist, I. (2009). Optimal feature integration in visual search. Journal of Vision, 9(5), 15-15. doi: 10.1167/9.5.15<br /> * Singh, A., Mihali, A., Chou, W. C., & Ma, W. J. (2023). A Computational Approach to Search in Visual Working Memory.<br /> * Mihali, A., & Ma, W. J. (2020). The psychophysics of visual search with heterogeneous distractors. BioRxiv, 2020-08.<br /> * Calder-Travis, J., & Ma, W. J. (2020). Explaining the effects of distractor statistics in visual search. Journal of Vision, 20(13), 11-11.

Reviewer #2 (Public review):

In this study, the authors measured extracellular electrical features of colliding APs travelling in different directions down an isolated earthworm axon. They then used these features to build a model of the potential ephaptic effects of AP annihilation, i.e. the electrical signals produced by colliding/annihilating APs that may influence neighbouring tissue. The model was then applied to some different hypothetical scenarios involving synaptic connections. In a revised version of the manuscript, it was also applied, with success, to published experimental data on the cerebellar basket cell-to-Purkinje cell pinceau connection. The conclusion is that an annihilating AP at a presynaptic terminal can emphatically influence the voltage of a postsynaptic cell (this is, presumably, the 'electrical coupling between neurons' of the title), and that the nature of this influence depends on the physical configuration of the synapse.

As an experimental neuroscientist who has never used computational approaches, I am unable to comment on the rigour of the analytical approaches that form the bulk of this paper. The experimental approaches appear very well carried out, and the data showing equal conduction velocity of anti- and orthodromically propagating APs in every preparation is now convincing.

The conclusions drawn from the synaptic modelling have been considerably strengthened by the new Figure 5. Here, the authors' model - including AP annihilation at a synaptic terminal - is used to predict the amplitude and direction of experimentally observed effects at the cerebellar basket cell-to-Purkinje cell synapse (Blot & Barbour 2014). One particular form of the model (RTM with tau=0.5ms and realistic non-excitability of the terminal) matches the experimental data extremely well. This is a much more convincing demonstration that the authors' model of ephaptic effects can quantitatively explain key features of experimental data pertaining to synaptic function. As such, the implications for the relevance of ephaptic coupling at different synaptic contacts may be widespread and important.

However, it appears that all of the models in the new Fig5 involve annihilating APs, yet only one fits the data closely. A key question, which should be addressed if at all possible, is what happens to the predictive power of the best-fitting model in Fig5 if the annihilation, and only the annihilation, is removed? In other words, can the authors show that it is specifically the ephaptic effects of AP annihilation, rather than other ephaptic effects of, say AP waveform/amplitude/propagation, that explain the synaptic effects measured in Blot & Barbour (2014)? This would appear to be a necessary demonstration to fully support the claims of the title.

Reviewer #2 (Public review):

Summary:

The authors used rats to determine the receptor for a food-related perception (kokumi) that has been characterized in humans. They employ a combination of behavioral, electrophysiological, and immunohistochemical results to support their conclusion that ornithine-mediated kokumi effects are mediated by the GPRC6A receptor. They complemented the rat data with some human psychophysical data. I find the results intriguing, but believe that the authors overinterpret their data.

Strengths:

The authors examined a new and exciting taste enhancer (ornithine). They used a variety of experimental approaches in rats to document the impact of ornithine on taste preference and peripheral taste nerve recordings. Further, they provided evidence pointing to a potential receptor for ornithine.

Weaknesses:

The authors have not established that the rat is an appropriate model system for studying kokumi. Their measurements do not provide insight into any of the established effects of kokumi on human flavor perception. The small study on humans is difficult to compare to the rat study because the authors made completely different types of measurements. Thus, I think that the authors need to substantially scale back the scope of their interpretations. These weaknesses diminish the likely impact of the work on the field of flavor perception.

Reviewer #2 (Public review):

Summary:

This is a well-written methodology paper applying a Bayesian framework to the statistics of cell counts in brain slices. A sharpening of the bounds on measured quantities is demonstrated over existing frequentist methods and therefore the work is a contribution to the field.

Strengths:

As well as a mathematical description of the approach, the code used is provided in a linked repository.

Weaknesses:

A clearer link between the experimental data and model-structure terminology would be a benefit to the non-expert reader.

Reviewer #2 (Public review):

Summary:

Koh et al. report an interesting manuscript studying dopamine binding in the lateral accumbens shell of rats across the course of conditioned taste aversion. The question being asked here is how does the dopamine system respond to aversion? The authors take advantage of unique properties of taste aversion learning (notably, within-subjects remapping of valence to the same physical stimulus) to address this.

They combine a well controlled behavioural design (including key, unpaired controls) with fibre photometry of dopamine binding via GrabDA and of dopamine neuron activity by gCaMP, careful analyses of behaviour (e.g., head movements; home cage ingestion), the authors show that, 1) conditioned taste aversion of sucrose suppresses the activity of VTA dopamine neurons and lateral shell dopamine binding to subsequent presentations of the sucrose tastant; 2) this pattern of activity was similar to the innately aversive tastant quinine; 3) dopamine responses were negatively correlated with behavioural (inferred taste reactivity) reactivity; and 4) dopamine responses tracked the contingency of between sucrose and illness because these responses recovered across extinction of the conditioned taste aversion.

Strengths:

There are important strengths here. The use of a well-controlled design, the measurement of both dopamine binding and VTA dopamine neuron activity, the inclusion of an extinction manipulation; and the thorough reporting of the data. I was not especially surprised by these results, but these data are a potentially important piece of the dopamine puzzle (e.g., as the authors note, salience-based argument struggles to explain these data).

Weaknesses for consideration:

(1) The focus here is on the lateral shell. This is a poorly investigated region in the context of the questions being asked here. Indeed, I suspect many readers might expect a focus on the medial shell. So, I think this focus is important. But, I think it does warrant greater attention in both the introduction and discussion. We do know from past work that there can be extensive compartmentalisation of dopamine responses to appetitive and aversive events and many of the inconsistent findings in the literature can be reconciled by careful examination of where dopamine is assessed. I do think readers would benefit from acknowledgement this - for example it is entirely reasonable to suppose that the findings here may be specific to the lateral shell.

(2) Relatedly, I think readers would benefit from an explicit rationale for studying the lateral shell as well as consideration of this in the discussion. We know that there are anatomical (PMID: 17574681), functional (PMID: 10357457), and cellular (PMID: 7906426) differences between the lateral shell and the rest of the ventral striatum. Critically, we know that profiles of dopamine binding during ingestive behaviours there can be highly dissimilar to the rest of ventral striatum (PMID: 32669355). I do think these points are worth considering.

(3) I found the data to be very thoughtfully analysed. But in places I was somewhat unsure:<br /> (a) Please indicate clearly in the text when photometry data show averages across trials versus when they show averages across animals.<br /> (b) I did struggle with the correlation analyses, for two reasons.<br /> (i) First, the key finding here is that the dopamine response to intraoral sucrose is suppressed by taste aversion. So, this will significantly restrict the range of dopamine transients, making interpretation of the correlations difficult.

(ii) Second, the authors report correlations by combining data across groups/conditions. I understand why the authors have done this, but it does risk obscuring differences between the groups. So, my question is: what happens to this trend when the correlations are computed separately for each group? I suspect other readers will share the same question. I think reporting these separate correlations would be very helpful for the field - regardless of the outcome.

(4) Figure 1A is not as helpful as it might be. I do think readers would expect a more precise reporting of GCaMP expression in TH+ and TH- neurons. I also note that many of the nuances in terms of compartmentalisation of dopamine signalling discussed above apply to ventral tegmental area dopamine neurons (e.g. medial v lateral) and this is worth acknowledging when interpreting.

Reviewer #2 (Public review):

Summary:

The authors used deep full-length single-cell sequencing to study human photoreceptor development, with a particular emphasis on the characteristics of photoreceptors that may contribute to retinoblastoma.

Strengths:

This single-cell study captures gene regulation in photoreceptors across different developmental stages, defining post-mitotic cone and rod populations by highlighting their unique gene expression profiles through analyses such as RNA velocity and SCENIC. By leveraging full-length sequencing data, the study identifies differentially expressed isoforms of NRL and THRB in L/M cone and rod precursors, illustrating the dynamic gene regulation involved in photoreceptor fate commitment. Additionally, the authors performed high-resolution clustering to explore markers defining developing photoreceptors across the fovea and peripheral retina, particularly characterizing SYK's role in the proliferative response of cones in the RB loss background. The study provides an in-depth analysis of developing human photoreceptors, with the authors conducting thorough analyses using full-length single-cell RNA sequencing. The strength of the study lies in its design, which integrates single-cell full-length RNA-seq, long-read RNA-seq, and follow-up histological and functional experiments to provide compelling evidence supporting their conclusions. The model of cell type-dependent splicing for NRL and THRB is particularly intriguing. Moreover, the potential involvement of the SYK and MYC pathways with RB in cone progenitor cells aligns with previous literature, offering additional insights into RB development.

Weaknesses:

The manuscript feels somewhat unfocused, with a lack of a strong connection between the analysis of developing photoreceptors, which constitutes the bulk of the manuscript, and the discussion on retinoblastoma. Additionally, given the recent publication of several single-cell studies on the developing human retina, it is important for the authors to cross-validate their findings and adjust their statements where appropriate.

Reviewer #2 (Public review):

This is a great paper. Yoshida et al. convincingly show that DnaA does not exclusively do loading of the replicative helicase at the E. coli oriC, but that PriC can also perform this function. Importantly, PriC seems to contribute to helicase loading even in wt cells albeit to a much lesser degree than DnaA. On the other hand, PriC takes a larger role in helicase loading during aberrant initiation, i.e. when the origin sequence is truncated or when the properties of initiation proteins are suboptimal. Here highlighted by mutations in dnaA or dnaC.

This is a major finding because it clearly demonstrates that the two roles of DnaA in the initiation process can be separated into initially forming an open complex at the DUE region by binding/nucleation onto DnaA-boxes and second by loading of the helicase. Whereas these two functions are normally assumed to be coupled, the present data clearly show that they can be separated and that PriC can perform at least part of the helicase loading provided that an area of duplex opening is formed by DnaA.

This puts into question the interpretation of a large body of previous work on mutagenesis of oriC and dnaA to find a minimal oriC/DnaA complex in many bacteria. In other words, mutants in which oriC is truncated/mutated may support the initiation of replication and cell viability only in the presence of PriC. Such mutants are capable of generating single-strand openings but may fail to load the helicase in the absence of PriC. Similarly, dnaA mutants may generate an aberrant complex on oriC that trigger strand opening but are incapable of loading DnaB unless PriC is present.

In the present work, the sequence of experiments presented is logical and the manuscript is clearly written and easy to follow. The very last part regarding PriC in cSDR replication does not add much to the story and may be omitted.

Reviewer #2 (Public review):

Summary:

The authors develop a computational model (and a simplified version thereof) to treat an extremely important issue regarding tumor growth. Specifically, it has been argued that fibroblasts have the ability to support tumor growth by creating physical conditions in the tumor microenvironment that prevent the relevant immune cells from entering into contact with, and ultimately killing, the cancer cells. This inhibition is referred to as immune exclusion. The computational approach follows standard procedures in the formulation of models for mixtures of different material species, adapted to the problem at hand by making a variety of assumptions as to the activity of different types of fibroblasts, namely "normal" versus "cancer-associated". The model itself is relatively complex, but the authors do a convincing job of analyzing possible behaviors and attempting to relate these to experimental observations.

Strengths:

As mentioned, the authors do an excellent job of analyzing the behavior of their model both in its full form (which includes spatial variation of the concentrations of the different cellular species) and in its simplified mean field form. The model itself is formulated based on established physical principles, although the extent to which some of these principles apply to active biological systems is not clear (see Weaknesses). The results of the model do offer some significant insights into the critical factors which determine how fibroblasts might affect tumor growth; these insights could lead to new experimental ways of unraveling these complex sets of issues and enhancing immunotherapy.

Weaknesses:

Models of the form being studied here rely on a large number of assumptions regarding cellular behavior. Some of these seemed questionable, based on what we have learned about active systems. The problem of T cell infiltration as well as the patterning of the extracellular matrix (ECM) by fibroblasts necessarily involve understanding cell motion and cell interactions due e.g. to cell signaling. Adopting an approach based purely on physical systems driven by free energies alone does not consider the special role that active processes can play, both in motility itself and in the type of self-organization that can occur due to these cell-cell interactions. This to me is the primary weakness of this paper.

A separate weakness concerns the assumption that fibroblasts affect T cell behavior primarily by just making a more dense ECM. There are a number of papers in the cancer literature (see, for some examples, Carstens, J., Correa de Sampaio, P., Yang, D. et al. Spatial computation of intratumoral T cells correlates with survival of patients with pancreatic cancer. Nat Commun 8, 15095 (2017); Sun, Xiujie, Bogang Wu, Huai-Chin Chiang, Hui Deng, Xiaowen Zhang, Wei Xiong, Junquan Liu et al. "Tumour DDR1 promotes collagen fibre alignment to instigate immune exclusion." Nature 599, no. 7886 (2021): 673-678) that seem to indicate that density alone is not a sufficient indicator of T cell behavior. Instead, the organization of the ECM (for example, its anisotropy) could be playing a much more essential role than is given credit for here. This possibility is hinted at in the Discussion section but deserves much more emphasis.

Finally, the mixed version of the model is, from a general perspective, not very different from many other published models treating the ecology of the tumor microenvironment (for a survey, see Arabameri A, Asemani D, Hadjati J (2018), A structural methodology for modeling immune-tumor interactions including pro-and anti-tumor factors for clinical applications. Math Biosci 304:48-61). There are even papers in this literature that specifically investigate effects due to allowing cancer cells to instigate changes in other cells from being tumor-inhibiting to tumor-promoting. This feature occurs not only for fibroblasts but also for example for macrophages which can change their polarization from M1 to M2. There needed to be some more detailed comparison with this existing literature.

Reviewer #2 (Public review):

Summary:

The study by Obray et al. entitled "Adolescent alcohol exposure promotes mechanical allodynia and alters synaptic function at inputs from the basolateral amygdala to the prelimbic cortex" investigated how adolescent intermittent ethanol exposure (AIE) affects the BLA -> PL circuit, with an emphasis on PAG projecting PL neurons, and how AIE changes mechanical and thermal nociception. The authors found that AIE increased mechanical, but not thermal nociception, and an injection of an inflammatory agent did not produce changes in an ethanol-dependent manner. Physiologically, a variety of AIE-specific effects were found in PL neuron firing at BLA synapses, suggestive of AIE-induced alterations in neurotransmission at BLA-PVIN synapses.

Strengths:

This was a comprehensive examination of the effects of AIE on this neural circuit, with an in-depth dissection of the various neuronal connections within the PL.

Sex was included as a biological variable, yet there were little to no sex differences in AIE's effects, suggestive of similar adaptations in males and females.

Reviewer #2 (Public Review):

In this manuscript, Otero-Coronel and colleagues use a combination of acoustic stimuli and electrical stimulation of the tectum to study MSI in the M-cells of adult goldfish. They first perform a necessary piece of groundwork in calibrating tectal stimulation for maximal M-cell MSI, and then characterize this MSI with slightly varying tectal and acoustic inputs. Next, they quantify the magnitude and timing of FFI that each type of input has on the M-cell, finding that both the tectum and the auditory system drive FFI, but that FFI decays more slowly for auditory signals. These are novel results that would be of interest to a broader sensory neuroscience community. By then providing pairs of stimuli separated by 50ms, they assess the ability of the first stimulus to suppress responses to the second, finding that acoustic stimuli strongly suppress subsequent acoustic responses in the M-cell, that they weakly suppress subsequent tectal stimulation, and that tectal stimulation does not appreciably inhibit subsequent stimuli of either type. Finally, they show that M-cell physiology mirrors previously reported behavioural data in which stronger stimuli underwent less integration.

The manuscript is generally well-written and clear. The discussion of results is appropriately broad and open-ended. It's a good document. Our major concerns regarding the study's validity are captured in the individual comments below. In terms of impact, the most compelling new observation is the quantification of the FFI from the two sources and the logical extension of these FFI dynamics to M-cell physiology during MSI. It is also nice, but unsurprising, to see that the relationship between stimulus strength that MSI is similar for M-cell physiology to what has previously been shown for behavior. While we find the results interesting, we think that they will be of greatest interest to those specifically interested in M-cell physiology and function.

Reviewer #2 (Public Review):

This work by Knights et al., makes use of the Cam-CAN dataset to investigate functional compensation during a fluid processing task in older adults, in a fairly large sample of approximately 200 healthy adults ranging from 19 to 87. Using univariate methods, the authors identify two brain regions in which activity increases as a function of both age and performance and conduct further investigations to assess whether the activity of these regions provides information regarding task difficulty. The authors conclude that the cuneal cortex - a region of the brain previously implicated in visual attention - shows evidence of compensation in older adults.

The conclusions of the paper are well supported by the data, and the authors use appropriate statistical analyses. The use of multivariate methods over the last 20 years has demonstrated many effects that would have been missed using more traditional univariate analysis techniques. The data set is also of an appropriate size, and as the authors note, fluid processing is an extremely important domain in the field of cognition in aging, due to its steep decline over aging.

Comment from Reviewing Editor: It would have been nice to see an analysis of a more crystallised intelligence task included too, as a contrast since this is an area that does not demonstrate such a decline (and perhaps continues to improve over aging). This comment does not take away the important contributions of the manuscript.

Reviewer #2 (Public review):

Summary:

In this work, the authors performed a CRISPR knockout screen in the presence of floxuridine, a chemotherapeutic agent that incorporates uracil and fluoro-uracil into DNA, and identified unexpected factors, such as the RNA m6A methyltransferase METTL3, as required to overcome floxuridine-driven cytotoxicity in mammalian cells. Interestingly, the observed N6-methyladenosine was embedded in DNA, which has been reported as DNA 6mA in mammalian genomes and is currently confirmed with mass spectrometry in this model. Therefore, this work consolidated the functional role of mammalian genomic DNA 6mA, and supported with solid evidence to uncover the METTL3-6mA-UNG2 axis in response to DNA base damage.

Strengths:

In this work, the authors took an unbiased, genome-wide CRISPR approach to identify novel factors involved in uracil repair with potential clinical interest.

The authors designed elegant experiments to confirm the METTL3 works through genomic DNA, adding the methylation into DNA (6mA) but not the RNA (m6A), in this base damage repair context. The authors employ different enzymes, such as RNase A, RNase H, DNase, and liquid chromatography coupled to tandem mass spectrometry to validate that METTL3 deposits 6mA in DNA in response to agents that increase genomic uracil.

They also have the Mettl3-KO and the METTL3 inhibition results to support their conclusion.

Weaknesses:

Although this study demonstrates that METTL3-dependent 6mA deposition in DNA is functionally relevant to DNA damage repair in mammalian cells, there are still several concerns and issues that need to be improved to strengthen this research.

First, in the whole paper, the authors never claim or mention the mammalian cell lines contamination testing result, which is the fundamental assay that has to be done for the mammalian cell lines DNA 6mA study.

Second, in the whole work, the authors have not supplied any genomic sequencing data to support their conclusions. Although the sequencing of DNA 6mA in mammalian models is challenging, recent breakthroughs in sequencing techniques, such as DR-Seq or NT/NAME-seq, have lowered the bar and improved a lot in the 6mA sequencing assay. Therefore, the authors should consider employing the sequencing methods to further confirm the functional role of 6mA in base repair.

Third, the authors used the METTL3 inhibitor and Mettl3-KO to validate the METTL3-6mA-UNG2 functional roles. However, the catalytic mutant and rescue of Mettl3 may be the further experiments to confirm the conclusion.

Reviewer #2 (Public review):

Summary:

The authors build a colossal anatomical model of juvenile rat non-barrel primary somatosensory cortex, including inputs from the thalamus. This enhances past models by incorporating information on the shape of the cortex and estimated densities of various types of excitatory and inhibitory neuron across layers. This is intended to enable analysis of the micro- and mesoscopic organisation of cortical connectivity and to be a base anatomical model for large-scale simulations of physiology.

Strengths:

• The authors incorporate many diverse data sources on morphology and connectivity.<br /> • This paper takes on the challenging task of linking micro- and meso-scale connectivity<br /> • By building in the shape of the cortex, the authors were able to link cortical geometry to connectivity. In particular they make an unexpected prediction that cortical conicality affects the modularity of local connectivity, which should be testable.<br /> • The author's analysis of the model led to the interesting prediction that layer 5 neurons' connect local modules, which may be testable in the future, and provide a basis to link from detailed anatomy to functional computations.<br /> • The visualisation of the anatomy in various forms is excellent<br /> • The model is openly shared

Weaknesses:

• There is no effort to determine how specific or generalisable the findings here are to other parts of cortex.<br /> • Although there is a link to physiological modelling in another paper, there is no clear pathway to go from this type of model to understanding how the specific function of the modelled areas may emerge here (and not in other cortical areas).<br /> • Some of the decisions seem a little ad-hoc, and the means to assess those decisions is not always easily available to the reader<br /> • The shape of the juvenile cortex - a key novelty of this work - was based on merely a scalar reduction of the adult cortex. This is very surprising, and surely an oversimplification. Huge efforts have gone into modelling the complex nonlinear development of cortex, by teams including the developing Human Connectome Project. For such a fundamental aspect of this work, why isn't it possible to reconstruct the shape of this relatively small part of juvenile rat cortex?<br /> • The same relative laminar depths are used for all subregions. This will have a large impact on the model. However, relative laminar depths can change drastically across the cortex (see e.g. many papers by Palomero-Gallagher, Zilles and colleagues). The authors should incorporate the real laminar depths, or, failing that, show evidence to show that the laminar depth differences across the subregions included in the model are negligible.<br /> • The authors perform an affine mapping between mouse and rat cortex. This is again surprising. In human imaging, affine mappings are insufficient to map between two individual brains of the same species, and nonlinear transformations are instead used. That an affine transformation should be considered sufficient to map between two different species is then very surprising. For some models, this may be fine, but there is a supposed emphasis here on biological precision in terms of anatomical location.<br /> o Live nature of the model. This is such a colossal model, and effort, that I worry that it may be quite difficult to update in light of new data. For example, how much person and compute time would it take to update the model to account for different layer sizes across subregions? Or to more precisely account for the shape of juvenile rat cortex?

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors investigate how epithelia maintain intercellular barrier function despite and during cellular rearrangements upon e.g. apoptotic extrusion in simple epithelia or regenerative turnover in stratified epithelia like this epidermis. A fundamental question in epithelial biology. Previous literature has shown that Rho-mediated local regulation of actomyosin is essential not only for cellular rearrangement itself but also for directly controlling tight junction barrier function. The molecular mechanics however remained unclear. Here the authors use extensive fluorescent imaging of fixed and live cells together with genetic and drug-mediated interference to show that Rho activation is required and sufficient to form novo tight junctional strands at intercellular contacts in epidermal keratinocytes (HaCat) and mammary epithelial cells. After having confirmed previous literature they then show that Rho activation activates the transmembrane protease Matriptase which cleaves EpCAM and TROP2, two claudin-binding transmembrane proteins, to release claudins and enable claudin strand formation and therefore tight junction barrier function.

Strengths:

The presented mechanism is shown to be relevant for epithelial barriers being conserved in simple and stratifying epithelial cells and mainly differs due to tissue-specific expression of EpCAM and TROP2. The authors present careful state-of-the-art imaging and logical experiments that convincingly support the statements and conclusion. The manuscript is well-written and easy to follow.

Weaknesses:

Whereas the in vitro evidence of the presented mechanism is strongly supported by the data, the in vivo confirmation is mostly based on the predicted distribution of TROP2. Whereas the causality of Rho-mediated Matriptase activation has been nicely demonstrated it remains unclear how Rho activates Matriptase.

Reviewer #2 (Public review):

Summary:

The author developed a new device to overcome current limitations in the imaging process of 3D spheroidal structures. In particular, they created a system to follow in real-time tumour spheroid formation, fusion and cell migration without disrupting their integrity. The system has also been exploited to test the effects of a therapeutic agent (chemotherapy) and immune cells.

Strengths:

The system allows the in situ observation of the 3D structures along the 3 axes (x,y and z) without disrupting the integrity of the spheroids; in a time-lapse manner it is possible to follow the formation of the 3D structure and the spheroids fusion from multiple angles, allowing a better understanding of the cell aggregation/growth and kinetic of the cells.

Interestingly the system allows the analysis of cell migration/ escape from the 3D structure analysing not only the morphological changes in the periphery of the spheroids but also from the inner region demonstrating that the proliferating cells in the periphery of the structure are more involved in the migration and dissemination process. The application of the system in the study of the effects of doxorubicin and NK cells would give new insights in the description of the response of tumor 3D structure to killing agents.

Reviewer #2 (Public review):

Summary:

The role of PRC2 in post-neural crest induction was not well understood. This work developed an elegant mouse genetic system to conditionally deplete EED upon SOX10 activation. Substantial developmental defects were identified for craniofacial and bone development. The authors also performed extensive single-cell RNA sequencing to analyze differentiation gene expression changes upon conditional EED disruption.

Strengths:

(1) Elegant genetic system to ablate EED post neural crest induction.

(2) Single-cell RNA-seq analysis is extremely suitable for studying the cell type-specific gene expression changes in developmental systems.

Weaknesses:

(1) Although this study is well designed and contains state-of-the-art single-cell RNA-seq analysis, it lacks the mechanistic depth in the EED/PRC2-mediated epigenetic repression. This is largely because no epigenomic data was shown.

(2) The mouse model of conditional loss of EZH2 in neural crest has been previously reported, as the authors pointed out in the discussion. What is novel in this study to disrupt EED? Perhaps a more detailed comparison of the two mouse models would be beneficial.

(3) The presentation of the single-cell RNA-seq data may need improvement. The complexity of the many cell types blurs the importance of which cell types are affected the most by EED disruption.

(4) While it's easy to identify PRC2/EED target genes using published epigenomic data, it would be nice to tease out the direct versus indirect effects in the gene expression changes (e.g Figure 4e).

Reviewer #2 (Public review):

Summary

Croshagen et al develop a range of tools based on selection-linked integration (SLI) to study PfEMP1 function in P. falciparum. PfEMP1 is encoded by a family of ~60 var genes subject to mutually exclusive expression. Switching expression between different family members can modify the binding properties of the infected erythrocyte while avoiding the adaptive immune response. Although critical to parasite survival and Malaria disease pathology, PfEMP1 proteins are difficult to study owing to their large size and variable expression between parasites within the same population. The SLI approach previously developed by this group for genetic modification of P. falciparum is employed here to selectively and stably activate the expression of target var genes at the population level. Using this strategy, the binding properties of specific PfEMP1 variants were measured for several distinct var genes with a novel semi-automated pipeline to increase throughput and reduce bias. Activation of similar var genes in both the common lab strain 3D7 and the cytoadhesion competent FCR3/IT4 strain revealed higher binding for several PfEMP1 IT4 variants with distinct receptors, indicating this strain provides a superior background for studying PfEMP1 binding. SLI also enables modifications to target var gene products to study PfEMP1 trafficking and identify interacting partners by proximity-labeling proteomics, revealing two novel exported proteins required for cytoadherence. Overall, the data demonstrate a range of SLI-based approaches for studying PfEMP1 that will be broadly useful for understanding the basis for cytoadhesion and parasite virulence.

Comments

(1) While the capability of SLI to actively select var gene expression was initially reported by Omelianczyk et al., the present study greatly expands the utility of this approach. Several distinct var genes are activated in two different P. falciparum strains and shown to modify the binding properties of infected RBCs to distinct endothelial receptors; development of SLI2 enables multiple SLI modifications in the same parasite line; SLI is used to modify target var genes to study PfEMP1 trafficking and determine PfEMP1 interactomes with BioID. Curiously, Omelianczyk et al activated a single var (Pf3D7_0421300) and observed elevated expression of an adjacent var arranged in a head-to-tail manner, possibly resulting from local chromatin modifications enabling expression of the neighboring gene. In contrast, the present study observed activation of neighboring genes with head-to-head but not head-to-tail arrangement, which may be the result of shared promoter regions. The reason for these differing results is unclear although it should be noted that the two studies examined different var loci.

(2) The IT4var19 panned line that became binding-competent showed increased expression of both paralogs of ptp3 (as well as a phista and gbp), suggesting that overexpression of PTP3 may improve PfEMP1 display and binding. Interestingly, IT4 appears to be the only known P. falciparum strain (only available in PlasmoDB) that encodes more than one ptp3 gene (PfIT_140083100 and PfIT_140084700). PfIT_140084700 is almost identical to the 3D7 PTP3 (except for a ~120 residue insertion in 3D7 beginning at residue 400). In contrast, while the C-terminal region of PfIT_140083100 shows near-perfect conservation with 3D7 PTP3 beginning at residue 450, the N-terminal regions between the PEXEL and residue 450 are quite different. This may indicate the generally stronger receptor binding observed in IT4 relative to 3D7 results from increased PTP3 activity due to multiple isoforms or that specialized trafficking machinery exists for some PfEMP1 proteins.

Reviewer #2 (Public review):

Summary:

Lai and collaborators use a previously published RNAseq dataset derived from an experimental evolution set up to compare the pleiotropic properties of genes whose expression evolved in response to fluctuating temperature for over 100 generations. The authors correlate gene pleiotropy with the degree of parallelisms in the experimental evolution set up to ask: are genes that evolved in multiple replicates more or less pleiotropic?

They find that, maybe counter to expectation, highly pleiotropic genes show more replicated evolution. Such an effect seems to be driven by direct effects (which the authors can only speculate on) and indirect effects through low variance in pleiotropic genes (which the authors indirectly link to genetic variation underlying gene expression variance).

Weaknesses:

The results offer new insights into the evolution of gene expression and into the parameters that constrain such evolution, i.e., pleiotropy. Although the conclusions are supported by the data, I find the interpretation of the results a little bit complicated.

Major comment:

The major point I ask the authors to address is whether the connection between polygenic adaptation and parallelism can indeed be used to interpret gene expression parallelism. If the answer is not, please rephrase the introduction and discussion, if the answer is yes, please make it explicit in the text why it is so.

The authors' argument: parallelism in gene expression is the same as parallelism in SNP allele frequency (AFC) (see L389-383 here they don't mention that this explanation is derived from SNP parallelism and not trait parallelism, and see Figure 1 b). In previous publications, the authors have explained the low level of AFC parallelism using a polygenic argument. Polygenic traits can reach a new trait optimum via multiple SNPs and therefore although the trait is parallel across replicates, the SNPs are not necessarily so.

In the current paper, they seem to be exchanging SNP AFC by gene expression, and to me, those are two levels that cannot be interchanged. Gene expression is a trait, not an SNP, and therefore the fact that a gene expression doesn't replicate cannot be explained by a polygenic basis, because again the trait is gene expression itself. And, actually, the results of the simulations show that high polygenicity = less trait parallelism (Figure 4).

Now, if the authors focus on high parallel genes (present in e.g. 7 or more replicates) and they show that the eQTLs for those genes are many (highly polygenic) and the AFC of those eQTLs are not parallel, then I would agree with the interpretation. But, given that here they just assess gene expression and not eQTL AFC, I do not think they can use the 'highly polygenic = low parallelism' explanation.

The interpretation of the results to me, should be limited to: genes with low variance and high pleiotropy tend to be more parallel, and the explanation might be synergistic pleiotropy.

Reviewer #2 (Public review):

Summary:

In this single center, single arm, open label non-randomised study the authors tested the use of paclitaxel at 180-220 mg/m2 and cisplatin at 60mg/m2 in patients with squamous NSCLC and pemetrexed at 500mg/m2 and cisplatin at 60mg/m2 in adenocarcinoma of lung origin in the neoadjuvant setting. The chemotherapy appears to have been given at a relatively standard dose; though the platin dose at 60mg/m2 is somewhat lower than has been used in the checkmate 816 trial (75mg/m2/dose), this is a well-established dose for NSCLC.

Key differences to currently approved neoadjuvant chemo-ICI treatment is that anti-PD1 antibody sintilimab (at 200mg/dose) was given on day 5 and that only 2 cycles of chemotherapy were given pre surgery, but then repeated on two occasions post surgery. Between May/2020 and Nov/2023 50 patients were screened, 38 went on to have this schedule of tx, 31 (~82%) went on to have surgery and 27 had the adjuvant treatment. The rate of surgery is entirely consistent with the checkmate 816 data.

Question to the authors:

It would be very helpful to understand why 7 (~18% of the population) patients did not make it to surgery and whether this is related to disease progression, toxicity or other reasons for withdrawal.

The key clinical endpoints were pCR and mPR rates. 2/38 patients are reported to have achieved a radiological pCR but only 31 patients underwent surgery with histological verification. Supp table2 suggests that 10/31 patients achieved a pCR, 6/31 additional patients achieved a major pathological response and that 13/31 did not achieve a major pathological response

It would be really helpful for understanding the clinical outcome to present the histopathological findings in the text in a bit more detail and to refer the outcome to the radiological findings. I note that the reference for pathological responses incorrectly is 38 patients as only 31 patients underwent surgery and were evaluated histologically.

The treatment was very well tolerated with only 1 grade 3 AE reported. The longer term outcome will need to be assessed over time as the cohort is very 'young'. It is not clear what the adjuvant chemo-ICI treatment would add and how this extra treatment would be evaluated for benefit - if all the benefit is in the neoadjuvant treatment then the extra post-operative tx would only add toxicity

Please consider what the two post-operative chemo-ICI cycles might add to the outcome and how the value of these cycles would be assessed. Would there be a case for a randomised assessment in the patients who have NOT achieved a mPR histologically?

While the clinical dataset identifies that the proposed reduced chemo-ICI therapy has clinical merit and should be assessed in a randomized study, the translational work is less informative.

The authors suggest that the treatment has a positive impact on T lymphocytes. Blood sampling was done at day 0 and day 5 of each of the four cycle of chemotherapy with an additional sample post cycle 4. The authors state that data were analysed at each stage.

The data in Figure 3B are reported for three sets of pairs: baseline to pre day 5 in cycle 1, day 5 to day 21 in cycle 1, baseline of cycle to to day 5. It remains unclear whether the datasets contain the same top 20 clones and it would be very helpful to show kinetic change for the individual 'top 20 clones' throughout the events in individual patients; as it stands the 'top20 clones' may vary widely from timepoint to timepoint. Of note, the figures do not demonstrate that the top 20 TCR clones were 'continuously increased'.

Instead, the data suggest that there are fluctuations in the relative distributions over time but that may simply be a reflection of shifts in T cell populations following chemotherapy rather than of immunological effects in the cancer tissue.<br /> Consistent with this the authors conclude (line 304/5): "No significant difference was observed in the diversity, evenness, and clonality of TCR clones across the whole treatment procedure" and this seems to be a more persuasive conclusion than the statement 'that a positive effect on T lymphocytes was observed' - where it is also not clear what 'positive' means.

The text needs a more balanced representation of the data: only a small subset of four patients appear to have been evaluated to generate the data for figure 3B and only three patients (P5, P6, P7) can have contributed to figure 3C if the sample collection is represented accurately in Figure 3A.

The text refers to flow cytometric results in SF3. However, no information is given on the flow cytometry in M&M, markers or gating strategy.

Please consider changing the terminology of the 'phases' into something that is easier to understand. One option would be to use a reference to a more standard unit (cycle 1-4 of chemotherapy and then d0/d5/d21).

Please make it explicit in the text that molecular analyses were undertaken for some patients only, and how many patients contribute to the data in figures 3B-F. Figure 3A suggests paired mRNA data were obtained in 2 patients (P2 and P5) but I cannot find the results on these analyses; four individual blood samples to assess TCR changes int PH1/PH2/PH3and PH4 were only available in four patients (P4,P5,P7,P9). Only three patients seem to have the right samples collected to allow the analysis for 'C3' in figure 3C.

Please display for each of the 'top 20 clones' at any one timepoint how these clones evolve throughout the study; I expect that a clone that is 'top 20' at a given timepoint may not be among the 'top twenty' at all timepoints.

Please also assess if the expanded clonotypes are present (and expanded) in the cancer tissue at resection, to link the effect in blood to the tumour. Given that tissue was collected for 31 patients, mRNA sequencing to generate TCR data should be possible to add to the blood analyses in the 12 patients in Figure 3A. Without this data no clear link can be made to events in the cancer.

Please provide in M&M the missing information on the flow cytometry methodology (instrument, antibody clones, gating strategy) and what markers were used to define T cell subsets (naïve, memory, central memory, effector memory).

The authors also describe that ctDNA reduces after chemo-ICI treatment. This is well documented in their data but ultimately irrelevant: if the cancer volume is reduced to the degree of a radiological or pathological response /complete response then the quantity of circulating DNA from the cancer cells must reduce. More interesting would be the question whether early changes predict clinical outcome and whether recurrent ct DNA elevations herald recurrence.

Please probe whether the molecular data identify good radiological or pathological outcomes before cycle 2 is started and whether the ctDNA levels identify patients who will have a poor response and/or who relapse early.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors follow up on their previous work showing that in the absence of the Sir2 deacetylase the MCM replicative helicase at the rDNA spacer region is repositioned to a region of low nucleosome occupancy. Here they show that the repositioned displaced MCMs have increased firing propensity relative to non-displaced MCMs. In addition, they show that activation of the repositioned MCMs and low nucleosome occupancy in the adjacent region depend on the chromatin remodeling activity of Fun30.

Strengths:

The paper provides new information on the role of a conserved chromatin remodeling protein in regulation of origin firing and in addition provides evidence that not all loaded MCMs fire and that origin firing is regulated at a step downstream of MCM loading.

Comments on revisions:

The authors have addressed my concerns with the addition of new experiments and analysis.

Reviewer #2 (Public review):

Summary:

The study explores the functional consequence of CDK12 loss in prostate cancer. While CDK12 loss has been shown to confer homologous recombination (HR) deficiency through premature intronic polyadenylation of HR genes, the response of PARPi monotherapy has failed. This study therefore performed an in-depth analysis of genomic sequencing data from mCRPC patient tumors, and showed that tumors with CDK12 loss lack pertinent HR signatures and scars. Furthermore, functional exploration in human prostate cancer cell lines showed that while the acute inhibition of CDK12 resulted in aberrant polyadenylation of HR genes like BRCA1/2, HR-specific effects were overall modest or absent in cell lines or xenografts adapted to chronic CDK12 loss. Instead, vulnerability to genetically targeting CDK13 resulted in a synthetic lethality in tumors with CDK12 loss, as shown in vivo with SR4825, a CDK12/13 inhibitor - thus serving as a potential therapeutic avenue.

The evidence supporting this study is based on in-depth genomic analyses of human patients, acute knockdown studies of CDK12 using a CDK12/13 inhibitors SR4835, adaptive knockout of CDK12 using LuCaP 189.4_CL and inducible re-expression of CDK12, CDK12 single clones in 22Rv1 (KO2 and KO5) and Skov3 (KO1), Tet-inducible knockdown of BRCA2 or CDK12 followed by ionizing radiation and measurement of RAD51 foci, lack of sensitivity generally to PARPi and platinum chemotherapy in cells adapted to CDK12 loss, loss of viability of CDK13 knockout in CDK12 knockout cells, and in vivo testing of SE4825 in LuCaP xenografts with intact and CDK12 loss.

Strengths:

Overall, this study is robust and of interest to the broader homologous recombination and CDK field. First, the topic is clinically relevant given the lack of PARPi response in CDK12 loss tumors. Second, the strength of the genomic analysis in CDK12 lost PCa tumors is robust with clear delineation that BRCA1/2 genes and maintenance of most genes regulating HR are intact. Specifically, the authors find that there is no mutational signature or genomic features suggestive of HR, such as those found in BRCA1/2 tumors. Lastly, novel lines are generated in this study, including de novo LuCaP 189.4_CL with CDK12 loss that can be profound for potential synthetic lethalities.

Weakness:

One caveat that continues to be unclear as presented, is the uncoupling of cell cycle/essentiality of CDK12/13 from HR-directed mechanisms. Is this purely a cell cycle arrest phenotype acutely with associated down-regulation of many genes?

While the RAD51 loading ssRNA experiments are informative, the Tet-inducible knockdown of BRCA2 and CDK12 is confusing as presented in Figure 5, shBRCA2 + and -dox are clearly shown. However, were the CDK12_K02 and K05 also knocked down using inducible shRNA or a stable knockout? The importance of this statement is the difference between acute and chronic deletion of CDK12. Previously, the authors showed that acute knockdown of CDK12 led to an HR phenotype, but here it is unclear whether CDK12-K02/05 are acute knockdowns of CDK12 or have been chronically adapted after single cell cloning from CRISPR-knockout.

Given the multitude of lines, including some single-cell clones with growth inhibitory phenotypes and ex-vivo derived xenografts, the variability of effects with SR4835, ATM, ATR, and WEE1 inhibitors in different models can be confusing to follow. Overall, the authors suggest that the cell lines differ in therapeutic susceptibility as they may have alternate and diverse susceptibilities. It may be possible that the team could present this more succinctly and move extraneous data to the supplement.

The in-vitro data suggests that SR4835 causes growth inhibition acutely in parental lines such as 22RV1. However, in vivo, tumor attenuation appears to be observed in both CDK12 intact and deficient xenografts, LuCAP136 and LuCaP 189.4 (albeit the latter is only nominally significant). Is there an effect of PARPi inhibition specifically in either model? What about the the 22RV1-K02/05? Do these engraft? Given the role of CDK12/13 in RNAP II, these data might suggest that the window of susceptibility in CDK12 tumors may not be that different from CDK12 intact tumors (or intact tissue) when using dual CDK12/13 inhibitors but rather represent more general canonical essential functions of CDK12 and CDK13 in transcription. From a therapeutic development strategy, the authors may want to comment in the discussion on the ability to target CDK13 specifically.

Reviewer #3 (Public review):

The manuscript presents interesting findings on the role of gut microbiota in gout, focusing on the interplay between age-related changes, inflammation, and microbiota-derived metabolites, particularly butyrate. The study provides valuable insights into the therapeutic potential of microbiota interventions and metabolites for managing hyperuricemia and gout.

The manuscript has improved with the revisions made, particularly regarding clarifications on experimental design and the inclusion of supplementary data.

Comments on latest version:

The authors have addressed many previous concerns; however, some areas still require clarification and improvement to support more definitive conclusions.

(1) This study suggests that microbiota interventions, particularly butyrate, show promising therapeutic potential for hyperuricemia and gout. While the authors discuss the functions of certain butyrate-producing bacteria, I recommend further validating the gut microbiota-butyrate pathway by supplementing germ-free animal models with a single butyrate-producing strain, such as Clostridium butyricum. To strengthen the manuscript, I suggest the authors make further revisions to address these key issues.

(2) Additionally, I was unable to locate the full-length, uncropped Western blot images in the manuscript or supplementary materials. Could the authors please provide these?

Reviewer #3 (Public review):

Previous work (Chouhan et al., 2022) from the Sehgal group investigated the relationship between sleep and long-term memory formation by dissecting the role of mushroom body intrinsic neurons, extrinsic neurons, and output neurons during sleep-dependent and sleep-independent memory consolidation. In this manuscript, Li et al., profiled transcriptome in the anterior-posterior (ap) α'/β' neurons and identified genes that are differentially expressed after training in fed condition, which supports sleep-dependent memory formation. By knocking down candidate genes systematically, the authors identified Polr1F and Regnase-1 as two important hits that play potential roles in sleep and memory formation. What is the function of sleep and how to create a memory are two long-standing questions in science. The present study used a new approach to identify novel components that may link sleep and memory consolidation in a specific type of neuron. Importantly, these components implicated that RNA processing may play a role in these processes.

I am enthusiastic about the innovative approach employed to identify RNA processing genes involved in sleep regulation and memory consolidation. During the revision process, the authors fully addressed major concerns raised by reviewers. First, the author used the Gal80ts to restrict the knockdown of Regnase-1 in adult animals and concluded that Regnase-1 RNAi appears to affect sleep through development. Second, the author showed that Regnase-1 knockdown produced robust phenotypes for both sleep-dependent and sleep-independent memory, as well as a severe short-term memory phenotype. The author cautiously concluded that flies with constitutive Regnase-1 knockdown could be poor learners, thereby exhibiting a memory phenotype. Although we don't yet have a strong link between sleep and long-term memory consolidation, the interpretation presented in the manuscript is sufficiently justified by the data. This work presents a novel strategy to explore the link between sleep and memory consolidation.

Reviewer #2 (Public review):

This study utilizes an extensive series of neurulation human embryos to address several open questions about the similarities and differences between human primary and secondary neurulation in the tail. Results are compared to other model systems, such as the chicken and rodent. Histology, in situ hybridization, and apoptosis analysis provide molecular data about how the tail regresses in the human embryo. The number of embryos utilized for the analysis and the quality of the histological analysis provide robustness to the findings.

Comments on revised version:

The authors have meticulously addressed all the concerns raised by the reviewers, using new data and modifications to the text to further strengthen the quality of the manuscript.

This is a fabulous manuscript. I have nothing more scientifically to critique.

Reviewer #2 (Public review):

Summary:

The current work investigates the neural signature of category representation in infancy. Neural responses during steady-state visually-evoked potentials (ssVEPs) were recorded in four age groups of infants between 3 and 15 months. Stimuli (i.e., faces, limbs, corridors, characters, and cars) were presented at 4.286 Hz with category changes occurring at a frequency of 0.857 Hz. Results of the category frequency analyses showed that reliable responses to faces emerge around 4-6 months, whereas response to libs, corridors, and characters emerge around 6-8 months. Additionally, the authors trained a classifier for each category to assess how consistent the responses were across participants (leave-one-out approach). Spatiotemporal responses to faces were more consistent than the responses to the remaining categories and increased with increasing age. Faces showed an advantage over other categories in two additional measures (i.e., representation similarity and distinctiveness). Together, these results suggest a different developmental timing of category representation.

Strengths:

The study design is well organized. The authors described and performed analyses on several measures of neural categorization, including innovative approaches to assess the organization of neural responses. Results are in support of one of the two main hypotheses on the development of category representation described in the introduction. Specifically, the results suggest a different timing in the formation of category representations, with earlier and more robust responses emerging for faces over the remaining categories. Graphic representations and figures are very useful when reading the results. The inclusion of the adult sample and results further validate the approach utilized with infants.

Comments on revised submission:

The revised manuscript satisfactorily addressed all my previous comments.

Reviewer #2 (Public review):

Summary:

This is an outstanding piece of work on the potential of FLO as a viable analgesic biologic for the treatment of postsurgical pain. The authors purified the HC-HA/PTX3 from FLO and demonstrated its potential as an effective non-opioid therapy for postsurgical pain. They further unraveled the mechanisms of action of the compound at cellular and molecular levels.

Strengths:

Prominent strengths include the incorporation of behavioral assessment, electrophysiological and imaging recordings, the use of knockout and knockdown animals, and the use of antagonist agents to verify biological effects. The integrated use of these techniques, combined with the hypothesis-driven approach and logical reasoning, provides compelling evidence and novel insight into the mechanisms of the significant findings of this work.

Weaknesses:

I did not find any significant weaknesses even with a critical set of mind. The only minor suggestion is that the Results section may focus on the results from this study and minimize the discussions of background information.

Comments on revisions:

The authors have adequately addressed all the points raised in the last round of review. Thanks!

Reviewer #2 (Public review):

Summary:

The goal of this work is to define the functions of T-box transcription factors Tbx3 and Tbx5 in the adult mouse ventricular cardiac conduction system (VCS) using a novel conditional mouse allele in which both genes are targeted in cis. A series of studies over the past 2 decades by this group and others have shown that Tbx3 is a transcriptional repressor that patterns the conduction system by repressing genes associated with working myocardium, while Tbx5 is a potent transcriptional activator of "fast" conduction system genes in the VCS. In a previous work, the authors of the present study further demonstrated that Tbx3 and Tbx5 exhibit an epistatic relationship whereby the relief of Tbx3-mediated repression through VCS conditional haploinsufficiency allows better toleration of Tbx5 VCS haploinsufficiency. Conversely, excess Tbx3-mediated repression through overexpression results in disruption of the fast-conduction gene network despite normal levels of Tbx5. Based on these data the authors proposed a model in which repressive functions of Tbx3 drive the adoption of conduction system fate, followed by segregation into a fast-conducting VCS and slow-conduction AVN through modulation of the Tbx5/Tbx3 ratio in these respective tissue compartments.

The question motivating the present work is: If Tbx5/Tbx3 ratio is important for slow versus fast VCS identity, what happens when both genes are completely deleted from the VCS? Is conduction system identity completely lost without both factors and if so, does the VCS network transform into a working myocardium-like state? To address this question, the authors have generated a novel mouse line in which both Tbx5 and Tbx3 are floxed on the same allele, allowing complete conditional deletion of both factors using the VCS-specific MinK-CreERT2 line, convincingly validated in previous work. The goal is to use these double conditional knockout mice to further explore the model of Tbx3/Tbx5 co-dependent gene networks and VCS patterning. First, the authors demonstrate that the double conditional knockout allele results in the expected loss of Tbx3 and Tbx5 specifically in the VCS when crossed with Mink-CreERT2 and induced with tamoxifen. The double conditional knockout also results in premature mortality. Detailed electrophysiological phenotyping demonstrated prolonged PR and QRS intervals, inducible ventricular tachycardia, and evidence of abnormal impulse propagation along the septal aspect of the right ventricle. In addition, the mutants exhibit downregulation of VCS genes responsible for both fast conduction AND slow conduction phenotypes with upregulation of 2 working myocardial genes including connexin-43. The authors conclude that loss of both Tbx3 and Tbx5 results in "reversion" or "transformation" of the VCS network to a working myocardial phenotype, which they further claim is a prediction of their model and establishes that Tbx3 and Tbx5 "coordinate" transcriptional control of VCS identity.

Overall Appraisal:

As noted above, the present study does not further explore the Tbx5/Tbx3 ratio concept since both genes are completely knocked out in the VCS. Instead, the main claims are that the absence of both factors results in a transcriptional shift of conduction tissue towards a working myocardial phenotype, and that this shift indicates that Tbx5 and Tbx3 "coordinate" to control VCS identity and function. However, only limited data are presented to support the claim of transcriptional reprogramming since the knockout cells are not directly compared to working myocardial cells at the transcriptional level and only a small number of key genes are assessed (versus genome-wide assessment). In addition, the optical mapping dataset is incomplete and has alternative interpretations that are not excluded or thoroughly discussed.

In sum, while this study adds an elegantly constructed genetic model to the field, the data presented fit well within the existing paradigm of established functions of Tbx3 and Tbx5 in the VCS and in that sense do not decisively advance the field. Moreover, the authors' claims about the implications of the data are not always strongly supported by the data presented and do not fully explore alternative possibilities.

Strengths:

(1) Successful generation of a novel Tbx3-Tbx5 double conditional mouse model.

(2) Successful VCS-specific deletion of Tbx3 and Tbx5 using a VCS-specific inducible Cre driver line.

(3) Well-powered and convincing assessments of mortality and physiological phenotypes.

(4) Isolation of genetically modified VCS cells using flow.

Weaknesses:

(1) In general, the data is consistent with a long-standing and well-supported model in which Tbx3 represses working myocardial genes and Tbx5 activates the expression of VCS genes, which seem like distinct roles in VCS patterning. However, the authors move between different descriptions of the functional relationship and epistatic relationship between these factors, including terms like "cooperative", "coordinated", and "distinct" at various points. In a similar vein, sometimes terms like "reversion" are used to describe how VCS cells change after Tbx3/Tbx5 conditional knockout, and other times "transcriptional shift" and at other times "reprogramming". But these are all different concepts. The lack of a clear and consistent terminology for describing the phenomena observed makes the overarching claims of the manuscript more difficult to evaluate.

(2) A more direct quantitative comparison of Tbx5 Adult VCS KO with Tbx5/Tbx3 Adult VCS double KO would be helpful to ascertain whether deletion of Tbx3 on top of Tbx5 deletion changes the underlying phenotype in some discernable way beyond mRNA expression of a few genes. Superficially, the phenotypes look quite similar at the EKG and arrhythmia inducibility level and no optical mapping data from a single Tbx5 KO is presented for comparison to the double KO.

(3) The authors claim that double knockout VCS cells transform to working myocardial fate, but there is no comparison of gene expression levels between actual working myocardial cells and the Tbx3/Tbx5 DKO VCS cells so it's hard to know if the data reflect an actual cell state change or a more non-specific phenomenon with global dysregulation of gene expression or perhaps dedifferentiation. I understand that the upregulation of Gja1 and Smpx is intended to address this, but it's only two genes and it seems relevant to understand their degree of expression relative to actual working myocardium. In addition, the gene panel is somewhat limited and does not include other key transcriptional regulators in the VCS such as Irx3 and Nkx2-5. RNA-seq in these populations would provide a clearer comparison among the groups.

(4) From the optical mapping data, it is difficult to distinguish between the presence of (a) a focal proximal right bundle branch block due to dysregulation of gene expression in the VCS but overall preservation of the right bundle and its distal ramifications; from (b) actual loss of the VCS with reversion of VCS cells to a working myocardial fate. Related to this, the authors claim that this experiment allows for direct visualization of His bundle activation, but can the authors confirm or provide evidence that the tissue penetration of their imaging modality allows for imaging of a deep structure like the AV bundle as opposed to the right bundle branch which is more superficial? Does the timing of the separation of the sharp deflection from the subsequent local activation suggest visualization of more distal components of the VCS rather than the AV bundle itself? Additional clarification would be helpful.

Impact:

The present study contributes a novel and elegantly constructed mouse model to the field. The data presented generally corroborate existing models of transcriptional regulation in the VCS but do not, as presented, constitute a decisive advance.

Reviewer #2 (Public review):

Summary:

This study by Sanchez-Vasquez is a very innovative approach to inducing aneuploidy and then studying the contribution of treated cells to different lineages, including post-implantation. It connects well to the authors' previous work to induce mosaic aneuploidies. The authors identify sensitivity to HIF1a loss in treated embryos with likely aneuploidy. This work is part of an important line of work with evaluates the consequences of aneuploidy in the mammalian embryo.

Weaknesses:

Given that this is a study on the induction of aneuploidy, it would be meaningful to assess aneuploidy immediately after induction, and then again before implantation. This is also applicable to the competition experiments on page 7/8. What is shown is the competitiveness of treated cells. Because the publication centers around aneuploidy, the inclusion of such data in the main figure at all relevant points would strengthen it. There is some evaluation of karyotypes only in the supplemental - why? It would be good not to rely on a single assay that the authors appear to not give much importance.

Reviewer #2 (Public review):

Gaertner and colleagues present a study examining the transcriptomic diversity and spatial location of dopaminergic neurons from mice and examine the changes in gene expression resulting from knock-in of the Parkinson's LRRK G2019S risk variant. Overall, I found the manuscript presented their study very clearly, well written with very clear figures for the most part. I am not an expert on mouse neuroanatomy but found their classification reasonably well justified and the spatial orientation of dopaminergic neurons within the mouse brain informative and clear. While trends were clear and well presented, the apparent spatial heterogeneity suggests that knowledge of the functional connections and roles of these neurons will be required to better interpret the results presented, but nonetheless their findings exposed significant detail that is required for further understanding.

The study of the transcriptional effects of the LRRK2 KI was also informative and clearly framed in terms of a focused analysis on the effects of the KI only on dopaminergic neurons. However, I think there are issues here in both methodology, narrative, and clarity.

(1) In the GO pathway analyses (both GSEA and DEG GO), I did not see a correction applied to the gene background considered. The study focusses on dopaminergic neurons and thus the gene background should be restricted to genes expressed in dopaminergic neurons, rather than all genes in the mouse genome. The problem arises that if we randomly sample genes from dopaminergic neurons instead of the whole genome, we are predisposed to sampling genes enriched in relevant cell-type-specific roles (and their relevant GO terms) and correspondingly depleted in genes enriched in functions not associated with this cell type. Thus, I am unsure whether the results presented in Figures 8 and 9 may be more likely to be obtained just by randomly sampling genes from a dopaminergic neuron. The background should be limited and these functional analyses rerun.

(2) In the scRDS results, I am unsure what is significant and what isn't. The authors refer to relative measures in the text ("highest") but I do not know whether these differences are significant nor whether any associations are significantly unexpected. Can the x-axis of scRDS results presented in Figure 9 H and I be replaced with a corrected p-value instead of the scRDS score?

(3) The results discussed at the bottom of page 13 state that 48.82% of the proteins encoded by the Calb1 DEGs have pre-synaptic localisations as opposed to 45.83% of the SOX6 DEGs, which does not support the statement that "greater proportions of DEGs are associated with presynaptic locations in cells from vulnerable DA neurons (Sox6 family, [and in particular,Sox6^tafa1]), compared to less vulnerable ones (Calb1 family)".

(4) While an interest in the Sox6^tafa1 subtype is explained through their expression of Anxa1 denoting a previously identified subtype associated with locomotory behaviours, it was unclear to me how to interpret the functional associations made to DEGs in this subtype taken out of context of other subtypes. Given all the other subtypes, it is not possible to ascertain how specific and thus how interesting these results are unless other subtypes are analysed in the same way and this Sox6^tafa1 subtype is demonstrated as unusual given results from other subtypes.

(5) On p12, the authors highlight Mir124a-1hg that encodes miR-124. This is upregulated in Figure 8D but the authors note this has been to be downregulated in PD patients and some PD mouse models. Can the authors comment on the directional difference?

(6) Lastly, can the authors comment on the selection of a LogFC cut-off of 0.15 for their DEG selection? I couldn't see this explained (apologies if I missed it).

Reviewer #2 (Public review):

Summary:

The goal of this study was to develop a model for CDHR1-based Con-rod dystrophy and study the role of this cadherin in cone photoreceptors. Using genetic manipulation, a cell binding assay, and high-resolution microscopy the authors find that like rods, cones localize CDHR1 to the lateral edge of outer segment (OS) discs and closely oppose PCDH15b which is known to localize to calyceal processes (CPs). Ectopic expression of CDHR1 and PCDH15b in K652 cells indicates these cadherins promote cell aggregation as heterophilic interactants, but not through homophilic binding. This data suggests a model where CDHR1 and PCDH15b link OS and CPs and potentially stabilize cone photoreceptor structure. Mutation analysis of each cadherin results in cone structural defects at late larval stages. While pcdh15b homozygous mutants are lethal, cdhr1 mutants are viable and subsequently show photoreceptor degeneration by 3-6 months.

Strengths:

A major strength of this research is the development of an animal model to study the cone-specific phenotypes associated with CDHR1-based CRD. The data supporting CDHR1 (OS) and PCDH15 (CP) binding is also a strength, although this interaction could be better characterized in future studies. The quality of the high-resolution imaging (at the light and EM levels) is outstanding. In general, the results support the conclusions of the authors.

Weaknesses:

While the cellular phenotyping is strong, the functional consequences of CDHR1 disruption are not addressed. While this is not the focus of the investigation, such analysis would raise the impact of the study overall. This is particularly important given some of the small changes observed in OS and CP structure. While statistically significant, are the subtle changes biologically significant? Examples include cone OS length (Figures 4F, 6E) as well as other morphometric data (Figure 7I in particular). Related, for quantitative data and analysis throughout the manuscript, more information regarding the number of fish/eyes analyzed as well as cells per sample would provide confidence in the rigor. The authors should also note whether the analysis was done in an automated and/or masked manner.

Reviewer #2 (Public review):

Summary:

Identifying an important role for the Integrator complex in repressing HIV transcription and suggesting that by targeting subunits of this complex specifically, INTS12, reversal of latency with and without latency reversal agents can be enhanced.

Strengths:

The strengths of the paper include the general strategy for screening targets that may activate HIV latency and the rigor of exploring the mechanism of INTS12 repression of HIV transcriptional elongation. I found the mechanism of INTS12 interesting and maybe even the most impactful part of the findings.

Weaknesses:

I have two minor comments:

There was an opportunity to examine a larger panel of latency reversal agents that reactivate by different mechanisms to determine whether INTS12 and transcriptional elongation are limiting for a broad spectrum of latency reversal agents.

I felt the authors could have extended their discussion of how exquisitely sensitive HIV transcription is to pausing and transcriptional elongation and the insights this provides about general HIV transcriptional regulation.

Reviewer #2 (Public review):

Summary:<br /> In their manuscript, Fang and colleagues make a notable contribution to the field of oncology, particularly in advancing our understanding of triple-negative breast cancer (TNBC). The research delineates the role of STAMBPL1 in promoting angiogenesis in TNBC through its interaction with FOXO1 and the subsequent activation of the GRHL3/HIF1A/VEGFA axis. The evidence presented is robust, with a combination of in vitro experiments, RNA sequencing, and in vivo studies providing a comprehensive view of the molecular mechanisms at play. The strength of the evidence is anchored in the systematic approach and the utilization of multiple methodologies to substantiate the findings.

Strengths:<br /> The manuscript presents a methodologically robust framework, incorporating RNA-sequencing, chromatin immunoprecipitation (ChIP) assays, and a suite of in vitro and in vivo model systems, which collectively substantiate the claims regarding the pro-angiogenic role of STAMBPL1 in TNBC. The employment of multiple cellular models, conditioned media to assess HUVEC functional responses, and xenograft tumor models in murine hosts offers a comprehensive evaluation of STAMBPL1's impact on angiogenic processes.A salient strength of this work is the identification of GRHL3 as a transcriptional target of STAMBPL1 and the demonstration of a physical interaction between STAMBPL1 and FOXO1, which modulates GRHL3-driven HIF1A transcription. The study further suggests a potential therapeutic strategy by revealing the synergistic inhibitory effects of combined VEGFR and FOXO1 inhibitor treatment on TNBC tumor growth.

Weaknesses:<br /> A potential limitation of the study is the reliance on specific cellular and animal models, which may constrain the extrapolation of these findings to the broader spectrum of human TNBC biology. Furthermore, while the study provides evidence for a novel regulatory axis involving STAMBPL1, FOXO1, and GRHL3, the multifaceted nature of angiogenesis may implicate additional regulatory factors not exhaustively addressed in this research.

Appraisal of Achievement and Conclusion Support:<br /> The authors have successfully demonstrated that STAMBPL1 promotes HIF1A transcription and activates the HIF1α/VEGFA axis in a non-enzymatic manner, leading to increased angiogenesis in TNBC. The results are generally supportive of their conclusions, with clear evidence that STAMBPL1 upregulates HIF1α expression and enhances the activity of HUVECs. The study also shows that STAMBPL1 interacts with FOXO1 to promote GRHL3 transcription, which in turn activates HIF1A.

Impact on the Field and Utility:<br /> This research is poised to exert a substantial impact on the oncological research community by uncovering the role of STAMBPL1 in TNBC angiogenesis and by identifying the STAMBPL1/FOXO1/GRHL3/HIF1α/VEGFA axis as a potential therapeutic target. The findings could pave the way for the development of novel therapeutic strategies for TNBC, a subtype characterized by a paucity of effective treatment options. The methodologies utilized in this study are likely to be valuable to the research community, offering a paradigm for investigating the role of deubiquitinating enzymes in oncogenic processes.

Additional Context:<br /> It would be beneficial for readers to understand the broader context of TNBC research and the current challenges in treating this aggressive cancer subtype. The significance of this work is heightened by the lack of effective treatments for TNBC, making the identification of new therapeutic targets particularly important. Furthermore, understanding the specific mechanisms by which STAMBPL1 regulates HIF1α expression could provide insights into hypoxia signaling in other cancer types as well.

Reviewer #2 (Public review):

Summary:

Chronic inflammation of the bone microenvironment conferred by T2DM and obesity may inhibit bone formation and bone strength by decreasing the ratio of Wnt ligands/Wnt inhibitors.

The authors studied 63 postmenopausal women (age >65 years) undergoing hip replacement for osteoarthritis. These were grouped into T2DM and obesity, obesity only, and normal subjects. A set of inflammatory markers was measured in the serum and gene expression of members of the Wnt system in the bone tissue. Bone samples were assessed by micro-CT.

While TNF-α serum levels were higher in T2DM, IL-6 levels were higher in obesity as compared to control. In the bone compartment the most consistent finding was decreased mRNA levels for WNt10b and increased sclerostin mRNA levels, translating into a suppressed Wnt-to-Wnt inhibitor ratio, which was associated with low bone strength.

Strengths:

The study includes clinically well-characterized subjects of three defined subgroups. The analyses were comprehensive.

Weaknesses:

Including data or information on the Wnt inhibitor Dkk1 would be instructive. Analysis were limited to mRNA studies. Validation of protein levels would be supportive (although technically challenging).

Reviewer #2 (Public review):

Summary:

In this study, the authors investigated the chemotaxis of E. coli swimming close to the bottom surface in gradients of attractant in channels of increasingly smaller width but fixed height = 30 µm and length ~160 µm. In relatively large channels, they find that on average the cells drift in response to the gradient, despite cells close to the surface away from the walls being known to not be chemotactic because they swim in circles.

They find that this average drift is due to the cell localization close to the side walls, where they slide along the wall. Whereas the bacteria away from the walls have no chemotaxis (as shown before), the ones on the left side wall go down-gradient on average, but the ones on the right side wall go up-gradient faster, hence the average drift. They then study the effect of reducing channel width. They find that chemotaxis is higher in channels with a width of about 8 µm, which approximately corresponds to the radius of the circular swimming R. This higher chemotactic drift is concomitant to an increased density of cells on the RSW. They do simulations and modeling to suggest that the disruption of circular swimming upon collision with the wall increases the density of cells on the RSW, with a maximal effect at w = ~ 2/3 R, which is a good match for their experiments.

Strengths:

The overall result that confinement at the edge stabilises bacterial motion and allows chemotaxis is very interesting although not entirely unexpected. It is also important for understanding bacterial motility and chemotaxis under ecologically relevant conditions, where bacteria frequently swim under confinement (although its relevance for controlling infections could be questioned). The experimental part of the study is nicely supported by the model.

Weaknesses:

Several points of this study, in particular the interpretation of the width effect, need better clarification:

(1) Context:

There are a number of highly relevant previous publications that should have been acknowledged and discussed in relation to the current work:<br /> https://pubs.rsc.org/en/content/articlehtml/2023/sm/d3sm00286a<br /> https://link.springer.com/article/10.1140/epje/s10189-024-00450-7<br /> https://doi.org/10.1016/j.bpj.2022.04.008<br /> https://doi.org/10.1073/pnas.1816315116<br /> https://www.pnas.org/doi/full/10.1073/pnas.0907542106<br /> https://doi.org/10.1038/s41467-020-15711-0<br /> http://doi.org/10.1038/s41467-020-15711-0<br /> http://doi.org/10.1039/c5sm00939a

(2) Experimental setup:

a) The channels are built with asymmetric entrances (Figure 1), which could trigger a ratchet effect (because bacteria swim in circle) that could bias the rate at which cells enter into the channel, and which side they follow preferentially, especially for the narrow channel. Since the channel is short (160 µm), that would reflect on the statistics of cell distribution. Controls with straight entrances or with a reversed symmetry of the channel need to be performed to ensure that the reported results are not affected by this asymmetry.

b) The authors say the motile bacteria accumulate mostly at the bottom surface. This is strange, for a small height of 30 µm, the bacteria should be more-or-less evenly spread between the top and bottom surface. How can this be explained?

c) At the edge, some of the bacteria could escape up in the third dimension (http://doi.org/10.1039/c5sm00939a). What is the magnitude of this phenomenon in the current setup? Does it have an effect?

d) What is the cell density in the device? Should we expect cell-cell interactions to play a role here? If not, I would suggest to de-emphasize the connection to chemotaxis in the swarming paper in the introduction and discussion, which doesn't feel very relevant here, and rather focus on the other papers mentioned in point 1.

e) We are not entirely convinced by the interpretation of the results in narrow channels. What is the causal relationship between the increased density on the RSW and the higher chemotactic drift? The authors seem to attribute higher drift to this increased RSW density, which emerges due to the geometric reasons. But if there is no initial bias, the same geometric argument would induce the same increased density of down-gradient swimmers on the LSW, and so, no imbalance between RSW and LSW density. Could it be the opposite that the increased RSW density results from chemotaxis (and maybe reinforces it), not the other way around? Confinement could then deplete one wall due to the proximity of the other, and/or modify the swimming pattern - 8 µm is very close to the size of the body + flagellum. To clarify this point, we suggest measuring the bacterial distributions in the absence of a gradient for all channel widths as a control.

(3) Simulations:

The simulations treat the wall interaction very crudely. We would suggest treating it as a mechanical object that exerts elastic or "hard sphere" forces and torques on the bacteria for more realistic modeling. Notably, the simulations have a constant (chemotaxis independent) rate of wall escape by tumbling. We would expect that reduced tumbling due to up-gradient motility induces a longer dwell time at the wall.

Reviewer #2 (Public review):

Summary:

In this work, the authors use a theoretical model to study the potential impact of Horizontal Gene Transfer on the number of alternative stable states of microbial communities. For this, they use a modified version of the competitive Lotka Volterra model-which accounts for the effects of pairwise, competitive interactions on species growth-that incorporates terms for the effects of both an added death (dilution) rate acting on all species and the rates of horizontal transfer of mobile genetic elements-which can in turn affect species growth rates. The authors analyze the impact of horizontal gene transfer in different scenarios: bistability between pairs of species, multistability in communities, and a modular structure in the interaction matrix to simulate multiple niches. They also incorporate additional elements to the model, such as spatial structure to simulate metacommunities and modification of pairwise interactions by mobile genetic elements. In almost all these cases, the authors report an increase in either the number of alternative stable states or the parameter region (e.g. growth rate values) in which they occur.

In my opinion, understanding the role of horizontal gene transfer in community multistability is a very important subject. This manuscript is a useful approach to the subject, but I'm afraid that a thorough analysis of the role of different parameters under different scenarios is missing in order to support the general claims of the authors. The authors have extended their analysis to increase their biological relevance, but I believe that the analysis still lacks comprehensiveness.

Understanding the origin of alternative stable states in microbial communities and how often they may occur is an important challenge in microbial ecology and evolution. Shifts between these alternative stable states can drive transitions between e.g. a healthy microbiome and dysbiosis. A better understanding of how horizontal gene transfer can drive multistability could help predict alternative stable states in microbial communities, as well as inspire novel treatments to steer communities towards the most desired (e.g. healthy) stable states.

Strengths:

(1) Generality of the model: the work is based on a phenomenological model that has been extensively used to predict the dynamics of ecological communities in many different scenarios.

(2) The question of how horizontal gene transfer can drive alternative stable states in microbial communities is important and there are very few studies addressing it.

Weaknesses:

(1) There is a need for a more comprehensive analysis of the relative importance of the different model parameters in driving multistability. For example, there is no analysis of the effects of the added death rate in multistability. This parameter has been shown to determine whether a given pair of interacting species exhibits bistability or not (see e.g. Abreu et al 2019 Nature Communications 10:2120). Similarly, each scenario is analyzed for a unique value of species interspecies interaction strength-with the exception of the case for mobile genetic elements affecting interaction strength, which considers three specific values. Considering heterogeneous interaction strengths (e.g. sampling from a random distribution) could also lead to more realistic scenarios - the authors generally considered that all species pairs interact with the same strength. Analyzing a larger range of growth rates effects of mobile genetic elements would also help generalize the results. In order to achieve a more generic assessment of the impact of horizontal gene transfer in driving multistability, its role should be systematically compared to the effects of the rest of the parameters of the model.

(2) The authors previously developed this theoretical model to study the impact of horizontal gene transfer on species coexistence. In this sense, it seems that the authors are exploring a different (stronger interspecies competition) range of parameter values of the same model, which could potentially limit novelty and generality.

(3) The authors analyze several scenarios that, in my opinion, naturally follow from the results and parameter value choices in the first sections, making their analysis not very informative. For example, after showing that horizontal gene transfer can increase multistability both between pairs of species and in a community context, the way they model different niches does not bring significantly new results. Given that the authors showed previously in the manuscript that horizontal gene transfer can impact multistability in a community in which all species interact with each other, one might expect that it will also impact multistability in a larger community made of (sub)communities that are independent of (not interacting with) each-which is the proposed way for modelling niches. A similar argument can be made regarding the analysis of (spatially structured) metacommunities. It is known that, for smaller enough dispersal rates, space can promote regional diversity by enabling each local community to remain in a different stable state. Therefore, in conditions in which the impact of horizontal gene transfer drives multistability, it will also drive regional diversity in a metacommunity.

(4) In some cases, the authors consider that mobile genetic elements can lead to ~50% growth rate differences. In the presence of an added death rate, this can be a relatively strong advantage that makes the fastest grower easily take over their competitors. It would be important to discuss biologically relevant examples in which such growth advantages driven by mobile genetic elements could be expected, and how common such scenarios might be.

Reviewer #2 (Public review):

Summary:

Weiler et al use retrograde tracers, two-photon tomography, and automatic cell detection to provide a detailed quantitative description of the laminar and area sources of ipsi- and contralateral cortico-cortical inputs to two primary sensory areas and a primary motor area. They found considerable bilateral symmetry in the areas providing cortico-cortical inputs. However, although the same regions in both hemispheres tended to supply inputs, a larger proportion of inputs from contralateral areas originated from deeper layers (L5 and L6).

Strengths:

The study applies state-of-the-art anatomical methods, and the data is very effectively presented and carefully analyzed. The results provide many novel insights into the similarities and differences of inputs from the two hemispheres. While over the past decade there have been many studies quantitively and comprehensively describing cortico-cortical connections, by directly comparing inputs from the ipsi and contralateral hemispheres, this study fills in an important gap in the field. It should be of great utility and an important reference for future studies on inter-hemispheric interactions.

Weaknesses:

Overall, I do not find any major weakness in the analyses or their interpretation. However, one must keep in mind that the study only analyses inputs projecting to three areas. This is not an inherent flaw of the study; however, it warrants caution when extrapolating the results to callosal projections terminating in other areas. As inputs to two primary sensory areas and one is the primary motor cortex are studied, some of the conclusions could potentially be different for inputs terminating in high-order sensory and motor areas. Given that primary areas were injected, there are few instances of feedforward connections sampled in the ipsilateral hemisphere. The study finds that while ipsi-projections from the visual cortex to the barrel cortex are feedforward given its fILN values, those from the contralateral visual cortex are feedback instead. One is left to wonder whether this is due to the cross-modal nature of these particular inputs and whether the same rule (that contralateral inputs consistently exhibit feedback characteristics regardless of the hierarchical relationship of their ipsilateral counterparts with the target area,) would also apply to feedforward inputs within the same sensory cortices.

Another issue that is left unexplored is that, in the current analyses the barrel and primary visual cortex are analyzed as a uniform structure. It is well established that both the laminar sources of callosal inputs and their terminations differ in the monocular and binocular areas of the visual cortex (border with V2L). Similarly, callosal projections differ when terminating the border of S1 (a row of whiskers), and then in other parts of S1. Thus, some of the conclusions regarding the laminar sources of callosal inputs might depend on whether one is analyzing inputs terminating or originating in these border regions.

Finally, while the paper emphasizes that projections from L6 "dominate" intra and contralateral cortico-cortical inputs, the data shows a more nuanced scenario. While it is true that the areas for which L6 neurons are the most common source of cortico-cortical projections are the most abundant, the picture becomes less clear when considering the number of neurons sending these connections. In fact, inputs from L2/3 and L5 combined are more abundant than those from L6 (Figure 3B), challenging the view that projections from L6 dominate ipsi- and contralateral projecting cortico-cortical inputs.

Reviewer #2 (Public review):

Summary:

The manuscript 'Recognition and Cleavage of Human tRNA Methyltransferase TRMT1 by the SARS-CoV-2 Main Protease' from Angel D'Oliviera et al., uncovers that TRMT1 can be cleaved by SARS-CoV-2 main protease (Mpro) and defines the structural basis of TRMT1 recognition by Mpro. They use both recombinant TRMT1 and Mpro as well as endogenous TRMT1 from HEK293T cell lysates to convincingly show cleavage of TRMT1 by the SARS-CoV-2 protease. Using in vitro assays, the authors demonstrate that TRMT1 cleavage by Mpro blocks its enzymatic activity leading to hypomodification of RNA. To understand how Mpro recognizes TRMT1, they solved a co-crystal structure of Mpro bound to a peptide derived from the predicted cleavage site of TRMT1. This structure revealed important protein-protein interfaces and highlights the importance of the conserved Q530 for cleavage by Mpro. They then compare their structure with previous X-ray crystal structures of Mpro bound to substrate peptides derived from the viral polyprotein and propose the concept of two distinct binding conformations to Mpro: P3´-out and P3´-in conformations (here P3´ stands for the third residue downstream of the cleavage site). It remains unknown what is the physiological role of these two binding conformations on Mpro function, but the authors established that Mpro has dramatically different cleavage efficiencies for three distinct substrates. In an effort to rationalize this observation, a series of mutations in Mpro's active site and the substrate peptide were tested but unexpectedly had no significant impact on cleavage efficiency. While molecular dynamic simulations further confirmed the propensity of certain substrates to adopt the P3´-out or P3´-in conformation, it did not provide additional insights into the dramatic differences in cleavage efficiencies between substrates. This led the authors to propose that the discrimination of Mpro for preferred substrates might occur at a later stage of catalysis after binding of the peptide. Overall, this work will be of interest to biologists studying proteases and substrate recognition by enzymes and RNA modifications as well as help efforts to target Mpro with peptide-like drugs.

Strengths:

• The authors' statements are well supported by their data, and they used relevant controls when needed. Indeed, they used the Mpro C145A inactive variant to unambiguously show that the TRMT1 cleavage detected in vitro is solely due to Mpro's activity. Moreover, they used two distinct polyclonal antibodies to probe TRMT1 cleavage.<br /> • They demonstrate the impact of TRMT1 cleavage on RNA modification by quantifying both its activity and binding to RNA.<br /> • Their 1.9 Å crystal structure is of high quality and increases the confidence in the reported protein-protein contacts seen between TRMT1-derived peptide and Mpro.<br /> • Their extensive in vitro kinetic assay was performed in ideal conditions although it is sometimes unclear how many replicates were performed.<br /> • They convincingly show how Mpro cleavage is conserved among most but not all mammalian TRMT1 bringing an interesting evolutionary perspective on virus-host interactions.<br /> • The authors test multiple hypotheses to rationalize the preference of Mpro for certain substrates.<br /> • While this reviewer is not able to comment on the rigor of the MD simulations, the interpretations made by the authors seem reasonable and convincing.<br /> • The concept of two binding conformations (P3´-out or P3´-in) for the substrate in the active site of Mpro is significant and can guide drug design.

Weaknesses:

• The two polyclonal antibodies used by the authors seem to have strong non-specific binding to proteins other than TRMT1 but did not impact the author's conclusions or statements. This is a limitation of the commercially available antibodies for TRMT1.<br /> • Despite the reasonable efforts of the authors, it remains unknown why Mpro shows higher cleavage efficiency for the nsp4/5 sequence compared to TRMT1 or nsp8/9 sequences. This is a challenging problem that will take substantially more effort by several labs to decipher mechanistically.<br /> • The peptide cleavage kinetic assay used by the authors relies on a peptide labelled with a fluorophore (MCA) on the N-terminus and a quencher (Dpn) on the C-terminus. This design allows high-throughput measurements compatible with plate readers and is a robust and convenient tool. Nevertheless, the authors did not control for the impact of the labels (MCA and Dpn) on the activity of Mpro. While in most cases the introduced fluorophore/quencher do not impact activity, sometimes it can.<br /> • An unanswered question not addressed by the authors is if the peptides undergo conformational changes upon Mpro binding or if they are pre-organized to adopt the P3´-out and P3´-in conformations. This might require substantially more work outside the scope of this immediate article.

Reviewer #3 (Public review):

This study provides valuable insights into the cellular responses to complex aneuploidy in human preimplantation embryos. The authors have significantly expanded their sample size and conducted additional analysis and experiments to address previous concerns. The revised manuscript presents stronger evidence for gene dosage-dependent effects of aneuploidy on stress responses and lineage segregation. Overall, the findings contribute important knowledge to our understanding of how human embryos respond to chromosomal abnormalities.

Overall, the revision has substantially improved the manuscript and addressed the major concerns raised in the initial review.

Reviewer #2 (Public review):

Summary:

This work introduces a new method of depleting the ribosomal reads from the single-cell RNA sequencing library prepared with one of the prokaryotic scRNA-seq techniques, PETRI-seq. The advance is very useful since it allows broader access to the technology by lowering the cost of sequencing. It also allows more transcript recovery with fewer sequencing reads. The authors demonstrate the utility and performance of the method for three different model species and find a subpopulation of cells in the E.coli biofilm that express a protein, PdeI, which causes elevated c-di-GMP levels. These cells were shown to be in a state that promotes persister formation in response to ampicillin treatment.

Strengths:

The introduced rRNA depletion method is highly efficient, with the depletion for E.coli resulting in over 90% of reads containing mRNA. The method is ready to use with existing PETRI-seq libraries which is a large advantage, given that no other rRNA depletion methods were published for split-pool bacterial scRNA-seq methods. Therefore, the value of the method for the field is high. There is also evidence that a small number of cells at the bottom of a static biofilm express PdeI which is causing the elevated c-di-GMP levels that are associated with persister formation. This finding highlights the potentially complex role of PdeI in regulation of c-di-GMP levels and persister formation in microbial biofilms.

Comments on revised version:

The authors edited the manuscript thoroughly in response to the comments, including both performing new experiments and showing more data and information. Most of the major points raised between both reviewers were addressed. The authors explained the seeming contradiction between c-di-GMP levels and PdeI expression.

Reviewer #2 (Public review):

FOXP3 has been known to form diverse complexes with different transcription factors and enzymes responsible for epigenetic modifications, but how extracellular signals timely regulate FOXP3 complex dynamics remains to be fully understood. Histone H3K4 tri-methylation (H3K4me3) and CXXC finger protein 1 (CXXC1), which is required to regulate H3K4me3, also remain to be fully investigated in Treg cells. Here, Meng et al. performed a comprehensive analysis of H3K4me3 CUT&Tag assay on Treg cells and a comparison of the dataset with the FOXP3 ChIP-seq dataset revealed that FOXP3 could facilitate the regulation of target genes by promoting H3K4me3 deposition.

Moreover, CXXC1-FOXP3 interaction is required for this regulation. They found that specific knockdown of Cxxc1 in Treg leads to spontaneous severe multi-organ inflammation in mice and that Cxxc1-deficient Treg exhibits enhanced activation and impaired suppression activity. In addition, they have also found that CXXC1 shares several binding sites with FOXP3 especially on Treg signature gene loci, which are necessary for maintaining homeostasis and identity of Treg cells.

The findings of the current study are pretty intriguing, and it would be great if the authors could fully address the following comments to support these interesting findings.

Major points:

(1) There is insufficient evidence in the first part of the Results to support the conclusion that "FOXP3 functions as an activator by promoting H3K4Me3 deposition in Treg cells". The authors should compare the results for H3K4Me3 in FOXP3-negative conventional T cells to demonstrate that at these promoter loci, FOXP3 promotes H3K4Me3 deposition.

(2) In Figure 3 F&G, the activation status and IFNγ production should be analyzed in Treg cells and Tconv cells separately rather than in total CD4+ T cells. Moreover, are there changes in autoantibodies and IgG and IgE levels in the serum of cKO mice?

(3) Why did Cxxc1-deficient Treg cells not show impaired suppression than WT Treg during in vitro suppression assay, despite the reduced expression of Treg cell suppression assay -associated markers at the transcriptional level demonstrated in both scRNA-seq and bulk RNA-seq?

(4) Is there a disease in which Cxxc1 is expressed at low levels or absent in Treg cells? Is the same immunodeficiency phenotype present in patients as in mice?

Reviewer #2 (Public review):

Summary:

The authors comprehensively assess differences in the TCRB and TCRA repertoires in the fetal and adult mouse thymus by deep sequencing of sorted cell populations. For TCRB and TCRA they observed biased gene segment usage, less diversity, and greater repertoire sharing among individuals in fetal thymocytes. The TCRB repertoire was less evenly distributed and displayed more evidence of clonal expansions in fetal thymocytes. Both fetal and adult thymocytes demonstrated repertoire skewing in CD4 and CD8 as compared to DP thymocytes, which was attributed to MHC-I- vs MHC-II-restriction during positive selection. Effects of MHC-restriction were notably weaker in fetal thymocytes. The authors conclude that in multiple respects fetal repertoires are distinct from adult repertoires.

Strengths:

The analyses of the F18.5 and adult thymic repertoires are comprehensive with respect to the cell populations analyzed and the diversity of statistical approaches used to characterize the repertoires. Because repertoires were analyzed in pre- and post-selection thymocyte subsets, the data allowed assessment of repertoire selection at different developmental stages. Intriguing differences between fetus and adult are identified.

Weaknesses:

Some of the repertoire characteristics reported are already fairly well documented in the literature. Moreover, an unaddressed limitation of the study is that fetal thymocytes were analyzed at single time-point in their development. As a result, at least some of the conclusions about the fetal repertoire may be viewed not as general conclusions, but rather, due to the synchronous development of fetal thymocytes, as pertaining to the one day of fetal/early neonatal development assayed. Statements suggesting that (1) "progressive TCRa rearrangements occur less frequently in foetal DP cells" (Abstract), (2) "One possible explanation for this bias is that in the foetus progressive rounds of TCRa rearrangement are less common than in young adult" (Discussion), and (3) "Overall, the differences between the foetal and adult thymus TCR repertoires are consistent with the foetal thymus producing abT-cells ... with preference for particular gene segment usage" (Discussion), are oversimplified and potentially misleading.

Reviewer #2 (Public Review):

In this manuscript, the authors revealed that genetic deficiencies of ACK1 and BRK are associated with human SLE. First, the authors found that compound heterozygous deleterious variants in the kinase domains of the non-receptor tyrosine kinases (NRTK) TNK2/ACK1 in one multiplex family and PTK6/BRK in another family. Then, by an experimental blockade of ACK1 or BRK in a mouse SLE model, they found an increase in glomerular IgG deposits and circulating autoantibodies. Furthermore, they reported that ACK and BRK variants from the SLE patients impaired the MERTK-mediated anti-inflammatory response to apoptotic cells in human induced pluripotent stem cells (hiPSC)-derived macrophages. This work identified new SLE-associated ACK and BRK variants and a role for the NRTK TNK2/ACK1 and PTK6/BRK in efferocytosis, providing a new molecular and cellular mechanism of SLE pathogenesis.

Reviewer #3 (Public Review):

Heparan Sulphate is a general association factor in the extracellular matrix which assists in host cell entry for a multitude of viral and bacterial pathogens by concentrating them in the vicinity of cellular membranes. The neurotropic picornavirus, EV-71 utilizes a protein receptor SCARB-2, in conjunction with Heparan Sulfate, in order to enter cells through the endo-lysosomal pathway. The uncoating and release of viral genome requires both receptor binding and late endosomal pH conditions. The authors have attempted to address a seeming contradiction in the in vitro and in vivo infectivity of strain MP4 variants of EV-71. One of the cell culture adapted strains MP4-L97R/E167G has stronger association with HS, which translates to higher infectivity in cell culture models; however, viral virulence is significantly lower in animal models.

Using an elegant and methodical set of experiments, the authors have probed the steps in the cellular entry pathway of MP4 and its L97R/E167G variant. Their experiments strongly suggest a difference in capsid uncoating mechanisms in the variant, with the L97R/E167G variant being significantly less robust and prone to destabilize earlier in the pathway. While this confers an advantage in terms of cell culture based infectivity, it is posited that the particles will not survive the gastric pH intact, which compromises virulence in the animal model. While the cell culture based uncoating experiments somewhat support this hypothesis, the main weakness of this work is a lack of explanation for the mechanism(s) of capsid destabilization conferred by overall increased positive charge. The structural bioinformatics study in the supplementary section does not explain how receptor binding, pocket factor expulsion, subunit interactions and low pH based capsid dynamics may be influenced by the mutations. Capsid destabilization could be an outcome in alteration of any or all of these processes. It is also unclear whether it is suggested that all mutations enhancing the net positive charge of VP1, or any other structural protein, will cause capsid destabilization by similar pathways. A clearer analysis of the influence of overall charge alterations, or individual mutations, on subunit interaction or particle conformation is needed. The enhancement in cell culture infectivity of the L97R/E167G variant under elevated endosomal pH is also unclear and requires further experimentation.

It has been suggested earlier that increased HS binding in vivo results in virus "trapping" and decreased infectivity. This may still be a major reason for reduced infectivity in vivo, in addition to the capsid destabilization as proposed in this work.

Reviewer #2 (Public Review):

Many naturally occurring networks are assumed to have a power-law (PL) degree distribution. This assumption has certainly been widely held in the field of protein interactomes (PPIs), although important studies around 2010 have conclusively shown that many of these PL distributions are either the result of data mis-handling or of sloppy statistical procedures (see e.g. Porter and Stumpf in Science around 2014, which I would advise the authors to cite). The value of the present study is to introduce a new mechanism, experiment bias, to explain the appearance of such distributions in the PPI case, and in particular to show how correcting empirically for this mechanism can lead to a reappraisal of which proteins are genuine hubs in these networks. The claims are well supported by empirical evidence and some theoretical analysis. Overall, this is a worthwhile contribution although its significance is somewhat dented by the fact that the PL enthusiasm of many had already been tempered by the studies mentioned above.

Reviewer #2 (Public review):

Summary:

This was a well-executed and well-written paper. The authors have provided important new datasets that expand on previous investigations substantially. The discovery that changes in diet are not so closely correlated with the presence of alkaloids (based on the expanded sampling of non-defended species) is important, in my opinion.

Strengths:

Provision of several new expanded datasets using cutting edge technology and sampling a wide range of species that had not been sampled previously. A conceptually important paper that provides evidence for the importance of intermediate stages in the evolution of chemical defense and aposematism.

Weaknesses:

There were some aspects of the paper that I thought could be revised. One thing I was struck by is lack of discussion of the potentially negative effects of toxin accumulation, and how this might play out in terms of different levels of toxicity in different species. Further, are there aspects of ecology or evolutionary history that might make some species less vulnerable to the accumulation of toxins than others? This could be another factor that strongly influences the ultimate trajectory of a species in terms of being well-defended. I think the authors did a good job in terms of describing mechanistic factors that could affect toxicity (e.g. potential molecular mechanisms), but did not make much of an attempt to describe potential ecological factors that could impact trajectories of the evolution of toxicity. This may have been done on purpose (to avoid being too speculative), but I think it would be worth some consideration.

In the discussion, the authors make the claim that poison frogs don't (seem to) suffer from eating alkaloids. I don't think this claim has been properly tested (the cited references don't adequately address it). To do so would require an experimental approach, ideally obtained data on both lifespan and lifetime reproductive success.

Update: Revised version: The authors carefully addressed the comments and suggestions on the first draft of the manuscript. In my opinion, these revisions were sufficient and the authors have adequately addressed the previously noted weaknesses in the manuscript.

Reviewer #2 (Public review):

This study builds upon the team's recent discovery that antibiotic treatment and other disturbances favours the persistence of bacteria with genomes that encode complete modules for the synthesis of essential metabolites (Watson et al. 2023). Veseli and collaborators now provide an in-depth analysis of metabolic pathway completeness within microbiomes, finding strong evidence for an enrichment of bacteria with high metabolic independence in the microbiomes associated with IBD and other gastrointestinal disorders. Importantly, this study provides a new open-source software to facilitate the reconstruction of metabolic pathways, estimate their completeness and normalize their results according to species diversity. Finally, this study also shows that metabolic independence of microbial communities can be used as a marker of dysbiosis. The function-based health index proposed here is more robust to individual's lifestyles and geographic origin than previously proposed methods based on bacterial taxonomy.

The implications of this study have the potential to spur a paradigm shift in the field. It shows that certain bacterial taxa that have been consistently associated with disease might not be harmful to their host as previously thought. These bacteria seem to be the only species that are able to survive in a stressed gut environment. They might even be important to rebuild a healthy microbiome (although the authors are careful in not making this speculation).

This paper provides an in-depth discussion of the results, and limitations are clearly addressed throughout the manuscript (see also the supplementary files for an in-depth assessment of the robustness of the methods). Some of the potential limitations relate to the use of large publicly available datasets, where sample processing and the definition of healthy status varies between studies. The authors have recognised these issues and their results were robust to analyses performed at a per-cohort basis. The potential limitations therefore are unlikely to have affected the conclusions of this study.

Overall, this is manuscript is a magnificent contribution to the field, likely to inspire many other studies to come.

Comments on revisions:

The authors have performed a detailed assessment of the accuracy and robustness of their new methods, and included an informative session comparing their new approach with existing ones. The new analyses have strengthened the manuscript, and the results support the biological interpretations of the study.<br /> I commend the authors for the effort and the excellent research.

Reviewer #2 (Public Review):

In this manuscript, Senn, Lipinski, and colleagues report on the structure and function of the conserved spliceosomal protein Fyv6. Pre-mRNA splicing is a critical gene expression step that occurs in two steps, branching and exon ligation. Fyv6 had been recently identified by the Hoskins' lab as a factor that aids exon ligation (Lipinski et al., 2023), yet the mechanistic basis for Fyv6 function was less clear. Here, the authors combine yeast genetics, transcriptomics, biochemical assays, and structural biology to reveal the function of Fyv6. Specifically, they describe that Fyv6 promotes the usage of distal 3'SSs by stabilizing a network of interactions that include the RNA helicase PRP22 and the spliceosome subunit SYF1. They discuss a generalizible mechanism for splice site proofreading by spliceosomsal RNA helicases that could be modulated by other, regulatory splicing factors.

This is a very high quality study, which expertly combines various approaches to provide new insights into the regulation of 3'SS choice, docking, and undocking. The cryo-EM data is also of excellent quality, which substantially extends on previous yeast P complex structures. This is also supported by the authors use of the latest data analysis tools (Relion-5, AlphaFold2 multimer predictions, Modelangelo). The authors re-evaluate published EM densities of yeast spliceosome complexes (B*, C,C*,P) for the presence or absence of Fyv6, substantiate Fyv6 as a 2nd step specific factor, confirm it as the homolog of the human protein FAM192A, and provide a model for how Fyv6 may fit into the splicing pathway. The biochemical experiments on probing the splicing effects of BP to 3'SS distances after Fyv6 KO, genetic experiments to probe Fyv6 and Syf1 domains, and the suppressor screening add substantially to the study and are well executed. The manuscript is clearly written and we particularly appreciated the nuanced discussions, for example for an alternative model by which Prp22 influences 3'SS undocking. The research findings will be of great interest to the pre-mRNA splicing community.

Comments on revisions:

I'm satisfied with the changes.

Reviewer #2 (Public review):

HIV infection is characterized by viral integration into permissive host cells - an event that occurs very early in viral-host encounter. This constitutes the HIV proviral reservoir and is a feature of HIV infection that provides the greatest challenge for eradicating HIV-1 infection once an individual is infected.

This study looks at how starting HIV treatment very early after infection, which substantially reduces the peak viral load detectable (compared to untreated infection), affects the amount and characteristics of the viral reservoir. The authors studied 35 women in South Africa who were at high risk of getting HIV. Some of these women started HIV treatment very soon after getting infected, while others started later. This study is well-designed and has as its focus a very well characterized cohort. Comparison groups are appropriately selected to address proviral DNA characterization and dynamics in the context of acute and chronic treated HIV-1. The amount of HIV and various characteristics of the genetic makeup of the virus (intact/defective proviral genome) was evaluated over one year of treatment. Methods employed for proviral DNA characterization are state-of-the-art and provide in-depth insights into the reservoir in peripheral blood.

While starting treatment early didn't reduce the amount of HIV DNA at the outset, it did lead to a gradual decrease in total HIV DNA quantity over time. In contrast, those who started treatment later didn't see much change in this parameter. Starting treatment early led to a faster decrease in intact provirus (a measure of replication-competence), compared to starting treatment later. Additionally, early treatment reduced genetic diversity of the viral DNA and resulted in fewer immune escape variants within intact genomes. This suggests that collectively having a smaller intact replication-competent reservoir, less viral variability, and less opportunity for virus to evade the immune system - are all features that are likely to facilitate more effective clearance of viral reservoir, especially when combined with other intervention strategies.

Major strengths of the study include the cohort of very early treated persons with HIV and the depth of study. These are important findings, particularly as the study was conducted in HIV-1 subtype C infected women (more cure studies have focussed on men and with subtype B infection)- and in populations most affected by HIV and in need of HIV cure interventions. This is highly relevant because it cannot be assumed that any interventions employed for reducing/clearing the HIV reservoir would perform similarly in men and women or across different populations. Other factors also deserve consideration and include age, and environment (e.g. other comorbidities and coinfections).

Reviewer #2 (Public review):

Summary:

In this manuscript by Torelli et al., the authors propose that the major function of MYR1 and MYR1-dependent secreted proteins is to contribute to parasite survival in a paracrine manner rather than to protect parasites from cell-autonomous immune response. The authors conclude that these paracrine effects rescue ∆MYR1 or knockouts of MYR1-dependent effectors within pooled in vivo CRISPR screens.

Strengths:

The authors raised a more general concern that pooled CRISPR screens (not only in Toxoplasma but also other microbes or cancers) would miss important genes by "paracrine masking effect". Although there is no doubt that pooled CRISPR screens (especially in vivo CRISPR screens) are powerful techniques, I think this topic could be of interest to those fields and researchers.

Weaknesses:

In this version, the reviewer is not entirely convinced of the 'paracrine masking effect' because the in vivo experiments should include appropriate controls (see major point 2) in the first submission.

After the revision, although no experiments were added, this reviewer considered that the points have been sufficiently discussed and commented on.