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Reply to the reviewers

1. Description of the planned revisions

Insert here a point-by-point reply that explains what revisions, additional experimentations and analyses are planned to address the points raised by the referees.

Reviewer 1:

Major comments (numbers correspond to the numbering made by the reviewer):

It is unclear what the TEM in Fig8 is trying to clarify. Since SMCs and elastic fibers are supposed to be bound, it would be better to show the binding site. In addition, the p-MLC in Fig8D-F is a qualitative evaluation, so the difference is not clear, and it is necessary to verify whether there is a difference in Myh11CreERT2;Loxfl/fl mice between aneurysmal (pathogenic) and non-aneurysmal lesions. Overall, this is an associated study that this only speculation since the causal relationship between aneurysm development and Lox functions, which authors found is unclear.

A: While no one has thus far carried out an in vivo deletion of LOX specifically in the smooth muscle cells to demonstrate that in a like manner to the BAPN treatment following its deletion aneurysms occur, the focus of this manuscript is to highlight the as yet undescribed intracellular cytoskeletal phenotypes in the LOX mutant smooth muscle cells and not the related ECM abnormalities. The TEM images in Figure 8 aim to show with high resolution the abnormal cytoskeleton and mitochondria in mice with a specific deletion of Lox in their SMC. Notably, these mice were not induced with AngII and therefore have not developed hypertension. Accordingly, they do not have any aneurysms yet they do display disrupted cytoskeleton and mitochondria within their aortic smooth muscle cells. As suggested by the reviewer, we will monitor SMC interaction with the elastic fibers using TEM. These findings will be presented.

With respect to phosphorylated Myosin Light Chain (p-MLC) - the analysis was carried out on 4 mice, and 6 sections from each mouse from non-aneurysmal regions. In this analysis we plotted the distribution of p-MLC expression which was calculated by quantifying 'intensity x area'. Statistical analysis of the distribution of the histograms (Kolmogorov Smirnov test) depicting p-MLC expression demonstrates they are significantly different (p=6.6E-16). In the mutant aortas, distribution is more dispersed and less organized. We have now elaborated on these findings within the text.

• *

In the discussion (lines 332-334), the Authors described that "Since TGFb signaling is implicated in aneurysm formation..." but the effect of TGFb signal in these Lox-deficient mice has not been examined at all. The effects of pSmad2/3 staining, Western, etc on TGFb activation should be examined and discussed.

A: We agree with the reviewer that we have not monitored TGFβ signaling throughout this manuscript however we and others have previously demonstrated that tight interactions take place between LOX and this signal transduction pathway in multiple processes, in health and disease including within the vasculature (e.g., Taylor MA et al., 2011 Lysyl oxidase contributes to mechanotransduction-mediated regulation of transforming growth factor-beta signaling in breast cancer cells. PMID: 21532881; Atsawasuwan P et al., 2008. Lysyl oxidase binds transforming growth factor-beta and regulates its signaling via amine oxidase activity. PMID: 18835815; Kutchuk L et al., 2015. Muscle composition is regulated by a lox-TGFβ feedback loop. PMID: 25715398; Xu XH et al., 2019. Downregulation of lysyl oxidase and lysyl oxidase-like protein 2 suppressed the migration and invasion of trophoblasts by activating the TGF-β/collagen pathway in preeclampsia. PMID: 30804321; Grunwald H et al., 2021. Lysyl oxidase interactions with transforming growth factor-β during angiogenesis are mediated by endothelin 1. PMID: 34370353). Notably, the effects of LOX on TGFβ signaling has not been the focus of this research and therefore we relate to it only in the Discussion, however as requested by the reviewer, we are now gearing up towards testing activation of the pathway is affected in the LOX mutant SMCs. Should we be unsuccessful we will tone down this statement.

• *

• *

Minor comments (numbers correspond to the numbering made by the reviewer):

1. What is the baseline group in Fig1A? and should be required a minimum 3 of animals in each group. A: The baseline for measuring blood pressure was Tamoxifen-treated Loxfl/fl. This was mentioned in the legend but not in the figure. We apologize for this. However since we only had 2 mice of this genotype, we *have replaced them with Myh11CreERT2; Loxfl/fl and have set additional mice that will be added that are Loxfl/fl. Essentially, all 'baseline' mice will have received tamoxifen yet have not been induced with AngII. A minimum of 4 animals per group will be in this figure. *

Reviewer 2:

Major comments (numbers correspond to the order written by the reviewer):

1. All three key conclusions are supported by data throughout the manuscript. However, the evidence is often based on data originating from western-blotting or immunofluorescent experiments and lack depth and rigidity. For example, figure 4 shows a change of cytoskeletal organization upon LOX KO in HAOSMCs but the authors lack to quantify or further analyse these exact differences in actin/tubulin organization. A: We thank the reviewer for stating that our conclusions are supported by data throughout the manuscript. As requested, we will analyze the organization of the cytoskeleton using image analyses software that enable dissecting linearity, number, length and angle of the cytoskeletal elements. We have already acquired the images and these analyses will be added to the manuscript upon their completion.

2. *

Description of the revisions that have already been incorporated in the transferred manuscript

Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. If no revisions have been carried out yet, please leave this section empty.

• *

__Reviewer 1: __

Major comments (numbers correspond to the numbering by the reviewer):

1. The number of mice used and a number of experiments ("n" number) are not described in each figure or its legends in an overall experiment. Also, there is no information on the statistical analysis, which makes it impossible to judge the validity of the results. A: The minimal number of mice used per analysis in each experiment was 4 apart from the blood pressure measurements for which we have now increased the number (see reply to Minor comment 1 by this Reviewer). These numbers have either been added to the legends or throughout the text. We further added the numbers of cells quantified in the different experiments as well as the p value stemming from the statistical assays (T, Kolmogorov-Smirnov or ANOVA where appropriate).

The Phenotype of Lox-deficient mice is unclear; the picture in Fig1C is not clear and a high-magnified view should be provided. Also, which part (aortic arch or abdominal aorta?) is histologically analyzed? It should be described. In addition to the morphological analysis, it cannot be called "aneurysm" unless the internal diameter is enlarged more than 1.5 times compared to the control aorta. The histological images seem to show only dissection, which is unclear since statistical analysis is not feasible with only 2-3 animals.

A: The images shown in Figure 1C are now larger and of a better resolution so that the various deformities could be easily observed. With respect to the histological analyses - they were carried out on both the thoracic and abdominal aortic sections as reflected by the quantifications in Figure 1E-H. Specifically, the representative histological stainings shown in Figure 1D are of the abdominal regions and this is now mentioned in both the legend and figure. We thank the reviewer for correcting the mistake in our annotation and we have now replaced the images adding higher magnification of aneurysmal and non-pathological regions to demonstrate the relative normal ECM (elastic fibers and collagen) in the non-pathological regions of mutant aortas even though they were derived from hypertensive mice.

• *

Immunostaining in Figs. 4-6 should add nuclei (DAPI) to all experiments. It is unclear how many cells are being looked at. For example, in the staining of Fig4A, the stained nuclei are slightly visible in the shLox group, but not at all in the control above. Phenotypes should be compared under the same conditions. A: *All phenotypes were analyzed under the same conditions and were taken with DAPI. We have added DAPI to all images. As mentioned in comment #1, we have now added to the legends the number of cells analyzed in each experiment. *

• *

For ROCK and RhoA analysis (in Fig4-6), immunostaining and Western alone are not convincing and not sufficient evidence for activation. Other factors, such as methods to measure activation and focal adhesion molecules should be considered.

A: The analyses of ROCK and RhoA are shown in Figure 6. As suggested, we have quantified focal adhesion numbers and size by monitoring vinculin. Our findings demonstrate there are more focal adhesions that form in control cells than in the LOX-devoid ones and that in the latter, those that do form are significantly smaller. These results suggest that the adhesions that form in the mutant cells are weaker and less mature. We have added this data and it will now be presented as Supp. Figure 5. Therefore the previous Supp. Figures 5 and 6 will be shifted accordingly. We have related to these findings in the text.

*As mentioned above, we will use image analysis to quantify the alterations in the cytoskeletal elements such as those shown in Figure 4.

*

It is unclear what the TEM in Fig8 is trying to clarify. Since SMCs and elastic fibers are supposed to be bound, it would be better to show the binding site. In addition, the p-MLC in Fig8D-F is a qualitative evaluation, so the difference is not clear, and it is necessary to verify whether there is a difference in Myh11CreERT2;Loxfl/fl mice between aneurysmal (pathogenic) and non-aneurysmal lesions. Overall, this is an associated study that this only speculation since the causal relationship between aneurysm development and Lox functions, which authors found is unclear.

A: The first part of the comment refers to the TEM images. These have been addressed in the previous section (planned revision) and as mentioned, we will monitor the SMC binding sites to the elastic fibers. The comment raised by the reviewer on p-MLC was not clear to us. As mentioned, we primarily focused on the non-aneurysmal regions whether in AngII-induced hypertensive mice or in non-hypertensive mice as our results suggest that even in the lack of hypertension where no aneurysms develop, cytoskeletal organization is lost following the reduction of Lox activity. In the images shown in Figure 8 (and the associated quantifications) we focused on such regions from mice that were not treated with AngII. We find that even in what appears as a "healthy" region, disrupted p-MLC is observed. Notably, this disruption is not that the cells do not respond, but rather that the coordinated response is lost in the mutant mice. This lack of coordination is shown in the quantification where the two histograms depicting p-MLC expression have distinct distributions (Kolmogorov-Smirnov test p value=0). We have rephrased the relevant text in the manuscript.

Minor comments (numbers correspond to the numbering by the reviewer):

Please indicate scale bar in Fig1D, Fig2D, Fig3A-B, D-F, Fig4A-C, Fig5A-E, Fig8D-E.

A: We apologize for omitting the scale bars. They have now been added to all figures.

What the bars in the Fig2A-B graphs indicate? Information on the number of experiments and statistical analysis should be included in Figure or its legend.

A: *The bars in Figure 2A are qRT-PCR results of 3 independent biological samples showing expression of LOX family members in the HAOSMC. In Figure 2B, we set to monitor whether the expression of other member of the LOX family is modified in the shLOX cells. The graph shown the relative genes' expression in relation to shCtrl cells. The error bars in both Fig. 2A and B relate to the results of the 3 independent repeats the experiments were performed. As seen in Fig. 2A, the predominant member of the LOX family expressed in SMC is LOX. Further, the expression of other members of the family is not significantly changed in its loss (Fig. 2B). *

Similarly, Fig3C should include information on the number of analyzed cells and statics in the figure legend.

A: The data has been added.

5. What is the reason for separating Fig4F-G? It is not clear how many times the experiment was conducted. Fig1C, Fig6A-B, F-G should also describe the number of experiments and statistical analysis.

A: We have added all repeat numbers and statistical analysis to the legends. We are not clear as to the separation of Fig. 4F-G as there is no such figure. If the reviewer refers to Fig. 5 F-G, then we simply aimed to show that although the immunostaining results demonstrate that the two proteins are mislocalized, their levels are not affected in the LOX mutant cells.

Please describe the administration of treatment and concentration of drugs such as Calyculin A, in figure legend.

A: Drug concentrations have now been added to the figure legend. A more detailed description is available in the Methods section.

• *

Reviewer 2:

Major comments (numbers correspond to the order written by the reviewer):

• *

The authors state in the introduction "Our results therefore highlight a missing link between the three distinct gene groups associated with aneurysms, thus serving as a molecular paradigm for the development of phenotypes that culminate in aneurysm.", referring to the groups of genes in ECM structural proteins, members of the TGFb signaling pathway and genes involved in VSMC contractile apparatus. However, they do not provide data on the complex interplay between all of these groups and LOX. Therefore, the authors should add more nuance to this statement or change it altogether.

A: We agree with the reviewer that we have not shown any link between TGFβ signaling and LOX, even though these interactions have been previously demonstrated by us and others (see reply to Reviewer 1 comment #6). We are gearing up towards testing the TGFβ pathway also in the LOX devoid SMCs. Should we be unsuccessful, we will tone down this statement.

The authors have provided data on the phenotypic modulation with regards to expression of LOX and the contractile apparatus of VSMCs. However, to support the claim mentioned in the previous point, the authors should add experiments that show the relationship between LOX expression and specific genes involved in ECM structure and/or members of the TGFb family.

A: In a recent manuscript (Melamed et al., Cell Reports, 2023; PMID 37148241) we specifically focused on LOX and Fibronectin and we demonstrated that the LOX-devoid HAOSMC build an abnormal Fibronectin matrix which serves as a scaffold for ECM buildup. Along these lines, Supp. Figure 3A shows LC-MS/MS data of changes in ECM structural proteins' presence in the matrix of cells following LOX knockdown in cultured HAOSMC. As requested in the above comment, we are gearing up towards assessing TGFb signaling in the mutant cells.

In general, the authors provide a detailed description of the experimental setup in the methods section of the manuscript. However, the authors fail to provide methodology on some of their experiments. Per example, in text-line 152 the authors describe removing the cells from the ECM whilst leaving the ECM behind, but do not provide information on how this was done.

A: We thank the reviewer for the comment. We have added the details of the experiment.

The authors partially fail to provide n# for experiments throughout the manuscript and which statistical test was used for the comparisons in the figure.

A: We have now added to the figure legends all the n# and statistical tests that were used.

Minor comments (numbers correspond to the order written by the reviewer):

1. The authors make limited use of referring to appropriate literature. A: We have added additional relevant references.

2. *

The figures including images often lack scalebars. Moreover, the figure description is often incomplete. A: We thank the reviewer for the comment. As mentioned in the replies to Reviewer 1, we will add all the data and bars to the relevant figures.

Use Graphad Prism (or another well designed software) for figure illustration. A: *Graphs and histograms were generated using Matlab, excel and R and the figures were put together using Adobe Illustrator, all of which are designed for such illustrations. *

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Referee #2

Evidence, reproducibility and clarity

The paper of Rohtem Aviram and colleagues describes the "Coordination between cytoskeletal organization, cell contraction and extracellular development, which is depended on LOX for aneurysm prevention."

The manuscript examines the role of LOX, a collagen/elastin crosslinker, in its mechanism underlying aortic aneurysm, by using an myh11-positive cell inducible KO mouse model or in vitro VSMC culture. The authors confirmed a link between LOX activity and ECM remodeling in the aorta of hypertensive mice and reported a ECM-independent role of LOX in regulating VSMC cytoskeletal organization.

• Are the key conclusions convincing? The key conclusions of this manuscript are:
  • LOX plays a crucial role in regulating VSMCs cytoskeleton, affecting their contractile machinery and viability, independent of its ECM-modifying functions.
  • The study highlights an additional intracellular role for LOX in VSMCs, shedding light on its importance in maintaining aortic tissue integrity and preventing aneurysm formation.
  • LOX is implicated in various processes related to aneurysms, serving as a key player in the vasculature and its inhibition leading to ECM defects that promote thoracic aortic disease.

All three key conclusions are supported by data throughout the manuscript. However, the evidence is often based on data originating from western-blotting or immunofluorescent experiments and lack depth and rigidity. For example, figure 4 shows a change of cytoskeletal organization upon LOX KO in HAOSMCs but the authors lack to quantify or further analyse these exact differences in actin/tubulin organization. - Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

The authors state in the introduction "Our results therefore highlight a missing link between the three distinct gene groups associated with aneurysms, thus serving as a molecular paradigm for the development of phenotypes that culminate in aneurysm.", referring to the groups of genes in ECM structural proteins, members of the TGFb signaling pathway and genes involved in VSMC contractile apparatus. However, they do not provide data on the complex interplay between all of these groups and LOX. Therefore, the authors should add more nuance to this statement or change it altogether. - Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

The authors have provided data on the phenotypic modulation with regards to expression of LOX and the contractile apparatus of VSMCs. However, to support the claim mentioned in the previous point, the authors should add experiments that show the relationship between LOX expression and specific genes involved in ECM structure and/or members of the TGFb family. - Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

Yes, the experiments can be formed using the same VSMCs already used in the manuscript and protein- and/or gene expression can be determined by the same methods already used throughout the manuscript. - Are the data and the methods presented in such a way that they can be reproduced?

In general, the authors provide a detailed description of the experimental setup in the methods section of the manuscript. However, the authors fail to provide methodology on some of their experiments. Per example, in text-line 152 the authors describe removing the cells from the ECM whilst leaving the ECM behind, but do not provide information on how this was done. - Are the experiments adequately replicated and statistical analysis adequate?

The authors partially fail to provide n# for experiments throughout the manuscript and which statistical test was used for the comparisons in the figure.

Minor comments:

• Specific experimental issues that are easily addressable.

See major comments - Are prior studies referenced appropriately?

The authors make limited use of referring to appropriate literature. - Are the text and figures clear and accurate?

The figures including images often lack scalebars. Moreover, the figure description is often incomplete. - Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

Use Graphad Prism (or another well designed software) for figure illustration.

Significance

• Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.

This manuscript brings forward a basic conceptual advantage with regards to the relationship between LOX expression in VSCMs and aortic aneurysm formation. - Place the work in the context of the existing literature (provide references, where appropriate).

Current literature mainly focusses on the role of LOX in ECM-oriented remodeling, this manuscript shows that LOX also plays a role in VSMC phenotypic alterations regardless of its ECM-altering role. - State what audience might be interested in and influenced by the reported findings.

Basic scientist with an interest in aneurysm or VSMC remodeling. - Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

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Referee #1

Evidence, reproducibility and clarity

Summary:

Dr. Aviram et al investigate the deletion of Lysyl oxidase (LOX) in vascular smooth muscle cells SMCs) leading to aortic aneurysm development. The authors performed in vito assay using primary SMCs and found that cytoskeletal organization and extracellular matrix (ECM) assembly are lost in Lox-deleted SMCs independent of Lox activity manner. The authors concluded that the novel intracellular function of Lox contributes to aortic aneurysm formation. The strength of this study is that it attempts to explain the underlying principle of aortic aneurysm development due to Lox deficiency using a cultured cell-based system, however, it lacks reliability of data due to insufficient technical problems in several experiments. In particular, the causal relationship between the discovered Lox function and the development of aortic aneurysms is unclear and remains a matter of conjecture. My comments are following.

Major comments:

1. The number of mice used and a number of experiments ("n" number) are not described in each figure or its legends in an overall experiment. Also, there is no information on the statistical analysis, which makes it impossible to judge the validity of the results.
2. The Phenotype of Lox-deficient mice is unclear; the picture in Fig1C is not clear and a high-magnified view should be provided. Also, which part (aortic arch or abdominal aorta?) is histologically analyzed? It should be described. In addition to the morphological analysis, it cannot be called "aneurysm" unless the internal diameter is enlarged more than 1.5 times compared to the control aorta. The histological images seem to show only dissection, which is unclear since statistical analysis is not feasible with only 2-3 animals.
3. Immunostaining in Figs. 4-6 should add nuclei (DAPI) to all experiments. It is unclear how many cells are being looked at. For example, in the staining of Fig4A, the stained nuclei are slightly visible in the shLox group, but not at all in the control above. Phenotypes should be compared under the same conditions.
4. For ROCK and RhoA analysis (in Fig4-6), immunostaining and Western alone are not convincing and not sufficient evidence for activation. Other factors, such as methods to measure activation and focal adhesion molecules should be considered.
5. It is unclear what the TEM in Fig8 is trying to clarify. Since SMCs and elastic fibers are supposed to be bound, it would be better to show the binding site. In addition, the p-MLC in Fig8D-F is a qualitative evaluation, so the difference is not clear, and it is necessary to verify whether there is a difference in Myh11CreERT2;Loxfl/fl mice between aneurysmal (pathogenic) and non-aneurysmal lesions. Overall, this is an associated study that this only speculation since the causal relationship between aneurysm development and Lox functions, which authors found is unclear.
6. In the discussion (lines 332-334), the Authors described that "Since TGFb signaling is implicated in aneurysm formation..." but the effect of TGFb signal in these Lox-deficient mice has not been examined at all. The effects of pSmad2/3 staining, Western, etc on TGFb activation should be examined and discussed.

Minor comments:

1. What is the baseline group in Fig1A? and should be required a minimum 3 of animals in each group.
2. Please indicate scale bar in Fig1D, Fig2D, Fig3A-B, D-F, Fig4A-C, Fig5A-E, Fig8D-E.
3. What the bars in the Fig2A-B graphs indicate? Information on the number of experiments and statistical analysis should be included in Figure or its legend.
4. Similarly, Fig3C should include information on the number of analyzed cells and statics in the figure legend.
5. What is the reason for separating Fig4F-G? It is not clear how many times the experiment was conducted. Fig1C, Fig6A-B, F-G should also describe the number of experiments and statistical analysis.
6. Please describe the administration of treatment and concentration of drugs such as Calyculin A, in figure legend.
7. Please show the data "shLox cell death (not shown)" in text, line 248.

Significance

While many studies have shown that the enzymatic activity of Lox is important for aneurysm formation, the focus on intracellular functions such as cytoskeleton remodeling, other than enzymatic activity is a novel point. However, the study is limited to speculation due to insufficient phenotypic analysis of aneurysms and the number of animals, as well as the inability to clearly prove a causal relationship. Revisions are needed to add significant additional data and to conduct more accurate analyses.

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eLife assessment

This study represents a useful description of a third interaction site between melanophilin and myosin-5a which has a role in regulating the distribution of pigment granules in melanocytes. While much of the data forms a solid case for this interaction, the inclusion of controls for the cellular studies and measurement of interaction affinities would have been helpful.

Reviewer #1 (Public Review):

Interactions known to be important for melanosome transport include exon F and the globular tail domain (GTD) of MyoVa with Mlph. Motivated by a discrepancy between in vitro and cell culture results regarding necessary interactions for MyoVa to be recruited to the melanosome, the authors used a series of pull-down and pelleting assays experiments to identify an additional interaction that occurs between exon G of MyoVa and Mlph. This interaction is independent of and synergistic with the interaction of Mlph with exon F. However, the interaction of the actin-binding domain of Mlph can occur either with exon G or with the actin filament, but not both simultaneously. These data lead to a modified recruitment model where both exon F and exon G enhance binding of Mlph to auto-inhibited MyoVa, and then via an unidentified switch (PKA?) the actin-binding domain of Mlph dissociates from MyoVa and interacts with the actin filament to enhance MyoVa processivity.

The only weakness noted is that the authors could have had a more complete story if they pursued whether PKA phosphorylation/dephosphorylation of Mlph is indeed the switch for the actin-binding domain of Mlph to interact with exon G versus the actin filament.

Reviewer #2 (Public Review):

The authors identify a third component in the interaction between myosin Va and melanophilin- an interaction between a 32-residue sequence encoded by exon-g in myosin Va and melanophilin's actin binding domain. This interaction has implications for how melanosome motility may be regulated.

The authors have now included some necessary controls that were requested. In terms of adding new information to increase the significance and impact of the paper, they added a single affinity measurement. Unfortunately, it did not involve Exon G specifically. Moreover, they did not add any new mechanistic or functional data to provide a more conceptual advance. For example, is the Exon G interaction regulated by phosphorylation? Is this what dictates the choice between Mlph's actin binding domain (ABD) binding to actin or to exon-G. How does local actin concentration influence this decision. What changes regarding melanosome dynamics in cells between these two alternatives? Do in vitro reconstitution assays show that binding to Exon-G instead of actin affects the processivity of a Rab27a/Myosin 5a/Mlph transport complex? Finally, while the authors make clear in the abstract and text that they are just identifying a third component that mediates the Melanophilin-dependent association of myosin-5a with melanosomes, the title gives the impression that they identified all three in this manuscript. I really think the title should be changed to something like Identification of a third component that mediates the Melanophilin-dependent association of myosin-5a with melanosomes, as this accurately reflects what is new in this work.

Author response:

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

We appreciate your comments and suggestions on our manuscript.

In particular, we have measured the affinity between the middle tail domain of myosin-5a (Myo5a-MTD) and the actin-binding domain of melanophilin (Mlph-ABD) using microscale thermophoresis, and obtained the Kd of ~0.56 uM, which is similar to the Kd of the globular tail domain of myosin-5a (Myo5a-GTD) to the GTD-binding motif of melanophilin (Mlph-GTBM). Moreover, we have performed Western blot of the lysate of transfected cells, showing that the proteins of the dominant negative construct and the negative control were expressed at similar lever without noticeable degradation.

We appreciate the editors’ and reviewers’ comment on how melanophilin might be regulated in binding to the exon-G of myosin-5 and to actin filaments. Phosphorylation of melanophilin by protein kinase A is one possible mechanism. We will investigate this issues in our future study.

We also took this opportunity to correct several minor errors in the manuscript. Textual alterations can be viewed in the “tracked change” version of the manuscript. Below is the comments from the editors and the two reviewers together with our point-by-point responses.

eLife assessment

This study represents a useful description of a third interaction site between melanophilin and myosin-5a which is important in regulating the distribution of pigment granules in melanocytes. While much of the data forms a solid case for this interaction, the inclusion of important controls for the cellular studies and measurement of interaction affinities would have been helpful.

Public Reviews:

Reviewer #1 (Public Review):

Interactions known to be important for melanosome transport include exon F and the globular tail domain (GTD) of MyoVa with Mlph. Motivated by a discrepancy between in vitro and cell culture results regarding necessary interactions for MyoVa to be recruited to the melanosome, the authors used a series of pull-down and pelleting assays experiments to identify an additional interaction that occurs between exon G of MyoVa and Mlph. This interaction is independent of and synergistic with the interaction of Mlph with exon F. However, the interaction of the actin-binding domain of Mlph can occur either with exon G or with the actin filament, but not both simultaneously. These data lead to a modified recruitment model where both exon F and exon G enhance the binding of Mlph to auto-inhibited MyoVa, and then via an unidentified switch (PKA?) the actin-binding domain of Mlph dissociates from MyoVa and interacts with the actin filament to enhance MyoVa processivity.

The only weakness noted is that the authors could have had a more complete story if they pursued whether PKA phosphorylation/dephosphorylation of Mlph is indeed the switch for the actin-binding domain of Mlph to interact with exon G versus the actin filament.

We thank Reviewer #1 for careful reading of the manuscript and appreciation of the study. We agree with the Reviewer that it is important to understand how the actin-binding domain of Mlph switch its interaction with the exon-G of Myo5a and actin filament. We would like to pursue this direction in our future research.

Reviewer #2 (Public Review):

The authors identify a third component in the interaction between myosin Va and melanophilin- an interaction between a 32-residue sequence encoded by exon-g in myosin Va and melanophilin's actin-binding domain. This interaction has implications for how melanosome motility may be regulated.

While this work is largely well done and certainly publishable following needed revisions (e.g. some affinity measurements, necessary controls for the dominant negative experiments), I believe that additional work would be required to make a more compelling case. First, the study provides just one more piece to a well-developed story (the role of exon-F and the GTD in myosin Va: melanophilin (Mlph) interaction), much of which was published 20 years ago by several labs. Second, the study does not demonstrate a physiological significance for their findings other than that exon-G plays an auxiliary role in the binding of myosin Va to Mlph. For example, what dictates the choice between Mlph's actin binding domain (ABD) binding to actin or to exon-G. Is it a PTM or local actin concentration? It is unlikely to be alternative splicing as exon-G is present in all spliced isoforms of myosin Va. And what changes re melanosome dynamics in cells between these two alternatives? Similarly, the paper does not provide any in vitro evidence that binding to exon-G instead of actin effects the processivity of a Rab27a/Myosin Va/Mlph transport complex. For example, if the ABD sticks to exon-G instead of actin, does that block Mlph's ability to promote processivity through its interaction with the actin filament during transport? In summary, given that the authors did not directly test their model either in vitro or in cells, I do not think this story represent a significant conceptual advance.

We thank Reviewer #2 for careful reading of the manuscript and the suggestions of improving the manuscript. As suggested by the reviewer, we have measured the affinity between the middle tail domain of Myo5a (Myo5a-MTD) and Mlph-ABD (Kd ~0.562 uM), which is similar to that between the globular tail domain of Myo5a (Myo5a-GTD) and the GTBM of Mlph. In addition, we have performed additional experiments showing the integrity and the expression level of the dominant negative constructs in the transfected cells.

We believe more extensive experiments are required to address other questions raised by the reviewer. For example, what dictates the choice between Mlph's actin binding domain (ABD) binding to actin or to exon-G is an open question. As we proposed, phosphorylation by protein kinase A is only one possible mechanism. We would like to pursue them in our future research.

Recommendations for the authors:

The reviewing editor feels strongly that addressing some of the points raised by the reviewers would make this a more compelling manuscript. In particular, a measurement of the affinity of the relevant fragments from melanophilin and myosin-5a would indicate that the interaction might be physiologically relevant. Concerning the dominant negative experiments, the lack of effect of an expressed fragment could be that the expressed fragments were simply degraded or expressed at too low of a level to be competing. The reviewer gives guidelines on how to address this. Reviewer #2 made a point that it would be compelling if the effect of phosphorylation as suggested in the model was tested, but we all agree that this could well be the subject of a later study. In addition, the authors make a very interesting proposal for how protein kinase A could be involved in this regulation as has been suggested previously. Perhaps the use of phosphomimetic mutations could give some insight into this. Such experiments, if consistent with the proposed model would certainly raise the impact of this study. Finally, a very clear periodicity in hydrophobic amino acids is apparent in the interacting sequences of both Myo5 (yrisLykrMidLmeqLekqdktVrkLkkqLkvFakkIgeLevgqmen) and Mlph (tdeeLseMedrVamtAseVqqAeseIsdIesrIaaLra). This is strongly suggesting a leucine-zipper-like coiled coil, rather than an interaction mediated solely by charge. Recent softwares (and easily accessible too) like AlphaFold multimer might yield important structural insight into the binding configuration and might help rationalize the effect of the mutations herein.

We thank the editors and the reviewers for their suggestions of improving the manuscript. We have performed the several essential experiments to address the concerns raised by the reviewers.

(1) Regarding the affinity of the relevant fragments from melanophilin and myosin-5a. We have measured the affinity between Mlph-ABD and Myo5a-MTD using MST (Kd ~562 nM) (see revised Figure 3A).

(2) Regarding the concerns on the dominant negative experiments. We have examined the molecular sizes and expression levels of  Mlph or Myo5a constructs by Western blots. First, we show that all constructs have correct molecular size in transfected cells (see revised Figure 6C and 7D), indicating that the inability of Myo5a or Mlph truncations to generate dilute-like phenotypes was not due to the intracellular degradation of the EGFP fusion protein. Second, by correcting for the percentage of transfected cells, we show that the overall expression levels of the wild-type construct and the mutants are roughly equal. Third, we categorized the expression levels into high and low, and calculated percentage of the DN phenotype in high and low expression levels. The results are consistent with the percentage of DN phenotype in total EGFP fusion protein cells.

(3) Regarding the suggestion to investigate the effect of phosphorylation by protein kinase A on Mlph-ABD’s interaction with Myo5a and actin filament. We understand that it is important to elucidate the mechanism by which the actin-binding domain of Mlph switch its interaction with the exon-G of Myo5a and actin filament. However, as we proposed, phosphorylation by protein kinase A is one possible mechanism, and more extensive experiments are required to address this question. Therefore, we would like to pursue it in our future research.

(4) Regarding the suggestion to predict the interaction between the exon-G of myosin-5a and Mlph-ABD using AlphaFold. We have used AlphaFold multimer to predict the Myo5a-MTD/Mlph-ABD interaction. Remarkably, the AlphaFold predicted that the binding of Myo5a-MTD with Mlph-ABD is mediated by an antiparallel coiled-coil formed by Myo5a (1430-1467) and Mlph (450-481), just as predicted by the editors. This prediction is also consistent with our finding that the exon-G of Myo5a interacts with Mlph-ABD. However, the predicted model cannot explain our mutagenesis results. We will pursue this point in the future research. Nevertheless, we are grateful to the editors for bringing this idea to our attention, because it will help us to design experiments to investigate the nature of Myo5a-exon-G/Mlph-ABD interaction.

Reviewer #1 (Recommendations For The Authors):

Specific minor comments

Q1: In figs 6-7 an overlay between DAPI and EGFP would be helpful for the reader to see perinuclear distribution.

As suggested, we have added the merged images of DAPI and EGFP in the revised Figure 6 and 7.

Q2: The delta symbol in the pdf text was corrupted.

The corrupted delta symbol has been fixed in the revised manuscript.

Reviewer #2 (Recommendations For The Authors):

Q1: Please explain in detail early in the text what exon-G is - length, position in the tail, and evidence that it is a coiled coil (CC). Of note, is it only long enough for about 4 heptad repeats? Has it been shown biochemically to form a CC? Is the CC irreversible? What would be the consequence of removing the exon-G CC on the ability of surrounding regions to bind Mlph (exon-F and the GTD)?

We thank the reviewer for this suggestion. In the revision, we added a new paragraph (the first paragraph in the results section) and revised Figure 1A to introduce the middle tail domain and alternatively spliced exons of Myo5a.

Exon-G is 32 amino acids in length, located at the C-terminal region of the middle tail domain, immediately before the globular tail domain. Exon-G region was predicted to form a short coiled-coil by using on-line tools (such as paircoil), and this prediction has not been tested biochemically. Moreover, we do not know whether the exon-G coiled-coil is reversible or not.

We have not examined the effect of removing the whole exon-G on the interaction between the GTD and Mlph-GTBM. The exon-G (residues 1436-1467) and the GTD core (residues 1498-1877) are separated by a long loop of 31 residues. We therefore expect that the removing the exon-G will not affect the GTD/Mlph-GTBM interaction.

Physically, exon-F is immediately followed by exon-G, and those two regions might interfere with each other. In our preliminary study, we found that removing the whole exon-G abolished the interaction between exon-F and Mlph-EFBD. On the other hand, removing the C-terminal half (residues 1454-1467) of exon-G had little effect the interaction between exon-F and Mlph-EFBD (see Figure 2C). In this work, we intentionally selected the later construct for functional analysis of the exon-G/Mlph-ABD interaction, because removing the C-terminal half of exon-G abolishes the interaction with Mlph-ABD, but does not affect the exon-F/Mlph-EFBD interaction.

Q2: Figures 1-3. While the pulldown experiments demonstrating an interaction between Mlph-ABD residues 446-571 and Myo5a-MTD are a good start, one would like to see affinity measurements to gauge the likelihood that this interaction is physiologically relevant. The same goes for the pulldown experiments demonstrating an interaction between (i) the C-terminal half of exon-G (residues 1453-1467) and the Mlph-ABD, (ii) between residues 1411-1467 (a short peptide containing exon-F and exon-G) and the Mlph-ABD, and (iii) between residues 1436-1467 (a short peptide containing exon-G) and the Mlph-ABD. This would also apply to the pulldowns in 3C-3E where versions of the proteins with charge residue changes were tested.

We agree the reviewer’s opinion that determination of the affinities between Mlph-ABD and Myo5a-MTD and their variants will be helpful in understanding the physiological relevance of Exon-G/Mlph-ABD interaction. However, the extensive experiments suggested by the reviewer require many high quality, purified proteins, which are not trivial.

Nevertheless, we think it is important to know the affinity between Myo5a-MTD and Mlph-ABD (both wild-type), as this parameter can be used for the comparison of the three interactions between Myo5a and Mlph. Therefore, we have obtained the affinity between Myo5a-MTD and Mlph-ABD using microscale thermophoresis (MST). The dissociation constant (Kd) of Myo5a-MTD to Mlph-ABD is 0.562±0.169 uM, which is similar to that between Myo5a-GTD and Mlph-GTBM (~1 uM) (Geething & Spudich (2007) JBC 282:21518). Consistent with GST pulldown results, MST shows that deletion of C-terminal half of exon-G (1453-1467) greatly decreases the MST signals (see revised Figure 3A).

Q3: While the domain negative (DN) approach to testing functional significance is OK, rescuing dilute/myosin Va null melanocytes with full-length myosin Va containing the various deletions would have been more convincing. Also, the authors must show (i) that the DN constructs are the correct size in transfected cells (i.e. are not degraded), and (ii) that they are expressed at roughly equal levels (either by doing Westerns and correcting for the percent of transfected cells, or by measuring total cellular fluorescence in transfected cells). Without this information, it remains possible that constructs not exhibiting a DN effect are simply degraded or poorly expressed. This applies to all the DN data in Figures 6 and 7.

We agree with the reviewer that Myo5a null melanocytes is ideal for investigating exon G function. Unfortunately, we do not have Myo5a null melanocytes derived from dilute mice.

To confirm the integrity of the overexpressed proteins in the transfected cells, we performed Western blot of those proteins, including  EGFP-Mlph-RBD (wild-type and two mutants) and Myo5a-Tail (wild-type and G mutant), in the lysate of the transfected cells. Western blots show that all those proteins have correct molecular masses, indicating no degradation of those overexpressed proteins (see revised Figure 6C and 7C). Moreover, by correcting for the percentage of transfected cells, we show that the overall expression levels in each transfected cell of the wild-type construct and the mutants are roughly equal. This information is included in the revised manuscript (Line 222-225; 237-241).

Q4: The authors scored the DN phenotype as yes/no but it mostly likely varies depending on the degree of over-expression. Showing that the degree of melanosome centralization scales with the degree of overexpression, and that the correlation between expression level and phenotype varies depending on the construct would strengthen the results.

We agree with the reviewer’s prediction that the degree of DN phenotype should depend on the of over-expression level. We analyzed the EGFP signals of transfected cells and found very few cells with medium expression level. Therefore, we simply categorized the expression levels into high and low, and calculated the DN phenotype in each categories as shown in the table below. These results are consistent with the expectation that the degree of DN phenotype depends on the over-expression level of the transfected constructs.

Author response table 1.

Percentage of the EGFP-expressing cells with perinuclear aggregation of melanosomes

Q5: The conclusion from the data in Figure 8A- "the presence of both exon-F and exon-G is insufficient for binding to the Mlph occupied by Myo5a, but sufficient for binding to the unoccupied Mlph"- should be verified by also doing the experiment in myosin Va knockdown cells.

We agree. Unfortunately, our RNAi knockdown of Myo5a in melanocytes by RNAi is not ideal and we do not have Myo5a knockout melanocytes. We will pursue this point in the future.

Q6: Line 213 "three Mlph-binding regions, i.e., exon-F, exon-F, and GTD (Figure 7A)" has a typo.

This typo has been corrected.

Q7: The authors should provide high mag insets for the images in Figure 8.

As suggested, we have revised Figure 8 by including high mag insets for the images.

Onderliggende gegevens? Python code voor de plot? Misschien wel aardig om ook het 10e en 90e percentiel te geven

The soil moisture to evaporation arrow in fact includes transpiration and soil evaporation.

Similarly interception evaporation and open water evaporation from the depression is not highlighted as process. We should think about including this (in correspondance to previous chapter). @Pierre, let's discuss before making changes.

Four

Infilitration I (In case we are looking at short timescales infiltation is a loss, yet for longer timescales infiltration is only a loss if evaporated by transpiration or soil evaporation, but it is not a loss in case it recharges the groundwater and contributes to baseflow)

delete sentence, I think this only leads to confusion

effective preciitation

@Pierre, please check groundwater chapter

@Pierre, can you check the groundwater chapter?

Author response

Reviewer #1 (Public Review):

Summary:

The authors aimed to modify the characteristics of the extracellular matrix (ECM) produced by immortalized mesenchymal stem cells (MSCs) by employing the CRISPR/Cas9 system to knock out specific genes. Initially, they established VEGF-KO cell lines, demonstrating that these cells retained chondrogenic and angiogenic properties. Additionally, lyophilized carriage tissues produced by these cells exhibited retained osteogenic properties.

Subsequently, the authors established RUNX2-KO cell lines, which exhibited reduced COLX expression during chondrogenic differentiation and notably diminished osteogenic properties in vitro. Transplantation of lyophilized carriage tissues produced by RUNX2-KO cell lines into osteochondral defects in rat knee joints resulted in the regeneration of articular cartilage tissues as well as bone tissues, a phenomenon not observed with tissues derived from parental cells. This suggests that gene-edited MSCs represent a valuable cell source for producing ECM with enhanced quality.

Strengths:

The enhanced cartilage regeneration observed with ECM derived from RUNX2-KO cells supports the authors' strategy of creating gene-edited MSCs capable of producing ECM with superior quality. Immortalized cell lines offer a limitless source of off-the-shelf material for tissue regeneration.

We thank the reviewer for the interest in our work. We however want to clarify that the present manuscript does not report the generation of ECM with “superior quality”, but rather of modulated composition and thus function.

Weaknesses:

Most data align with anticipated outcomes, offering limited novelty to advance scientific understanding. Methodologically, the chondrogenic differentiation properties of immortalized MSCs appeared deficient, evidenced by Safranin-O staining of 3D tissues and histological findings lacking robust evidence for endochondral differentiation. This presents a critical limitation, particularly as authors propose the implantation of cartilage tissues for in vivo experiments. Instead, the bulk of data stemmed from type I collagen scaffold with factors produced by MSCs stimulated by TGFβ.

The chondrogenic differentiation of our MSOD-B line and their capacity of undergoing endochondral ossification has been robustly demonstrated in previous studies (Pigeot et al., Advanced Materials 2021 and Grigoryan et al., Science Translational Medicine 2022). In the present manuscript, we thus compare the chondrogenic capacity of newly established VEGF-KO and RUNX-KO lines to those of MSOD-B cells. We demonstrate by qualitative (Safranin-O staining, Collagen type 2 and Collagen type X immuno-stainings) and quantitative (glycosaminoglycans assay) assays that the generated tissues consist in cartilage grafts of similar quality than the MSOD-B counterpart. Of note, the safranin-O stainings were performed on lyophilized tissues, which can alter the staining quality/intensity. We will thus provide additional stainings of generated tissues pre-lyophilization.

The rationale behind establishing VEGF-KO cell lines remains unclear. What specific outcomes did the authors anticipate from this modification?

VEGF is a known master regulator of angiogenesis and a key mediator of endochondral ossification. It has also been extensively used in bone tissue engineering studies as a supplemented factor – primarily in the form of VEGFα – to increase the vascularization and thus outcome of bone formation of engineered grafts (https://www.nature.com/articles/s42003-020-01606-9, https://www.sciencedirect.com/science/article/pii/S8756328216301752). In our study, it was thus identified as a natural candidate to demonstrate the possibility to generate VEGF-KO cartilage and subsequently assess the functional impact on both the angiogenic and osteogenic potential of resulting cartilage tissue.

Insufficient depth was given to elucidate the disparity in osteogenic properties between those observed in ectopic bone formation and those observed in transplantation into osteochondral defects. While the regeneration of articular cartilage in RUNX2-KO ECM presents intriguing results, the study lacked an exploration into underlying mechanisms, such as histological analyses at earlier time points.

Using RUNX2-KO ECM, we aimed at demonstrating the impact on cartilage remodeling and bone formation. This was performed ectopically but also in the rat osteochondral defect as a regenerative set-up of higher clinical relevance. We agree with the reviewer that additional experimental groups and time-points (not only earlier but also longer ones) would offer a better mechanistic understanding of the ECM contribution to the joint repair. However, as stated in our manuscript this is a proof-of-concept study that successfully demonstrated the influence of the cartilage ECM modification on the in vivo skeletal regeneration. A follow-up study would need to be performed to complement existing evidence and strengthen the relevance of our approach for cartilage repair.

Reviewer #2 (Public Review):

The manuscript submitted by Sujeethkumar et al. describes an alternative approach to skeletal tissue repair using extracellular matrix (ECM) deposited by genetically modified mesenchymal stromal/stem cells. Here, they generate a loss of function mutations in VEGF or RUNX2 in a BMP2-overexpressing MSC line and define the differences in the resulting tissue-engineered constructs following seeding onto a type I collagen matrix in vitro, and following lyophilization and subcutaneous and orthotopic implantation into mice and rats. Some strengths of this manuscript are the establishment of a platform by which modifications in cell-derived ECM can be evaluated both in vitro and in vivo, the demonstration that genetic modification of cells results in complexity of in vitro cell-derived ECM that elicits quantifiable results, and the admirable goal to improve endogenous cartilage repair. However, I recommend the authors clarify their conclusions and add more information regarding reproducibility, which was one limitation of primary-cell-derived ECMs.

We thank the reviewer for the positive evaluation of our work.

Overcoming the limitations of native/autologous/allogeneic ECMs such as complete decellularization and reduction of batch-to-batch variability was not specifically addressed in the data provided herein. For the maintenance of ECM organization and complexity following lyophilization, evidence of complete decellularization was not addressed, but could be easily evaluated using polarized light microscopy and quantification of human DNA for example in constructs pre and post-lyophilization.

We will clarify the experiments and characterization performed with lyophilized tissues versus those performed with decellularized ones. We will also provide evidence of DNA removal in our decellularized ECMs.

It would be ideal to see minimization of batch-to-batch variability using this approach, as mitigation of using a sole cell line is likely not sufficient (considering that the sole cell line-derived Matrigel does exhibit batch-to-batch and manufacturer-to-manufacturer variability). I recommend adding details regarding experimental design and outcomes not initially considered. Inter- and intra-experimental reproducibility was not adequately addressed. The size of in vitro-derived cartilage pellets was not quantified, and it is not clear that more than one independent 'differentiation' was performed from each gene-edited MSC line to generate in vitro replicates and constructs that were implanted in vivo.

We thank the Reviewer for the comment on variability/reproducibility concern. Using a cell line does confer higher robustness but indeed does not grant unlimited consistency of batch production. We will temper our claims in the discussion and mention the need to regularly re-characterize cell lines properties upon passages.

In our study, our grafts have been generated from various batches and tested in more than one experimental repeat. This will be further described in the revised version of our manuscript. We will also implement data on the size variability of generated tissues.

The use of descriptive language in describing conclusions may mislead the reader and should be modified accordingly throughout the manuscript. For example, although this reviewer agrees with the comparative statements made by the authors regarding parental and gene-edited MSC lines, non-quantifiable terms such as 'frank' 'superior' (example, line 242) are inappropriate and should rather be discussed in terms of significance. Another example is 'rich-collagenous matrix,' which was not substantiated by uniform immunostaining for type II collagen (line 189).

I have similar recommendations regarding conclusive statements from the rat implantation model, which was appropriately used for the purpose of evaluating the response of native skeletal cells to the different cell-derived ECMs. Interpretations of these results should be described with more accuracy. For example, increased TRAP staining does not indicate reduced active bone formation (line 237). Many would not conclude that GAGs were retained in the RUNX2-KO line graft subchondral region based on the histology. Quantification of % chondral regeneration using histology is not accurate as it is greatly influenced by the location in the defect from which the section was taken. Chondral regeneration is usually semi-quantified from gross observations of the cartilage surface immediately following excision. The statements regarding integration (example line 290) are not founded by histological evidence, which should show high magnification of the periphery of the graft adjacent to the native tissue.

We thank the Reviewer for the constructive suggestions. We will revise language accordingly throughout the manuscript.

Reviewer #3 (Public Review):

Summary:

In this study, the authors have started off using an immortalized human cell line and then gene-edited it to decrease the levels of VEGF1 (in order to influence vascularization), and the levels of Runx2 (to decrease chondro/osteogenesis). They first transplanted these cells with a collagen scaffold. The modified cells showed a decrease in vascularization when VEGF1 was decreased, and suggested an increase in cartilage formation.

In another study, the matrix generated by these cells was subsequently remodeled into a bone marrow organ. When RUNX2 was decreased, the cells did not mineralize in vitro, and their matrices expressed types I and II collagen but not type X collagen in vitro, in comparison with unedited cells. In vivo, the author claims that remodeling of the matrices into bone was somewhat inhibited. Lastly, they utilized matrices generated by RUNX2 edited cells to regenerate chondro-osteal defects. They suggest that the edited cells regenerated cartilage in comparison with unedited cells.

Strengths:

-The notion that inducing changes in the ECM by genetically editing the cells is a novel one, as it has long been thought that ECM composition influences cell activity.

-If successful, it may be possible to make off-the-shelf ECMS to carry out different types of tissue repair.

We thank the Reviewer for the critical evaluation of our work and the highlighted novelty of it.

Weaknesses:

-The authors have not generated histologically identifiable cartilage or bone in their transplants of the cells with a type I scaffold.

The chondrogenic differentiation of our MSOD-B line and their capacity of undergoing endochondral ossification has been robustly demonstrated in previous studies (Pigeot et al., Advanced Materials 2021 and Grigoryan et al., Science Translational Medicine 2022). In the present manuscript, we thus compare the chondrogenic capacity of newly established VEGF-KO and RUNX-KO lines to those of MSOD-B. We demonstrate by qualitative (Safranin-O staining, Collagen type 2 and Collagen type X immuno-stainings) and quantitative (glycosaminoglycans assay) assays that the generated tissues consist in cartilage tissue of similar quality than the MSOD-B. However, the safranin-O stainings were performed on lyophilized tissues, which can alter the staining quality/intensity. We will thus provide additional stainings of generated tissues pre-lyophilization.

On the contested formation of bone in vivo by our ECMs grafts, we have provided compelling qualitative evidence via Masson´s Trichrome stainings and quantification of mineralized volume by µCT. Both cortical bone and trabecular structures were identified ectopically. Those are standard evaluation methods in the field, we would be happy to receive additional suggestions by the Reviewer.

-In many cases, they did not generate histologically identifiable cartilage with their cell-free-edited scaffold. They did generate small amounts of bone but this is most likely due to BMPs that were synthesized by the cells and trapped in the matrix.

We now appreciate that the Reviewer agrees on the successful formation of bone induced by our engineered grafts. We however still respectfully disagree with the “small amount of bone” statement since our MSOD-B and MSOD-B VEGF KO cartilage grafts led to the full generation of a mature ectopic bone organ (that is, also composed of extensive marrow). This has been assessed qualitatively and quantitatively.

We agree with the Reviewer on the key role of BMP-2 in the remodeling process into bone and bone marrow, which we have extensively described in our previous publication (Pigeot et al., Advanced Materials 2021). We previously demonstrated that the low amount of BMP-2 (in the dozens of nanogram/tissue range) embedded in the matrix is not sufficient per se to induce ectopic endochondral ossification. It is the combined presence of GAGs in the matrix -thus cartilage- that allows the success of bone formation. Since we have already demonstrated in the present manuscript that the GAGs content is the same in MSOD-B and MSOD-B edited ECMs, we will provide additional data demonstrating the maintenance of BMP-2 content in all generated cartilage tissues.

-There is a great deal of missing detail in the manuscript.

We will provide additional information on the MSOD-B line and the overall methodology in our revised version.

-The in vivo study is underpowered, the results are not well documented pictorially, and are not convincing.

We will provide additional information and pictures related to our in vivo studies. We believe our group size supports our conclusions confirmed by statistical assessment.

-Given the fact that they have genetically modified cells, they could have done analyses of ECM components to determine what was different between the lines, both at the transcriptome and the protein level. Consequently, the study is purely descriptive and does not provide any mechanistic understanding of what mixture of matrix components and growth factors works best for cartilage or bone. But this presupposes that they actually induced the formation of bona fide cartilage, at least.

We thank the Reviewer for the suggestion. However, our study did not aim at understanding what ECM graft composition work best for cartilage nor bone regeneration respectively. Instead, we propose the exploitation of our cellular tools to interrogate the function of key ECM constituents and their impact in skeletal regeneration. We once more confirm that we generated lyophilized cartilage grafts which will be more evidently supported by histological assessment before lyophilization.

Author response:

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

Public Reviews:

Reviewer #1 (Public Review):

Summary:

Chen and colleagues first compared the cartilage tissues collected from OA and HA patients using histology and immunostaining. Then, a genome-wide DNA methylation analysis was performed, which informed the changes of a novel gene, TNXB. IHC confirmed that TNXB has a lower expression level in HA cartilage than OA. Next, the authors demonstrated that TNXB levels were reduced in the HA animal model, and intraarticular injection of AAV carrying TNXB siRNA induced cartilage degradation and promoted chondrocyte apoptosis. Based on KEGG enrichment, histopathological analysis, and western blot, the authors also showed the relationship between TNXB and AKT phosphorylation. Lastly, AKT agonist, specifically SC79 in this study, was shown to partially rescue the changes of in vitro-cultured chondrocytes induced by Tnxb knock-down. Overall, this is an interesting study and provided sufficient data to support their conclusion.

Strengths:

(1) Both human and mouse samples were examined.

(2) The HA model was used.

(3) Genome-wide DNA methylation analysis was performed.

Weaknesses:

(1) In some experiments, the selection of the control groups was not ideal.

Thank you for comments. The reviewer raised the concerns about using human OA cartilage as control, instead of health cartilage. This is an important detail we didn’t describe in the previous version. We have added our explanation in revised Methods.

(2) More details on analyzing methods and information on replicates need to be included.

We greatly appreciate your careful review and helpful suggestions. We have added detailed information to our revised draft.

(3) Discussion can be improved by comparing findings to other relevant studies.

Thank the reviewer very much for the opportunity to improve our manuscript. We have improved discussions as reviewer suggested in Recommendation 13.

(4) The use of transgenic mice with conditional Tnxb depletion can further define the physiological roles of Tnxb.

Thanks for this valuable comment. We understand that conditional Tnxb-KO mice is much helpful for the study of biological roles of Tnxb, and it will be constructed and used in our future studies.

Recommendations For the Authors:

(1) Please add more information about HA such as incidence to highlight the importance of the study.

We greatly appreciate your careful review and helpful suggestions. We have provided more information about the importance of HA study in revised Introduction. Please see lines 90-93 and 103-112.

(2) Please justify the use of OA cartilage, instead of normal tissues, as the control.

Thanks for your suggestion. We certainly would have liked to use healthy cartilage as control, but we were extremely difficult to obtain enough control samples from healthy individuals. Despite the mechanistic and phenotypic differences between HA and OA, OA is often used as “disease” control to reveal the characteristics in HA 1,2. Thus, we measured cartilage degeneration and DNA methylation difference in HA and OA patients. We have provided the statement and evidence in revised manuscript. Please see lines 144-145.

(3) Please provide details of how to calculate the Cartilage wear area ratio in Figure 1D, and measure the positive staining area in Figure 1F.

We apologize for the issue you pointed out. Here, we provide detailed information for how positively stained areas are calculated. Specifically, in Figure 1D, we obtained the cartilage area ratio by calculating the ratio of blue cartilage staining area to the whole tissue area by using image J software. In Figure 1F, the area of positive staining was determined upon secondary antibody treatment and color development using DAB chromogen (brown stain). We then obtained the positive staining area ratio by calculating the ratio of positive staining area to the whole cartilage area by using image J software.

(4) Please label the location of hemorrhagic ferruginous deposits in Figure 1.

Thank you for your valuable suggestion. We have used black arrows to indicate hemorrhagic ferruginous deposits in revised Figure 1A.

(5) Please define the meaning of "n" in all figure legends, such as technical or biological replicates.

Thanks for your suggestion. We have defined the meaning of "n" in all figure legends in revised manuscript.

(6) In Figure 3, please increase the font size of B, D, F, H, and J. The same applies to other figures.

Thank you for your valuable suggestion. We have increased the font size of figures in our revised manuscript.

(7) Line 327, "(Figure 1, F and G)" should be Figure 2F, G.

Thanks for your reminding. We have corrected it in the revision. Please see lines 347.

(8) Reduced TNXB levels in human HA cartilage are one of the major findings in this study. Currently, only semi-quatative IHC was used to draw the conclusion. A second method, such as real-time PCR or western blot, is required.

Thanks for your suggestion. We feel very sorry that we did not have enough samples of human HA cartilages for qPCR and WB experiments, due to severe erosion of the HA cartilage. We have pointed out this limitation in revised drafts. Please see lines 445-448.

(9) Figure 3 shows that reduced Tnxb was accompanied by the increased Dnmt1. In addition, this study is about methylation. Have the authors tested the change of Dnmt1 levels when Tnxb was knocked down?

Thanks for your suggestion. According to the reviewer's suggestion, we have tested the expression of Dnmt1 in Tnxb-KD chondrocytes, and no significant alteration was observed. Please see the following Figure.

Author response image 1.

Figure Legend: Representative IHC staining of Dnmt1 in articular cartilage from Tnxb-KD HA mice. Corresponding quantification of the proportion of Dnmt1 positive regions. Red arrows indicate positive cells. Scale bar: 100 μm. Data were presented as means ± SD; n = 5 in each group. ns = no significance by unpaired Student’s t test.

(10) Also, is there a causal relationship between Tnxb levels and the distribution of methylation levels? Any related study was performed?

Following the valuable suggestion of the reviewer, we used two well-known DNA methyltransferase inhibitors (RG108 or 5-Aza-dc) 3 to examine whether DNA methylation regulates transcriptional expression of TNXB. We found that both inhibitors significantly up-regulated Tnxb mRNA level. We have added this result to the revised Supplementary Figure 4 and draft (lines 292-296 and 369-374).

(11) In Figure 6, what was the control of "AKT agnost" group?

Thank you for your suggestion. We feel sorry for our negligence and we have added the vehicle group as a control for AKT agonists in Figure 6 in our revised manuscript.

(12) Previous studies have reported the involvement of TNXB in TGF-β signaling. Have the authors examined the effect of TNXB on TGF-β signaling in chondrocytes?

Thank you for your suggestion. Here, we examined the expression of TGF-β signaling in Tnxb-KD chondrocyte and no significant changes were observed. We have discussed this result in revised draft (lines 475-479). We have added this result to the revised Supplementary Figure 7.

(13) Discussion can be improved. For example, have previous studies reported the association between TNXB and methylation in other cells/tissues? In addition to apoptosis, are there other potential mechanisms underlying the protective role of TNXB in chondrocytes?

Thank you for your valuable comments. Previous studies have shown the different DNA methylation of TNXB in whole blood from rheumatoid arthritis patients and in retinal pigment epithelium from patients with age-related macular degeneration 4,5. Herein, we were the first to report the association between DNA methylation of TNXB and HA cartilage degeneration. As for TNXB, there are limited public studies regarding physiological function of TNXB, among which mostly report the effect of TNXB on extracellular matrix organization 6,7. In our work, we found that TNXB regulated the phosphorylation of AKT. Since previous reports showed AKT controlled the expression of Mmp13 8, we thought that TNXB might regulated the chondrocyte extracellular matrix organization, in addition to its function on apoptosis. We have discussed these in revised manuscript (lines 462-464, and 495-501).

(14) The manuscript writing needs to be improved. Typos and grammar issues were noted.

Thanks. We have modified and polished our language and we hope the revised version could be acceptable for you.

Reviewer #2 (Public Review):

Summary:

This manuscript mainly studied the biological effect of tenascin XB (TNXB) on hemophilic arthropathy (HA) progression. Using bioinformatic and histopathological approaches, the authors identified the novel candidate gene TNXB for HA. Next, the authors showed that TNXB knockdown leads to chondrocyte apoptosis, matrix degeneration, and subchondral bone loss in vivo/vitro. Furthermore, AKT agonists promoted extracellular matrix synthesis and prevented apoptosis in TNXB knockdown chondrocytes.

Strengths:

In general, this study significantly advances our understanding of HA pathogenesis. The authors utilize comprehensive experimental strategies to demonstrate the role of TNXB in cartilage degeneration associated with HA. The results are clearly presented, and the conclusions appear appropriate.

Weaknesses:

Additional clarification is required regarding the gender of the F8-/- mouse in the study. Is the mouse male or female?

We feel sorry that we did not provide enough information about the gender of the F8-/- mouse in the previous draft. Here, we used male F8-/- mice as the study subjects for our experiments. Hemophilia A is predominantly seen in males because of the X chromosome linkage 9.

Recommendations For The Authors:

Some issues need to be addressed in the manuscript:

(1) During the progression of HA, in addition to cartilage degeneration, synovial hypertrophy and inflammation are also significant symptoms. How is the expression of TNXB in HA synovium?

Thank you for your valuable comments. According to the reviewer's suggestion, we tested the expression of TNXB in the synovium, and there was no statistically significant difference in the expression level of TNXB in the synovium (Supplementary Figure. 2) Please see lines 347-349.

(2) Lines 183-188. The methods of virus infection should be more detailed. What was the concentration of the AAVs injected? And how many doses were administrated?

Thank you for your suggestion. We have added an explanation of virus infection and injected doses in revised methods section (lines 205-206).

(3) Line 197-198. Could the author double-check the decalcification time for human cartilage samples? Is it for 3 months? Or for 3 weeks?

Thank you for your suggestion. We have reconfirmed the decalcification of human cartilage samples for 3 months.

(4) Line 343-344 "Above results suggest that TNXB might be protective against HA and its cartilage suppression is closely related to HA development." The conclusion is inappropriate, please revise it.

Thanks for your suggestion. We have revised this conclusion into “Above results suggest that the suppression of TNXB in cartilage promotes the HA development”. Please see lines 365-366.

(5) Line 326-327, the IHC staining for human samples is shown in Figure 2, not Figure 1. Please double check and revise it.

Thanks for your reminding. We feel sorry for our negligence and we have corrected it in the revision.

(6) For Figure 1B, it shows the MRI images of knee joints. However, the method section lacks details regarding the MRI imaging scan and analysis. Could the author include this information in the method section?

Thank you for your valuable comments. We have added the method of MRI imaging scan and analysis in revised Methods. Please see lines 154-163.

(7) In Figure 5, The statistical result of Bcl-2 is inconsistent with its Western blot band. Please check.

Thanks for your reminding. We have modified it in the revision.

(8) Please read through the text carefully to check for language problems. For example, in Line 68 "Our" not "our".

Thanks for your reminding. In revision, we have corrected it. Please see Line 68.

Reviewer #3 (Public Review):

Summary:

The manuscript by Dr. Chen et al. investigates the genes that are differentially methylated and associated with cartilage degeneration in hemophilia patients. The study demonstrates the functional mechanisms of the TNXB gene in chondrocytes and F8-/- mice. The authors first showed significant DNA methylation differences between hemophilic arthritis (HA) and osteoarthritis through genome-wide DNA methylation analysis. Subsequently, they showed a decreased expression of the differentially methylated TNXB gene in cartilage from HA patients and mice. By knocking down TNXB in vivo and in vitro, the results indicated that TNXB regulates extracellular matrix homeostasis and apoptosis by modulating p-AKT. The findings are novel and interesting, and the study presents valuable information in blood-induced arthritis research.

Strengths:

The authors adopted a comprehensive approach by combining genome-wide DNA methylation analysis, in vivo and in vitro experiments using human and mouse samples to illustrate the molecular mechanisms involved in HA progression, which is crucial for developing targeted therapeutic strategies. The study identifies Tenascin XB (TNXB) as a central mediator in cartilage matrix degradation. It provides mechanistic insights into how TNXB influences cartilage matrix degradation by regulating the activation of AKT. It opens avenues for future research and potential therapeutic interventions using AKT agonists for cartilage protection in hemophilic arthropathy. The conclusions drawn from the study are clear and directly tied to the findings.

Weaknesses:

(1) The study utilizes a small sample size (N=5 for both osteoarthritis and hemophilic arthropathy). A larger sample size would enhance the generalizability and statistical power of the findings.

Thank you for pointing out this deficiency. Indeed, our sample size is relatively small, although the overall sample size was sufficient for statistical analyses. And we have added this limitation in discussion in revised manuscript. Please see line 445-448. Considering the small sample size, we subsequently performed functional validation study for TNXB, one of the most significant genes, and demonstrated that TNXB exerted critical impacts on chondrocytes apoptosis in HA pathogenesis in vivo and in vitro.

(2) The use of an animal model (F8-/- mouse) to investigate the role of TNXB may not fully capture the complexity of human hemophilic arthropathy. Differences in the biology between species may affect the translatability of the findings to human patients.

Thank you for your valuable comments. We recognize that biological differences between species can affect the clinical translation of research findings. In our work, we sequenced human cartilage samples to obtain the differentially methylated gene-TNXB. Meanwhile, we demonstrated that protein expression of TNXB protein was significantly down-regulated in HA human cartilage and F8-/- transgenic mouse cartilage. The F8-/- transgenic mouse serves as a well-accepted model for the study of hemophilia, which is phenotypically similar to that of human patients suffering from the disease and spontaneously bleeds into the joints and soft tissues. Besides, this model mouse has been widely used in the study of hemophilia and hemophilic arthritis 9-11.

(3) The study primarily focuses on TNXB as a central mediator, but it might overlook other potentially relevant factors contributing to cartilage degradation in hemophilic arthropathy. A more holistic exploration of genetic and molecular factors could provide a broader understanding of the condition.

Thanks for your suggestion. Since our human sample size is relatively small, we should interpret differentially methylated genes cautiously. Therefore, we mainly focused on the most top significant gene TNXB for functional study. In our further study, we will expand the sample size to more comprehensively explore the molecular mechanisms of HA.

Recommendations For The Authors:

The following are my suggestions:

(1) Why do the authors choose to concentrate on the knee joint in the introduction when hemophilia, characterized by a deficiency in clotting factor F8, is recognized as a systemic disease?

Thank you for your valuable comments. Although hemophilia a systemic disease, approximately 80%-90% of bleeding episodes in patients with hemophilia occur within the musculoskeletal system, especially in the knee joint 12.

(2) While Figure 1 illustrates distinct expressions of Dnmt1 and Dnmt3a, only Dnmt1 results are presented in HA mice models in Figure 3. To address this, it is suggested that the expression of Dnmt3a be explored in animal models.

Thank you for your suggestion. According to the reviewer's suggestion, we examined the expression of Dnmt3a in mouse articular cartilage, and the expression level of Dnmt3a was significantly up-regulated in both the 4W and 8W model groups compared with the control group (Figure 3). Please see line 364.

(3) In Figure 3, the sample size for Dnmt1 is smaller than the other indicators; therefore, supplementing the sample count is recommended.

Thanks for your reminding. We have corrected it in the revision.

(4) Regarding Figure 4G, a few apoptotic cells were observed in the AAV NC group. It is advised that this figure be reviewed for accuracy.

Thanks for your suggestion. In Figure 5D, the AAV-NC group is the case of needle-injected with AAV. Therefore, it is normal for apoptotic cells to appear in the cartilage layer.

(5) The authors concluded that TNXB plays a role in apoptosis and AKT signaling. Providing expression data for Caspase9 would be valuable to strengthen this assertion, as PI3K/AKT signaling directly influences its activation during apoptosis.

Thank you for your comments. We have examined the expression of Cleaved-Caspase9 protein, and found that knockdown of TNXB resulted in upregulation of Cleaved-Caspase9 protein expression, which was reversed by addition of SC79. This result has added in revised Figure 6 and manuscript. Please see line 414.

(6) Quantitative analysis of the differences between the two groups in Supplemental Figures is necessary.

Thank you for your suggestion. We have added the quantitative analysis of the differences between the two groups in Supplemental Figures.

(7) With three major isoforms (homologs) of AKT in mammals-AKT1, 2, and 3 - why did the authors specifically focus on AKT1?

Thank you for your comments. Based on the results of the KEGG enrichment analysis of differential methylated genes, we investigated the role of PI3K/AKT pathway in apoptosis of HA chondrocytes. AKT is universally acknowledged as a core factor in the PI3K/AKT pathway that plays critical roles in various cellular activities such as cell proliferation, cell differentiation, cell apoptosis, metabolism and so on 13,14, More notably, several studies demonstrated that in AKT family, Akt1 primarily was involved in regulation of chondrocyte survival and proteoglycan synthesis 15. Therefore, we detected phosphorylation of AKT1 in HA cartilages and TNXB-KD chondrocytes, and found that TNXB regulation chondrocytes ECM and apoptosis by AKT1. Reference:

(1) Cooke, E.J., Zhou, J.Y., Wyseure, T., Joshi, S., Bhat, V., Durden, D.L., Mosnier, L.O., and von Drygalski, A. (2018). Vascular Permeability and Remodelling Coincide with Inflammatory and Reparative Processes after Joint Bleeding in Factor VIII-Deficient Mice. Thromb Haemost 118, 1036-1047. 10.1055/s-0038-1641755.

(2) Kleiboer, B., Layer, M.A., Cafuir, L.A., Cuker, A., Escobar, M., Eyster, M.E., Kraut, E., Leavitt, A.D., Lentz, S.R., Quon, D., et al. (2022). Postoperative bleeding complications in patients with hemophilia undergoing major orthopedic surgery: A prospective multicenter observational study. J Thromb Haemost 20, 857-865. 10.1111/jth.15654.

(3) Weiland, T., Weiller, M., Kunstle, G., and Wendel, A. (2009). Sensitization by 5-azacytidine toward death receptor-induced hepatic apoptosis. J Pharmacol Exp Ther 328, 107-115. 10.1124/jpet.108.143560.

(4) Anaparti, V., Agarwal, P., Smolik, I., Mookherjee, N., and El-Gabalawy, H. (2020). Whole Blood Targeted Bisulfite Sequencing and Differential Methylation in the C6ORF10 Gene of Patients with Rheumatoid Arthritis. J Rheumatol 47, 1614-1623. 10.3899/jrheum.190376.

(5) Porter, L.F., Saptarshi, N., Fang, Y., Rathi, S., den Hollander, A.I., de Jong, E.K., Clark, S.J., Bishop, P.N., Olsen, T.W., Liloglou, T., et al. (2019). Whole-genome methylation profiling of the retinal pigment epithelium of individuals with age-related macular degeneration reveals differential methylation of the SKI, GTF2H4, and TNXB genes. Clin Epigenetics 11, 6. 10.1186/s13148-019-0608-2.

(6) Mao, J.R., Taylor, G., Dean, W.B., Wagner, D.R., Afzal, V., Lotz, J.C., Rubin, E.M., and Bristow, J. (2002). Tenascin-X deficiency mimics Ehlers-Danlos syndrome in mice through alteration of collagen deposition. Nat Genet 30, 421-425. 10.1038/ng850.

(7) Zhang, K., Wang, X., Zeng, L.T., Yang, X., Cheng, X.F., Tian, H.J., Chen, C., Sun, X.J., Zhao, C.Q., Ma, H., and Zhao, J. (2023). Circular RNA PDK1 targets miR-4731-5p to enhance TNXB expression in ligamentum flavum hypertrophy. FASEB J 37, e22877. 10.1096/fj.202200022RR.

(8) Guo, H., Yin, W., Zou, Z., Zhang, C., Sun, M., Min, L., Yang, L., and Kong, L. (2021). Quercitrin alleviates cartilage extracellular matrix degradation and delays ACLT rat osteoarthritis development: An in vivo and in vitro study. J Adv Res 28, 255-267. 10.1016/j.jare.2020.06.020.

(9) Weitzmann, M.N., Roser-Page, S., Vikulina, T., Weiss, D., Hao, L., Baldwin, W.H., Yu, K., Del Mazo Arbona, N., McGee-Lawrence, M.E., Meeks, S.L., and Kempton, C.L. (2019). Reduced bone formation in males and increased bone resorption in females drive bone loss in hemophilia A mice. Blood Adv 3, 288-300. 10.1182/bloodadvances.2018027557.

(10) Haxaire, C., Hakobyan, N., Pannellini, T., Carballo, C., McIlwain, D., Mak, T.W., Rodeo, S., Acharya, S., Li, D., Szymonifka, J., et al. (2018). Blood-induced bone loss in murine hemophilic arthropathy is prevented by blocking the iRhom2/ADAM17/TNF-alpha pathway. Blood 132, 1064-1074. 10.1182/blood-2017-12-820571.

(11) Vols, K.K., Kjelgaard-Hansen, M., Ley, C.D., Hansen, A.K., and Petersen, M. (2019). Bleed volume of experimental knee haemarthrosis correlates with the subsequent degree of haemophilic arthropathy. Haemophilia 25, 324-333. 10.1111/hae.13672.

(12) Lobet, S., Peerlinck, K., Hermans, C., Van Damme, A., Staes, F., and Deschamps, K. (2020). Acquired multi-segment foot kinematics in haemophilic children, adolescents and young adults with or without haemophilic ankle arthropathy. Haemophilia 26, 701-710. 10.1111/hae.14076.

(13) Garcia, D., and Shaw, R.J. (2017). AMPK: Mechanisms of Cellular Energy Sensing and Restoration of Metabolic Balance. Mol Cell 66, 789-800. 10.1016/j.molcel.2017.05.032.

(14) Johnson, J., Chow, Z., Lee, E., Weiss, H.L., Evers, B.M., and Rychahou, P. (2021). Role of AMPK and Akt in triple negative breast cancer lung colonization. Neoplasia 23, 429-438. 10.1016/j.neo.2021.03.005.

(15) Rao, Z., Wang, S., and Wang, J. (2017). Peroxiredoxin 4 inhibits IL-1beta-induced chondrocyte apoptosis via PI3K/AKT signaling. Biomed Pharmacother 90, 414-420. 10.1016/j.biopha.2017.03.075.

Reviewer #2 (Public Review):

Summary:

This manuscript mainly studied the biological effect of tenascin XB (TNXB) on hemophilic arthropathy (HA) progression. Using bioinformatic and histopathological approaches, the authors identified the novel candidate gene TNXB for HA. Next, authors showed that TNXB knockdown lead to chondrocyte apoptosis, matrix degeneration and subchondral bone loss in vivo/vitro. Furthermore, AKT agonist promoted extracellular matrix synthesis and prevented apoptosis in TNXB knockdown chondrocytes.

Strengths:

In general, this study significantly advances our understanding of HA pathogenesis. The authors utilize comprehensive experimental strategies to demonstrate the role of TNXB in cartilage degeneration associated with HA. The results are clearly presented, and the conclusions appear appropriate.

Weaknesses:

Additional clarification is required regarding the gender of the F8-/- mouse in the study. Is the mouse male or female?

eLife assessment

This important study identifies the TNXB-AKT pathway as a potential mechanism underlying hemophilia-associated cartilage degeneration. The evidence supporting the conclusions is convincing, with murine and human patient evidence as well as genome-wide DNA methylation analysis. This paper would be of interest to cell biologists and biochemists working on the field of musculoskeletal disorders.

Reviewer #1 (Public Review):

Summary:

Chen and colleagues first compared the cartilage tissues collected from OA and HA patients using histology and immunostaining. Then, a genome-wide DNA methylation analysis was performed, which informed the changes of a novel gene, TNXB. IHC confirmed that TNXB has a lower expression level in HA cartilage than OA. Next, the authors demonstrated that TNXB levels were reduced in HA animal model, and intraarticular injection of AAV carrying TNXB siRNA induced cartilage degradation and promoted chondrocyte apoptosis. Based on KEGG enrichment, histopathological analysis, and western blot, the authors also showed the relationship between TNXB and AKT phosphorylation. Lastly, AKT agonist, specifically SC79 in this study, was shown to partially rescue the changes of in vitro-cultured chondrocytes induced by Tnxb knock-down. Overall, this is an interesting study and provided sufficient data to support their conclusion.

Strengths:

(1) Both human and mouse samples were examined.<br /> (2) The HA model was used.<br /> (3) genome-wide DNA methylation analysis was performed.

Weaknesses:

(1) In some experiments, the selection of the control groups was not ideal.<br /> (2) More details on analyzing methods and information on replicates need to be included.<br /> (3) Discussion can be improved by comparing findings to other relevant studies.<br /> (4) The use of transgenic mice with conditional Tnxb depletion can further define the physiological roles of Tnxb.

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Author response:

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

We thank the reviewers for their thorough review of and overall positive comments on our manuscript. We have revised the manuscript to address the one remaining concern raised by one of the reviewers. This is described below.

Fig.1B-C: To give a standard deviation from 2 data points has no statistical significance. In this case it would be better to define as range/difference of the 2 data points.

We have modified the legend for Figure 1 to now read, “The average of two experiments is plotted with the bars representing the range of each time point.”

eLife assessment

This important study contributes insights into the regulatory mechanisms of a protein governing cell migration at the membrane. The integration of approaches revealing protein structure and dynamics provides convincing data for a model of regulation and suggests a new allosteric role for a solubilized phospholipid headgroup. The work will be interesting to researchers focusing on signaling mechanisms, cell motility, and cancer metathesis.

Reviewer #1 (Public Review):

Summary:

The authors perform a multidisciplinary approach to describe the conformational plasticity of P-Rex1 in various states (autoinhibited, IP4 bound and PIP3 bound). Hydrogen-deuterium exchange (HDX) is used to reveal how IP4 and PIP3 binding affect intramolecular interactions. While IP4 is found to stabilize autoinhibitory interactions, PIP3 does the opposite, leading to deprotection of autoinhibitory sites. Cryo-EM of IP4 bound P-Rex1 reveals a structure in the autoinhibited conformation, very similar to the unliganded structure reported previously (Chang et al. 2022). Mutations at observed autoinhibitory interfaces result in a more open structure (as shown by SAXS), reduced thermal stability and increased GEF activity in biochemical and cellular assays. Together their work portrays a dynamic enzyme that undergoes long-range conformational changes upon activation on PIP3 membranes. The results are technically sound and the conclusions are justified. The main drawback is the limited novelty due to the recently published structure of unliganded P-Rex1, which is virtually identical to the IP4 bound structure presented here. Novel aspects suggest a regulatory role for IP4, but the exact significance and mechanism of this regulation has not been explored.

Strengths:

The authors use a multitude of techniques to describe the dynamic nature and conformational changes of P-Rex1 upon binding to IP4 and PIP3 membranes. The different approaches together fit well with the overall conclusion that IP4 binding negatively regulates P-Rex1, while binding to PIP3 membranes leads to conformational opening and catalytic activation. The experiments are performed very thoroughly and are technically sound. The results are clear and support the conclusions.

Weaknesses:

(1) The novelty of the study is compromised due to the recently published structure of unliganded P-Rex1 (Chang et al. 2022). The unliganded and IP4 bound structure of P-Rex1 appear virtually identical, however, no clear comparison is presented in the manuscript. In the same paper a very similar model of P-Rex1 activation upon binding to PIP3 membranes and Gbeta-gamma is presented.

(2) The authors demonstrate that IP4 binding to P-Rex1 results in catalytic inhibition and increased protection of autoinhibitory interfaces, as judged by HDX. The relevance of this in a cellular setting is not clear and is not experimentally demonstrated. Further, mechanistically, it is not clear whether the biochemical inhibition by IP4 of PIP3 activated P-Rex1 is due to competition of IP4 with activating PIP3 binding to the PH domain of P-Rex1, or due to stabilizing the autoinhibited conformation, or both.

Reviewer #2 (Public Review):

Summary:

In this new paper, the authors used biochemical, structural, and biophysical methods to elucidate the mechanisms by which IP4, the PIP3 headgroup, can induce an autoinhibit form of P-Rex1 and propose a model of how PIP3 can trigger long-range conformational changes of P-Rex1 to relieve this autoinhibition. The main findings of this study are that a new P-Rex1 autoinhibition is driven by an IP4-induced binding of the PH domain to the DH domain active site and that this autoinhibit form stabilized by two key interactions between DEP1 and DH and between PH and IP4P 4-helix bundle (4HB) subdomain. Moreover, they found that the binding of phospholipid PIP3 to the PH domain can disrupt these interactions to relieve P-Rex1 autoinhibition.

Strengths:

The study provides good evidence that binding of IP4 to the P-Rex1 PH domain can make the two long-range interactions between the catalytic DH domain and the first DEP domain, and between the PH domain and the C-terminal IP4P 4HB subdomain that generate a novel P-Rex1 autoinhibition mechanism. This valuable finding adds an extra layer of P-Rex1 regulation (perhaps in the cytoplasm) to the synergistic activation by phospholipid PIP3 and the heterotrimeric Gβγ subunits at the plasma membrane. Overall, this manuscript's goal sounds interesting, the experimental data were carried out carefully and reliably.

Weakness:

The set of experiments with the disulfide bond S235C/M244C caused a bit of confusion for interpretation, it should be moved into the supplement, and the text and Figure 4 were altered accordingly.

Reviewer #3 (Public Review):

Summary:

In this report, Ravala et al demonstrate that IP4, the soluble head-group of phosphatiylinositol 3,4,5 - trisphosphate (PIP3), is an inhibitor of pREX-1, a guanine nucleotide exchange factor (GEF) for Rac1 and related small G proteins that regulate cell cell migration. This finding is perhaps unexpected since pREX-1 activity is PIP3-dependent. By way of Cryo-EM (revealing the structure of the p-REX-1/IP4 complex at 4.2Å resolution), hydrogen-deuterium mass spectrometry and small angle X-ray scattering, they deduce a mechanism for IP4 activation, and conduct mutagenic and cell-based signaling assays that support it. The major finding is that IP4 stabilizes two interdomain interfaces that block access of the DH domain, which conveys GEF activity towards small G protein substrates. One of these is the interface between the PH domain that binds to IP4 and a 4-helix bundle extension of the IP4 Phosphatase domain and the DEP1 domain. The two interfaces are connected by a long helix that extends from PH to DEP1. Although the structure of fully activated pREX-1 has not been determined, the authors propose a "jackknife" mechanism, similar to that described earlier by Chang et al (2022) (referenced in the author's manuscript) in which binding of IP3 relieves a kink in a helix that links the PH/DH modules and allows the DH-PH-DEP triad to assume an extended conformation in which the DH domain is accessible. While the structure of the activated pREX-1 has not been determined, cysteine mutagenesis that enforces the proposed kink is consistent with this hypothesis. SAXS and HDX-MS experiments suggest that IP4 acts by stiffening the inhibitory interfaces, rather than by reorganizing them. Indeed, the cryo-EM structure of ligand-free pREX-1 shows that interdomain contacts are largely retained in the absence of IP4.

Strengths:

The manuscript thus describes a novel regulatory role for IP4 and is thus of considerable significance to our understanding of regulatory mechanisms that control cell migration, particularly in immune cell populations. Specifically, they show how the inositol polyphosphate IP4 controls the activity of pREX-1, a guanine nucleotide exchange factor that controls the activity of small G proteins Rac and CDC42. In their clearly-written discussion, the authors explain how PIP3, the cell membrane and the Gbeta-gamma subunits of heterotrimeric membranes together localize pREX-1 at the membrane and induce activation. The quality of experimental data is high and both in vitro and cell-based assays of site-directed mutants designed to test the author's hypotheses are confirmatory. The results strongly support the conclusions. The combination of cryo-EM data, that describe the static (if heterogeneous) structures with experiments (small angle x-ray scattering and hydrogen-deuterium exchange-mass spectrometry) that report on dynamics are well employed by the authors

Manuscript revision:

The reviewers noted a number of weaknesses, including error analysis of the HDX data, interpretation of the mutagenesis data, the small fraction of the total number of particles used to generate the EM reconstruction, the novelty of the findings in light of the previous report by Cheng et al, 2022, various details regarding presentation of structural results and questions regarding the interpretation of the inhibition data (Figure 1D). The authors have responded adequately to these critiques. It appears that pREX-1 is a highly dynamic molecule, and considerable heterogeneity among particles might be expected.

While, indeed, the conformation of pREX presented in this report is not novel, the finding that this inactive conformational state is stabilized by IP4 is significant and important. The evidence for this is both structural and biochemical, as indicated by micromolar competition of IP4 with PI3-enriched vesicles resulting in the inhibition of pREX-1 GEF activity.

eLife assessment

This study provides valuable insight into the role of miR-199a/b-5p in cartilage formation. The evidence supporting the significance of the identified miRNA and its target mRNA transcripts is convincing. This paper will likely primarily benefit scientists focused on diseases related to this biological process, such as osteoarthritis. Furthermore, researchers interested in miRNAs as a broader subject may find the computational model development methodology helpful.

Reviewer #1 (Public Review):

The comments below are from my review of the first submission of this article. I would now like to thank the authors for their hard work in responding to my comments. I am happy with the changes they have made, in particular the inclusion of further experimental evidence in Figures 2 and 4. I have no further comments to make.

In 'Systems analysis of miR-199a/b-5p and multiple miR-199a/b-5p targets during chondrogenesis', Patel et al. present a variety of analyses using different methodologies to investigate the importance of two miRNAs in regulating gene expression in a cellular model of cartilage development. They first re-analysed existing data to identify these miRNAs as one of the most dynamic across a chondrogenesis development timecourse. Next, they manipulated the expression of these miRNAs and showed that this affected the expression of various marker genes as expected. An RNA-seq experiment on these manipulations identified putative mRNA targets of the miRNAs which were also supported by bioinformatics predictions. These top hits were validated experimentally and, finally, a kinetic model was developed to demonstrate the relationship between the miRNAs and mRNAs studied throughout the paper.

I am convinced that the novel relationships reported here between miR-199a/b-5p and target genes FZD6, ITGA3 and CAV1 are likely to be genuine. It is important for researchers working on this system and related diseases to know all the miRNA/mRNA relationships but, as the authors have already published work studying the most dynamic miRNA (miR-140-5p) in this biological system I was not convinced that this study of the second miRNA in their list provided a conceptual advance on their previous work.

I was also concerned with the lack of reporting of details of the manipulation experiments. The authors state that they have over-expressed miR-199a-5p (Figure 2A) and knocked down miR-199b-5p (Figure 2B) but they should have reported their proof that these experiments had worked as predicted, e.g. showing the qRT-PCR change in miRNA expression. Similarly, I was concerned that one miRNA was over-expressed while the other was knocked down - why did the authors not attempt to manipulate both miRNAs in both directions? Were they unable to achieve a significant change in miRNA expression or did these experiments not confirm the results reported in the manuscript?

I had a number of issues with the way in which some of the data is presented. Table 1 only reported whether a specific pathway was significant or not for a given differential expression analysis but this concealed the extent of this enrichment or the level of statistical significance reported. Could it be redrawn to more similarly match the format of Figure 3A? The various shades of grey in Figure 2 and Figure 4 made it impossible to discriminate between treatments and therefore identify whether these data supported the conclusions made in the text. It also appeared that the same results were reported in Figure 3B and 3C and, indeed, Figure 3B was not referred to in the main text. Perhaps this figure could be made more concise by removing one of these two sets of panels?

Overall, while I think that this is an interesting and valuable paper, I think its findings are relatively limited to those interested in the role of miRNAs in this specific biomedical context.

Reviewer #2 (Public Review):

Summary:

This study represents an ambitious endeavor to comprehensively analyze the role of miR-199a/b-5p and its networks in cartilage formation. By conducting experiments that go beyond in vitro MSC differentiation models, more robust conclusions can be achieved.

Strengths:

This research investigates the role of miR-199a/b-5p during chondrogenesis using bioinformatics and in vitro experimental systems. The significance of miRNAs in chondrogenesis and OA is crucial, warranting further research, and this study contributes novel insights.

Weaknesses:

While miR-140 and miR-455 are used as controls, these miRNAs have been demonstrated to be more relevant to Cartilage Homeostasis than chondrogenesis itself. Their deficiency has been genetically proven to induce Osteoarthritis in mice. Therefore, the results of this study should be considered in comparison with these existing findings.

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

In 'Systems analysis of miR-199a/b-5p and multiple miR-199a/b-5p targets during chondrogenesis', Patel et al. present a variety of analyses using different methodologies to investigate the importance of two miRNAs in regulating gene expression in a cellular model of cartilage development. They first re-analysed existing data to identify these miRNAs as one of the most dynamic across a chondrogenesis development time course. Next, they manipulated the expression of these miRNAs and showed that this affected the expression of various marker genes as expected. An RNA-seq experiment on these manipulations identified putative mRNA targets of the miRNAs which were also supported by bioinformatics predictions. These top hits were validated experimentally and, finally, a kinetic model was developed to demonstrate the relationship between the miRNAs and mRNAs studied throughout the paper.

I am convinced that the novel relationships reported here between miR-199a/b-5p and target genes FZD6, ITGA3, and CAV1 are likely to be genuine. It is important for researchers working on this system and related diseases to know all the miRNA/mRNA relationships but, as the authors have already published work studying the most dynamic miRNA (miR-140-5p) in this biological system I was not convinced that this study of the second miRNA in their list provided a conceptual advance on their previous work.

We believe this study is an enhancement on our previous work for two reasons, which have been alluded to in new text within the introduction. Firstly, our previous work used experimental and bioinformatic analysis to identify microRNAs with significant regulatory roles during chondrogenesis. This new manuscript additionally uses  a systems biology approaches to identify novel miRNA-mRNA interactions and capture these within an in silico model. Secondly, this work was initiated by the analysis of our previously generated data – using a novel tool we developed for this type of data (Bioconductor - TimiRGeN).  

I was also concerned with the lack of reporting of details of the manipulation experiments. The authors state that they have over-expressed miR-199a-5p (Figure 2A) and knocked down miR-199b-5p (Figure 2B) but they should have reported their proof that these experiments had worked as predicted, e.g. showing the qRT-PCR change in miRNA expression. Similarly, I was concerned that one miRNA was over-expressed while the other was knocked down - why did the authors not attempt to manipulate both miRNAs in both directions? Were they unable to achieve a significant change in miRNA expression or did these experiments not confirm the results reported in the manuscript?

We agree with the reviewer that some additional data were needed to demonstrate the effective regulation of miR-199-5p.  Hence, Supplementary Figure 1 is now included which provides validation of the effects of miR-199a-5p overexpression (Supplementary Figure 1A) and inhibition of miR-199a/b-5p (Supplementary Figure 1B). Within the main manuscript, Figure 2B has been amended to include the consequences of inhibition of miR-199a-5p, with 2C showing the consequences of miR-199b-5p inhibition. Further, we include new data with regards to miR-199a/b-5p inhibition on CAV1 (Figure 4A). 

I had a number of issues with the way in which some of the data was presented. Table 1 only reported whether a specific pathway was significant or not for a given differential expression analysis but this concealed the extent of this enrichment or the level of statistical significance reported. Could it be redrawn to more similarly match the format of Figure 3A? The various shades of grey in Figure 2 and Figure 4 made it impossible to discriminate between treatments and therefore identify whether these data supported the conclusions made in the text. It also appeared that the same results were reported in Figure 3B and 3C and, indeed, Figure 3B was not referred to in the main text. Perhaps this figure could be made more concise by removing one of these two sets of panels.

We agree with all points made here and have amended these within the manuscript. Figure 1A is now pathway enrichment plots from the TimiRGeN R Bioconductor package, and the table which previously showed the pathways enriched at each time point is now in the supplementary materials (supp. Table 1). Figure 2 and 4 now have color instead of shades of grey. Figure 3C has now been moved to supplementary materials (Supplementary Figure 2) and is referenced in the text. 

Overall, while I think that this is an interesting and valuable paper, I think its findings are relatively limited to those interested in the role of miRNAs in this specific biomedical context.

Reviewer #2 (Public review):

Summary:

This study represents an ambitious endeavor to comprehensively analyze the role of miR199a/b-5p and its networks in cartilage formation. By conducting experiments that go beyond in vitro MSC differentiation models, more robust conclusions can be achieved.

Strengths:

This research investigates the role of miR-199a/b-5p during chondrogenesis using bioinformatics and in vitro experimental systems. The significance of miRNAs in chondrogenesis and OA is crucial, warranting further research, and this study contributes novel insights.

Weaknesses:

While miR-140 and miR-455 are used as controls, these miRNAs have been demonstrated to be more relevant to Cartilage Homeostasis than chondrogenesis itself. Their deficiency has been genetically proven to induce Osteoarthritis in mice. Therefore, the results of this study should be considered in comparison with these existing findings.

We agree with the reviewers comments. miR-455-null mice develop normally but miR-140-null (or mutated) mice and humans do have skeletal abnormalities (e.g. Nat Med. 2019 Apr;25(4):583-590. doi: 10.1038/s41591-019-0353-2), indicating a role in chondrogenesis.  We have made an addition in the description to point towards the need to assess the roles miR-199a/b-5p may play during skeletogenesis and OA. We anticipate miR-199a/b-5p to be relevant in OA and have ongoing additional work for this – but this beyond the scope of this manuscript. 

Recommendations to Authors:

Reviewer #1 (Recommendations to authors):

Beyond the issues raised in the public review, I had a few minor recommendations that are largely designed to help improve the understanding of the manuscript as it is currently written.

(1) Please provide the statistical tests used to obtain p-values in the Figure 2 and 4 legends.

We have now added statistical test information to the figure legends of figures 2 and 4.

(2) It is stated on p. 9 that both miRNAs may share a functional repertoire because 25 and 341 genes are interested between their inhibition experiments. Please provide statistical support that this overlap is an enrichment over the null background in this experiment. Total DE genes – chi squared. Expected / Observed. 

A chi-squared test is now presented in the manuscript which shows that the number of significant genes which were found in common between miR-199a-5p knockdown and miR-199b-5p knockdown were significantly more than expected for day 0 or day 1 of the experiments. 

(3) The final sentence on p. 12 (beginning 'Size of the points reflect...') seemed out of place - is it part of a legend?

Thank you for pointing out this mistake - it was part of figure 3C and now is in the supplementary materials.

(4) A sentence on p. 14 reads that 'FZD6 and ITGA3 levels increased significantly' but this should read decreased, rather than increased. Quite an important typo!

Thank you for pointing this error out. It has been corrected.

(5) Theoretical transcripts are mentioned in the legend of Figure 5A but these were not present in the figure. Please include these or remove them from the legend.

This error has been removed form Figure 5A.

(6) On p 20, the references 22 and 27 should I think be moved to earlier in the sentence (after 'miR-199a-5p-FZD6 has been predicted previously'). Currently, it reads as if these references support your luciferase assays which you claim are the first evidence for this target relationship.

We agree with this change and have corrected the manuscript.

(7) The reference to Figure 5D on p. 20 should be a reference to Figure 5C.

Thank you for pointing this error out – this has been corrected.

Reviewer #2 (Recommendations to authors):

(1) The paper is based on the importance of miR-140 and miR-455 as miRNAs in chondrogenesis, citing only Barter, M. J. et al. Stem Cells 33, (2015). Considering the scope and results of this study, this citation is insufficient.

We agree with this reviewers comments. For many year miR-140 and miR-455 have been experimented on and their importance in OA research has become apparent. We included additional references within the introduction to address this.

(2) Analyzing chondrogenesis solely through differentiation experiments from MSCs is inadequate. It is essential to perform experiments involving the network within normal cartilage tissue and/or the generation of knockout mice to understand the precise role of miR199a/b-5p in chondrogenesis.

We have added an additional paragraph in the discussion to state this, and do believe it is highly important that miR-199a/b-5p be tested in OA samples – however this would be beyond the intended scope of this article.

(3) In light of the above points, it is imperative to investigate the role of miR-199a/b-5p beyond the in vitro differentiation model from MSCs, encompassing mouse OA models or human disease samples.

In tangent with the previous address, we agree with the pretense and believe additional experiments should be performed to gain more insight to the mechanism of how miR-199a/b-5p regulate OA. But development of a new mouse line to investigate this is not in the scope of this manuscript.

Lone wolf attack refers to violent acts committed by a person who plans and executes the attack without direct support from large organizations or groups. But he/she still can causes significant harm and generate widespread fear to the public.

The article reports that dentist Walter Palmer, who killed the famous lion Cecil, hunted and killed a protected wild ram four years later. This has sparked outrage and condemnation, raising questions about the enforcement of wildlife protection laws.

Dogpiling is an online harassment tactic where a group of harassers target a single victim. The abuse can involve various methods including flaming (hostile and insulting interaction), doxing (publishing private or identifying information), impersonation, and public shaming. The term is most often associated with internet discussions and is used to describe situations similar to those seen in high-profile online harassment campaigns. An example cited is its usage during the Gamergate harassment campaign, indicating its relevance in major online controversies.

Doxing can be extremely dangerous as it violates others' security and privacy. By providing someone else's personal information, individuals can be threatened, leading to fear of potential danger. I believe that any form of doxing should not be tolerated

I think the article explores the seriousness of cyberstalking and the lasting impact of this practice on victims, while also revealing the inadequacy of legal and social protection measures to protect victims. This personal narrative highlights the importance of cybersecurity and privacy rights, as well as the need for more proactive engagement and updating of social and legal systems when dealing with such incidents.

I remembered when I was in high school, my English teacher told us that there was a man trying to rape her at the gas station because she is a black woman that seems easy to be harassed.

Personal opinion but home/about should be merged. The landing page of any website communicates what the website is about, maybe merge the two and keep the about page as the homepage.

These navigation buttons are a little small

This text isn't legible at all in front of the meringue, really hard to read

Needs more bottom-padding to offset from the bottom of the page

I like these buttons but could be a bit more accessibility freindly if the button background had some sort of tint on it.

Sant, men det hävdades väll aldrig från början

bra för oss att nämna

känns inte som ett syfte

jag har ingen förförståelse

取消/折叠 chapter 显示

???

???

学习集

eLife assessment

This study demonstrates that neurons receiving inputs from auditory cortex in the inferior colliculus widely encode the outcome of a sound detection task independant of the presence of auditory cortex. This valuable study based on imaging of transynaptically labelled neurons provides convincing evidence that auditory cortex is necessary neither for sound detection, nor to channel information related to behavioral outcome to the subcortical auditory system. This study will be of wide interest for sensory neuroscientists.

Reviewer #1 (Public Review):

The inferior colliculus (IC) is the central auditory system's major hub. It integrates ascending brainstem signals to provide acoustic information to the auditory thalamus. The superficial layers of the IC ("shell" IC regions as defined in the current manuscript) also receive a massive descending projection from the auditory cortex. This auditory cortico-collicular pathway has long fascinated the hearing field, as it may provide a route to funnel "high-level" cortical signals and impart behavioral salience upon an otherwise behaviorally agnostic midbrain circuit.

Accordingly, IC neurons can respond differently to the same sound depending on whether animals engage in a behavioral task (Ryan and Miller 1977; Ryan et al., 1984; Slee & David, 2015; Saderi et al., 2021; De Franceschi & Barkat, 2021). Many studies also report a rich variety of non-auditory responses in the IC, far beyond the simple acoustic responses one expects to find in a "low-level" region (Sakurai, 1990; Metzger et al., 2006; Porter et al., 2007). A tacit assumption is that the behaviorally relevant activity of IC neurons is inherited from the auditory cortico-collicular pathway. However, this assumption has never been tested, owing to two main limitations of past studies:

(1) Prior studies could not confirm if data were obtained from IC neurons that receive monosynaptic input from the auditory cortex.

(2) Many studies have tested how auditory cortical inactivation impacts IC neuron activity; the consequence of cortical silencing is sometimes quite modest. However, all prior inactivation studies were conducted in anesthetized or passively listening animals. These conditions may not fully engage the auditory cortico-collicular pathway. Moreover, the extent of cortical inactivation in prior studies was sometimes ambiguous, which complicates interpreting modest or negative results.

Here, the authors' goal is to directly test if the auditory cortex is necessary for behaviorally relevant activity in IC neurons. They conclude that surprisingly, task relevant activity in cortico-recipient IC neuron persists in absence of auditory cortico-collicular transmission. To this end, a major strength of the paper is that the authors combine a sound-detection behavior with clever approaches that unambiguously overcome the limitations of past studies.

First the authors inject a transsynaptic virus into the auditory cortex, thereby expressing a genetically encoded calcium indicator in the auditory cortex's postsynaptic targets in the IC. This powerful approach enables 2-photon Ca2+ imaging from IC neurons that unambiguously receive monosynaptic input from auditory cortex. Thus, any effect of cortical silencing should be maximally observable in this neuronal population. Second, they abrogate auditory cortico-collicular transmission using lesions of auditory cortex. This "sledgehammer" approach is arguably the most direct test of whether cortico-recipient IC neurons will continue to encode task-relevant information in absence of descending feedback. Indeed, their method circumvents the known limitations of more modern optogenetic or chemogenetic silencing, e.g. variable efficacy.

The authors have revised their manuscript and adequately addressed the major concerns. Although more in depth analyses of these rich datasets are definitely possible, the current results nevertheless stand on their own. Indeed, the work serves as a beacon to move away from the idea that cortico-collicular projections function primarily to impart behavioral relevance upon auditory midbrain neurons. This knowledge inspires a search for alternative explanations as to the role of auditory cortico-collicular synapses in behavior.

Reviewer #2 (Public Review):

Summary:

This study takes a new approach to studying the role of corticofugal projections from auditory cortex to inferior colliculus. The authors performed two-photon imaging of cortico-recipient IC neurons during a click detection task in mice with and without lesions of auditory cortex. In both groups of animals, they observed similar task performance and relatively small differences in the encoding of task-response variables in the IC population. They conclude that non-cortical inputs to the IC provide can substantial task-related modulation, at least when AC is absent.

Strengths:

This study provides valuable new insight into big and challenging questions around top-down modulation of activity in the IC. The approach here is novel and appears to have been executed thoughtfully. Thus, it should be of interest to the community.

Weaknesses:

There are however, substantial concerns about the interpretation of the findings and limitations to the current analysis. In particular, Analysis of single unit activity is absent, making interpretation of population clusters and decoding less interpretable. These concerns should be addressed to make sure that the results can be interpreted clearly in an active field that already contains a number of confusing and possibly contradictory findings.

Reviewer #3 (Public Review):

Summary:

This study aims to demonstrate that cortical feedback is not necessary to signal behavioral outcome to shell neurons of the inferior colliculus during a sound detection task. The demonstration is achieved in a very clear manner by the observation of the activity of cortico-recepient neurons in animals which have received lesions of the auditory cortex. The experiment shows that neither behavior performance nor neuronal responses are significantly impacted by cortical lesions except for the case of partial lesions which seem to have a disruptive effect on behavioral outcome signaling.

Strengths:

The demonstration of the main conclusions is based on state-of-the-art, carefully controlled methods and is highly convincing. There is an in depth discussion of the different effects of auditory cortical lesions on sound detection behavior.

Weaknesses:

The description of feedback signals could be more detailed although it is difficult to achieve good temporal resolution with the calcium imaging technique necessary for targeting cortico-recipient neurons.

eLife assessment

The study, from the group that pioneered migrasome, describes a novel vaccine platform derived from this newly discovered organelle. Using these cleverly engineered migrasomes - that behave like natural migrasomes - as a novel vaccine platform has the potential to overcome obstacles such as cold chain issues for vaccines like messenger RNA. Although the findings are important with practical implications for the vaccine technology, and the evidence, based on appropriate and validated methodology is solid and convincing and is in line with current state-of-the-art, there are some critical issues that need to be addressed. Amongst others, these include a head-to-head comparison with proven vaccine platforms, for example, a SARS-CoV-2 mRNA vaccine or an adjuvanted recombinant spike protein.

Reviewer #1 (Public Review):

Summary:<br /> This is an excellent study by a superb investigator who discovered and is championing the field of migrasomes. This study contains a hidden "gem" - the induction of migrasomes by hypotonicity and how that happens. In summary, an outstanding fundamental phenomenon (migrasomes) en route to becoming transitionally highly significant.

Strengths:

Innovative approach at several levels. Migrasomes - discovered by Dr Yu's group - are an outstanding biological phenomenon of fundamental interest and now of potentially practical value.

Weaknesses:

I feel that the overemphasis on practical aspects (vaccine), however important, eclipses some of the fundamental aspects that may be just as important and actually more interesting. If this can be expanded, the study would be outstanding.

Reviewer #2 (Public Review):

Summary:

The authors' report describes a novel vaccine platform derived from a newly discovered organelle called a migrasome. First, the authors address a technical hurdle in using migrasomes as a vaccine platform. Natural migrasome formation occurs at low levels and is labor intensive, however, by understanding the molecular underpinning of migrasome formation, the authors have designed a method to make engineered migrasomes from cultured, cells at higher yields utilizing a robust process. These engineered migrasomes behave like natural migrasomes. Next, the authors immunized mice with migrasomes that either expressed a model peptide or the SARS-CoV-2 spike protein. Antibodies against the spike protein were raised that could be boosted by a 2nd vaccination and these antibodies were functional as assessed by an in vitro pseudoviral assay. This new vaccine platform has the potential to overcome obstacles such as cold chain issues for vaccines like messenger RNA that require very stringent storage conditions.

Strengths:

The authors present very robust studies detailing the biology behind migrasome formation and this fundamental understanding was used to form engineered migrasomes, which makes it possible to utilize migrasomes as a vaccine platform. The characterization of engineered migrasomes is thorough and establishes comparability with naturally occurring migrasomes. The biophysical characterization of the migrasomes is well done including thermal stability and characterization of the particle size (important characterizations for a good vaccine).

Weaknesses:

With a new vaccine platform technology, it would be nice to compare them head-to-head against a proven technology. The authors would improve the manuscript if they made some comparisons to other vaccine platforms such as a SARS-CoV-2 mRNA vaccine or even an adjuvanted recombinant spike protein. This would demonstrate a migrasome-based vaccine could elicit responses comparable to a proven vaccine technology. Additionally, understanding the integrity of the antigens expressed in their migrasomes could be useful. This could be done by looking at functional monoclonal antibodies binding to their migrasomes in a confocal microscopy experiment.

eLife assessment

This study provides valuable evidence that differentiated cells of the zebrafish skin form membrane protrusions called cytonemes, that contact and potentially transmit Notch signals to cells of the intermediate layer below. Evidence that periderm cells send out cytoneme-like protrusions is solid, and perturbations that affect cytoneme number clearly affect periderm structure and gene expression. However, evidence that these effects are directly due to cytoneme mediated-Notch signaling is incomplete.

Reviewer #1 (Public Review):

Summary:

In this paper, Wang et al show that differentiated peridermal cells of the zebrafish epidermis extend cytoneme-like protrusions toward the less differentiated, intermediate layer below. They present evidence that expression of a dominant-negative cdc42, inhibits cytoneme formation and leads to elevated expression of a marker of undifferentiated keratinocytes, krtt1c19e, in the periderm layer. Data is presented suggesting the involvement of Delta-Notch signaling in keratinocyte differentiation. Finally, changes in expression of the inflammatory cytokine IL-17 and its receptors is shown to affect cytoneme number and periderm structure in a manner similar to Notch and cdc42 perturbations.

Strengths:

Overall, the idea that differentiated cells signal to underlying undifferentiated cells via membrane protrusions in skin keratinocytes is interesting and novel, and it is clear that periderm cells send out thin membrane protrusions that contain a Notch ligand. Further, perturbations that affect cytoneme number, Notch signaling, and IL-17 expression clearly lead to changes in periderm structure and gene expression.

Weaknesses:

More work is needed to determine whether the effects on keratinocyte differentiation are due to a loss of cytonemes themselves, or to broader effects of inhibiting cdc42. Moreover, more evidence is needed to support the claim that periderm cytonemes deliver Delta ligands to induce Notch signaling below. Without these aspects of the study being solidified, understanding how IL-17 affects these processes seems premature.

Reviewer #2 (Public Review):

Summary:

The aim of the study was to understand how cells of the skin communicate across dermal layers. The research group has previously demonstrated that cellular connections called airinemes contribute to this communication. The current work builds upon this knowledge by showing that differentiated keratinocytes also use cytonemes, specialized signaling filopodia, to communicate with undifferentiated keratinocytes. They show that cytonemes are the more abundant type of cellular extension used for communication between the differentiated keratinocyte layer and the undifferentiated keratinocytes. Disruption of cytoneme formation led to the expansion of the undifferentiated keratinocytes into the periderm, mimicking skin diseases like psoriasis. The authors go on to show that disruption of cytonemes results in perturbations in Notch signaling between the differentiated keratinocytes of the periderm and the underlying proliferating undifferentiated keratinocytes. Further, the authors show that Interleukin-17, also known to drive psoriasis, can restrict the formation of periderm cytonemes, possibly through the inhibition of Cdc42 expression. This work suggests that cytoneme-mediated Notch signaling plays a central role in normal epidermal regulation. The authors propose that disruption of cytoneme function may be an underlying cause of various human skin diseases.

Strengths:

The authors provide strong evidence that periderm keratinocytes cytonemes contain the notch ligand DeltaC to promote Notch activation in the underlying intermediate layer to regulate accurate epidermal maintenance.

Weaknesses:

The impact of the study would be increased if the mechanism by which Interlukin-17 and Cdc42 collaborate to regulate cytonemes was defined. Experiments measuring Cdc42 activity, rather than just measuring expression, would strengthen the conclusions.

Reviewer #3 (Public Review):

Summary:

Leveraging zebra fish as a research model, Wang et al identified "cytoneme-like structures" as a mechanism for mediating cell-cell communications among skin epidermal cells. The authors further demonstrated that the "cytoneme-like structures" can mediate Notch signaling, and the "cytoneme-like structures" are influenced by IL17 signaling.

Strengths:

Elegant zebrafish genetics, reporters, and live imaging.

Weaknesses: (minor)<br /> This paper focused on characterizing the "cytoneme-like structures" between different layers and the NOTCH signaling. However, these "cytoneme-like structures" observed in undifferentiated KC (Figure 2B), although at a slightly lower frequency, were not interpreted. In addition, it is unclear if these "cytoneme-like structures" can mediate other signaling pathways than NOTCH.

Overall, this is a solid paper with convincing data reporting the "cytoneme-like structures" in vivo, and with compelling data demonstrating the roles in NOTCH signaling and the regulation by IL17.

These findings provide a foundation for future work exploring the "cytoneme-like structures" in the mammalian system and other epithelial tissue types. This paper also suggests a potential connection between the "cytoneme-like structures" and psoriasis, which needs to be further explored in clinical samples.

eLife assessment

In this important study, Li and others identified cell membrane receptors for juvenile hormone (JH), a terpenoid hormone in insects that regulates their development and reproduction. While intracellular receptors for JH have been well characterized, membrane receptors for JH have remained elusive. Although the authors provide convincing evidence to indicate that the receptor tyrosine kinases they identified bind to JH in vitro and induce responses in cultured cells, their loss-of-function phenotypes are not consistent with known JH functions, leaving obscure the physiological roles of these receptors in mediating in vivo JH function.

Reviewer #1 (Public Review):

Summary:

Juvenile Hormone (JH) plays a key role in insect development and physiology. Although the intracellular receptor for JH was identified long ago, a number of studies have shown that part of JH functions should be fulfilled through binding to an unknown membrane receptor, which was proposed to belong to the RTK family. In this study, the authors screened all RTKs from the H. armigera genome for their ability to mediate responses to JH III treatment both in cultured cells and in developing animals. They also present convincing evidence that CAD96CA and FGFR1 directly bind JH III, and that their role might be conserved in other insect species.

Strengths:

Altogether, the experimental approach is very complete and elegant, providing evidence for the role of CAD96CA and FGFR1 in JH signalling using different techniques and in different contexts. I believe that this work will open new perspectives to study the role of JH and better understand what is the contribution of signalling through membrane receptors for JH-dependent developmental processes.

Weaknesses:

I don't see major weaknesses in this study. However, I think that the manuscript would benefit from further information or discussion regarding the relationship between the two newly identified receptors. Experiments (especially in HEK-293T cells) suggest that CAD96CA and FGFR1 are sufficient on their own to transduce JH signalling. However, they are also necessary since loss-of-function conditions for each of them are sufficient to trigger strong effects (while the other is supposed to be still present).

In addition, despite showing different expression patterns, the two receptors seem to display similar developmental functions according to loss-of-function phenotypes. It is therefore unclear how to draw a model for membrane receptor-mediated JH signalling that includes both CAD96CA and FGFR1.

Reviewer #2 (Public Review):

Summary:

Juvenile hormone (JH) is a pleiotropic terpenoid hormone in insects that mainly regulates their development and reproduction. In particular, its developmental functions are described as the "status quo" action, as its presence in the hemolymph (the insect blood) prevents metamorphosis-initiating effects of ecdysone, another important hormone in insect development, and maintains the juvenile status of insects.

While such canonical functions of JH are known to be mediated by its intracellular receptor complex composed of Met and Tai, there have been multiple reports suggesting the presence of cell membrane receptor(s) for JH, which mediate non-genomic effects of this terpenoid hormone. In particular, the presence of receptor tyrosine kinase(s) that phosphorylate Met/Tai in response to JH and thus indirectly affect the canonical JH signaling pathway has been strongly suggested. Given the importance of JH in insect physiology and the fact that the JH signaling pathway is a major target of insect growth regulators, elucidating the identification and functions of putative JH membrane receptors is of great significance from both basic and applied perspectives.

In the present study, the authors identified candidate receptors for such cell membrane JH receptors, CAD96CA and FGFR1, in the cotton bollworm Helicoverpa armigera.

Strengths:

Their in vitro analyses are conducted thoroughly using multiple methods, which overall supports their claim that these receptors can bind to JH and mediate their non-genomic effects.

Weaknesses:

Results of their in vivo experiments, particularly those of their loss-of-function analyses using CRISPR mutants are still preliminary, and the results rather indicate that these membrane receptors do not have any physiologically significant roles in vivo. More specifically, previous studies in lepidopteran species have clearly and repeatedly shown that precocious metamorphosis is the hallmark phenotype for all JH signaling-deficient larvae. In contrast, the present study showed that Cad96ca and Fgfr1 G0 mutants only showed a slight acceleration in their pupation timing, which is not a typical phenotype one would expect from JH signaling deficiency. This is inconsistent with their working model provided in Figure 6, which indicates that these cell membrane JH receptors promote the canonical JH signaling by phosphorylating Met/Tai.

If the authors argue that this slight acceleration of pupation is indeed a major JH signaling-deficient phenotype in Helicoverpa, they need to provide more data to support their claim by analyzing CRISPR mutants of other genes involved in JH signaling, such as Jhamt and Met. An alternative explanation is that there is functional redundancy between CAD96CA and FGFR1 in mediating phosphorylation of Met/Tai. This possibility can be tested by analyzing double knockouts of these two receptors.

Currently, the validity of their calcium imaging analysis in Figure 5 is also questionable. When performing calcium imaging in cultured cells, it is critically important to treat all the cells at the end of each experiment with a hormone or other chemical reagents that universally induce calcium increase in each particular cell line. Without such positive control, the validity of calcium imaging data remains unknown, and readers cannot properly evaluate their results.

Reviewer #3 (Public Review):

Summary:

In this study, Li et al. identified CAD96CA and FGF1 among 20 receptor tyrosine kinase receptors as mediators of JH signaling. By performing a screen in HaEpi cells with overactivated JH signaling, the authors pinpointed two main RTKs that contribute to the transduction of JH. Using the CRISPR/Cas9 system to generate mutants, the authors confirmed that these RTKs are required for normal JH activation, as precocious pupariation was observed in their absence. Additionally, the authors demonstrated that both CAD96CA and FGF1 exhibit a high affinity for JH, and their activation is necessary for the proper phosphorylation of Tai and Met, transcription factors that promote the transcriptional response. Finally, the authors provided evidence suggesting that the function of CAD96CA and FGF1 as JH receptors is conserved across insects.

Strengths:

The data provided by the authors are convincing and support the main conclusions of the study, providing ample evidence to demonstrate that phosphorylation of the transducers Met and Tai mainly depends on the activity of two RTKs. Additionally, the binding assays conducted by the authors support the function of CAD96CA and FGF1 as membrane receptors of JH. The study's results validate, at least in H. amigera, the predicted existence of membrane receptors for JH.

Weaknesses:

The study has several weaknesses that need to be addressed. Firstly, it is not clear what criteria were used by the authors to discard several other RTKs that were identified as repressors of JH signaling. For example, while NRK and Wsck may not fulfill all the requirements to become JH receptors, other evidence, such as depletion analysis and target gene expression, suggests they are involved in proper JH signaling activation.

Secondly, the expression of the six RTKs, which, when knocked down, were able to revert JH signaling activation, was mainly detected in the last larval stage of H. amigera. However, since JH signaling is active throughout larval development, it is unclear whether these RTKs are completely required for pathway activation or only needed for high activation levels at the last larval stage.<br /> Additionally, the mechanism by which different RTKs exert their functions in a specific manner is not clear. According to the expression profile of the different RTKs, one might expect some redundant role of those receptors. In fact the no reversion of phosphorilation of tai and met upon depletion of Wsck in cells with overactivated JH signalling seems to support this idea.

Nevertheless, and despite the overlapping expression of the different receptors, all RTKs seem to be required for proper pathway activation, even in the case of FGF1 which seems to be only expressed in the midgut. This is an intriguing point unresolved in the study.

Finally, the study does not explain how RTKs with known ligands could also bind JH and contribute to JH signaling activation. in Drosophila, FGF1 is activated by pyramus and thisbe for mesoderm development, while CAD96CA is activated by collagen during wound healing. Now the authors claim that in addition to these ligands, the receptors also bind to JH. However, it is unclear whether these RTKs are activated by JH independently of their known ligands, suggesting a specific binding site for JH, or if they are only induced by JH activation when those ligands are present in a synergistic manner. Alternatively, another explanation could be that the RTK pathways by their known ligands activation may induce certain levels of JH transducer phosphorylation, which, in the presence of JH, contributes to the full pathway activation without JH-RTK binding being necessary.

什么意思？不能用32bit的访问方式吗？

A person of wide knowledge or learning

Die Übernahme von Anglo American durch die BHP-Gruppe ist vorerst gescheitert. Die Knappheit an Rohstoffen aufgrund der Energiewende betrifft Kupfer am stärksten. Deshalb sind weitere Übernahmeversuche bei Bergbaukonzernen zu erwarten. Die Gesellschaften versuchen sich vorhandene Minen zu sichern, um möglichst wenig in neue Bergwerke zu investieren. Hintergrundbericht in der Repubblica. https://www.repubblica.it/economia/2024/05/13/news/prezzo_rame_miniere_bhp_anglo_american_fusioni-422904602/

Die Kupferpreise müssen um 20% steigen, um Investitionen in neue Kupferminen attraktiv zu machen.

ytn

The section highlights the pervasive issue of harassment, now amplified by social media. It raises concerns about the ease with which individuals can exploit technology to invade others' privacy and safety, making it a significant modern problem that demands more robust protective measures.

These methods underscore the complex and insidious ways in which social media can be weaponized against individuals. Addressing these issues requires comprehensive solutions that involve legal, social, and technological responses to protect individuals and maintain the integrity of online spaces.

Cyberbullying is very common in our daily lives. People post mean messages to others to make them feel bad and hurt, gaining satisfaction through this process. However, these actions should be banned. People might resort to extreme actions if they are deeply hurt. Intentionally hurting someone should be condemned, and individuals should face the consequences of their actions.

to - youtube - https://hyp.is/tw84PBKAEe-8zL-2G5dN0Q/docdrop.org/video/9g6w2f3ttMM/

for - post comment - Linked I - Daniel Schmachtenberger - why good people comply with evil - direct citizen action - networked commons

summary - A great short video that is a teaser to a longer podcast conversation on the topic of confirmation bias, and recognizing it to empower citizens during this time of rapid whole system change.

Die amerikanische Behörde zur energieregulation Regulierung hat die Bestimmung für Stromnetze radikal reformiert um die Produktion erneuerbarer Energien zu fördern. Unter anderem müssen Netzbetreiber für den voraussichtlichen Bedarf in 20 Jahren planen. Einer neu einen neuen Bericht zufolge werden 50% der positiven Effekte des Inflation reduction Act für die Senkung der Emissionen verloren gehen wenn die amerikanischen Stromnetze nicht grundsätzlich gründlich überholt werden. https://www.nytimes.com/2024/05/13/climate/electric-grid-overhaul-ferc.html?region=BELOW_MAIN_CONTENT&block=storyline_flex_guide_recirc&name=styln-climate&variant=show&pgtype=Article

Matt Stoller denies racism is why Democrats lost the South.

.meta.doHow - development on the margins - continue with the practice of articulating on the margin - start to develop on the margins, the software needed to creat & morph annotations

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.real.soon - intent to be able to process annotations

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.card - title:'IPFS Fetch - Mauve - YouTube' .- src: 'IPFS Fetch - Mauve - YouTube' - stub: - talking about ipfs fetch: - how i've combined some of the usual webby interfaces we're used to with some of these cool peer-to-peer interface

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Luddites, a group of 19th-century English textile workers who resisted the adoption of new machinery that they believed threatened their jobs. Between 1811 and 1817, in Nottingham and later in areas like the North West and Yorkshire, these workers, named after the mythical figure Ned Ludd, engaged in destroying machinery as a form of protest. They aimed to protect their livelihoods against what they saw as the use of machines to undercut skilled labor and reduce wages. The movement was eventually suppressed through legal and military action. The term "Luddite" has since evolved to describe someone who opposes industrialization, automation, or new technologies.

How Social Media Works is lile recognizing the mechanisms behind content distribution, including algorithms that dictate what you see based on user engagement and preferences. Emotional and Mental Impact is like being aware of how content can influence your feelings and mental state, whether through positive reinforcement or negative interactions. Data Privacy and Security is like knowing how your data is collected, used, and potentially misused by platforms or third parties, which is crucial for protecting your personal information.

Das EU-Parlament hat einer Aufweichung der wichtigsten Bestimmungen zum Schutz der Biodiversität als Voraussetzung für Agrarsubventionen zugestimmt. Greenpeace bezeichnet die Entscheidung als schockierend. 13% der in Deutschland imitierten Treibhausgase stammen laut Umweltbundesamt aus der Landwirtschaft. https://taz.de/Rollback-bei-EU-Agrarsubventionen/!6003674/

Timmy Facciola Twitter bio The Intercept, New York Times, New York Magazine, Time Union (New York), Commonwealth Magazine, Boston College newspaper (The Heights), The River Hudson Valley Newsroom.

Hernán Cortés, born in Medellín, Spain in 1485, was a Spanish conquistador known for leading the expedition that led to the fall of the Aztec Empire in the early 16th century. As a young man from a family of lesser nobility, Cortés sought adventure and fortune in the New World, initially settling in Hispaniola and later in Cuba where he founded the second Spanish town and received an encomienda. In 1519, he led a daring expedition to mainland Mexico, defying orders from his superiors, and through a combination of military strategy, alliances with local indigenous groups, and the use of native translator Malinche (Doña Marina), he was able to capture the Aztec capital, Tenochtitlan. Following his conquest, Cortés was appointed as the first Governor of New Spain, leaving a complex legacy marked by significant territorial expansion for Spain but also the dramatic decline of indigenous populations and cultures.

The decision-making and strategic directions of social media platforms are significantly influenced by capitalism. These platforms often make decisions that prioritize profitability and market dominance, sometimes at the expense of user experience or ethical considerations. This can lead to practices that may harm users or alienate them, as the primary aim is often to maximize returns for shareholders rather than focusing solely on user satisfaction.

Die EU verzichtet auf die wichtigsten bisher vorgesehenen Umweltauflagen für die Landwirtschaft, vor allem auf die Verpflichtung zur Vergrößerung der Brachflächen Punkt der deutsche Umweltminister özdemir leistete dagegen kein Widerstand. https://taz.de/Agrarsubventionen-fuer-Bauern/!6007450/

I.e., identity system should be ledger-agnostic.

John Hudson Twitter account profile diplomacy & national security for Washington Post

Eine neue Studie zeigt, dass nur wenige Baumarten sowohl den klimatischen Bedingungen der Gegenwart wie den zu erwartenden und 2100 gewachsen sind. Von den gängigsten 69 Baumarten können nur neun pro Quadratmeter unter den zukünftigen Bedingungen überleben was aufforstungsprojekte viel mehr erschwert als bisher angenommen. Bei dieser Studie werden allerdings die Interaktionen zwischen den Bäumen und eine dadurch möglicherweise vergrößerte Anpassungsfähigkeit nicht berücksichtigt. https://www.liberation.fr/environnement/biodiversite/forets-quels-arbres-planter-pour-quils-resistent-au-climat-daujourdhui-mais-aussi-a-celui-de-2100-20240515_XZHQZ32BNJALHMOVJEOAKOU4WQ/

Face refers to a set of behaviors and customs related to the morality, honor, and authority of an individual or group, playing a significant role in their social image and interactions. the concept of face is linked to the dignity and prestige that a person holds within their social relationships. It holds a particularly profound significance in Chinese culture.

The handling of these emotions is crucial in shaping children's understanding of personal responsibility and integrity, guiding them towards mature emotional responses that separate their worth from their actions. This process underscores the developmental journey from reacting with shame to responding with guilt, facilitated by supportive parenting that emphasizes correction and reassurance rather than rejection.

2050 werden vulnerable Gruppen, vor allem Menschen über 60 Jahren, doppelt so vielen Tagen mit extremer Hitze ausgesetzt sein wie in der vorindustriellen Zeit.

Temperaturdaten aus der Messung von Baumringen zeigen, dass der Sommer 2023 in den nicht tropischen Gebieten der Nordhalbkugel eindeutig der heißeste Sommer seit mindestens 2000 Jahren war. Einer neuen Studie von forschenden um Jan esper zu Folge lagen die Temperaturen um 2 Grad über dem vorindustriellen Mittel, nicht nur, wie vom europäischen Wetterdienst Kopernikus publiziert, um 1,5 Grad https://www.derstandard.at/story/3000000219984/schnellster-co2-anstieg-in-der-luft-seit-50000-jahren

Die Studie einer Gruppe um Kathleen Wendt aufgrund von Eisbohrkernen ergibt, dass der CO2 Anstieg in den vergangenen Jahrzehnten zehnmal so schnell war wie in den 50.000 Jahren davor.

Transparent Peer Review

Download the complete Review Process [PDF] including:

• reviews
• authors' reply
• editorial decisions

</br>

I have to agree with this statement. From first glance the paintings did not look like much nor do I understand in detail what the picture is about or trying to convey. Per the text however, once you look deeper into the story unfolding you get a better understanding of what is actually going on. It wasn’t until I went back to review that I actually was able to identify and analyze some of the things pointed out in the text I may have overlooked at first glance. For example, the use of elephants, especially with the materials on the elephant can help us in realizing that the picture must take place in or around India. Also if we analyze further we can see that there are people using materials to build something while others apparently have the role of watching over the workers. This may suggest that the workers are slaves from India working on the Great Mosque of Samarqand (the description of the painting).

named and amenable to the put to be interpreted in specific context/environment dependent way

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title

Editors Assessment:

This work is part of a series of papers from the Hong Kong Biodiversity Genomics Consortium sequencing the rich biodiversity of species in Hong Kong. This example This work is part of a series of papers from the Hong Kong Biodiversity Genomics Consortium sequencing the rich biodiversity of species in Hong Kong. This example presenting the first whole genome assembly of Dacryopinax spathularia, an edible mushroom-forming fungus that is used in the food industry to produce natural preservatives. Using PacBio and Omni-C data a 29.2 Mb genome was assembled, with a scaffold N50 of 1.925 Mb and 92.0% BUSCO score demonstrating the quality (review pushing the authors to provide more detail and QC stats to help better convince on this). This data providing a useful resource for further phylogenomic studies in the family Dacrymycetaceae and investigations on the biosynthesis of glycolipids with potential applications in the food industry.

This evaluation refers to version 1 of the preprint

This work has been published in GigaByte Journal under a CC-BY 4.0 license (https://doi.org/10.46471/gigabyte.120), and has published the reviews under the same license. This is part of a thematic series presenting Data Releases from the Hong Kong Biodiversity Genomics consortium (https://doi.org/10.46471/GIGABYTE_SERIES_0006). These are as follows.

Reviewer 1. Anton Sonnenberg

Is the language of sufficient quality? Yes.

Are all data available and do they match the descriptions in the paper? Yes.

Is the data acquisition clear, complete and methodologically sound? Yes.

Is there sufficient detail in the methods and data-processing steps to allow reproduction? Yes.

Figure 1E could be improved by eliminating in the pie-chart the non-repeat sequences or bar-plot the repeats. That will visualize better the frequencies of each type of repeats.

Reviewer 2. Riccardo Iacovelli

Is the language of sufficient quality? No.

There are several typos spread across the text, and some sentences are written in an unclear manner. I provide some suggestions in the attachment.

Are all data available and do they match the descriptions in the paper?

Yes, but some of the data shown is rather unclear and/or not supported by sufficient explanation. For example, what is actually Fig. 1C showing? Because the reference in the text (which contains a typo, line 197) refers to something else. What is the second set of stats in Fig. 1B? This other organism is not mentioned at all anywhere in the manuscript.

Are the data and metadata consistent with relevant minimum information or reporting standards?

No. NCBI TaxID of the sequenced species object of this work is missing.

Is there sufficient detail in the methods and data-processing steps to allow reproduction?

No. In my opinion, some of the procedures described for the processing of the sample and library prep for sequencing are reported in an unclear way. For example, lines 100-103: no details on RNAse A treatment; how do you define chloroform:IAA (24:1) washes? how much supernatant is added to how much H1 buffer to have the final volume of 6 ml? Another example, lines 180-175: what parameters did you use for EvidenceModeler to generate the final consensus genes model? The weight given to each particular prediction set is important.

Is there sufficient data validation and statistical analyses of data quality?

No/ While sufficient data validation and statistical analyses have been carried out with respect to DNA sequencing and genome assembly, nothing is reported about DNA extraction and quality. The authors mention several times throughout the text that DNA preps are checked via NanoDrop, Qubit, gel electrophoresis, etc. But none of this is shown in the main body or in the supplementary information. Without this information, it is difficult to assess directly the efficacy of DNA extraction and preparation methods. I recommend including this type of data.

Additional Comments:

In this article, the authors report the first whole genome assembly of Dacryopinax spathularia, an edible mushroom-forming fungus that is used in the food industry to produce natural preservatives. In general, I find the data of sufficiently high quality for release, and I do agree with the authors in that it will prove useful to gain further insights into the ecology of the fungus, and to better understand the genetic basis of its ability to decay wood and produce valuable compounds. This can ultimately lead to discoveries with applications in biotech and other industries.

Nevertheless, during the review process I noticed several shortcomings with respect to unclear language, insufficient description of the experimental procedures and/or results presented, and missing data altogether. These are all discussed within the checklist available in the ReView portal. For minor comments line-by-line, see below:

1: Dacrymycetaceae should be italicized (throughout the whole manuscript). This follows the convention established by The International Code of Nomenclature for algae, fungi, and plants (https://www.iaptglobal.org/icn). Although not binding, this allows easy recognition of taxonomic ranks when reading an article. 49: other fungus -> other fungi 56: photodynamic injury -> UV damage/radiation (photodynamic is used with respect to light-activated therapies etc.) 60: in food industry as natural preservatives in soft drinks -> in food industry to produce natural preservatives for soft drinks 68: cultivated in industry as food additives -> cultivated in industry to produce food additives 69: isolated fungal extract -> the isolated fungal extract 71: What do you mean by Pacific? It’s unclear 71-72: the genomic resource -> genomic data/ genome sequence 72: I would remove “with translational values”, it is very vague and does not add anything to the statement 78: genomic resource -> genomic data/ genome sequence 78-81: this could be rephrased in a smoother manner: e.g. something like “the genomic data will be useful to gain a better understanding of the fungus’ ecology as well as the genetic basis of its wood-decaying ability and…” 85: fruit bodies -> fruiting bodies 88-89: Grown hyphae from >2 week-old was transferred  Fungal hyphae from 2-week old colonies were transferred 90-91: validated with the DNA barcode of Translation  assigned by DNA barcoding using the sequence of Translation… 95: ~ -> Approximately (sentences are not usually started with symbols or numbers) 101-3: Procedure is not clear enough (see other comments through ReView portal) 124: for further cleanup the library -> to further clean up the library / for further cleanup of the library 132: as line 95 152: as lines 95, 132 181-5: Insufficient description of methods, see comments through ReView portal 197: Figure and 1C; Table 2 -> Figure 1C and Table 2 200: average protein length of 451 bp -> average protein-coding gene length / average protein length of ~150 amino acids 211: via the fermentation process with applications in the food industry -> via the fermentation process with potential applications in the food industry

As a fungal biologist myself interested in fungal genomics and biotechnology, I would like to thank the authors for carrying out this work and the editor for the opportunity to review it. I am looking forward to reading the revised version of the manuscript.

Riccardo Iacovelli, PhD GRIP, Chemical and Pharmaceutical Biology department University of Groningen, Groningen - The Netherlands

The European Sociery of Clinical Microbiology and Infectious Diseases has authority over this because is an organization that specializes against viruses and bacterias that causes hard to the human body. In this case, there are more than 180 senior scientist, with decades of experience and knowledge, agreeing that the next flu is going to be a virus.

The purpose of the article is to inform and to warn everyone of the possible dangers of a new strain of flu virus. This is very informative and is something to be aware of, especially approaching flu season.

New York Times fails

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Malala's character is defined by her individual experiences and her role as a symbol of hope and inspiration for millions of girls denied access to education. Her narrative culminates in a powerful declaration of her continued advocacy for peace and education.

Using this language the rhetor conveys a tone of seriousness in which we must deal the matter.

In this passage she uses the story of one of her female friends and converts her as the act or purpose as a part of her cause. She discusses how this story inspired her to speak up using the experience of others the rhetor gives us a sense of how she imagines others might feel too.

In this passage the rhetor asserts the agency of the reading. Of what she is fighting for. If we're to apply Burke's rhetorical model to this portion of the speech and make use of the five elements of the pentad, then Malala's story leading to her Nobel Prize Speech she claims the agency - which is her experience to getting shot that allowed her a space to open up and have an area of advocacy.

While Malala does bring to light the suffrages of her and her female friends many critics could believe that she highlighted in in a way that focused on superiority rather than equality. This article focuses in the way that Malala's take on feminism was controversial. Malala's Take on Feminism

Critics might also point out that Malala's story represents only one narrative of feminism, primarily focused on education and empowerment, which might not fully capture the experiences and struggles of all women, particularly those from marginalized communities or different cultural contexts.

This image is a clear visual demonstration of the scene that Malala was placed in. As discussed previously stated the scene includes an insight in which Malala's activism took a turn. Malala's environment was dangerous and under a circumstance that hadn't allowed her to receive education without the danger. She was one who suffered the consequences of simply having the privilege to be educated and living where she does. This image not only speaks volumes about what she with through but also evokes ethos. Allowing us the audience to feel for her and only imagine what time of pain other women go through.

I feel that seeing these behaviors as part of the culture may also require a deeper analysis. These behaviors may be more accurately described as manifestations of political or social structural problems rather than purely cultural phenomena. Culture usually refers to the values, beliefs, customs and expressions shared by a group of people, and the use of "culture" in this sentence may be too broad.

If a person were fleeing slavery,they would have been running to save their life or the life/lives of their offspring. What kind of people could not only enact these types of laws but also support them as well.

I think that this is disgusting! How dare these people think it is okay to exclude everyone who is not a "free white male" who is 21 and pay no attention to the indigenous persons who were already living on the land. What about the women or the widows?

I think that as more people speak up and push for a better education there will be a change to the curriculum that helps all students. However, I think that many fear that they will be the only ones who speak up. when this is not actually the case as many people do want a change in education for the better of our future.

I very much agree that these labels create a sense of lack when these students really do excel in specific areas of their lives. Instead, there should be a focus on the abilities that they do have because of what they have been through. I am also sure that growing up it may be tough to constantly hear these labels throughout education.

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It is disappointing that parents have to pay this much money to get a fulfilling education for their children. This shows how more money needs to be spent on these aspects of education that special needs children need support with. Especially if some are coming from low-income areas where they can not afford to find the right facilities.

This is something that can be quite concerning as we need to make sure that all students are receiving the resources and gu8idnace that they require. This shows that students may continue to be behind because their progress is not being continuously monitored. Thus, making us question where the funding is truly going to.

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This sentence really reiterates the importance of funding and how these issues could be solved through better resources and the hiring of more minority groups. As these minority groups will help students feel more confident in their abilities. Overall, more funding is what can solve the lack of resources and better become an accessible space for all students in education.

This is extremely disheartening to think about as students who are probably misplaced into these groups are missing out on living life at their fullest potential. As people with special needs or disabilities already have a tough time with higher ed. Being a minority and being misplaced into that group would cause even greater hurdles. This is why students should be better evaluated and race should never play a factor in these situations.

Because altering frequency, duration, or setting might severely impact the efficacy of a service or support, i

wow interesting I never thought of this

Failing to include all of a student’s educational needs in the PLAAFP:

is there a list somewhere that explains what falls under "educational need"

inally, IEP teams should not make placements based on factors such as disability category, the availability of educational or related services, or administrative convenience (i.e., logistics, space, or budgetary considerations).

while I totally agree, I feel like there isn't enough funding or something because resource teachers are already overworked-- at least that seems to be what I here.

A thorough evaluation should include vision and hearing screenings, even though the primary area of concern is academics.

so could we include depression/anxiety screenings?>

the evaluation must address all areas of student need, even if the area does not seem to be related to a student’s suspected disability.

including social emotional? I feel like this part is almost never on IEPs and could be greatly used.

IDEA specifies the required members of a properly constituted IEP team

this usually consists of LEA, classroom teacher, Sped teacher, School psychologist, parent,-- sometimes OT and Speech are included.

Procedural Errors • Predetermining a student’s placement or services:

I feel like I hear the comment, "They qualify" or "they don't qualify" before IEPs ever take place.

Can parents insist on their child being on an IEP even if testing doesn't determine qualification?

A procedural error that may occur at any point during the IEP process is failure to involve parents

I have 3 of my own children on IEPs. How come I am not notified in advance the kind of testing that is to be done and what it is for?

Usually I just show up to the IEP meeting they they say, well we did this testing -- I may or may not understand what it was for or why it was given.

Also, can parents suggest different tests? Like why can't my school do a dyslexia screener?

School administrators play a critical role in ensuring the development and implementation of high-quality individualized education programs

it isn't until the last couple of year that I have seen people other than the special education teacher play a role in creating IEPs.

The school I am at now has a team that discusses what students need as far as interventions before students are ever recommended for IEP testing. Admin are included in this meeting. That said, I've never had my principal make any kind of suggestion, although they have helped to facilitate discussion on how to help.

I think the team is called the intervention team.

What if we include all except financial experts in the creation of sustainability standards?

Including a diverse range of experts, excluding only financial experts, can lead to more comprehensive and balanced sustainability standards. Here are some potential benefits:

1. Holistic Perspective:

   • Non-financial experts (e.g., environmentalists, social scientists, ethicists) bring unique viewpoints.
   • They consider ecological, social, and ethical aspects beyond profit.

   • Addressing Externalities:

   • Non-financial experts focus on externalities (impacts beyond financial metrics).

   • Including them ensures standards account for broader consequences.

   • Long-Term Vision:

   • Sustainability requires long-term thinking.

   • Non-financial experts emphasize intergenerational equity and planetary health.

   • Transparency and Trust:

   • Involving diverse experts builds trust.

   • Transparency in standard-setting enhances credibility.

Remember, collaboration among experts from various fields fosters robust and inclusive sustainability standards

DOI: 10.1016/j.celrep.2021.108887

Resource: RRID:Addgene_12259

Curator: @Naa003

SciCrunch record: RRID:Addgene_12259

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DOI: 10.1016/j.celrep.2021.108896

Resource: (BD Biosciences Cat# 559565, RRID:AB_397274)

Curator: @Naa003

SciCrunch record: RRID:AB_397274

What is this?

DOI: 10.1016/j.isci.2024.109691

Resource: MATLAB (RRID:SCR_001622)

Curator: @evieth

SciCrunch record: RRID:SCR_001622

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DOI: 10.17179/excli2023-6878

Resource: (RCB Cat# RCB1189, RRID:CVCL_0006)

Curator: @evieth

SciCrunch record: RRID:CVCL_0006

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DOI: 10.1016/j.isci.2024.109747

Resource: (IMSR Cat# JAX_007914,RRID:IMSR_JAX:007914)

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DOI: 10.1016/j.isci.2024.109747

Resource: (IMSR Cat# JAX_008069,RRID:IMSR_JAX:008069)

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DOI: 10.1016/j.isci.2024.109747

Resource: (IMSR Cat# JAX_003025,RRID:IMSR_JAX:003025)

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DOI: 10.1016/j.isci.2024.109747

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DOI: 10.1016/j.isci.2024.109747

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DOI: 10.1016/j.isci.2024.109747

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DOI: 10.1016/j.isci.2024.109747

Resource: (IMSR Cat# JAX_017482,RRID:IMSR_JAX:017482)

Curator: @evieth

SciCrunch record: RRID:IMSR_JAX:017482

What is this?

DOI: 10.3390/cancers16091664

Resource: (BCRJ Cat# 0223, RRID:CVCL_0019)

Curator: @evieth

SciCrunch record: RRID:CVCL_0019

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DOI: 10.3390/cancers16091664

Resource: (KCB Cat# KCB 2011100YJ, RRID:CVCL_0346)

Curator: @evieth

SciCrunch record: RRID:CVCL_0346

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DOI: 10.3390/diagnostics14090914

Resource: IBM SPSS Statistics (RRID:SCR_016479)

Curator: @evieth

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DOI: 10.3389/fimmu.2024.1355130

Resource: (Thermo Fisher Scientific Cat# MA1-10346, RRID:AB_11154825)

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SciCrunch record: RRID:AB_11154825

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DOI: 10.3389/fimmu.2024.1355130

Resource: (BioLegend Cat# 103044, RRID:AB_2650923)

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SciCrunch record: RRID:AB_2650923

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DOI: 10.3389/fimmu.2024.1355130

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DOI: 10.3389/fimmu.2024.1355130

Resource: (BioLegend Cat# 324212, RRID:AB_756086)

Curator: @evieth

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What is this?

DOI: 10.3389/fcell.2023.1301913

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @maulamb

SciCrunch record: RRID:SCR_006457

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DOI: 10.3389/fcell.2023.1301913

Resource: (BDSC Cat# 32860,RRID:BDSC_32860)

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SciCrunch record: RRID:BDSC_32860

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