this shouldn't be necessary, you can encode the remediation id in the workflow id and that provides the same thing

this can be extracted from the remediation report

this doesn't cover the client's internal package management, we would need to have a vpn as well as a network policy per client.

we talked about why we can't do this and why it's not a good idea. we have 0 sandboxing functionality, and most of our clients use their own internal package hosting. all package managers have some level of arbitrary code execution, if we're not running the installs and version numbers ourselves we do run the risk of exposure to completely unknown supply chain attacks if this is done completely agentically. I would stick to simple version bumps in the manifests, and fixing code that needs to be fixed.

People start leaving Everglades because of the warnings they’ve been given.

A huge hurricane strikes the Everglades. Janie and Tea Cake try to escape the dangerous flooding. During the chaos, Tea Cake saves Janie from a rabid dog but gets bitten in the process. Many people die in the storm.

Tea cake is jealous and hits her and he does it to feel in power.

Tea Cake sometimes feels insecure because Janie is wealthier and admired by other men. To show control, he beats Janie lightly, which sadly earns approval from some people around them. Despite this, Janie still deeply loves Tea Cake.

They got stuck in a hurricane and almost lost each other.

Janie and Tea Cake arrive in the Everglades, also called “the muck.” They work on bean farms with other migrant workers. Janie enjoys the lively community, music, storytelling, and the equal relationship she has with Tea Cake.

Author response:

Both reviewers noted that some published studies question the association of HPV types with cervical cancer survival {PMIDs 36207323 and 33117670}, while others did not observe that {REFS 69-74 in Chakravarty}. We appreciate both reviewers pushing us to discuss and hypothesize (even speculate) on our finding that HPV types not in phylogenetic clade α9 types (including HPV18) had more recurrences than α9 types (including HPV16). The most likely explanation is that we analyzed 225 HPV types not just the most prevalent types. Specifically, each of the 5 recurrences in our pilot study had different HPV types (α7’s: 18, 39, 45, 70 & α5: 69). Similarly, on re-examination of the TCGA data set, we found that 80% of the 181 α9 samples had HPV16, while 52.5% of the non-α9 samples had HPV18, consistent with a broader variety of types in the latter. We note that PMID: 36207323 did assess a broad number of HPV types, but these were classified into three non-cladistic categories, HPV16, HPV18 and Other for comparison. More in line with the main point of that study, HPV18 was enriched, though not significantly, in the more pathogenic C2 group (which was defined by a deep analysis of specific genomic alterations). It can be speculated that perhaps α9 types are less proficient at effecting or interacting with some C2 characteristic(s). Overall, we suggest that these observations emphasize the importance of examining the full spectrum of HPV types including phylogenetic relationships in cervical cancers induced by these viruses.

Reviewer #1:

The detection of “non-tumor HPVs” was noted as a potential limitation. The highly multiplexed, HC+SEQ methodology that we use obviously detects many HPV types and thus can identify lesions with multiple HPV types as occurred in Patient 16 and in other HPV cancers. It is unclear what role multiple HPV types might play in tumorigenesis if any. Regardless of whether broad detection of HPV types proves to be a limitation or an advantage, it will be interesting. Our approach in this study focused on integration of HPV DNAs into human DNA, as this is a key event in cervical tumorigenesis. We believe that detection of clonally expanded cells with an integrated URR-E6-E7 DNA segment of any HPV type (whether high-risk, low-risk, or intermediate, or even perhaps non α-clade {PMID:40742260}) should be viewed with suspicion. For the small fraction of cervical cancers that contain only unintegrated HPV DNA, it will be interesting to see if these viral DNAs share any particular properties.

The reviewer asked for details of the HPV DNA capture probes used. All were from the proprietary Roche Nimblegen SeqCap EZ System. They encompassed all HPV types from HPV1 through HPV225.

The reviewer questioned why the data verifying the viral-human DNA junctions in primary tumor tissue by the orthogonal approach of PCR assays PCR assays were not shown. The data summary and the approach used for PCR are in Figure 1, Table 1 and Supplementary Table 1. Only the dozens of agarose gel photographs were not shown. PCR assays that addressed key issues comparing primary and metastatic sites and confirming HPV16 + HPV18 coinfection are shown in Figure 2 and Figures 4A & 4B, respectively.

Reviewer #2:

The reviewer raised general issues about data quantification and statistical adequacy. Regarding data quantification, we used a strict, conservative guideline of a 10 read minimum per junction in the DNA from tumor samples. This was based on the sequence analysis pipeline design and on our requirement that some clonal expansion of cells containing specific junctions must have occurred. Extensive complications to comparing quantified read counts in different studies are detailed below in the responses to specific comments. The statistical methods used were based on the dichotomous variable of detection versus no detection of integrated HPV DNA. For this study, we also used the orthogonal method of verifying every junction by PCR with one primer in viral DNA and the other in flanking human DNA followed by Sanger sequencing. The statistical methods used were entirely appropriate for this dichotomous variable and time to event analyses. Nonetheless, we concur that quantification of HPV DNA integration would be an interesting variable to consider once carefully controlled methodologies are applied considering the issues detailed below.

Regarding the first point about variability in HPV-human junction number in different studies: The number of HPV DNA genome and junction read counts obtained from a sample are subject to numerous technical and biological variables. Extensive caution should be applied when comparing quantitative results among different studies, and this particularly includes the number HPV-human DNA junctions detected. Among the factors that can be involved among different studies are the following: 1) inadequate deduplication of sequence reads; 2) “barcode-hopping” or “bleed-through” from one sample to another and thus cross-contamination of one sample with another during multiplexed short-read sequencing; 3) variation in the fraction of cells that are tumor cells in the post-clinical analysis sample of tissue obtained; 4) artifactual ligation of HPV and human DNA segments occurring at the adaptor ligation step of short-read sequencing; 5) variability in the mismatch settings of computational sequence aligners used; 6) perhaps most importantly, the level of genomic instability of each particular integration locus; and 7) subclonal variation in proliferation or survival of cells containing specific junctions within a lesion. The reviewer correctly implied that our requirement for a minimum of 10 sequence reads at each junction excludes low level, subclonal variants. Nonetheless, one tumor did have two integrations (Table 1). More importantly, we emphasize that all five tumor-recurrences at distant metastatic sites in our study had the exact same integration event as the primary tumor (determined at single nucleotide resolution at both ends). We judge this to be compelling evidence that the approach we use correctly identifies the key integration event underlying each cancer.

Regarding the second point about ratios between genomic DNA copy numbers and junction read counts: Both human genome and HPV genome copy numbers deserve mention in regard to this issue. HPV HC+SEQ highly enriches for viral DNA, with the advantage gained of high read depth for viral sequences, but with human DNA largely excluded (except for the junction reads). Thus, ratios of junctions to the rest of the human genome cannot be assessed as they can be with whole genome sequencing methodologies. While HPV genome read depth can be ascertained with HC+SEQ reads (as in Figure 1C, 1D, 1E), and the reviewer’s suggestion raises the possibility of using junction to viral read ratios to normalize data to compare different integration loci and even perhaps different studies, there are nonetheless additional, biomedically relevant complications. HPV DNA segments are sometimes often present as tandem units with or without human DNA segments in tumors (Figure 1E shows the former), and this affects the ratio of junctions to viral genomes. Thus, using the suggested ratios would require additional normalization for tandem copy numbers, and thus, it would be difficult to use them in a manner analogous to gene-specific read counts per million total read ratios in RNA-seq.

Regarding the third point about comparing read counts from primary tumor tissue with those from cfDNA: Ours was a retrospective study using archived samples that were available, and the HPV genome coverage obtained by HC+SEQ using cfDNA varied (Table 1). Assessment of viral DNA genome and human junction reads in a quantitatively reliable manner by HC+SEQ will require application of precise collection, storage, and processing of cfDNA samples. Nonetheless, the results presented in this study, while variable among the different samples, were entirely sufficient to test the dichotomous variable analyzed. We note that this included orthogonal, PCR verification of junctions, based on the straightforward, abundant identification of the junctions by HC+SEQ in the primary tumor samples. We emphasize that examination of HPV DNA integration directly interrogates a key, likely causal event in HPV cervical tumorigenesis.

Regarding the fourth point about many of the initial cancer samples harboring no junction breakpoints: 100% of the 16 initial, cervical, primary tumor tissue samples harbored an integration (one sample had two). The reviewer is correct that many of the initial cfDNA samples lacked HPV DNA integration as assessed by HC+SEQ and by PCR based on the junctions detected in the primary tumor tissue. We interpret this to mean that these cancers were not spilling genomic DNA containing the integrated HPV DNA into serum at sufficient levels to be detected, and judge this to be due to underlying, unidentified, biomedically-relevant effects.

Regarding the fifth point about HPV-human DNA junctions being used as a measure of tumor heterogeneity and subclonal variation: We concur with the reviewer that this is an interesting, important issue. We noted it in the response to the “first” point (numbers 6 and 7) above. Again, one of the samples had two integrations, and this patient did not suffer a recurrence (Table 1, Figure 1). Based on our ongoing experience, to take findings of junction subclonality beyond just detection of multiple integration junctions, we believe that development of in situ, single cell approaches are necessary to reveal the full meaningful picture of subclonality.

Beyond these quantitative issues that we raise in response to Reviewer #2’s comments, the Reviewers’ comments point at important, incompletely understood aspects about HPV tumorigenesis. Our finding of the identical viral DNA insertions in primary tumors and metastases point to a central, constant role for these structures in viral tumorigenesis. Nonetheless, the issues raised point to key questions concerning subclonality, detailed structures and quantification of HPV and human tandem DNA units, intrachromosomal DNA vs. ecDNA, genomic instability of integrated HPV DNA loci, and cell-to-cell variation, and what roles these might play in tumorigenesis.

Regarding the point about cell-free DNA breakpoints, we note the field of circulating tumor DNA fragmentomics that examines the sequences and a host of structural properties of circulating DNAs derived from tumors including specific, short sequences at the ends (breakpoints) of DNA fragments circulating in blood. These are of emerging significance as biomarkers for cancer {PMIDs:40038442 and 41043439}. We note that cell free DNA breakpoints are not synonymous with DNA junctions. We stress again that the main point of our manuscript was to investigate HPV-human DNA junctions in cfDNA, as this directly addresses a likely causal mechanism underlying HPV cervical tumorigenesis. Additional, future studies would be required to assess the effectiveness of our targeted, individualized approach relative to other aspects of fragmentomics in cervical cancer.

In summary, we restate one of the reviewers’ points. “This study provides important foundational evidence for further evaluating the clinical utility of HPV DNA detection from cfDNA and specifically assessing for integration junctions.” Both reviewers raised thoughtful points about DNA integration and HPV tumorigenesis, and prospective studies are required to refine and evaluate clinical utility of the new findings presented here.

Janie goes back to the Eatenville

muy interesante, me imagino si por defecto mi sistema Yocto hace lo mismo, y sino, si es posible configurarlo

UN peacekeepers are required to be fair and balanced, keeping all sides on a conflict from fighting with each other.

eLife Assessment

This important study probes the long-standing failure to resolve evolutionary relationships between the classical "spiralian" taxa-i.e., annelids, molluscs, brachiopods, platyhelminths and nemerteans-and provides convincing evidence that the branches leading to them are so short as to be unreliable guides to their relationships. This, in turn, has wide-ranging implications for our understanding of animal body plan evolution and the interpretation of early animal fossils.

Reviewer #1 (Public review):

[Editors' note: this version has been assessed by the Reviewing Editor without further input from the original reviewers. The revised version adequately addresses the relatively minor comments from the previous round of review.]

Summary:

This interesting paper probes the problematic relationships between the classical "spiralian" taxa, i.e., annelids, molluscs, brachiopods, platyhelminths and nemerteans, and shows that the branches leading to them are so short as to be unreliable guides to their relationships. This, in turn, has important implications for how we view the origin of the animal phyla.

Strengths:

A very careful analysis of a famous old problem with quite significant results. The results seem to be robust and support their conclusions.

It often passes uncommented that many different trees are published about animal relationships, yet some parts of the tree seem extremely difficult to resolve; the spiralians are perhaps the most difficult case. More recently, problems about sponges or ctenophores as sister groups to the rest of the animals have alerted us to major areas of uncertainty in large-scale phylogenetic reconstruction; this paper is a welcome reminder that other, perhaps even harder, problems exist which may be difficult to ever resolve with the (molecular) data we have.

Reviewer #2 (Public review):

Summary:

The relationships among the phyla making up Spiralia - a major clade of animals including molluscs, annelids, flatworms, nemerteans and brachiopods - have been challenging from a phylogenomic perspective despite decades of molecular phylogenetic effort. Every topology uniting subsets of these phyla has been recovered with apparent support in at least one study, yet no consensus has emerged even from large-scale genomic datasets. Serra Silva and Telford set out to determine whether this instability reflects a genuine biological signal being obscured by analytical limitations, or whether it reflects a rapid, near-simultaneous origin of these phyla that has left behind in modern genomes far too little phylogenetic information to resolve. They focused deliberately on five phyla, reducing the problem to a tractable set of 15 unrooted and 105 rooted topologies, and applied a suite of complementary approaches across two independent datasets and multiple substitution models to test whether any topology is significantly preferred over alternatives.

Strengths:

(1) The conceptual framing of the problem is excellent, and the study makes a convincing case across several lines of evidence. By enumerating all possible topologies and demonstrating empirically that every one of the 15 unrooted arrangements has been recovered as the preferred solution in at least one published study, the authors make a strong argument about the state of the field. The use of two entirely independent datasets as a consistency check is great, and convergence between them, where it occur,s substantially strengthens confidence in the conclusions.

(2) It is my view that the simulation framework is a particular strength. Generating data on a fully unresolved star tree and scoring those data under both correctly-specified and misspecified substitution models provides convincing evidence that the strong preference for rooting Spiralia on the flatworm branch is, at least partly, an analytical artefact driven by the exceptionally long branch in combination with compositional heterogeneity across sites. This is an important methodological demonstration with implications beyond spiralian phylogenetics, as the same issue is likely to affect other deep, long-branched lineages in the animal tree of life.

(3) The randomised taxon-jackknifing approach is a very nice addition here. The demonstration that preferred topologies shift depending on which species happen to be sampled (even within the same phylum) is a convincing indicator of weak signal, and provides a practical caution for future studies that may report strong support for a particular spiralian arrangement based on a fixed taxon sample.

(4) The branch-length analyses, benchmarking internal interphylum branches against the already disputed and extremely short branch uniting deuterostomes (work also by this group), are well-conceived and solid.

(5) I think it is worth highlighting the notable intellectual honesty throughout the paper: the authors do not overstate their results, correctly acknowledging that while the unrooted topology grouping molluscs with brachiopods and flatworms with nemerteans emerges most consistently, this preference is not statistically significant under more adequate substitution models and may itself carry some artefactual component.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public review):

Summary:

This interesting paper probes the problematic relationships between the classical "spiralian" taxa, i.e., annelids, molluscs, brachiopods, platyhelminths and nemerteans, and shows that the branches leading to them are so short as to be unreliable guides to their relationships. This, in turn, has important implications for how we view the origin of the animal phyla.

Strengths:

A very careful analysis of a famous old problem with quite significant results. The results seem to be robust and support their conclusions.

It often passes uncommented that many different trees are published about animal relationships, yet some parts of the tree seem extremely difficult to resolve; the spiralians are perhaps the most difficult case. More recently, problems about sponges or ctenophores as sister groups to the rest of the animals have alerted us to major areas of uncertainty in large-scale phylogenetic reconstruction; this paper is a welcome reminder that other, perhaps even harder, problems exist which may be difficult to ever resolve with the (molecular) data we have.

Weaknesses:

The paper could have perhaps drawn out some of the implications of its results in a clearer manner.

Reviewer #2 (Public review):

Summary:

The relationships among the phyla making up Spiralia - a major clade of animals including molluscs, annelids, flatworms, nemerteans and brachiopods - have been challenging from a phylogenomic perspective despite decades of molecular phylogenetic effort. Every topology uniting subsets of these phyla has been recovered with apparent support in at least one study, yet no consensus has emerged even from large-scale genomic datasets. Serra Silva and Telford set out to determine whether this instability reflects a genuine biological signal being obscured by analytical limitations, or whether it reflects a rapid, near-simultaneous origin of these phyla that has left behind in modern genomes far too little phylogenetic information to resolve. They focused deliberately on five phyla, reducing the problem to a tractable set of 15 unrooted and 105 rooted topologies, and applied a suite of complementary approaches across two independent datasets and multiple substitution models to test whether any topology is significantly preferred over alternatives.

Strengths:

(1) The conceptual framing of the problem is excellent, and the study makes a convincing case across several lines of evidence. By enumerating all possible topologies and demonstrating empirically that every one of the 15 unrooted arrangements has been recovered as the preferred solution in at least one published study, the authors make a strong argument about the state of the field. The use of two entirely independent datasets as a consistency check is great, and convergence between them, where it occur,s substantially strengthens confidence in the conclusions.

(2) It is my view that the simulation framework is a particular strength. Generating data on a fully unresolved star tree and scoring those data under both correctly-specified and misspecified substitution models provides convincing evidence that the strong preference for rooting Spiralia on the flatworm branch is, at least partly, an analytical artefact driven by the exceptionally long branch in combination with compositional heterogeneity across sites. This is an important methodological demonstration with implications beyond spiralian phylogenetics, as the same issue is likely to affect other deep, long-branched lineages in the animal tree of life.

(3) The randomised taxon-jackknifing approach is a very nice addition here. The demonstration that preferred topologies shift depending on which species happen to be sampled (even within the same phylum) is a convincing indicator of weak signal, and provides a practical caution for future studies that may report strong support for a particular spiralian arrangement based on a fixed taxon sample.

(4) The branch-length analyses, benchmarking internal interphylum branches against the already disputed and extremely short branch uniting deuterostomes (work also by this group), are well-conceived and solid.

(5) I think it is worth highlighting the notable intellectual honesty throughout the paper: the authors do not overstate their results, correctly acknowledging that while the unrooted topology grouping molluscs with brachiopods and flatworms with nemerteans emerges most consistently, this preference is not statistically significant under more adequate substitution models and may itself carry some artefactual component.

Weaknesses:

(1) The restriction to five phyla is the most significant limitation, as the authors acknowledge this and give a clear computational justification, but readers should be aware that the paper's convincing conclusions apply specifically to the five focal phyla and the evidence remains incomplete with respect to spiralian phylogeny as a whole.

(2) The treatment of substitution model adequacy, while commendably thorough for site-heterogeneous models, is necessarily bounded. The authors note that models accounting for non-stationarity, across-lineage compositional heterogeneity, or mixtures of tree histories might yield different results, and that even the most sophisticated currently available approaches have not produced consistent spiralian topologies across studies. This is not a criticism of what has been done here - the analytical scope is reasonable and well-implemented - but it means the paper cannot be read as a definitive demonstration that no model will ever resolve these relationships. The distinction between a true hard polytomy and a radiation that is effectively unresolvable given current data and methods could be drawn more sharply in the discussion.

(3) The reticulation-aware coalescent analyses are presented somewhat briefly relative to the likelihood-based topology scoring. The finding that flatworms are recovered within a paraphyletic jaw-bearing animal clade in both summary trees - interpreted as long-branch attraction - is striking, and its implications for gene-tree-based approaches to spiralian rooting deserve more discussion than they currently receive.

(4) The central conclusions - that interphylum branches in Spiralia are extraordinarily short, that topological preferences are strongly model-dependent and taxon-sampling-sensitive, and that an ancient rapid radiation is the most parsimonious explanation - are convincingly supported by the evidence presented. The identification of flatworm long-branch attraction as an important confounding factor in rooting analyses is itself an important and well-demonstrated result.

Conclusion:

This paper clearly makes an important contribution to the ongoing debate about spiralian relationships and, more broadly, to methodological discussions about how to handle anciently diversified clades where phylogenetic signal is genuinely limited. The exhaustive topology-scoring framework combined with taxon-jackknifing and simulation under unresolved trees is a valuable methodological template that could usefully be applied to other notoriously difficult nodes in the animal tree. I thoroughly enjoyed the discussion of the implications of these findings for interpreting Cambrian fossils and the evolutionary history of shells, segmentation, larval types and other characters - it is both thoughtful and thought-provoking and will be of broad interest well beyond the phylogenomics and zoology communities. From a very practical perspective, the data and scripts provided make the work useful to researchers wishing to apply similar approaches to other groups.

Reviewer #3 (Public review):

Summary:

This paper addresses the controversial internal relationships within the Spiralia, a major clade of invertebrate animals including molluscs, annelids, brachiopods and flatworms.

Strengths:

Performs a range of empirical analyses and simulations that address the core question. Although a favoured unrooted topology finds some support, this is not strongly endorsed in the paper.

Weaknesses:

(1) Only considers a subset of relevant phyla (e.g. gastrotrichs are relevant to the phylogenetic position of Platyhelminthes), although how this would change the scale of the analyses (i.e. number of topologies) is addressed in the paper.

(2) Discussion of Spiralia evolution and broader context, particularly the relevance for the fossil record. Line 448: our current understanding of the early spiralian fossil record is quite consistent with the main results of this paper. For example, there are very few claims for fossils that sit on the short branch leading to Spiralia (or Lophotrochozoa as defined here) that this paper discusses. Many of the key fossils that inform on the characters discussed in the introduction, which have unusual character combinations, have an apomorphy of one of the phyla discussed, and so are resolved as members of the stem lineages of particular phyla.

(3) This is what you would expect with long phylum stem lineages (line 148) and a short spiralia stem lineage. For example, the mollusc Wiwaxia has chaetae, but a mollusc like Radula (Smith 2012), the conchiferan mollusc Pelagiella has chaetae and a coiled shell (Thomas et al. 2020). The only fossil groups that are routinely discussed as belonging to the stem lineage of more than one phylum are the tommotiids, which have chaetae, segmentation and a complex mineralised skeleton (but not shells in the brachiopod/mollusc sense, see Guo et al 2023) but they sit on the lophophorate stem lineage, a synapomorphy rich group the monophyly of which the present paper endorses (e.g. line 435). The fossil record is consistent with the scenario presented in line 442, e.g. convergent loss or reduction of chaetae and segmentation and convergent evolution of shells in molluscs and brachiopods.

We thank the reviewers for their kind comments. Please see below for detailed responses to all identified weaknesses.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

Some minor comments that might help improve the paper:

(1) Abstract L17. "Most analyses on the 15 unrooted trees showed a preference for the same topology but the support over other solutions was non significant" - I don't really understand this sentence in the context of the paper; it makes it sound as if the tree is, after all, well resolved! Non-significant, or not significant better than non significant?

Having read the rest of the paper I see what this refers to (uT4), but still I don't understand the second clause.

Re-written to clarify.

(2) Introduction L31. This makes it sound as if phoronids are actually part of brachiopods, and while that was recovered by Cohen and Weydmann 2005, I'm not sure if it's really a general result. In addition, rather than using "brachiopods plus phoronids" everywhere, you could use "Brachiozoa" (Cavalier-Smith 1998, Biol. Rev).

We have updated our text and figures to use Brachiozoa.

(3) L36-37. Yes, but the presence of Chaetagnatha in this clade is suggestive that their primitive body size is not small.

Have made clear that chaetognaths are not all tiny.

(4) L85. Kumar et al. may have claimed that Spiralia are as old as 670, but many other analyses would suggest a range of different results. Why choose just this one? In addition, this age seems rather incompatible with your results.

We agree this maximum age is highly improbable (the principal point remains the deep age of the protostomes). We have used a different reference and refer to a generally acceptable minimum age only.

(5) L88. The key part of this sentence, "proving a hard polytomy", comes at the end of a long set of references that makes it hard to connect to the lead-in "given the age of", so I would suggest rephrasing.

Rephrased for clarity.

(6) L109. It is unclear what this means in the context: "and even support multiple topologies".

Re-worded for clarity.

(7) Figure 1. Why did you choose to indicate brachiopods plus phoronids as a larval form, unlike the other clades? Perhaps it's because we don't know what the last common ancestor of the two looked like (unless P is an ingroup of B), but that's arguably true for some of the other clades as well!

Apologies, this was laziness as we already had a line drawing of an actinotroch larva. Have improved the images in figures 1 and 5 where required.

(8) L164. Reticulation-aware analyses. As I understand it, this would include introgression, hybridization, etc. However, incomplete lineage sorting has also been invoked, not just for Cambrian-explosion age events but also for other major radiations, such as for angiosperms and birds. How significant might ILS be for generating the results you get?

Section title amended. Results section updated to reflect this. We now explicitly mention the potential impact of ILS and introgression on spiralian relationships in our discussion.

Unrooted trees analysis:

(9) L405 on. Maybe it would be worth including a figure showing the relative branch lengths of uT4. All the images of trees show similar-length branches, which gives off the wrong impression within the context of the paper!

We understand the motivation, but we worry that showing uT4 as the sole phylogram may end up with this being interpreted by a casual reader as being the main result of the paper. Hopefully the figures with branch lengths encompass this information well enough and with no danger of misinterpretation.

(10) L430 on. Why is this a "conservative" interpretation?

Yes agreed not clear. Have changed to “We interpret our results as showing that…”

(11) You mention synapomorphy accumulation time and implicitly equate shortness of branches with shortness of time. However, other options are available under varying diversification rate models (e.g. ClaDs, Barido-Sottani et al. 2023 Syst. Biol.; CET, Budd and Mann 2025, Syst.Biol.). In particular, the latter paper shows that when unusually large clades are selected for study (as is arguably the case here), then those clades are likely to have started with very high "evolutionary tempo", which speeds up all aspects of evolution, including diversification rates.

In the Budd and Mann scenario large clades begin with high tempo of cladogenesis, high substitution rate and high diversification rate (rapid origin of new characters). This would suggest that the period of the radiation was extra rapid (even less time than in a ‘normal’ period during which smaller clades emerge) so we feel the point stands.

(12) L449. Maybe refer to the Song et al. paper again here on scaphopods plus bivalves, as it makes the same sort of points, albeit in a slightly different context.

We thank the reviewer for the suggestion and have added the citation where relevant.

(13) Finally, to return to L20. You mention implications for the Cambrian fossil record, but then fail to deliver any!

We have hopefully addressed this remark in the discussion better (at least to the extent we are qualified to).

Yet if you are correct, then synapomorphy accumulation would unite groups of phyla, and would surely lead to a scenario highly incompatible with clock models suggesting deep origins of clades (as they would all be more fossilisable).

Apologies but we don’t completely understand this point as ‘synapomorphy accumulation would unite groups of phyla’ is a little ambiguous. Of course, this is generally true, but our results suggest there was little opportunity to accumulate identifiable synapomorphies linking pairs, triplets or quartets of our 5 spiralian phyla.

In addition, clock results suggest rather long periods of time leading to the phyla, which would imply that there would have to be extremely slow rates of molecular evolution to yield the short early branches here. Also, it might be worth referring to papers compatible with this view, such as Wernström, J.V. et al., EvoDevo 13, 17 (2022). https://doi.org/10.1186/s13227-022-00202-8 or some of the palaeo literature, such as Budd and Jackson 2016, Phil Trans.

The referee refers to clock results suggesting a (deep) Ediacaran origin of Lophotrochozoa/Spiralia. We interpret the spiralian radiation itself as rapid but, in the absence of a clock analysis, we cannot comment on when it took place.

Reviewer #2 (Recommendations for the authors):

(My not very) Major points - as I feel this is an excellent paper.

(1) The coalescent-based summary tree analyses warrant expansion. The recovery of flatworms within a paraphyletic jaw-bearing animal clade in both summary trees is a striking result attributed to long-branch attraction, but this interpretation would be strengthened by examining whether pruning or downweighting the longest-branching taxa within those groups affects the outcome, or by reporting per-node quartet scores more fully. This would make the reticulation-aware results more directly informative and would bring this section into better balance with the detailed likelihood-based analyses.

We thank the reviewer for the suggestion of the expanded analyses. We have now done these, and they yielded essentially the same results as the unpruned analyses. Additionally, while not discussed, we ran the Astral analyses on the subset of gene-trees where all groups of interest (spiralian phyla and superphyletic Ecdysozoa, Deuterostomia, etc.) were monophyletic and found no changes to interphylum quartet scores beyond those due to enforced (super)phylum monophyly, with Platyhelminths still recovered within Gnathifera.

We have expanded our description of the results slightly as well as our discussion. Location of the tables with detailed quartet scores and local posterior probabilities has been added to Fig. S1’s legend.

(2) It would strengthen the paper to include at least a brief analysis or explicit discussion of whether any currently available models accounting for non-stationary or across-lineage compositional heterogeneity show any change in the pattern of support, even if only tested on a subset of topologies. A null result here would itself be informative and would make the conclusions more robust to the concern that unexamined model classes might behave differently.

We thank the reviewer for the suggestion, but this represents a considerable amount of new work and we think it falls outside the scope of the present work. We have, as suggested, included this as a discussion point.

(3) The authors note that topologies grouping flatworms with ribbon worms appear among the higher-scoring arrangements even under model misspecification in simulations. It would be helpful to comment explicitly on whether the apparent signal for this grouping should therefore be regarded with particular scepticism, or whether it survives artefact correction in any of the analyses, as this is a grouping that has appeared repeatedly in the literature and readers will want guidance on how to interpret it.

We do state that the nemertean+platyhelminth grouping seems likely to be at the least emphasised by an artefact (as the referee points out it is common to the higher scoring trees in the star tree simulations). We state that this suggests “…that this grouping derives some support from systematic errors.” We now return briefly to this in the discussion.

Writing and presentation

(1) The abstract states that rooting Spiralia on the flatworm branch "is a long-branch artefact" - this is slightly stronger than the language used in the body of the paper, where the authors correctly write that this preference is "at least enhanced by" the artefact. The abstract phrasing should be softened to reflect the more nuanced conclusion in the text.

Good point. Done.

(2) A brief signposting sentence near the start of the Results, setting out the overall analytical logic before the individual sections begin, would help orient readers. The strategy - score all topologies, test robustness to model choice and taxon sampling, then use simulation to identify artefactual signals - is clear in retrospect but would benefit from being made explicit upfront.

We have taken this suggestion on board. The summary seemed in the end better placed as the final part of the introduction.

(3) Figure 3 is complex and would be easier to interpret with a brief explanatory note in the legend clarifying what a wide versus narrow range of log-likelihood scores across topologies means in practical terms for statistical resolution between trees.

Added sentence to legend.

Minor Corrections:

(1) The Figure 2 legend contains a typographical error: "shorter than the short, disputed deuterostome branch" should read "shorter than."

Done

(2) At least one reference appears to carry a future publication year (Ishii et al., 2026) and should be verified for accuracy before final submission.

This reference is correct per the journal’s website. We did find Google Scholar to list it as being from 2025.

Reviewer #3 (Recommendations for the authors):

(1) Abstract/SI definitions of Spiralia/Lophotrochozoa

While I don't have strong feelings about this, if Spiralia is being used as an apomorphy-based name, then it still might be equivalent to Lophotrochozoa, as spiral cleavage in Gnathostoniula jenneri was illustrated by Riedl (1969). Although no other studies have replicated this observation, this should at least be mentioned.

Sorry this reference to gnathostomulid spiral cleavage was included in a longer version of the discussion of nomenclature. This was first reduced in length (which was when the mention of gnathostomulid spiral cleavage was dropped) then finally moved to the supplementary material. We have now re-included mention of this in the discussion in supplementary info.

The SI text suggests that the name Lophotrochozoa, as used in its original form by Halanych et al. (1995), was a node-based definition, and that this name is for the sister group of Ecdysozoa. However, in that paper, the name is actually defined as "as the last common ancestor of the three traditional lophophorate taxa, the molluscs, and the annelids, and all of the descendants of that common ancestor". This definition would exclude Gnathifera, and depending on the internal relationships of the non-Gnathiferan phyla, may be equivalent (or not) to the usage of the name Spiralia adopted in the present paper. The perils of mixing node and apomorphy-based definitions of clades are clear, and the situation is less straightforward than the paper suggests, and (somewhat unhelpfully given the subject of the paper) may only become clearer if the relationships of non-ecdysozoan protostomes are resolved.

We believe that the community universally understood the definition of Lophotrochozoa following the 1997 paper (by the authors who also provided the original 1995 definition). This 1997 definition included both chaetognaths and rotifers as examples of the Gnathifera. The Spiralia, in contrast, began life not even as a name for a clade but a description of a character shared by some apparently unrelated taxa – similar to a grouping of ‘carnivores’. The introduction of a new name was, we suggest, unhelpful. We hope that by defining our terms up front the meaning in the current paper is clear.

(2) Introduction

Line 76. Some references needed regarding claims that there was a polymeric brachiopod ancestor, e.g. Gutman (1978), Temereva and Malakhov (2011), Guo et al. (2023). Likewise for the chaetae of brachiopods, annelids and molluscs, e.g. Schiemann (2017), as it's key to trace where these ideas originated.

Added

Figure 1. This is a nice illustration of the uncertainty in the relationships of these groups. However, I kept checking which thumbnail image was which for nemerteans and annelids. A minor suggestion, but perhaps a polychaete instead for the annelid?

We have replaced the rather poor image of an earthworm with a polychaete and also now include labels. We hope the improved images are more helpful. Good point.

(3) Results

Branch length comparison. I understand why the deuterostome stem was chosen as the branch for comparison from the point of view of phylogenetic uncertainty. However, what about the branch leading to ecdysozoa or the branch subtending lophotrochozoan and/or gnathifera? Given that the short internodes are used as an argument underpinning uncertain relationships, can we be sure that Gnathifera is not nested within the group of interest, especially given that Gnathifera contains many long-branched taxa and the root may be misplaced within the group?

We have added the Lophotrochozoa and Ecdysozoa median lengths to our plots and now discuss both the lophotrochozoan branch in our results.

Line 249. Given that Spiralia is the group of interest, why were the Gnathiferans also chosen at random?

The point of the experiment was to see the effect of taxon sampling on the consistency of the resulting topology. Random sampling across the tree seems helpful in this context. We chose Gnathifera as one group to sample from as this ensured they would be present in all trees. This seems appropriate as they are the sister group of the clade of interest and as such their inclusion reflects a choice a typical investigator might make when choosing which species to include. Additionally, as noted in the reviewer’s earlier comment, Gnathifera includes many long-branched taxa and we wanted to ensure our root-placement results were robust to this aspect of taxon sampling.

(4) Discussion

Line 448. Our current understanding of the early spiralian fossil record is quite consistent with the main results of this paper. For example, there are very few claims for fossils that sit on the short branch leading to Spiralia (or Lophotrochozoa as defined here) that this paper discusses. Many of the key fossils that inform on the characters discussed in the introduction that have unusual character combinations have an apomorphy of one of the phyla discussed, and so are resolved as members of the stem lineages of particular phyla.

This is what you would expect with long phylum stem lineages (line 148) and a short spiralia stem lineage. For example, the mollusc Wiwaxia has chaetae, but a mollusc like radula (Smith 2012), the conchiferan mollusc Pelagiella has chaetae and a coiled shell (Thomas et al. 2020). The only fossil groups that are routinely discussed as belonging to the stem lineage of more than one phylum are the tommotiids, which have chaetae, segmentation and a complex mineralised skeleton (but not shells in the brachiopod/mollusc sense, see Guo et al 2023) but they sit on the lophophorate stem lineage, a synapomorphy rich group the monophyly of which the present paper endorses (e.g. line 435). The fossil record is consistent with the scenario presented in line 442, e.g. convergent loss or reduction of chaetae and segmentation and convergent evolution of shells in molluscs and brachiopods.

We accept these points (though are clearly not experts on these fossils). We have (slightly tentatively given our lack of expertise) expanded our discussion to include these fossil taxa with their combinations of characters.

focus is on the location of a chunk of data

emergent autopoietic data-constucts

not model take any complete raph of what has been elaborated it could be viewed and used as a data model

if we hide the intent trails that spanned them

But then you realize a data model is just a limited idea

it supposed to promote stability reuse and agreement

but in fact it goes against the grain of things tht are dynamic emergent autopoietic

delight in alternaitves and make it easy to change 1s' minds

refactor root and branch the more consequential the better

AI and the Coming Deluge of Noise | Frankly 128

no need to move server with loss of network and even data

a clear case of a "better spot" -

the dual of a sweat spot

one that is clearly bellow the threshold of what is required

again it should have said sweet spot

creative dislexia

eLife Assessment

This study presents a useful database resource containing protein conformations generated through molecular dynamics simulations, with extensive quality evaluation and benchmarking. While the database is well-constructed and professionally organized, the evidence supporting its claimed representation of protein conformational landscapes is incomplete, as the short simulation times and starting structure bias prevent true Boltzmann sampling of the conformational space.

Reviewer #1 (Public review):

Summary:

The authors describe a new database that rigorously explores protein conformations.

Strengths:

It is extremely well done, using state-of-the-art tools by a group at the top of the field of structural modeling. The evaluation of qualities and the benchmarking of the structures are outstanding, and it is expected that the new database will have a significant impact on the field.

Weaknesses:

The authors are using MD simulation to generate some of the structure, and therefore should have access to standard MD energies. I am surprised that no evaluation is provided based on these energies that can be extended to free energies.

Reviewer #2 (Public review):

Summary:

The authors developed a dataset of protein conformations by running molecular dynamics simulations starting from both native and decoy conformations for a large number of proteins. These conformations were put together as a dataset for querying and downloading, along with their energies under different force fields. The authors suggest that such conformations represent the proteins' conformational landscape, so that they will be useful for evaluating methods generating multiple conformations of proteins.

Strengths:

The dataset is online and working. It has good documentation for others to use.

Weaknesses:

The biggest weakness is that the collected conformations very likely do not represent the true conformational landscape. To represent the conformational landscape, the structures need to be sampled based on the Boltzmann distribution. However, in this study, conformations are generated by running very short (125ps to 375ps) MD simulations starting from near-native conformations and decoys. Such short simulations will produce small fluctuations around the starting conformations, so the distribution of conformations is largely dominated by the distribution of the initial conformations, which by one means are Boltzmann distributed. A conformation might be physically plausible, but it might have very small weight in the Boltzmann distribution. On the other hand, conformations with large weights might not be in the dataset.

Reviewer #3 (Public review):

Summary:

This manuscript describes a web-based tool that allows researchers to compare large numbers of representative ("plausible") conformations of proteins. It also includes energetic analysis from multiple widely used structure-prediction methods.

Strengths:

This tool will likely be useful for students who want to learn more about the ensemble properties of proteins. The resource is well organized and it represents a large amount of computing resources.

Weaknesses:

It is not entirely clear how the database may be utilized by other groups to advance research. It could be helpful if the authors add a short section that provides example use cases that illustrate how this database can support new strategies for studying protein dynamics.

eLife Assessment

This is an important study uncovering a new role of the SETD6-PPARγ axis in the regulation of hepatic lipid metabolism. The data convincingly demonstrate that methylation of PPARγ by SETD6 plays a key role in this process, linking lysine methylation to transcriptional control of lipid storage genes.

Reviewer #1 (Public review):

Summary:

In this manuscript from the Levy lab, the authors investigate whether SETD6 regulates hepatic lipid accumulation through direct methylation of PPARγ. They show that SETD6 binds and mono-methylates PPARγ at K170, and provide evidence that this modification enhances PPARγ occupancy at target promoters, promotes expression of lipid metabolism genes, as well as facilitates lipid droplet accumulation in HepG2 cells. The authors also find a positive feedback loop or circuit in which PPARγ activates SETD6 transcription in a methylation-dependent manner, thereby reinforcing this lipogenic program. Overall, the work presents a novel SETD6-PPARγ regulatory axis linking lysine methylation to transcriptional control of lipid storage genes, with possible relevance to NAFLD-associated biology.

In all, I find this to be an important paper that describes and advances a new regulatory pathway that has significance to human health and disease. It would also be of interest to a broad audience. That said, there are also some concerns that the authors should address, as outlined below.

Major concerns (pertains to rigor - highest priority)

(1) Overall, the work presented is of high quality, and the data nicely support the conclusions; however, a few panels should be strengthened that have missing controls or information:<br /> a. The co-IP panel in Figure 1B lacks a lane where HA SETD6 is expressed without PPARγ. This control is needed to verify that the SEDT6-HA signal depends on PPARγ.<br /> b. In Figure 1C, the authors should show that the co-IP works in both directions (include IP for PPARγ/blot for SETD6). I am a bit confused also over the labeling with IP on the left and on top of the panel next to the beads label. More importantly, the data would be stronger if the authors took advantage of a deletion line to validate that the co-IP is specific to the presence of both.<br /> c. The same IP labeling issue exists for Figure 3B (label is on the same and on top).<br /> d. Antibody information (e.g., where the pan-methyl Ab comes from and at what dilutions they are used at) is missing.

Nice to have experiments (medium priority - strongly consider)

(2) A missing gap is how K170me1 contributes to DNA binding and gene transcription. One possibility is that methylation enhances the DNA-binding activity of PPARγ. Given that the authors have all of the reagents, it would be possible to perform a gel shift assay (or other approach) with and without SETD6-mediated methylation. Is DNA binding affected/enhanced?

(3) Along these lines, I wonder if there is another possibility: could SETD6-mediated methylation of PPARγ drive SETD6-PPARγ interaction? In other words, in the K170R, is SETD6 still even associated with PPARγ, and this interaction is required for promoter recruitment? Alternatively, would a catalytic dead version of SETD6 fail to associate with PPARγ? Currently, no experiments test the impact of an unmethylatable version of PPARγ or a catalytic dead version of SETD6 on SETD6-PPARγ interaction or SETD6 recruitment to promoters.

Minor concerns (text and figure display)

(4) The text has multiple typos and grammatical errors, and there are some issues with the figure display.

Reviewer #2 (Public review):

Summary:

In this work, the authors investigated the regulation of the transcription factor PPARγ by the post-translational modification lysine methylation. The data demonstrate that the lysine methyltransferase SETD6 targets PPARγ for methylation using biochemical and cell-based assays. Methylation of PPARγ occurs in its DNA binding domain, and the authors demonstrate that loss of methylation limits PPARγ chromatin binding, particularly to lipid storage and metabolism gene promoters. As a physiological output, the authors demonstrate that deletion of SETD6 and loss of PPARγ methylation also disrupt lipid droplet accumulation in hepatocytes. In addition, the authors uncover a positive feedback loop in which SETD6 methylation of PPARγ also regulates its binding to the SETD6 promoter and expression of the gene.

Strengths:

One of the key strengths of this manuscript is the novelty of the findings in terms of identifying a new mode of regulation of PPARγ that modulates its chromatin association in cells and thereby regulates lipid metabolism genes. The authors nicely combine biochemical studies of SETD6 activity with cell-based assays investigating PPARγ and SETD6 function in regulating lipid storage. Data supporting this conclusion is largely convincing, and frequently, multiple assays are used to provide sufficient support to the conclusions. This work therefore expands regulatory modes of PPARγ and identifies a new target for SETD6, an enzyme that targets a number of other transcription factors. Furthermore, the regulatory loop that controls SETD6 expression via PPARγ methylation is likely important for understanding SETD6 function in different cell types that have high levels of lipid accumulation or regulation. The gene expression and lipid accumulation assays are useful for testing the physiological outcome of loss of SETD6 activity or PPARγ methylation directly.

Weaknesses:

The data presented in the manuscript are largely convincing in support of the authors' conclusions; however, there are some errors in the presentation of the figures and some issues in the text that would benefit from editing. Furthermore, there are some important questions not fully addressed in the results or discussion. It would be great if the authors could speculate more on the diverse roles of SETD6 in methylated transcription factors and/or provide more context regarding the conditions that are likely to support methylation of PPARγ by SETD6. Also, while a potential cross-talk between methylation and phosphorylation is described in the discussion, it would be great to provide more structural insight into how this might regulate DNA binding of PPARγ and/or discuss whether there are other possibilities given the location of the target lysine in the DNA binding domain.

eLife Assessment

In this useful manuscript, Yang et al attempt to show that platelet recruitment to the liver via macrophages contributes to APAP-induced liver injury, but there were many areas where the data supporting the conclusions were incomplete. For example, the idea that platelets only affected KC glycolysis, but not the metabolism of other cells, to mediate the phenotype after injury is not adequately supported by the evidence. It is recommended to perform additional experiments to strengthen the conclusions.

Reviewer #1 (Public review):

Summary:

In this manuscript, Yang et al expand on their previous work showing that platelet recruitment to the liver via liver macrophages is important for APAP-induced liver injury. Here, they show that platelets induce a glycolytic switch in liver non-parenchymal cells, including Kupffer cells, and that this is mediated by the protein Aldolase A produced by platelet-derived extracellular vesicles (PEV). They show that targeting Aldolase A may be a valid therapeutic strategy for severe APAP injury.

Strengths:

(1) They nicely showed that platelet effects in APAP are mediated by Aldoa via platelet-derived extracellular vesicles.

(2) Their data show that one of the effects of platelets in APAP liver injury is inducing metabolic switch to the glycolytic pathway, including in KCs.

(3) Their data points to the therapeutic potential of targeting ALDOA in severe APAP liver injury.

Weaknesses:

(1) They have not shown that the platelet-induced glycolytic switch is only in KCs.

(2) They also have not shown that KC's role in APAP injury is primarily mediated by their interaction with platelets and the subsequent glycolytic switch.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors have investigated the role of platelet-derived ALDOA in liver injury induced acetaminophen (APAP) induced acute liver injury. There are some major flaws in data interpretation as described below. While a decrease in liver injury due to platelet depletion and lower injury in platelet-specific ALDOA KO mice seems real, the claims related to EVs and Platelet-KC crosstalk are not well supported.

Strengths:

Core findings are interesting and supported by the data

Weaknesses:

(1) At least two additional timepoints, one at 6 hr and another at 24 hr should be performed in the APAP model to better understand the dynamics of liver injury, especially after platelet depletion.

(2) Interpretation of the experiments in Figure 2 with clodronate is flawed. 2-DG pretreatment and CLDN administration alone both seem to decrease liver injury substantially, so it is not surprising to see very little injury in the 2-DG+CLDN group.

(3) Since both 2-DG and CLDN were administered pre-APAP, it is possible that they may interfere with APAP metabolism. This should be checked by looking at GSH depletion at 30 min post APAP treatment. The same question goes for S2 figure data.

(4) There are no data on specific steps of APAP toxicity, such as GSH depletion, JNK activation, mitochondrial injury, etc., which are all well characterized in any of the studies. Rather, only injury endpoints are measured. It is critical to measure the mechanistic steps. This applies to all studies, but most importantly to the ALDOA-PF-KO mice in Figure 6.

(5) Interpretation of data in Figure 5F is flawed. Since depletion of platelets also decreases liver injury along with the platelets, it can not be deduced that the decrease in ALDOA is only in platelets. Many other things are changing.

Reviewer #3 (Public review):

Summary:

The authors address the possibility that platelet (PLT) derived EVs are important mediators of acute liver injury. Furthermore, KCs are important mediators of inflammation and are noted to need to undergo metabolic reprogramming to achieve their effects during injury. They use an APAP-induced liver injury model (AILI). They show that PLTs are recruited and that they interact with KCs in this model system. RNA-seq of KCs showed upregulation of glycolysis and gluconeogenesis. PLT depletion led to reduced liver injury. RNA-seq of KCs showed downregulation of glycolysis. In vitro co-culture of KCs and pets recapitulated the glycolysis findings. In vivo, 2DG inhibited liver injury, but not in the setting of KC depletion. They went on to show that PLT-derived EVs mediate this effect on KCs using a mix of in vitro and in vivo assays, although control EVs were lacking. After doing mass spec on EVs, they find that ALDOA is the critical payload of the PEVs that mediates the pro-glycolytic effect in vivo. They both delete ALDOA from PLTs, and they use an ALDOA inhibitor to show that injury in AILI requires ALDOA.

Strengths:

This is generally an interesting series of observations with an elegant mechanism. Many of the experiments are done in vivo with highly rigorous KO models. However, in many of the EV experiments, there are concerns about a lack of appropriate controls that might limit the rigor of those aspects of the study. 

Weaknesses:

(1) There is strong variability in the gene expression between mice in Figure 1B. I worry that the signals may not be statistically significant. The authors should assess the statistical significance.

(2) In Figure 2B, the necrosis areas that are circled in the image do not seem to resemble the quantitation on the right. For example, I don't see 60% necrosis in the APAP PBS group. Also, I don't see 5-10% necrosis in the CLDN APAP group. More images that are clearer are needed, and circled necrosis areas should be shown.

(3) In Figure 2D, a higher N should be shown. The number of mice (3) is different from the other experiments, so the exclusion of those mice should be explained.

(4) In general, control EVs from a non-PLT source should be used for all EV-related experiments. EVs derived from AML12 hepatocytes would seem to be a reasonable control for some of the experiments. Otherwise, it is hard to know if this is a general EV effect or one that is specific to PLT-derived EVs. In Figure 3B, EVs from non-PLTs should be used as a control. Since it is possible that all EVs express some level of TSG101 or CD63. In addition, control EVs should be used to test effects on KC metabolism, since the claim is that the effects are specific to PLT-derived EVs. Similarly, Figure 4 needs some kind of EV control that is not from PLTs.

(5) Figure 5B should include an EV control in the blot. Most of the blots need controls from AML12 EVs or from another in vivo source.

(6) It is a little difficult to imagine how enough ALDOA protein could be transmitted from PEVs to influence KC glycolysis on the gene expression level. It is possible that ALDOA is required for PLT-induced activation of KCs, or that EVs from PLTs can induce a metabolic shift in KCs. However, it has not been definitively shown that ALDOA from PEVs is directly causing the KC activation. Ultimately, it would be good to obtain PEVs from ALDOA WT and KO mice, then provide these PEVs to AILI mice without PLTs to see if they have differential effects on the AILI model. This would really demonstrate that the ALDOA in the PEVs is mediating the glycolytic, injurious effect.

In a field dominated by philosophy, the Coldewey article about the 2017 Reddit subreddit bans are a relatively few pieces of empirical evidence. While banning toxic communities may have decreased overall hate speech across platforms (i.e., users did not simply migrate and reestablish at the same levels) this was empirically measured; therefore, this will provide an objective measure that does mitigate the motivated reasoner problem when evaluating moderation actions from a Rawlsian perspective. However, there are limitations to the consequences-based "escape" mechanism - while the Coldewey article quantifies the reduction in hate speech as a result of the ban, it does not determine whether or not those who were banned had their attitudes toward hate speech changed as a result of being banned. That is a far more difficult measurement to make.

This study was conducted by Georgia Institute of Technology. The finding was that because Reddit banned toxic subreddits in 2015, it successfully reduced hate speech on the platform by up to 80 - 90% among users. The article also shows that while many offenders migrated to other communities like Voat and Gab, they didn't significantly increase the amount of hate speech in the subreddits that they joined. Researchers of this study concluded that by making bigotry more difficult to practice, they can effectively discourages it and eventually yield lasting positive results.

The Know Your Meme source explains that copypasta is copied text spread repeatedly by people, not always by bots. I think this matters for moderation because harmful spam is not only automated. Human users can also flood platforms, annoy others, or spread harmful messages, so moderation tools need to consider behavior and context

This article by Elizabeth de Luna summarizes the decline of the social media site known as Tumblr via interviewing a former Tumblr employee. Beginning with the company's acquisition by Yahoo in 2013, the article details how the new (often rotating) management of Tumblr failed to create a sustainable business strategy- instead chasing short-term profitability that alienated Tumblr's users (the "Porn Ban" being a famous example). Because of this, the site also failed to innovate- with many cumbersome outdated features lingering. However, the article ends on a positive note- pointing out that, despite its many issues, Tumblr as a site is still alive as "creative culture platform" with many niche communities- therefore, the interviewee is rather confident that the site can still continue to survive in its own niche.

This article explains how FB's AI systems helped the platform grow but also made the negative actions become harder to control. These negative actions being things like spreading misinformation or hate speech. The AI focuses on how FB's algorithms were designed to maximize engagement by showing users content they were likely to click and share. This means that more extreme content that gets views and clicks get more engagement and end up rewarding these negative actions within posts. The article also argues that the AI team at FB focuses more on the algorithmic bias than fixing the recommendation system that spreads misinformation because it could hurt FB overall.

The First Amendment source made me think about the role of the government, not just social media companies. I learned that free speech protection mainly limits what the government can do. This matters because it raises a harder question: what happens if the government pressures a private platform to remove certain posts? In that case, moderation may no longer be only a company decision. I think this shows why the line between platform rules and government censorship can sometimes become complicated.

This article talks about how Tumblr was mismanaged by the many different acquisitions it went through. Many of the employees didn't feel like their voices were being heard about the user experience and where they thought the platform should go, and many decisions were made by the leadership that was only interested in Tumblr as a business rather than understanding Tumblr in context. It talks about Tumblr's irrelevancy and how it got there through bad leadership.

The Coldewey TechCrunch article on Reddit subreddit bans provides a lot of insight regarding the issues raised in this chapter on moderation tradeoffs. In a study conducted by Georgia Tech, the results showed that banning subreddits on the basis of hate speech actually helped in reducing the occurrence of such activities on Reddit. People who remained after the ban greatly decreased their usage of hate speech while people migrating to other subreddits did not show an increase in such activities. However, what I find to be understated in this chapter is the effectiveness of the policy itself. Banning the hate subreddits may have 'solved' the issue, but the banned users merely migrated to sites such as Voat and Gab, which openly encourage such activities.

I found this really interesting as it does show our own biases. The "don't read the comments" line is interesting because it highlights how big tech exploits the algorithm by ensuring maximium attention (which often means negative content is pushed upwards). This can create a cycle of hate that hurts the original creator. But now "dont read the comments" has been reformed into a more "corporate" thing. These companies are happy to profit, but not actually contribute for meaningful good. I wonder if there will be any future redesigns of social media sites to reduce this spread of hate and fear.

This article basically argues that Facebook can’t really solve its misinformation and hate speech problem without changing its engagement‑first business model. Even when they created a “Responsible AI” team, leadership steered it away from tackling the core recommendation algorithms, so a lot of the work feels like PR rather than actually changing what users see.

I read this article which talked about how Tumblr went from a very large sharing platform to its now more dead site. Tumblr grew in popularity with creative users and fandoms, but once Yahoo bought the site it began to die out. This was due to aggressive moderation and then a later ban on adult content which steered many off the site.

The amount of power these social media sites have with our data is something to ponder about. Facebook had a recent data breach that allowed certain parties to feed content to certain users that could influence them politically. With social media being prevalent now more than ever, this is especially dangerous. I think that these companies should have harsher consequences when these data breaches happen and there should be a government-adjacent but separate entity that ensures the privacy of all social media users.

The section title of “Origins for Moderation” is quiet in its performance of the very problem described, i.e., it illustrates how a series of moderation’s ancestry (Confucius→Aristotle→Kant→Rawls) is also a way of demonstrating which rationality can determine the golden mean. Critics have argued for some time that Rawl's "veil of ignorance" was created to remove from consideration just those forms of self-interested thinking (the wealthy man's "rational" argument against paying taxes) that are identified in the chapter; however, at least as many critics argue that the veil still includes within it certain assumptions regarding what a rational person values. The chapter defines this problem as having been resolved by Rawls; yet, the bigger question remains that moderation-as-a-virtue and moderation-as-content policies suffer from the same fundamental flaw -- each assumes there will exist a mediator/moderator standing apart from all other participants involved in the moderation process.

It's really interesting to see how Reddit operates on a moderation system that relies heavily on its community. The fact that volunteers who manage subreddits are unpaid provokes major debates. I think this is completely reasonable, especially when Reddit is a multi-billion-dolllar company who can do better than exploiting the free mods. Moreover, everyday users on Reddit also act like moderators who help filter content by upvoting good posts and downvoting bad ones. Thanks to this, unpopular comments are hidden. Even though the authority to shut down any group still eventually falls on the hand of Reddit itself, those moderators have contributed immensesly to the development and operation of this app.

I personally don't see too much of a problem with unpaid Reddit moderators. Yes, they're unpaid, but they're also volunteers who "work" whenever they want and the like.

I also honestly don't think it would be a good idea for Reddit to get more involved in the moderation of communities- as that would only further separate regular users from the decision making process of their subreddits (any user can theoretically become a mod).

There are dangers of people using that language as a loophole to spread misinformation, hate, or any type of normally restricted content. If Facebook does not have the ability to properly regulate posts that are written in a language unknown to moderators, then the people who's first language is that are at risk for negative influences.

Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

Kim and Parsons present a timely overview of the NTR/prodrug system and its applications in regenerative biology research, with particular emphasis on tissue-specific cell ablation. The system has substantially advanced the field by enabling non-invasive, conditional cell elimination, and has proven especially powerful in zebrafish, though applications in other classical model organisms are also noted. The review covers the historical origins of the NTR system, its use in regeneration studies, small molecule screening, and genetic and CRISPR-based screening, as well as future directions, including the development of the highly efficient NTR2 enzyme variant.

Strengths:

This is a useful and well-structured contribution. The manuscript is a valuable resource for the regeneration biology community.

Weaknesses:

The impact and scientific value of this paper could be meaningfully enhanced by addressing several points outlined below. The concerns centre on completeness, conceptual precision, and the depth of mechanistic discussion.

(1) Title: Species specificity.

Given that the review's primary focus is the zebrafish model, it would be appropriate to include the species name in the title. This would improve discoverability and accurately set the scope of the article for prospective readers.

Thank you for this suggestion. In revising the review, we have substantially expanded the content to address the reviewers' comments, including adding more detail on the use of NTR in other species. We agree that the majority of published work, and the research we cover, has been conducted in zebrafish, and we have clarified this in the abstract and introduction. However, our aim in writing the review was also to highlight that there is no intrinsic barrier to adopting this technique more broadly in other systems. Notably, NTR was first developed in mice, but with a prodrug that proved difficult to use, and it was not widely pursued. In mouse models, the development of DTR offered an alternative, though that approach carries risks of kidney toxicity and is incompatible with chronic ablation due to immunogenicity. Given this context, we would prefer to retain a title that does not limit the scope exclusively to zebrafish, so as not to discourage readers working in other model systems who might benefit from considering the NTR system.

(2) Subchapter: Physical injury.

The subchapter enumerates different types of physical injury models but would benefit from a more substantive comparative discussion. In particular, the authors are encouraged to address the following:

(2.1) Outcome comparison: Surgical and other invasive approaches cause damage to entire tissue structures comprising multiple cell types, whereas tissue-specific genetic ablation eliminates a defined cell population while leaving the surrounding architecture largely intact. This fundamental distinction has direct implications for the interpretation of regenerative outcomes and should be clearly articulated.

We appreciate the reviewer raising these important points, as well as those noted in Section 2.2. We addressed the concerns from Sections 2.1 and 2.2 throughout multiple parts of our review, specifically in the following sections:

• Physical injury – where we highlight the importance of precisely characterizing the nature and extent of tissue damage in order to appropriately interpret subsequent biological responses.

• Chemogenetic cell-specific ablation – where we expand on this theme by discussing the advantages of selectively eliminating discrete cell populations and how this improves mechanistic interpretation of regeneration.

• Development of NTR as a suicide gene – where we examine apoptotic pathways and their relevance to nitroreductase-mediated cell ablation.

• NTR/prodrug systems in regenerative studies – where we compare what is currently known about immune activation and inflammatory responses across different NTR-based ablation paradigms.

(2.2) Inflammatory response: Invasive injuries typically trigger a robust inflammatory response, which itself can be a potent driver of regeneration. By contrast, genetic cell ablation may elicit a qualitatively different inflammatory reaction. A comparative discussion of this distinction would help readers appreciate a critical limitation of genetic ablation systems relative to models of natural, accidental tissue damage.

Please see above response 2.1

(3) Subchapter: Cell-specific toxins.

This subchapter would benefit from several targeted expansions:

(3.1) Off-target effects: The authors should include evidence that the exemplified drugs have known off-target activities, with a discussion of how these confounded the interpretation of experimental data. At least a few concrete published examples should be cited.

Thank you very much for the comments. We have strengthened the discussion of off-target effects by adding concrete published examples. We now note that MPTP/MPP⁺ can affect noradrenergic and serotonergic systems in addition to dopaminergic neurons, that aminoglycoside antibiotics can damage support cells and afferent neurons at higher concentrations with compound-specific differences in ototoxicity, and that streptozotocin exhibits hepatotoxicity beyond pancreatic β-cells.

(3.2) Completeness of the toxin list: The current list appears illustrative rather than comprehensive. A more complete enumeration would be valuable, particularly for neurotoxins and drugs targeting sensory cells, as these are highly relevant to the zebrafish regeneration field.

We have now consolidated the toxins discussed throughout the review into Table 1, which includes additional entries alongside the previously listed agents. We have explicitly noted that this list is representative rather than exhaustive, as the full range of cell-specific toxins used across species is extensive.

(3.3) Interspecies differences: It would be informative to specify whether drug specificity differs across species, as this is a practical consideration for researchers working in organisms other than zebrafish.

We appreciate the reviewer’s question regarding potential interspecies differences in prodrug performance. Early work using NTR in mammals was conducted in mice, and all five published mouse studies relied exclusively on CB1954. No other NTR-activating prodrugs have been reported in mouse models, so direct comparisons are not available. Likewise, all published Xenopus studies used MTZ and thus do not provide internal comparisons across prodrugs. The Nematostella study employed NFP (citing rationale from a zebrafish study) and the approach yielded effective ablation.

The only non-zebrafish study that directly compared prodrugs is the Drosophila work, which evaluated MTZ, RNZ, and NFP and reported lower activity for MTZ relative to the other compounds. Because it is not clear whether the authors were aware of the batch variability of MTZ or the need for freshly prepared solutions, interpreting this specific comparison is difficult.

To address the reviewer’s comment, we have expanded the section on non-zebrafish organisms to clearly state which prodrug was successfully used in each species. However, given the limited number of studies, the absence of titration experiments, and the lack of standardized conditions across laboratories, we do not feel that the available evidence supports drawing conclusions about interspecies differences in prodrug performance.

Consistent with our original discussion and based on the broader biochemical and empirical data available, we continue to recommend RNZ as the starting point for new experiments.

(4) Subchapter: Optogenetic cell ablation.

The authors note that optogenetic cell ablation has not yet been applied in conventional regeneration studies. It would strengthen this section to include a discussion of the underlying reasons for this gap, whether technical or biological, so that readers can appreciate the barriers and potential for future adoption.

We thank the reviewer for this helpful suggestion. As recommended, we have added a concise, explicitly speculative statement discussing potential technical factors that may explain why optogenetic cell ablation has not yet been widely applied in regeneration studies. Specifically, we note that KillerRed-based ablation requires localized light delivery and ROS generation, making it best suited for discrete, optically accessible cells and less practical for targeting large or deep tissues. We also highlight that the dependence on microscopy-based illumination inherently limits throughput. This new text clarifies possible barriers to broader adoption while acknowledging that these points remain speculative.

(5) Terminology: "Suicide gene".

The use of the term "suicide gene" to nitroreductase is conceptually imprecise and merits reconsideration. Strictly speaking, a suicide gene is one whose expression alone is sufficient to kill the cell, as in the case of genes encoding direct triggers of apoptosis or the catalytic A subunit of diphtheria toxin (DTA). NTR does not meet this criterion: it requires the exogenous administration of a prodrug (e.g., metronidazole) to produce a cytotoxic metabolite and is therefore only conditionally lethal.

It is worth noting that nitroreductases evolved in bacteria and fungi as enzymes involved in chemoprotection and detoxification, converting potentially toxic and mutagenic nitroaromatic compounds into less harmful metabolites (PMID: 18355273). This biological context further underscores that NTR is not inherently a lethal protein. The authors are encouraged to replace or qualify the term "suicide gene" and instead adopt terminology that more accurately reflects the conditional, prodrug-dependent nature of the system.

We appreciate the reviewer’s thoughtful attention to terminology. We agree that, in its most classical and stringent sense, a suicide gene is one whose expression alone is sufficient to induce cell death. We also recognize that NTR does not meet this strict criterion.

At the same time, we note that the term has broadened in contemporary usage, particularly within applied and translational contexts, to encompass prodrug-dependent systems. For example, the National Cancer Institute Thesaurus defines a suicide gene as “a gene which will cause a cell to kill itself, typically through interaction with a prodrug,” and Taber’s Medical Dictionary likewise states that it is “a gene that causes a cell to kill itself, usually by encoding an enzyme that converts a nontoxic prodrug into a toxic metabolite.” Under these widely used definitions, NTR is included within the scope of suicide gene systems.

Nevertheless, we appreciate that terminology in this area is not universally standardized. To ensure clarity for all readers, we have added a brief definition in the revised manuscript explicitly noting the conditional, prodrug-dependent nature of NTR-mediated ablation. We are grateful to the reviewer for prompting this clarification.

(6) NTR/MTZ in regenerative studies: Mechanistic depth.

While the review catalogues several studies employing the NTR/MTZ system, it lacks mechanistic depth regarding the cellular basis of ablation. The following questions should be addressed, where evidence exists in the literature:

(6.1) Temporal dynamics of cell death: What is known about the kinetics of NTR/MTZ induced lethality across different tissue types in larval and adult zebrafish, as well as other organisms? Are there age- and tissue-specific differences in the speed or completeness of ablation?

Thank you for this important question. We have added text noting that the kinetics and completeness of NTR/prodrug-mediated ablation vary across experimental contexts, including with differences in NTR expression, enzyme/prodrug pairing, dose, cell type, and developmental stage. Published studies illustrate that the time course of ablation can differ substantially between models. Because most studies were designed to optimize ablation within individual tissues rather than for direct side-by-side comparison, the literature does not yet support broad quantitative conclusions about age- or tissue-specific differences across systems.

(6.2) Mechanism of cell death: What is the cellular basis of NTR/MTZ-induced cytotoxicity in zebrafish? In particular, do the toxic metabolites preferentially cause mitochondrial damage or nuclear DNA damage, and what downstream death pathways are engaged?

Thank you for the comments. We have added text discussing the mechanism of NTR/MTZ-induced cell death. We now note that NTR-mediated reduction of MTZ generates reactive intermediates that cause DNA damage and oxidative stress, with cell death occurring predominantly through apoptosis. We have also more strongly emphasized that in dopaminergic neurons, mitochondrial damage was identified as the primary cytotoxic mechanism. We acknowledge that the relative contribution of these pathways is likely to vary by cell type and remains an important area for future study.

(6.3) Proliferative versus post-mitotic cells: Are proliferating and non-proliferating cells equally sensitive to the NTR/MTZ system, or does the proliferative status of a cell influence susceptibility? This is a practically important question for researchers designing ablation experiments in tissues with mixed cell populations.

We appreciate the reviewer’s insightful question. We have now added a brief clarification to this section explaining that the NTR/MTZ system has been shown to act in a cell-cycle–independent manner, and both proliferating and post-mitotic cells can be ablated effectively.

(6.4) Ablation of progenitor cells: Are there published examples demonstrating that co-ablation of differentiated functional cells and organ-specific progenitor cells abolishes regenerative capacity? Such examples would be highly informative in illustrating the system's power to dissect the cellular requirements for regeneration.

To our knowledge, the zebrafish lateral line currently provides the clearest example in which NTR-mediated ablation of progenitor populations results in a loss of regenerative capacity. In this system, targeted ablation of support-cell progenitors severely reduces hair-cell regeneration, illustrating how NTR enables direct testing of cellular requirements for tissue repair.

Addressing the points above, particularly the comparative discussion of injury models and inflammatory responses, the clarification of terminology, and the mechanistic discussion of NTR/MTZ-induced cell death would substantially strengthen the review's scientific contribution and utility.

Reviewer #2 (Public review):

Summary:

Kim and Parsons reviewed the nitroreductase (NTR)/prodrug system: when engineered cells expressing the enzyme NTR are treated with prodrug (e.g. metronidazole), NTR converts the prodrug into a cytotoxic compound that kills these cells. The review covers how the system has been developed, spatiotemporal control of targeted cell ablation, and its broad utility to study regenerative mechanisms, model human diseases, and screen chemicals to discover pro-regenerative and protective compounds. They further discussed the newer version of NTR, a more potent prodrug, and experimental design, which not only expands the possible utility of the NTR/prodrug system, but also allows the research community to develop a precise, reproducible and versatile platform.

Strengths:

The review summarized landmark work application of the NTR/prodrug system, and recent studies, with focus on the model organism zebrafish. The review provides a good gateway to understanding the system and considering regenerative studies.

Weaknesses:

No weaknesses were identified by this reviewer.

Reviewer #3 (Public review):

Summary:

This manuscript by Kim and Parsons presents an overview of the nitroreductase/metronidazole (NTR/MTZ) cell ablation system.

Strengths:

This manuscript nicely places the NTR/MTZ system in the context of other cell ablation methods, with a discussion of their respective advantages and disadvantages. This review is particularly useful for highlighting the many ways the NTR/MTZ system has been applied to study the regeneration of multiple cell types and to model different degenerative human diseases. The review concludes with a discussion on recent improvements made to the system and practical considerations and "best practices" for NTR-based experiments. This review could be a helpful resource, especially for researchers new to regeneration or cell ablation studies.

Weaknesses:

Although the NTR/MTZ system has been used in other model organisms, this review is primarily focused on its uses in zebrafish. While this is understandable given the wide adoption of NTR/MTZ in the zebrafish field, discussion of the unique considerations and/or challenges for non-zebrafish systems would be an interesting addition and could broaden the potential audience for this review. Additional minor revisions, as suggested below, could also improve readability.

Recommendations for the authors:

Reviewer #2 (Recommendations for the authors):

Since the lab mouse is an important mammalian model system, with certain tissues harbouring some regenerative capabilities, including the peripheral nervous system (e.g., sciatic nerve regeneration after crush), and myelin, etc., it would be great if a section could be included to discuss the potential adoption of the NTR/prodrug system in future mouse studies.

We appreciate the reviewer’s suggestion to discuss the potential future use of the NTR/prodrug system in mouse models. In surveying the literature, we identified only five mouse studies employing NTR, all of which used CB1954. These early studies were conducted primarily as proof-of-principle work in the context of gene-directed enzyme prodrug therapy (GDEPT) for cancer, rather than for regenerative or lineage-specific ablation applications. We added this point to the text.

Since those reports, we have not found additional examples of NTR use in mice. We do not know the precise reasons for this limited adoption, but it may reflect the availability of alternative ablation systems that are widely established in mouse research, such as the diphtheria toxin receptor (DTR) system.

We agree that certain mouse tissues exhibit regenerative capacity and that targeted ablation tools can be valuable in such contexts. To address the reviewer’s point, we have added text noting the very limited historical use of NTR/CB1954 in mouse. We have no explanation as to why no one moved onto using NTR/MTZ in the mouse but note in two places in the text that DTR is preferred method to use in mouse ablation experiments (even though DT does cause kidney damage and is incompatible with chronic studies!).

Minor:

(1) Line 174-176, the sentence was repeated.

(2) Figure 1, for the transgenic line, please be consistent with the line name in italics.

Reviewer #3 (Recommendations for the authors):

(1) In the abstract as well as in the main text, the authors note that the NTR/MTZ system has been used in multiple model systems. Yet, most of the review, and especially the practical advice given at the end, is very zebrafish-focused. Although this is understandable given the wide adoption of NTR/MTZ in the zebrafish field, the authors might consider revising the abstract to make it clearer that this review is primarily concerned with the use of the NTR/MTZ system in zebrafish.

Thanks for the suggestion. We have changed last half of first paragraph in abstract

That said, a brief discussion of any unique considerations and/or challenges for non-zebrafish systems would be an interesting addition and could broaden the potential audience for this review.

Agreed and we have expanded in several places in the text to discuss more about the NTR system in non-zebrafish. We especially expanded our discussion about NTR in the mouse.

(2) Line 176: There is a repetition of the sentence, "NTR/MTZ-mediated ablation has also been adapted for other model organisms."

Found and deleted. Thank you!

(3) Line 177: To improve clarity, the authors should include species names to prevent confusion. For example, both Xenopus laevis and Xenopus tropicalis are commonly used model organisms. Similarly, multiple Drosophila species are used by researchers.

Added melanogaster and laevis to text.

(4) Can the authors address whether alternatives to MTZ (RNZ, etc.) have the same issues with batch-to-batch variability? That might be an important consideration for potential users. It would also be useful to include practical guidance for accounting for batch variability, for example, how to determine optimal prodrug concentrations, whether effective concentrations need to be determined for every batch/replicate/experiment, etc.

Added text that discusses that, it is not yet known whether RNZ exhibits batch-to-batch variability similar to MTZ, as this has not been systematically reported. Given the potential for variability, it would be prudent for researchers to titrate each new batch of RNZ or, alternatively, adopt a dosing strategy that exceeds the minimum effective concentration to ensure consistent ablation results.

(5) For the last section ("Experimental design: Practical and technical considerations"), readability would be improved by applying a consistent bullet point format.

Made the changes as requested.

(6) Figure 1: Asterisks are not defined.

The asterisks where to link to two boxes depicting the same transgene without rewriting the name of the transgene. Clearly, this wasn’t clear, so we have added explanation to legend too.

(7) Figure 3: Given that the schematics specify expression of NTR1 and NTR1.1, I assume this figure is adapted or based on previous published report(s). If so, the reference(s) should be noted in the figure legend or on the figure itself (as done for Figure 1). If the schematic is meant to depict only in general terms how binary expression vectors can be used, a more inclusive "NTR" label might be less confusing.

Changed figure legend and figure

(8) Figure 4: To improve readability and accessibility, the authors should consider modifying panels C-N to use a more colorblind-friendly palette (e.g., green/magenta) or to present each channel as separate grayscale images.

eLife Assessment

This Review Article nicely synthesizes the development, applications, and recent technical advances of the nitroreductase/prodrug system, highlighting how it enables precise spatiotemporal cell ablation and experimental platforms for studying regenerative mechanisms and screening for pro-regenerative or protective compounds. Together, the article provides a conceptual and practical overview that will help researchers adopt and further develop this versatile approach in regenerative biology. It will be of interest to researchers studying regeneration, disease modelling, and targeted cell ablation, particularly those working with zebrafish and other genetic model systems.

Reviewer #1 (Public review):

Summary:

Kim and Parsons present a timely overview of the NTR/prodrug system and its applications in regenerative biology research, with particular emphasis on tissue-specific cell ablation. The system has substantially advanced the field by enabling non-invasive, conditional cell elimination, and has proven especially powerful in zebrafish, though applications in other classical model organisms are also noted. The review covers the historical origins of the NTR system, its use in regeneration studies, small-molecule screening, and genetic and CRISPR-based screening, as well as future directions including the development of the highly efficient NTR2 enzyme variant.

Strengths:

This is a useful and well-structured contribution. The manuscript is a valuable resource for the regeneration biology community.

Weaknesses:

The revised manuscript shows significant improvements; however, two points remain insufficiently addressed and should be resolved in the final version.

(1) The term 'suicide gene'

As noted in my first round of revisions, the term 'suicide gene' as applied to bacterial nitroreductase remains unaddressed in the revised manuscript, despite being scientifically inappropriate and a potential source of confusion regarding the NTR/Mtz mechanism.

'Suicide' implies an intrinsic, cell-autonomous programme of self-destruction. This is incompatible with the NTR/Mtz system, in which cell death is experimentally induced through exogenous administration of metronidazole (Mtz) by the investigator. While the 'suicide gene' framing may have utility in the cancer therapy literature, likely to aid communication with non-specialist and clinical audiences, however, it is not standard in the zebrafish field, where NTR is more accurately described as a conditional toxigene. Since this review focuses predominantly on zebrafish models, its terminology should reflect that of the relevant literature.

A further conceptual problem with the 'suicide gene' framing is that it obscures the pharmacological nature of Metronidazole. Mtz is a pharmaceutical agent with intrinsic baseline toxicity: extended exposure or modestly elevated concentrations cause toxic side effects and lethality even in non-transgenic (wild-type) zebrafish (PMID: 24428354). NTR-expressing cells do not self-destruct; rather, they are rendered selectively hypersensitive to Mtz relative to other eukaryotic cells by virtue of expressing the enzyme. This distinction is mechanistically important and should be reflected in the language used throughout the manuscript.

In summary, the term 'suicide gene' does not accurately capture enzyme-mediated bioactivation of an exogenous prodrug and should be removed from the manuscript.

(2) Barriers to using the NTR/Mtz system in non-aquatic model organisms

In response to my suggestion that the title should include "zebrafish" to accurately convey the scope of the review to prospective readers, the authors stated that "there is no intrinsic barrier to adopting this technique more broadly in other systems," citing the example that "NTR was first developed in mice, but with a prodrug that proved difficult to use, and it was not widely pursued." These two statements are, however, contradictory: if the prodrug proved difficult to use, this constitutes precisely the kind of practical barrier the authors claim does not exist. The authors should clarify and reconcile this inconsistency, and provide a more thorough discussion of why the NTR/Mtz system has seen limited adoption in classical model organisms, such as mice and Drosophila.

Reviewer #2 (Public review):

Summary:

Kim and Parsons reviewed the nitroreductase (NTR)/prodrug system: when engineered cells expressing the enzyme NTR are treated with prodrug (e.g. metronidazole), NTR converts the prodrug into cytotoxic compound which kill these cells. The review covers how the system has been developed, spatiotemporal control of targeted cell ablation, and its broad utility to study regenerative mechanisms, model human diseases, and screen chemicals to discover pro-regenerative and protective compounds. They further discussed the newer version of NTR, more potent prodrug, and experimental design, which not only expand the possible utility of the NTR/prodrug system, but allow the research community to develop a precise, reproducible and versatile platform.

Strengths:

The review summarized landmark work application of the NTR/prodrug system, and recent studies in model organisms, with focus on the model organism zebrafish. The review provides a good gateway to understanding the system and considering regenerative studies.

Weaknesses:

None.

Comments on revisions:

The authors have addressed the previous points, and the manuscript has been greatly improved.

Reviewer #3 (Public review):

Summary:

This manuscript by Kim and Parsons presents an overview of the nitroreductase/metronidazole (NTR/MTZ) cell ablation system.

Strengths:

This manuscript nicely places the NTR/MTZ system in context of other cell ablation methods, with a discussion of their respective advantages and disadvantages. This review is particularly useful for highlighting the many ways the NTR/MTZ system has been applied to study regeneration of multiple cell types and to model different degenerative human diseases. The review concludes with a discussion on recent improvements made to the system and practical considerations and "best practices" for NTR-based experiments. This review could be a helpful resource, especially for researchers new to regeneration or cell ablation studies.

Comments on revised version:

I thank the reviewers for revising the manuscript to expand their discussion of using the prodrug/NTR system in other model organisms while also revising the abstract to make it clear this review will be zebrafish focused. With these revisions, this review provides an informative overview of how the prodrug/NTR system has not only been an important tool for regeneration studies and but also for elevating the zebrafish as a regeneration model. That said, including other model organisms could have been a nice addition to the last section on experimental considerations, especially in the context of discussing potential barriers to wider adoption of the NTR system. However, given that the vast majority of studies using the NTR system are in zebrafish, the current scope of this review is understandable.

The paper is now published and available online. Below is the link: https://pubs.acs.org/doi/10.1021/jacsau.6c00003

DOI: 10.1016/j.nlm.2026.108167

Resource: Waxholm Space Atlas of the Sprague Dawley Rat Brain (RRID:SCR_017124)

Curator: @scibot

SciCrunch record: RRID:SCR_017124

What is this?

The Future of Text 2017

Session 2.

session 2

map the expanding frontier

doing he job

our life

perusia

kingdom of the spirit

find something not pairwise

Add the site vectorisation index

I think I have participated in crowd sourcing when liking and engaging with TikTok's that go towards something important. A lot of times the video will include a caption saying, "Stay for 20 seconds so I can make enough money to pay for my dog's surgery". And although I don't know if I fully believe, a lot of other people will stay as well.

I disagree with this. I think it should be done at the beginning with the rest of the CPT

concerns of this if everyone just wants to join execution team. i think you dont need to inform of the new system - you should join being prepared to do service, whether its planning or execution

agreed - it should run with when the sponsors open up their applications

concern for general members recruited later - how to make sure people don't join for the sake of just carrying other people's hard work out? - this planning & execution team distinction should not be made apparent when recruiting

Could this also indicate something else is affecting the rat's behavior? Especially since they were deprived, it would be unusual for them to not drink more than that

Measured response via preference ratio and access ratio

Important to avoid as much bias as possible within the experimental set up

Water deprivation increased motivation to drink during the two-bottle test

Exposure to taste solutions (umami or bitter) began at 4 weeks of age immediately after weaning

Purpose of the study to provide more insights into taste preferences and how this correlates to neuronal activation

Interesting how each of the five taste qualities has a distinct signaling pathway, such so that impairing one would likely not implicate the other taste qualities

Shows gap in research in specifically understanding the mechanisms behind the preferences exhibited

Humans and rats show comparable behaviors in response to exposure to either sweet/umami or bitter/sour

This chapter's section discussing the idea of "quality control" was really interesting because it shows that content moderation isn't only about removing harmful content, but also about shaping what kind of space a platform wants to be. Even with platforms that claim to support free speech also moderate spam because it could provide a platform that would be unusable for normal users trying to find variety of content. This connects to the bigger idea that moderation is never really neutral, there's always some bias. Every rule that a platform implements should be upheld and reflect the companies values that are reflected in the platform, like providing a safe space or legal protection. There should always been transparency between platform and users about the real reasons why they moderate certain types of content.

The fact that 'quality' is dependent on the target audience of the platform is a perspective I need to take into consideration when thinking about moderation in the future. Before reading the article, I assumed that content moderation was an absolute necessity for all platforms since certain content could be classified as 'bad'. However, the 4chan example showed that content moderation may also involve a very different form of moderation based on a new definition of quality. This brings up the question of whether content moderation is truly ethical or simply another aspect of doing business with one's own target market demographic.

The later preferences (past infancy) of the participants of this specific study was not explored

Most important finding from study. Longer exposure (3-8 months) to ePHF led to more savory brother being eaten and at a quicker rate, supporting hypothesis

Length of time consuming ePHF (1, 3, or 8 months) was the experimental variable

Table 1 shows no significant differences in the characteristics of infants and mothers measured, indicating no glaring confounding variables or other patterns

This information is definitely relevant to childrearing and advising parents on nutrition and introduction of varied flavors being beneficial

While there does seem to be a significant effect of exposure (and duration of exposure), this study so far does not fully establish a critical period for when an infant is most sensitive to the exposure and/or at what point is no longer sensitive to the exposure

This is the major implication that study draws from its results

Really interesting that their tastes can be influenced by flavor in the milk and not just directly from food they consume and allows for exposure to more kinds of food to occur before solid food-eating begins

This might have to do with adapting to the environment the mother is raising the child is and adjusting preferences based on what food will be available in that specific environment is beneficial to survival

Minimum length of time of exposure to see this specific effect on preference months later is between 1 and 3 months based on the experimental groups

This is the main finding from the study: longer exposure to ePHF increased acceptance of savory broth later

Based on Figure 2, they did differ just not statistically significantly

Noticed in this figure that the group that was 8 months on ePHF started at 2 weeks of age compared to the other groups that started at 6 weeks. Wonder why this was done, especially since it could impact the length of a potential critical period for developing acceptance of flavor

Dependent variable was response to broth, including total intake of broth, rate of eating broth, and parental rating of enjoyment

Analyzed data using ANOVA and Bonferroni corrections to understand significance of results

Interesting to get parent perspective on measuring infant enjoyment, less objective but helpful in understanding typical behavior in infant (i.e. infant might not typically eat a lot but eats more in experiment, even if it's lower than average)

Enjoyment of broth was experimentally associated with greater amount of broth consumed by the infant

All groups were measured for response at 8.5 months regardless of length of ePHF exposure, tested twice to have separate exposure to plain vs savory broth

Specific behaviors led to ending of feeding, rejection limit seems arbitrary but also reasonable

Explains why there is a gap in research and less attention placed on taste in comparison to other sensory systems

General research question and goal of study to identify a sensitive (critical) period for savory taste

I think auto-detect moderation can be useful, but it also makes me worry about mistakes. A computer program may flag a post without fully understanding the context, humor, or meaning behind it. This could cause normal posts to be removed or reviewed unfairly. Because of this, I think platforms should use automatic detection as a first step, but humans should still check important cases before making serious decisions.

Awareness, Trust, and Value are the words that trigger much thought for me. Articles are easily created on a variety for subjects; anyone using charismatic words could pull an audience or incite either of these words. From experience I have learned that no matter the source, to question what I am reading, verify sources, and not immediately presume that all stated in the article. It is then anybody's responsibility to ensure proper references and cites are mentioned, to provide credibility and support to our created content.

test

This is showing how Janie feels overwhelmed by the weight of memories and judgement once she has returned home.

She truly loved tea cake and because the sickness made him aggressive she had to shoot him and she deeply regretted it but she had to do it

The hurricane winds were strong enough to blow some peoples clothes off (or they were already naked before)

Janie wished that she died so she wouldn’t have to shoot Tea Cake, she knew he loved her and she didn’t want him to die like that.

Tea Cake becomes sick from rabies after the dog bite. He grows paranoid and dangerous as the disease worsens. When he tries to shoot Janie, she is forced to shoot him first in self-defense. Tea Cake dies, and Janie is heartbroken but knows she acted to save herself.

Tea cake is crazy from the dog bite so Janie had no choice but to protect herself

I’m especially interested in this project because I’ve previously worked with water samples in other biology labs, so I think it will be interesting to compare how studying soil microbes differs from working with microorganisms found in water environments. I’m curious to see whether the techniques and results will be similar or completely different.

I like that the specifications grading system allows revisions and retakes because it gives students opportunities to improve instead of being stuck with one bad grade.

Here, Martha pushes back against the anxiety of authorship by refusing to be "crippled by the debilitating alternatives her culture offers her," (17). Despite Martha's outward silence, she internally rejects God's notion of what her life should have been, instead of succumbing to it.

The comment of Martha's height seems extremely intentional to me, and calls to mind the racialized misogyny experienced by black women, a topic with which likely Martha, and Butler, have unfortunate experience. In particular, the masculinization of black women is a prevailing issue, one in which characteristics like height are used to demean and disrespect a Black woman's identity and womanhood. While it is unclear how Martha was affected by this racialized violence, it seems intentional on Butler's part to mention this, and as Gilbert and Gubar say, "Learning to become a beautiful object, the girl learns anxiety about— perhaps even loathing of— her own flesh." (15) This is one of the many ways in which the sickness of misogyny in the literary tradition may manifest and contribute to the anxiety of authorship, and may well have contributed to Martha.

Gilbert and Gubar recall Satan when speaking of Bloom, arguing that he sees Milton's "fiercely masculine fallen Satan as the type of the poet in our culture, and he metaphorically defines the poetic process as a sexual encounter between a male poet and his female muse." (10). With such a quote as a framework, Martha's elective comparison makes sense; here, God, as progenitor and ultimate creator is representative of "the" poet, and encounters Martha through a sexual lens (sexual in the sense of pertaining to biological sex, not sexuality); God's gender, or perhaps lack thereof, as well as his position as head of the patriarchal standard and probable omnipotence, creates conflict with Martha.

Yet another expression of Martha's anxiety of authorship; as Gilbert and Gubar say, "This anxiety [of authorship] is, of course, exacerbated by her fear that not only can she not fight a male precursor on “his” terms and win, she cannot “beget” art upon the (female) body of the muse." (11-12). In this moment, Martha embodies this first anxiety; to fight God on "his" terms would be untenable, and thus, her willingness to oppose him outright, by making her own, utterly independent ideas, is diminished. Regardless of whether God would retaliate against her, she fears the inability to withstand an onslaught predicated upon her desire for independent thought and creativity.

I would argue that this is one of the necessary, but unfortunate, growing pains in the process of overcoming the "anxiety of authorship." In a real, literary sense, being a trailblazer of female literary tradition would be a disheartening, isolating experience, and, in its own way, an "infection in the sentence" as Emily Dickinson said (13). Martha faces not only a gender-related anxiety here - and gendered it certainly is, because God, despite later appearing to Martha as a woman, evidently exists beyond such bounds - but a species one, too, as she is - as far as she has been told - the first human to be given this responsibility.

In this moment, Martha works towards the destruction of the anxiety of authorship that plagues her; by choosing to see God as female, she rejects the influence of her forefathers, searching instead to "legitimize her own rebellious endeavors," (12) through the use of a female muse or sounding-board.

everyone has “social views”that they feel this way about, and everyone has “social views” that they think should be up for pleasant debate over a cold seltzer. where folks disagree is what goes on what point on the line between, and it is tedious to get cute about conflating lesser and greater evils

this is aesthetics

this is interesting. the logic is solid. but I am not sure that it’s good to want those people to trust you. or, rather: fine to want them to trust you, but not good to take actions informed by those people’s preferences? like if the Human Biodiversity Crowd is shaping my comms, something’s gone wrong. (point about condescension would itself be enough, ofc)

Chairman of the State Council of East Germany

1 day after the Fall of the Berlin Wall

I think with the development of AI, moderation could be automated and not require people at all. Traumatizing jobs like moderation could then be done with AI, and people wouldn't have to do those jobs anymore. I think it then poses an issue that some things could get past automated or AI moderation, and it wouldn't be as good at catching everything, but I think if the AI is well trained enough it wouldn't be a problem.

[hope]

🥲

This article is about how social media filters and photo editing apps can negatively affect people's body image, and it called "Snapchat dysmorphia". People tried to focus on their appearance on social media before they post it like using filter and editing their body perfectly. It explains Snapchat and Instagram can create unrealistic beauty standards, making they feel insecure about their natural looks. Also it concerns from psychologists and medical professionals about the mental health effects of comparing real appearances to unrealistic online images.

A part of this article that really stuck out to me was simply how it explains what the spectrum that is referred to when people say "autism is a spectrum" actually looks like. Despite having some autistic family members, I was under the impression that the meaning was simply referring to the gradient that the article mentions and that it simply wasn't based on facts. Because of this, I believe that this labeled spectrum is extremely helpful to understand what autism actually is and should be taught to the wider public.

The good side is that I can stay updated with the news every day and learn useful tips from other people for daily life, work, and studying. I can also stay connected with my family and friends when they are far away by messaging, calling, and seeing their posts. The bad side is that social media can make us victims of cyberbullying at any time, even because of a small mistake, such as leaving a comment that others disagree with or posting a photo and then being body-shamed and this still happens quite often.

I have found that social media has distinctive positive and negative effects. The major positive for me is that it allows me to stay connected and in-touch with other people. I am personally more introverted, so being able to communicate on a social media platform is much easier. I have found many negatives with social media, such as feeling negative emotions from comments and getting sucked into social media algorithms.

this is a strong claim ("most under-modeled") -- what's it based on? Reasoning transparency please. Provide support and links to this, tooltips etc. I want to make sure this is well-backed before I post and ~"co-sign" it !

wait -- are you sharing the beliefs here? we didn't wnt to do that yet!

skip/remove this -- no one showed up

you keep pushing this -- it's ok to mention but the way it's written it seems like you are calling them out for hypocrisy; too harsh, and there can be other explanations

I wonder if the hired moderators are still a thing in this day and age, with the rise of AI. Perhaps maybe a combinition of the two would be best for cost and effectiveness. The people mods have their own bias's towards posts, so although they are likely trained, there still will be that unconscious bias. I guess however though, there are also bias's in the data sets that AI are trained off of. But yeah, overall this was making me thinking about the benefits and drawbacks of using hired moderators versus bots.

I’ve seen people argue that moderating or banning hate just “drives it underground” and doesn’t actually solve anything, so this result surprised me. It lines up more with my experience that when a site actually enforces rules, it does somewhat work. It makes me more skeptical of platforms that claim they “can’t” moderate because it won’t work; this suggests it can work if they’re willing to take the hit.

We know that, "uptake of atmospheric CO2 should react not to a change in emissions, but to a change in concentrations" (Knorr 2009). So if our current anthropogenic emissions of 4.9 ±0.5 ppmv/yr of fossil CO2 were to suddenly cease, the CO2 trend would be reduced by that same amount: 4.9 ±0.5 ppmv/yr. That means it would be negative instead of positive: initially averaging about -2.4 ±0.6 ppmv/year instead of the current average of +2.5 ±0.1 ppmv/year.

In other words, were it not for ongoing anthropogenic CO2 emissions the atmospheric CO2 concentration would currently be falling, rather than rising.

That obviously means that the current upward trend in atmospheric CO2 concentration is entirely due to anthropogenic emissions.

● Mankind is ADDING CO2 to the atmosphere

● Nature (the net sum of all non-anthropogenic carbon fluxes) is REMOVING CO2 from the atmosphere

● The atmospheric CO2 level is currently rising because mankind is currently adding CO2 faster than nature is removing it.

The TOA radiative imbalance does not affect CO2 fluxes, directly. It only does so indirectly, by affecting temperatures. We know from ice core data that both over glaciation cycles and over shorter climate cycles such as the RWP 🡖 DACP 🡕 MWP 🡖 LIA, 1°C of average temperature change could only affect atmospheric CO2 levels by at most 10-15 ppmv (and even that was only after long equilibration periods).

But the atmospheric CO2 level has risen by 112 ppmv just since 1958 (when precise measurements began).

Obviously we haven't had 112/15 = 7.5°C of warming since 1958 (and we also haven't had hundreds of years to equilibrate).

What's more, CO2 levels continued to rise (at an accelerating pace!) during the 1950s through 1970s, as temperatures FELL. So obviously the rise in CO2 levels wasn't caused by rising temperatures. (It was caused by the post-WWII boom in industrial production.)

What's more, we know that both the oceans and the soils (as well as the terrestrial biosphere) are currently net removers of CO2 from the air, not net producers of it.

The sole reason for the ongoing increase in CO2 level is that human CO2 emissions currently exceed the net rate of natural CO2 removals.

The so-called "airborne fraction" (AF) doesn't represent anything physical. It is just a computed ratio between the observed rate of increase in the amount of CO2 in the atmosphere, and the rate at which human CO2 emissions add CO2 to the atmosphere.

The AF is currently about 50%, meaning that nature is removing CO2 at about half the rate at which humans are adding it. So if we were to suddenly halve human CO2 emissions, the AF would be zero. If we were to more than halve human CO2 emissions the AF would be negative, and the atmospheric CO2 concentration would be decreasing, rather than increasing.

That does mean that "net zero" is unscientific. But it does not affect the conclusion that the sole reason that the atmospheric CO2 concentration is increasing is that the rate of anthropogenic CO2 emissions exceeds the rate of natural CO2 removals.

The "bomb spike" e-folding lifetime is a little over 20 years. When it is mistakenly reported as 16-17 years, it's usually due to failure to account for the Suess Effect. When 14C-depleted fossil carbon is added to the atmosphere, that reduces Δ14C without reducing the total amount of carbon in the atmosphere. That's explained in (Burton 2024).

We know that, "uptake of atmospheric CO2 should react not to a change in emissions, but to a change in concentrations" (Knorr 2009). So if our current anthropogenic emissions of 4.9 ±0.5 ppmv/yr of fossil CO2 were to suddenly cease, the CO2 trend would be reduced by that same amount: 4.9 ±0.5 ppmv/yr. That means it would be negative instead of positive: initially averaging about -2.4 ±0.6 ppmv/year instead of the current average of +2.5 ±0.1 ppmv/year.

In other words, were it not for ongoing anthropogenic CO2 emissions the atmospheric CO2 concentration would currently be falling, rather than rising.

That obviously means that the current upward trend in atmospheric CO2 concentration is entirely due to anthropogenic emissions.

● Mankind is ADDING CO2 to the atmosphere

● Nature (the net sum of all non-anthropogenic carbon fluxes) is REMOVING CO2 from the atmosphere

● The atmospheric CO2 level is currently rising because mankind is currently adding CO2 faster than nature is removing it.

The TOA radiative imbalance does not affect CO2 fluxes, directly. It only does so indirectly, by affecting temperatures. We know from ice core data that both over glaciation cycles and over shorter climate cycles such as the RWP 🡖 DACP 🡕 MWP 🡖 LIA, 1°C of average temperature change could only affect atmospheric CO2 levels by at most 10-15 ppmv (and even that was only after long equilibration periods).

But the atmospheric CO2 level has risen by 112 ppmv just since 1958 (when precise measurements began).

Obviously we haven't had 112/15 = 7.5°C of warming since 1958 (and we also haven't had hundreds of years to equilibrate).

What's more, CO2 levels continued to rise (at an accelerating pace!) during the 1950s through 1970s, as temperatures FELL. So obviously the rise in CO2 levels wasn't caused by rising temperatures. (It was caused by the post-WWII boom in industrial production.)

What's more, we know that both the oceans and the soils are currently net removers of CO2 from the air, not net producers of it.

The reason for the ongoing increase in CO2 level is that human CO2 emissions currently exceed the net rate of natural CO2 removals.

The so-called "airborne fraction" (AF) doesn't represent anything physical. It is just a computed ratio between the observed rate of increase in the amount of CO2 in the atmosphere, and the rate at which human CO2 emissions add CO2 to the atmosphere.

The AF is currently about 50%, meaning that nature is removing CO2 at about half the rate at which humans are adding it. So if we were to suddenly halve human CO2 emissions, the AF would be zero. If we were to more than halve human CO2 emissions the AF would be negative, and the atmospheric CO2 concentration would be decreasing, rather than increasing.

That does mean that "net zero" is unscientific. But it does not affect the conclusion that the sole reason that the atmospheric CO2 concentration is increasing is that the rate of anthropogenic CO2 emissions exceeds the rate of natural CO2 removals.

The "bomb spike" e-folding lifetime is a little over 20 years. When it is mistakenly reported as 16-17 years, it's usually due to failure to account for the Suess Effect. When 14C-depleted fossil carbon is added to the atmosphere, that reduces Δ14C without reducing the total amount of carbon in the atmosphere. That's explained in (Burton 2024).

I'd reverse the order - first describe what the resistance profile is, and then how it is controlled.

I would consider removing the components that aren't relevant for the particular step. E.g. for (1), can just have 'patient generator' and 'patient'

I would leave this out of this section - I think it's a bit too specific

Again - looks like two papers. First to do #1. And second to do #2 and compare results to #1.

This has two messages (and again makes me wonder if there are two papers). One is that the simulator allow comparative policy analysis. The second is that RL can out-perforam static presecription rules.

I wouldn't be so explicit about noting 'novelty'- can lead that for reviewers/editors to decide. Could rub some folks the wrong way - particularly if they have published a paper that they think is even more novel. I'd rather emphasize how it builds/extends existing literature and findings, adresses gaps, etc.

Great paragraph

Move to methods

Great paragraph for motivating the benefit (and novelty) of AS simulation.

I would be careful about highlighting these as challenges early in the intro as it sets the paper up as a 'solution' to these challenges. I would rather emphasize why simulation is useful by itself (even if you had perfect observation / mechanistic understanding). Then you can introduce the limitations mentioned here as constraints (rather than challenges) that need to be incorporated into simulations.

briefly describe the distinction

Feels you have mixed messages here. On the one hand you are 'evaluating the impact of stewardship strategies' (which sounds like you are focusing on evaluating current strategies0 and on the other you are developing novel RL strategies. I'm still of the mind these are two separate papers! But I'll keep reading :)

I would lead with this, and then follow with the approach

Homepage Complexity: The homepage may create accessibility challenges because the large number of articles, images, headlines, and advertisements can make the webpage feel visually cluttered and overwhelming at first glance. This could make it more difficult for some users to navigate the webpage efficiently or focus on important information. Accessible webpages should avoid unnecessary complexity and present content in a simpler, more structured way.

Navigation / Header Menus: The navigation menus support the “Operable” principle of the Web Content Accessibility Guidelines because users can easily move through different sections of the website and access information in an organized way. The clear layout and dropdown menus make it easier for users to quickly find news topics and content they are interested in.

Clear Colour Contrast: This webpage demonstrates good accessibility through strong colour contrast between the black text and white background. This connects to the “Perceivable” principle of the Web Content Accessibility Guidelines because clear contrast helps users with visual impairments read content more easily.

not power but righteousness

Since, then, a blessed life consists of these two things, and is known to all, and dear to all; what can we think to be the cause why, when they cannot have both, men choose, out of these two, to have all things that they will, rather than to will all things well, even although they do not have them? Is it the depravity itself of the human race, in such wise that, while they are not unaware that neither is he blessed who has not what he wills, nor he who has what he wills wrongly, but he who both has whatsoever good things he wills, and wills no evil ones, yet, when both are not granted of those two things in which the blessed life consists, that is rather chosen by which one is withdrawn the more from a blessed life (since he certainly is further from it who obtains things which he wickedly desired, than he who only does not obtain the things which he desired); whereas the good will ought rather to be chosen, and to be preferred, even if it do not obtain the things which it seeks? For he comes near to being a blessed man, who wills well whatsoever he wills, and wills things, which when he obtains, he will be blessed. And certainly not bad things, but good, make men blessed, when they do so make them. And of good things he already has something, and that, too, a something not to be lightly esteemed — namely, the very good will itself; who longs to rejoice in those good things of which human nature is capable, and not in the performance or the attainment of any evil; and who follows diligently, and attains as much as he can, with a prudent, temperate, courageous, and right mind, such good things as are possible in the present miserable life; so as to be good even in evils, and when all evils have been put an end to, and all good things fulfilled, then to be blessed.

Accessibility Options on Videos: The videos on the website offer a variety of accessibility features, including closed captions, full-screen mode, and adjustable playback speed. Closed captions improve accessibility for users who are deaf or hard of hearing, while playback controls and full-screen options allow users to customize how they view content based on their individual needs. This connects to accessibility concepts related to the importance of creating more inclusive and adaptable web content.

Listen to this Article Feature: The “Listen to this article” feature improves accessibility for users who may have visual impairments, reading difficulties, or who prefer audio-based content. This demonstrates the concept of assistive technology, particularly text-to-speech accessibility tools that help make web content more accessible to a wider range of users.

Cute!!!

I found this source extremely interesting because it made me realize how little I know about content moderation. I have always assumed that when posts get taken down or people get banned, it is because of some algorithm that detects this content as harmful. However, I now know that there are real people behind this moderation. When reading about this, I was really shocked. The author made it clear that these workers are meant to be "invisible" on social media, and they have done a good job at this because I had no idea.

Janie and Tea Cake continue growing closer in Eatonville. Tea Cake teaches Janie how to play checkers, fish, and shoot a gun. The townspeople gossip because Tea Cake is younger and poorer, but Janie enjoys finally feeling happy and free.

Tea Cake leaves with Janie’s money for a day, making her nervous that he may have tricked her. He later returns with gifts and explains he spent the money at a party while working railroad jobs. Janie forgives him because she trusts and loves him.

Janie decides to leave Eatonville and marry Tea Cake. They move to Jacksonville and later travel south together. Janie feels excited about starting a new life away from the judgment of her old town.

I have reported posts on TikTok for violating the platform's rules. The most recent posts that I have been reporting are these ads I started seeing that were advertising for users to click a link to visit a different website that had sexual content on it. In most of these videos, the person urging the viewers to click the link is generated by AI. I reported these because inappropriate content, like what was being advertised, is not allowed on the platform.

The people who have the least power are minorities. Minorities are often harassed on social media, but due to moderation, hate speech is usually reported and taken down. If there was no moderation, I think the amount of hate online, especially towards minority groups, will grow exponentially.

Complete the space with supportive lumbar cushions and thoughtfully designed accent pillows: adding comfort, balance, and quiet character.

The bed is more than a place to rest. It is a space that holds your body, your breath, and your nervous system for hours each night.

By softening the space, choosing clean materials, and layering with intention, the bed becomes a place where the body can unwind, and the mind can settle.

The Conscious Bed was born from a simple, personal desire: to create a bedroom that truly supported rest, healing, and well-being. I was searching for a fabric headboard, something soft, grounding, and inviting. But every option felt like a compromise. Upholstered headboards attracted dust and aggravated my allergies, with no way to properly clean them. Rigid headboards were visually structured but uncomfortable to lean against. And the solution most beds relied on, layers of pillows, only added clutter, constantly ending up on the floor instead of supporting the body. Nothing felt intentional. Nothing felt conscious. I realized that the bed, the place where we spend a third of our lives, was being designed for appearance, not for how we actually rest. That realization became the beginning of The Conscious Bed. Instead of a single rigid structure, I envisioned a layered, adaptable system that could support the body, calm the nervous system, and stay clean over time. A bed designed with the understanding that materials, colors, and textures directly affect how we sleep, breathe, and heal. The Conscious Bed replaces fixed headboards and excess pillows with washable, interchangeable cushions crafted from organic, breathable fabrics. Each layer serves a purpose: comfort, support, ease of care, and energetic harmony. Nothing is decorative without intention. Nothing is added without function. This system allows the bed to evolve through changing needs, sensitivities, seasons, and moods, while remaining grounded in simplicity and cleanliness. At its core, The Conscious Bed is about reclaiming the bedroom as a space of restoration. A place where the body feels supported. Where the air feels clean. Where the environment works with you, not against you. Because true rest begins with intention, and the space we sleep in matters more than we’ve been taught to believe.

Every element is carefully considered, how it feels, how it breathes, how it supports both the body and the atmosphere of the room.

this section has a different style than other categories' Fabric Benefits sections

Timeless, soft, and sustainably made, our Organic Cotton Box-Stitch Quilt is a staple for every bedroom, designed to layer beautifully and mix effortlessly with the rest of your bedding.

Celebrate the natural beauty and healing benefits of linen with our 100% Linen Accent Pillow Covers, breathable, textured, and crafted for clean, calming, and toxin-free comfort in your home.

Please help me to find this paper

I wonder how they did that

Can you explain more on this?

Maybe we can add the link: https://www.parfumdreams.de/Umwelt

At the beginning of the bullet list, the letters should be written in lowercase.

Kuration und Exklusivität, nachhaltige Markenauswahl, vegane und tierversuchsfreie Produkte, Refill-Angebote, Loyalty und Service, Unternehmenstransparenz sowie Preis-Leistung.

Replace the word Kuration = Produktauswahl

The sentence is very long, better:

Nach intensiver Recherche der sieben wichtigsten Premium-Beauty-Händler im deutschsprachigen Raum werden deutliche Unterschiede sichtbar. Oft zeigt nicht die große Nachhaltigkeitsseite, wie ernst ein Unternehmen das Thema nimmt, sondern das tatsächliche Sortiment und der Umgang mit Verantwortung.

The same as above, the sentences are too long. Better: Im Vergleich fällt Niche Beauty vor allem durch die Auswahl nachhaltiger Marken und das starke Loyalty-Programm auf. Der Shop macht bewusstes Einkaufen einfacher und übersichtlicher statt komplizierter.

truth

This passage shows how migration can completely change the culture and ethnic makeup of a region over time. I thought it was interesting that the Turkic migrations did not just influence Central Asia politically, but also changed the languages and identity of the people living there.

MLB Scorebook Sales Are Rising as Baseball Scorekeeping Has a Moment<br /> by [[Jacob Feldman]]<br /> accessed on 2026-05-12T10:27:43

being able to map a plan instead of hoping for the best as you go.

using tools to intentional write rather than hoping to get it right on the first try.

It turns out our friend Tom has been scoring games with a typewriter since at least 2013!

Keeping score at a baseball game with a typewriter is not only possible but is also a much more detailed record of the match. (ORTEGA. Full count! Fouled back three in a row ... OH, THAT BALL’S LANDIN’ WHERE THE FANS ARE STANDIN’!!! Walk. Off. Home. Run. Thanks for your attendance and drive safely.) —Tom Hanks in "I Am TOM. I Like to TYPE. Hear That?" on Aug. 3, 2013 in the New York Times https://www.nytimes.com/2013/08/04/opinion/sunday/i-am-tom-i-like-to-type-hear-that.html?pagewanted=all&_r=0

cc: u/lou_spirito<br /> u/Informal-Writer-1140<br /> u/oogieball<br /> u/joe_skidiachi_irl

Other examples of his scoring efforts: - https://www.reddit.com/r/BaseballScorecards/comments/1jn2475/yes_tom_hanks_does_schlep_a_typewriter_to_ball/ - https://www.reddit.com/r/BaseballScorecards/comments/1jm0l8k/tom_hanks_keeps_score/ - https://www.reddit.com/r/BaseballScorecards/comments/1jm4pie/tom_hanks_scorecard/

Posted at https://reddit.com/r/BaseballScorecards/comments/1tb7fbw/tom_hanks_scoring_baseball_games_via_typewriter/

Hanks, Tom. 2013. “I Am TOM. I Like to TYPE. Hear That?” The New York Times. https://www.nytimes.com/2013/08/04/opinion/sunday/i-am-tom-i-like-to-type-hear-that.html (May 12, 2026).

This is basically applied common good ethics bringing everyone affected into the process, not just decision makers at the top.