Reviewer #2 (Public Review):

Ehring et al. analyze contributions of Dispatched, Scube2, serum lipoproteins and Sonic Hedgehog lipid modifications to the generation of different Shh release forms. Hedgehog proteins are anchored in cellular membranes by N-terminal palmitate and C-terminal cholesterol modifications, yet spread through tissues and are released into the circulation. How Hedgehog proteins can be released, and in which form, remains controversial. The authors systematically dissect contributions of several previously identified factors, and present evidence that Disp, Scube2 and lipoproteins concertedly act to release a novel Shh variant that is cholesterol-modified but not palmitoylated. The results provide new insights into the function of Disp and Scube2 in Hedgehog release. The findings concerning the function of lipoproteins and cholesterol in Hedgehog release are largely confirmatory (PMID 23554573, 20685986). However, in light of the multitude of competing models for Hedgehog release, the present study is a valuable contribution that provides further insights into the relevance of lipoproteins in this process.

A novel and surprising finding of the present study is the differential removal of Shh N- or C-terminal lipid anchors depending on the presence of HDL and/or Disp. In particular, the identification of a non-palmitoylated but cholesterol-modified Shh variant that associates with lipoproteins is potentially important. The authors use RP-HPLC and defined controls to assess the properties of processed Shh forms, but their precise molecular identity remains to be defined. A caveat is the strong reliance on over-expression of Shh in a single cell line. The authors detect Shh variants that are released independently of Disp and Scube2 in secretion assays, which however are excluded from interpretation as experimental artifacts. Thus, it would be important to demonstrate key findings in cells that secrete Shh endogenously.

Reviewer #2 (Public Review):

In this work, Sarkar et al. investigated the potential ability of adenosine triphosphate (ATP) as a solubilizer of protein aggregates by combining MD simulations and ThT/TEM experiments. They explored how ATP influences the conformational behaviors of Trp-cage and β-amyloid Aβ40 proteins. Currently, there are no experiments in the literature supporting their simulation results of ATP on Trp-cage. The simulation protocol employed for the Aβ40 monomer system is conventional MD simulation, while REMD simulation (an enhanced sampling method) is used for the Aβ monomer + ATP system. It is not clear whether the conformational difference is caused by ATP or by the different simulation methods used. ThT/TEM experiments should be performed on Aβ40 fibrils rather than on Aβ(16-22) aggregates. Moreover, to elucidate their experimental results that ATP can dissolve preformed Aβ fibrils, the authors need to study the influence of ATP on Aβ fibrils instead of on Aβ dimer in their MD simulations. The novelty of this study is limited. The role of ATP in inhibiting Aβ fibril formation and dissolving preformed Aβ fibrils has been reported in previous experimental and computational studies (Journal of Alzheimer's Disease, 2014, 41: 561; Science 2017, 2017, 356, 753-756 J. Phys. Chem. B 2019, 123, 9922−9933; Scientific Reports, 2024, 14: 8134). However, most of those papers are not discussed in this manuscript. Additionally, some details of MD simulations and data analysis are missing in the manuscript, including the initial structures of all the simulations, the method for free energy calculation, the dielectric constant used, etc.

Reviewer #2 (Public Review):

Summary:<br /> This paper presents a model of out-of-distribution (OOD) generalization that focuses on modeling an analogy task, in which translation or scaling is tested with training in one part of the space and testing in other areas of the space progressively more distant from the training location. Similar tests were performed on arithmetic including addition and multiplication, and similarly impressive results appear for addition but not multiplication. The authors show that a grid cell coding scheme helps performance on these analogy and arithmetic tasks, but the most dramatic increase in performance is provided by a complex algorithm for distributional point-process attention (DPP-A) based on maximizing the determinant of the covariance matrix of the grid embeddings.

Strengths:<br /> The results appear quite impressive. The results for generalization appear quite dramatic when compared to other coding schemes (i.e. one-hot) or when compared to the performance when ablating the DPP-A component but retaining the same inference modules using LSTM or transformers. This appears to be an important result in terms of generalization of results in an analogy space.

Weaknesses:<br /> There are a number of ways that its impact and connection to grid cells could be enhanced. From the neuroscience perspective, the major comments concern making a clearer and stronger connection to the actual literature on grid cells and grid cell modeling, and discussing the relationship of the complex DPP-A algorithm to biological circuits.

Major comments:<br /> 1. They should provide more citations to other groups that have explored analogy using this type of task. Currently, they only cite one paper (Webb et al., 2020) by their own group in their footnote 1 which used the same representation of behavioral tasks for generalization of analogy. It would be useful if they could cite other papers using this simplified representation of analogy and also show the best performance of other algorithms from other groups in their figures, so that there is a sense of how their results compare to the best previous algorithm by other groups in the field (or they can identify which of their comparison algorithms corresponds to the best of previously published work).

2. While the grid code they use is very standard and based on grid cell researchers (Bicanski and Burgess, 2019), the rest of the algorithm doesn't have a clear claim on biological plausibility. It has become somewhat standard in the field to ignore the problem of how the brain could biologically implement the latest complex algorithm, but it would be useful if they at least mention the problem (or difficulty) of implementing DPP-A in a biological network. In particular, does maximizing the determinant of the covariance matrix of the grid code correspond to something that could be tested experimentally?

3. Related to major comment 2., it would be very exciting if they could show what the grid code looks like after the attentional modulation inner product xT w has been implemented. This could be highly useful for experimental researchers trying to connect these theoretical simulation results to data. This would be most intuitive to grid cell researchers if it is plotted in the same format as actual biological experimental data - specifically which grid cell codes get strengthened the most (beyond just the highest frequencies).

4. To enhance the connection to biological systems, they should cite more of the experimental and modeling work on grid cell coding (for example on page 2 where they mention relational coding by grid cells). Currently, they tend to cite studies of grid cell relational representations that are very indirect in their relationship to grid cell recordings (i.e. indirect fMRI measures by Constaninescu et al., 2016 or the very abstract models by Whittington et al., 2020). They should cite more papers on actual neurophysiological recordings of grid cells that suggest relational/metric representations, and they should cite more of the previous modeling papers that have addressed relational representations. This could include work on using grid cell relational coding to guide spatial behavior (e.g. Erdem and Hasselmo, 2014; Bush, Barry, Manson, Burges, 2015). This could also include other papers on the grid cell code beyond the paper by Wei et al., 2015 - they could also cite work on the efficiency of coding by Sreenivasan and Fiete and by Mathis, Herz, and Stemmler.

Reviewer #2 (Public Review):

Summary:

This manuscript explores a DNA fluorescent light-up aptamer (FLAP) with the specific goal of comparing activity in vitro to that in bacterial cells. In order to achieve expression in bacteria, the authors devise an expression strategy based on retrons and test four different constructs with the aptamer inserted at different points in the retron scaffold. They only observe binding for one scaffold in vitro, but achieve fluorescence enhancement for all four scaffolds in bacterial cells. These results demonstrate that aptamer performance can be very different in these two contexts.

Strengths:

-Given the importance of FLAPs for use in cellular imaging and the fact that these are typically evolved in vitro, understanding the difference in performance between a buffer and a cellular environment is an important research question.

-The return strategy utilized by the authors is thoughtful and well-described.

-The observation that some aptamers fail to show binding in vitro but do show enhancement in cells is interesting and surprising.

Weaknesses:

-This study hints toward an interesting observation, but would benefit from greater depth to more fully understand this phenomenon. Particularly challenging is that FLAP performance is measured in vitro by affinity and in cells by enhancement, and these may not be directly proportional. For example, it may be that some constructs have much lower affinity but a greater enhancement and this is the explanation for the seemingly different performance.

-The authors only test enhancement at one concentration of fluorophore in cells (and this experimental detail is difficult to find and would be helpful to include in the figure legend). This limits the conclusions that can be drawn from the data and limits utility for other researchers aiming to use these constructs.

-The FLAP that is used seems to have a relatively low fluorescence enhancement of only 2-3 fold in cells. It would be interesting to know if this is also the case in vitro. This is lower than typical FLAPs and it would be helpful for the authors to comment on what level of enhancement is needed for the FLAP to be of practical use for cellular imaging.

Reviewer #2 (Public Review):

It is challenging to study the biophysical properties of organelle channels using conventional electrophysiology. The conventional reconstitution methods require multiple steps and can be contaminated by endogenous ionophores from the host cell lines after purification. To overcome this challenge, in this manuscript, Larmore et al. described a fully synthetic method to assay the functional properties of the TRPP channel family. The TRPP channels are an important organelle ion channel family that natively traffic to primary cilia and ER organelles. The authors utilized cell-free protein expression and reconstitution of the synthetic channel protein into giant unilamellar vesicles (GUV), the single channel properties can be measured using voltage-clamp electrophysiology. Using this innovative method, the authors characterized their membrane integration, orientation, and conductance, comparing the results to those of endogenous channels. The manuscript is well-written and may present broad interest to the ion channel community studying organelle ion channels. Particularly because of the challenges of patching native cilia cells, the functional characterization is highly concentrated in very few labs. This method may provide an alternative approach to investigate other channels resistant to biophysical analysis and pharmacological characterization.

Reviewer #2 (Public Review):

Summary:

The authors set out to study whether the cooling agent binding site in TRPM8, which is located between the S1-S4 and the TRP domain, is conserved within the TRPM family of ion channels. They specifically chose the TRPM4 channel as the model system, which is directly activated by intracellular Ca2+. Using electrophysiology, the authors characterized and compared the Ca2+ sensitivity and the voltage dependence of TRPM4 channels in the absence and presence of synthetic cooling agonist icilin. They also analyzed the mutational effects of residues (A867G and R901H; equivalent mutations in TRPM8 were shown involved in icilin sensitivity) on Ca2+ sensitivity and voltage-dependence of TRPM4 in the absence and presence of Ca2+. Based on the results as well as structure/sequence alignment, the authors concluded that icilin likely binds to the same pocket in TRPM4 and suggested that this cooling agonist binding pocket is conserved in TRPM channels.

Strengths:

The authors gave a very thorough introduction to the TRPM channels. They have nicely characterized the Ca2+ sensitivity and the voltage-dependence of TRPM4 channels and demonstrated icilin potentiates the Ca2+ sensitivity and diminishes the outward rectification of TRPM4. These results indicate icilin modulates TRPM4 activation by Ca2+.

Weaknesses:

The reviewer has a few concerns. First, icilin alone (at 25µM) and in the absence of Ca2+ does not activate the TRPM4 channel. Have the authors titrated a wide range of icilin concentrations (without Ca2+ present) for TRPM4 activation? It raises the question that whether icilin is indeed an agonist for TRPM4 channel. This has not been tested so it is unclear. One may argue that icilin needs Ca2+ as a co-factor for channel activation just like in TRPM8 channel. This leads to the second concern, which is a complication in the experimental design and data interpretation. TRPM4 itself requires Ca2+ for activation to begin with, thus it is hard to dissect whether the current observed here for TRPM4 is activated by Ca2+ or by icilin plus its cofactor Ca2+. This is the difference between TRPM8 and TRPM4, as TRPM8 itself is not activated by Ca2+, thus TRPM8 activation is through icilin and Ca2+ acts as a prerequisite for icilin activation.

The results presented in this study are only sufficient to show that icilin modulates the Ca2+-dependent activation of TRPM4 and icilin at best may act as an allosteric modulator for TRPM4 function. One cannot conclude from the current work that icilin is an agonist or even specifically a cooling agonist for TRPM4. Icilin is a cooling agonist for TRPM8, but it does not mean that if icilin modulates TRPM4 activity then it serves as a cooling agonist for TRPM4.

For the mutation data on A867G, Figure 4A-B, left panels, it looks like A867G has stronger Ca2+ sensitivity compared to the WT in the absence of icilin and the onset of current activation is faster than the WT, or this is simply due to the scale of the data figure are different between A867G and the WT. Overall the mutagenesis data are weak to support the conclusion that icilin binds to the S1-S4 pocket. The authors need to mutate more residues that are involved in direct interaction with icilin based on the available structural information, including but limited to residues equivalent to Y745 and H845 in human TRPM8.

The authors set out to study the conservation of the cooling agonist binding site in TRPM family, but only tested a synthetic cooling agonist icilin on TRPM4. In order to draw a broad conclusion as the title and the discussion have claimed, the authors need to more cooling compounds, including the most well-known natural cooling agonist menthol, and other cooling agonists such as WS-12 and/or C3, and test their effects on several TRPM channels, not just TRPM4. With the current data, the authors need to significantly tone down the claim of a conserved cooling agonist binding pocket in the TRPM family.

On page 11, the authors suggest based on the current data, that TRPM2 and TRPM5 may also be sensitive to cooling agonists because the key residues are conserved. TRPM2 is the closest homolog to TRPM8 but is menthol-insensitive. There are studies that attempted to convert menthol sensitivity to TRPM2, for example, Bandell 2006 attempted to introduce S2 and TRP domains from TRPM8 into TRPM2 but failed to make TRPM2 a menthol-sensitive channel. The sequence conservation or structural similarity is not sufficient for the authors to suggest a shared cooling agonist sensitivity or even a common binding site in the TRPM2 and TRPM5 channels. Again, as pointed out above, the authors need to establish the actual activation of other TRPM channels by these agonists first, before proceeding to functionally probe whether other TRPM channels adopt a conserved agonist binding site.

Taken together, this current work presents data to show the modulatory effects of icilin on the Ca2+ dependent activation and voltage dependence of the TRPM4 channel.

Reviewer #2 (Public Review):

Summary:

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

Strengths:

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

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

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

Weaknesses:

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

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

Reviewer #2 (Public Review):

Summary:

In this study, Sha and Zhang et al. reported that androgen deprivation therapy (ADT) induces a switch to a basal-stemness status, driven by the TNF-CCL2-CCR2 axis. Their results also reveal that enhanced CCL2 coincides with increased macrophages and decreased CD8 T cells, suggesting that ADT resistance may be related to the TNF/CCL2/CCR2-dependent immunosuppressive tumor microenvironment (TME). Overall, this is a very interesting study with a significant amount of data.

Strengths:

The strengths of the study include various clinically relevant models, cutting-edge technology (such as single-cell RNA-seq), translational potential (TNF and CCR2 inhibitors), and novel insights connecting stemness lineage switch to an immunosuppressive TME. Thus, I believe this work would be of significant interest to the field of prostate cancer and journal readership.

Weaknesses:

(1) One of the key conclusions/findings of this study is the ADT-induced basal-stemness lineage switch driving ADT resistance. However, most of the presented evidence supporting this conclusion only selects a couple of marker genes. What exacerbates this issue is that different basal-stemness markers were often selected with different results. For example, Figure S1A uses CD166/EZH2 as markers, while Figure S1B uses ITGb1/EZH2. In contrast, Figure 1D uses Sca1/CD49, and Figure 2B-C uses CD49/CD166. Since many basal-stemness lineage gene signatures have been previously established, the study should examine various basal-stemness gene signatures rather than a couple of selected markers. Moreover, why were none of the stemness/basal-gene signatures significantly changed in the GO enrichment analysis in Figure 6A/B?

(2) A related weakness is the lack of functional results supporting the stemness lineage switch. Although the authors present colony formation assay results, these could be influenced simply by promoted cell proliferation, which is not a convincing indicator of stemness. To support this key conclusion, widely accepted stemness assays, such as the prostasphere formation assay (in vitro) and Extreme Limiting Dilution Analysis (ELDA) xenograft assay (in vivo), should be carried out.

(3) Another significant concern is that this study uses concurrency to demonstrate a causal relationship in many key results, which is entirely different. For example, Figure S4A and S4B only show increased CCL2 and TNF secretion simultaneously, which cannot support that CCL2 is dependent on TNF. Similarly, Figure 5A only shows that CCL2 increased coincidently with a rise in TNF, which cannot support a causal relationship. To support the causal relationship of this conclusion, it is necessary to show that TNF-KO/KD would abolish the increased CCL2 secretion.

(4) Some of the selective data presentations are not explained and are difficult to understand. For example, why does CD49 staining in Figure S3A have data for all four time points, while CD166 in Figure S3D only has data for the last time point (day 21)? Similarly, although several TNF_UP gene signatures were highlighted in Figure 4B, several TNF_DN signatures were also enriched in the same table, such as RUAN_RESPONSE_TO_TNF_DN. What is the explanation for these contrasting results?

Reviewer #2 (Public Review):

Summary:

The manuscript of Patton et al. shows that in mice in which both FXR and SHP are knocked out, the sex difference in liver cancer risk is recapitulated. Authors show that the protection against tumor development seen in female mice is dependent upon ovarian hormone secretion and higher fecal bile acid excretion in females compared to males. The female liver-specific gene signature correlates with low-grade tumors and better survival in human HCC patients.

The combination of the use of the double knockout mice together with ovariectomy in female mice and using a bile acid raisin in male mice to underscore their conclusion is strong. However, there are also some shortcomings, that should be addressed.

Strengths:

(1) Using computational modelling, Patton and colleagues correlate mouse DKO transcriptome data to the clinical outcomes of HCC patients using HCC transcriptome datasets.

(2) The dependence of female protection on ovarian hormones and increased fecal bile acid excretion is nicely shown by combining ovariectomy and bile acid raisin with the use of double knockout mice.

Weaknesses:

(1) The translational value to human HCC is not so strong yet. Authors show that there is a correlation between the female-selective gene signature and low-grade tumors and better survival in HCC patients overall. However, these data do not show whether this signature is more highly correlated with female tumor burden and survival. In other words, whether the mechanisms of female protection may be similar between humans and mice. In that respect, it would also be good to elaborate on whether women have higher fecal BA excretion and lower serum BA concentration.

(2) The authors should perform a thorough spelling and grammar check.

(3) There are quite some errors and inaccuracies in the result section, figures, and legends. The authors should correct this.

Reviewer #2 (Public Review):

Summary:

Wilson's disease is a rare genetic disorder caused by mutations in the ATP7B gene. Previous studies have documented that ATP7B mutations can disrupt copper metabolism, affecting brain and liver function. In this paper, the authors performed a retrospective clinical study and found that Wilson's disease has a high incidence of cholecystitis. Single-cell RNA-seq analysis revealed changes in the immune microenvironment, including the activation of immune responses and the exhaustion of natural killer cells.

Strengths:

A key finding of this study is that the predominant ATP7B gene mutation in the Chinese population is the 2333G>T (p. R778L) mutation. The authors reported associations between Wilson's disease and cholecystitis, as well as the exhaustion of natural killer cells.

Weaknesses:

The underlying mechanisms linking ATP7B mutations to cholecystitis and natural killer cell exhaustion remain unclear. Specifically, it is not yet determined whether copper metabolism alterations directly cause cholecystitis and natural killer cell exhaustion, or if these effects are secondary to liver dysfunction.

DOI: 10.1038/s41598-024-60269-2

Resource: (BDSC Cat# 30137,RRID:BDSC_30137)

Curator: @bandrow

SciCrunch record: RRID:BDSC_30137

What is this?

Reviewer #2 (Public Review):

This is an important and very interesting report on a change in newborns' neural abilities to distinguish auditory signals as a function of the gestational age (GA) of the infant at birth (from 35 weeks GA to 40 weeks GA). The authors tested neural discrimination of sounds that were labeled 'happy' vs 'neutral' by listeners that represent two categories of sound, either human voices or auditory signals that mimic only certain properties of the human vocal signals. The finding is that a change occurs in neural discrimination of the happy and neutral auditory signals for infants born at or after 37 weeks of gestation, and not prior (at 35 or 36 weeks of gestation), and only for discrimination of the human vocal signals; no change occurs in discrimination of the nonhuman signals over the 35- to 40-week gestational ages tested. The neural evidence of discrimination of the vocal happy-neutral distinction and the absence of the discrimination of the control signals is convincing. The authors interpret this as a 'landmark' in infants' ability to detect changes in emotional vocal signals, and remark on the potential value of the test as a marker of the infants' interest in emotional signals, underscoring the fact that children at risk for autism spectrum disorder may not show the discrimination. Although the finding is novel and interesting, additional discussion is essential so that readers understand two potential caveats affecting this interpretation.

Comments on the revised version:

The revised manuscript does discuss the limitations of the control stimuli, as well as the limitations with regard to conclusions that can be drawn from this data set. I therefore expected the authors to temper a bit their recommendation that this could be a 'screening' signal for autism because these data are not sufficiently strong to make that recommendation. Also, in the same vein, perhaps the title might be adjusted somewhat to suggest less certainty, for example, by using the word "change" rather than "milestone"'? The data are of interest, but the limitations are genuine limitations.

Reviewer #2 (Public Review):

Summary:

Dubicka et al. in their paper entitled " Biocalcification in porcelaneous foraminifera" suggest that in contrast to the traditionally claimed two different modes of test calcification by rotallid and porcelaneous miliolid formaminifera, both groups produce calcareous tests via the intravesicular mineral precursors (Mg-rich amorphous calcium carbonate). These precursors are proposed to be supplied by endocytosed seawater and deposited in situ as mesocrystals formed at the site of new wall formation within the organic matrix. The authors did not observe the calcification of the needles within the transported vesicles, which challenges the previous model of miliolid mineralization. Although the authors argue that these two groups of foraminifera utilize the same calcification mechanism, they also suggest that these calcification pathways evolved independently in the Paleozoic.

Comments on the revised version

In my reply to the author's rebuttal letter, I will focus on one key point. The main observation supporting the author's conclusion, as expressed in the abstract, is:

"We found that both groups [i.e., rotaliids and miliolids, the latter documented in the reviewed paper] produced calcareous shells via the intravesicular formation of unstable mineral precursors (Mg-rich amorphous calcium carbonates) supplied by endocytosed seawater and deposited at the site of new wall formation within the organic matrix. Precipitation of high-Mg calcitic mesocrystals took place in situ and formed a dense, chaotic meshwork of needle-like crystallites."

In my review, I pointed out that there is no support for the existence of an intracellular, vesicular intermediate amorphous phase.

The authors replied:

"We used laser line 405 nm and multiphoton excitation to detect ACCs. These wavelengths (partly) permeate the shell to excite ACCs autofluorescence. The autofluorescence of the shells is present as well but not clearly visible in movie S4 as the fluorescence of ACCs is stronger. This may be related to the plane/section of the cell which is shown. The laser permeates the shell above the ACCs (short distance) but to excite the shell CaCO3 around foraminifera in the same three-dimensional section where ACCs are shown, the light must pass a thick CaCO3 area due to the three-dimensional structure of the foraminiferan shell. Therefore, the laser light intensity is reduced. In a revised version, a movie/image with reduced threshold is shown."

This reply does not address the reviewer's concerns. Detection of ACC with 405 nm excitation is not sufficient; many organic components can fluoresce under violet light excitation. For example, Delvene et al. (2002) (https://doi.org/10.18261/let.55.4.7) showed that "the Pleistocene and Jurassic microborings emit in the blue-yellow spectral region (420-600 nm) with a laser excitation of 405 nm, which coincides with the emission due to NADPH [nicotinamide adenine dinucleotide], FAD [flavin adenine dinucleotide], and riboflavin pigments characteristic of some cyanobacteria." Traditionally, in geological or biogenic calcium carbonate samples, Raman spectroscopic characterization of ACC and its magnesium content can be used (e.g., Wang, D., Hamm, L. M., Bodnar, R. J. & Dove, P. M. Raman spectroscopic characterization of the magnesium content in amorphous calcium carbonates. J. Raman Spectrosc. 43, 543-548 (2012); Perrin, J. et al. Raman characterization of synthetic magnesian calcites. Am. Mineral. 101, 2525-2538 (2016)). However, in biological, living-cell systems, Mehta et al. (2022) (doi: 10.1016/j.saa.2022.121262) successfully used FTIR spectroscopy to identify ACC by two characteristic FTIR vibrations at ca. 860 cm-1 and ca. 306 cm-1. Other methods such as STXM analyses at the C K-edge (Monteil et al. 2021, doi: 10.1038/s41396-020-00747-3) are also available. Because the core of the authors' interpretation (i.e., detection of ACC in vesicles) is not supported by hard evidence, the claim that the study represents a "paradigm shift" is far-fetched and the whole model is based on speculations. If the authors are able to unequivocally confirm the presence of ACC within the vesicles and its subsequent transformation into calcitic needles, the other problems noted in the paper will be relatively trivial.

Reviewer #2 (Public Review):

Summary:

In their manuscript, Daniel Spari et al. explored the dual role of ATP in exacerbating sepsis, revealing that ATP from both host and bacteria significantly impacts immune responses and disease progression.

Strengths:

The study meticulously examines the complex relationship between ATP release and bacterial growth, membrane integrity, and how bacterial ATP potentially dampens inflammatory responses, thereby impairing survival in sepsis models. Additionally, this compelling paper implies a concept that bacterial OMVs act as vehicles for the systemic distribution of ATP, influencing neutrophil activity and exacerbating sepsis severity.

Weaknesses:

(1) The researchers extracted and cultivated abdominal fluid on LB agar plates, then randomly picked 25 colonies for analysis. However, they didn't conduct 16S sequencing on the fluid itself. It's worth noting that the bacterial species present may vary depending on the individual patients. It would be beneficial if the authors could specify whether they've verified the existence of unculturable species capable of secreting high levels of Extracellular ATP.

(2) Do mice lacking commensal bacteria show a lack of Extracellular ATP following cecal ligation puncture?

(3) The authors isolated various bacteria from abdominal fluid, encompassing both Gram-negative and Gram-positive types. Nevertheless, their emphasis appeared to be primarily on the Gram-negative E. coli. It would be beneficial to ascertain whether the mechanisms of Extracellular ATP release differ between Gram-positive and Gram-negative bacteria. This is particularly relevant given that the Gram-positive bacterium E. faecalis, also isolated from the abdominal fluid, is recognized for its propensity to release substantial amounts of Extracellular ATP.

(4) The authors observed changes in the levels of LPM, SPM, and neutrophils in vivo. However, it remains uncertain whether the proliferation or migration of these cells is modulated or inhibited by ATP receptors like P2Y receptors. This aspect requires further investigation to establish a convincing connection.

(5) Additionally, is it possible that the observed in vivo changes could be triggered by bacterial components other than Extracellular ATP? In this research field, a comprehensive collection of inhibitors is available, so it is desirable to utilize them to demonstrate clearer results.

(6) Have the authors considered the role of host-derived Extracellular ATP in the context of inflammation?

(7) The authors mention that Extracellular ATP is rapidly hydrolyzed by ectonucleotases in vivo. Are the changes of immune cells within the peritoneal cavity caused by Extracellular ATP released from bacterial death or by OMVs?

(8) In the manuscript, the sample size (n) for the data consistently remains at 2. I would suggest expanding the sample size to enhance the robustness and rigor of the results.

Reviewer #2 (Public Review):

Wang, He et al. shed insight into the molecular mechanisms of deep-sea chemosymbiosis at the single-cell level. They do so by producing a comprehensive cell atlas of the gill of Gigantidas platifrons, a chemosymbiotic mussel that dominates the deep-sea ecosystem. They uncover novel cell types and find that the gene expression of bacteriocytes, the symbiont-hosting cells, supports two hypotheses of host-symbiont interactions: the "farming" pathway, where symbionts are directly digested, and the "milking" pathway, where nutrients released by the symbionts are used by the host. They perform an in situ transplantation experiment in the deep sea and reveal transitional changes in gene expression that support a model where starvation stress induces bacteriocytes to "farm" their symbionts, while recovery leads to the restoration of the "farming" and "milking" pathways.

A major strength of this study includes the successful application of advanced single nucleus techniques to a non-model, deep sea organism that remains challenging to sample. I also applaud the authors for performing an in situ transplantation experiment in a deep sea environment. From gene expression profiles, the authors deftly provide a rich functional description of G. platifrons cell types that is well-contextualized within the unique biology of chemosymbiosis. These findings offer significant insight into the molecular mechanisms of deep-sea host-symbiont ecology, and will serve as a valuable resource for future studies into the striking biology of G. platifrons.

The authors' conclusions are generally well-supported by their results. However, I recognize that the difficulty of obtaining deep-sea specimens may have impacted experimental design and no replicates were sampled.

It is notable that the Fanmao cells were much more sparsely sampled. It appears that fewer cells were sequenced, resulting in the Starvation and Reconstitution conditions having 2-3x more cells after doublet filtering. These discrepancies also are reflected in the proportion of cells that survived QC, suggesting a distinction in quality or approach. However, the authors provide clear and sufficient evidence via bootstrapping that batch effects between the three samples are negligible. While batch effect does not appear to have affected gene expression profiles, the proportion of cell types may remain sensitive to sampling techniques, and thus interpretation of Fig. S12 must be approached with caution.

Reviewer #2 (Public Review):

Summary:

The authors seek to elucidate the early evolution of cnidarians through computer modeling of fluid flow in the oral region of very small, putative medusozoan polyps. They propose that the evolutionary advent of the free-swimming medusoid life stage was preceded by a sessile benthic life stage equipped with circular muscles that originally functioned to facilitate feeding and that later became co-opted for locomotion through jet propulsion.

Strengths:

Assumptions of the modeling exercise laid out clearly; interpretations of the results of the model runs in terms of functional morphology plausible. An intriguing investigation that should stimulate further discussion and research.

Weaknesses:

Speculation on the origin of the medusoid life stage in cnidarians heavily dependent on prior assumptions concerning the soft part anatomy and material properties of the skeleton of the modeled fossil organism that may be open to alternative interpretations. Logically, of course, the hypothesis that cnidarian medusae originated from benthic polyps must be evaluated along with the alternative hypotheses that the medusa came first and that the ancestral cnidarian exhibited both life stages.

Reviewer #3 (Public Review):

In the present manuscript, Abdelaziz and colleagues interrogate the gating mechanisms of Kv10.1, an important voltage-gated K+ channel in cell cycle and cancer physiology. At the molecular level, Kv10.1 is regulated by voltage and Ca-CaM. Structures solved using Cryo-EM for Kv10.1 as well as other members of the KCNH family (Kv11 and Kv12) show channels that do not contain a structured S4-S5 linker imposing therefore a non-domain swapped architecture in the transmembrane region. However, the cytoplasmatic N- and C- terminal domains interact in a domain swapped manner forming a gating ring. The N-terminal domain (PAS domain) of one subunit is located close to the intracellular side of the voltage sensor domain and interacts with the C-terminal domain (CNBHD domain) of the neighbor subunit. Mutations in the intracellular domains has a profound effect in the channel gating. The complex network of interactions between the voltage-sensor and the intracellular domains makes the PAS domain a particularly interesting domain of the channel to study as responsible for the coupling between the voltage sensor domains and the intracellular gating ring.

The coupling between the voltage-sensor domain and the gating ring is not fully understood and the authors aim to shed light into the details of this mechanism. In order to do that, they use well established techniques such as site-directed mutagenesis, electrophysiology, biochemistry and mathematical modeling. In the present work, the authors propose a two open state model that arises from functional experiments after introducing a deletion on the PAS domain (ΔPAS Cap) or a point mutation (E600R) in the CNBHD domain. The authors measure a bi-phasic G-V curve with these mutations and assign each phase as two different open states, one of them not visible on the WT and only unveiled after introducing the mutations. The hypothesis proposed by the authors could change the current paradigm in the current understanding for Kv10.1 and it is quite extraordinary; therefore, it requires extraordinary evidence to support it.

STRENGTHS: The authors use adequate techniques such as electrophysiology and site-directed mutagenesis to address the gating changes introduced by the molecular manipulations. They also use appropriate mathematical modeling to build a Markov model and identify the mechanism behind the gating changes.

WEAKNESSES: The results presented by the authors do not fully support their conclusions since they could have alternative explanations. The authors base their primary hypothesis on the bi-phasic behavior of a calculated G-V curve that do not match the tail behavior, the experimental conditions used in the present manuscript introduce uncertainties, weakening their conclusions and complicating the interpretation of the results. Therefore, their experimental conditions need to be revisited

I have some concerns related to the following points:

(1) Biphasic gating behavior<br /> The authors use the TEVC technique in oocytes extracted surgically from Xenopus Leavis frogs. The method is well established and is adequate to address ion channel behavior. The experiments are performed in chloride-based solutions which present a handicap when measuring outward rectifying currents at very depolarizing potentials due to the presence of calcium activated chloride channel expressed endogenously in the oocytes; these channels will open and rectify chloride intracellularly adding to the outward rectifying traces during the test pulse.<br /> The authors calculate their G-V curves from the test pulse steady-state current instead of using the tail currents. The conductance measurements are normally taken from the 'tail current' because tails are measured at a fix voltage hence maintaining the driving force constant. Calculating the conductance from the traces should not be a problem, however, in the present manuscript, the traces and the tail currents do not agree. The tail traces shown in Fig1E do not show an increasing current amplitude in the voltage range from +50mV to +120mV, they seem to have reached a 'saturation state', suggesting that the traces from the test pulse contain an inward chloride current contamination. In addition, this second component identified by the authors as a second open state appears after +50mV and seems to never saturate. The normalization to the maximum current level during the test pulse, exaggerates this second component on the calculated G-V curve. It's worth noticing that the ΔPASCap mutant experiments on Fig 5 in Mes based solutions do not show that second component on the G-V.

Because these results are the foundation for their two open state hypotheses, I will strongly suggest the authors to repeat all their Chloride-based experiments in Mes-based solutions to eliminate the undesired chloride contribution to the mutants current and clarify the contribution of the mutations to the Kv10.1 gating.

(2) Two step gating mechanism.<br /> The authors interpret the results obtained with the ΔPASCap and the E600R as two step gating mechanisms containing two open states (O1 and O2) and assign them to the voltage sensor movement and gating ring rotation respectively. It is not clear, however how the authors assign the two open states.<br /> The results show how the first component is conserved amongst mutations; however, the second one is not. The authors attribute the second component, hence the second open state to the movement of the gating ring. This scenario seems unlikely since there is a clear voltage-dependence of the second component that will suggest an implication of a voltage-sensing current.

The split channel experiment is interesting but needs more explanation. I assume the authors expressed the 2 parts of the split channel (1-341 and 342-end), however Tomczak et al showed in 2017 how the split presents a constitutively activated function with inward currents that are not visible here, this point needs clarification.

Moreover, the authors assume that the mutations introduced uncover a new open state, however the traces presented for the mutations suggest that other explanations are possible. Other gating mechanisms like inactivation from the closed state, can be introduced by the mutations. The traces presented for ΔPASCap but specially E600R present clear 'hooked tails', a direct indicator of a populations of inactive channels during the test pulse that recover from inactivation upon repolarization (Tristani-Firouzi M, Sanguinetti MC. J Physiol. 1998). The results presented by the authors can be alternatively explained with a change in the equilibrium between the close to inactivated/recovery from inactivation to the open state. Finally, the authors state that they do not detect "cumulative inactivation after repeated depolarization" but that is considering inactivation only from the open state and ignoring the possibility of the existence of close state inactivation or, that like in hERG, that the channel inactivates faster that what it activates (Smith PL, Yellen G. J Gen Physiol. 2002).

(3) Single channel conductance.<br /> The single channels experiments are a great way to assess the different conductance of single channel openings, unfortunately the authors cannot measure accurately different conductances for the two proposed open states. The Markov Model built by the authors, disagrees with their interpretation of the experimental results assigning the exact same conductance to the two modeled open states. To interpret the mutant data, it is needed to add data with the WT for comparison and in presence of specific blockers.

Reviewer #2 (Public Review):

"Plasticity of the proteasome-targeting signal Fat10 enhances substrate degradation" is a nice study where the authors have shown the differences between two protein degradation tags namely, FAT10 and ubiquitin. Even though these tags are closely related in terms of folds, they have differential efficiency in degrading the substrates covalently attached to them. The authors have utilised extensive MD simulations combined with biophysics and cell biology to show the structural dynamics these tags provide for proteasomal degradation.

Reviewer #2 (Public Review):

This paper presents a modeling analysis of a diffusing morphogen (hh) that patterns the wing disk by controlling the expression of dpp and col. Two modes of gene expression control/interpretation are analyzed and presented, one is a response using a steady state threshold (col), which could be robust (defined as a small spatial shift of the gene expression when hh dosage changes) by a ptch mediated negative feedback mechanism; the other is the "overshoot" where an earlier hh gradient profile pre-steady state is read at a threshold to activate the gene (dpp), which is less robust to dosage changes but has better boundary features. Experimental measurements of pattern widths of col and dpp were performed under different hh dosage to test the models. How these different modes were achieved by each gene was unclear.

The reviewer found this study presents at best incremental advances to the field. It doesn't provide substantial progress conceptually or experimentally from Eldar et al., 2003, Adleman et al., 2022 and particularly Nahmad and Stathopoulos, 2009. The experimental data and interpretation appear to lack the rigor needed to challenge the model predictions.

The authors pitched the difference between dpp and col in their response to hh dosage change as a tradeoff between robustness and precision. Specifically, the robustness refers to positioning and the precision refers to sharpness, which are somewhat arbitrary - as robustness could also refer to maintaining the sharpness of a expression boundary and precision can also refer to the position. Particularly for dpp, whose developmental significance of stripe position and sharpness is not analyzed (disc growth, pSmad, etc, for example - does a sharper but more mislocated dpp domain help the tissue?). The relationship between positioning and sharpness of a pattern in a morphogen system has been extensively discussed by many authors on a theoretcial level. The authors' theoretical analysis is clear and simple but not new. Experimental evidence indicates that dpp and col are regulated very differently by hh, particularly in terms of timing of response (Nahmad and Stathopoulos, 2009). No comparison of the GRNs from hh to these two genes was made or experimentally tested. It is difficult to conclude that their behaviors in response to hh dosage change are indeed from the hh gradient profile. It is also difficult to speculate if either of these genes (particularly dpp) is facing a true biological tradeoff or tuning back and forth between positioning and sharpness during evolution.

Methods 4.5: To measure widths of gene expression patterns, the authors used a background subtraction, followed by normalization and then thresholded the boundary at 0.2 - this approach firstly is oversimplifying the profile of the expression gradient/profile which could be informative in model testing (e.g., sharpness of dpp?). Secondly, the sequence of the analysis steps may introduce larger errors to lower signal-to-noise images where the subtraction narrows the pattern more than those with higher signal-to-noise (e.g., the 18 degree vs 25 degree images, Fig.6A), this would result in errors in the width measurements. Importantly, disk size and wing size controls are not reported.

Reviewer #2 (Public Review):

In their article titled, van Kerkoerle et al address the timely question of whether non-human primates (rhesus macaques) possess the ability for reverse symbolic inference as observed in humans. Through an fMRI experiment in both humans and monkeys, they analyzed the bold signal in both species while observing audio-visual and visual-visual stimuli pairs that had been previously learned in a particular direction. Remarkably, the findings pertaining to humans revealed that a broad brain network exhibited increased activity in response to surprises occurring in both the learned and reverse directions. Conversely, in monkeys, the study uncovered that the brain activity within sensory areas only responded to the learned direction but failed to exhibit any discernible response to the reverse direction. These compelling results indicate that the capacity for reversible symbolic inference may be specific to humans, even though it remains to be tested in other species.

In general, the manuscript is skillfully crafted and highly accessible to readers. The experimental design exhibits originality, and the analyses are tailored to effectively address the central question at hand. Although the first experiment raised a number of methodological inquiries, the subsequent second experiment thoroughly addresses these concerns and effectively replicates the initial findings, thereby significantly strengthening the overall study. Overall, this article is of high quality and brings new insight into human cognition.

The main limitation of the studies is the sample size of the non-human primate group (n=2 and n=3). Nevertheless, this limitation is carefully addressed and discussed in the manuscript.

Reviewer #2 (Public Review):

This work aims at answering whether activity in primate visual cortex is modulated by locomotion, as was reported for mouse visual cortex. The finding that the activity in mouse visual cortex is modulated by running has changed the concept of primary sensory cortical areas. However, it was an open question whether this modulation generalizes to primates.

To answer this fundamental question the authors established a novel paradigm in which a head-fixed marmoset was able to run on a treadmill while watching a visual stimulus on a display. In addition, eye movements and running speed were monitored continuously and extracellular neuronal activity in primary visual cortex recorded using high-channel-count electrode arrays. This paradigm uniquely permitted to investigate whether locomotion modulates sensory evoked activity in visual cortex of marmoset. Moreover, to directly compare the responses in marmoset visual cortex to responses in mouse visual cortex the authors made use of a publicly-available mouse dataset from the Allen Institute. In this dataset the mouse was also running on a treadmill and observing a set of visual stimuli on a display. The authors took extra care to have the marmoset and mouse paradigms as comparable as possible.

To characterize the visually driven activity the authors present a series of moving gratings and estimate receptive fields with sparse noise. To estimate the gain modulation by running the authors split the dataset into epochs of running and non-running which allowed them to estimate the visually evoked firing rates in both behavioral states.

Strengths:

The novel paradigm of head-fixed marmosets running on a treadmill while being presented with a visual stimulus is unique and ideally tailored to answering the question that the authors aimed to answer. Moreover, the authors took extra care to ensure that the paradigm in marmoset matched as closely as possible to the conditions in the mouse experiments such that the results can be directly compared. To directly compare their data the authors re-analyzed publicly available data from visual cortex of mice recorded at the Allen Institute. Such a direct comparison, and reuse of existing datasets, is another strong aspect of the work. Finally, the presented new marmoset dataset appears to be of high quality, the comparison between mouse and marmoset visual cortex is well done and the results and interpretation straightforward.

Weaknesses:

It is known that the locomotion gain modulation varies with layer in mouse visual cortex, with neurons in the infragranular layers expressing a diversity of modulations (Erisken et al. 2014 Current Biology). However, for the marmoset dataset the layer information was unfortunately not recorded, leaving this point open for future studies.

Nonetheless, the aim of comparing the locomotion induced modulation of activity in primate and mouse primary visual cortex was convincingly achieved by the authors. The results shown in the figures support the conclusion that locomotion modulates the activity in primate and mouse visual cortex differently. While mice show a profound gain increase, neurons in primate visual cortex show little modulation or even a reduction in response strength.

This work will have a strong impact on the field of visual neuroscience but also on neuroscience in general. It revives the debate of whether results obtained in the mouse model system can be simply generalized to other mammalian model systems, such as non-human primates. Based on the presented results, the comparison between the mouse and primate visual cortex is not as straightforward as previously assumed. This will likely trigger more comparative studies between mice and primates in the future, which is important and absolutely needed to advance our understanding of the mammalian brain.

Moreover, the reported finding that neurons in primary visual cortex of marmosets do not increase their activity during running is intriguing, as it makes you wonder why neurons in the mouse visual cortex do so. The authors discuss a few ideas in the paper which can be addressed in future experiments. In this regard it is worth noting that the authors report an interesting difference between the foveal and peripheral part of the visual cortex in marmoset. It will be interesting to investigate these differences in more detail in future studies. Likewise, while running might be an important behavioral state for mice, other behavioral states might be more relevant for marmosets and do modulate the activity of primate visual cortex more profoundly. Future work could leverage the opportunities that the marmoset model system offers to reveal new insights about behavioral related modulation in the primate brain.

Reviewer #2 (Public Review):

This paper addresses the empirical demonstration of "distractor effects" in multi-attribute decision-making. It continues a debate in the literature on the presence (or not) of these effects, which domains they arise in, and their heterogeneity across subjects. The domain of the study is in a particular type of multi-attribute decision-making: choices over risky lotteries. The paper reports a re-analysis of lottery data from multiple experiments run previously by the authors and other labs involved in the debate.

Methodologically, the analysis assumes a number of simple forms for how attributes are aggregated (adaptively, or multiplicatively, or both) and then applies a "reduced form" logistic regression to the choices with a number of interaction terms intended to control for various features of the choice set. One of these interactions, modulated by ternary/binary treatment, is interpreted as a "distractor effect."

The claimed contribution of the re-analysis is to demonstrate correlation in the strength/sign of this treatment effect with another estimated parameter: the relative mixture of additive/multiplicative preferences.

Major Issues

(1) How to Interpret GLM 1 and 2

This paper, and others before it, have used a binary logistic regression with a number of interaction terms to attempt to control for various features of the choice set and how they influence choice. It is important to recognize that this modelling approach is not derived from a theoretical claim about the form of the computational model that guides decision-making in this task, nor an explicit test for a distractor effect. This can be seen most clearly in the equations after line 321 and its corresponding log-likelihood after 354, which contain no parameter or test for "distractor effects". Rather the computational model assumes a binary choice probability, and then shoehorns the test for distractor effects via a binary/ternary treatment interaction in a separate regression (GLM 1 and 2). This approach has already led to multiple misinterpretations in the literature (see Cao & Tsetsos, 2022; Webb et al., 2020). One of these misinterpretations occurred in the datasets the authors study, in which the lottery stimuli contained a confound with the interaction that Chau et al., (2014) were interpreting as a distractor effect (GLM 1). Cao & Tsetsos (2022) demonstrated that the interaction was significant in binary choice data from the study, therefore it can not be caused by a third alternative. This paper attempts to address this issue with a further interaction with the binary/ternary treatment (GLM 2). Therefore the difference in the interaction across the two conditions is claimed to now be the distractor effect. The validity of this claim brings us to what exactly is meant by a "distractor effect."

The paper begins by noting that "Rationally, choices ought to be unaffected by distractors" (line 33). This is not true. There are many normative models which allow for the value of alternatives (even low-valued "distractors") to influence choices, including a simple random utility model. Since Luce (1959), it has been known that the axiom of "Independence of Irrelevant Alternatives" (that the probability ratio between any two alternatives not depend on a third) is an extremely strong axiom, and only a sufficiency axiom for a random utility representation (Block and Marschak, 1959). It is not a necessary condition of a utility representation, and if this is our definition of rational (which is highly debatable), not necessary for it either. Countless empirical studies have demonstrated that IIA is falsified, and a large number of models can address it, including a simple random utility model with independent normal errors (i.e. a multivariate Probit model). In fact, it is only the multinomial Logit model that imposes IIA. It is also why so much attention is paid to the asymmetric dominance effect, which is a violation of a necessary condition for random utility (the Regularity axiom).

So what do the authors even mean by a "distractor effect." It is true that the form of IIA violations (i.e. their path through the probability simplex as the low-option varies) tells us something about the computational model underlying choice (after all, different models will predict different patterns). But we do not know how the interaction terms in the binary logit regression relate to the pattern of the violations because there is no formal theory that relates them. Any test for relative value coding is a joint test of the computational model and the form of the stochastic component (Webb et al,. 2020). These interaction terms may simply be picking up substitution patterns that can be easily reconciled with some form of random utility. While we can not check all forms of random utility in these datasets (because the class of such models is large), this paper doesn't even rule any of these models out.

(2) How to Interpret the Composite (Mixture) model?

On the other side of the correlation is the results from the mixture model for how decision-makers aggregate attributes. The authors report that most subjects are best represented by a mixture between additive and multiplicative aggregation models. The authors justify this with the proposal that these values are computed in different brain regions and then aggregated (which is reasonable, though raises the question of "where" if not the mPFC). But an equally reasonable interpretation is that the improved fit of the mixture model simply reflects a misspecification of two extreme aggregation process (additive and EV), so the log-likelihood is maximized at some point in between them.

One possibility is a model with utility curvature. How much of this result is just due to curvature in valuation? There are many reasonable theories for why we should expect curvature in utility for human subjects (for example, limited perception: Robson, 2001, Khaw, Li Woodford, 2019; Netzer et al., 2022) and of course many empirical demonstrations of risk aversion for small stakes lotteries. The mixture model, on the other hand, has parametric flexibility.

There is also a large literature on testing expected utility jointly with stochastic choice, and the impact of these assumptions on parameter interpretation (Loomes & Sugden, 1998; Apesteguia & Ballester, 2018; Webb, 2019). This relates back to the point above: the mixture may reflect the joint assumption of how choice departs from deterministic EV.

(3) So then how should we interpret the correlation that the authors report?

On one side we have the impact of the binary/ternary treatment which demonstrates some impact of the low value alternative on a binary choice probability. This may reflect some deep flaw in existing theories of choice, or it may simply reflect some departure from purely deterministic expected value maximization that existing theories can address. We have no theory to connect it to, so we cannot tell. On the other side of the correlation with have the mixture between additive and multiplicative preferences over risk. This result may reflect two distinct neural processes at work, or it may simply reflect a misspecification of the manner in which humans perceive and aggregate attributes of a lottery (or even just the stimuli in this experiment) by these two extreme candidates (additive vs. EV). Again, this would entail some departure from purely deterministic expected value maximization that existing theories can address.

It is entirely possible that the authors are reporting a result that points to the more exciting of these two possibilities. But it is also possible (and perhaps more likely) that the correlation is more mundane. The paper does not guide us to theories that predict such a correlation, nor reject any existing ones. In my opinion, we should be striving for theoretically-driven analyses of datasets, where the interpretation of results is clearer.

(4) Finally, the results from these experiments might not have external validity for two reasons. First, the normative criterion for multi-attribute decision-making differs depending on whether the attributes are lotteries or nor (i.e. multiplicative vs additive). Whether it does so for humans is a matter of debate. Therefore if the result is unique to lotteries, it might not be robust for multi-attribute choice more generally. The paper largely glosses over this difference and mixes literature from both domains. Second, the lottery information was presented visually and there is literature suggesting this form of presentation might differ from numerical attributes. Which is more ecologically valid is also a matter of debate.

Minor Issues:

The definition of EV as a normative choice baseline is problematic. The analysis requires that EV is the normative choice model (this is why the HV-LV gap is analyzed and the distractor effect defined in relation to it). But if the binary/ternary interaction effect can be accounted for by curvature of a value function, this should also change the definition of which lottery is HV or LV for that subject!

Comments on latest version: the authors did respond to some of my comments with discussion points in the paper.

References

Apesteguia, J. & Ballester, M. Monotone stochastic choice models: The case of risk and time preferences. Journal of Political Economy (2018).

Block, H. D. & Marschak, J. Random Orderings and Stochastic Theories of Responses. Cowles Foundation Discussion Papers (1959).

Khaw, M. W., Li, Z. & Woodford, M. Cognitive Imprecision and Small-Stakes Risk Aversion. Rev. Econ. Stud. 88, 1979-2013 (2020).

Loomes, G. & Sugden, R. Testing Different Stochastic Specifications of Risky Choice. Economica 65, 581-598 (1998).

Luce, R. D. Indvidual Choice Behaviour. (John Wiley and Sons, Inc., 1959).

Netzer, N., Robson, A. J., Steiner, J. & Kocourek, P. Endogenous Risk Attitudes. SSRN Electron. J. (2022) doi:10.2139/ssrn.4024773.

Robson, A. J. Why would nature give individuals utility functions? Journal of Political Economy 109, 900-914 (2001).

Webb, R. The (Neural) Dynamics of Stochastic Choice. Manage Sci 65, 230-255 (2019).

Reviewer #2 (Public Review):

This paper examined how the activity of neurons in the entopeduncular nucleus (EPN) of mice relates to kinematics, value, and reward. The authors recorded neural activity during an auditory-cued two-alternative choice task, allowing them to examine how neuronal firing relates to specific movements like licking or paw movements, as well as how contextual factors like task stage or proximity to a goal influence the coding of kinematic and spatiotemporal features. The data shows that the firing of individual neurons is linked to kinematic features such as lick or step cycles. However, the majority of neurons exhibited activity related to both movement types, suggesting that EPN neuronal activity does not merely reflect muscle-level representations. This contradicts what would be expected from traditional action selection or action specification models of the basal ganglia.

The authors also show that spatiotemporal variables account for more variability compared to kinematic features alone. Using demixed Principal Component Analysis, they reveal that at the population level, the three principal components explaining the most variance were related to specific temporal or spatial features of the task, such as ramping activity as mice approached reward ports, rather than trial outcome or specific actions. Notably, this activity was present in neurons whose firing was also modulated by kinematic features, demonstrating that individual EPN neurons integrate multiple features. A weakness is that what the spatiotemporal activity reflects is not well specified. The authors suggest some may relate to action value due to greater modulation when approaching a reward port, but acknowledge action value is not well parametrized or separated from variables like reward expectation.

A key goal was to determine whether activity related to expected value and reward delivery arose from a distinct population of EPN neurons or was also present in neurons modulated by kinematic and spatiotemporal features. In contrast to previous studies (Hong & Hikosaka 2008 and Stephenson-Jones et al., 2016), the current data reveals that individual neurons can exhibit modulation by both reward and kinematic parameters. Two potential differences may explain this discrepancy: First, the previous studies used head-fixed recordings, where it may have been easier to isolate movement versus reward-related responses. Second, those studies observed prominent phasic responses to the delivery or omission of expected rewards - responses largely absent in the current paper. This absence suggests a possibility that neurons exhibiting such phasic "reward" responses were not sampled, which is plausible since in both primates and rodents, these neurons tend to be located in restricted topographic regions. Alternatively, in the head-fixed recordings, kinematic/spatial coding may have gone undetected due to the forced immobility.

Overall, this paper offers needed insight into how the basal ganglia output encodes behavior. The EPN recordings from freely moving mice clearly demonstrate that individual neurons integrate reward, kinematic, and spatiotemporal features, challenging traditional models. However, the specific relationship between spatiotemporal activity and factors like action value remains unclear.

Reviewer #2 (Public Review):

Summary:

This is a fundamental and elegant study showing the role of BMP signaling in cerebellar development. This is an important question because there are multiple diseases, including aggressive childhood cancers, which involve granule cell precursors. Thus understanding of the factors that govern the formation of the granule cell layer is important both from a basic science and a disease perspective.

Overall, the manuscript is clear and well-written. The figures are extremely clear, wonderfully informative, and overall quite beautiful.

Figures 1-3 show the experimental design and report how BMP activity is altered over development in both the chick and the human developing cerebellum. Both data is very impressive and convincing.

They then go on to modulate BMP activity in the developing chick, using a complex electroporation paradigm that allows them to label cells with GFP as well as with cell-specific reporters of BMP activity levels. They bidirectionally modulate BMP levels and then can look at both cell-specific and non-specific alterations in the formation of the external and internal granule cell layer, across different developmental timepoints. These are really elegant and rigorous experiments, as they look at both sagittal and transverse sections to collect this data. This makes the data extremely compelling. With these rigorous techniques, they show that BMP signaling serves more than one function across development: it is involved in the initial tangential migration from the rhombic lip, but at a later time, both up- and down-regulation of BMP activity reduces density of amplifying cells in the external granule cell layer.

Strengths:

Overall, I think the paper is interesting and important and the data is strong. The use of both chick and human tissue strengthens the findings. They are extremely rigorous, analyzing data from multiple planes at multiple ages, which also really strengthens their findings. The dual electroporation approach is extremely elegant, providing beautiful visual representations of their findings.

Weaknesses:

I find no significant weaknesses.

Reviewer #2 (Public Review):

Summary:

The authors have addressed the majority of my comments, and I believe the revised manuscript has improved significantly.

The escape behavior of Drosophila larvae includes rolling followed by fast crawling, but the neural mechanism of this sequence was unclear. The authors determined the function of SeIN128, a group of descending neurons that terminate rolling and shorten crawling latency. SeIN128 receives inputs from Basin-2 and A00c neurons, which facilitate rolling, and makes reciprocal inhibitory synapses onto Basin-2 and A00c. SeIN128 shows a delayed activity peak upon Basins or A00c stimulation. Gad staining indicates that SeIN128 neurons are GABAergic, and blocking of SeIN128 function caused increased rolling probability and prolonged rolling. RNAi knockdown of GABA receptors in Basins suggests that several GABA receptors, especially GABA-A-R, mediate the SeIN128 to Basins inhibition. Among Basins subtypes, both Basin-2 and Basin-4 facilitate rolling but SeIN128 specifically terminates rolling elicited by Basin-2 activation. Overall, SeIN128 forms a feedback inhibition ensemble with Basin-2 and A00c that terminates rolling and shifts the animal to crawling.

Overall, this study discovered a neural mechanism that serves as a switch from rolling to fast crawling behaviors in Drosophila larvae. It addressed important open questions of how neural circuits determine the sequence of locomotor behaviors and how animals switch from one behavior to another. Its results support the conclusions and are backed up with proper control experiments.

Strengths:

- The question (i.e., the neural circuitry of action selection) addressed by this study is important.<br /> - Larval and adult Drosophila is a powerful model system in neuroscience study, with rich genetic tools, diverse behaviors, and well-studied nervous systems. This study makes good use of them.<br /> - The experiments, analyses, and results are rigorous and support the major claims. This study combined multiple innovative approaches, such as automated, machine-learning-based behavioral assays, EM reconstruction of larval CNS neurons, and genetic manipulation of specific neurons. A wide range of control experiments enhanced the credibility of the results.<br /> - The graphical representations are clear and mindfully arranged.

Weaknesses:

I believe "Corkscrew-like rolling" is not an accurate term for larval rolling. The neuromuscular basis of rolling was recently studied by Cooney et. al., showing that rolling is the circumferential propagation of muscle activity where all segments contract similarly and synchronously. So using another term instead of "Corkscrew-like rolling" may help.

Reviewer #2 (Public Review):

Summary

The interplay between environmental factors and cognitive performance has been a focal point of neuroscientific research, with illuminance emerging as a significant variable of interest. The hypothalamus, a brain region integral to regulating circadian rhythms, sleep, and alertness, has been posited to mediate the effects of light exposure on cognitive functions. Previous studies have highlighted the role of the hypothalamus in orchestrating bodily responses to light, implicating specific neural pathways such as the orexin and histamine systems, which are crucial for maintaining wakefulness and processing environmental cues. Despite advancements in our understanding, the specific mechanisms through which varying levels of light exposure influence hypothalamic activity and, in turn, cognitive performance, remain inadequately explored. This gap in knowledge underscores the need for high-resolution investigations that can dissect the nuanced impacts of illuminance on different hypothalamic regions. Utilizing state-of-the-art 7 Tesla functional magnetic resonance imaging (fMRI), the present study aims to elucidate the differential effects of light on hypothalamic dynamics and establish a link between regional hypothalamic activity and cognitive outcomes in healthy young adults. By shedding light on these complex interactions, this research endeavours to contribute to the foundational knowledge necessary for developing innovative therapeutic strategies aimed at enhancing cognitive function through environmental modulation.

Strengths:

(1) Considerable Sample Size and Detailed Analysis: The study leverages a robust sample size and conducts a thorough analysis of hypothalamic dynamics, which enhances the reliability and depth of the findings.<br /> (2) Use of High-Resolution Imaging: Utilizing 7 Tesla fMRI to analyze brain activity during cognitive tasks offers high-resolution insights into the differential effects of illuminance on hypothalamic activity, showcasing the methodological rigour of the study.<br /> (3) Novel Insights into Illuminance Effects: The manuscript reveals new understandings of how different regions of the hypothalamus respond to varying illuminance levels, contributing valuable knowledge to the field.<br /> (4) Exploration of Potential Therapeutic Applications: Discussing the potential therapeutic applications of light modulation based on the findings suggests practical implications and future research directions.

The current version of the manuscript addresses previous weaknesses, including details about the illuminance levels, light spectral characteristics used in the MRI study, and light patterns during behavioural tasks. The authors effectively tackle open questions in the field and provide solid evidence that enhances our understanding of the mechanisms underlying the effects of light on cognition.

Reviewer #2 (Public Review):

It is controversial whether liver gremlin-1 expression correlates with liver fibrosis in metabolic dysfunction-associated steatohepatitis (MASH). Horn et al. developed an anti-Gremlin-1 antibody in-house and tested its ability to neutralize gremlin-1 and treat liver fibrosis. This article has the advantage of testing its hypothesis with different animal and human liver fibrosis models and using a variety of research methodologies.

The experimental design and results support the conclusion that the anti-gremlin-1 antibody had no therapeutic effect on treating liver fibrosis, so there are no other suggestions for new experiments:

(1) The authors used RNAscope in situ hybridization to establish the correlation between Gremlin-1 expression and NMSH livers or cell lines.

(2) A luminescent oxygen channelling immunoassay was used to measure circulating Gremlin-1 concentration. They found that Gremlin-1 binds to heparin very efficiently, preventing Gremlin-1 from entering circulation, and restricting Gremlin-1's ability to mediate organ cross-communication.

(3) The authors developed a suitable NMSH rat model which is a choline-deficient, L-amino acid defined high fat 1% cholesterol diet (CDAA-HFD) fed rat model of NMSH, and created a selective anti-Gremlin-1 antibody which is heparin-displacing 0030:HD antibody. They also used human cirrhotic precision-cut liver slices to test their hypotheses. They demonstrated that neutralization of Gremlin-1 activity with monoclonal therapeutic antibodies does not reduce liver inflammation or liver fibrosis.

One concern is that several reagents and assays are made in-house without external validation. Also, will those in-house reagents and assays be available to the science community?

Overall this manuscript provides useful information that gremlin-1 has a limited role in liver fibrosis pathogenesis and treatment.

Reviewer #2 (Public Review):

Summary:

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

The manuscript stands out for its methodological advancements in quantifying thoughts over time and its aim to study the implementation of thoughts in the brain during naturalistic movie watching. However, the conceptualization of thoughts remains vague, its distinction from other concepts like attention is unclear, and interindividual differences are not sufficiently addressed, limiting the study's insights into brain function.

Strengths:

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

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

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

(4) The figures are highly informative, accessible, and consistent, aiding comprehension.

Weaknesses:

(1) The dimensions of thought seem to distinguish between sensory and executive processing states. However, it is unclear if this effect primarily pertains to thinking. I could imagine highly intrusive distractions in movie segments to correlate with stagnating plot development, little change in scenery, or incomprehensible events. Put differently, it may primarily be the properties of the movies that evoke different processing modes, but these properties are not accounted for. For example, I'm wondering whether a simple measure of engagement with stimulus materials could explain the effects just as much. How can the effects of thinking be distinguished from the perceptual and semantic properties of the movie, as well as attentional effects? Is the measure used here capturing thought processes beyond what other factors could explain?

(2) I'm skeptical about taking human thought ratings at face value. Intrusive distraction might imply disengagement from stimulus materials, but it could also be an intended effect of the movie to trigger higher-level, abstract thinking. Can a label like intrusive distraction be misleading without considering the actual thought and movie content?

(3) A jittered sampling approach is used to acquire thought ratings every 15 seconds. Are ratings for the same time point averaged across participants? If so, how consistent are ratings among participants? High consistency would suggest thoughts are mainly stimulus-evoked. Low consistency would question the validity of applying ratings from one (group of) participant(s) to brain-related analyses of another participant.

(4) Using three different movies to conclude that different genres evoke different thought patterns (e.g., line 277) seems like an overinterpretation with only one instance per genre.

(5) I see no indication that results were cross-validated, and no effect sizes are reported, leaving the robustness and strength of effects unknown.

Reviewer #2 (Public Review):

Summary:

This is a very elegant and important EEG study that unifies within a single set of behaviorally equated experimental conditions conscious access (and therefore also conscious access failures) during visual masking and attentional blink (AB) paradigms in humans. By a systematic and clever use of multivariate pattern classifiers across conditions, they could dissect, confirm, and extend a key distinction (initially framed within the GNWT framework) between 'subliminal' and 'pre-conscious' unconscious levels of processing. In particular, the authors could provide strong evidence to distinguish here within the same paradigm these two levels of unconscious processing that precede conscious access : (i) an early (< 80ms) bottom-up and local (in brain) stage of perceptual processing ('local contrast processing') that was preserved in both unconscious conditions, (ii) a later stage and more integrated processing (200-250ms) that was impaired by masking but preserved during AB. On the basis of preexisting studies and theoretical arguments, they suggest that this later stage could correspond to lateral and local recurrent feedback processes. Then, the late conscious access stage appeared as a P3b-like event.

Strengths:

The methodology and analyses are strong and valid. This work adds an important piece in the current scientific debate about levels of unconscious processing and specificities of conscious access in relation to feed-forward, lateral, and late brain-scale top-down recurrent processing.

Weaknesses:

- The authors could improve clarity of the rich set of decoding analyses across conditions.<br /> - They could also enrich their Introduction and Discussion sections by taking into account the importance of conscious influences on some unconscious cognitive processes (revision of traditional concept of 'automaticity'), that may introduce some complexity in Results interpretation<br /> - They should discuss the rich literature reporting high-level unconscious processing in masking paradigms (culminating in semantic processing of digits, words or even small group of words, and pictures) in the light of their proposal (deeper unconscious processing during AB than during masking).

Reviewer #2 (Public Review):

Summary:

The biologically realistic model of the locomotor circuits developed by this group continues to define the state of the art for understanding spinal genesis of locomotion. Here the authors have achieved a new level of analysis of this model to generate surprising and potentially transformative new insights. They show that these circuits can operate in three very distinct states and that, in the intact cord, these states come into successive operation as the speed of locomotion increases. Equally important, they show that in spinal injury the model is "stuck" in the low speed "state machine" behavior.

Strengths:

There are many strengths for the simulation results presented here. The model itself has been closely tuned to match a huge range of experimental data and this has a high degree of plausibility. The novel insight presented here, with the three different states, constitutes a truly major advance in the understanding of neural genesis of locomotion in spinal circuits. The authors systematically consider how the states of the model relate to presently available data from animal studies. Equally important, they provide a number of intriguing and testable predictions. It is likely that these insights are the most important achieved in the past 10 years. It is highly likely proposed multi-state behavior will have a transformative effect on this field.

Weaknesses:

I have no major weaknesses. A moderate concern is that the authors should consider some basic sensitivity analyses to determine if the 3 state behavior is especially sensitive to any of the major circuit parameters - e.g. connection strengths in the oscillators or?

Reviewer #2 (Public Review):

Summary:

The stated ambition of the authors in this manuscript is to thoroughly analyze the complete neural connectome of the three-day larva of the marine annelid Platynereis. This manuscript follows several previous publications by the same group on the same volume of serial EM data, addressing several specialized functional circuits, and supersedes a previous preprint published in 2020. To this end, the authors have annotated the whole cell complement of the larva, including non-neural cells, with the collaborative tool CATMAID, traced the whole neurite extensions of neural cells, and annotated all synapses. The connectome has been algorithmically analyzed to extract the principal modules, adding several new, so far unexplored neural circuits to the list.

Strengths:

This remarkable study adds a third species to the list of animals in which the full connectome and functional modules have been analyzed, alongside C. elegans and Ciona intestinalis. It represents a leap in phylogeny, with Platynereis being a representative of the lophotrochozoans. Also, Platynereis has considerably more neurons than the latter species. The study provides a complete picture of the set of neural modules that are necessary for the survival of an autonomous marine larva with an active lifestyle.

The analysis is particularly impressive for revealing the complete innervation of the entire set of effector cells in the Platynereis larva, including muscle fibers, glands, pigment cells, ciliated cells, and helping understand the overall control of the organism's behavior through multiple sensory pathway integrations. It also reveals layers of neuronal intercalation in sensory-effector pathways that allow further integration even in a larva with limited behavioral complexity. The structure of the developing mushroom bodies, proposed ancestral bilaterian brain sensory integrative units, is detailed, as well as a complex mechanosensory module specific to a swimming larva.

A key new aspect of this connectome study is the thorough analysis of segmental cell types and intersegmental connectivity. Metameric organization is widespread in bilaterians and is nowhere clearer than in annelids. This metameric organization is even proposed by some authors to be an ancestral trait of bilaterians. Here, the authors show that homologous cell types and connectivity are shared not only by all segments of the animal but also by its non-segmental terminal parts (anterior prostomium and posterior pygidium). They suggest, in turn, that the entire body of the annelid may be formed of ancestral metameric units, an idea proposed before but here strongly supported by a list of homologous cell types. This is the most thorough evidence obtained so far for this provocative and stimulating evolutionary hypothesis.

Reviewer #2 (Public Review):

Summary:

The authors repeatedly measured the behavior of individual flies across several environmental situations in custom-made behavioral phenotyping rigs.

Strengths:

The study uses several different behavioral phenotyping devices to quantify individual behavior in a number of different situations and over time. It seems to be a very impressive amount of data. The authors also make all their behavioral phenotyping rig design and tracking software available, which I think is great and I'm sure other folks will be interested in using and adapting it to their own needs.

Weaknesses/Limitations:

I think an important limitation is that while the authors measured the flies under different environmental scenarios (i.e. with different lighting and temperature) they didn't really alter the "context" of the environment. At least within behavioral ecology, context would refer to the potential functionality of the expressed behaviors so for example, an anti-predator context, a mating context, or foraging. Here, the authors seem to really just be measuring aspects of locomotion under benign (relatively low-risk perception) contexts. This is not a flaw of the study, but rather a limitation to how strongly the authors can really say that this demonstrates that individuality is generalized across many different contexts. It's quite possible that rank order of locomotor (or other) behaviors may shift when the flies are in a mating or risky context.

The analytical framework in terms of statistical methods is lacking. It appears as though the authors used correlations across time/situations to estimate individual variation; however, far more sophisticated and elegant methods exist. The paper would be a lot stronger, and my guess is, much more streamlined if the authors employ hierarchical mixed models to analyse these data these models could capture and estimate differences in individual behavior across time and situations simultaneously. Along with this, it's currently unclear whether and how any statistical inference was performed. Right now, it appears as though any results describing how individuality changes across situations are largely descriptive (i.e. a visual comparison of the strengths of the correlation coefficients?).

Another pretty major weakness is that right now, I can't find any explicit mention of how many flies were used and whether they were re-used across situations. Some sort of overall schematic showing exactly how many measurements were made in which rigs and with which flies would be very beneficial.

I don't necessarily doubt the robustness of the results and my guess is that the author's interpretations would remain the same, but a more appropriate modeling framework could certainly improve their statistical inference and likely highlight some other cool patterns as these methods could better estimate stability and covariance in individual intercepts (and potentially slopes) across time and situation.

Reviewer #2 (Public Review):

Summary:

Yonk and colleagues show that the posterior medial thalamus (POm), which is interconnected with sensory and motor systems, projects directly to major categories of neurons in the striatum, including direct and indirect pathway MSNs, and PV interneurons. Activity in POm-striatal neurons during a sensory-based learning task indicates a relationship between reward expectation and arousal. Inhibition of these neurons slows reaction to stimuli and overall learning. This circuit is positioned to feed salient event activation to the striatum to set the stage for effective learning and action selection.

Strengths:

The results are well presented and offer interesting insight into an understudied thalamostriatal circuit. In general, this work is important as part of a general need for an increased understanding of thalamostriatal circuits in complex learning and action selection processes, which have generally received less attention than corticostriatal systems.

Weaknesses:

There could be a stronger connection between the connectivity part of the data - showing that POm neurons context D1, D2, and PV neurons in the striatum but with some different properties - and the functional side of the project. One wonders whether the POm neurons projecting to these subtypes or striatal neurons have unique signaling properties related to learning, or if there is a uniform, bulk signal sent to the striatum. This is not a weakness per se, as it's reasonable for these questions to be answered in future papers.

All the in vivo activity-related conclusions stem from data from just 5 mice, which is a relatively small sample set. Optogenetic groups are also on the small side.

Reviewer #2 (Public Review):

Summary

The study investigated whether memory retrieval followed soon by extinction training results in a short-term memory deficit when tested - with a reinstatement test that results in recovery from extinction - soon after extinction training. Experiment 1 documents this phenomenon using a between-subjects design. Experiment 2 used a within-subject control and saw that the effect was also observed in a control condition. In addition, it also revealed that if testing is conducted 6 hours after extinction, there is no effect of retrieval prior to extinction as there is recovery from extinction independently of retrieval prior to extinction. A third group also revealed that retrieval followed by extinction attenuates reinstatement when the test is conducted 24 hours later, consistent with previous literature. Finally, Experiment 3 used continuous theta-burst stimulation of the dorsolateral prefrontal cortex and assessed whether inhibition of that region (vs a control region) reversed the short-term effect revealed in Experiments 1 and 2. The results of the control groups in Experiment 3 replicated the previous findings (short-term effect), and the experimental group revealed that these can be reversed by inhibition of the dorsolateral prefrontal cortex.

Strengths

The work is performed using standard procedures (fear conditioning and continuous theta-burst stimulation) and there is some justification for the sample sizes. The results replicate previous findings - some of which have been difficult to replicate and this needs to be acknowledged - and suggest that the effect can also be observed in a short-term reinstatement test.

The study establishes links between memory reconsolidation and retrieval-induced forgetting (or memory suppression) literature. The explanations that have been developed for these are distinct and the current results integrate these, by revealing that the DLPFC activity involved in retrieval-extinction short-term effect. There is thus some novelty in the present results, but numerous questions remain unaddressed.

Weakness

The fear acquisition data is converted to a differential fear SCR and this is what is analysed (early vs late). However, the figure shows the raw SCR values for CS+ and CS- and therefore it is unclear whether the acquisition was successful (despite there being an "early" vs "late" effect - no descriptives are provided).

In Experiment 1 (Test results) it is unclear whether the main conclusion stems from a comparison of the test data relative to the last extinction trial ("we defined the fear recovery index as the SCR difference between the first test trial and the last extinction trial for a specific CS") or the difference relative to the CS- ("differential fear recovery index between CS+ and CS-"). It would help the reader assess the data if Figure 1e presents all the indexes (both CS+ and CS-). In addition, there is one sentence that I could not understand "there is no statistical difference between the differential fear recovery indexes between CS+ in the reminder and no reminder groups (P=0.048)". The p-value suggests that there is a difference, yet it is not clear what is being compared here. Critically, any index taken as a difference relative to the CS- can indicate recovery of fear to the CS+ or absence of discrimination relative to the CS-, so ideally the authors would want to directly compare responses to the CS+ in the reminder and no-reminder groups. The latter issue is particularly relevant in Experiment 2, in which the CS- seems to vary between groups during the test and this can obscure the interpretation of the result.

In Experiment 1, the findings suggest that there is a benefit of retrieval followed by extinction in a short-term reinstatement test. In Experiment 2, the same effect is observed on a cue that did not undergo retrieval before extinction (CS2+), a result that is interpreted as resulting from cue-independence, rather than a failure to replicate in a within-subjects design the observations of Experiment 1 (between-subjects). Although retrieval-induced forgetting is cue-independent (the effect on items that are suppressed [Rp-] can be observed with an independent probe), it is not clear that the current findings are similar. Here, both cues have been extinguished and therefore been equally exposed during the critical stage.

The findings in Experiment 2 suggest that the amnesia reported in Experiment 1 is transient, in that no effect is observed when the test is delayed by 6 hours. The phenomena whereby reactivated memories transition to extinguished memories as a function of the amount of exposure (or number of trials) is completely different from the phenomena observed here. In the former, the manipulation has to do with the number of trials (or the total amount of time) that the cues are exposed to. In the current study, the authors did not manipulate the number of trials but instead the retention interval between extinction and test. The finding reported here is closer to a "Kamin effect", that is the forgetting of learned information which is observed with intervals of intermediate length (Baum, 1968). Because the Kamin effect has been inferred to result from retrieval failure, it is unclear how this can be explained here. There needs to be much more clarity on the explanations to substantiate the conclusions.

There are many results (Ryan et al., 2015) that challenge the framework that the authors base their predictions on (consolidation and reconsolidation theory), therefore these need to be acknowledged. Similarly, there are reports that failed to observe the retrieval-extinction phenomenon (Chalkia et al., 2020), and the work presented here is written as if the phenomenon under consideration is robust and replicable. This needs to be acknowledged.

The parallels between the current findings and the memory suppression literature are speculated in the general discussion, and there is the conclusion that "the retrieval-extinction procedure might facilitate a spontaneous memory suppression process". Because one of the basic tenets of the memory suppression literature is that it reflects an "active suppression" process, there is no reason to believe that in the current paradigm, the same phenomenon is in place, but instead, it is "automatic". In other words, the conclusions make strong parallels with the memory suppression (and cognitive control) literature, yet the phenomena that they observed are thought to be passive (or spontaneous/automatic).<br /> Ultimately, it is unclear why 10 mins between the reminder and extinction learning will "automatically" suppress fear memories. Further down in the discussion, it is argued that "For example, in the well-known retrieval-induced forgetting (RIF) phenomenon, the recall of a stored memory can impair the retention of related long-term memory and this forgetting effect emerges as early as 20 minutes after the retrieval procedure, suggesting memory suppression or inhibition can occur in a more spontaneous and automatic manner". I did not follow with the time delay between manipulation and test (20 mins) would speak about whether the process is controlled or automatic.

Among the many conclusions, one is that the current study uncovers the "mechanism" underlying the short-term effects of retrieval extinction. There is little in the current report that uncovers the mechanism, even in the most psychological sense of the mechanism, so this needs to be clarified. The same applies to the use of "adaptive".

Whilst I could access the data on the OFS site, I could not make sense of the Matlab files as there is no signposting indicating what data is being shown in the files. Thus, as it stands, there is no way of independently replicating the analyses reported.

The supplemental material shows figures with all participants, but only some statistical analyses are provided, and sometimes these are different from those reported in the main manuscript. For example, the test data in Experiment 1 is analysed with a two-way ANOVA with the main effects of group (reminder vs no-reminder) and time (last trial of extinction vs first trial of the test) in the main report. The analyses with all participants in the sup mat used a mixed two-way ANOVA with a group (reminder vs no reminder) and CS (CS+ vs CS-). This makes it difficult to assess the robustness of the results when including all participants. In addition, in the supplementary materials, there are no figures and analyses for Experiment 3.

One of the overarching conclusions is that the "mechanisms" underlying reconsolidation (long term) and memory suppression (short term) phenomena are distinct, but memory suppression phenomena can also be observed after a 7-day retention interval (Storm et al., 2012), which then questions the conclusions achieved by the current study.

References:

Baum, M. (1968). Reversal learning of an avoidance response and the Kamin effect. Journal of Comparative and Physiological Psychology, 66(2), 495.<br /> Chalkia, A., Schroyens, N., Leng, L., Vanhasbroeck, N., Zenses, A. K., Van Oudenhove, L., & Beckers, T. (2020). No persistent attenuation of fear memories in humans: A registered replication of the reactivation-extinction effect. Cortex, 129, 496-509.<br /> Ryan, T. J., Roy, D. S., Pignatelli, M., Arons, A., & Tonegawa, S. (2015). Engram cells retain memory under retrograde amnesia. Science, 348(6238), 1007-1013.<br /> Storm, B. C., Bjork, E. L., & Bjork, R. A. (2012). On the durability of retrieval-induced forgetting. Journal of Cognitive Psychology, 24(5), 617-629.

Reviewer #2 (Public Review):

Summary

The authors present multiple machine-learning methodologies to predict post-stroke epilepsy (PSE) from admission clinical data.

Strengths

The Statistical Approach section is very well written. The approaches used in this section are very sensible for the data in question.

Weaknesses

There are many typos and unclear statements throughout the paper.

There are some issues with SHAP interpretation. SHAP in its default form, does not provide robust statistical guarantees of effect size. There is a claim that "SHAP analysis showed that white blood cell count had the greatest impact among the routine blood test parameters". This is a difficult claim to make.

The Data Collection section is very poorly written, and the methodology is not clear.

There is no information about hyperparameter selection for models or whether a hyperparameter search was performed. Given this, it is difficult to conclude whether one machine learning model performs better than others on this task.

The inclusion and exclusion criteria are unclear - how many patients were excluded and for what reasons?

There is no sensitivity analysis of the SMOTE methodology: How many synthetic data points were created, and how does the number of synthetic data points affect classification accuracy?

Did the authors achieve their aims? Do the results support their conclusions?

The paper does not clarify the features' temporal origins. If some features were not recorded on admission to the hospital but were recorded after PSE occurred, there would be temporal leakage.

The authors claim that their models can predict PSE. To believe this claim, seeing more information on out-of-distribution generalisation performance would be helpful. There is limited reporting on the external validation cohort relative to the reporting on train and test data.

For greater certainty on all reported results, it would be most appropriate to perform n-fold cross-validation, and report mean scores and confidence intervals across the cross-validation splits

The likely impact of the work on the field

If this model works as claimed, it will be useful for predicting PSE. This has some direct clinical utility.

Analysis of features contributing to PSE may provide clinical researchers with ideas for further research on the underlying aetiology of PSE.

Additional context that might help readers

The authors show force plots and decision plots from SHAP values. These plots are non-trivial to interpret, and the authors should include an explanation of how to interpret them.

Reviewer #2 (Public Review):

Summary:

The study aims to probe the neural correlates of visual serial dependence - the phenomenon that estimates of a visual feature (here motion direction) are attracted towards the recent history of encoded and reported stimuli. The authors utilize an established retro-cue working memory task together with magnetoencephalography, which allows to probe neural representations of motion direction during encoding and retrieval (retro-cue) periods of each trial. The main finding is that neural representations of motion direction are not systematically biased during the encoding of motion stimuli, but are attracted towards the motion direction of the previous trial's target during the retrieval (retro-cue period), just prior to the behavioral response. By demonstrating a neural signature of attractive biases in working memory representations, which align with attractive behavioral biases, this study highlights the importance of post-encoding memory processes in visual serial dependence.

Strengths:

The main strength of the study is its elegant use of a retro-cue working memory task together with high temporal resolution MEG, enabling to probe neural representations related to stimulus encoding and working memory. The behavioral task elicits robust behavioral serial dependence and replicates previous behavioral findings by the same research group. The careful neural decoding analysis benefits from a large number of trials per participant, considering the slow-paced nature of the working memory paradigm. This is crucial in a paradigm with considerable trial-by-trial behavioral variability (serial dependence biases are typically small, relative to the overall variability in response errors). While the current study is broadly consistent with previous studies showing that attractive biases in neural responses are absent during stimulus encoding (previous studies reported repulsive biases), to my knowledge it is the first study showing attractive biases in current stimulus representations during working memory. The study also connects to previous literature showing reactivations of previous stimulus representations, although the link between reactivations and biases remains somewhat vague in the current manuscript. Together, the study reveals an interesting avenue for future studies investigating the neural basis of visual serial dependence.

Weaknesses:

The main weakness of the current manuscript is that the authors could have done more analyses to address the concern that their neural decoding results are driven by signals related to eye movements. The authors show that participants' gaze position systematically depended on the current stimuli's motion directions, which together with previous studies on eye movement-related confounds in neural decoding justifies such a concern. The authors seek to rule out this confound by showing that the consistency of stimulus-dependent gaze position does not correlate with (a) the neural reconstruction fidelity and (b) the repulsive shift in reconstructed motion direction. However, both of these controls do not directly address the concern. If I understand correctly the metric quantifying the consistency of stimulus-dependent gaze position (Figure S3a) only considers gaze angle and not gaze amplitude. Furthermore, it does not consider gaze position as a function of continuous motion direction, but instead treats motion directions as categorical variables. Therefore, assuming an eye movement confound, it is unclear whether the gaze consistency metric should strongly correlate with neural reconstruction fidelity, or whether there are other features of eye movements (e.g., amplitude differences across participants, and tuning of gaze in the continuous space of motion directions) which would impact the relationship with neural decoding. Moreover, it is unclear whether the consistency metric, which does not consider history dependencies in eye movements, should correlate with attractive history biases in neural decoding. It would be more straightforward if the authors would attempt to (a) directly decode stimulus motion direction from x-y gaze coordinates and relate this decoding performance to neural reconstruction fidelity, and (b) investigate whether gaze coordinates themselves are history-dependent and are attracted to the average gaze position associated with the previous trials' target stimulus. If the authors could show that (b) is not the case, I would be much more convinced that their main finding is not driven by eye movement confounds.

I am not convinced by the across-participant correlation between attractive biases in neural representations and attractive behavioral biases in estimation reports. One would expect a correlation with the behavioral bias amplitude, which is not borne out. Instead, there is a correlation with behavioral bias width, but no explanation of how bias width should relate to the bias in neural representations. The authors could be more explicit in their arguments about how these metrics would be functionally related, and why there is no correlation with behavioral bias amplitude.

The sample size (n = 10) is definitely at the lower end of sample sizes in this field. The authors collected two sessions per participant, which partly alleviates the concern. However, given that serial dependencies can be very variable across participants, I believe that future studies should aim for larger sample sizes.

It would have been great to see an analysis in source space. As the authors mention in their introduction, different brain areas, such as PPC, mPFC, and dlPFC have been implicated in serial biases. This begs the question of which brain areas contribute to the serial dependencies observed in the current study. For instance, it would be interesting to see whether attractive shifts in current representations and pre-stimulus reactivations of previous stimuli are evident in the same or different brain areas.

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What is this?

Reviewer #2 (Public Review):

Summary:

In this study, Yagasaki et al. describe an organoid system to study the interactions between smooth muscle cells (SMCs) and interstitial cells of Cajal (ICCs). While these interactions are essential for the control of rhythmic intestinal contractility (i.e., peristalsis), they are poorly understood, largely due to the complexity of and access to the in vivo environment and the inability to co-culture these cell types in vitro for long term under physiological conditions. The "gut contractile organoids" organoids described herein are reconstituted from stromal cells of the fetal chicken hindgut that rapidly reorganize into multilayered spheroids containing an outer layer of smooth muscle cells and an inner core of interstitial cells. The authors demonstrate that they contract cyclically and additionally use calcium imagining to show that these contractions occur concomitantly with calcium transients that initiate in the interstitial cell core and are synchronized within the organoid and between ICCs and SMCs. Furthermore, they use several pharmacological inhibitors to show that these contractions are dependent upon non-muscle myosin activity and, surprisingly, independent of gap junction activity. Finally, they develop a 3D hydrogel for the culturing of multiple organoids and found that they synchronize their contractile activities through interconnecting smooth muscle cells, suggesting that this model can be used to study the emergence of pacemaking activities. Overall, this study provides a relatively easy-to-establish organoid system that will be of use in studies examining the emergence of rhythmic peristaltic smooth muscle contractions and how these are regulated by interstitial cell interactions. However, further validation and quantification will be necessary to conclusively determine show the cellular composition of the organoids and how reproducible their behaviors are.

Strengths:

This work establishes a new self-organizing organoid system that can easily be generated from the muscle layers of the chick fetal hindgut to study the emergence of spontaneous smooth muscle cell contractility. A key strength of this approach is that the organoids seem to contain few cell types (though more validation is needed), namely smooth muscle cells (SMCs) and interstitial cells of Cajal (ICCs). These organoids are amenable to live imaging of calcium dynamics as well as pharmacological perturbations for functional assays, and since they are derived from developing tissues, the emergence of the interactions between cell types can be functionally studied. Thus, the gut contractile organoids represent a reductionist system to study the interactions between SMCs and ICCs in comparison to the more complex in vivo environment, which has made studying these interactions challenging.

Weaknesses:

The study falls short in the sense that it does not provide a rigorous amount of evidence to validate that the gut organoids are made of bona fide smooth muscle cells and ICCs. For example, only two "marker" proteins are used to support the claims of cell identity of SMCs and ICCs. At the same time, certain aspects of the data are not quantified sufficiently to appreciate the variance of organoid rhythmic contractility. For example, most contractility plots show the trace for a single organoid. This leads to a concern for how reproducible certain aspects of the organoid system (e.g. wavelength between contractions/rhythm) might be, or how these evolve uniquely over time in culture. Furthermore, while this study might be able to capture the emergence of ICC-SMC interactions as they related to muscle contraction and pacemaking, it is unclear how these interactions relate to adult gastrointestinal physiology given that the organoids are derived from fetal cells that might not be fully differentiated or might have distinct functions from the adult. Finally, despite the strength of this system, discoveries made in it will need to be validated in vivo.

Reviewer #2 (Public Review):

Summary:

The manuscript by Dearlove et al. entitled "DTX3L ubiquitin ligase ubiquitinates single-stranded nucleic acids" reports a novel activity of a DELTEX E3 ligase family member, DTX3L, which can conjugate ubiquitin to the 3' hydroxyl of single-stranded oligonucleotides via an ester linkage. The findings that unmodified oligonucleotides can act as substrates for direct ubiquitylation and the identification of DTX3 as the enzyme capable of performing such oligonucleotide modification are novel, intriguing, and impactful because they represent a significant expansion of our view of the ubiquitin biology. The authors perform a detailed and diligent biochemical characterization of this novel activity, and key claims made in the article are well supported by experimental data. However, the studies leave room for some healthy skepticism about the physiological significance of the unique activity of DTX3 and DTX3L described by the authors because DTX3/DTX3L can also robustly attach ubiquitin to the ADP ribose moiety of NAD or ADP-ribosylated substrates. The study could be strengthened by a more direct and quantitative comparison between ubiquitylation of unmodified oligonucleotides by DTX3/DTX3L with the ubiquitylation of ADP-ribose, the activity that DTX3 and DTX3L share with the other members of the DELTEX family.

Strengths:

The manuscript reports a novel and exciting observation that ubiquitin can be directly attached to the 3' hydroxyl of unmodified, single-stranded oligonucleotides by DTX3L. The study builds on the extensive expertise and the impactful previous studies by the Huang laboratory of the DELTEX family of E3 ubiquitin ligases. The authors perform a detailed and diligent biochemical characterization of this novel activity, and all claims made in the article are well supported by experimental data. The manuscript is clearly written and easy to read, which further elevates the overall quality of submitted work. The findings are impactful and will help illuminate multiple avenues for future follow-up investigations that may help establish how this novel biochemical activity observed in vitro may contribute to the biological function of DTX3L. The authors demonstrate that the activity is unique to the DTX3/DTX3L members of the DELTEX family and show that the enzyme requires at least two single-stranded nucleotides at the 3' end of the oligonucleotide substrate and that the adenine nucleotide is preferred in the 3' position. Most notably, the authors describe a chimeric construct containing RING domain of DTX3L fused to the DTC domain DTX2, which displays robust NAD ubiquitylation, but lacks the ability to ubiquitylate unmodified oligonucleotides. This construct will be invaluable in the future cell-based studies of DTX3L biology that may help establish the physiological relevance of 3' ubiquitylation of nucleic acids.

Weaknesses:

The main weakness of the study is in the lack of direct evidence that the ubiquitylation of unmodified oligonucleotides reported by the authors plays any role in the biological function of DTX3L. The study leaves plenty of room for natural skepticism regarding the physiological relevance of the reported activity, because, akin to other DELTEX family members, DTX3 and DTX3L can also catalyze attachment of ubiquitin to NAD, ADP ribose and ADP-ribosylated substrates. Unfortunately, the study does not offer any quantitative comparison of the two distinct activities of the enzyme, which leaves plenty of room for doubt. One is left wondering, whether ubiquitylation of unmodified oligonucleotides is just a minor and artifactual side activity owing to the high concentration of the oligonucleotide substrates and E2~Ub conjugates present in the in-vitro conditions and the somewhat lower specificity of the DTX3 and DTX3L DTC domains (compared to DTX2 and other DELTEX family members) for ADP ribose over other adenine-containing substrates such as unmodified oligonucleotides, ADP/ATP/dADP/dATP, etc. The intriguing coincidence that DTX3L, which is the only DTX protein capable of ubiquitylating unmodified oligonucleotides, is also the only family member that contains nucleic acid interacting domains in the N-terminus, is suggestive but not compelling. A recently published DTX3L study by a competing laboratory (PMID: 38000390), which is not cited in the manuscript, suggests that ADP-ribose-modified nucleic acids could be the physiologically relevant substrates of DTX3L. That competing hypothesis appears more convincing than ubiquitylation of unmodified oligonucleotides because experiments in that study demonstrate that ubiquitylation of ADP-ribosylated oligos is quite robust in comparison to ubiquitylation of unmodified oligos, which is undetectable. It is possible that the unmodified oligonucleotides in the competing study did not have adenine in the 3' position, which may explain the apparent discrepancy between the two studies. In summary, a quantitative comparison of ubiquitylation of ADP ribose vs. unmodified oligonucleotides could strengthen the study.

Reviewer #2 (Public Review):

SUMMARY

This manuscript by Knudsen-Palmer et al. describes and models the contribution of MUT-16 and RDE-10 in the silencing through RNAi by the Argonaute protein NRDE-3 or others. The authors show that MUT-16 and RDE-10 constitute an intersecting network that can be redundant or not depending on the gene being targeted by RNAi. In addition, the authors provide evidence that increasing dsRNA processing can compensate for NRDE-3 mutants. Overall, the authors provide convincing evidence to understand the factors involved in RNAi in C. elegans by using a genetic approach.

MAJOR STRENGTHS

The author's work presents a compelling case for understanding the intricacies of RNA interference (RNAi) within the model organism Caenorhabditis elegans through a meticulous genetic approach. By harnessing genetic manipulation, they delve into the role of MUT-16 and RDE-10 in RNAi, offering a nuanced understanding of the molecular mechanisms at play in two independent case study targets (unc-22 and bli-1).

MAJOR WEAKNESSES

(1) It is unclear how the molecular mechanisms of amplification are different under the MUT-16 and RDE-10 branches of the regulatory pathway, since they are clearly distinct proteins structurally. It would be interesting to do some small-RNA-seq of products generated from unc-22 and bli-1, on wild-type conditions and some of the mutants studied (eg. mut-16, rde-10 and mut-16 + rde-10). That would provide some insights on whether the products of the 2 amplifications are the same in all conditions, just changing in abundance, or whether they are distinct in sequence patterns.

(2) In the same line, Figure 5 aims to provide insights to the sequence determinants that influence on the RNAi of bli-1. It is unclear whether the changes in transcript stability dictated by the 3'UTR are the sole factor governing the preference for the MUT-16 and RDE-10 branches of the regulatory pathway. In line with the mutant jam297, it might be interesting to test whether factors like codon optimality, splicing, ... of the ORF region upstream from bli-1-dsRNA can affect its sensitivity to the MUT-16 and RDE-10 branches of the regulatory pathway.

Reviewer #2 (Public Review):

Summary:

This study presents a significant finding that enhances our understanding of spermatogenesis. TMC7 belongs to a family of transmembrane channel-like proteins (TMC1-8), primarily known for their role in the ear. Mutations to TMC1/2 are linked to deafness in humans and mice and were originally characterized as auditory mechanosensitive ion channels. However, the function of the other TMC family members remains poorly characterized. In this study, the authors begin to elucidate the function of TMC7 in acrosome biogenesis during spermatogenesis. Through analysis of transcriptomics datasets, they elevated levels of TMC7 in round spermatids in both mouse and human testis. They then generate Tmc7-/- mice and find that male mice exhibit smaller testes and complete infertility. Examination of different developmental stages reveals spermatogenesis defects, including with reduced sperm count, elongated spermatids and large vacuoles. Additionally, abnormal acrosome morphology are observed beginning at the early-stage Golgi phase, indicating TMC7's involvement in proacrosomal vesicle trafficking and fusion. They observed localization of TMC7 in the cis-Golgi and suggest that its presence is required for maintaining Golgi integrity, with Tmc7-/- leading to reduced intracellular Ca2+, elevated pH and increased ROS levels, likely resulting in spermatid apoptosis. Overall, the work delineates a new function of TMC7 in spermatogenesis and the authors propose that its ion channel and/or scramblase activity is likely important for Golgi homeostasis. This work is of significant interest to the community and is of high quality.

Strengths:

The biggest strength of the paper is the phenotypic characterization of the TMC7-/- mouse model, which has clear acrosome biogenesis/spermatogenesis defects. This is the main claim of the paper and it is supported with the data that are presented.

Weaknesses:

It isn't clear whether TMC7 functions as an ion channel from the current data presented in this paper, but the authors are careful in their interpretation and present this merely as a hypothesis supporting this idea.

Reviewer #2 (Public Review):

In the manuscript "Modulation of α-Synuclein Aggregation Amid Diverse Environmental Perturbation", Wasim et al describe coarse-grained molecular dynamics (cgMD) simulations of α-Synuclein (aSyn) at several concentrations and in the presence of molecular crowding agents or high salt. They begin by bench-marking their cgMD against all-atom simulations by Shaw. They then carry 2.4-4.3 µs cgMD simulations under the above-noted conditions and analyze the data in terms of protein structure, interaction network analysis, and extrapolated fluid mechanics properties. This is an interesting study because a molecular scale understanding of protein droplets is currently lacking.

Reviewer #3 (Public Review):

Fister and colleagues use regeneration of the larval zebrafish caudal fin to compare the effects of two modes of tissue damage-transection and burn-on cutaneous sensory axon regeneration. The authors found that restoration of sensory axon density and function is delayed following burn injury compared to transection.

The authors hypothesized that thermal injury triggers signals within the wound microenvironment that impair sensory neuron regeneration. The authors identify differences in the responses of epithelial keratinocytes to the two modes of injury: keratinocytes migrate in response to burn but not transection. Inhibiting keratinocyte migration with a small-molecule inhibitor of Arp2/3 (CK666) resulted in decreased production of reactive oxygen species (ROS) at early, but not late, timepoints. Preventing keratinocyte migration by wounding in isotonic media resulted in increased sensory function 24 hours after burn.

Strengths of the study include the beautiful imaging and rigorous statistical approaches used by the authors. The ability to assess both axon density and axon function during regeneration is quite powerful. The touch assay adds a unique component to the paper and strengthens the argument that burns are more damaging to sensory structures and that different treatments help to ameliorate this.

A weakness of the study is the lack of genetic and cell autonomous manipulations. Additional comparisons between transection and burns, in particular with manipulations that specifically modulate ROS generation or cell migration without potentially confounding effects on other cell types or processes would help to strengthen the manuscript. In terms of framing their results, the authors refer to "sensory neurons" and "sensory axons" throughout the text - it should be made clear what type of neuron(s)/axon(s) are being visualized/assayed. Along these lines, a broader discussion of how burn injuries affect sensory function in other systems-and how the authors' results might inform our understanding of these injury responses-would be beneficial to the reader.

In summary, the authors have established a tractable vertebrate system to investigate different sensory axon wound healing outcomes in vivo that may ultimately allow for the identification of improved treatment strategies for human burn patients. Although the study implicates differences in keratinocyte migration and associated ROS production in sensory axon wound healing outcomes, the links between these processes could be more rigorously established.

Reviewer #2 (Public Review):

Summary:

Proteins that bind to double-stranded RNA regulate various cellular processes, including gene expression and viral recognition. Such proteins often contain multiple double-stranded RNA-binding domains (dsRBDs) that play an important role in target search and recognition. In this work, Chug and colleagues have characterized the backbone dynamics of one of the dsRBDs of a protein called TRBP2, which carries two tandem dsRBDs. Using solution NMR spectroscopy, the authors characterize the backbone motions of dsRBD2 in the absence and presence of dsRNA and compare these with their previously published results on dsRBD1. The authors show that dsRBD2 is comparatively more rigid than dsRBD1 and claim that these differences in backbone motions are important for target recognition.

Strengths:

The strengths of this study are multiple solution NMR measurements to characterize the backbone motions of dsRBD2. These include 15N-R1, R2, and HetNOE experiments in the absence and presence of RNA and the analysis of these data using an extended-model-free approach; HARD-15N-experiments and their analysis to characterize the kex. The authors also report differences in binding affinities of dsRBD1 and dsRBD2 using ITC and have performed MD simulations to probe the differential flexibility of these two domains.

Weaknesses:

While it may be true that dsRBD2 is more rigid than dsRBD1, the manuscript lacks conclusive and decisive proof that such changes in backbone dynamics are responsible for target search and recognition and for the diffusion of TRBP2 along the RNA molecule.

Reviewer #2 (Public Review):

Summary:

Wu et al. explores the role of the histone reader protein SntB in Aspergillus flavus. They not only studied its function related to the growth, development, and secondary metabolite through gene knockout and complement, but also explored the underlying potential mechanisms by RNA-seq and ChIP-seq. The response of oxidative stress in ΔsntB strain and ΔcatC strain were further analyzed. Their study revealed a potential machinery that SntB regulated fungal morphogenesis, mycotoxin anabolism, and fungal virulence through the axle of from epigenetic modification to fungal virulence and mycotoxin bio-synthesis via SntB, i.e. H3K36me3 modification-SntB-Peroxisomes-Lipid hydrolysis-fungal virulence and mycotoxin bio-synthesis. This work is of great significance in revealing the regulatory mechanisms of pathogenic fungi in toxin production, pathogenicity, and in its prevention and pollution control.

Strengths:

One of the main advantages of this study is that the author constructed HA fused strains for ChIP seq analysis, rather than using antibodies related to epigenetic modifications. Nancy et al. reported the functions of sntB as a histone methylation regulator, but in addition to being an epigenetic regulator, there are also reports that it has transcriptional regulatory activity. Through integration analysis with RNA-seq data, it was found that SntB played key roles in oxidative stress response of A. flavus. This study can increase our understanding of more functions of the SntB in A. flavus.

Weaknesses:

The authors only studied the function of catC among the 7 genes related to oxidative response listed in Table S14.

Reviewer #2 (Public Review):

This study established an alternate way of p53 inactivation and proposed PITAR as a potential therapeutic target, so the impact is high. In addition, this manuscript has apparent strengths, including a logically designed research strategy, in vitro and in vivo study, and well-designed control.

This manuscript identified a long noncoding RNA, PITAR (p53 Inactivating TRIM28 associated RNA), as an inhibitor of p53. PITAR is highly expressed in glioblastoma (GBM) and glioma stem-like cells (GSC). The authors found that TRIM28 mRNA, which encodes a p53-specific E3 ubiquitin ligase, is a direct target of PITAR. PITAR interaction with TRIM28 RNA stabilized TRIM28 mRNA, which resulted in increased TRIM28 protein levels, enhanced p53 ubiquitination, and attenuated DNA damage response. While PITAR silencing inhibited the growth of WT p53 containing GSCs in vitro and reduced glioma tumor growth in vivo, its overexpression enhanced the tumor growth and promoted resistance to Temozolomide. DNA damage also activated PITAR, in addition to p53, thus creating an incoherent feedforward loop. Together, this study established an alternate way of p53 inactivation and proposed PITAR as a potential therapeutic target.

P53 is a well-established tumor suppressor gene contributing to cancer progression in many human cancers. It plays a vital role in preserving genome integrity and inhibiting malignant transformation. p53 is mutated in more than 50% of human cancers. In cancers that do not carry mutations in p53, the inactivation occurs through other genetic or epigenetic alterations. Therefore, further study of the mechanism of regulation of wt-p53 remains vital in cancer research. This study identified a novel LncRNA PITAR, which is highly expressed in glioblastoma (GBM) and glioma stem-like cells (GSCs) and interacts with and stabilizes TRIM28 mRNA, which encodes a p53-specific E3 ubiquitin ligase. TRIM28 can inhibit p53 through HDAC1-mediated deacetylation and direct ubiquitination in an MDM2-dependent manner. Thus, the overall impact of this study is high because of the identification of a novel mechanism in regulating wt-p53.

The other significant strengths of this manuscript included an apparent research strategy design and a clearly outlined and logically organized research approach. They provided both the in vitro and in vivo studies to evaluate the effect of PITAR. They offered reasonable control of the study by validating the results in cells with mutant p53. They also performed a rescue experiment to confirm the PITAR and TRIM28 relationship regulating p53. The conclusions were all supported by solid results. The overall data presentation is clear and convincing.

Reviewer #2 (Public Review):

Summary:

In this paper, the authors train a simple machine learning to improve the ability of AlphaFold-multimers ability to separate interacting from non-interacting pairs. The improvement is small compared with the default AlphaFold score (AUROC from 0.84 to 0.88).

Strengths:

The dataset seems to be carefully constructed.

Weaknesses:

The comparison with the state of the art is limited.<br /> - pDockQ comparison is (likely) incorrect (v2.1 should be used, not v1.0).<br /> - Comparison with ipTM should be complemented with RankingConfidence (the default AF2-score).<br /> - Several other scores than pDockQ have been developed for this task.<br /> - Other methods (by Jianlin Chen) to "improve" quality assessment of AF2-models have been presented - these should at least be cited.

Lack of ablation studies:

- Quite likely the most significant contributor is the ipTM (and other scores from AF2). This should be analyzed and discussed.

Lack of data:

- The GitHub repository does not contain the models - so the data can not be examined carefully. Nor can the model be retrained.

- No license is provided for the code in the Git repository.

Reviewer #2 (Public Review):

Tunneling nanotubes (TNT) are common cellular protrusions that allow the transfer of multiple types of cargo between mammalian cells. TNTs are fragile, and lack any known unique marker, making it challenging to isolate and study them. Therefore, the content of TNTs is mostly unknown, and there are only a handful of proteins known to play a role in TNT formation or function.

In this paper, the authors developed a new protocol to isolate TNT fragments from a culture of adherent mammalian cells in a way that is distinctive of extracellular vesicle and identify the proteins within the TNT (referred to as TNTome) by mass spectrometry. The authors provide an analysis of the results in comparison to the extracellular vesicle (EV) proteome, and validate a few examples, thus providing valuable data for the TNT field. However, there is a big overlap between TNTome and EV proteome.

The authors further focus on two proteins, CD9 and CD81, that are enriched in TNTs. Using cells that are knocked out (KO) or over-expressing (OE) these proteins, the authors study their role in TNT formation and function. The authors focus on two major parameters, which are the percent of cells connected by TNT, and the percent of acceptor cells containing fluorescently labeled transferred vesicles. The authors use various assays, which are properly controlled, to measure these parameters. Their analysis provides convincing evidence that CD9 plays a partial role in TNT formation or stabilization and CD81 plays a partial role in forming fully elongated/connected TNT.

However, the authors overstate the importance of these proteins, since their absence only partially affects TNT formation and function, similar to what is seen when knocking out most any other protein implicated in TNT formation. Even their best results show just a 50% reduction for TNT formation and 70% vesicle transfer (in the double KO). Thus, these are not "key" regulators as the title suggests - no more than many other factors, some of them identified by the authors in previous publications. The model presented in Figure 7D is thus misleading, as it states that CD9 KO has "No TNT" which is incorrect (only a slight decrease according to Figure 3C), and states that CD81 KO has "Non-functional TNT" whereas there is still 50% vesicle transfer in this mutant.

In addition, the authors use vesicle transfer as a measure of function, but this is just one type of cargo amongst many others: ions, proteins, RNA, various organelles, and pathogens like viruses and bacteria. Since the authors clearly cannot test every type of cargo, the authors should at least be more accurate in their statements regarding functionality and mention the possibility that other types of cargo transfer could be less or more affected by the KO or OE of these proteins.

It is not completely clear from the text why the authors decided to focus on CD9 and CD81, which are also found in EV, instead of focusing on TNT-unique proteins, and in particular the cytoskeleton-related ones.

In summary, it is a good paper, that provides valuable data on the composition of TNT, and the role of additional players, bringing us closer to understanding the mechanism of TNT formation.

Reviewer #2 (Public Review):

Summary:

The current work by Banwait et al. reports a fluorescence-based single turnover method based on protein-induced fluorescence enhancement (PIFE) to show that ClpB is a processive motor. The paper is a crucial finding as there has been ambiguity on whether ClpB is a processive or non-processive motor. Optical tweezers-based single-molecule studies have shown that ClpB is a processive motor, whereas previous studies from the same group hypothesized it to be a non-processive motor. As co-chaperones are needed for the motor activity of the ClpB, to isolate the activity of ClpB, they have used a 1:1 ratio ATP and ATPgS, where the enzyme is active even in the absence of its co-chaperones, as previously observed. A sequential mixing stop-flow protocol was developed, and the unfolding and translocation of RepA-TitinX, X = 1,2,3 repeats was monitored by measuring the fluorescence intensity with the time of Alexa F555 which was labelled at the C-terminal Cysteine. The observations were a lag time, followed by a gradual increase in fluorescence due to PIFE, and then a decrease in fluorescence plausibly due to the dissociation from the substrate allowing it to refold. The authors observed that the peak time depends on the substrate length, indicating the processive nature of ClpB. In addition, the lag and peak times depend on the pre-incubation time with ATPgS, indicating that the enzyme translocates on the substrates even with just ATPgS without the addition of ATP, which is plausible due to the slow hydrolysis of ATPgS. From the plot of substrate length vs peak time, the authors calculated the rate of unfolding and translocation to be ~0.1 aas-1 in the presence of ~1 mM ATPgS and increases to 1 aas-1 in the presence of 1:1 ATP and ATPgS. The authors have further performed experiments at 3:1 ATP and ATPgS concentrations and observed ~5 times increase in the translocation rates as expected due to faster hydrolysis of ATP by ClpB and reconfirming that processivity is majorly ATP driven. Further, the authors model their results to multiple sequential unfolding steps, determining the rate of unfolding and the number of amino acids unfolded during each step. Overall, the study uses a novel method to reconfirm the processive nature of ClpB.

Strengths:

(1) Previous studies on understanding the processivity of ClpB have primarily focused on unfolded or disordered proteins; this study paves new insights into our understanding of the processing of folded proteins by ClpB. They have cleverly used RepA as a recognition sequence to understand the unfolding of titin-I27 folded domains.

(2) The method developed can be applied to many disaggregating enzymes and has broader significance.

(3) The data from various experiments are consistent with each other, indicating the reproducibility of the data. For example, the rate of translocation in the presence of ATPgS, ~0.1 aas-1 from the single mixing experiment and double mixing experiment are very similar.

(4) The study convincingly shows that ClpB is a processive motor, which has long been debated, describing its activity in the presence of only ATPgS and a mixture of ATP and ATPgS.

(5) The discussion part has been written in a way that describes many previous experiments from various groups supporting the processive nature of the enzyme and supports their current study.

Weaknesses:

(1) The authors model that the enzyme unfolds the protein sequentially around 60 aa each time through multiple steps and translocates rapidly. This contradicts our knowledge of protein unfolding, which is generally cooperative, particularly for titinI27, which is reported to unfold cooperatively or utmost through one intermediate during enzymatic unfolding by ClpX and ClpA.

(2) It is also important to note that the unfolding of titinI27 from the N-terminus (as done in this study) has been reported to be very fast and cannot be the rate-limiting step as reported earlier(Olivares et al, PNAS, 2017). This contradicts the current model where unfolding is the rate-limiting step, and the translocation is assumed to be many orders faster than unfolding.

(3) The model assumes the same time constant for all the unfolding steps irrespective of the secondary structural interactions.

(4) Unlike other single-molecule optical tweezer-based assays, the study cannot distinguish the unfolding and translocation events and assumes that unfolding is the rate-limiting step.

Reviewer #2 (Public Review):

Summary:

In this report Abidi et al. use an antibody against Jag2, a Notch1 ligand, to inhibit its activity in skin. A single dose of this treatment leads to an impairment of sebocyte differentiation and an accumulation of basal sebocytes. Consistently Notch1 activity, measured as cleaved form of the Notch1 intracellular domain, is detected in basal sebocytes together with the expression of Jag2. Interestingly the phenotype caused by the antibody treatment is reversible.

Strengths:

The quality of the histological data with a clear phenotype, together with the quantification represents a solid base for the authors' claims.

This work identifies that the ligand Jag2 is the Notch1 ligand required for sebocyte differentiation.

From a therapeutic point of view, it is interesting that the treatment with anti-Jag2 is reversible.

Weaknesses:

The authors use a single approach to support their claims.

In this report, the analysis of the potential anti-Jag2 effect on the sebaceous ducts, the second cellular component of the sebaceous gland, is neglected.

Reviewer #2 (Public Review):

Summary:

Vladimir Khayenko et al. discovered two novel binding pockets on HBc with in vitro binding and electron microscopy experiments. While the geranyl dimer targeting a central hydrophobic pocket displayed a micromolar affinity, the P1-dimer binding to the spike tip of HBc has a nanomolar affinity. In the turbidity assay and at the cellular level, an HBc aggregation from peptide crosslinking was demonstrated.

Strengths:

The study identifies two previously unexplored binding pockets on HBc capsids and develops novel binders targeting these sites with promising affinities.

Weaknesses:

While the in vitro and cellular HBc aggregation effects are demonstrated, the antiviral potential against HBV infection is not directly evaluated in this study.

Reviewer #2 (Public Review):

Barsukov and his colleagues investigate the interaction mechanism between the EB1 C-terminal domain (EBH) and its binding motif, "SxIP," from MACF. From the crystal structure of the C-terminus of EB1 and SxIP, it has been postulated that complex formation is a simple protein-peptide interaction, achieved by only four residues. The authors demonstrate that the post-SxIP region is involved in EBH interactions using NMR and ITC, and propose that a more complex system exists - a two-step "dock-and-lock" model. The CEST data clearly show that EBH possesses two structural conformations and that the C-terminal EBH conformation undergoes a change upon binding to 11MACF. The authors then mutate the 11MACF peptide sequence and identify peptides with much higher affinities for EBH. These findings may contribute to the development of peptide drugs targeting EB1/microtubules.

This work provides a novel structural insight into EB1 and its binding proteins, and the authors present solid experimental evidence to support the idea. One thing the authors should do is, I think, to use the longer EB1 construct. As the authors describe in the Introduction, each domain of EB1 has a distinct function. The C-terminal tail of EB1, which is adjacent to EBH and is not analyzed in this study, is highly acidic and plays an important role in protein interactions. If the authors discuss the C-terminus of EB1, they should analyze the whole C-terminus of EB1, which would strengthen the conclusion they have made.

Reviewer #2 (Public Review):

Summary:

Velichko, Artem, et al. investigate the role played by the long intrinsically disordered protein Trecle in nucleolar morphology and function, with an interest in its potential ability to undergo liquid-liquid phase separation. The authors explore Treacle's role in core functions of the nucleolus (rRNA biogenesis and DNA repair), which has been a subject of continual investigation since it was identified that truncation of Treacle is the major genetic cause of Treacher-Collins syndrome. They show that knock out of Treacle leads to de-mixing of canonical markers of the FC (UBF, RPA194) and DFC (FBL) phases of the nucleolus. They also show that replacing Treacle with mutants that disrupt its bulk dynamics leads to the de-mixing of FBL. These mutants either remove the central region of Treacle (∆83-1121) or, more subtly, reduce the segregation of charged residues by scrambling them (CS- Charge Scrambled). The observed morphological disruptions mirrored disruptions to the production of rRNA and the ability to recruit the DNA-damage response factor TOPBP1. These data give new insight into the role played by the central region of Treacle in affecting its bulk dynamics and the potential effects of disruptions therein to nucleolar morphology and function.

Strengths:

The characterizations of changes to nuclear morphology upon Treacle knockout is the major strength of this study (Figure 1). Methodologically the CRISPR knockout appears sound. The characterized effects on the canonical markers of the FC and DFC phases support the idea that Treacle has a scaffolding function. While the effect of Treacle perturbations has been studied before, this has often been phenotyped in the context of development or rRNA biogenesis, and less often on the sub-cellular level.

The other major strength of this study is its characterization of the effects of the charge scramble mutant. The authors find that replacing endogenous Treacle with this mutant reduces the bulk dynamics of Treacle (Figure 3K-M), de-mixes FBL from the DFC (Figure 4C-D), lowers pre-rRNA synthesis (Figure 4E-G), and abolishes the recruitment of the DNA-damage response factor TOPBP1 (Figure 5).

Weaknesses:

Clarity around the reagents used and deeper analyses would bolster the author's claims about the condensation behavior of Treacle.

Limited characterization and sparse methodological details regarding recombinant Treacle lead to a concern about the observation that Treacle condenses in vitro. The concerns are offset by the fact that most of the paper uses cellular data to draw conclusions.

The authors ascribe liquid-like behavior to Treacle based on spherical morphology and fusion events of Treacle-Katushka2S condensates as well as fluorescence recovery after a photobleaching (FRAP); these are accepted characterizations in the biomedical field. Nonetheless, the authors only use FRAP to characterize mutants, which limits conclusions about their apparent material state. Overall, FRAP data are better interpreted as a readout of bulk dynamics. For example, the FRAP traces of Treacle plateau at a recovery percentage between 40 and 60%, indicating complex bulk dynamics and the possibility of an immobile pool that is not liquid-like.

Lastly, the Treacle-Katushka2S construct is the predominant construct used throughout the paper. The known tetrameric nature of Katushka2S contrasts with the presumptively monomeric Treacle-FusionRed-Cry2 construct. This is relevant because multi-valance is known to increase the driving forces for condensation and affect condensate material properties. The authors report that the Treacle-FusionRed-Cry2 construct (monomeric) exhibits less condensation than the Treacle-Katushka2S construct (tetrameric). Thus, one is left concerned that the latter construct is not wholly representative of intrinsic Treacle condensation behavior.

Reviewer #2 (Public Review):

Summary:

The authors used a yeast model for analyzing Parkinson's disease-associated synphilin-1 inclusion bodies (SY1 IBs). In this model system, large SY1 IBs are efficiently formed from smaller potentially more toxic SY1 aggregates. Using a genome-wide approach (synthetic genetic array, SGA, combined with a high content imaging approach), the authors identified the sphingolipid metabolic pathway as pivotal for SY1 IBs formation. Disturbances of this pathway increased SY1-triggered growth deficits, loss of mitochondrial membrane potential, increased production of reactive oxygen species (ROS), and decreased cellular ATP levels pointing to an increased energy crisis within affected cells. Notably, SY1 IBs were found to be surrounded by mitochondrial membranes using state-of-the-art super-resolution microscopy. Finally, the effects observed in the yeast for SY1 IBs turned out to be evolutionary conserved in mammalian cells. Thus, sphingolipid metabolism might play an important role in the detoxification of misfolded proteins by large IBs formation at the mitochondrial outer membrane.

Strengths:

• The SY1 IB yeast model is very suitable for the analysis of genes involved in IB formation.<br /> • The genome-wide approach combining a synthetic genetic array (SGA) with a high content imaging approach is a compelling approach and enabled the reliable identification of novel genes. The authors tightly checked the output of the screen.<br /> • The authors clearly showed, including a couple of control experiments, that the sphingolipid metabolic pathway is crucial for SY1 IB formation and cytotoxicity.<br /> • The localization of SY1 IBs at mitochondrial membranes has been clearly demonstrated with state-of-the-art super-resolution microscopy and biochemical methods.<br /> • Pharmacological manipulation of the sphingolipid pathway influenced mitochondrial function and cell survival.

Weaknesses:

• It remains unclear how sphingolipids are involved in SY1 IB formation.

Reviewer #2 (Public Review):

Summary:

The aim of this paper was to elucidate the role of the dorsal HP and intermediate HP (dHP and iHP) in value-based spatial navigation through behavioral and pharmacological experiments using a newly developed VR apparatus. The authors inactivated dHP and iHP by muscimol injection and analyzed the differences in behavior. The results showed that dHP was important for spatial navigation, while iHP was critical for both value judgments and spatial navigation. The present study developed a new sophisticated behavioral experimental apparatus and proposed a behavioral paradigm that is useful for studying value-dependent spatial navigation. In addition, the present study provides important results that support previous findings of differential function along the dorsoventral axis of the hippocampus.

Reviewer #2 (Public Review):

Summary:

This manuscript proposes a modeling approach to capture nonlinear processes of photocurrents in mammalian (mouse, primate) rod and cone photoreceptors. The ultimate goal is to separate these nonlinearities at the level of photocurrent from subsequent nonlinear processing that occurs in retinal circuitry. The authors devised a strategy to generate stimuli that cancel the major nonlinearities in photocurrents. For example, modified stimuli would generate genuine sinusoidal modulation of the photocurrent, whereas a sinusoidal stimulus would not (i.e., because of asymmetries in the photocurrent to light vs. dark phases of a sinusoidal stimulus); and modified stimuli that could cancel the effects of light adaptation at the photocurrent level. Using these modified stimuli, one could record downstream neurons, knowing that any nonlinearities that emerge must happen beyond the stage of the photocurrent. This could be a useful method for separating nonlinear mechanisms across different stages of retinal processing and may be useful in vivo.

Strengths:

(1) This is a very quantitative and thoughtful approach and addresses a long-standing problem in the field: determining the location of nonlinearities within a complex circuit, including asymmetric responses to different polarities of contrast, adaptation, etc.<br /> (2) The study presents data for two primary models of mammalian retina, mouse and primate, and shows that the basic strategy works in each case.<br /> (3) Ideally, the present results would generalize to the work in other labs and possibly other sensory systems. The authors do provide evidence that a photocurrent model constructed from data in one set of cells can be used in a second set of cells.

Weaknesses:

(1) The model is limited to describing photoreceptor responses at the level of photocurrents, as opposed to the output of the cell, which takes into account voltage-dependent mechanisms, horizontal cell feedback, etc., as the authors acknowledge. It could be interesting to expand the model in the future to include factors that affect photoreceptor output beyond the stage of the photocurrent.<br /> (2). It will be interesting to eventually test the impact of this work for in vivo experiments.

Reviewer #2 (Public Review):

Here I submit my previous review and a great deal of additional information following on from the initial review and the response by the authors.

* Initial Review *

Assessment:

This manuscript is based upon the unprecedented identification of an apparently highly unusual trigeminal nuclear organization within the elephant brainstem, related to a large trigeminal nerve in these animals. The apparently highly specialized elephant trigeminal nuclear complex identified in the current study has been classified as the inferior olivary nuclear complex in four previous studies of the elephant brainstem. The entire study is predicated upon the correct identification of the trigeminal sensory nuclear complex and the inferior olivary nuclear complex in the elephant, and if this is incorrect, then the remainder of the manuscript is merely unsupported speculation. There are many reasons indicating that the trigeminal nuclear complex is misidentified in the current study, rendering the entire study, and associated speculation, inadequate at best, and damaging in terms of understanding elephant brains and behaviour at worst.

Original Public Review:

The authors describe what they assert to be a very unusual trigeminal nuclear complex in the brainstem of elephants, and based on this, follow with many speculations about how the trigeminal nuclear complex, as identified by them, might be organized in terms of the sensory capacity of the elephant trunk.<br /> The identification of the trigeminal nuclear complex/inferior olivary nuclear complex in the elephant brainstem is the central pillar of this manuscript from which everything else follows, and if this is incorrect, then the entire manuscript fails, and all the associated speculations become completely unsupported.

The authors note that what they identify as the trigeminal nuclear complex has been identified as the inferior olivary nuclear complex by other authors, citing Shoshani et al. (2006; 10.1016/j.brainresbull.2006.03.016) and Maseko et al (2013; 10.1159/000352004), but fail to cite either Verhaart and Kramer (1958; PMID 13841799) or Verhaart (1962; 10.1515/9783112519882-001). These four studies are in agreement, the current study differs.

Let's assume for the moment that the four previous studies are all incorrect and the current study is correct. This would mean that the entire architecture and organization of the elephant brainstem is significantly rearranged in comparison to ALL other mammals, including humans, previously studied (e.g. Kappers et al. 1965, The Comparative Anatomy of the Nervous System of Vertebrates, Including Man, Volume 1 pp. 668-695) and the closely related manatee (10.1002/ar.20573). This rearrangement necessitates that the trigeminal nuclei would have had to "migrate" and shorten rostrocaudally, specifically and only, from the lateral aspect of the brainstem where these nuclei extend from the pons through to the cervical spinal cord (e.g. the Paxinos and Watson rat brain atlases), the to the spatially restricted ventromedial region of specifically and only the rostral medulla oblongata. According to the current paper the inferior olivary complex of the elephant is very small and located lateral to their trigeminal nuclear complex, and the region from where the trigeminal nuclei are located by others, appears to be just "lateral nuclei" with no suggestion of what might be there instead.

Such an extraordinary rearrangement of brainstem nuclei would require a major transformation in the manner in which the mutations, patterning, and expression of genes and associated molecules during development occurs. Such a major change is likely to lead to lethal phenotypes, making such a transformation extremely unlikely. Variations in mammalian brainstem anatomy are most commonly associated with quantitative changes rather than qualitative changes (10.1016/B978-0-12-804042-3.00045-2).

The impetus for the identification of the unusual brainstem trigeminal nuclei in the current study rests upon a previous study from the same laboratory (10.1016/j.cub.2021.12.051) that estimated that the number of axons contained in the infraorbital branch of the trigeminal nerve that innervate the sensory surfaces of the trunk is approximately 400 000. Is this number unusual? In a much smaller mammal with a highly specialized trigeminal system, the platypus, the number of axons innervating the sensory surface of the platypus bill skin comes to 1 344 000 (10.1159/000113185). Yet, there is no complex rearrangement of the brainstem trigeminal nuclei in the brain of the developing or adult platypus (Ashwell, 2013, Neurobiology of Monotremes), despite the brainstem trigeminal nuclei being very large in the platypus (10.1159/000067195). Even in other large-brained mammals, such as large whales that do not have a trunk, the number of axons in the trigeminal nerve ranges between 400 000 and 500 000 (10.1007/978-3-319-47829-6_988-1). The lack of comparative support for the argument forwarded in the previous and current study from this laboratory, and that the comparative data indicates that the brainstem nuclei do not change in the manner suggested in the elephant, argues against the identification of the trigeminal nuclei as outlined in the current study. Moreover, the comparative studies undermine the prior claim of the authors, informing the current study, that "the elephant trigeminal ganglion ... point to a high degree of tactile specialization in elephants" (10.1016/j.cub.2021.12.051). While clearly the elephant has tactile sensitivity in the trunk, it is questionable as to whether what has been observed in elephants is indeed "truly extraordinary".

But let's look more specifically at the justification outlined in the current study to support their identification of the unusual located trigeminal sensory nuclei of the brainstem.

(1) Intense cytochrome oxidase reactivity<br /> (2) Large size of the putative trunk module<br /> (3) Elongation of the putative trunk module<br /> (4) Arrangement of these putative modules correspond to elephant head anatomy<br /> (5) Myelin stripes within the putative trunk module that apparently match trunk folds<br /> (6) Location apparently matches other mammals<br /> (7) Repetitive modular organization apparently similar to other mammals.<br /> (8) The inferior olive described by other authors lacks the lamellated appearance of this structure in other mammals

Let's examine these justifications more closely.

(1) Cytochrome oxidase histochemistry is typically used as an indicative marker of neuronal energy metabolism. The authors indicate, based on the "truly extraordinary" somatosensory capacities of the elephant trunk, that any nuclei processing this tactile information should be highly metabolically active, and thus should react intensely when stained for cytochrome oxidase. We are told in the methods section that the protocols used are described by Purkart et al (2022) and Kaufmann et al (2022). In neither of these cited papers is there any description, nor mention, of the cytochrome oxidase histochemistry methodology, thus we have no idea of how this histochemical staining was done. In order to obtain the best results for cytochrome oxidase histochemistry, the tissue is either processed very rapidly after buffer perfusion to remove blood or in recently perfusion-fixed tissue (e.g., 10.1016/0165-0270(93)90122-8). Given: (1) the presumably long post-mortem interval between death and fixation - "it often takes days to dissect elephants"; (2) subsequent fixation of the brains in 4% paraformaldehyde for "several weeks"; (3) The intense cytochrome oxidase reactivity in the inferior olivary complex of the laboratory rat (Gonzalez-Lima, 1998, Cytochrome oxidase in neuronal metabolism and Alzheimer's diseases); and (4) The lack of any comparative images from other stained portions of the elephant brainstem; it is difficult to support the justification as forwarded by the authors. It is likely that the histochemical staining observed is background reactivity from the use of diaminobenzidine in the staining protocol. Thus, this first justification is unsupported.<br /> Justifications (2), (3), and (4) are sequelae from justification (1). In this sense, they do not count as justifications, but rather unsupported extensions.

(4) and (5) These are interesting justifications, as the paper has clear internal contradictions, and (5) is a sequelae of (4). The reader is led to the concept that the myelin tracts divide the nuclei into sub-modules that match the folding of the skin on the elephant trunk. One would then readily presume that these myelin tracts are in the incoming sensory axons from the trigeminal nerve. However, the authors note that this is not the case: "Our observations on trunk module myelin stripes are at odds with this view of myelin. Specifically, myelin stripes show no tapering (which we would expect if axons divert off into the tissue). More than that, there is no correlation between myelin stripe thickness (which presumably correlates with axon numbers) and trigeminal module neuron numbers. Thus, there are numerous myelinated axons, where we observe few or no trigeminal neurons. These observations are incompatible with the idea that myelin stripes form an axonal 'supply' system or that their prime function is to connect neurons. What do myelin stripe axons do, if they do not connect neurons? We suggest that myelin stripes serve to separate rather than connect neurons." So, we are left with the observation that the myelin stripes do not pass afferent trigeminal sensory information from the "truly extraordinary" trunk skin somatic sensory system, and rather function as units that separate neurons - but to what end? It appears that the myelin stripes are more likely to be efferent axonal bundles leaving the nuclei (to form the olivocerebellar tract). This justification is unsupported.

(6) The authors indicate that the location of these nuclei matches that of the trigeminal nuclei in other mammals. This is not supported in any way. In ALL other mammals in which the trigeminal nuclei of the brainstem have been reported they are found in the lateral aspect of the brainstem, bordered laterally by the spinal trigeminal tract. This is most readily seen and accessible in the Paxinos and Watson rat brain atlases. The authors indicate that the trigeminal nuclei are medial to the facial nerve nucleus, but in every other species the trigeminal sensory nuclei are found lateral to the facial nerve nucleus. This is most salient when examining a close relative, the manatee (10.1002/ar.20573), where the location of the inferior olive and the trigeminal nuclei matches that described by Maseko et al (2013) for the African elephant. This justification is not supported.

(7) The dual to quadruple repetition of rostro-caudal modules within the putative trigeminal nucleus as identified by the authors relies on the fact that in the neurotypical mammal, there are several trigeminal sensory nuclei arranged in a column running from the pons to the cervical spinal cord, these include (nomenclature from Paxinos and Watson in roughly rostral to caudal order) the Pr5VL, Pr5DM, Sp5O, Sp5I, and Sp5C. But, these nuclei are all located far from the midline and lateral to the facial nerve nucleus, unlike what the authors describe in the elephants. These rostrocaudal modules are expanded upon in Figure 2, and it is apparent from what is shown is that the authors are attributing other brainstem nuclei to the putative trigeminal nuclei to confirm their conclusion. For example, what they identify as the inferior olive in figure 2D is likely the lateral reticular nucleus as identified by Maseko et al (2013). This justification is not supported.

(8) In primates and related species, there is a distinct banded appearance of the inferior olive, but what has been termed the inferior olive in the elephant by other authors does not have this appearance, rather, and specifically, the largest nuclear mass in the region (termed the principal nucleus of the inferior olive by Maseko et al, 2013, but Pr5, the principal trigeminal nucleus in the current paper) overshadows the partial banded appearance of the remaining nuclei in the region (but also drawn by the authors of the current paper). Thus, what is at debate here is whether the principal nucleus of the inferior olive can take on a nuclear shape rather than evince a banded appearance. The authors of this paper use this variance as justification that this cluster of nuclei could not possibly be the inferior olive. Such a "semi-nuclear/banded" arrangement of the inferior olive is seen in, for example, giraffe (10.1016/j.jchemneu.2007.05.003), domestic dog, polar bear, and most specifically the manatee (a close relative of the elephant) (brainmuseum.org; 10.1002/ar.20573). This justification is not supported.

Thus, all the justifications forwarded by the authors are unsupported. Based on methodological concerns, prior comparative mammalian neuroanatomy, and prior studies in the elephant and closely related species, the authors fail to support their notion that what was previously termed the inferior olive in the elephant is actually the trigeminal sensory nuclei. Given this failure, the justifications provided above that are sequelae also fail. In this sense, the entire manuscript and all the sequelae are not supported.

What the authors have not done is to trace the pathway of the large trigeminal nerve in the elephant brainstem, as was done by Maseko et al (2013), which clearly shows the internal pathways of this nerve, from the branch that leads to the fifth mesencephalic nucleus adjacent to the periventricular grey matter, through to the spinal trigeminal tract that extends from the pons to the spinal cord in a manner very similar to all other mammals. Nor have they shown how the supposed trigeminal information reaches the putative trigeminal nuclei in the ventromedial rostral medulla oblongata. These are but two examples of many specific lines of evidence that would be required to support their conclusions. Clearly tract tracing methods, such as cholera toxin tracing of peripheral nerves cannot be done in elephants, thus the neuroanatomy must be done properly and with attention to details to support the major changes indicated by the authors.

So what are these "bumps" in the elephant brainstem?

Four previous authors indicate that these bumps are the inferior olivary nuclear complex. Can this be supported?

The inferior olivary nuclear complex acts "as a relay station between the spinal cord (n.b. trigeminal input does reach the spinal cord via the spinal trigeminal tract) and the cerebellum, integrating motor and sensory information to provide feedback and training to cerebellar neurons" (https://www.ncbi.nlm.nih.gov/books/NBK542242/). The inferior olivary nuclear complex is located dorsal and medial to the pyramidal tracts (which were not labelled in the current study by the authors but are clearly present in Fig. 1C and 2A) in the ventromedial aspect of the rostral medulla oblongata. This is precisely where previous authors have identified the inferior olivary nuclear complex and what the current authors assign to their putative trigeminal nuclei. The neurons of the inferior olivary nuclei project, via the olivocerebellar tract to the cerebellum to terminate in the climbing fibres of the cerebellar cortex.

Elephants have the largest (relative and absolute) cerebellum of all mammals (10.1002/ar.22425), this cerebellum contains 257 x109 neurons (10.3389/fnana.2014.00046; three times more than the entire human brain, 10.3389/neuro.09.031.2009). Each of these neurons appears to be more structurally complex than the homologous neurons in other mammals (10.1159/000345565; 10.1007/s00429-010-0288-3). In the African elephant, the neurons of the inferior olivary nuclear complex are described by Maseko et al (2013) as being both calbindin and calretinin immunoreactive. Climbing fibres in the cerebellar cortex of the African elephant are clearly calretinin immunopositive and also are likely to contain calbindin (10.1159/000345565). Given this, would it be surprising that the inferior olivary nuclear complex of the elephant is enlarged enough to create a very distinct bump in exactly the same place where these nuclei are identified in other mammals?

What about the myelin stripes? These are most likely to be the origin of the olivocerebellar tract and probably only have a coincidental relationship to the trunk. Thus, given what we know, the inferior olivary nuclear complex as described in other studies, and the putative trigeminal nuclear complex as described in the current study, is the elephant inferior olivary nuclear complex. It is not what the authors believe it to be, and they do not provide any evidence that discounts the previous studies. The authors are quite simply put, wrong. All the speculations that flow from this major neuroanatomical error are therefore science fiction rather than useful additions to the scientific literature.

What do the authors actually have?<br /> The authors have interesting data, based on their Golgi staining and analysis, of the inferior olivary nuclear complex in the elephant.

* Review of Revised Manuscript *

Assessment:

There is a clear dichotomy between the authors and this reviewer regarding the identification of specific structures, namely the inferior olivary nuclear complex and the trigeminal nuclear complex, in the brainstem of the elephant. The authors maintain the position that in the elephant alone, irrespective of all the published data on other mammals and previously published data on the elephant brainstem, these two nuclear complexes are switched in location. The authors maintain that their interpretation is correct, this reviewer maintains that this interpretation is erroneous. The authors expressed concern that the remainder of the paper was not addressed by the reviewer, but the reviewer maintains that these sequelae to the misidentification of nuclear complexes in the elephant brainstem renders any of these speculations irrelevant as the critical structures are incorrectly identified. It is this reviewer's opinion that this paper is incorrect. I provide a lot of detail below in order to provide support to the opinion I express.

Public Review of Current Submission:

As indicated in my previous review of this manuscript (see above), it is my opinion that the authors have misidentified, and indeed switched, the inferior olivary nuclear complex (IO) and the trigeminal nuclear complex (Vsens). It is this specific point only that I will address in this second review, as this is the crucial aspect of this paper - if the identification of these nuclear complexes in the elephant brainstem by the authors is incorrect, the remainder of the paper does not have any scientific validity.

The authors, in their response to my initial review, claim that I "bend" the comparative evidence against them. They further claim that as all other mammalian species exhibit a "serrated" appearance of the inferior olive, and as the elephant does not exhibit this appearance, that what was previously identified as the inferior olive is actually the trigeminal nucleus and vice versa.

For convenience, I will refer to IOM and VsensM as the identification of these structures according to Maseko et al (2013) and other authors and will use IOR and VsensR to refer to the identification forwarded in the study under review.<br /> The IOM/VsensR certainly does not have a serrated appearance in elephants. Indeed, from the plates supplied by the authors in response (Referee Fig. 2), the cytochrome oxidase image supplied and the image from Maseko et al (2013) shows a very similar appearance. There is no doubt that the authors are identifying structures that closely correspond to those provided by Maseko et al (2013). It is solely a contrast in what these nuclear complexes are called and the functional sequelae of the identification of these complexes (are they related to the trunk sensation or movement controlled by the cerebellum?) that is under debate.

Elephants are part of the Afrotheria, thus the most relevant comparative data to resolve this issue will be the identification of these nuclei in other Afrotherian species. Below I provide images of these nuclear complexes, labelled in the standard nomenclature, across several Afrotherian species.

(A) Lesser hedgehog tenrec (Echinops telfairi)

Tenrecs brains are the most intensively studied of the Afrotherian brains, these extensive neuroanatomical studies undertaken primarily by Heinz Künzle. Below I append images (coronal sections stained with cresol violet) of the IO and Vsens (labelled in the standard mammalian manner) in the lesser hedgehog tenrec. It should be clear that the inferior olive is located in the ventral midline of the rostral medulla oblongata (just like the rat) and that this nucleus is not distinctly serrated. The Vsens is located in the lateral aspect of the medulla skirted laterally by the spinal trigeminal tract (Sp5). These images and the labels indicating structures correlate precisely with that provide by Künzle (1997, 10.1016/S0168- 0102(97)00034-5), see his Figure 1K,L. Thus, in the first case of a related species, there is no serrated appearance of the inferior olive, the location of the inferior olive is confirmed through connectivity with the superior colliculus (a standard connection in mammals) by Künzle (1997), and the location of Vsens is what is considered to be typical for mammals. This is in agreement with the authors, as they propose that ONLY the elephants show the variations they report.

Review image 1.

(B) Giant otter shrew (Potomogale velox)

The otter shrews are close relatives of the Tenrecs. Below I append images of cresyl violet (left column) and myelin (right column) stained coronal sections through the brainstem with the IO, Vsens and Sp5 labelled as per standard mammalian anatomy. Here we see hints of the serration of the IO as defined by the authors, but we also see many myelin stripes across the IO. Vsens is located laterally and skirted by the Sp5. This is in agreement with the authors, as they propose that ONLY the elephants show the variations they report.

Review image 2.

(C) Four-toed sengi (Petrodromus tetradactylus)

The sengis are close relatives of the Tenrecs and otter shrews, these three groups being part of the Afroinsectiphilia, a distinct branch of the Afrotheria. Below I append images of cresyl violet (left column) and myelin (right column) stained coronal sections through the brainstem with the IO, Vsens and Sp5 labelled as per standard mammalian anatomy. Here we see vague hints of the serration of the IO (as defined by the authors), and we also see many myelin stripes across the IO. Vsens is located laterally and skirted by the Sp5. This is in agreement with the authors, as they propose that ONLY the elephants show the variations they report.

Review image 3.

(D) Rock hyrax (Procavia capensis)

The hyraxes, along with the sirens and elephants form the Paenungulata branch of the Afrotheria. Below I append images of cresyl violet (left column) and myelin (right column) stained coronal sections through the brainstem with the IO, Vsens and Sp5 labelled as per the standard mammalian anatomy. Here we see hints of the serration of the IO (as defined by the authors), but we also see evidence of a more "bulbous" appearance of subnuclei of the IO (particularly the principal nucleus), and we also see many myelin stripes across the IO. Vsens is located laterally and skirted by the Sp5. This is in agreement with the authors, as they propose that ONLY the elephants show the variations they report.

Review image 4.

(E) West Indian manatee (Trichechus manatus)

The sirens are the closest extant relatives of the elephants in the Afrotheria. Below I append images of cresyl violet (top) and myelin (bottom) stained coronal sections (taken from the University of Wisconsin-Madison Brain Collection, https://brainmuseum.org, and while quite low in magnification they do reveal the structures under debate) through the brainstem with the IO, Vsens and Sp5 labelled as per standard mammalian anatomy. Here we see the serration of the IO (as defined by the authors). Vsens is located laterally and skirted by the Sp5. This is in agreement with the authors, as they propose that ONLY the elephants show the variations they report.

Review image 5.

These comparisons and the structural identification, with which the authors agree as they only distinguish the elephants from the other Afrotheria, demonstrate that the appearance of the IO can be quite variable across mammalian species, including those with a close phylogenetic affinity to the elephants. Not all mammal species possess a "serrated" appearance of the IO. Thus, it is more than just theoretically possible that the IO of the elephant appears as described prior to this study.

So what about elephants? Below I append a series of images from coronal sections through the African elephant brainstem stained for Nissl, myelin, and immunostained for calretinin. These sections are labelled according to standard mammalian nomenclature. In these complete sections of the elephant brainstem, we do not see a serrated appearance of the IOM (as described previously and in the current study by the authors). Rather the principal nucleus of the IOM appears to be bulbous in nature. In the current study, no image of myelin staining in the IOM/VsensR is provided by the authors. However, in the images I provide, we do see the reported myelin stripes in all stains - agreement between the authors and reviewer on this point. The higher magnification image to the bottom left of the plate shows one of the IOM/VsensR myelin stripes immunostained for calretinin, and within the myelin stripes axons immunopositive for calretinin are seen (labelled with an arrow). The climbing fibres of the elephant cerebellar cortex are similarly calretinin immunopositive (10.1159/000345565). In contrast, although not shown at high magnification, the fibres forming the Sp5 in the elephant (in the Maseko description, unnamed in the description of the authors) show no immunoreactivity to calretinin.

Review image 6.

Peripherin Immunostaining

In their revised manuscript the authors present immunostaining of peripherin in the elephant brainstem. This is an important addition (although it does replace the only staining of myelin provided by the authors which is unusual as the word myelin is in the title of the paper) as peripherin is known to specifically label peripheral nerves. In addition, as pointed out by the authors, peripherin also immunostains climbing fibres (Errante et al., 1998). The understanding of this staining is important in determining the identification of the IO and Vsens in the elephant, although it is not ideal for this task as there is some ambiguity. Errante and colleagues (1998; Fig. 1) show that climbing fibres are peripherin-immunopositive in the rat. But what the authors do not evaluate is the extensive peripherin staining in the rat Sp5 in the same paper (Errante et al, 1998, Fig. 2). The image provided by the authors of their peripherin immunostaining (their new Figure 2) shows what I would call the Sp5 of the elephant to be strongly peripherin immunoreactive, just like the rat shown in Errant et al (1998), and more over in the precise position of the rat Sp5! This makes sense as this is where the axons subserving the "extraordinary" tactile sensitivity of the elephant trunk would be found (in the standard model of mammalian brainstem anatomy). Interestingly, the peripherin immunostaining in the elephant is clearly lamellated...this coincides precisely with the description of the trigeminal sensory nuclei in the elephant by Maskeo et al (2013) as pointed out by the authors in their rebuttal. Errante et al (1998) also point out peripherin immunostaining in the inferior olive, but according to the authors this is only "weakly present" in the elephant IOM/VsensR. This latter point is crucial. Surely if the elephant has an extraordinary sensory innervation from the trunk, with 400 000 axons entering the brain, the VsensR/IOM should be highly peripherin-immunopositive, including the myelinated axon bundles?! In this sense, the authors argue against their own interpretation - either the elephant trunk is not a highly sensitive tactile organ, or the VsensR is not the trigeminal nuclei it is supposed to be.

Summary:

(1) Comparative data of species closely related to elephants (Afrotherians) demonstrates that not all mammals exhibit the "serrated" appearance of the principal nucleus of the inferior olive.

(2) The location of the IO and Vsens as reported in the current study (IOR and VsensR) would require a significant, and unprecedented, rearrangement of the brainstem in the elephants independently. I argue that the underlying molecular and genetic changes required to achieve this would be so extreme that it would lead to lethal phenotypes. Arguing that the "switcheroo" of the IO and Vsens does occur in the elephant (and no other mammals) and thus doesn't lead to lethal phenotypes is a circular argument that cannot be substantiated.

(3) Myelin stripes in the subnuclei of the inferior olivary nuclear complex are seen across all related mammals as shown above. Thus, the observation made in the elephant by the authors in what they call the VsensR, is similar to that seen in the IO of related mammals, especially when the IO takes on a more bulbous appearance. These myelin stripes are the origin of the olivocerebellar pathway, and are indeed calretinin immunopositive in the elephant as I show.

(4) What the authors see aligns perfectly with what has been described previously, the only difference being the names that nuclear complexes are being called. But identifying these nuclei is important, as any functional sequelae, as extensively discussed by the authors, is entirely dependent upon accurately identifying these nuclei.

(4) The peripherin immunostaining scores an own goal - if peripherin is marking peripheral nerves (as the authors and I believe it is), then why is the VsensR/IOM only "weakly positive" for this stain? This either means that the "extraordinary" tactile sensitivity of the elephant trunk is non-existent, or that the authors have misinterpreted this staining. That there is extensive staining in the fibre pathway dorsal and lateral to the IOR (which I call the spinal trigeminal tract), supports the idea that the authors have misinterpreted their peripherin immunostaining.

(5) Evolutionary expediency. The authors argue that what they report is an expedient way in which to modify the organisation of the brainstem in the elephant to accommodate the "extraordinary" tactile sensitivity. I disagree. As pointed out in my first review, the elephant cerebellum is very large and comprised of huge numbers of morphologically complex neurons. The inferior olivary nuclei in all mammals studied in detail to date, give rise to the climbing fibres that terminate on the Purkinje cells of the cerebellar cortex. It is more parsimonious to argue that, in alignment with the expansion of the elephant cerebellum (for motor control of the trunk), the inferior olivary nuclei (specifically the principal nucleus) have had additional neurons added to accommodate this cerebellar expansion. Such an addition of neurons to the principal nucleus of the inferior olive could readily lead to the loss of the serrated appearance of the principal nucleus of the inferior olive, and would require far less modifications in the developmental genetic program that forms these nuclei. This type of quantitative change appears to be the primary way in which structures are altered in the mammalian brainstem.

Reviewer #2 (Public Review):

Summary:

In summary, the manuscript describes life-cycle-related morphologies of primitive vesicle-like states (Em-P) produced in the laboratory from the Gram-positive bacterium Exiguobacterium Strain-Molly) under assumed Archean environmental conditions. Em-P morphologies (life cycles) are controlled by the "native environment". In order to mimic Archean environmental conditions, soy broth supplemented with Dead Sea salt was used to cultivate Em-Ps. The manuscript compares Archean microfossils and biofilms from selected photos with those laboratory morphologies. The photos derive from publications on various stratigraphic sections of Paleo- to Neoarchean ages. Based on the similarity of morphologies of microfossils and Em-Ps, the manuscript concludes that all Archean microfossils are in fact not prokaryotes, but merely "sacks of cytoplasm".

Strengths:

The approach of the authors to recognize the possibility that "real" cells were not around in the Archean time is appealing. The manuscript reflects the very hard work by the authors composing the Em-Ps used for comparison and selecting the appropriate photo material of fossils.

Weaknesses:

While the basic idea is very interesting, the manuscript includes flaws and falls short in presenting supportive data. The manuscript makes too simplistic assumptions on the "Archean paleoenvironment". First, like in our modern world, the environmental conditions during the Archean time were not globally the same. Second, we do not know much about the Archean paleoenvironment due to the immense lack of rock records. More so, the Archean stratigraphic sections from where the fossil material derived record different paleoenvironments: shelf to tidal flat and lacustrine settings, so differences must have been significant. Finally, the Archean spanned 2.500 billion years and it is unlikely that environmental conditions remained the same. Diurnal or seasonal variations are not considered. Sediment types are not considered. Due to these reasons, the laboratory model of an Archean paleoenvironment and the life therein is too simplistic. Another aspect is that eucaryote cells are described from Archean rocks, so it seems unlikely that prokaryotes were not around at the same time. Considering other fossil evidence preserved in Archean rocks except for microfossils, the many early Archean microbialites that show baffling and trapping cannot be explained without the presence of "real cells". With respect to lithology: chert is a rock predominantly composed of silica, not salt. The formation of Em-Ps in the "salty" laboratory set-up seems therefore not a good fit to evaluate chert fossils. Formation of structures in sediment is one step. The second step is their preservation. However, the second aspect of taphonomy is largely excluded in the manuscript, and the role of fossilization (lithification) of Em-Ps is not discussed. This is important because Archean rock successions are known for their tectonic and hydrothermal overprint, as well as recrystallization over time. Some of the comparisons of laboratory morphologies with fossil microfossils and biofilms are incorrect because scales differ by magnitudes. In general, one has to recognize that prokaryote cell morphologies do not offer many variations. It is possible to arrive at the morphologies described in various ways including abiotic ones.

Reviewer #2 (Public Review):

Summary:

The authors establish a close spatial relationship between RMS neurons and blood vessels. They demonstrated that high blood flow was correlated with migratory speed. In vitro, they demonstrate that Ghrelin functions as a motogen that increases migratory speed through augmentation of actin cup formation. The authors proceed to demonstrate through the knockdown of the Ghrelin receptor that fewer RMS neurons reach the OB. They show the opposite is true when the animal is fasted.

Strengths:

Compelling evidence of close association of RMS neurons with blood vessels (tissue clearing 3D), preferentially arterioles. Good use of 2-photon imaging to demonstrate migratory speed and its correlation with blood flow. In vitro analysis of Ghrelin administration to cultured RMS neurons, actin visualization, Ghsr1KD, is solid and compelling.

Weaknesses:

(1) Novelty of findings attenuated due to prior work, especially Li et al., Experimental Neurology 2014. Here, the authors demonstrated that Ghrelin enhances migration in adult-born neurons in the SVZ and RMS.

(2) The evidence for blood delivery of Ghrelin is not very convincing. Fluorescently-labeled Ghrelin appears to be found throughout the brain parenchyma, irrespective of the distance from vessels. It is also not clear from the data whether there is a link between increased blood flow and Ghrelin delivery.

(3) The in vivo link between Ghsr1KD and migratory speed is not established. Given the strong work to open the study on blood flow and migratory speed and the in vitro evidence that migratory speed is augmented by Ghrelin, the paper would be much stronger with direct measurement of migration speed upon Ghsr1KD. Indeed, blood flow should also be measured in this experiment since it would address concerns in 2. If blood flow and ghrelin delivery are linked, one would expect that Ghsr1KD neurons would not exhibit increased migratory speed when associated with slow or fast blood flow vessels.

Reviewer #2 (Public Review):

Summary:

Spns1 is a recently identified lysosomal transporter of lysophospholipids and sphingosine and its mutations in humans lead to neurodegeneration with white matter dysplasia. Since global Spns1 deficiency is embryonic lethal, the role of this particular lipid transporter in the nervous system remained unclear. In this study, Ichimura et al generated and analyzed nervous system-specific Spns1 knockout mice. The mutant mice showed epilepsy, growth retardation, demyelination, and early death, with accumulation of various LPC, LPE, and LPI species as well as sphingosine in specific areas of the brain. Probably due to impaired lysosomal efflux of sphingosine, brain levels of sphingolipids (ceramides, sulfatides, and glycolipids), which are main myelin components, were markedly reduced in the KO brain.

Strengths:

This study has provided convincing evidence for the first time that nervous system-specific deletion of Spns1 in mice leads to neurodegeneration, with disturbed lysophospholipid and sphingolipid metabolism in the brain. The results support the idea that the defective transport of lysosomal sphingosine by loss of Spns1 leads to a marked reduction of sphingolipid species required for myelin formation. This study significantly contributes to the research fields of neurodegeneration, lysosomal biology, and lipid biology.

Weaknesses:

It remains unclear why oligodendrocytes but not neurons are specifically damaged and how astroglia are affected by Spns1 deficiency. Lysosomal efflux of lysophospholipids and sphingosine by Spns1 relied solely on the knowledge from published studies and was not addressed in this study. The expression of key lipid-metabolizing genes and molecular markers should be examined more deeply. Several images lack quantification.

Reviewer #2 (Public Review):

Hensel investigated the implications of SARS-CoV-2 RNA secondary structure in synonymous and nonsynonymous mutation frequency. The analysis integrated estimates of mutational fitness generated by Bloom and Neher (from publicly available patient sequences) and a population-averaged model of RNA basepairing from Lan et al (from DMS mutational profiling with sequencing, DMS-MaPseq).

The results show that base-pairing limits the frequency of some synonymous substitutions (including the most common CT), but not all: GA and AG substitutions seem unaffected by base-pairing.

The author then addressed nonsynonymous CT substitutions at base-paired positions. While there is still a generally higher estimated mutational fitness at unpaired positions, they propose a coarse adjustment to disentangle base-pairing from inherent mutational fitness at a given position. This adjustment reveals that nonsynonymous substitutions at base-paired positions, which define major variants, have higher mutational fitness.

Overall, this manuscript highlights the importance of considering RNA secondary structure in viral evolution studies.

The conclusions of this work are generally well supported by the data presented. Particularly, the author acknowledges most limitations of the analyses, and addresses them. Even though no new sequencing results were generated, the author used available data generated from the analysis of roughly seven million sequenced patient samples. Finally, the author discusses ways to improve the current available models.

There are a number of limitations of this work that should be highlighted, specifically in regard to the secondary structure data used in this paper. The Lan et al. dataset was generated using a multiplicity of infection (MOI) of 0.05, 24 hours post-infection (h.p.i.). At such a low MOI and late timepoint, viral replication is not synchronous and sequencing artifacts might be generated by cell debris and viral RNA degradation, therefore impacting the population-averaged results. In addition, the nonsynonymous base-paired positions in Figure 2 have relatively high population-averaged DMS reactivity, which suggests those positions are dynamic. Therefore, the proposed adjustment could result in an incorrect estimation of their inherent mutational fitness.

Additionally, like all such RNA probing experiments within cells, it remains difficult to deconvolve DMS/SHAPE low reactivity with RNA accessibility (e.g. from protein binding).

This work presents clear methods and an easy-to-access bioinformatic pipeline, which can be applied to other RNA viruses. Of note, it can be readily implemented in existing datasets. Finally, this study raises novel mechanistic questions on how mutational fitness is not correlated to secondary structure in the same way for every substitution.

Overall, this work highlights the importance of studying mutational fitness beyond an immune evasion perspective. On the other hand, it also adds to the viral intrinsic constraints to immune evasion.

Reviewer #2 (Public Review):

In this manuscript by Floeder et al., the authors report a correlation between ITI duration and the strength of a dopamine ramp occurring in the time between a predictive conditioned stimulus and a subsequent reward. They found this relationship occurring within two different tasks with mice, during both a Pavlovian task as well as an instrumental virtual visual navigation task. Additionally, they observed this relationship only in conditions when using a dynamic predictive stimulus. The authors relate this finding to their previously published model ANCCR in which the time constant of the eligibility trace is proportionate to the reward rate within the task.

The relationship between ITI duration and the extent of a dopamine ramp which the authors have reported is very intriguing and certainly provides an important constraint for models for dopamine function. As such, these findings are potentially highly impactful to the field. I do have a few questions for the authors which are written below.

(1) I was surprised to see a lack of counterbalance within the Pavlovian design for the order of the long vs short ITI. Ramping of the lick rate does increase from the long-duration ITIs to the short-duration ITI sessions. Although of course, this increase in ramping of the licking across the two conditions is not necessarily a function of learning, it doesn't lend support to the opposite possibility that the timing of the dynamic CS hasn't reached asymptotic learning by the end of the long-duration ITI. The authors do reference papers in which overtraining tends to result in a reduction of ramping, which would argue against this possibility, yet differential learning of the dynamic CS would presumably be required to observe this effect. Do the authors have any evidence that the effect is not due to heightened learning of the timing of the dynamic CS across the experiment?

(2) The dopamine response, as measured by dLight, seems to drop after the reward is delivered. This reduction in responding also tends to be observed with electrophysiological recordings of dopamine neurons. It seems possible that during the short ITI sessions, particularly on the shorter ITI duration trials, that dopamine levels may still be reduced from the previous trial at the onset of the CS on the subsequent trial. Perhaps the authors can observe the dynamics of the recovery of the dopamine response following a reward delivery on longer-duration ITIs in order to determine how quickly dopamine is recovering following a reward delivery. Are the trials with very short ITIs occurring within this period that dopamine is recovering from the previous trial? If so, how much of the effect may be due to this effect? It should be noted that the lack of observance of a ramp on the condition of short-duration ITIs with fixed CSs provides a potential control for this effect, yet the extent to which a natural ramp might occur following sucrose deliveries should be investigated.

(3) The authors primarily relate the finding of the correlation between the ITI and the slope of the ramp to their ANCCR model by suggesting that shorter time constants of the eligibility trace will result in more precisely timed predictors of reward across discrete periods of the dynamic cue. Based on this prediction, would the change in slope be more gradual, and perhaps be more correlated with a broader cumulative estimate of reward rate than just a single trial?

Reviewer #2 (Public Review):

Summary:

This report describes a new "Repix" device for collecting stable, long-term recordings from chronically implanted Neuropixels probes in freely behaving rodents. The device follows the "docking module with payload" design of other similar devices that allows probe explantation and reuse but requires minimal components and is robust to a wide range of rodent behaviors. The docking module is a set of metal posts that are screwed into the payload module (cassette carrying the probe) at one end and cemented to the skull of the animal during surgery at the other end to reversibly anchor the probe to the skull. Loosening of the screws allows the cassette to travel off the posts for explantation. An additional headstage holder and cover are also available for further protection of the implant from mechanical damage during freely moving behaviors. Usage data from almost 200 procedures across multiple labs and users showcase high success rates at all stages of implementation (implantation, data collection, and explantation), even from users without direct training from the original developer of Repix. Device proficiency, defined by the authors as three successive full procedures without failure, was typically achieved within five attempts. Hundreds of neurons were consistently recorded from multiple brain regions, irrespective of animal behavior, Neuropixels probe type, and probe reuse. Impressively, neurophysiological data using Repix has already been published in two studies (one in mice and the other in rats). These findings demonstrate the intended functioning of the device as well as its ease of adoption. The effort to make the Repix system as straightforward as possible (e.g., minimal components and detailed protocols) is evident and will likely be appreciated by new adopters. Furthermore, the cell yield and procedures-to-proficiency data collected from a variety of experiments provide useful data for new adopters to plan their own studies with realistic expectations.

Strengths:

The main claims that the Repix device is "reliable, reusable, [and] versatile" are well-supported.

Weaknesses:

(1) The methodology used to quantify cell yields is concerning, potentially leading to an overestimation of "good" units and a misleading amount of "total" units. The authors define "good" unit yield as the amount of simultaneously recorded neurons labeled "good" by the automated spike sorter Kilosort without post-hoc manual curation. This definition was used to standardize cell yield between users who would otherwise manually curate cells and introduce individual variability as to what is considered a "good" unit. However, manual curation of spike sorted output is typically necessary to eliminate false positive units and "merge" spikes belonging to the same neuron that Kilosort identified as belonging to two separate neurons (i.e., spikes that share a refractory period, waveform shape, and localized to the same channels). As such, one may reasonably expect the yield for actual "good" units to be lower than what is reported. Furthermore, including units labeled by Kilosort as multi-unit activity in the "total" yield does not lend itself, by definition, to accurate quantification of individual neurons.

(2) For transparency's sake, restatement of whether the cell yield data came from mice or rats, and from one lab or multiple labs, in the figure or figure captions would be helpful. Based on the introduction of the paper, one gets the impression that the Repix system was designed for mice and rats and, therefore, that data from mice and rats were to be roughly equally represented. This is not the case, as only 1/3 of the reported Repix users were implanted in rats, and cell yield data was shown for only two brain regions in rats (compared with four in mice). The authors state that Repix was designed "... to record neural activities during social interaction of mice" in the Discussion section. It would be helpful for this statement to appear in the Introduction so that it is clear to the reader that Repix was designed for mice but also works well for rats.

(3) Regarding Figure 2, it would be informative to separate this data by species. Does Repix fail more in a procedural stage depending on whether the user is working with mice or rats?

Reviewer #2 (Public Review):

Vangl2, a core planar cell polarity protein involved in Wnt/PCP signaling, cell proliferation, differentiation, homeostasis, and cell migration. Vangl2 malfunctioning has been linked to various human ailments, including autoimmune and neoplastic disorders. Interestingly, it was shown that Vangl2 interacts with the autophagy regulator p62, and autophagic degradation limits the activity of inflammatory mediators, such as p65/NF-κB. However, the possible role of Vangl2 in inflammation has not been investigated. In this manuscript, Lu et al. describe that Vangl2 expression is upregulated in human sepsis-associated PBMCs and that Vangl2 mitigates experimental sepsis in mice by negatively regulating p65/NF-κB signaling in myeloid cells. Their mechanistic studies further revealed that Vangl2 recruits the E3 ubiquitin ligase PDLIM2 to promote K63-linked poly-ubiquitination of p65. Vangl2 also facilitated the recognition of ubiquitinated p65 by the cargo receptor NDP52. These molecular processes caused selective autophagic degradation of p65. Indeed, abrogation of PDLIM2 or NDP52 functions rescued p65 from autophagic degradation, leading to extended p65/NF-κB activity in myeloid cells. Overall, the manuscript presents convincing evidence for novel Vangl2-mediated control of inflammatory p65/NF-kB activity. The proposed pathway may expand interventional opportunities restraining aberrant p65/NF-kB activity in human ailments.

IKK is known to mediate p65 phosphorylation, which instructs NF-kB transcriptional activity. In this manuscript, Vangl2 deficiency led to an increased accumulation of phosphorylated p65 and IKK also at 30 minutes post-LPS stimulation; however, autophagic degradation of p-p65 may not have been initiated at this early time point. Therefore, this set of data put forward the exciting possibility that Vangl2 could also be regulating the immediate early phase of inflammatory response involving the IKK-p65 axis - a proposition that may be tested in future studies.

Reviewer #2 (Public Review):

The authors screened 21 E2 enzymes for their role in HTTExon1Q72-mCherry (HTT) aggregation in the Drosophila eye. They identified UBE2D, whose knockdown leads to increased HTT aggregation that can be rescued by ectopic expression of the human homolog. The protein levels of UBE2D decrease with aging and knockdown of UBED2 leads to an accumulation of ubiquitinated proteins and a shortened lifespan that can be rescued by ectopic expression of the human homolog. Knockdown of UBE2D leads to proteomic changes with up- and down-regulated proteins that include both components of the proteostasis network.

Comments on revised version:

The authors have not addressed a single critical point experimentally. Their explanations are not resolving my concerns and hence the following critical points remain:

• The readout of HTT aggregation (with methods that are not suitable) as proxy for the role of UBE2D in proteostasis is not convincing.

• UBE2D knockdown increases the number of HTT foci (Fig. 1A), but the quantification is less convincing as depicted in Fig. 1B and other E2 enzymes show a stronger effect (e.g. Ubc6 that is only studied in Figs. 1 + 2 without an explanation and Ubc84D). It does not help or add anything to this study that the authors refer to a previous publication. This review assesses this manuscript.

• The quantification of the HTT fluorescence cannot be used as proxy for HTT aggregation. The authors should assess HTT aggregation by e.g. SDD-AGE, FRAP, filter retardation etc. The quantification of the higher MW species of HTT in the SDS-PAGE is not ideal either as this simply reflects material that is stuck in the wells that could not enter the gel. Aggregation and hence high MW size could be one reason, but it can also be HTT trapped in cell debris etc. This point is critical and I disagree with the response of the authors.

• Does UBE2D ubiquitinate HTT? And thus, is HTT accumulation a suitable readout for the functional assessment of the E2 enzyme UBE2D? The authors state that UBE2D does not ubiquitinate HTT. Thus, HTT accumulation is an indirect consequence of perturbed proteostasis. There are certainly better readouts for the role of UBE2D once they have identified substrates.

• The proteomic analyses could help to identify potential substrates for UBE2D. I think its is a missed chance to not follow up on the proteomic analysis to identify substrates and define the role of UBE2D in maintainig proteostasis.

• Are there mutants available for UBE2D or conditional mutants? One caveat of RNAi are: first not complete knockdown and second, variable knockdown efficiencies that increases variability. So mutants are available and yet the authors refuse to use those.

• The analysis of the E3 enzymes does not add anything to this manuscript and the author's response that this manuscript is a follow-up study on a previous publication of the lab is certainly not a valid argument.

• The manuscript remains at this stage rather descriptive.

Reviewer #2 (Public Review):

Summary:

The authors in this study previously reported that BYL719, an inhibitor of PI3Kα, suppressed heterotopic ossification in mice model of a human genetic disease, fibrodysplasia ossificans progressive, which is caused by the activation of mutant ACVR1/R206H by Activin A. The aim of this study is to identify the mechanism of BYL719 for the inhibition of heterotopic ossification. They found that BYL719 suppressed heterotopic ossification in two ways: one is to inhibit the specification of precursor cells for chondrogenic and osteogenic differentiation and the other is to suppress the activation of inflammatory cells.

Strengths:

This study is based on authors' previous reports and the experimental procedures including the animal model are established. In addition, to confirm the role of PI3Kα, authors used the conditional knock-out mice of the subunit of PI3Kα. They clearly demonstrated the evidence indicating that the targets of PI3Kα are not members of TGFBR by a newly established experimental method.

Weaknesses:

Overall, the presented data were closely related to those previously published by authors' group or others and there were very few new findings. The molecular mechanisms through which BYL719 inhibits HO remain unclear, even in the revised manuscript.

Heterotopic ossification in the mice model was not stable and inappropriate for the scientific evaluation.

The method for chondrogenic differentiation was not appropriate, and the scientific evidence of successful differentiation was lacking.

The design of the gene expression profile comparison was not appropriate and failed to obtain the data for the main aim of this study.

The experiments of inflammatory cells were performed in cell lines without ACVR1/R206H mutation, and therefore the obtained data were not precisely related to the inflammation in FOP.

Reviewer #2 (Public Review):

Summary:

This paper reports the structures of two human biotin-dependent carboxylases. The authors used endogenously purified proteins and solved the structures in high resolutions. Based on the structures, they defined the binding site for acyl-CoA and biotin and reported the potential conformational changes in biotin position.

Strengths:

The authors effectively utilized the biotin of the two proteins and obtained homogeneous proteins from human cells. They determined the high-resolution structures of the two enzymes in apo and substrate-bound states.

Comments and questions to the manuscripts:

(1) I'm quite impressed with the protein purification and structure determination, but I think some functional characterization of the purified proteins should be included in the manuscript. The activity of enzymes should be the foundation of all structures and other speculations based on structures.

(2) In Figure 1B, the structure of MCC is shown as two layers of beta units and two layers of alpha units, while there is only one layer of alpha units resolved in the density maps. I suggest the authors show the structures resolved based on the density maps and show the complete structure with the docked layer in the supplementary figure.

(3) In the introduction, I suggest the author provide more information about the previous studies about the structure and reaction mechanisms of BDCs, what is the knowledge gap, and what problem you will resolve with a higher resolution structure. For example, you mentioned in line 52 that G437 and A438 are catalytic residues, are these residues reported as catalytic residues or this is based on your structures? Has the catalytic mechanism been reported before? Has the role of biotin in catalytic reactions revealed in previous studies?

(4) In the discussion, the authors indicate that the movement of biotin could be related to the recognition of acyl-CoA in BDCs, however, they didn't observe a change in the propionyl-CoA bound MCC structure, which is contradictory to their speculation. What could be the explanation for the exception in the MCC structure?

(5) In the discussion, the authors indicate that the selectivity of PCC to different acyl-CoA is determined by the recognition of the acyl chain. However, there are no figures or descriptions about the recognition of the acyl chain by PCC and MCC. It will be more informative if they can show more details about substrate recognition in Figures 3 and 4.

(6) How are the solved structures compared with the latest Alphafold3 prediction?

Reviewer #2 (Public Review):

This manuscript by Xue et al. describes the effects of a long noncoding RNA, lncDACH1, on the localization of Nav channel expression, the magnitude of INa, and arrhythmia susceptibility in the mouse heart. Because lncDACH1 was previously reported to bind and disrupt membrane expression of dystrophin, which in turn is required for proper Nav1.5 localization, much of the findings are inferred through the lens of dystrophin alterations.

The results report that cardiomyocyte-specific transgenic overexpression of lncDACH1 reduces INa in isolated cardiomyocytes; measurements in the whole heart show a corresponding reduction in conduction velocity and enhanced susceptibility to arrhythmia. The effect on INa was confirmed in isolated WT mouse cardiomyocytes infected with a lncDACH1 adenoviral construct. Importantly, reducing lncDACH1 expression via either a cardiomyocyte-specific knockout or using shRNA had the opposite effect: INa was increased in isolated cells, as was conduction velocity in the heart. Experiments were also conducted with a fragment of lnDACH1 identified by its conservation with other mammalian species. Overexpression of this fragment resulted in reduced INa and greater proarrhythmic behavior. Alteration of expression was confirmed by qPCR.

The mechanism by which lnDACH1 exerts its effects on INa was explored by measuring protein levels from cell fractions and immunofluorescence localization in cells. In general, overexpression was reported to reduce Nav1.5 and dystrophin levels and knockout or knockdown increased them.

The strengths of this manuscript include convincing evidence of a link between lncDACH1 and Na channel function. The identification of a lncDACH1 segment conserved among mammalian species is compelling. The observation that lncDACH1 is increased in a heart failure model and provides a plausible hypothesis for disease mechanism.

Reviewer #2 (Public Review):

Summary:

This work addresses a puzzling finding in the viral forecasting literature: high-frequency viral variants evince signatures of neutral dynamics, despite strong evidence for adaptive antigenic evolution. The authors explicitly model interactions between the dynamics of viral adaptations and of the environment of host immune memory, making a solid theoretical and simulation-based case for the essential role of host-pathogen eco-evolutionary dynamics. While the work does not directly address improved data-driven viral forecasting, it makes a valuable conceptual contribution to the key dynamical ingredients (and perhaps intrinsic limitations) of such efforts.

Strengths:

This paper follows up on previous work from these authors and others concerning the problem of predicting future viral variant frequency from variant trajectory (or phylogenetic tree) data, and a model of evolving fitness. This is a problem of high impact: if such predictions are reliable, they empower vaccine design and immunization strategies. A key feature of this previous work is a "traveling fitness wave" picture, in which absolute fitnesses of genotypes degrade at a fixed rate due to an advancing external field, or "degradation of the environment". The authors have contributed to these modeling efforts, as well as to work that critically evaluates fitness prediction (references 11 and 12). A key point of that prior work was the finding that fitness metrics performed no better than a baseline neutral model estimate (Hamming distance to a consensus nucleotide sequence). Indeed, the apparent good performance of their well-adopted "local branching index" (LBI) was found to be an artifact of its tendency to function as a proxy for the neutral predictor. A commendable strength of this line of work is the scrutiny and critique the authors apply to their own previous projects. The current manuscript follows with a theory and simulation treatment of model elaborations that may explain previous difficulties, as well as point to the intrinsic hardness of the viral forecasting inference problem.

This work abandons the mathematical expedience of traveling fitness waves in favor of explicitly coupled eco-evolutionary dynamics. The authors develop a multi-compartment susceptible/infected model of the host population, with variant cross-immunity parameters, immune waning, and infectious contact among compartments, alongside the viral growth dynamics. Studying the invasion of adaptive variants in this setting, they discover dynamics that differ qualitatively from the fitness wave setting: instead of a succession of adaptive fixations, invading variants have a characteristic "expiring fitness": as the immune memories of the host population reconfigure in response to an adaptive variant, the fitness advantage transitions to quasi-neutral behavior. Although their minimal model is not designed for inference, the authors have shown how an elaboration of host immunity dynamics can reproduce a transition to neutral dynamics. This is a valuable contribution that clarifies previously puzzling findings and may facilitate future elaborations for fitness inference methods.

The authors provide open access to their modeling and simulation code, facilitating future applications of their ideas or critiques of their conclusions.

Weaknesses:

The current modeling work does not make direct contact with data. I was hoping to see a more direct application of the model to a data-driven prediction problem. In the end, although the results are compelling as is, this disconnect leaves me wondering if the proposed model captures the phenomena in detail, beyond the qualitative phenomenology of expiring fitness. I would imagine that some data is available about cross-immunity between strains of influenza and sarscov2, so hopefully some validation of these mechanisms would be possible.

After developing the SIR model, the authors introduce an effective "expiring fitness" model that avoids the oscillatory behavior of the SIR model. I hoped this could be motivated more directly, perhaps as a limit of the SIR model with many immune groups. As is, the expiring fitness model seems to lose the eco-evolutionary interpretability of the SIR model, retreating to a more phenomenological approach. In particular, it's not clear how the fitness decay parameter nu and the initial fitness advantage s_0 relate to the key ecological parameters: the strain cross-immunity and immune group interaction matrices.

Reviewer #2 (Public Review):

Summary:

In this study, Bisen et al. characterized the state-dependency of insulin-producing cells in the brain of *Drosophila melanogaster*. They successfully established that IPC activity is modulated by the nutritional state and age of the animal. Interestingly, they demonstrate that IPCs respond to the ingestion of glucose, rather than to perfusion with it, an observation reminiscent of the incretin effect in mammals. The study is well conducted and presented and the experimental data convincingly support the claims made.

Strengths:

The study makes great use of the tools available in *Drosophila* research, demonstrating the effect that starvation and subsequent refeeding have on the physiological activity of IPCs as well as on the behavior of flies to then establish causal links by making use of optogenetic tools.

It is particularly nice to see how the authors put their findings in context to published research and use for example TDC2 neuron activation or DH44 activity to establish baselines to relate their data to.

Weaknesses:

I find the inability of SD to rescue the IPC starvation effect in Figure 1G&H surprising, given that the fully fed flies were raised and kept on that exact diet. Did the authors try to refeed flies with SD for longer than 24 hours? I understand that at some point the age effect would also kick in and counteract potential IPC activity rescue. I think the manuscript would benefit if the authors could indicate the exact age of the SD refed flies and expand a bit on the discussion of that point.

The incretin-like effect is exciting and it will be interesting in the future to find out what might be the signal mediating this effect. It is interesting that IPCs in explants seem to be responsive to glucose. I think it would help if the authors could briefly discuss possible sources for the different findings between these in fact very different preparations. Could the the absence of the inhibitory DH44 feedback in the *ex-vivo* recordings for example play a role?

The incretin-like effect the authors observed seems to start only after 5h which seems longer than in mammals where, as far as I know, insulin peaks around 1h. Do the authors have ideas on how this timescale relates to ingestion and glucose dynamics in flies?

The authors mention "a decrease in the FV of IPC-activated starved flies even before the first optogenetic stimulation (Figure 2I),". Could this be addressed by running an experiment in darkness, only using the IR illumination of their behavioral assay?

The authors show an inhibitory effect of DH44 neuron activation on IPC activity. They further demonstrate that DH44PI neurons are not the ones driving this and thus conclude that "...IPCs are inhibited by DH44Ns outside the PI.". As the authors mentioned the broad expression of the DH44-Gal4 line, can they be sure that the cells labeled outside the PI are actually DH44+? If so they should state this more clearly, if not they should adapt the discussion accordingly.

Reviewer #2 (Public Review):

Summary:

In this study the authors have used pull-down experiments in a cell line overexpressing tagged SERPINE1 mRNA binding protein 1 (SERBP1) followed by mass spectrometry-based proteomics, to establish its interactome. Extensive analyses are performed to connect the data to published resources. The authors attempt to connect SERBP1 to stress granules and Alzheimer's disease-associated tau pathology. Based on the interactome, the authors propose a cross-talk between SERBP1 and PARP1 functions.

Strengths:

The main strength of this study lies in the proteomics data analysis, and its effort to connect the data to published studies.

Weaknesses:

While the authors propose a feedback regulatory model for SERBP1 and PARP1 functions, strong evidence for PARylation modulating SERBP1 functions is lacking. PARP inhibition decreasing the amount of PARylated proteins associated with SERBP1 and likely all other PARylated proteins is expected. This study is also incomplete in its attempt to establish a connection to Alzheimer's disease related tauopathy. A single AD case is not sufficient, and frozen autopsy tissue shows unexplained punctate staining likely due to poor preservation of cellular structures for immunohistochemistry. There is a lack of essential demographic data, source of the tissue, brain regions shown, and whether there was an IRB protocol for the human brain tissue. The presence of phase-separated transient stress granules in an autopsy brain is unlikely, even if G3BP1 staining is present. Normally, stress granule proteins move to the cytoplasm under cellular stress, whereas SERBP1 becomes nuclear. The co-localization of abundant cytoplasmic G3BP1 and SERBP1 under normal conditions does not indicate an association with stress granules.

Reviewer #2 (Public Review):

Summary:

The authors have collected a significant amount of data from the literature on the flow regimes associated with microorganisms whose propulsion is achieved through the action of cilia or flagella, with particular interest in the competition between sessile and motile lifestyles. They then use several distinct hydrodynamic models for the cilia-driven flows to quantify the nutrient uptake and clearance rate, reported as a function of the Peclet number. Among the interesting conclusions the authors draw concerns the question of whether, for certain ciliates, there is a clear difference in nutrient uptake rates in the sessile versus motile forms. The authors show that this is not the case, thereby suggesting that the evolutionary pressure associated with such a difference is not present. The analysis also includes numerical calculations of the uptake rate for spherical swimmers in the regime of large Peclet numbers, where the authors note an enhancement due to advection-generated thinning of the solutal boundary layer around the organism.

Strengths:

In addressing the whole range of organism sizes and Peclet numbers the authors have achieved an important broad perspective on the problem of nutrient uptake of ciliates, with implications for understanding evolutionary driving forces toward particular lifestyles (e.g. sessile versus motile).

Weaknesses:

The authors appear to be unaware of rather similar calculations that were done some years ago in the context of Volvox, in which the issue of the boundary layer size and nutrient uptake enhancement were clearly recognized [M.B. Short, et al., Flows Driven by Flagella of Multicellular Organisms Enhance Long-Range Molecular Transport, PNAS 103, 8315-8319 (2006)]. This reference also introduced the model of a fixed shear stress at the surface of the sphere as a representation of the action of the cilia, which may be more realistic than the squirmer-type boundary condition, although the two lead to similar large-Pe scalings.

The findings reported in Figure 4, that the uptake rate is robust to variations in cilia coverage and absorption fraction, are similar in spirit to an observation made recently in the context of the somatic cell neighbourhood areas in Vovox [Day, et al., eLife 11, e72707 (2022)]. There, it was found that while there is a broad distribution of those areas, and hence of the coarse-grained tangential flagellar force acting on the fluid, the propulsion speed is rather insensitive to those variations.

Reviewer #2 (Public Review):

Summary:

Translation of CGG repeats leads to the accumulation of poly G, which is associated with neurological disorders. This is a valuable paper in which the authors sought out proteins that modulate RAN translation. They determined which proteins in Hela cells bound to CGG repeats and affected levels of polyG encoded in the 5'UTR of the FMR1 mRNA. They then showed that siRNA depletion of ribosomal protein RPS26 results in less production of FMR1polyG than in control. There are data supporting the claim that RPS26 depletion modulates RAN translation in this RNA, although for some results, the Western results are not strong. The data to support increased aggregation by polyG expression upon S26 KD are incomplete.

Strengths:

The authors have proteomics data that show the enrichment of a set of proteins on FMR1 RNA but not a related RNA.

Weaknesses:

-It is insinuated that RPS26 binds the RNA to enhance CGG-containing protein expression. However, RPS26 reduction was also shown previously to affect ribosome levels, and reduced ribosome levels can result in ribosomes translating very different RNA pools.

-A significant claim is that RPS26 KD alleviates the effects of FMR polyG expression, but those data aren't presented well.

Reviewer #3 (Public Review):

Summary:

This article is a direct follow-up to the paper published last year in eLife by the same group. In the previous article, the authors discovered a zinc finger protein, Kipferl, capable of guiding the HP1 protein Rhino towards certain genomic regions enriched in GRGGN motifs and packaged in heterochromatin marked by H3K9me3. Unlike other HP1 proteins, Rhino recruitment activates the transcription of heterochromatic regions, which are then converted into piRNA source loci. The molecular mechanism by which Kipferl interacts specifically with Rhino (via its chromodomain) and not with other HP1 proteins remained enigmatic.

In this latest article, the authors go a step further by elucidating the molecular mechanisms important for the specific interaction of Rhino and not other HP1 proteins with Kipferl. A phylogenetic study carried out between the HP1 proteins of 5 Drosophila species led them to study the importance of an AA Glycine at position 31 located in the Rhino chromodomain, an AA different from the AA (aspartic acid) found at the same position in the other HP1 proteins. The authors then demonstrate, through a series of structure predictions, biochemical and genetic experiments, that this specific AA in the Rhino-specific chromodomain explains the difference in the chromatin binding pattern between Rhino and the other Drosophila HP1 proteins. Importantly, the G31D conversion of the Rhino protein prevents interaction between Rhino and Kipferl, phenocopying a Kipfer mutant.

Strengths:

The strength of this study is to test at the molecular and genetic level whether the difference in the AA sequence- encovered by phylogenetic analysis of HP1 proteins including Rhino combined with structure prediction- can explain the difference in chromatin binding patterns between HP1 proteins and Rhino.<br /> To do so they have created a Rhino mutant by introducing a point mutation into the endogenous rhino gene, reverting the Glycine in position 31 to the aspartic acid found in all other HP1 proteins. Even if the Rhino G31D mutant retains its ability to interact with H3K9me3 (predictive and biochemistry approaches that I'm less familiar with) it does not localize correctly on the chromatin preventing certain regions such as locus 80F from being converted into piRNA source loci. However other regions such as satellite regions attract the Rhino mutant protein converting them into super piRNA source loci, phenocopying the effects observed in a Kipferl mutant. Why Rhino when not bound to Kipferl concentrates in satellite regions is a question that remains unanswered.

Weaknesses:

In this new version of the manuscript, the authors have answered all the questions and weaknesses raised previously.

Reviewer #2 (Public Review):

Summary:

Jellinger, Suthard, et al. investigated the transcriptome of positive and negative valence engram cells in the ventral hippocampus, revealing anti- and pro-inflammatory signatures of these respective valences. The authors further reactivated the negative valence engram ensembles to assay the effects of chronic negative memory reactivation in young and old mice. This chronic re-activation resulted in differences in aspects of working memory, fear memory, and caused morphological changes in glia. Such reactivation-associated changes are putatively linked to GABA changes and behavioral rumination.

Strengths:

Much the content of of this manuscript is of benefit to the community, such as the discovery of differential engram transcriptomes dependent on memory valence. The chronic activation of neurons, and the resultant effects on glial cells and behavior, also provide the community with important data. Laudable points of this manuscript include the comprehensiveness of behavioral experiments, as well as the cross-disciplinary approach.

Weaknesses:

Weaknesses noted in the previous version of the manuscript have been accounted for.

Reviewer #3 (Public Review):

Summary:

In their article Jack Lindsey and Ashok Litwin-Kumar describe a new model for systems memory consolidation. Their idea is that a short-term memory acts not as a teacher for a long-term memory - as is common in most complementary learning systems -, but as a selection module that determines which memories are eligible for long term storage. The criterion for the consolidation of a given memory is a sufficient strength of recall in the short term memory.

The authors provide an in-depth analysis of the suggested mechanism. They demonstrate that it allows substantially higher SNRs than previous synaptic consolidation models, provide an extensive mathematical treatment of the suggested mechanism, show that the required recall strength can be computed in a biologically plausible way for three different learning paradigms, and illustrate how the mechanism can explain spaced training effects.

Strengths:

The suggested consolidation mechanism is novel and provides a very interesting alternative to the classical view of complementary learning systems. The analysis is thorough and convincing.

Weaknesses:

The main weakness of the paper is the equation of recall strength with the synaptic changes brought about by the presentation of a stimulus. In most models of learning, synaptic changes are driven by an error signal and hence cease once the task has been learned. The suggested consolidation mechanism would stop at that point, although recall is still fine. The authors should discuss other notions of recall strength that would allow memory consolidation to continue after the initial learning phase. Aside from that, I have only a few technical comments that I'm sure the authors can address with a reasonable amount of work.

Reviewer #2 (Public Review):

The paper from Liu et al shows a mechanism by which axons can change direction during development. They use the sLNv neurons as a model. They find that the appearance of a new group of neurons (DNs) during post-embryonic proliferation secretes netrins and repels horizontally towards the midline, the axonal tip of the LNvs. The experiments are well done and the results are conclusive.

Reviewer #2 (Public Review):

Summary:

Here the effect of overall transcription blockade, and then specifically depletion of YAP/TAZ transcription factors was tested on cytoskeletal responses, starting from a previous paper showing YAP/TAZ-mediated effects on the cytoskeleton and cell behaviors. Here, primary endothelial cells were assessed on substrates of different stiffness and parameters such as migration, cell spreading, and focal adhesion number/length were tested upon transcriptional manipulation. Zebrafish subjected to similar manipulations were also assessed during the phase of intersegmental vessel elongation. The conclusion was that there is a feedback loop of 4 hours that is important for the effects of mechanical changes to be translated into transcriptional changes that then permanently affect the cytoskeleton.

The idea is intriguing and a previous paper contains data supporting the overall model. The fish washout data is quite interesting and supports the kinetics conclusions. New transcriptional profiling in this version supports that cytoskeletal genes are differentially regulated with YAP/TAZ manipulations.

Major strengths: The combination of in vitro and in vivo assessment provides evidence for timing in physiologically relevant contexts, and rigorous quantification of outputs is provided. The idea of defining temporal aspects of the system is quite interesting. New RNA profiling supports the model.

Weaknesses:

Actinomycin D blocks most transcription so exposure for hours likely leads to secondary and tertiary effects and perhaps effects on viability.

Reviewer #2 (Public Review):

The manuscript by Okholm and colleagues identified an interesting new instance of ceRNA involving a circular RNA. The data are clearly presented and support the conclusions. Quantification of the copy number of circRNA and quantification of the protein were performed, and this is important to support the ceRNA mechanism.

This is the second rebuttal and the authors further improved the manuscript. The data are of interest to the large spectrum of readers of the journal.

Comments on revision:

The authors explain that they have compared primer efficiencies of two linear Laccase version amplicons and their divergent primers targeting circHIPK3 using amplification standard curves (not shown). They claim that all amplicons were found to be directly comparable, ensuring that their estimation of cirRNA:lineal ratio estimation by RT-qPCR was accurate. I agree that this is not a technically trivial experiment. However, for this measurement to be valid, it is not enough to compare the efficiencies of primers using cDNA/DNA standard curves in the context of the qPCR reaction alone. Instead, one should perform the full RT-qPCR tandem reactions in the context of standard curves of the specific RNAs (for example, obtained by in vitro synthesis). RNA absolute amounts in these standard curves should be known in order to compare the different RNA species (linear or circular).

I do not have major concerns about this issue.

Reviewer #2 (Public Review):

The authors of this study have investigated how oscillations may promote fear learning using a network model. They distinguished three types of rhythmic activities and implemented an STDP rule to the network aiming to understand the mechanisms underlying fear learning in the BLA. My comments are the following.

(1) Gamma oscillations are generated locally; thus, it is appropriate to model in any cortical structure. However, the generation of theta rhythms is based on the interplay of many brain areas therefore local circuits may not be sufficient to model these oscillations. Moreover, to generate the classical theta, a laminal structure arrangement is needed (where neurons form layers like in the hippocampus and cortex)(Buzsaki, 2002), which is clearly not present in the BLA. To date, I am not aware of any study which has demonstrated that theta is generated in the BLA. All studies that recorded theta in the BLA performed the recordings referenced to a ground electrode far away from the BLA, an approach that can easily pick up volume conducted theta rhythm generated e.g., in the hippocampus or other layered cortical structure. To clarify whether theta rhythm can be generated locally, one should have conducted recordings referenced to a local channel (see Lalla et al., 2017 eNeuro). In summary, at present, there is no evidence that theta can be generated locally within the BLA. Though, there can be BLA neurons, firing of which shows theta rhythmicity, e.g., driven by hippocampal afferents at theta rhythm, this does not mean that theta rhythm per se can be generated within the BLA as the structure of the BLA does not support generation of rhythmic current dipoles. This questions the rationale of using theta as a proxy for BLA network function which does not necessarily reflect the population activity of local principal neurons in contrast to that seen in the hippocampus.

(2) The authors distinguished low and high theta. This may be misleading, as the low theta they refer to is basically a respiratory-driven rhythm typically present during an attentive state (Karalis and Sirota, 2022; Bagur et al., 2021, etc.). Thus, it would be more appropriate to use breathing-driven oscillations instead of low theta. Again, this rhythm is not generated by the BLA circuits, but by volume conducted into this region. Yet, the firing of BLA neurons can still be entrained by this oscillation. I think it is important to emphasize the difference.

(3) The authors implemented three interneuron types in their model, ignoring a large fraction of GABAergic cells present in the BLA (Vereczki et al., 2021). Recently, the microcircuit organization of the BLA has been more thoroughly uncovered, including connectivity details for PV interneurons, firing features of neurochemically identified interneurons (instead of mRNA expression-based identification, Sosulina et al., 2010), synaptic properties between distinct interneuron types as well as principal cells and interneurons using paired recordings. These recent findings would be vital to incorporate into the model instead of using results obtained in the hippocampus and neocortex. I am not sure that a realistic model can be achieved by excluding many interneuron types.

(4) The authors set the reversal potential of GABA-A receptor-mediated currents to -80 mV. What was the rationale for choosing this value? The reversal potential of IPSCs has been found to be -54 mV in fast-spiking (i.e., parvalbumin) interneurons and around -72 mV in principal cells (Martina et al., 2001, Veres et al., 2017).

(5) Proposing neuropeptide VIP as a key factor for learning is interesting. Though, it is not clear why this peptide is more important in fear learning in comparison to SST and CCK, which are also abundant in the BLA and can effectively regulate the circuit operation in cortical areas.

Reviewer #2 (Public Review):

The authors of this study have investigated how oscillations may promote fear learning using a network model. They distinguished three types of rhythmic activities and implemented an STDP rule to the network aiming to understand the mechanisms underlying fear learning in the BLA.

After the revision, the fundamental question, namely, whether the BLA networks can or cannot intrinsically generate any theta rhythms, is still unanswered. The author added this sentence to the revised version: "A recent experimental paper, (Antonoudiou et al., 2022), suggests that the BLA can intrinsically generate theta oscillations (3-12 Hz) detectable by LFP recordings under certain conditions, such as reduced inhibitory tone." In the cited paper, the authors studied gamma oscillations, and when they applied 10 uM Gabazine to the BLA slices observed rhythmic oscillations at theta frequencies. 10 uM Gabazine does not reduce the GABA-A receptor-mediated inhibition but eliminates it, resulting in rhythmic populations burst driven solely by excitatory cells. Thus, the results by Antonoudiou et al., 2022 contrast with, and do not support, the present study, which claims that rhythmic oscillations in the BLA depend on the function of interneurons. Thus, there is still no convincing evidence that BLA circuits can intrinsically generate theta oscillations in intact brain or acute slices. If one extrapolates from the hippocampal studies, then this is not surprising, as the hippocampal theta depends on extra-hippocampal inputs, including, but not limited to the entorhinal afferents and medial septal projections (see Buzsaki, 2002). Similarly, respiratory related 4 Hz oscillations are also driven by extrinsic inputs. Therefore, at present, it is unclear which kind of physiologically relevant theta rhythm in the BLA networks has been modelled.

Reviewer #2 (Public Review):

Summary:

Molecular dynamics (MD) data is deposited in public, non-specialist repositories. This work starts from the premise that these data are a valuable resource as they could be used by other researchers to extract additional insights from these simulations; it could also potentially be used as training data for ML/AI approaches. The problem is that mining these data is difficult because they are not easy to find and work with. The primary goal of the authors was to discover and index these difficult-to-find MD datasets, which they call the "dark matter of the MD universe" (in contrast to data sets held in specialist databases).

The authors developed a search strategy that avoided the use of ill-defined metadata but instead relied on the knowledge of the restricted set of file formats used in MD simulations as a true marker for the data they were looking for. Detection of MD data marked a data set as relevant with a follow-up indexing strategy of all associated content. This "explore-and-expand" strategy allowed the authors for the first time to provide a realistic census of the MD data in non-specialist repositories.

As a proof of principle, they analyzed a subset of the data (primarily related to simulations with the popular Gromacs MD package) to summarize the types of simulated systems (primarily biomolecular systems) and commonly used simulation settings.

Based on their experience they propose best practices for metadata provision to make MD data FAIR (findable, accessible, interoperable, reusable).

A prototype search engine that works on the indexed datasets is made publicly available. All data and code are made freely available as open source/open data.

Strengths:

- The novel search strategy is based on relevant data to identify full datasets instead of relying on metadata and thus is likely to have many true positives and few false positives.

- The paper provides a first glimpse at the potential hidden treasures of MD simulations and force field parametrizations of molecules.

- Analysis of parameter settings of MD simulations from how researchers *actually* run simulations can provide valuable feedback to MD code developers for how to document/educate users. This approach is much better than analyzing what authors write in the Methods sections.

- The authors make a prototype search engine available.

- The guidelines for FAIR MD data are based on experience gained from trying to make sense of the data.

Weaknesses:

- So far the work is a proof-of-concept that focuses on MD data produced by Gromacs (which was prevalent under all indexed and identified packages).

As discussed in the manuscript, some types of biomolecules are likely underrepresented because different communities have different preferences for force fields/MD codes (for example: carbohydrates with AMBER/GLYCAM using AMBER MD instead of Gromacs).

- Materials sciences seem to be severely under-represented - commonly used codes in this area such as LAMMPS are not even detected, and only very few examples could be identified. As it is, the paper primarily provides an insight into the *biomolecular* MD simulation world.

The authors succeed in providing a first realistic view on what MD data is available in public repositories. In particular, their explore-expand approach has the potential to be customized for all kinds of specialist simulation data, whereby specific artifacts are<br /> used as fiducial markers instead of metadata. The more detailed analysis is limited to Gromacs simulations and primarily biomolecular simulations (even though MD is also widely used in other fields such as the materials sciences). This restricted view may simply be correlated with the user community of Gromacs and hopefully, follow-up studies from this work will shed more light on this shortcoming.

The study quantified the number of trajectories currently held in structured databases as ~10k vs ~30k in generalist repositories. To go beyond the proof-of-principle analysis it would be interesting to analyze the data in specialist repositories in the same way as the one in the generalist ones, especially as there are now efforts underway to create a database for MD simulations (Grant 'Molecular dynamics simulation for biology and chemistry research' to establish MDDB' DOI 10.3030/101094651). One should note that structured databases do not invalidate the approach pioneered in this work; if anything they are orthogonal to each other and both will likely play an important role in growing the usefulness of MD simulations in the future.

Reviewer #2 (Public Review):

Summary:

The paper sought to determine the number of myosin 10 molecules per cell and localized to filopodia, where they are known to be involved in formation, transport within, and dynamics of these important actin-based protrusions. The authors used a novel method to determine the number of molecules per cell. First, they expressed HALO tagged Myo10 in U20S cells and generated cell lysates of a certain number of cells and detected Myo10 after SDS-PAGE, with fluorescence and a stained free method. They used a purified HALO tagged standard protein to generate a standard curve which allowed for determining Myo10 concentration in cell lysates and thus an estimate of the number of Myo10 molecules per cell. They also examined the fluorescence intensity in fixed cell images to determine the average fluorescence intensity per Myo10 molecule, which allowed the number of Myo10 molecules per region of the cell to be determined. They found a relatively small fraction of Myo10 (6%) localizes to filopodia. There are hundreds of Myo10 in each filopodia, which suggests some filopodia have more Myo10 than actin binding sites. Thus, there may be crowding of Myo10 at the tips, which could impact transport, the morphology at the tips, and dynamics of the protrusions themselves. Overall, the study forms the basis for a novel technique to estimate the number of molecules per cell and their localization to actin-based structures. The implications are broad also for being able to understand the role of myosins in actin protrusions, which is important for cancer metastasis and wound healing.

Strengths:

The paper addresses an important fundamental biological question about how many molecular motors are localized to a specific cellular compartment and how that may relate to other aspects of the compartment such as the actin cytoskeleton and the membrane. The paper demonstrates a method of estimating the number of myosin molecules per cell using the fluorescently labeled HALO tag and SDS-PAGE analysis. There are several important conclusions from this work in that it estimates the number of Myo10 molecules localized to different regions of the filopodia and the minimum number required for filopodia formation. The authors also establish a correlation between number of Myo10 molecules filopodia localized and the number of filopodia in the cell. There is only a small % of Myo10 that tip localized relative to the total amount in the cell, suggesting Myo10 have to be activated to enter the filopodia compartment. The localization of Myo10 is log-normal, which suggests a clustering of Myo10 is a feature of this motor.

One of the main critiques of the manuscript was that the results were derived from experiments with overexpressed Myo10 and therefore are hard to extrapolate to physiological conditions. The authors counter this critique with the argument that their results provide insight into a system in which Myo10 is a limiting factor for controlling filopodia formation. They demonstrate that U20S cells do not express detectable levels of Myo10 (supplementary Figure 1E) and thus introducing Myo10 expression demonstrates how triggering Myo10 expression impacts filopodia. An example is given how melanoma cells often heavily upregulation Myo10.

In addition, the revised manuscript addresses the concerns about the method to quantitate the number of Myo10 molecules per cell and therefore puncta in the cell. The authors have now made a good faith effort to correct for incomplete labeling of the HALO tag (Figure 2A-C, supplementary Figure 2D-E). The authors also address the concerns about variability in transfection efficiency (Figure 1D-E).

A very interesting addition to the revised manuscript was the quantitation of the number of Myo10 molecules present during an initiation event when a newly formed filopodia just starts to elongate from the plasma membrane. They conclude that 100s of Myo10 molecules are present during an initiation event. They also examined other live cell imaging events in which growth occurs from a stable filopodia tip and correlated with elongation rates.

Weaknesses:

The authors acknowledge that a limitation of the study is that all of the experiments were performed with overexpressed Myo10. They address this limitation in the discussion but also provide important comparisons for how their work relates to physiological conditions, such as melanoma cells that only express large amounts of Myo10 when they are metastatic. Also, the speculation about how fascin can outcompete Myo10 should include a mechanism for how the physiological levels of fascin can complete with the overabundance of Myo10 (page 10, lines 401-408).

Reviewer #2 (Public Review):

Summary:

Most polymerases and nucleases use two or three divalent metal ions in their catalytic functions. The family of His-Me nucleases, however, use only one divalent metal ion, along with a conserved histidine, to catalyze DNA hydrolysis. The mechanism has been studied previously but, according to the authors, it remained unclear. By use of a time resolved X-ray crystallography, this work convincingly demonstrated that only one M2+ ion is involved in the catalysis of the His-Me I-PpoI 19 nuclease, and proposed concerted functions of the metal and the histidine.

Strengths:

This work performs mechanistic studies, including the number and roles of metal ion, pH dependence, and activation mechanism, all by structural analyses, coupled with some kinetics and mutagenesis. Overall, it is a highly rigorous work. This approach was first developed in Science (2016) for a DNA polymerase, in which Yang Cao was the first author. It has subsequently been applied to just 5 to 10 enzymes by different labs, mainly to clarify two versus three metal ion mechanisms. The present study is the first one to demonstrate a single metal ion mechanism by this approach.

Furthermore, on the basis of the quantitative correlation between the fraction of metal ion binding and the formation of product, as well as the pH dependence, and the data from site-specific mutants, the authors concluded that the functions of Mg2+ and His are a concerted process. A detailed mechanism is proposed in Figure 6.

Even though there are no major surprises in the results and conclusions, the time-resolved structural approach and the overall quality of the results represent a significant step forward for the Me-His family of nucleases. In addition, since the mechanism is unique among different classes of nucleases and polymerases, the work should be of interest to readers in DNA enzymology, or even mechanistic enzymology in general.

Weaknesses:

Two relatively minor issues are raised here for consideration:<br /> p. 4, last para, lines 1-2: "we next visualized the entire reaction process by soaking I-PpoI crystals in buffer....". This is a little over-stated. The structures being observed are not reaction intermediates. They are mixtures of substrates and products in the enzyme-bound state. The progress of the reaction is limited by the progress of the soaking of the metal ion. Crystallography has just been used as a tool to monitor the reaction (and provide structural information about the product). It would be more accurate to say that "we next monitored the reaction progress by soaking....".

p. 5, the beginning of the section. The authors on one hand emphasized the quantitative correlation between Mg ion density and the product density. On the other hand, they raised the uncertainty in the quantitation of Mg2+ density versus Na+ density, thus they repeated the study with Mn2+ which has distinct anomalous signals. This is a very good approach. However, there is still no metal ion density shown in the key Figure 2A. It will be clearer to show the progress of metal ion density in a figure (in addition to just plots), whether it is Mg or Mn.

Reviewer #2 (Public Review):

Summary:

The authors demonstrate that a low parenteral glucose regimen can lead to improved bacterial clearance and survival from Staph epi sepsis in newborn pigs without inducing hypoglycemia, as compared to a high glucose regimen. Using RNA-seq, metabolomic, and proteomic data, the authors conclude that this is primarily mediated by altered hepatic metabolism.

Strengths:

Well-defined controls for every time point, with multiple time points and biological replicates.

The authors used different experimental strategies to arrive at the same conclusion, which lends credibility to their findings.

The authors have published the negative findings associated with their study, including the inability to reverse sepsis-related mortality after switching from SE-high to SE-low at 3h or 6h and after administration of hIAIP.

Weaknesses:

(1) The authors mention, and it is well-known, that Staph epi is primarily involved in late-onset sepsis. The model of S. epi sepsis used in this study clearly replicates early-onset sepsis, but S. epi is extremely rare in this time period. How do the authors justify the clinical relevance of this model?

(2) The authors find that the neutrophil subset of the leukocyte population is diminished significantly in the SE-low and SE-high populations. However, they conclude on page 10 that "modulations of hepatic, but not circulating immune cell metabolism, by reduced glucose supply..." and this is possible because the authors have looked at the entire leukocyte transcriptome. I am curious about why the authors did not sequence the neutrophil-specific transcriptome.

(3) The authors use high (30g/k/d) and low (7.2g/k/d) glucose regimens. These translate into a GIR of 21 and 5 mg/k/min respectively. A normal GIR for a preterm infant is usually 5-8, and sometimes up to 10. Do the authors have a "safe GIR" or a threshold they think we cannot cross? Maybe a point where the metabolism switch takes place? They do not comment on this, especially as GIR and glucose levels are continuous variables and not categorical.

(4) In Figures 2B and C the authors show that SE-high and SE-low animals have differences in the oxphos, TCA, and glycolytic pathways. The authors themselves comment in the Supplementary Table S1B, E-F that these same metabolic pathways are also different in the Con-Low and Con-high animals, it is just the inflammatory pathways that are not different in the non-infected animals. How can they then justify that it is these metabolic pathways specifically which lead to altered inflammatory pathways, and not just the presence of infection along with some other unfound mechanism?

(5) The authors mention in Figure 1F that SE-low animals had lower bacterial burdens than SE-high animals, but then go on to infer that the inflammatory cytokine differences are attributed to a rewiring of the immune response. However, they have not normalized the cytokine levels to the bacterial loads, as the differences in the cytokines might be attributed purely to a difference in bacterial proliferation/clearing.

(6) The authors mention that switching from SE-high to SE-low at 3 or 6 h time points does not reduce mortality. Have the authors considered the reverse? Does hyperglycemia after euglycemia initially, worsen mortality? That would really conclude that there is some metabolic reprogramming happening at the very onset of sepsis and it is a lost battle after that.

Reviewer #2 (Public Review):

Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria and plays a critical role in bacterial virulence. The LPS export mechanism is a potential target for new antibiotics. Inhibiting this process can render bacteria more susceptible to the host immune system or other antibacterial agents. Given the rise of antibiotic-resistant bacteria, novel targets are urgently needed. The seven LPS transport (Lpt) proteins, A-G, move LPS from the inner to the outer membrane. This study investigated the conformational changes in the LptB2FG-LptC complex using site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy, revealing how ATP binding and hydrolysis affect the LptF β-jellyroll domain and lateral gates. The findings highlight the role of LptC in regulating LPS entry, ensuring efficient and unidirectional transport across the periplasm.

The β-jellyrolls are not fully resolved in the vanadate-trapped structure of LptB2FG and LptB2FGC. Therefore, the current study provides valuable information on the functional dynamics of these periplasmic domains, their interactions, and their roles in the unidirectional transport of LPS. Additionally, the dynamic perspective of the lateral gates in LptFG in the presence and absence of LptC is another strength of this study. Moreover, at least in detergent samples, more comprehensive intermediates of the ATP turnover cycle are studied than in the available structures, providing crucial missing mechanistic details.

Other major strengths of the study include high-quality DEER distance measurements in both detergent and proteoliposomes, the latter providing valuable dynamics information in the lipid environment. However, lipid composition is not mentioned. The proteoliposome study is crucial since the previous structural study (Li, Orlando & Liao 2019) was done in rather small-diameter nanodiscs, which might affect the overall dynamics of the complex. It would have been beneficial if the investigators had reconstituted the complex in lipid nanodiscs with the same composition as proteoliposomes. The mixed lipid/detergent micelles provide an alternative. It seems the ATPase activity of the protein complex is much lower in detergent compared with lipid nanodiscs (Li, Orlando & Liao 2019). In the current study, ATPase activity in proteoliposomes is not provided. Also, the reviewer assumes cysteine-less (CL) constructs of the complex components were utilized. The ATPase assay on CL complex is not presented.

Additionally, from previous structural studies and the mass spectrometry data presented here, LPS co-purifies and is already bound to the complex, thus the Apo state may represent the LPS-bound state without nucleotides.

The selection of sites to probe lateral gate 2, which forms the main LPS entry site, may pose an issue. Although the authors provide justification based on the available structures, one site (position 325 in LptF) is located on a flexible loop, and position 52 in LptG is on the neighboring transmembrane helix, separated by a potentially flexible loop from the gating TM1. These labeling sites could exhibit significant local dynamics, resulting in a broader distribution of distances and potentially masking the gating-related conformational changes.

Reviewer #2 (Public Review):

Summary:

Using an innovative task design and analysis approach, the authors set out to show that the activity patterns in the hippocampus related to the development of social relationships with multiple partners in a virtual game. While I found the paper highly interesting (and would be thrilled if the claims made in the paper turned out to be true), I found many of the analyses presented either unconvincing or slightly unconnected to the claims that they were supposed to support. I very much hope the authors can alleviate these concerns in a revision of the paper.

Strengths & Weaknesses:

(1) The innovative task design and analyses, and the two independent samples of participants are clear strengths of the paper.

(2) The RSA analysis is not what I expected after I read the abstract and tile of the result section "The hippocampus represents abstract dimensions of affiliation and power". To me, the title suggests that the hippocampus has voxel patterns, which could be read out by a downstream area to infer the affiliation and power value, independent of the exact identity of the character in the current trial. The presented RSA analysis however presents something entirely different - namely that the affiliation trials and power trials elicit different activity patterns in the area indicated in Figure 3. What is the meaning of this analysis? It is not clear to me what is being "decoded" here and alternative explanations have not been considered. How do affiliation and power trials differ in terms of the length of sentences, complexity of the statements, and reaction time? Can the subsequent decision be decoded from these areas? I hope in the revision the authors can test these ideas - and also explain how the current RSA analysis relates to a representation of the "dimensions of affiliation and power".

(3) Overall, I found that the paper was missing some more fundamental and simpler RSA analyses that would provide a necessary backdrop for the more complicated analyses that followed. Can you decode character identity from the regions in question? If you trained a simple decoder for power and affiliation values (using the LLE, but without consideration of the sequential position as used in the spline analysis), could you predict left-out trials? Are affiliation and power represented in a way that is consistent across participants - i.e. could you train a model that predicts affiliation and power from N-1 subjects and then predict the Nth subject? Even if the answer to these questions is "no", I believe that they are important to report for the reader to get a full understanding of the nature of the neural representations in these areas. If the claim is that the hippocampus represents an "abstract" relationship space, then I think it is important to show that these representations hold across relationships. Otherwise, the claim needs to be adjusted to say that it is a representation of a relationship-specific trajectory, but not an abstract social space.

(4) To determine that the location of a specific character can be decoded from the hippocampal activity patterns, the authors use a sequential analysis in a low-dimensional space (using local linear embedding). In essence, each trial is decoded by finding the pair of two temporally sequential trials that is closest to this pattern, and then interpolating the power/affiliation values linearly between these two points. The obvious problem with this analysis is that fMRI pattern will have temporal autocorrelation and the power and affiliation values have temporal autocorrelation. Successful decoding could just reflect this smoothness in both time series. The authors present a series of control analyses, but I found most of them to not be incisive or convincing and I believe that they (and their explanation of their rationale) need to be improved. For example, the circular shifting of the patterns preserves some of the autocorrelation of the time series - but not entirely. In the shifted patterns, the first and last items are considered to be neighboring and used in the evaluation, which alone could explain the poor performance. The simplest way that I can see is to also connect the first and last item in a circular fashion, even when evaluating the veridical ordering. The only really convincing control condition I found was the generation of new sequences for every character by shuffling the sequence of choices and re-creating new artificial trajectories with the same start and endpoint. This analysis performs much better than chance (circular shuffling), suggesting to me that a lot of the observed decoding accuracy is indeed simply caused by the temporal smoothness of both time series.

(5) Overall, I found the analysis of the brain-behavior correlation presented in Figure 5 unconvincing. First, the correlation is mostly driven by one individual with a large network size and a 6.5 cluster. I suspect that the exclusion of this individual would lead to the correlation losing significance. Secondly, the neural measure used for this analysis (determining the number of optimal clusters that maximize the overlap between neural clustering and behavioral clustering) is new, non-validated, and disconnected from all the analyses that had been reported previously. The authors need to forgive me for saying so, but at this point of the paper, would it not be much more obvious to use the decoding accuracy for power and affiliation from the main model used in the paper thus far? Does this correlate? Another obvious candidate would be the decoding accuracy for character identity or the size of the region that encodes affiliation and power. Given the plethora of candidate neural measures, I would appreciate if the authors reported the other neural measures that were tried (and that did not correlate). One way to address this would have been to select the method on the initial sample and then test it on the validation sample - unfortunately, the measure was not pre-registered before the validation sample was collected. It seems that the correlation was only found and reported on the validation sample?

Reviewer #2 (Public Review):

Summary:

Lamination is a layered neuronal arrangement that provides a basic frame to establish functional connectivity in the brain. The formation of a layered structure requires a highly coordinated interaction between migrating neurons and the developing microenvironment. Earlier studies revealed that to reach specific locations, migrating neurons typically follow various morphogen gradients. Here, Hallada et al. showed that cerebellar granule neurons (CGNs) could navigate via adhesive interaction with Junctional Adhesion Molecule C (JAM-C) followed by recruitment and distribution of intercellular partners (Pard3 and debris) at the contact sites. These results show that neuronal migration could be structured by specific interactions with adhesion molecules and spatial re-arrangements of downstream effectors.

Strengths:

The authors concluded that cis/trans binding sites of JAM-C on CGNs are crucial for contact formation with cerebellar glial cells (Bergman glial cells, BGs) and recruitment of Pard3 and drebrin to contact sites. This conclusion was based on the data obtained utilizing several advanced tools and technical approaches, such as cutting-edge microscopy, detailed visualization of cell-cell recognition, and a new correlation analysis.

Weaknesses:

(1) Despite multiple advanced methodologies, the study has weaknesses related primarily to the lack of specific evidence in support of findings and data interpretation issues. For example, it is unclear how JAM-C-mediated adhesion facilitates the entry of CGNs into the cerebellar molecular layer (ML). The authors described that CGN-CGN JAM recognition recruits more Pard3 and drebrin compared to CGN-BG recognition, which could increase the dwelling time of CGNs before moving to ML. However, such a mechanism does not explain what would initiate the entry of CGNs into ML. Perhaps the authors could provide a detailed explanation of this phenomenon in the Discussion (but certainly not in the Abstract). Also, the authors could consider revising the content of the Abstract, emphasizing their findings, and leaving out the speculations.

(2) To allow for comparison, it would be very helpful to indicate specific numerical values for each data point throughout the manuscript. For example, the authors stated that a change in instantaneous migration angle due to JAM-C silencing negatively affects CGNs movement to the ML (Figure 2) and that spatial distribution of negative JAM-Drebrin correlation is altered at CGN-CGN contacts (Figure 7). However, without specific values, it remains unclear what the magnitude of the discussed changes is or whether they were actually significant. It was not certainly straightforward to make specific conclusions based on graphical presentation alone.

Reviewer #2 (Public Review):

I think this is a very promising paper. The combination of EEG and fMRI is unique and original. However, I also have some suggestions that I think could help improve the manuscript.

This manuscript reports the findings of an EEG-fMRI study (n = 50) on the effects of expectations on pain. The combination of EEG with fMRI is extremely original and well-suited to study the transition from expectation to perception. However, I think that the current treatment of the data, as well as the way that the manuscript is currently written, does not fully capitalize on the potential of this unique dataset. Several findings are presented but there is currently no clear message coming out of this manuscript.

First, one positive point is that the experimental manipulation clearly worked. However, it should be noted that the instructions used are not typical of studies on placebo/nocebo. Participants were not told that the stimulations would be of higher/lower intensity. Rather, they were told that objective intensities were held constant, but that EEG recordings could be used to predict whether they would perceive the stimulus as more or less intense. I think that this is an interesting way to manipulate expectations, but there could have been more justification in the introduction for why the authors have chosen this unusual procedure.

Also, the introduction mentions that little is known about potential cerebral differences between expectations of high vs. low pain expectations. I think the fear conditioning literature could be cited here. Activations in ACC, SMA, Ins, parahippocampal gyrus, PAG, etc. are often associated with upcoming threat, whereas activations vmPFC/default mode network are associated with safety.

The fact that the authors didn't observe a clearer distinction between high and low expectations here could be related to their specific instructions that imply that the stimulus is the same and that it is the subjective perception that is expected to change. In any case, this is a relatively minor issue that is easy to address.

Towards the end of the introduction, the authors present the aims of the study in mainly exploratory terms:<br /> (1) What are the differences between anticipation and perception?<br /> (2) What regions display a difference between high and low expectations (high > low or low < high) vs. an effect of expectation regardless of the direction (high and low different than neutral)?<br /> I think these are good questions, but the authors should provide more justification, or framework, for these questions. More specifically, what will they be able to conclude based on their observations?

For instance (note that this is just an example to illustrate my point. I encourage the authors to come up with their own framework/predictions) :

(1) Possibility #1: A certain region encodes expectations in a directed fashion (high > low) and that same region also responds to perception in the same direction (high > low). This region would therefore modulate pain by assimilating perception towards expectations.<br /> (2) Possibility # 2: different regions are involved in expectation and perception. Perhaps this could mean that certain regions influence pain processing through descending facilitation for instance...

Regarding analyses, I think that examining the transition from expectations to perception is a strong angle of the manuscript given the EGG-fMRI nature of the study. However, I feel that more could have been done here. One problem is that the sequence of analyses starts by identifying an fMRI signal of interest and then attempts to find its EEG correlates. The problem is that the low temporal resolution of fMRI makes it difficult to differentiate expectation from perception, which doesn't make this analysis a good starting point in my opinion. Why not start by identifying an EEG signal that differentiates perception vs expectation, and then look for its fMRI correlates?

Finally, I found the hypotheses on "valenced" vs. "absolute" effects a little bit more difficult to follow. This is because "neutral" is not really neutral: it falls in between low and high. If I follow correctly, participants know that the temperature is always the same. Therefore, if they are told that the machine cannot predict whether their perception is going to be low or high, then it must be because it is likely to be in between. Ratings of expectation and pain ratings confirm that. The neutral condition is not "devoid" of expectations as the authors suggest. Therefore, it would make sense to look at regions with the following pattern low > neutral > high, or vice-versa, low < neutral < high. Low & high being different than neutral is more difficult to interpret. I don't think that you can say that it reflects "absolute" expectations because neutral is also the expectation of a medium temperature. Perhaps it reflects "certainty/uncertainty" or something like that, but it is not clear that it reflects "expectations".

Reviewer #2 (Public Review):

Background and Summary:

This study addresses the intriguing question of whether and how tumors can develop in the freshwater polyp hydra and how they influence the fitness of the animals. Hydra is notable for its significant morphogenetic plasticity and nearly unlimited capacity for regeneration. While its growth through asexual reproduction (budding) and the associated processes of pattern formation have been extensively studied at the cellular level, the occurrence of tumors was only recently described in two strains of Hydra oligactis (Domazet-Lošo et al, 2014). In that research, an arrest in the differentiation of female germ cells led to an accumulation of germline cells that failed to develop into eggs. In hydra, fertile egg cells typically incorporate nurse cells, which originate from large interstitial stem cells (ISCs) restricted to the germline, through apoptosis. However, this increase in apoptosis activity is absent in "germline tumors," and germline ISCs instead form slowly growing patches that do not compromise tissue integrity. Despite the upregulation of certain genes associated with mammalian neoplasms (such as tpt1 and p23) in this tissue, determining whether this differentiation arrest and the resulting egg patches truly constitute neoplasms remains a challenge.

The authors have recently published two papers on the ecological and evolutionary aspects of hydra tumor formation (Boutry et al 2022, 2023), which is also the focus of this manuscript. They transplanted tissues derived from animals with germline tumors to wildtype animals and analyzed their growth patterns, specifically the number of tentacles in the host tissue. They observed that such tissues induced the growth of additional tentacles compared to tissues without germline tumors. The authors conclude that this growth pattern (increased number of tentacles) is correlated with "reducing the burden on the host by (over-)compensating for the reproductive costs of tumors" and claim that "transmissible tumors in hydra have evolved strategies to manipulate the phenotype of their host". While it might be stimulating to add a fresh view from other disciplines (here, ecological and evolutionary aspects), the authors completely ignore the current knowledge of the underlying cell biology of the processes they analyze.

Strengths:

The study focuses on intriguing questions. Whether and how tumors can develop in the freshwater polyp hydra, and how they influence the fitness of the animals?

Weaknesses:

Concept of germline tumors.<br /> The conceptual foundation of their experiments on germline tumors was the study of Domazet-Lošo et al (2014) introducing the concept of germline tumors in hydra (see above). While this is an intriguing hypothesis, there has been little advancement in comprehending the molecular mechanisms underlying tumor formation in hydra beyond this initial investigation. Germline tumors in hydra do not fully meet the typical criteria for neoplasms observed in mammalian tissues. More importantly, a similar phenotype was already reported by the work of Paul Brien and described as "crise gametique" (Brien, 1966, Biologie de la reproduction animale - Blastogenèse, Gamétogenèse, Sexualisation, ed. Masson & Cie, Paris). This phenomenon of gametic crisis is unique to Hydra oligactis, a stenotherm, cold-adapted cosmopolitan species. In this species, gametogenesis severely impacts the vitality of the polyps, often leading to complete exhaustion and death (Tardent, 1974). Animals can only be rescued during the initial phase of the cold-induced sexual period (see also the research of Littlefield (1984, 1985, 1986, 1991). The observed arrest in differentiation arrest in germline tumors might represent an epigenetically established consequence of surviving gametogenesis. Regrettably, this important work was not mentioned by the authors or by Domazet-Lošo et al. (2014), highlighting a notable gap in the recognition of basic research in this area that might challenge the hydra tumor hypothesis.

"Super-nummary" tentacles in graft experiments.<br /> The authors describe that after grafting tissue from animals with germline tumors to wild-type animals, the number of tentacles in the host tissue increased when the donor tissue had germline tumors. A maximum effect of four additional tentacles was found with donor strain H. oligactis robusta and three additional tentacles with donor strain H.oligactis St Petersburg. In general, H.oligactis wild-type host strains had fewer tentacles than H.oligactis St Petersburg strains. This is consistent with the results of Domazet-Lošo et al (2014) who showed that the number of tentacles increased in the strains with germline tumors. What conclusions can be drawn from these experiments? The authors might want to conclude that transmissible tumors in Hydra have developed strategies to manipulate the phenotype of their host. But there is no evidence for this, as essential controls are missing. It is known that the size of hydra polyps is proportion-regulated, i.e. the number of tentacles varies with the size and number of (epithelial) cells. Such controls are missing in the experiments. There is also a lack of controls from wild-type animals in gametogenesis: it is very likely that grafts with wild-type animals with egg spots of comparable size as the germline tumors (see above) will result in similar numbers of tentacles in host tissue.