Reviewer #1 (Public Review):

Summary:

In this study, a chromosome-level genome of the rose-grain aphid M. dirhodum was assembled with high quality, and A-to-I RNA-editing sites were systematically identified. The authors then demonstrated that: 1) Wing dimorphism induced by crowding in M. dirhodum is regulated by 20E (ecdysone signaling pathway); 2) an A-to-I RNA editing prevents the binding of miR-3036-5p to CYP18A1 (the enzyme required for 20E degradation), thus elevating CYP18A1 expression, decreasing 20E titer, and finally regulating the wing dimorphism of offspring.

Strengths:

The authors present both genome and A-to-I RNA editing data. An interesting finding is that a A-to-I RNA editing site in CYP18A1 ruin the miRNA binding site of miR-3036-5p. And loss of miR-3036-5p regulation lead to less 20E and winged offspring.

Weaknesses:

How crowding represses the miR-3036-5p is still unclear.

Reviewer #2 (Public Review):

Summary:

Environmental influences on development are ubiquitous, affecting many phenotypes in organisms. However molecular genetic and cellular mechanisms transducing environmental signals are still only barely understood. This study examines part of one such intracellular mechanism in a polyphenic (or dimorphic) aphid.

Strengths:

While other published reports have linked phenotypic plasticity to RNA editing before, this study reports such an interaction in insects. The study uses a wide array of molecular tools to identify connections upstream and downstream of the RNA editing to elucidate the regulatory mechanism, which is illuminating.

Weaknesses:

While this system is intriguing, this report does not foster confidence in its conclusions. Many of the analyses seem based on very small sample sizes. It is itself problematic that sample sizes are not obvious in most figures, although based on Methods section covering RNAseq, they seem to be either 3, 6 or 9, depending on whether stages were pooled, but that point is not made clear. With such small sample sizes, statistical tests of any kind are unreliable. Besides the ambiguity on sample sizes, it's unclear what error bars or whiskers show in plots throughout this study. When sample sizes are small estimates of variance are not reliable. Student's t-test is not appropriate for comparisons with such small sample sizes. Presently, it is not possible to replicate the tests shown in Figures 3, 4 and 6. (Besides the HT-seq reads, other data should also be made publicly available, following the journal's recommendations.) Regardless, effect sizes in some comparisons (Fig 3J, 4A-C, 6E,H) are clearly not large, making confidence in conclusions low. The authors should be cautious about over-interpreting these data.

eLife assessment

This study presents an important finding on the molecular mechanism for transduction of environmentally induced polyphenism. The evidence supporting the claims of the author is incomplete due to limited sample sizes and inadequate analysis. This paper would be of interest to those studying aphids wing dimorphism.

Reviewer #1 (Public Review):

This manuscript presents an extremely exciting and very timely analysis of the role that the nucleosome acidic patch plays in SWR1-catalyzed histone exchange. Intriguingly, SWR1 loses activity almost completely if any of the acidic patches are absent. To my knowledge, this makes SWR1 the first remodeler with such a unique and pronounced requirement for the acidic patch. The authors demonstrate that SWR1 affinity is dramatically reduced if at least one of the acidic patches is absent, pointing to a key role of the acidic patch in SWR1 binding to the nucleosome. The authors also pinpoint a specific subunit - Swc5 - that can bind nucleosomes and engage the acidic patch and obtain a cryo-EM structure of Swc5 bound to a nucleosome. They also identify a conserved arginine-rich motif in this subunit that is critical for nucleosome binding and histone exchange in vitro and for SWR1 function in vivo. The authors provide evidence that suggests a direct interaction between this motif and the acidic patch.

Strengths:

The manuscript is well-written and the experimental data are of outstanding quality and importance for the field. This manuscript significantly expands our understanding of the fundamentally important and complex process of H2A.Z deposition by SWR1 and would be of great interest for a broad readership.

eLife assessment

This manuscript presents an important analysis of the role that the nucleosome acidic patch plays in SWR1-catalyzed histone exchange. This manuscript contains convincing data which significantly expands our understanding of the complex process of H2A.Z deposition by SWR1 and therefore would be of interest to a broad readership.

eLife assessment

This study presents important new insights linking obesity to kidney disease using a Drosophila model. A series of compelling experiments demonstrated that a high-fat diet induces the excretion of a leptin-like JAK-STAT ligand from the fat body, driving the adipose-nephrocyte axis through activated JAK-STAT signaling and subsequently causing a functional defect in nephrocytes. While the combination of genetic tools and pharmacological intervention provides solid data and confirms the mechanistic link, the phenotypic analysis is restricted to tracer endocytosis and would benefit from immunofluorescence studies and higher animal numbers.

Reviewer #1 (Public Review):

Summary:

Zhao and colleagues employ Drosophila nephrocytes as a model to investigate the effects of a high-fat diet on these podocyte-like cells. Through a highly focused analysis, they initially confirm previous research in their hands demonstrating impaired nephrocyte function and move on to observe the mislocalization of a slit diaphragm-associated protein (pyd). Employing a reporter construct, they identify the activation of the JAK/STAT signaling pathway in nephrocytes. Subsequently, the authors demonstrate the involvement of this pathway in nephrocyte function from multiple angles, using a gain-of-function construct, silencing of an inhibitor, and ectopic overexpression of a ligand. Silencing the effector Stat92E via RNAi or inhibiting JAK/STAT with Methotrexate effectively restored impaired nephrocyte function induced by a high-fat diet, while showing no impact under normal dietary conditions.

Strengths:

The findings establish a link between JAK/STAT activity and the impact of a high-fat diet on nephrocytes. This nicely underscores the importance of organ crosstalk for nephrocytes and supports a potential role for JAK/STAT in diabetic nephropathy, as previously suggested by other models.

Weaknesses:

The analysis is overly reliant on tracer endocytosis and single lines. Immunofluorescence of slit diaphragm proteins would provide a more specific assessment of the phenotypes.

Reviewer #2 (Public Review):

Summary:

In their manuscript, Zhao et al. describe a link between JAK-STAT pathway activation in nephrocytes on a high-fat diet. Nephrocytes are the homologs to mammalian podocytes and it has been previously shown, that metabolic syndrome and obesity are associated with worse outcomes for chronic kidney disease. A study from 2021 (Lubojemska et al.) could already confirm a severe nephrocyte phenotype upon feeding Drosophila a high-fat diet and also linking lipid overflow by expressing adipose triglyceride lipase in the fat body to nephrocyte dysfunction. In this study, the authors identified a second pathway and mechanism, how lipid dysregulation impact on nephrocyte function. In detail, they show activation of JAK-STAT signaling in nephrocytes upon feeding them a high-fat diet, which was induced by Upd2 expression (a leptin-like hormone) in the fat body, and the adipose tissue in Drosophila. Further, they could show genetic and pharmacological interventions can reduce JAK-STAT activation and thereby prevent the nephrocyte phenotype in the high-fat diet model.

Strengths:

The strength of this study is the combination of genetic tools and pharmacological intervention to confirm a mechanistic link between the fat body/adipose tissue and nephrocytes. Inter-organ communication is crucial in the development of several diseases, but the underlying mechanisms are only poorly understood. Using Drosophila, it is possible to investigate several players of one pathway, here JAK-STAT. This was done, by investigating the functional role of Hop, Socs36E, and Stat92E in nephrocytes and has also been combined with feeding a high-fat diet, to assess restoration of nephrocyte function by inhibiting JAK-STAT signaling. Adding a translational approach was done by inhibiting JAK-STAT signaling with methotrexate, which also resulted in attenuated nephrocyte dysfunction. Expression of the leptin-like hormone upd2 in the fat body is a good approach to studying inter-organ communication and the impact of other organs/tissue on nephrocyte function and expands their findings from nephrocyte function towards whole animal physiology.

Weaknesses:

Although the general findings of this study are of great interest, there are some weaknesses in the study, which should be addressed. Overall, the number of flies investigated for the majority of the experiments is very low (6 flies) and it is not clear whether the flies used, are from independent experiments to exclude problems with food/diet. For the analysis, the mean values of flies should be calculated, as one fly can be considered a biological replicate, but not all individual cells. By increasing the number of flies investigated, statistical analysis will become more solid. In addition, the morphological assessment is rather preliminary, by only using a Pyd antibody. Duf or Sns should be visualized as well, also the investigation of the different transgenic fly strains studying the importance of JAK-STAT signaling in nephrocytes needs to include a morphological assessment. Moreover, the expected effect of feeding a high-fat diet on nephrocytes needs to be shown (e.g. by lipid droplet formation) and whether upd2 is actually increased here should also be assessed. The time points of assessment vary between 1, 3, and 7 days and should be consistent throughout the study or the authors should describe why they use different time points.

Reviewer #2 (Public Review):

This work deals with a very difficult physical problem: relating the assembly of building blocks on a molecular scale to the appearance of large, macroscopic assemblies. This problem is particularly difficult to treat, because of the large number of units involved, and of the complex way in which these units-monomers-interact with each other and with the solvent. In order to make the problem treatable, the authors recur to a number of approximations: Among these, there is the assumption that the system is spatially homogeneous, i.e., its features are the same in all regions of space. In particular, the homogeneity assumption may not hold in biologically relevant systems such as cells, where the behavior close to the cell membrane may strongly differ from the one in the bulk. As a result, this hypothesis calls for a cautious consideration and interpretation of the results of this work. Another notable simplification introduced by the authors is the assumption that the system can only follow two possible behaviors: In the first, each monomer interacts equally with the solvent; no matter the size of the cluster of which it is part. In the second case, monomers in the bulk of a cluster and monomers at the assembly boundary interact with the solvent in a different way. These two cases are considered not only because they simplify the problem, but also because they are inspired by biologically relevant proteins.

With these simplifications, the authors trace the phase diagram of the system, characterizing its phases for different fractions of the volume occupied by the monomers and solvent, and for different values of the temperature. The results qualitatively reproduce some features observed in recent experiments, such as an anomalous distribution of cluster sizes below the system saturation threshold, and the gelation of condensed phases above such threshold.

eLife assessment

The authors present an important theoretical framework that describes the interplay between liquid-liquid phase separation and protein aggregation within a mean-field model. This work will be of high interest to the biophysics and molecular biology communities, as it will understand and analyse assembly within biomolecular condensates in cells or in-vitro. Major strengths of this convincing work are the consideration of aggregates with various dimensionality and the possibility for protein gelation. A relative weakness is the lack of intuitive interpretation of some of the results and the work could be more accessible to non-experts.

Reviewer #1 (Public Review):

Summary:

The authors present a mean-field model that describes the interplay between (protein) aggregation and phase separation. Different classes of interaction complexity and aggregate dimensionality are considered, both in calculations concerning (equilibrium) phase behavior and kinetics of assembly formation.

Strengths:

The present work is, although purely theoretical, of high interest to understanding biological processes that occur as a result of a coupling between protein aggregation and phase separation. Of course, such processes are abundant, in the living cell as well as in in-vitro experiments. I appreciate the consideration of aggregates with various dimensionality, as well as the categorization into different "interaction classes", together with the mentioning of experimental observations from biology. The model is convincing and underlines the complexity associated with the distribution of proteins across phases and aggregates in the living cell.

Weaknesses:

There are a few minor weaknesses.

Reviewer #3 (Public Review):

Summary:

The authors combine classical theories of phase separation and self-assembly to establish a framework for explaining the coupling between the two phenomena in the context of protein assemblies and condensates. By starting from a mean-field free energy for monomers and assemblies immersed in solvent and imposing conditions of equilibrium, the authors derive phase diagrams indicating how assemblies partition into different condensed phases as temperature and the total volume fraction of proteins are varied. They find that phase separation can promote assembly within the protein-rich phase, providing a potential mechanism for spatial control of assembly. They extend their theory to account for the possibility of gelation. They also create a theory for the kinetics of self-assembly within phase separated systems, predicting how assembly size distributions change with time within the different phases as well as how the volumes of the different phases change with time.

Strengths:

The theoretical framework that the authors present is an interesting marriage of classic theories of phase separation and self-assembly. Its simplicity should make it a powerful general tool for understanding the thermodynamics of assembly coupled to phase separation, and it should provide a useful framework for analyzing experiments on assembly within biomolecular condensates.

The key advance over previous work is that the authors now account for how self-assembly can change the boundaries of the phase diagram.

A second interesting point is the explicit theoretical consideration for the possibility that gelation (i.e. self-assembly into a macroscopic aggregate) could account for widely observed solidification of condensates. While this concept has been broadly discussed, to date I have yet to see a rigorous theoretical analysis of the possibility.

The kinetic theory in sections 5 and 6 is also interesting as it extends on previous work by considering the kinetics of phase separation as well as those of self-assembly.

Weaknesses:

A key point the authors make about their theory is that it allows, as opposed to previous research, to study non-dilute limits. It is true that they consider gelation when the 3D assemblies become macroscopic. However, dilute solution theory assumptions seem to be embedded in many aspects of their theory, and it is not always clear where else the non-dilute limits are considered. Is it in the inter-species interaction \chi_{ij}? Why then do they never explore cases for which \chi_{ij} is nonzero in their analysis?

The connection between this theory and biological systems is described in the introduction but lost along the main text. It would be very helpful to point out, for instance, that the presence of phase separation might induce aggregation of proteins. This point is described formally at the end of Section 3, but a more qualitative connection to biological systems would be very useful here.

Building on the previous point, it would be helpful to give an intuitive sense of where the equations derived in the Appendices and presented in the main text come from and to spell out clear physical interpretations of the results. For example, it would be helpful to point out that Eq. 4 is a form of the law of mass action, familiar from introductory chemistry.

It would be useful to better explain how the current work extends on existing previous work from these authors as well as others. Along these lines, closely related work by W. Jacobs and B. Rogers [O. Hedge et al. 2023, https://arxiv.org/abs/2301.06134; T. Li et al. 2023, https://arxiv.org/abs/2306.13198] should be cited in the introduction.

The results discussed in the first paragraph of Section 3 on assembly size distributions in a homogeneous system are well-known from classic theories of self-assembly. This should be acknowledged and appropriate references should be added; see for instance Rev. Mod. Phys. 93, 025008 and Statistical Thermodynamics Of Surfaces, Interfaces, And Membranes by Sam Safran.

Equation 14 for the kinetic of volume fractions is given with a reference to Bauermann et al 2022, but it should be accompanied by a better intuitive interpretation of its terms in the main text. In particular, how should one understand the third term in this equation? Why does the change in volume impact the change of volume fraction in this way?

The discussion in the last paragraph of Section 6 should be clarified. How can the total amount of protein in both phases decrease? This would necessarily violate either mass or volume conservation. Also, the discussion of why the volume is non-monotonic in time is not clear.

Author response:

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

Reviewer #1 (Public Review):

Summary:

This paper provides a straightforward mechanism of how mycobacterial cAMP level is increased under stressful conditions and shows that the increase is important for the survival of the bacterium in animal hosts. The cAMP level is increased by decreasing the expression of an enzyme that degrades cAMP.

We thank the reviewer for these extremely encouraging comments.

Strengths:

The paper shows that under different stresses the response regulator PhoP represses a phosphodiesterase (PDE) that degrades cAMP specifically. Identification of PhoP as a regulator of cAMP is significant progress in understanding Mtb pathogenesis, as increase in cAMP apparently increases bacterial survival upon infection. On the practical side, reduction of cAMP by increasing PDE can be a means to attenuate the growth of the bacilli. The results have wider implications since PhoP is implicated in controlling diverse mycobacterial stress responses and many bacterial pathogens modulate host cell cAMP level. The results here are straightforward, internally consistent, and of both theoretical and applied interests. The results also open considerable future work, especially how increases in cAMP level help to increase survival of the pathogen.

Weaknesses:

It is not clear whether PhoP-PDE Rv0805 is the only pathway to regulate cAMP level under stress.

Reviewer 1 (Recommendations for the authors):

(1) L.1: "maintenance of" or 'regulating'- I thought change in cAMP level upon stress is the whole point of the paper. Also, can replace "intracellular survival" with 'survival in host macrophages' if you want to be more specific.

We agree with the reviewer, and therefore, we have now replaced “maintenance of” with “regulating cAMP level” in the title. However, we feel more comfortable with “intracellular survival” rather than being more specific with ‘survival in host macrophages’ as we have also shown animal experiments to demonstrate ‘in vivo’ effect in mice lung and spleen.

(2) L.26: ---requires the bacterial virulence regulator –

The suggested change has been made to the text.

(3) L.30: Replace "phoP locus since the" with 'PhoP since this'. (The product, not the locus, is the regulator). The same comment for l.113.

We agree with the reviewer. The suggested changes have been made to the text.

(4) L.31: Change represtsor to repressor.

We are sorry for the embarrassing spelling mistake. We have rectified the mistake in the revised version.

(5) L.32: "hydrolytically degrades" or hydrolyses? (lytic and degrade sound like tautology). Same comment for l.117.

We agree. The suggested change has been made to the text in both places of the revised manuscript.

(6) L.35: I would also suggest changing "intra-mycobacterial" to 'intra bacterial' because you are talking about one bacterium here. The same change is recommended in l.29.

Following reviewer’s recommendation, we have made the changes in the revised manuscript.

(7) L.37: bacillus unless use of the plural form is the norm in the field.

We agree. The suggested change has been made to the text.

(8) L.43: Delete "intracellular" and change "intracellular" to host in l.44.

The suggested changes have been made to the text.

(9) L.66: --that a burst--

We have corrected the mistake in the revised manuscript.

(10) L.76: Receptor or receptor?

We have corrected the mistake in the revised manuscript.

(11) L.86: -- mechanisms of regulation of mycobacterial cAMP level. (homeostasis needs to be introduced first, and not used in the concluding statement for the first time).

The suggested changes have been made to the text.

(12) L.96: "essential" or 'a requirement'. (reduction is not the same as elimination)

We understand the reviewer’s concern. However, several studies have independently established that phoPR remains an essential requirement for mycobacterial virulence.

(13) L.97: Moreover, a mutant

The suggested change has been made to the text.

(14) L.113: --locus since PhoP has been –

The suggested change has been made to the text.

(15) L.119: mechanism or manner? (you are stating a fact, not a mechanism)

We agree. We have now replaced ‘mechanism’ with ‘manner’ in the revised manuscript.

(16) L.130: --lacking copies of both phoP and phoR (I am assuming you don't have two copies of each gene)

We understand the reviewer’s concern. For better clarity, we have now clearly mentioned that the phoPR-KO mutant lacks both the single copies of phoP and phoR genes.

(17) L.156: Indicate why GroEL2? - cells as another cytoplasmic protein, GroEL2 was also undetectable

We have now mentioned it in the secretion experiments that mycobacterial cells did not undergo autolysis. To prove this point, we have used cytoplasmic GroEL2 as a marker protein. The absence of detectable GroEL2 in the culture filtrates (CFs) suggests absence of autolysis. To this end, we have modified the sentence in the revised manuscript (duplicated below):

“Fig. 1C confirms absence of autolysis of mycobacterial cells as GroEL2, a cytoplasmic protein, was undetectable in the culture filtrates (CF).”

(18) L.266: May delete "Together". Start with These data--, which would draw more attention to integrated view. In l.268-270, a reminder that intracellular pH is acidic in the normal course would enhance the physiological significance of the present results.

We agree. We have made the suggested changes to the text. In view of the second comment of the reviewer, we have modified the text (duplicated below):

“These data represent an integrated view of our results suggesting that PhoP-dependant repression of rv0805 regulates intra-mycobacterial cAMP level. In keeping with these results, activated PhoP under acidic pH conditions significantly represses rv0805, and intracellular mycobacteria most likely utilizes a higher level of cAMP to effectively mitigate stress for survival under hostile environment including acidic pH of the phagosome.”

(19) L.272: Delete "and intracellular survival" (?) (I am assuming the survival is due to stress tolerance; also the section talks about stress only). No period in l.273.

Following reviewer’s recommendations, the suggested changes have been made to the text.

(20) L.295: Start the sentence thus: It appears that at least one of ---. (This would put more emphasis on the inference)

We agree. We have now incorporated the recommended changes in the revised version.

(21) L.301: No parenthesis.

The parenthesis has been removed in the revised manuscript.

(22) L.306: Together already implies these. Either delete Together (which I would prefer) or say 'Together, the results suggest that strains expressing wild type and mutant----properties, and the results are

We agree. We have now deleted ‘Together’ in the revised manuscript.

(23) L.311: These results support our view that higher---- (to avoid repetition of l.266)

We agree. We have now incorporated the suggested change in the revised manuscript.

(24) L.316: Using or with?

We think “with” goes well with the statement.

(25) L.329: Rephrase thus: Effect of intra-bacterial cAMP level on in vivo--

The recommended change has been made to the text.

(26) L.333: I would use ~, if you want to indicate about.

We agree. We have now used ‘~’ in the revised version. Changes were incorporated in lines 328, 330 and 333 of the revised manuscript.

(27) L.350: Change "somewhat functionally" to phenotypically?

We thank the reviewer for this suggestion. We have changed “somewhat functionally” to “phenotypically” in the revised manuscript.

(28) L.361: Change "is connected to" to 'regulates'.

The suggested change has been made to the text.

(29) L.365: ACs (to be parallel with PDEs)

We agree. The suggested change has been made to the text.

(30) L.366: delete "very" (let the readers decide how recent from the reference date).

The suggested change has been made to the text.

(31) L.382: level remained unknown before the present study.

The recommended change has been made to the text.

(32) L.399: add at the end of the sentence 'under stress'. Also, represent, not represents.

The recommended changes have been made to the text.

(33) L.560 and 571: Section headings formatted differently from the rest. Similar problem in l.900.

We have rectified the issue and all of the section headings are now formatted in the same style.

Reviewer #2 (Public Review):

Summary:

In the manuscript, the authors have presented new mechanistic details to show how intracellular cAMP levels are maintained linked to the phosphodiesterase enzyme which in turn is controlled by PhoP. Later, they showed the physiological relevance linked to altered cAMP concentrations.

Strengths:

Well thought out experiments. The authors carefully planned the experiments well to uncover the molecular aspects of it diligently.

We thank the reviewer for these extremely encouraging comments.

Weaknesses:

Some fresh queries were made based on the author's previous responses and hope to get satisfactory answers this time.

We provide below a point-by-point response to the fresh queries.

(2) Line 134: please describe the complementation strain features as it is mentioned for the first time (plasmid, copy number, promoter etc.) in the manuscript. Especially under NO stress what could be the authors' justification regarding the high cAMP concentration in the complementation strain?

As recommended by the reviewer, the details of construction of the complemented strain have been incorporated in the 'Materials and Methods' section of the revised manuscript (duplicated below): "To complement phoPR expression, pSM607 containing a 3.6-kb DNA fragment of M. tuberculosis phoPR including 200-bp phoP promoter region, a hygromycin resistance cassette, attP site and the gene encoding phage L5 integrase, as detailed earlier (Walters et al., 2006) was used to transform phoPR mutant to integrate at the L5 attB site.

" To address the reviewer's other concern, we have now included the following sentence in the 'Results' section of the revised manuscript (duplicated below): "A higher cAMP level in the complemented strain under NO stress is possibly attributable to reproducibly higher phoP expression in the complemented mutant under specific stress condition (Khan et al., 2022)."

Reference: Khan et al. (2022) Convergence of two global regulators to coordinate expression of essential virulence determinants of Mycobacterium tuberculosis. eLife 2022, 11:e80965.

New query: The complemented gene (in pSM607 plasmid) becomes a single copy after chromosomal integration, so it should ideally behave like a WT strain. How could authors still justify the high cAMP concentration under NO stress?

We agree with the reviewer. We are unable to provide a cogent justification regarding this result. We speculate that PhoP is strikingly activated under NO stress by a non-canonical mechanism and strongly represses rv0805 expression. As a result, there is a significantly higher cAMP concentration in case of the complemented mutant under NO stress.

(13) Line 292: There is a difference between red and green bars. Authors should do statistical analysis and then comment on whether overexpression of WT and mutant pde are different or similar, to me they are different; also, explain why the WT-Rv0805 strain is different than the phoPR-KO strain in the context of cell wall metabolism.

As recommended by the reviewer, we have now included statistical significance of the data in the revised version, and modified the text accordingly in the manuscript.

New query: Authors are asked to put a statistical significance test between WT-Rv0805 and WT-Rv0805M.

We have included it in the modified figure. Also, to explain it we incorporated new text in the legend to Fig. 4C of the revised manuscript (duplicated below):

“Note that similar to phoPR-KO, WT-Rv0805 shows a comparably higher sensitivity to CHP relative to WT bacilli. However, WT-Rv0805M expressing a mutant Rv0805, shows a significantly lower sensitivity to CHP relative to WT-Rv0805, as measured by the corresponding CFU values.”

(14) Line 299-303: Authors should explain how the colocalization % are calculated. Also, in the figure 4D merge panel please highlight the difference.

As suggested by the reviewer, we have now explained the methodology used to calculate percent colocalization in greater details. Also, we have modified Figure 4D to highlight the difference between samples shown in merge panel. Please see our response to comment # 33 from the Reviewer 1.

New query: In the figure legend it should be mentioned that the white arrow indicates non-co-localization which is visibly higher in WT and WT Rvo805M.

We thank the reviewer for this very important suggestion. We have now included the following text in the legend to Fig. 4D of the revised manuscript.

“White arrowheads in the merge panels indicate non-colocalization, which remains higher in WT-H37Rv and WT-Rv0805M relative to phoPR-KO or WT-Rv0805.”

eLife assessment

This important study describes how PhoP regulates cyclic-AMP production in the human pathogen Mycobacterium tuberculosis. The authors provide convincing evidence that PhoP acts as a repressor of the cyclic-AMP-specific phosphodiesterase, Rv0805, which can degrade cyclic-AMP. The revised manuscript has addressed all outstanding comments and the work will be of interest to bacteriologists.

Reviewer #1 (Public Review):

Summary:

This paper provides a straightforward mechanism of how mycobacterial cAMP level is increased under stressful conditions and shows that the increase is important for the survival of the bacterium in animal hosts. The cAMP level is increased by decreasing the expression of an enzyme that degrades cAMP.

Strengths:

The paper shows that under different stresses the response regulator PhoP represses a phosphodiesterase (PDE) that degrades cAMP specifically. Identification of PhoP as a regulator of cAMP is significant progress in understanding Mtb pathogenesis, as an increase in cAMP apparently increases bacterial survival upon infection. On the practical side, reduction of cAMP by increasing PDE can be a means to attenuate the growth of the bacilli. The results have wider implications since PhoP is implicated in controlling diverse mycobacterial stress responses and many bacterial pathogens modulate host cell cAMP levels. The results here are straightforward, internally consistent, and of both theoretical and applied interests. The results also open considerable future work, especially how increases in cAMP level help to increase survival of the pathogen.

Weaknesses:

It is not clear whether PhoP-PDE Rv0805 is the only pathway to regulate cAMP level under stress.

Comments on revised submission:

The authors have addressed my comments adequately, actually except for all but one. I have only one comment to do with the last line of the abstract. First, "genetic manipulation" usually means changing DNA. In Mtb pathogenesis I hope there is no DNA modification or change in the bacterial DNA. Also, the authors did not really inactivate the whole PhoP- rv0805-cAMP pathway. It would be best if the last line is made more fact based: Thus, inactivation of PhoP decreases cAMP level, thereby stress tolerance and intracellular survival of the bacillus.

Reviewer #2 (Public Review):

Summary:

In the manuscript, the authors have presented new mechanistic details to show how intracellular cAMP levels are maintained and linked to the phosphodiesterase enzyme which in turn is controlled by PhoP. Later, they showed the physiological relevance linked to altered cAMP concentrations.

Strengths:

Well-thought-out experiments. The authors carefully planned the experiments well to uncover the molecular aspects of it diligently.

Weaknesses:

None. The authors have meticulously responded to all my queries and concerns through multiple rounds of review.

eLife assessment

This manuscript finds evidence for a latent capability in several members of the TMEM16 and OSCA/TMEM family of ion channels for lipid scramblase activity. The authors demonstrate that the introduction of lysine mutations in evolutionarily conserved areas of TM4 can confer constitutive ion conduction and scramblase activity. Although the significance and scope of the work are important, the strength of the evidence is incomplete and could be improved.

Reviewer #1 (Public Review):

Summary:

TMEM16, OSCA/TMEM63, and TMC belong to a large superfamily of ion channels where TMEM16 members are calcium-activated lipid scramblases and chloride channels, whereas OSCA/TMEM63 and TMCs are mechanically activated ion channels. In the TMEM16 family, TMEM16F is a well-characterized calcium-activated lipid scramblase that plays an important role in processes like blood coagulation, cell death signaling, and phagocytosis. In a previous study, the group demonstrated that lysine mutation in TM4 of TMEM16A can enable the calcium-activated chloride channel to permeate phospholipids too. Based on this they hypothesize that the energy barrier for lipid scramblase in these ion channels is low, and that modification in the hydrophobic gate region by introducing a charged side chain between the TM4/6 interface in TMEM16 and OSCA/TMEM63 family can allow lipid scramblase. In this manuscript, using scramblase activity via Annexin V binding to phosphatidylserine, and electrophysiology, the authors demonstrate that lysine mutation in TM4 of TMEM16F and TMEM16A can cause constitutive lipid scramblase activity. The authors then go on to show that analogous mutations in OSCA1.2 and TMEM63A can lead to scramblase activity.

Strengths:

Overall, the authors introduce an interesting concept that this large superfamily can permeate ions and lipids.

Weaknesses:

The electrophysiology data does not entirely support their claims.

Reviewer #2 (Public Review):

This concise and focused study by Lowry and colleagues identifies a motif in the pores of three families of channel/scramblase proteins that regulate exclusive ion permeation and lipid transport. These three ion channel families, which include the TMEM16s, the plant-expressed and stress-gated cation channel OSCA, and the mammalian homolog and mechanosensitive cation channel, TMEM63 share low sequence similarity between them and have seemingly differing functions, as anion (TMEM16s), or stress-activated cation channels (OSCA/TMEM63). The study finds that in all three families, mutating a single hydrophobic residue in the ion permeation pathway of the channels confers lipid transport through the pores of the channels, indicating that TMEM16 and the related OSCA and TMEM63 channels have a conserved potential for both ion and lipid permeation. The authors interpret the findings as revealing that these channel/scramblase proteins have a relatively low "energetic barrier for scramblase" activity. The experiments themselves seem to be done with a high level of rigor and the paper is well written. A weakness is the limited scope of the experiments which, if fixed, could open up a new line of inquiry.

Reviewer #1 (Public Review):

Using a combination of cutting-edge high-resolution approaches (expansion microscopy, SIM, and CLEM) and biochemical approaches (in vitro translocation of actin filaments, cargo uptake assays, and drug treatment), the authors revisit previous results about TbMyo1 and TbACT in the bloodstream form (BSF) of Trypanosoma brucei. They show that a great part of the myosin motor is cytoplasmic but the fraction associated with organelles is in proximity to the endosomal system. In addition, they show that TbMyo1 can move actin filaments in vitro and visualize for the first time this actomyosin system using specific antibodies, a "classical" antibody for TbMyo1, and a chromobody for actin. Finally, using latrunculin A, which sequesters G-actin and prevents F-actin assembly, the authors show the delocalization and eventually the loss of the filamentous actin signal as well as the concomitant loss of the endosomal system integrity. However, they do not assess the localization of TbMyo1 in the same conditions.

Overall the work is well conducted and convincing. The conclusions are not over-interpreted and are supported by the experimental results.

Reviewer #2 (Public Review):

Summary:

The study by Link et al. advances our understanding of the actomyosin system in T. brucei, focusing on the role of TbMyo1, a class I myosin, within the parasite's endosomal system. Using a combination of biochemical fractionation, in vitro motility assays, and advanced imaging techniques such as correlative light and electron microscopy (CLEM), this paper demonstrates that TbMyo1 is dynamically distributed across early and late endosomes, the cytosol, is associated with the cytoskeleton, and a fraction has an unexpected association with glycosomes. Notably, the study shows that TbMyo1 can translocate actin filaments at velocities suggesting an active role in intracellular trafficking, potentially higher than those observed for similar myosins in other cell types. This work not only elucidates the spatial dynamics of TbMyo1 within T. brucei but also suggests its broader involvement in maintaining the complex architecture of the endosomal network, underscoring the critical role of the actomyosin system in a parasite that relies on high rates of endocytosis for immune evasion.

Strengths:

A key strength of the study is its exceptional rigor and successful integration of a wide array of sophisticated techniques, such as in vitro motility assays, and advanced imaging methods, including correlative light and electron microscopy (CLEM) and immuno-electron microscopy. This combination of approaches underscores the study's comprehensive approach to examining the ultrastructural organization of the trypanosome endomembrane system. The application of functional data using inhibitors, such as latrunculin A for actin depolymerization, further strengthens the study by providing insights into the dynamics and regulatory mechanisms of the endomembrane system. This demonstrates how the actomyosin system contributes to cellular morphology and trafficking processes. Furthermore, the discovery of TbMyo1 localization to glycosomes introduces a novel aspect to the potential roles of myosin I proteins within the cell, particularly in the context of organelles analogous to peroxisomes. This observation not only broadens our understanding of myosin I functionality but also opens up new avenues for research into the cellular biology of trypanosomatids, marking a significant contribution to the field.

Weaknesses:

Certain limitations inherent in the study's design and scope render the narrative incomplete and make it challenging to reach definitive conclusions. One significant limitation is the reliance on spatial association data, such as colocalization of TbMyo1 with various cellular components-or the absence thereof-to infer functional relationships. Although these data suggest potential interactions, the authors do not confirm functional or direct physical interactions.

While TbMyo1's localization is informative, the authors do not directly demonstrate its biochemical or mechanical activities in vivo, leaving its precise role in cellular processes speculative. Direct assays that manipulate TbMyo1 levels, activity, and/or function, coupled with observations of the outcomes on cellular processes, would provide more definitive evidence of the protein's specific roles in T. brucei. A multifaceted approach, including genetic manipulations, uptake assays, kinetic trafficking experiments, and imaging, would offer a more robust framework for understanding TbMyo1's roles. This comprehensive approach would elucidate not just the "what" and "where" of TbMyo1's function but also the "how" and "why," thereby deepening our mechanistic insights into T. brucei's biology.

Reviewer #3 (Public Review):

Summary:

In this work, Link and colleagues have investigated the localization and function of the actomyosin system in the parasite Trypanosoma brucei, which represents a highly divergent and streamlined version of this important cytoskeletal pathway. Using a variety of cutting-edge methods, the authors have shown that the T. brucei Myo1 homolog is a dynamic motor that can translocate actin, suggesting that it may not function as a more passive crosslinker. Using expansion microscopy, iEM, and CLEM, the authors show that MyoI localizes to the endosomal pathway, specifically the portion tasked with internalizing and targeting cargo for degradation, not the recycling endosomes. The glycosomes also appear to be associated with MyoI, which was previously not known. An actin chromobody was employed to determine the localization of filamentous actin in cells, which was correlated with the localization of Myo1. Interestingly, the pool of actomyosin was not always closely associated with the flagellar pocket region, suggesting that portions of the endolysomal system may remain at a distance from the sole site of parasite endocytosis. Lastly, the authors used actin-perturbing drugs to show that disrupting actin causes a collapse of the endosomal system in T. brucei, which they have shown recently does not comprise distinct compartments but instead a single continuous membrane system with subdomains containing distinct Rab markers.

Strengths:

Overall, the quality of the work is extremely high. It contains a wide variety of methods, including biochemistry, biophysics, and advanced microscopy that are all well-deployed to answer the central question. The data is also well-quantitated to provide additional rigor to the results. The main premise, that actomyosin is essential for the overall structure of the T. brucei endocytic system, is well supported and is of general interest, considering how uniquely configured this pathway is in this divergent eukaryote and how important it is to the elevated rates of endocytosis that are necessary for this parasite to inhabit its host.

Weaknesses:

(1) Did the authors observe any negative effects on parasite growth or phenotypes like BigEye upon expression of the actin chromobody?

(2) The Garcia-Salcedo EMBO paper cited included the production of anti-actin polyclonal antibodies that appeared to work quite well. The localization pattern produced by the anti-actin polyclonals looks similar to the chromobody, with perhaps a slightly larger labeling profile that could be due to differences in imaging conditions. I feel that the anti-actin antibody labeling should be expressly mentioned in this manuscript, and perhaps could reflect differences in the F-actin vs total actin pool within cells.

(3) The authors showed that disruption of F-actin with LatA leads to disruption of the endomembrane system, which suggests that the unique configuration of this compartment in T. brucei relies on actin dynamics. What happens under conditions where endocytosis and endocyctic traffic is blocked, such as 4 C? Are there changes to the localization of the actomyosin components?

(4) Along these lines, the authors suggest that their LatA treatments were able to disrupt the endosomal pathway without disrupting clathrin-mediated endocytosis at the flagellar pocket. Do they believe that actin is dispensable in this process? That seems like an important point that should be stated clearly or put in greater context.

Olá a todos/as, creio ser este um ponto central na conceção de atividades de ensino em geral, em especial as e-atividades. Quando da construção de um planeamento de ensino - aprendizagem, as estratégias utilizadas devem considerar o perfil dos estudantes, os conteúdos previstos, as competências a serem desenvolvidas, mas, acima de tudo, devem fazer sentido ao estudante. As e-atividades devem ser fatores de motivação para a participação e desenvolvimento do estudante, motivação esta que desenvolve a partir das referências do estudante, ancorando a aprendizagem nestas referências. A aprendizagem ocorrerá, bem como a motivação do estudante para este movimento, a partir do significado que esta e-atividade terá para o estudante. Uma e-atividade perde seu sentido se passar a ser mais uma atividade por parte do estudante que será somente mais uma forma de obtenção de "notas" para aprovação, pouco trazendo de aprendizagem e de desenvolvimento de competências. Esta discussão tem especial importãncia na Educação de Adultos, na qual a utilizade do processo de aprendizagem tem maior ligação a aplicação dos conhecimentos e competências devem ter com a aplicabilidade no mundo do trabalho. Ricardo Rodrigues

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eLife assessment

This study presents a useful characterization of 3D chromosome conformation changes in activated T lymphocytes, linking risk variants for autoimmune disease to putative target genes. The study employs solid methods and approaches and demonstrates the utility of using chromatin conformation to understand gene regulatory processes. However, the same data modality (chromatin conformation) was previously generated by another group in the same model system, and a more in-depth comparison of results would have improved the utility of this study.

Reviewer #1 (Public Review):

Summary:

The authors profile gene expression, chromatin accessibility, and chromosomal architecture (by Hi-C) in activated CD4 T cells and use this information to link non-coding variants associated with autoimmune diseases with putative target genes. They find over 1000 genes physically linked with autoimmune disease loci in these cells, many of which are upregulated upon T cell activation. Focusing on IL2, they dissect the regulatory architecture of this locus, including the allelic effects of GWAS variants. They also intersect their variant-to-gene lists with data from CRISPR screens for genes involved in CD4 T cell activation and expression of inflammatory genes, finding enrichments for regulators. Finally, they showed that pharmacological inhibition of some of these genes impacts T-cell activation.

This is a solid study that follows a well-established canvas for variant-to-gene prioritisation using 3D genomics, applying it to activated T cells. The authors go some way in validating the lists of candidate genes, as well as exploring the regulatory architecture of a candidate GWAS locus. Jointly with data from previous studies performing variant-to-gene assignment in activated CD4 T cells (and other immune cells), this work provides a useful additional resource for interpreting autoimmune disease-associated genetic variation.

Suggestions for improvement:

Autoimmune disease variants were already linked with genes in CD28-stimulated CD4 T cells using chromosome conformation capture, specifically Promoter CHi-C and the COGS pipeline (Javierre et al., Cell 2016; Burren et al., Genome Biol 2017; Yang et al., Nat Comms 2020). The authors cite these papers and present a comparative analysis of their variant-to-gene assignments (in addition to scRNA-seq eQTL-based assignments). Furthermore, they find that the Burren analysis yields a higher enrichment for gold standard genes.

The obvious question that the authors don't venture into is why the results are quite different. In principle, this could be due to the differences between:<br /> (a) the cell stimulation procedure<br /> (b) the GWAS datasets used<br /> (c) the types of assay (Hi-C vs Capture Hi-C)<br /> (d) approaches for defining gene-linked regions (loops vs neighbourhoods)<br /> (e) how the GWAS signals at gene-linked regions are aggregated (e.g., the flavours of COGS in Javierre and Burren vs the authors' approach).

Re (a), I'm not sure the authors make it explicitly clear in the main text that the Capture Hi-C-based studies also use *stimulated* CD4 T cells, particularly in the section "Comparative predictive power...". So the cells used are pretty much the same, and the differences likely arise from points (b) to (e).

It would be useful for the community to understand more clearly what is driving these differences, ideally with some added data. Could the authors, for example, take the PCHi-C data from Javierre/Burren and use their GWAS data and variant-to-gene assignment algorithms?

In addition, given that the authors use Hi-C, a popular method for V2G prioritisation for this type of data is currently ABC (Nasser et al, Nature 2021). Could the authors provide a comparative analysis with respect to the V2G assignments in the paper and, if they see it appropriate, also run ABC-based GWAS integration on their own Hi-C data?

Reviewer #2 (Public Review):

Summary:

There is significant interest in characterizing the mechanisms by which genetic mutations linked to autoimmunity perturb immune processes. Pahl et al. collect information on dynamic accessible regions, genes, and 3D contacts in primary CD4+ T cell samples that have been stimulated ex vivo. The study includes a variety of analyses characterizing these dynamic changes. With TF footprinting they propose factors linked to active regulatory elements. They compare the performance of their variant mapping pipeline that uses their data versus existing datasets. Most compelling there was a deep dive into additional study of regulatory elements nearby the IL2 gene. Finally, they perform a pharmacological screen targeting several genes they suggest are involved in T cell proliferation.

Strengths:

The work done characterizing elements at the IL2 locus is impressive.

Weaknesses:

- Missing critical context to evaluate claims. There are extensive studies performed on resting and activated immune cell states (CD4+ T cells and other cell types) and some at multiple time points or concentrations of stimuli that collect ATAC-seq and/or RNA-seq that have been ignored by this study. How do conclusions from previous studies compare to what the authors conclude here? It is impossible to evaluate the claims without this additional context. These are a few studies I am familiar with (the authors should perform a more comprehensive search to be sure they're not ignoring existing observations) that would be important to compare/contrast conclusions:<br /> o Alasoo, K. et al. Shared genetic effects on chromatin and gene expression indicate a role for enhancer priming in immune response. Nat. Genet. 50, 424-431 (2018).<br /> o Calderon, D., Nguyen, M.L.T., Mezger, A. et al. Landscape of stimulation-responsive chromatin across diverse human immune cells. Nat Genet 51, 1494-1505 (2019).<br /> o Gate, R.E., Cheng, C.S., Aiden, A.P. et al. Genetic determinants of co-accessible chromatin regions in activated T cells across humans. Nat Genet 50, 1140-1150 (2018).<br /> o Glinos, D.A., Soskic, B., Williams, C. et al. Genomic profiling of T-cell activation suggests increased sensitivity of memory T cells to CD28 costimulation. Genes Immun 21, 390-408 (2020).<br /> o Gutierrez-Arcelus, M., Baglaenko, Y., Arora, J. et al. Allele-specific expression changes dynamically during T cell activation in HLA and other autoimmune loci. Nat Genet 52, 247-253 (2020).<br /> o Kim-Hellmuth, S. et al. Genetic regulatory effects modified by immune activation contribute to autoimmune disease associations. Nat. Commun. 8, 266 (2017).<br /> o Ye, C. J. et al. Intersection of population variation and autoimmunity genetics in human T cell activation. Science 345, 1254665 (2014).

- As a general point, I appreciate it when each claim includes a corresponding effect size and p-value, which helps me evaluate the strength of significance of supporting evidence.

Reviewer #3 (Public Review):

Summary:

This paper used RNAseq, ATACseq, and Hi-C to assess gene expression, chromatin accessibility, and chromatin physical associations for native CD4+ T cells as they respond to stimulation through TCR and CD28. With these data in hand, the author identified 423 GWAS signals to their respective target genes, where most of these were not in the proximal promoter, but rather distal enhancers. The IL-2 gene was used as an example to identify new distal cis-regulatory regions required for optimal IL-2 gene transcription. These distal elements interact with the proximal IL2 promoter region. When the distal enhancer contained an autoimmune SNP, it affected IL-2 gene transcription. The authors also identified genetic risk variants that were associated with genes upon activation. Some of these regulate proliferation and cytokine production, but others are novel.

Strengths:

This paper provides a wealth of data related to gene expression after CD4 T cells are activated through the TCR and CD28. An important strength of this paper is that these data were intensively analyzed to uncover autoimmune disease SNPs in cis-acting regions. Many of these could be assigned to likely target genes even though they often are in distal enhancers. These findings help to provide a better understanding concerning the mechanism by which GWAS risk elements impact gene expression.

Another strength of this study was the proof-of-principle studies examining the IL-2 gene. Not only were new cis-acting enhancers discovered, but they were functionally shown to be important in regulating IL-2 expression, including susceptibility to colitis. Their importance was also established with respect to such distal enhancers harboring disease-relevant SNPs, which were shown to affect IL-2 transcription.

The data from this study were also mined against past CRISPR screens that identified genes that control aspects of CD4 T cell activation. From these comparisons, novel genes were identified that function during T cell activation.

Weaknesses:

A weakness of this study is that few individuals were analyzed, i.e., RNAseq and ATACseq (n=3) and HiC (n=2). Thus, the authors may have underestimated potentially relevant risk associations by their chromatin capture-based methodology. This might account for the low overlap of their data with the eQTL-based approach or the HIEI truth set.

Impact:

This study indicates that defining distal chromatin interacting regions helps to identify distal genetic elements, including relevant variants, that contribute to gene activation.

Reviewer #1 (Public Review):

In this study, the authors examined the role of IBTK, a substrate-binding adaptor of the CRL3 ubiquitin ligase complex, in modulating the activity of the eiF4F translation initiation complex. They find that IBTK mediates the non-degradative ubiquitination of eiF4A1, promotes cap-dependent translational initiation, nascent protein synthesis, oncogene expression, and tumor cell growth. Correspondingly, phosphorylation of  IBTK by mTORC1/ S6K1 increases eIF4A1 ubiquitination and sustains oncogenic translation.

Strengths:

This study utilizes multiple biochemical, proteomic, functional and cell biology assays to substantiate their results.  Importantly, the work nominates IBTK as a unique substrate of mTORC1, and further validates eiF4A1 ( a crucial subunit of the ei44F complex) as a promising therapeutic target in cancer. Since IBTK interacts broadly with multiple members of the translational initial complex- it will be interesting to examine its role in eiF2alpha-mediated ER stress as well as eiF3-mediated translation. Additionally, since IBTK exerts pro-survival effects in multiple cell types, it will be of relevance to characterize the role of IBTK in mediating increased mTORC1 mediated translation in other tumor types, thus potentially impacting their treatment with eiF4F inhibitors.

Limitations/Weaknesses:

The findings are mostly well supported by data, but some areas need clarification and could potentially be enhanced with further experiments:

(1) Since eiF4A1 appears to function downstream of IBTK1, can the effects of IBTK1 KO/KD in reducing puromycin incorporation ( in Fig 3A),  cap-dependent luciferase reporter activity (Fig 3G), reduced oncogene expression ( Fig 4A) or 2D growth/ invasion assays (Fig 4) be overcome or bypassed by overexpressing eiF4A1? These could potentially be tested in future studies. <br /> (2) The decrease in nascent protein synthesis in puromycin incorporation assays in Figure 3A suggests that the effects of IBTK KO are comparable to and additive with silvesterol. It would be of interest to examine whether silvesterol decreases nascent protein synthesis or increases stress granules in the IBTK KO cells stably expressing IBTK as well. <br /> (3) The data presented in Figure 5 regarding the role of mTORC1 in IBTK-mediated eiF4A1 ubiquitination needs further clarification on several points:<br /> - It is not clear if the experiments in Figure 5F with Phos-tag gels are using the FLAG-IBTK deletion mutant or the peptide containing the mTOR sites as it is mentioned on line 517, page 19 "To do so, we generated an IBTK deletion mutant (900-1150 aa) spanning the potential mTORC1-regulated phosphorylation sites" This needs further clarification.<br /> -It may be of benefit to repeat the Phos tag experiments with full length FLAG-IBTK and/or endogenous IBTK with molecular weight markers indicating size of migrated bands.<br /> -Additionally, torin or Lambda phosphatase treatment may be used to confirm the specificity of the band in separate experiments.<br /> -Phos-tag gels with the IBTK CRISPR KO line would also help confirm that the non-phosphorylated band is indeed IBTK. <br /> -It is unclear why the lower, phosphorylated bands seem to be increasing ( rather than decreasing) with AA starvation/ Rapa in Fig 5H.

Reviewer #2 (Public Review):

Summary:

This study by Sun et al. identifies a novel role for IBTK in promoting cancer protein translation, through regulation of the translational helicase eIF4A1. Using a multifaceted approach, the authors demonstrate that IBTK interacts with and ubiquitinates eIF4A1 in a non-degradative manner, enhancing its activation downstream of mTORC1/S6K1 signaling. This represents a significant advance in elucidating the complex layers of dysregulated translational control in cancer.

Strengths:

A major strength of this work is the convincing biochemical evidence for a direct regulatory relationship between IBTK and eIF4A1. The authors utilize affinity purification and proximity labeling methods to comprehensively map the IBTK interactome, identifying eIF4A1 as a top hit. Importantly, they validate this interaction and the specificity for eIF4A1 over other eIF4 isoforms by co-immunoprecipitation in multiple cell lines. Building on this, they demonstrate that IBTK catalyzes non-degradative ubiquitination of eIF4A1 both in cells and in vitro through the E3 ligase activity of the CRL3-IBTK complex. Mapping IBTK phosphorylation sites and showing mTORC1/S6K1-dependent regulation provides mechanistic insight. The reduction in global translation and eIF4A1-dependent oncoproteins upon IBTK loss, along with clinical data linking IBTK to poor prognosis, support the functional importance. Finally, the impact of IBTK on eIF4A1 target gene expression in colon and lung cancer cell lines, strengthens these findings.

Weaknesses:

While the effects of IBTK knockout/over-expression on bulk protein synthesis are shown, the expression of several eIF4A1 target oncogenes remains unchanged.

Summary:

Overall, this study significantly advances our understanding of how aberrant mTORC1/S6K1 signaling promotes cancer pathogenic translation via IBTK and eIF4A1. The proteomic, biochemical and phosphorylation mapping approaches established here provide a blueprint for interrogating IBTK function. These data should galvanize future efforts to target the mTORC1/S6K1-IBTK-eIF4A1 axis as an avenue for cancer therapy, particularly in combination with eIF4A inhibitors.

eLife assessment

This valuable study contributes to the understanding of phase separation in Dishevelled (DVL) proteins, by investigating the endogenous complexes of DVL2 using ultracentrifugation and contrasting them with DVL1 and DVL3 behavior and the functional validation of the DVL2 intrinsically disordered regions mediating the protein condensate. The study is, however, incomplete due to the lack of several controls and its focus on overexpression and mutants lacking key domains.

Reviewer #2 (Public Review):

Summary:

The authors aimed to identify which regions of DVL2 contribute to its endogenous/basal clustering, as well as the relevance of such domains to condensate/phase separation and WNT activation.

Strengths:

A strength of the study is the focus on endogenous DVL2 to set up the research questions, as well as the incorporation of various techniques to tackle it. I found also quite interesting that DVL2-CFR addition to DVL1 increased its MW in density gradients.

Weaknesses:

I think that several of the approaches of the manuscript are subpar to achieve the goals and/or support several of the conclusions. For example:

(1) Although endogenous DVL2 indeed seems to form complexes (Figure 1A), neither the number of proteins involved nor whether those are homo-complexes can be determined with a density gradient. Super-resolution imaging or structural analyses are needed to support these claims.

(2) Follow-up analyses of the relevance of the DVL2 domains solely rely on overexpressed proteins. However, there were previous questions arising from o/e studies that prompted the focus on endogenous, physiologically relevant DVL interactions, clustering, and condensate formation. Although the title, conclusions, and relevance all point to the importance of this study for understanding endogenous complexes, only Figures 1A and B deal with endogenous DVL2.

(3) Mutants lacking activity/complex formation, e.g. DVL2_1-418, may need further validation. For instance, DVL2_1-506 (same mutant but with DEP) seems to form condensates and it is functional in WNT signalling (King et al., 20223). These differences could be caused by the lack of DEP domain in this particular construct and/or folding differences.

(4) The key mutants, DeltaCFR and VV/FF only show mild phenotypes. The authors' results suggest that these regions contribute but are not necessary for 1) complex formation (Density gradient Figures 7A and B), condensate formation (Figures 7C and D), and WNT activity (Figure 7E). Of note Figure 7C shows examples for the mutants with no condensates while the qualification indicates that 50% of the cells do have condensates.

(5) Most of the o/e analyses (including all reporter assays) should be performed in DVL1-3 KO cells in order to explore specifically the behaviour of the investigated mutants.

(6) How comparable are condensates found in the cytoplasm (usually for wt DVL) with those located in the nucleus (DEP mutants)?

Several studies in the last two decades have analysed the relevance of DVL homo - and hetero-clustering by relying on overexpressed proteins. Recent studies also explored the possibility of DVL undergoing liquid-liquid phase separation following similar principles. As highlighted by the authors in the introduction, there is a need to understand DVL dynamics under endogenous/physiological conditions. Recent super-resolution studies aimed at that question by characterising endogenously edited DVL2. The authors seemed to aim in the same direction with their initial findings (Figure 1A) but quickly moved to o/e proteins without going back to the initial question. This reviewer thinks that to support their conclusions and advance in this important question, the authors should introduce the relevant mutations in the endogenous locus (e.g. by Cas9+ donor template encoding the required 3' exons, as done by others before for WNT components, including DVL2) and determine their impact in the above-indicated processes.

eLife assessment

This is a valuable manuscript describing the competitive binding between the RING2 and phosphorylated Ubl domains within Parkin involved in the regulation of Parkin activity. The evidence supporting this conclusion is incomplete, as it primarily relies on a single biochemical assay and does not utilize more stringent, quantitative biophysical approaches to probe this competitive binding. This work will be of interest to the research communities focused on the molecular basis of ubiquitin ligase regulation, PINK-PARKIN-regulated mitophagy, and mitochondrial quality control.

Reviewer #1 (Public Review):

Summary:

The authors used structural and biophysical methods to provide insight into Parkin regulation. The breadth of data supporting their findings was impressive and generally well-orchestrated. Still, the impact of their results builds on recent structural studies and the stated impact is based on these prior works.

Strengths:

(1) After reading through the paper, the major findings are:<br /> - RING2 and pUbl compete for binding to RING0.<br /> - Parkin can dimerize.<br /> - ACT plays an important role in enzyme kinetics.

(2) The use of molecular scissors in their construct represents a creative approach to examining inter-domain interactions.

(3) From my assessment, the experiments are well-conceived and executed.

Weaknesses:

(1) The manuscript, as written, is NOT for a general audience. Admittedly, I am not an expert on Parkin structure and function, but I had to do a lot of homework to try to understand the underlying rationale and impact. This reflects, I think, that the work generally represents an incremental advance on recent structural findings.

(2) To this point, it is hard to understand the impact of this work without more information highlighting the novelty. There are several structures of Parkin in various auto-inhibited states, and it was hard to delineate how this is different.

(3) As noted, I appreciated the use of protease sites in the fusion protein construct. It is unclear how the loop region might affect the protein structure and function. The authors worked to demonstrate that this did not introduce artifacts, but the biological context is missing.

(4) While it is likely that the binding is competitive between the Ubl and RING2 domains, the data is not quantitative. Is it known whether the folding of the distinct domains is independent? Or are there interactions that alter folding? It seems plausible that conformational rearrangements may invoke an orientation of domains that would be incompatible. The biological context for the importance of this interaction was not clear to me.

(5) What is the rationale for mutating Lys211 to Asn? Were other mutations tried? Glu? Ala? Just missing the rationale. I think this may have been identified previously in the field, but not clear what this mutation represents biologically.

(6) I was confused about how the phospho-proteins were generated. After looking through the methods, there appear to be phosphorylation experiments, but it is unclear what the efficiency was for each protein (i.e. what % gets modified). In the text, the authors refer to phospho-Parkin (T270R, C431A), but not clear how these mutations might influence this process. I gather that these are catalytically inactive, but it is unclear to me how this is catalyzing the ubiquitination in the assay.

(7) The authors note that "ACT can be complemented in trans; however, it is more efficient in cis", but it is unclear whether both would be important or if the favored interaction is dominant in a biological context.

(8) The authors repeatedly note that this study could aid in the development of small-molecule regulators against Parkin to treat PD, but this is a long way off. And it is not clear from their manuscript how this would be achieved. As stated, this is conjecture.

Reviewer #2 (Public Review):

This manuscript uses biochemistry and X-ray crystallography to further probe the molecular mechanism of Parkin regulation and activation. Using a construct that incorporates cleavage sites between different Parkin domains to increase the local concentration of specific domains (i.e., molecular scissors), the authors suggest that competitive binding between the p-Ubl and RING2 domains for the RING0 domain regulates Parkin activity. Further, they demonstrate that this competition can occur in trans, with a p-Ubl domain of one Parkin molecule binding the RING0 domain of a second monomer, thus activating the catalytic RING1 domain. In addition, they suggest that the ACT domain can similarly bind and activate Parkin in trans, albeit at a lower efficiency than that observed for p-Ubl. The authors also suggest from crystal structure analysis and some biochemical experiments that the linker region between RING2 and repressor elements interacts with the donor ubiquitin to enhance Parkin activity.

Ultimately this manuscript challenges previous work suggesting that the p-Ubl domain does not bind to the Parkin core in the mechanism of Parkin activation. The use of the 'molecular scissors' approach to probe these effects is an interesting approach to probe this type of competitive binding. However, there are issues with the experimental approach manuscript that detract from the overall quality and potential impact of the work.

The competitive binding between p-Ubl and RING2 domains for the Parkin core could have been better defined using biophysical and biochemical approaches that explicitly define the relative affinities that dictate these interactions. A better understanding of these affinities could provide more insight into the relative bindings of these domains, especially as it relates to the in trans interactions.

I also have concerns about the results of using molecular scissors to 'increase local concentrations' and allow for binding to be observed. These experiments are done primarily using proteolytic cleavage of different domains followed by size exclusion chromatography. ITC experiments suggest that the binding constants for these interactions are in the µM range, although these experiments are problematic as the authors indicate in the text that protein precipitation was observed during these experiments. This type of binding could easily be measured in other assays. My issue relates to the ability of a protein complex (comprising the core and cleaved domains) with a Kd of 1 µM to be maintained in an SEC experiment. The off-rates for these complexes must be exceeding slow, which doesn't really correspond to the low µM binding constants discussed in the text. How do the authors explain this? What is driving the Koff to levels sufficiently slow to prevent dissociation by SEC? Considering that the authors are challenging previous work describing the lack of binding between the p-Ubl domain and the core, these issues should be better resolved in this current manuscript. Further, it's important to have a more detailed understanding of relative affinities when considering the functional implications of this competition in the context of full-length Parkin. Similar comments could be made about the ACT experiments described in the text.

Ultimately, this work does suggest additional insights into the mechanism of Parkin activation that could contribute to the field. There is a lot of information included in this manuscript, giving it breadth, albeit at the cost of depth for the study of specific interactions. Further, I felt that the authors oversold some of their data in the text, and I'd recommend being a bit more careful when claiming an experiment 'confirms' a specific model. In many cases, there are other models that could explain similar results. For example, in Figure 1C, the authors state that their crystal structure 'confirms' that "RING2 is transiently displaced from the RING0 domain and returns to its original position after washing off the p-Ubl linker". However, it isn't clear to me that RING2 ever dissociated when prepared this way. While there are issues with the work that I feel should be further addressed with additional experiments, there are interesting mechanistic details suggested by this work that could improve our understanding of Parkin activation. However, the full impact of this work won't be fully appreciated until there is a more thorough understanding of the regulation and competitive binding between p-Ubl and RIGN2 to RORB both in cis and in trans.

Reviewer #3 (Public Review):

Summary:

In their manuscript "Additional feedforward mechanism of Parkin activation via binding of phospho-UBL and RING0 in trans", Lenka et al present data that could suggest an "in trans" model of Parkin ubiquitination activity. Parkin is an intensely studied E3 ligase implicated in mitophagy, whereby missense mutations to the PARK2 gene are known to cause autosomal recessive juvenile parkinsonism. From a mechanistic point of view, Parkin is extremely complex. Its activity is tightly controlled by several modes of auto-inhibition that must be released by queues of mitochondrial damage. While the general overview of Parkin activation has been mapped out in recent years, several details have remained murky. In particular, whether Parkin dimerizes as part of its feed-forward signaling mechanism, and whether said dimerization can facilitate ligase activation, has remained unclear. Here, Lenka et al. use various truncation mutants of Parkin in an attempt to understand the likelihood of dimerization (in support of an "in trans" model for catalysis).

Strengths:

The results are bolstered by several distinct approaches including analytical SEC with cleavable Parkin constructs, ITC interaction studies, ubiquitination assays, protein crystallography, and cellular localization studies.

Weaknesses:

As presented, however, the storyline is very confusing to follow and several lines of experimentation felt like distractions from the primary message. Furthermore, many experiments could only indirectly support the author's conclusions, and therefore the final picture of what new features can be firmly added to the model of Parkin activation and function is unclear.

Major concerns:

(1) This manuscript solves numerous crystal structures of various Parkin components to help support their idea of in trans transfer. The way these structures are presented more resemble models and it is unclear from the figures that these are new complexes solved in this work, and what new insights can be gleaned from them.

(2) There are no experiments that definitively show the in trans activation of Parkin. The binding experiments and size exclusion chromatography are a good start, but the way these experiments are performed, they'd be better suited as support for a stronger experiment showing Parkin dimerization. In addition, the rationale for an in trans activation model is not convincingly explained until the concept of Parkin isoforms is introduced in the Discussion. The authors should consider expanding this concept into other parts of the manuscript.

2a. For the in trans activation experiment using wt Parkin and pParkin (T270R/C431A) (Figure 3D), there needs to be a large excess of pParkin to stimulate the catalytic activity of wt Parkin. This experiment has low cellular relevance as these point mutations are unlikely to occur together to create this nonfunctional pParkin protein. In the case of pParkin activating wt Parkin (regardless of artificial point mutations inserted to study specifically the in trans activation), if there needs to be much more pParkin around to fully activate wt Parkin, isn't it just more likely that the pParkin would activate in cis?

2ai. Another underlying issue with this experiment is that the authors do not consider the possibility that the increased activity observed is a result of increased "substrate" for auto-ubiquitination, as opposed to any role in catalytic activation. Have the authors considered looking at Miro as a substrate in order to control for this?

2b. The authors mention a "higher net concentration" of the "fused domains" with RING0, and use this to justify artificially cleaving the Ubl or RING2 domains from the Parkin core. This fact should be moot. In cells, it is expected there will only be a 1:1 ratio of the Parkin core with the Ubl or RING2 domains. To date, there is no evidence suggesting multiple pUbls or multiple RING2s can bind the RING0 binding site. In fact, the authors here even show that either the RING2 or pUbl needs to be displaced to permit the binding of the other domain. That being said, there would be no "higher net concentration" because there would always be the same molar equivalents of Ubl, RING2, and the Parkin core.

2c. A larger issue remaining in terms of Parkin activation is the lack of clarity surrounding the role of the linker (77-140); particularly whether its primary role is to tether the Ubl to the cis Parkin molecule versus a role in permitting distal interactions to a trans molecule. The way the authors have conducted the experiments presented in Figure 2 limits the possible interactions that the activated pUbl could have by (a) ablating the binding site in the cis molecule with the K211N mutation; (b) further blocking the binding site in the cis molecule by keeping the RING2 domain intact. These restrictions to the cis parkin molecule effectively force the pUbl to bind in trans. A competition experiment to demonstrate the likelihood of cis or trans activation in direct comparison with each other would provide stronger evidence for trans activation.

(3) A major limitation of this study is that the authors interpret structural flexibility from experiments that do not report directly on flexibility. The analytical SEC experiments report on binding affinity and more specifically off-rates. By removing the interdomain linkages, the accompanying on-rate would be drastically impacted, and thus the observations are disconnected from a native scenario. Likewise, observations from protein crystallography can be consistent with flexibility, but certainly should not be directly interpreted in this manner. Rigorous determination of linker and/or domain flexibility would require alternative methods that measure this directly.

(4) The analysis of the ACT element comes across as incomplete. The authors make a point of a competing interaction with Lys48 of the Ubl domain, but the significance of this is unclear. It is possible that this observation could be an overinterpretation of the crystal structures. Additionally, the rationale for why the ACT element should or shouldn't contribute to in trans activation of different Parkin constructs is not clear. Lastly, the conclusion that this work explains the evolutionary nature of this element in chordates is highly overstated.

(5) The analysis of the REP linker element also seems incomplete. The authors identify contacts to a neighboring pUb molecule in their crystal structure, but the connection between this interface (which could be a crystallization artifact) and their biochemical activity data is not straightforward. The analysis of flexibility within this region using crystallographic and AlphaFold modeling observations is very indirect. The authors also draw parallels with linker regions in other RBR ligases that are involved in recognizing the E2-loaded Ub. Firstly, it is not clear from the text or figures whether the "conserved" hydrophobic within the linker region is involved in these alternative Ub interfaces. And secondly, the authors appear to jump to the conclusion that the Parkin linker region also binds an E2-loaded Ub, even though their original observation from the crystal structure seems inconsistent with this. The entire analysis feels very preliminary and also comes across as tangential to the primary storyline of in trans Parkin activation.

eLife assessment

This useful study reports an unexpected phenotype of atrophy of the male reproductive system and infertility upon combined knockout in adult mice of the genes encoding the two kinases CDK8 and CDK19. While the morphological evidence and single-cell transcriptomic data are solid, the proposed mechanism remains unconvincing as there is little evidence for causality, and some controls are missing. This work will be of interest to reproductive biologists, developmental biologists, and andrologists.

Reviewer #1 (Public Review):

Summary:

In this paper, Bruter and colleagues report the effects of inducible deletion of the genes encoding the two paralogous kinases of the Mediator complex in adult mice. The physiological roles of these two kinases, CDK8 and CDK19, are currently rather poorly understood; although conserved in all eukaryotes, and among the most highly conserved kinases in vertebrates, individual knockouts of genes encoding CDK8 homologues in different species have revealed generally rather mild and specific effects, in contrast to Mediator itself. Here, the authors provide evidence that neither CDK8 nor CDK19 are required for adult homeostasis but they are functionally redundant for maintenance of reproductive tissue morphology and fertility in males.

Strengths:

The morphological data on the atrophy of the male reproductive system and the arrest of spermatocyte meiosis are solid and are reinforced by single-cell transcriptomics data, which is a challenging technique to implement in vivo. The main findings are important and will be of interest to scientists in the fields of transcription and developmental biology.

Weaknesses:

There are several major weaknesses.

The first is that data on the general health of mice with single and double knockouts is not shown, nor is there any data on effects in any other tissues. This gives the impression that the only phenotype is in the male reproductive system, which would be misleading if there were phenotypes in other tissues that are not reported. Furthermore, data for the genitourinary system in single knockouts are very sparse; data are described for fertility in Figure 1H, ploidy, and cell number in Figures 2B and C, plasma testosterone and luteinizing hormone levels in Figures 5C and 5D, and morphology of testis and prostate tissue for single Cdk8 knockout in Supplementary Figure 1C (although in this case the images do not appear very comparable between control and CDK8 KO, thus perhaps wider fields should be shown), but, for example, there is no analysis of different meiotic stages or of gene expression in single knockouts. It is worth mentioning that single knockouts seem to show a corresponding upregulation of the level of the paralogue kinase, indicating that any lack of phenotypes might be due to feedback compensation, which would be an interesting finding if confirmed; this has not been mentioned.

The second major weakness is that the correlation between double knockout and reduced expression of genes involved in steroid hormone biosynthesis is portrayed as a causal mechanism for the phenotypes observed. While this is a possibility, there are no experiments performed to provide evidence that this is the case. Furthermore, there is no evidence showing that CDK8 and/or CDK19 are directly responsible for the transcription of the genes concerned.

Finally, the authors propose that the phenotypes are independent of the kinase activity of CDK8 or CDK19 because treatment of mice for a month with an inhibitor does not recapitulate the effects of the knockout, and nor does expression of two steroidogenic genes change in cultured Leydig cells upon treatment with an inhibitor. However, there are no controls for effective target inhibition shown.

Reviewer #2 (Public Review):

Summary:

The authors tried to test the hypothesis that Cdk8 and Cdk19 stabilize the cytoplasmic CcNC protein, the partner protein of the Mediator complex including CDK8/19 and Mediator protein via a kinase-independent function by generating induced double knockout of Cdk8/19. However, the evidence presented suffers from a lack of focus and rigor and does not support their claims.

Strengths:

This is the first comprehensive report on the effect of a double knockout of CDK8 and CDK19 in mice on male fertility, hormones, and single-cell testicular cellular expression. The inducible knockout mice led to male sterility with severe spermatogenic defects, and the authors attempted to use this animal model to test the kinase-independent function of CDK8/19, previously reported for humans. Single-cell RNA-seq of knockout testis presented a high resolution of molecular defects of all the major cell types in the testes of the inducible double knockout mice. The authors also have several interesting findings such as reentry into cell cycles by Sertoli cells, and loss of Testosterone in induced dko that could be investigated further.

Weaknesses:

The claim of reproductive defects in the induced double knockout of CDK8/19 resulted from the loss of CCNC via a kinase-independent mechanism is interesting but was not supported by the data presented. While the construction and analysis of the systemic induced knockout model of Cdk8 in Cdk19KO mice is not trivial, the analysis and data are weakened by the systemic effect of Cdk8 loss, making it difficult to separate the systemic effect from the local testis effect.

The analysis of male sterile phenotype is also inadequate with poor image quality, especially testis HE sections. The male reproductive tract picture is also small and difficult to evaluate. The mice crossing scheme is unusual as you have three mice to cross to produce genotypes, while we could understand that it is possible to produce pups of desired genotypes with different mating schemes, such a vague crossing scheme is not desirable and of poor genetics practice. Also using TAM-treated wild type as control is ok, but a better control will be TAM-treated ERT2-cre; CDK8f/f or TAM-treated ERT2 Cre CDK19/19 KO, so as to minimize the impact from the well-recognized effect of TAM.

While the authors proposed that the inducible loss of CDK8 in the CDK19 knockout background is responsible for spermatogenic defects, it was not clear in which cells CDK8/19 genes are interested and which cell types might have a major role in spermatogenesis. The authors also put forward the evidence that reduction/loss of Testosterone might be the main cause of spermatogenic defects, which is consistent with the expression change in genes involved in steroigenesis pathway in Leydig cells of inducible double knockout. However it is not clear how the loss of Testosterone contributed to the loss of CcnC protein.

The authors should clarify or present the data on where CDK8 and CDK19 as well as CcnC are expressed so as to help the readers understand which tissues both CDK might be functioning in and cause the loss of CcnC. It should be easier to test the hypothesis of CDK8/19 stabilizing CcnC protein using double knock-out primary cells, instead of the whole testis.

Since CDK8KO and CDK19KO both have significantly reduced fertility in comparison with wildtype, it might be important to measure the sperm quantity and motility among CDK8 KO, CDK19KO, and induced DKO to evaluate spermatogenesis based on their sperm production.

Some data for the inducible knockout efficiency of Cdk8 were presented in Supplemental Figure 1, but there is no legend for the supplemental figures, it was not clear which band represented the deletion band, and which tissues were examined. Tail or testis? It seems that two months after the injection of Tam, all the Cdk8 were completely deleted, indicating extremely efficient deletion of Tam induction by two months post administration. Were the complete deletion of Cdk8 happening even earlier? An examination of time points of induced loss would be useful and instructional as to when is the best time to examine phenotypes.

The authors found that Sertoli cells re-entered the cell cycle in the inducible double knockout but stopped short of careful characterization other than increased expression of cell cycle genes.

Overall this work suffered from a lack of focus and rigor in the analysis and lack of sufficient evidence to support their main conclusions.

Minor:

Dko should be appropriately named iDKO (induced dKO).

"suppress spermatogenesis and male fertility" in the title does not fit the evidence presented.

"DKO males, had an understized and dedifferentiated reproductive system?" what is the evidence for "undifferentiated"?

We performed necropsy ? not the right wording here.

Colchicine-lke apoptotic bodies ? what does this mean? Not clear.

Images throughout the manuscript suffer from poor resolution and are often blurry and hard to evaluate.

To pinpoint the meiotic stage defect of iDKO, it is better to use the meiotic chromosome spread approach.

Reviewer #1 (Public Review):

Summary:

The authors presented here a novel 3D fibroblast culture and transdifferentiation approach for potential meat production with GelMA hydrogel.

Strengths:

(1) Reduced serum concentration for 3D chicken fibroblast culture and transdifferentiation is optimized.<br /> (2) Efficient myogenic transdifferentiation and lipogenesis as well as controlled fat deposition are achieved in the 3D GelMA.

Reviewer #2 (Public Review):

The manuscript by Ma et al. tries to develop a protocol for cell-based meat production using chicken fibroblasts as three-dimensional (3D) muscle tissues with fat accumulation. The authors used genetically modified fibroblasts, which can be forced to differentiate into muscle cells, and formulated 3D tissues with these cells and a biphasic material (hydrogel). The degrees of muscle differentiation and lipid deposition in culture were determined by immunohistochemical, biochemical, and molecular biological evaluations. Notably, the protocol successfully achieved the process of myogenic and lipogenic stimulation in the 3D tissues.

As addressed after the initial review process, the manuscript is clearly written with well-supportive figures. The study design is reasonable with adequate analysis. In the revised manuscript, the authors further discussed the ideas in terms of the approach using genetic modification for cell-based meat production. However, more careful considerations may still be helpful when actually using the technology for cultivated meat production.

difference of data samples and simulation samples

Genau genommen gründete Snell schon 1944 (Anregungen von Ernst Kapp aufgreifend) das „Archiv für griechische Lexikographie“ (vgl. dazu Lohse, Gerhard, Bruno Snell (1896–1986). Geisteswissenschaft und politische Erfahrung im 20. Jahrhundert, Göttingen 2023, 122–124), das 1950 zum Thesaurus linguae Graecae wurde, aus dem der Index Hippocraticus (1989 abgeschlossen) und das Lexikon des frühgriechischen Epos (2010 abgeschlossen) entstanden.

eLife assessment

This valuable work presents the latest version of CTFFIND, which is the most popular software for determination of the contrast transfer function (CTF) in cryo-electron microscopy. CTFFIND5 estimates and considers acquisition geometry and sample thickness, which leads to improved CTF determination. The paper describes convincing evidence that CTFFIND5 finds better CTF parameters than previous methods, in particular for tilted samples (e.g. for cryo-electron tomography) or where thickness is an issue (e.g. cellular samples, or electron microscopy at low voltages).

Reviewer #1 (Public Review):

This work presents CTFFIND5, a new version of the software for determination of the Contrast Transfer Function (CTF) that models the distortions introduced by the microscope in cryoEM images. CTFFIND5 can take acquisition geometry and sample thickness into consideration to improve CTF estimation.

To estimate tilt (tilt angle and tilt axis), the input image is split into tiles and correlation coefficients are computed between their power spectra and a local CTF model that includes the defocus variation according to a tilted plane. As a final step, by applying a rescaling factor to the power spectra of the tiles, an average tilt-corrected power spectrum is obtained and used for diagnostic purposes and to estimate the goodness of fit. This global procedure and the rescaling factor resemble those used in Bsoft, Warp, etc, with determination of the tilt parameters being a feature specific of CTFFIND5 (and formerly CTFTILT). The performance of the algorithm is evaluated with tilted 2D crystals and tilt-series, demonstrating accurate tilt estimation in some cases and some limitations in others. Further analysis of CTF determination with tilt-series, particularly showing whether there is accurate or stable estimation at high tilts, might be helpful to show the robustness of CTFFIND5 in cryoET.

CTFFIND5 represents the first CTF determination tool that considers the thickness-related modulation envelope of the CTF firstly described by McMullan et al. (2015) and experimentally confirmed by Tichelaar et al. (2020). To this end, CTFFIND5 uses a new CTF model that takes the sample thickness into account. CTFFIND5 thus provides more accurate CTF estimation and, furthermore, gives an estimation of the sample thickness, which may be a valuable resource to judge the potential for high resolution. To evaluate the accuracy of thickness estimation in CTFFIND5, the authors use the Lambert-Beer law on energy-filtered data and also tomographic data, thus demonstrating that the estimates are reasonable for images with exposure around 30 e/A2. While consideration of sample thickness in CTF determination sounds ideally suited for cryoET, practical application under the standard acquisition protocols in cryoET (exposure of 3-5 e/A2 per image) is still limited. In this regard, the authors are honest in the conclusions and clearly identify the areas where thickness-aware CTF determination will be valuable at present: e.g. in situ single particle analysis and in vitro single particle cryoEM of purified samples at low voltages.

In conclusion, the manuscript introduces novel methods inside CTFFIND5 that improve CTF estimation, namely acquisition geometry and sample thickness. The evaluation demonstrates the performance of the new tool, with fairly accurate estimates of tilt axis, tilt angle and sample thickness and improved CTF estimation. The manuscript critically defines the current range of application of the new methods in cryoEM.

Reviewer #2 (Public Review):

Summary:

This paper describes the latest version of the most popular program for CTF estimation for cryo-EM images: CTFFIND5. New features in CTFFIND5 are the estimation of tilt geometry, including for samples, like FIB-milled lamellae, that are pre-tilted along a different axis than the tilt axis of the tomographic experiment, plus the estimation of sample thickness from the expanded CTF model described by McMullan et al (2015). The results convincingly show the added value of the program for thicker and tilted images, such as are common in modern cryo-ET experiments. The program will therefore have a considerable impact on the field.

I have only minor suggestions for improvement below:

Abstract: "[CTF estimation] has been one of the key aspects of the resolution revolution"-> This is a bit over the top. Not much changed in the actual algorithms for CTF estimation during the resolution revolution.<br /> L34: "These parameters" -> Cs is typically given, only defocus (and if relevant phase shift) are estimated.<br /> L110-116: The text is ambiguous: are rotations defined clockwise or counter-clockwise? It would be good to explicitly state what subsequent rotations, in which directions and around which axes this transformation matrix (and the input/output angles in CTFFIND5) correspond to.<br /> L129-130: As a suggestion: it would be relatively easy, and possibly beneficial to the user, to implement a high-resolution limit that varies with the accumulated dose on the sample. One example of this exists in the tomography pipeline of RELION-5.<br /> Substituting Eq (7) into Eq (6) yields ksi=pi, which cannot be true. If t is the sample thickness, then how can this be a function of the frequency g of the first node of the CTF function? The former is a feature of the sample, the latter is a parameter of the optical system. This needs correction.

Reviewer #3 (Public Review):

In this manuscript, the authors detail improvements in the core CTFFIND (CTFFIND5 as implemented in cisTEM) algorithm that better estimates CTF parameters from titled micrographs and those that exhibit signal attenuation due to ice thickness. These improvements typically yield more accurate CTF values that better represent the data. Although some of the improvements result in slower calculations per micrograph, these can be easily overcome through parallelization.

There are some concerns outlined below that would benefit from further evaluation by the authors.

For the examples shown in Figure 3b, given the small differences in estimated defocus1 and 2, what type of improvements would be expected in the reconstructed tomograms? Do such improvements in estimates manifest in better tilt-series reconstruction?

Similarly, the data shown in Figure 3C shows minimal improvements in the CTF resolution estimate (e.g., 4.3 versus 4.2 Å), but exhibited several hundred Å difference in defocus values. How do such differences impact downstream processing? Is such a difference overcame by per-particle (local) CTF refinements (like the authors mention in the discussion, see below)?

At which point does the thickness of the specimen preclude the ice thickness modulation to be included for "accurate" estimate? 500Å? 1000Å? 2000Å? Based on the data shown in Figure 3B, as high as 969 Å thick specimens benefit moderately (4.6 versus 3.4 Å fit estimate), but perhaps not significantly, from the ice thickness estimation. Considering the increased computational time for ice thickness estimation, such an estimate of when to incorporate for single-particle workflows would be beneficial.

It would seem that this statement could be evaluated herein: "the analysis of images of purified samples recorded at lower acceleration voltages, e.g., 100 keV (McMullan et al., 2023), may also benefit since thickness-dependent CTF modulations will appear at lower resolution with longer electron wavelengths". There are numerous examples of 300kV, 200kV, and 100kV EMPIAR datasets to be compared and recommendations would be welcomed.

Although logical, this statement is not supported by the data presented in this manuscript: "The improvements of CTFFIND5 will provide better starting values for this refinement, yielding better overall CTF estimation and recovery of high-resolution information during 3D reconstruction."

Moreso, the lack of single-particle data evaluation does present a concern. Naively, these improvements would benefit all cryoEM data, regardless of modality.

eLife assessment

This work is of fundamental significance and has a compelling level of evidence for a new population that protects against obesity-induced hypothalamic inflammation. This topic will attract attention from a broad base of readers, from hypothalamic neuroscientists to immunologists with an interest in metabolism.

Reviewer #1 (Public Review):

Summary:

The present work from Velloso and collaborators investigated the transcription profiles of resident and recruited hypothalamic microglia. They found sex-dependent differences between males and females and identified the protective role of chemokine receptor CXCR3 against diet-induced obesity.

Strengths:

(1) Novelty<br /> (2) Relevance, since this work provides evidence about a subset of recruited microglia that has a protective effect against DIO. This provides a new concept in hypothalamic inflammation and obesity.

Weaknesses:

(1) Lack of mechanistic insight into the sex-dependent effects.<br /> (2) Analysis of indirect calorimetry data requires more depth.<br /> (3) A deeper analysis of hypothalamic inflammation and ER stress pathways would strengthen the manuscript.

Reviewer #2 (Public Review):

Summary:

This study by Mendes et al provides novel key insights into the role of chemotaxis and immune cell recruitment into the hypothalamus in the development of diet-induced obesity. Specifically, the authors reveal that although transcriptional changes in hypothalamic resident microglia following exposure to high-fat feeding are minor, there are compelling transcriptomic differences between resident microglia and microglia recruited to the hypothalamus, and these are sexually dimorphic. Using independent loss-of-function studies, the authors also demonstrate an important role of CXCR3 and hypothalamic CXCL10 in the hypothalamic recruitment of CCR2+ positive cells on metabolism following exposure to high-fat diet-feeding in mice. This manuscript puts forth conceptually novel evidence that inhibition of chemotaxis-mediated immune cell recruitment accelerates body weight gain in high-fat diet-feeding, suggesting that a subset of microglia that express CXCR3 may confer protective, anti-obesogenic effects.

Strengths:

The work is exciting and relevant given the prevalence of obesity and the consequences of inflammation in the brain on perturbations of energy metabolism and ensuant metabolic diseases. Hypothalamic inflammation is associated with disrupted energy balance, and activated microglia within the hypothalamus resulting from excessive caloric intake and saturated fatty acids are often thought to be mediators of impairment of hypothalamic regulation of metabolism. The present work reports a novel notion in which immune cells recruited into the hypothalamus that express chemokine receptor CXCR3 may have a protective role against diet-induced obesity. In vivo studies reported herein demonstrate that inhibition of CXCR3 exacerbates high-fat diet-induced body weight gain, increases circulating triglycerides and fasting glucose levels, worsens glucose tolerance, and increases the expression of orexigenic neuropeptides, at least in female mice.

This work provides a highly interesting and needed overview of preclinical and clinical brain inflammation, which is relevant to readers with an interest in metabolism and immunometabolism in the context of obesity.

Using flow cytometry, cell sorting, and transcriptomics including RNA-sequencing, the manuscript provides novel insights into transcriptional landscapes of resident and recruited microglia in the hypothalamus. Importantly, sex differences are investigated.

Overall, the manuscript is perceived to be highly interesting, relevant, and timely. The discussion is thoughtful, well-articulated, and a pleasure to read and felt to be of interest to a broad audience.

Weaknesses:

There were no major weaknesses perceived. Some comments for potential textual additions to the results/discussion are listed in recommendations to authors.

eLife assessment

This fundamental study advances our understanding of the cell specific treatment of cone photoreceptor degeneration by Txnip. The evidence supporting the conclusions is compelling with rigorous genetic manipulation of Txnip mutations. The work will be of broad interest to vision researchers, cell biologists and biochemists.

Reviewer #1 (Public Review):

Summary:

This is a follow-up study to the authors' previous eLife report about the roles of an alpha-arrestin called protein thioredoxin interacting protein (Txnip) in cone photoreceptors and in the retinal pigment epithelium. The findings are important because they provide new information about the mechanism of glucose and lactate transport to cone photoreceptors and because they may become the basis for therapies for retinal degenerative diseases.

Strengths:

Overall, the study is carefully done and, although the analysis is fairly comprehensive with many different versions of the protein analyzed, it is clearly enough described to follow. Figure 4 greatly facilitated my ability to follow, understand and interpret the study. The authors have appropriately addressed a few concerns about statistical significance and the relationship between their findings and previous studies of the possible roles of Txnip on GLUT1 expression and localization on the surfaces of RPE cells.

Reviewer #2 (Public Review):

The hard work of the authors is much appreciated. With overexpression of a-arrestin Txnip in RPE, cones and the combined respectively, the authors show a potential gene agnostic treatment that can be applied to retinitis pigmentosa. Furthermore, since Txnip is related to multiple intracellular signaling pathway, this study is of value for research in the mechanism of secondary cone dystrophy as well.

Strengths

- The follow-up study builds on innovative ground by exploring the impact of TxnipC247S and its combination with HSP90AB1 knockdown on cone survival, offering novel therapeutic pathways.<br /> - Testing of different Txnip deletion mutants provides a nuanced understanding of its functional domains, contributing valuable insights into the mechanism of action in RP treatment.<br /> - The findings regarding GLUT1 clearance and the differential effects of Txnip mutants on cone and RPE cells lay the groundwork for targeted gene therapy in RP.

Comments on revised version:

The researchers answered our questions and included additional discussion in the manuscript.

1 +

Connant is the physicist (cosmic ray specialist) and on night watch with the creature.

Những câu chuyện mang tính cảnh giác hoặc đạo đức, trong đó trẻ ngoan được khen thưởng và trẻ hư bị trừng phạt thích đáng, nhìn chung ít được quan tâm đến hình ảnh minh họa hơn so với truyện cổ tích. Nhiều cuốn sách về tôn giáo được viết dưới ảnh hưởng của những người theo đạo Tin lành Anh giáo, và chúng đã được xuất bản với số lượng lớn trong suốt thế kỷ 18 và đầu thế kỷ 19 bởi các công ty như Newbery và Marshall. Sự gia tăng nhanh chóng các ấn bản của những cuốn sách như Những bài hát thiêng liêng của Isaac Watts (1715; xem mèo. số 213) chứng tỏ sự phổ biến lâu dài của những tác phẩm biến các bài học tôn giáo thành một hình thức thú vị hơn. Trong số các tác giả nữ viết truyện sùng đạo hoặc truyện đạo đức đáng chú ý nhất ở Anh có Trimmer, Anna Laetitia Barbauld (1743–1825) và Mary Martha Sherwood.

Những câu chuyện cổ tích, cũng như những câu chuyện phiêu lưu nổi tiếng như Robinson Crusoe của Daniel Defoe (1719; cat. nos. 115, 121), thường gây ra nhiều lời chỉ trích trong thế kỷ 18 và đầu thế kỷ 19. Sarah Trimmer (1741–1810), một tác giả nổi tiếng về các cuốn sách dạy đạo đức, đã lên án “những sinh vật tưởng tượng dành cho trẻ em” trong bài phê bình Truyện cổ tích của Mẹ Bunch năm 1773.9 Thật vậy, mặc dù nhiều ấn bản sách giáo khoa của Perrault đã được xuất bản trong suốt thế kỷ 18, chúng nhìn chung bị lu mờ bởi những cuốn sách mang tính mô phạm hơn đề cập đến các vấn đề đạo đức hoặc tôn giáo. Mãi đến thế kỷ 19, truyện cổ tích mới thống trị thị trường sách thiếu nhi.

Hai thể loại quan trọng nhất của văn học thiếu nhi thế kỷ 18 là truyện cổ tích và truyện đạo đức. Truyện cổ tích được truyền từ thế hệ này sang thế hệ khác thông qua truyền miệng, lần đầu tiên được sưu tầm và in tại triều đình Pháp của Louis XIV bởi các nhà văn như Nữ bá tước d'Aulnoy, Madame de Villeneuve và Madame Le Prince de Beaumont. . Cuốn sách Histoires ou contes du temps passé năm 1697 của Charles Perrault (Những câu chuyện về cách đây đã lâu; xem cat. nos. 144, 146–48, 150, 192) chứa các phiên bản viết đầu tiên của "Cô bé lọ lem", "Người đẹp ngủ trong rừng", "Cô bé quàng khăn đỏ", “Blue Beard,” “Hop o' My Thumb,” và “Puss in Boots.”8 Phiên bản của Perrault về những câu chuyện này đã thống trị văn học thiếu nhi Anh và Mỹ kể từ thế kỷ thứ mười tám. Mặt trước của ấn bản gốc của ông (hình 11) là hình một bà già kể chuyện cho một nhóm trẻ em, với dòng chữ Contes de ma mère l'oye ("Truyện kể về mẹ ngỗng", một cách diễn đạt dân gian của Pháp gần tương đương với "chuyện xưa". chuyện của vợ”). Đây là lần xuất hiện đầu tiên của nhân vật mà sau này gắn liền với những bài đồng dao dành cho trẻ mẫu giáo khi hãng Newbery gắn tên vào một tuyển tập được xuất bản với tựa đề Giai điệu của Mẹ Ngỗng; hoặc, Sonnets for the Cradle (1781; cat. no. 154).

Thái độ mới đối với trẻ em và nền giáo dục của chúng bắt đầu phát triển vào cuối thế kỷ 17, khi nhiều nhà giáo dục kêu gọi quan tâm nhiều hơn đến những nhu cầu đặc biệt của trẻ và khi khái niệm về niềm vui học tập ngày càng được chấp nhận rộng rãi hơn.3 Biểu hiện rõ ràng nhất của sự phát triển ý tưởng này là các bài viết của triết gia John Locke (1632–1704) và Jean-Jacques Rousseau (1712–78). Năm 1693, Locke viết trong Một số suy nghĩ liên quan đến giáo dục rằng “trẻ em nên được đối xử như những sinh vật có lý trí…. Chúng không được bị cản trở để trở thành trẻ con, cũng như không được chơi và làm như trẻ con, mà là bị bệnh.” Rousseau coi tuổi thơ như một đứa trẻ. một trạng thái trong sáng và tự nhiên - khác biệt với trạng thái trưởng thành - và tin rằng mục tiêu trọng tâm của giáo dục phải là bảo tồn bản chất nguyên thủy của trẻ. Ông cũng tin rằng điều cần thiết là giáo viên phải nhìn sự việc như trẻ em nhìn.5 Các tác phẩm của Locke và Rousseau đã ảnh hưởng đến các nhà giáo dục Anh, và ý tưởng của họ cuối cùng đã dẫn tới một cách tiếp cận giáo dục nhân đạo hơn, trong đó niềm vui được coi là một phương tiện hỗ trợ cho việc học.

Một trong những thể loại tiểu thuyết lâu đời nhất, truyện ngụ ngôn (xem cat. nos. 15–32) ban đầu được đọc bằng tiếng Latinh trong lớp học hơn là để giải trí ở nhà. Những câu chuyện được cho là của Aesop (được cho là một người kể chuyện Hy Lạp ở thế kỷ thứ sáu trước Công nguyên nhưng gần như chắc chắn là một nhân vật huyền thoại) nằm trong số những câu chuyện được xuất bản và minh họa thường xuyên nhất (xem hình 5, 6). Truyện ngụ ngôn của Aesop được xuất bản trong bản dịch tiếng Anh đầu tiên của William Caxton (khoảng 1422–91) vào năm 1484. Nó nhanh chóng trở thành một trong những cuốn sách có tranh minh họa phổ biến nhất dành cho trẻ em, mặc dù trong nhiều ấn bản đầu tiên có rất ít nỗ lực chuyển thể các câu chuyện để tạo nên một tác phẩm hay. chúng dễ dàng hơn cho trẻ em hiểu và liên hệ với

Sách bảng chữ cái minh họa một trong những cách sử dụng hình ảnh sớm nhất trong sách hướng dẫn dành cho trẻ em (xem hình 2; mèo. số 33–77). Từ thế kỷ 16 cho đến tận thế kỷ 18, trẻ em học bảng chữ cái bằng cách nghiên cứu sách sừng (xem cat. nos. 33, 38, 39, 50), những mái chèo bằng gỗ có ghi các bảng chữ cái thường được kết hợp với các tác phẩm tôn giáo như Kinh Lạy Cha. Ngoài truyền thống sách kèn đã phát triển thêm battledore bằng hình ảnh (xem cat. nos. 41, 45, 47, 54, 56, 58, 59), một mảnh bìa cứng gấp lại với bảng chữ cái minh họa, được đặt tên theo một trò chơi truyền thống trong đó sách sừng dùng làm mái chèo. Battledore tồn tại cho đến giữa thế kỷ 19. Vào đầu thế kỷ 19, các loại trò chơi có hình ảnh minh họa khác đã được phát triển để dạy ABC cũng như toán, ngữ pháp và khoa học.

Para la actualidad la tecnología sigue evolucionando cada vez más y con ellos los programas educativos que se encuentran disponibles para cada usuario a menos de que sea una aplicación pagada, pero por lo general la mayoría de ellas están a nuestra disposición al momento que deseemos.

Can you add these as icons below this section so they stand out?

Like a logo icon bar where you list them side by side with a matching icon.

Can you possibly add a private podcast seeing that your students are busy mums? Just a thought. ;-)

Can you be more specific here around how many recipes and what type of recipes, as well as the benefits of having these?

This one also doesn't seem to apply

Delete this one too

This doesn't really apply because they get access to the replay, right? I'd just delete this and change it to something like... how long do I get access to the class for?

Change this to something like 'How the first aid skills you'll uncover have helped others'

I'd also delete this to keep it short and sharp

Change this to 'the xx-min medicinal first aid class video'

Shorten this to 'Get instant access to...'

delete this and change to 'Imagine solving those inevitable ouchies with the wisdom...'

And remove the code because it should link to the discounted checkout cart, right?

Cross out the $47 original price here

I'd delete the term 'the class' to keep it super concise

I would change the wording of the Deadline Funnel bar to 'Special One-Time Offer: SAVE $20 RIGHT NOW'

This comment is not properly reflected in Figure 2.6.1. Specifically, Figure 2.6.1 makes it appear that all n=3 electrons, whether s, p, or d, will shield at 0.35. While the table calls out that l less than the probe electron value (ie l=2 for 3d electron) receives a shielding constant of 1 (ie 3s and 3p electrons) even though the n value is the same for 3s, 3p, and 3d electrons.

Las investigaciones realizadas sobre el nivel de efectividad de la gamificación aumentaron de manera significativa, ya que muchos maestros empezaron a adoptar enfoques tecnológicos y mediante esta gamificación se logró identificar que los niños aumentaban el grado de motivación y rendimiento académico.

Imagery. Details of what is found around them, makes the reader feel like they are right there.

Personification. Stoves aren't actually capable of cooking themselves.

Genre: science fiction, dealt a lot with the idea of technology taking over. This story feels as if it was in the Postmodernist movement

Explaining what is going to be told in this collection of short stories. The stories that are on the illustrated man are the stories the narrator is going to see.

imagery, making the reader feel as if they are present in this part of the story.

Personification. Pictures don't actually move.

Overall I think this is my best source. They only reported actual facts and kept opinions out but still talked about the conspiracies but more importantly, why they started.

See this is an actual good quote. It isn't from a random internet sluth bored at home, but an actual royal spokesperson explaining things from their perspective.

Great fact to add. Seems like they are actually just reporting the facts.

This was a big part of the conspiracies.

Well of course if she was undergoing abdomen surgery I wouldn't want to be awake either. Weird thing to site.

Yes I am glad this was pointed out. When King Charles was sick he was still photographed all the time in public but Kate was not. I think that is what sparked the she was killed or kidnapped rumors.

Gossipy is a strong word choice. Could be pushing a narrative of people are too bored and nosey.

This wording was used in another article... weird choice and I do not know what it means.

Using old photos could be confusing for those who do not read the dates.

From what I know about Forbes is that they are a legitimate news source? Interested to see how this play out.

Los programas y herramientas tecnológicas han facilitado la forma de enseñar a los estudiante, por que hoy en día existe un sin número de actividades que se puede desarrollar de manera mas lúdica y creativa, de esta manera también logramos captar la concentración y atención de los estudiantes de forma mas sencilla y eficaz.

After a little more research, this article is not under the opinions page, which is just interesting to note based on the tones and wording used.

Again with these random quotes. This quote does not make me any more convinced because they asked a random Oklahoma lady.

Have not heard this yet. I think this article has some different information that the others.

These are some new theories not mentioned in the other articles.

A lot of big words used already two paragraphs in. I think they are trying to establish some kind of legitimacy?

What on Earth is this opening line.

Como se evidencia hoy en día, los avances tecnológicos son de gran ayuda en el ámbito educativo ya que son implementados en cada aula de clases para generar un mejor ambiente de aprendizaje, de igual manera cada docente busca mejorar su proceso de enseñanza-aprendizaje por medio de estos programas de innovación.

For a gossip site they do show a lot of evidence as to why they think what they think. Somewhat convincing.

The palace is a great source of evidence here.

No one ever mentioned that the palace said she would be out until Easter or else I think a lot of people would not have believed the conspiracies.

Most people know cosmo is not a "news" source in a sense, more just a gossip site, kind of like a better TMZ. But for those who don't know, this line could be misleading.

I know my topic is before people found out she has cancer and the truth, but this article looks back at the conspiracies which I think could be interesting.

I could see why the author may have took offense to the term "that" being used to describe the population of students at the school. It is dismissive, and almost others the population of students at the school, by labeling them as different from the "normal" or "typical" student bodies at other more affluent high schools. There is a largely negative connotation towards these students categorized as low-income in an urban classroom setting.

I'm more than halfway thought this article and this is the first mention of William. The title of the article is about what he says and thinks about all of this. Should have come up sooner.

This is the only article to say what actually happened and not in an accusatory tone. I was curious if the news sites just took it down because there was speculation so I am glad they cleared this up.

I get what Kate was trying to do but it does not take an expert to notice bad photoshop. Plus not putting her husband in the picture was a bad choice because it made more rumors.

I do like that this article is starting with the facts in the beginning and then listing the rumors.

A lot of the other article says jan 17th...

The journalist is saying we do these things and make up these elaborate stories because we are bored. Another factor of a conspiracy theory.

Making light of the situation and stating that these are not facts at all which is good because there are stupid people on the internet that would actually believe this.

Playing into the conspiracies by using the word ominous.

They are pointing out exactly what makes a conspiracy theory. I like that.

Niche audience, but this is exactly who reads Vogue though. They understand their audience. Also, makes it kinda known its an opinion piece right off the bat. Respectable.

This passage shed light on an interesting idea. Even though both of Alexander's parents felt that there may be racial prejudice in the school, Alexander's mom, Ms. Williams argued that Mr. Williams should not voice these concerns around Alexander. I could see how even if there was a concern about prejudice, Ms. Williams wanted Alexander to have an open mindset. She nonetheless kept a commitment to vigilance, as did Mr. Alexander.

The wording of this whole article seems harsh and like they want to prove a point...

This is one of the only article to say this. This is why the rumors started. Also, the royal family has updated the public every second of everyone else's health issues in the family, so I will admit since there were no updates that I believed in a conspiracy or two as well.

I remember people being way more suspicious after this video because she looked so different than before.

The whole article this Intelligencer magazine is so mean and critical of The Sun. Wonder what the relation is there.

Abbink, Jon. “Dam Controversies: Contested Governance and Developmental Discourse on the Ethiopian Omo River Dam.” Social Anthropology/Anthropologie Sociale 20, no. 2 (2012): 125–144.

Again a grasp for straws. I don't even see this one.

Bloom in March is such a grasp for straws because they clearly did not have ten things but wanted an article. Makes people look at the image and say "oh they are right" when it literally means nothing.

Why did they chose this man to quote? Anyone could look at the picture and say that. Also, why a professor of computer sciences? Why one at Cal Berkeley? Just a weird quote.

Yanking being the wording here is very harsh, making the news outlets that published the photos seem embarrassed or like they are covering something up.

Well not exactly... according to the post from Kate shown in the article, she did not clearly say this was photoshoped. She said she does experiment with editing sometimes but just says happy mother's day. Bad wording.

Abbink, Jon. “Dam Controversies: Contested Governance and Developmental Discourse on the Ethiopian Omo River Dam.” Social Anthropology/Anthropologie Sociale 20, no. 2 (2012): 125–144.

Leauthaud, Crystele, Stéphanie Duvail, Olivier Hamerlynck, Jean-Luc Paul, Hubert Cochet, Judith Nyunja, Jean Albergel, and Olivier Grünberger. “Floods and Livelihoods: The Impact of Changing Water Resources on Wetland Agro-Ecological Production Systems in the Tana River Delta, Kenya.” Global Environmental Change 23, no. 1 (February 1, 2013): 252–63. doi:10.1016/j.gloenvcha.2012.09.003.

"Water Harvesting has Added Benefit for Kenya - Less Flooding." AllAfrica.Com, Jan 03, 2020. https://ezproxy.lafayette.edu/login?url=https://www.proquest.com/wire-feeds/water-harvesting-has-added-benefit-kenya-less/docview/2334201005/se-2.

"Water Harvesting has Added Benefit for Kenya - Less Flooding." AllAfrica.Com, Jan 03, 2020. https://ezproxy.lafayette.edu/login?url=https://www.proquest.com/wire-feeds/water-harvesting-has-added-benefit-kenya-less/docview/2334201005/se-2.

"Water Harvesting has Added Benefit for Kenya - Less Flooding." AllAfrica.Com, Jan 03, 2020. https://ezproxy.lafayette.edu/login?url=https://www.proquest.com/wire-feeds/water-harvesting-has-added-benefit-kenya-less/docview/2334201005/se-2.

“Lead and Copper Rule.” EPA. Accessed April 9, 2024. https://www.epa.gov/dwreginfo/lead-and-copper-rule.

Katner, Adrienne, Kelsey J. Pieper, Yanna Lambrinidou, Komal Brown, Chih-Yang Hu, Howard W. Mielke, and Marc A. Edwards. “Weaknesses in Federal Drinking Water Regulations and Public Health Policies That Impede Lead Poisoning Prevention and Environmental Justice.” Environmental Justice 9, no. 4 (August 2016): 109–17. https://doi.org/10.1089/env.2016.0012.

Lubrano, Jonathon. “Water, Lead, and Environmental Justice: Easing the Flint Water Crisis With a Public Water Contamination Liability Fund.” William and Mary Environmental Law and Policy Review 42, no. 1 (January 1, 2017): 331. https://scholarship.law.wm.edu/cgi/viewcontent.cgi?article=1697&context=wmelpr.

Clark, Karen. “The Value of Water: The Flint Water Crisis as a Devaluation of Natural Resources, Not a Matter of Racial Justice.” Environmental Justice 9, no. 4 (August 2016): 99–102. https://doi.org/10.1089/env.2016.0007.

Rodgers, Dennis, and Bruce O’Neill. “Infrastructural Violence: Introduction to the Special Issue.” Ethnography 13, no. 4 (October 23, 2012): 401–12. https://doi.org/10.1177/1466138111435738.

London, J.K.; Fencl, A.L.; Watterson, S.; Choueiri, Y.; Seaton, P.; Jarin, J.; Dawson, M.; Aranda, A.; King, A.; Nguyen, P.; Pannu, C.; Firestone, L. and Bailey, C. “Disadvantaged unincorporated communities and the struggle for water justice in California.” Water Alternatives 14(2) (2021): 536. https://www.water-alternatives.org/index.php/alldoc/articles/vol14/v14issue2/626-a14-2-4/file

London, J.K.; Fencl, A.L.; Watterson, S.; Choueiri, Y.; Seaton, P.; Jarin, J.; Dawson, M.; Aranda, A.; King, A.; Nguyen, P.; Pannu, C.; Firestone, L. and Bailey, C. “Disadvantaged unincorporated communities and the struggle for water justice in California.” Water Alternatives 14(2) (2021): 534. https://www.water-alternatives.org/index.php/alldoc/articles/vol14/v14issue2/626-a14-2-4/file

London, J.K.; Fencl, A.L.; Watterson, S.; Choueiri, Y.; Seaton, P.; Jarin, J.; Dawson, M.; Aranda, A.; King, A.; Nguyen, P.; Pannu, C.; Firestone, L. and Bailey, C. “Disadvantaged unincorporated communities and the struggle for water justice in California.” Water Alternatives 14(2) (2021): 529. https://www.water-alternatives.org/index.php/alldoc/articles/vol14/v14issue2/626-a14-2-4/file

The United States Congress, Environmental Protection Agency, Safe Drinking Water Act, 104th Congress, Washington D.C., 1974 https://www.epa.gov/sdwa

Henry, Lois, “Contamination a looming possibility for groundwater bank to be built using public funds”, San Joaquin Valley Water (February 24, 2022). https://sjvwater.org/contamination-a-looming-possibility-for-groundwater-bank-to-be-built-using-public-funds/

“Actions for Cleaner Water., United States Environmental Protection Agency (May 17, 2023). https://www.epa.gov/sanjoaquinvalley/actions-cleaner-water.

“Actions for Cleaner Water., United States Environmental Protection Agency (May 17, 2023). https://www.epa.gov/sanjoaquinvalley/actions-cleaner-water.

The United States Congress, Environmental Protection Agency, Safe Drinking Water Act, 104th Congress, Washington D.C., 1974 https://www.epa.gov/sdwa

“Human Right to Water”, California Department of Water Resources, https://water.ca.gov/Programs/All-Programs/Human-Right-to-Water#:~:text=California%20was%20the%20first%20state,%2C%20cooking%2C%20and%20sanitary%20purposes.

Angel Santiago Fernandez-Bou, José M. Rodríguez-Flores, Alexander Guzman, J. Pablo Ortiz-Partida, Leticia M. Classen-Rodriguez, Pedro A. Sánchez-Pérez, Jorge Valero-Fandiño, Chantelise Pells, Humberto Flores-Landeros, Samuel Sandoval-Solís, Gregory W. Characklis, Thomas C. Harmon, Michael McCullough, Josué Medellín-Azuara. “Water, environment, and socioeconomic justice in California: A multi-benefit cropland repurposing framework” Science of The Total Environment, Volume 858, Part 3 (February 1, 2023), 340 and 343. https://doi.org/10.1016/j.scitotenv.2022.159963

“New Drinking Water Report: Communities of Color More Likely to Suffer Drinking Water Violations For Years” National Resources Defence Council, September 24, 2019 https://www.nrdc.org/press-releases/new-drinking-water-report-communities-color-more-likely-suffer-drinking-water

Huang, Ganlin, and Jonathan K. London. "Cumulative Environmental Vulnerability and Environmental Justice in California’s San Joaquin Valley" International Journal of Environmental Research and Public Health9, no. 5: 1600 (March 19, 2012). https://doi.org/10.3390/ijerph9051593

Huang, Ganlin, and Jonathan K. London. "Cumulative Environmental Vulnerability and Environmental Justice in California’s San Joaquin Valley" International Journal of Environmental Research and Public Health9, no. 5: 1600 (March 19, 2012). https://doi.org/10.3390/ijerph9051593

Boyden, Hollynd, Mayela Gillan, Javier Molina, Ashok Gadgil, and Winston Tseng. "Community Perceptions of Arsenic Contaminated Drinking Water and Preferences for Risk Communication in California’s San Joaquin Valley" International Journal of Environmental Research and Public Health 20, no. 1: 813 (January 1, 2023). https://doi.org/10.3390/ijerph20010813

Balazs, Caroline L., Rachel Morello-Frosch, Alan Hubbard, and Isha Ray. “Just Water?: Social Disparities and Drinking Water Quality in California’s San Joaquin Valley”, (University of California, Berkeley, 2011): 32. https://www.proquest.com/docview/1526000633?pq-origsite=gscholar&fromopenview=true&sourcetype=Dissertations%20&%20Theses

Cole, Luke, and Susan Senger Bowyer. “Pesticides and the Poor in California.” Race, Poverty & the Environment 2, no. 1 (1991): 17. http://www.jstor.org/stable/41555051.

“Farmworkers in California: A Brief Introduction”, California Research Bureau, October 2013, 1. https://latinocaucus.legislature.ca.gov/sites/latinocaucus.legislature.ca.gov/files/CRB%20Report%20on%20Farmworkers%20in%20CA%20S-13-017.pdf

London, J.K.; Fencl, A.L.; Watterson, S.; Choueiri, Y.; Seaton, P.; Jarin, J.; Dawson, M.; Aranda, A.; King, A.; Nguyen, P.; Pannu, C.; Firestone, L. and Bailey, C. “Disadvantaged unincorporated communities and the struggle for water justice in California.” Water Alternatives 14(2) (2021): 533. https://www.water-alternatives.org/index.php/alldoc/articles/vol14/v14issue2/626-a14-2-4/file

“New Drinking Water Report: Communities of Color More Likely to Suffer Drinking Water Violations For Years” National Resources Defence Council, September 24, 2019 https://www.nrdc.org/press-releases/new-drinking-water-report-communities-color-more-likely-suffer-drinking-water

U.S. Census Bureau, “Population by Race (San Joaquin County)”, San Joaquin Council of Governments, 2020. https://www.sjcog.org/236/Demographics

Matthew 6:34 “Take therefore no thought for the morrow: for the morrow shall take thought for the things of itself. Sufficient unto the day is the evil thereof.”

I did not previously think about how age impacts the way you experience poverty, but I can see how this may be true. When we are little kids, we are still unaware of a lot of the different facets of identity that set us apart, and are more likely to be open minded to more things-- we are still very easily impressionable. As we become older though, and cliques form, there is a way clearer understanding of what may be deemed as cool or desirable for teenagers and what is embarrassing or something to be ashamed of.

To me this passage ties back to flawed pedagogical structures in k-12 schools; so often the academics and even fun seeming extracurricular activities are tailored to a white middle class (nuclear families). They do not seem to be inclusive or take into consideration that certain children may not have or live with fathers, and that a Father's Day activity might not feel right to them.

Blake, Sarah B., “Spatial Relationships among Dairy Farms, Drinking Water Quality, and Maternal-Child Health Outcomes in the San Joaquin Valley.” Public Health Nursing, 31 (2014): 492. https://doi.org/10.1111/phn.12166

“Water & Health in the Valley: Nitrate Concentration of Drinking Water and the Health of San Joaquin Valley Residents”, California State Water Resources Control Board, 5408 and 5410. https://www.waterboards.ca.gov/public_notices/petitions/water_quality/docs/a2239/overview/Documents/AR-Docs%20(296).pdf

Gunier, Robert B., Martha E. Harnly, Peggy Reynolds, Andrew Hertz, and Julie Von Behren. “Agricultural Pesticide Use in California: Pesticide Prioritization, Use Densities, and Population Distributions for a Childhood Cancer Study.” Environmental Health Perspectives 109, no. 10 (2001): 1071–78. https://doi.org/10.2307/3454963.

Boyden, Hollynd, Mayela Gillan, Javier Molina, Ashok Gadgil, and Winston Tseng. "Community Perceptions of Arsenic Contaminated Drinking Water and Preferences for Risk Communication in California’s San Joaquin Valley" International Journal of Environmental Research and Public Health 20, no. 1: 813 (January 1, 2023). https://doi.org/10.3390/ijerph20010813

Meadows, Robin, “Living in California’s San Joaquin Valley May Harm Your Health”, The New Humanitarian, July 5, 2017. https://deeply.thenewhumanitarian.org/water/articles/2017/07/05/living-in-californias-san-joaquin-valley-may-harm-your-health

Meadows, Robin, “Living in California’s San Joaquin Valley May Harm Your Health”, The New Humanitarian, July 5, 2017. https://deeply.thenewhumanitarian.org/water/articles/2017/07/05/living-in-californias-san-joaquin-valley-may-harm-your-health

Cole, Luke, and Susan Senger Bowyer. “Pesticides and the Poor in California.” Race, Poverty & the Environment 2, no. 1 (1991): 16. http://www.jstor.org/stable/41555051.

“Water & Health in the Valley: Nitrate Concentration of Drinking Water and the Health of San Joaquin Valley Residents”, California State Water Resources Control Board, 5409. https://www.waterboards.ca.gov/public_notices/petitions/water_quality/docs/a2239/overview/Documents/AR-Docs%20(296).pdf

“Water & Health in the Valley: Nitrate Concentration of Drinking Water and the Health of San Joaquin Valley Residents”, California State Water Resources Control Board, 5410. https://www.waterboards.ca.gov/public_notices/petitions/water_quality/docs/a2239/overview/Documents/AR-Docs%20(296).pdf

Diekman, Kristine, “Sandra Garcia, Fighting for Clean Water”, Run Dry, last modified 2017, http://www.kristinediekman.net/run-dry-1

I thought this was a very interesting point! I can absolutely see the way that certain school cultures can shape a student's understanding of their capabilities or influence them to think they fit into a certain mold. I think there is definitely room for reform in these types of pedagogical structures and biases. Empowering students in underprivileged schools could be great way to help them realize their full potential and not feel as stuck in a box of label as being low SES.

the landlord laughs as because they are foreigners they do not understand the lands local currency, implying they are not to pay because they do not have money from the kingdom. This highlights the hospitability of the innkeeper and suggestive of the cultural norms within the society

highlights the value and principles of individual freedom, but also the king warns them of the challenges they will face if they attempt to leave venice.

the landlord laughs as because they are foreigners they do not understand the lands local currency, implying they are not to pay because they do not have money from the kingdom. This highlights the hospitability of the innkeeper and suggestive of the cultural norms within the society

抗议的结果

I think that it is important to have a team working together to ensure the best practices for the student. It take a village and everyone needs to working to ensure the best support.

This is very important and not just because it is in bold print. This statement helps to create a positive learning community where all learners feel valued. This leads to an enjoyment of learning, as well as, a culturally rich classroom which helps to promote diversity and inclusion.

This finding makes a lot of sense to me-- over 1/3 of families living in poverty experienced six or more risk factors. Thus, with risk factors happening, poverty a lot more likely to occur ( this made me think back to situational poverty, and how one risky or unpredictable situation can land an individual in poverty so quickly). I think all of the risk factors-- emotional and social challenges, acute and chronic stressors, cognitive lags, and health and safety issues-- can become escalated and have the potential to result in a family being in poverty.

I remember when everyone used to hate Rebecca Black because of this song. However, I have recently seen that her other music is extremely critically acclaimed and has one of the "best pop albums" of the 2000s. How do we alter social media to not allow something to impact an artists' impact and artistry?

This demonstrates that the government did not follow the constitution, because if they had, they would have known that this implied that slavery was unconstitutional.

Important because it describes what the federal government did enact.

South Carolina seceded from the United States.

In other words, if an African American flees to another state where they may have greater freedom, if captured, they will be returned to their masters in the state from which they escaped.

Yes

Real

Heck yeah

Rodgers, Dennis, and Bruce O’Neill. “Infrastructural Violence: Introduction to the Special Issue.” Ethnography 13, no. 4 (October 23, 2012): 401–12. https://doi.org/10.1177/1466138111435738., 402.

Rodgers, Dennis, and Bruce O’Neill. “Infrastructural Violence: Introduction to the Special Issue.”, 402

Fearnside, Philip M. “The Roles and Movements of Actors in the Deforestation of Brazilian Amazonia.”, 23.

Fearnside, Philip M. “The Roles and Movements of Actors in the Deforestation of Brazilian Amazonia.” Ecology and Society 13, no. 1 (2008).