The perception of disability is alwayws displayed eith a negative connotation. If people are disabled, they looked poorly, not strong, not normal"

How could this idea change how scientists model or plan for the ecological effects of climate change? Specifically, since the decline in the murre population was due to behavior changes—such as earlier mosquito emergence and altered polar bear foraging—rather than species migration, what challenges does this create for conservation planning and prediction models that usually focus on range shifts or habitat loss?

This section about bear predation highlights the ecological effects of climate change at different levels. The bears’ move from feeding in the ocean to foraging on land shows that the loss of sea ice is not only a physical change; it also affects behavior and ecology, disrupting entire food webs. The researchers noted that gull and fox hunting patterns changed because of the scavenging opportunities presented by bears. This indicates an unexpected temporary buffer on murre egg predation, reminding us that the impacts of climate change are not always straightforward or fully negative in the short term. However, the long-term outlook is bleak. Bears’ ongoing dependence on seabird colonies is not sustainable for either species. This section encourages us to think about how apex predators, when displaced by climate change, can unintentionally increase stress on already fragile ecosystems

What evidence suggests that mosquito parasitism was the primary cause of adult murre deaths in 2011, and how might limited visibility of the colony have affected the accuracy of mortality estimates?

How might the location of northern Hudson Bay at the transition between the Low and High Arctic make its ecosystems more vulnerable to climate-driven shifts in species composition and food web structure?

This section about mosquito parasitism clearly shows how even small organisms like mosquitoes can become significant ecological stressors when climate conditions change. The murres’ strong attachment to their nest sites and their dedicated parenting, traits that are usually beneficial, became problems due to the new heat and insect pressure. What stands out is that this illustrates a behaviorally driven effect of climate change. The mosquitoes didn’t migrate to a new area; instead, they changed their timing and intensity because of warming. This challenges the simple idea that climate change primarily affects species through movements toward the poles. It also emphasizes the vulnerability of highly specialized Arctic species, whose breeding habits offer little flexibility to handle sudden, new stressors.

Researchers found unusually high bird mortality upon arrival, signaling a potential environmental or biological disturbance that year.

This shows how different ecological interactions occur together and are influenced by climate change. It creates the idea that multiple stressors can act at the same time, demonstrating the complex effects of environmental change on species.

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eLife Assessment

This study presents results supporting a model that tumorous germline stem cells (GSCs) in the Drosophila ovary mimic the stem cell niche and inhibit the differentiation of neighboring cells. The valuable findings show that GSC tumors often contain non-mutant cells whose differentiation is suppressed by the GSC tumorous cells. However, the evidence showing that the GSC tumors produce BMP ligands to suppress differentiation of non-mutant cells is incomplete. It could be strengthened by the use of sensitive RNA in situ hybridization approaches.

Reviewer #1 (Public review):

Summary:

This preprint from Shaowei Zhao and colleagues presents results that suggest tumorous germline stem cells (GSCs) in the Drosophila ovary mimic the ovarian stem cell niche and inhibit the differentiation of neighboring non-mutant GSC-like cells. The authors use FRT-mediated clonal analysis driven by a germline-specific gene (nos-Gal4, UASp-flp) to induce GSC-like cells mutant for bam or bam's co-factor bgcn. Bam-mutant or bgcn-mutant germ cells produce tumors in the stem cell compartment (the germarium) of the ovary (Figure 1). These tumors contain non-mutant cells - termed SGC for single-germ cells. 75% of SGCs do not exhibit signs of differentiation (as assessed by bamP-GFP) (Figure 2). The authors demonstrate that block in differentiation in SGC is a result of suppression of bam expression (Figure 2). They present data suggesting that in 73% of SGCs, BMP signaling is low (assessed by dad-lacZ) (Figure 3) and proliferation is less in SGCs vs GSCs. They present genetic evidence that mutations in BMP pathway receptors and transcription factors suppress some of the non-autonomous effects exhibited by SGCs within bam-mutant tumors (Figure 4). They show data that bam-mutant cells secrete Dpp, but this data is not compelling (see below) (Figure 5). They provide genetic data that loss of BMP ligands (dpp and gbb) suppresses the appearance of SGCs in bam-mutant tumors (Figure 6). Taken together, their data support a model in which bam-mutant GSC-like cells produce BMPs that act on non-mutant cells (i.e., SGCs) to prevent their differentiation, similar to what is seen in the ovarian stem cell niche. This preprint from Shaowei Zhao and colleagues presents results that suggest tumorous germline stem cells (GSCs) in the Drosophila ovary mimic the ovarian stem cell niche and inhibit the differentiation of neighboring non-mutant GSC-like cells. The authors use FRT-mediated clonal analysis driven by a germline-specific gene (nos-Gal4, UASp-flp) to induce GSC-like cells mutant for bam or bam's co-factor bgcn. Bam-mutant or bgcn-mutant germ cells produce tumors in the stem cell compartment (the germarium) of the ovary (Figure 1). These tumors contain non-mutant cells - termed SGC for single-germ cells. 75% of SGCs do not exhibit signs of differentiation (as assessed by bamP-GFP) (Figure 2). The authors demonstrate that block in differentiation in SGC is a result of suppression of bam expression (Figure 2). They present data suggesting that in 73% of SGCs, BMP signaling is low (assessed by dad-lacZ) (Figure 3) and proliferation is less in SGCs vs GSCs. They present genetic evidence that mutations in BMP pathway receptors and transcription factors suppress some of the non-autonomous effects exhibited by SGCs within bam-mutant tumors (Figure 4). They show data that bam-mutant cells secrete Dpp, but this data is not compelling (see below) (Figure 5). They provide genetic data that loss of BMP ligands (dpp and gbb) suppresses the appearance of SGCs in bam-mutant tumors (Figure 6). Taken together, their data support a model in which bam-mutant GSC-like cells produce BMPs that act on non-mutant cells (i.e., SGCs) to prevent their differentiation, similar to what in seen in the ovarian stem cell niche.

Strengths:

(1) Use of an excellent and established model for tumorous cells in a stem cell microenvironment.

(2) Powerful genetics allow them to test various factors in the tumorous vs non-tumorous cells.

(3) Appropriate use of quantification and statistics.

Weaknesses:

(1) What is the frequency of SGCs in nos>flp; bam-mutant tumors? For example, are they seen in every germarium, or in some germaria, etc, or in a few germaria?

(2) Does the breakdown in clonality vary when they induce hs-flp clones in adults as opposed to in larvae/pupae?

(3) Approximately 20-25% of SGCs are bam+, dad-LacZ+. Firstly, how do the authors explain this? Secondly, of the 70-75% of SGCs that have no/low BMP signaling, the authors should perform additional characterization using markers that are expressed in GSCs (i.e., Sex lethal and nanos).

(4) All experiments except Figure 1I (where a single germarium with no quantification) were performed with nos-Gal4, UASp-flp. Have the authors performed any of the phenotypic characterizations (i.e., figures other than Figure 1) with hs-flp?

(5) Does the number of SGCs change with the age of the female? The experiments were all performed in 14-day-old adult females. What happens when they look at a young female (like 2-day-old). I assume that the nos>flp is working in larval and pupal stages, and so the phenotype should be present in young females. Why did the authors choose this later age? For example, is the phenotype more robust in older females? Or do you see more SGCs at later time points?

(6) Can the authors distinguish one copy of GFP versus 2 copies of GFP in germ cells of the ovary? This is not possible in the Drosophila testis. I ask because this could impact the clonal analyses diagrammed in Figure 4A and 4G and in 6A and B. Additionally, in most of the figures, the GFP is saturated, so it is not possible to discern one vs two copies of GFP.

(7) More evidence is needed to support the claim of elevated Dpp levels in bam or bgcn mutant tumors. The current results with the dpp-lacZ enhancer trap in Figure 5A, B are not convincing. First, why is the dpp-lacZ so much brighter in the mosaic analysis (A) than in the no-clone analysis (B)? It is expected that the level of dpp-lacZ in cap cells should be invariant between ovaries, and yet LacZ is very faint in Figure 5B. I think that if the settings in A matched those in B, the apparent expression of dpp-lacZ in the tumor would be much lower and likely not statistically significant. Second, they should use RNA in situ hybridization with a sensitive technique like hybridization chain reactions (HCR) - an approach that has worked well in numerous Drosophila tissues, including the ovary.

(8) In Figure 6, the authors report results obtained with the bamBG allele. Do they obtain similar data with another bam allele (i.e., bamdelta86)?

Reviewer #2 (Public review):

While the study by Zhang et al. provides valuable insights into how germline tumors can non-autonomously suppress the differentiation of neighboring wild-type germline stem cells (GSCs), several conceptual and technical issues limit the strength of the conclusions.

Major points:

(1) Naming of SGCs is confusing. In line 68, the authors state that "many wild-type germ cells located outside the niche retained a GSC-like single-germ-cell (SGC) morphology." However, bam or bgcn mutant GSCs are also referred to as "SGCs," which creates confusion when reading the text and interpreting the figures. The authors should clarify the terminology used to distinguish between wild-type SGCs and tumor (bam/bgcn mutant) SGCs, and apply consistent naming throughout the manuscript and figure legends.

a) The same confusion appears in Figure 2. It is unclear whether the analyzed SGCs are wild-type or bam mutant cells. If the SGCs analyzed are Bam mutants, then the lack of Bam expression and failure to differentiate would be expected and not informative. However, if the SGCs are wild-type GSCs located outside the niche, then the observation would suggest that Bam expression is silenced in these wild-type cells, which is a significant finding. The authors should clarify the genotype of the SGCs analyzed in Figure 2C, as this information is not currently provided.

b) In Figures 4B and 4E, the analysis of SGC composition is confusing. In the control germaria (bam mutant mosaic), the authors label GFP⁺ SGCs as "wild-type," which makes interpretation unclear. Note, this is completely different from their earlier definition shown in line 68.

c) Additionally, bam⁺/⁻ GSCs (the first bar in Figure 4E) should appear GFP⁺ and Red⁺ (i.e., yellow). It would be helpful if the authors could indicate these bam⁺/⁻ germ cells directly in the image and clarify the corresponding color representation in the main text. In Figure 2A, although a color code is shown, the legend does not explain it clearly, nor does it specify the identity of bam⁺/⁻ cells alone. Figure 4F has the same issue, and in this graph, the color does not match Figure 4A.

(2) The frequencies of bam or bgcn mutant mosaic germaria carrying [wild-type] SGCs or wild-type germ cell cysts with branched fusomes, as well as the average number of wild-type SGCs per germarium and the number of days after heat shock for the representative images, are not provided when Figure 1 is first introduced. Since this is the first time the authors describe these phenotypes, including these details is essential. Without this information, it is difficult for readers to follow and evaluate the presented observations.

(3) Without the information mentioned in point 2, it causes problems when reading through the section regarding [wild-type] SGCs induced by impairment of differentiation or dedifferentiation. In lines 90-97, the authors use the presence of midbodies between cystocytes as a criterion to determine whether the wild-type GSCs surrounded by tumor GSCs arise through dedifferentiation. However, the cited study (Mathieu et al., 2022) reports that midbodies can be detected between two germ cells within a cyst carrying a branched fusome upon USP8 loss.

a) Are wild-type germ cell cysts with branched fusomes present in the bam mutant mosaic germaria? What is the proportion of germaria containing wild-type SGCs versus those containing wild-type germ cell cysts with branched fusomes?

b) If all bam mutant mosaic germaria carry only wild-type GSCs outside the niche and no germaria contain wild-type germ cell cysts with branched fusomes, then examining midbodies as an indicator of dedifferentiation may not be appropriate.

c) If, however, some germaria do contain wild-type germ cell cysts with branched fusomes, the authors should provide representative images and quantify their proportion.

d) In line 95, although the authors state that 50 germ cell cysts were analyzed for the presence of midbodies, it would be more informative to specify how many germaria these cysts were derived from and how many biological replicates were examined.

(4) Note that both bam mutant GSCs and wild-type SGCs can undergo division to generate midbodies (double cells), as shown in Figure 4H. Therefore, the current description of the midbody analysis is confusing. The authors should clarify which cell types were examined and explain how midbodies were interpreted in distinguishing between cell division and differentiation.

(5) The data in Figure 5 showing Dpp expression in bam mutant tumorous GSCs are not convincing. The Dpp-lacZ signal appears broadly distributed throughout the germarium, including in escort cells. To support the claim more clearly, the authors should present corresponding images for Figures 5D and 5E, in which dpp expression was knocked down in the germ cells of bam or bgcn mutant mosaic germaria. Showing these images would help clarify the localization and specificity of Dpp-lacZ expression relative to the tumorous GSCs.

(6) While Figure 6 provides genetic evidence that bam mutant tumorous GSCs produce Dpp to inhibit the differentiation of wild-type SGCs, it should be noted that these analyses were performed in a dpp⁺/⁻ background. To strengthen the conclusion, the authors should include appropriate controls showing [dpp⁺/⁻; bam⁺/⁻] SGCs and [dpp⁺/⁻; bam⁺/⁻] germ cell cysts without heat shock (as referenced in Figures 6F and 6I).

(7) Previous studies have reported that bam mutant germ cells cause blunted escort cell protrusions (e.g., Kirilly et al., Development, 2011), which are known to contribute to germ cell differentiation (e.g., Chen et al., Frontiers in Cell and Developmental Biology, 2022). The authors should include these findings in the Discussion to provide a broader context and to acknowledge how alterations in escort cell morphology may further influence differentiation defects in their model.

(8) Since fusome morphology is an important readout of SGCs vs differentiation. All the clonal analysis should have fusome staining.

(9) Figure arrangement. It is somewhat difficult to identify the figure panels cited in the text due to the current panel arrangement.

(10) The number of biological replicates and germaria analyzed should be clearly stated somewhere in the manuscript-ideally in the Methods section or figure legends. Providing this information is essential for assessing data reliability and reproducibility.

Reviewer #3 (Public review):

Summary:

Zhang et al. investigated how germline tumors influence the development of neighboring wild-type (WT) germline stem cells (GSC) in the Drosophila ovary. They report that germline tumors inhibit the differentiation of neighboring WT GSCs by arresting them in an undifferentiated state, resulting from reduced expression of the differentiation-promoting factor Bam. They find that these tumor cells produce low levels of the niche-associated signaling molecules Dpp and Gbb, which suppress bam expression and consequently inhibit the differentiation of neighboring WT GSCs non-cell-autonomously. Based on these findings, the authors propose that germline tumors mimic the niche to suppress the differentiation of the neighboring stem cells.

Strengths:

This study addresses an important biological question concerning the interaction between germline tumor cells and WT germline stem cells in the Drosophila ovary. If the findings are substantiated, they could provide valuable insights applicable to other stem cell systems.

Weaknesses:

Previous work from Xie's lab demonstrated that bam and bgcn mutant GSCs can outcompete WT GSCs for niche occupancy. Furthermore, a large body of literature has established that the interactions between escort cells (ECs) and GSC daughters are essential for proper and timely germline differentiation (the differentiation niche). Disruption of these interactions leads to arrest of germline cell differentiation in a status with weak BMP signaling activation and low bam expression, a phenotype virtually identical to what is reported here.

Thus, it remains unclear whether the observed phenotype reflects "direct inhibition by tumor cells" or "arrested differentiation due to the loss of the differentiation niche". Because most data were collected at a very late stage (more than 10 days after clonal induction), when tumor cells already dominate the germarium, this question cannot be solved. To distinguish between these two possibilities, the authors could conduct a time-course analysis to examine the onset of the WT GSC-like single-germ-cell (SGC) phenotype and determine whether early-stage tumor clones with a few tumor cells can suppress the differentiation of neighboring WT GSCs with only a few tumor cells present. If tumor cells indeed produce Dpp and Gbb (as proposed here) to inhibit the differentiation of neighboring germline cells, a small cluster or probably even a single tumor cell generated at an early stage might prevent the differentiation of their neighboring germ cells.

The key evidence supporting the claim that tumor cells produce Gpp and Gbb comes from Figures 5 and 6, which suggest that tumor-derived dpp and gbb are required for this inhibition. However, interpretation of these data requires caution.

In Figure 5, the authors use dpp-lacZ to support the claim that dpp is upregulated in tumor cells (Figure 5A and 5B). However, the background expression in somatic cells (ECs and pre-follicular cells) differs noticeably between these panels. In Figure 5A, dpp-lacZ expression in somatic cells in 5A is clearly higher than in 5B, and the expression level in tumor cells appears comparable to that in somatic cells (dpp-lacZ single channel). Similarly, in Figure 5B, dpp-lacZ expression in germline cells is also comparable to that in somatic cells. Providing clear evidence of upregulated dpp and gbb expression in tumor cells (for example, through single-molecular RNA in situ) would be essential.

Most tumor data present in this study were collected from the bam[86] null allele, whereas the data in Figure 6 were derived from a weaker bam[BG] allele. This bam[BG] allele is not molecularly defined and shows some genetic interaction with dpp mutants. As shown in Figure 6E, removal of dpp from homozygous bam[BG] mutant leads to germline differentiation (evidenced by a branched fusome connecting several cystocytes, located at the right side of the white arrowhead). In Figure 6D, fusome is likely present in some GFP-negative bam[BG]/bam[BG] cells. To strengthen their claim that the tumor produces Dpp and Gbb to inhibit WT germline cell differentiation, the authors should repeat these experiments using the bam[86] null allele.

It is well established that the stem niche provides multiple functional supports for maintaining resident stem cells, including physical anchorage and signaling regulation. In Drosophila, several signaling molecules produced by the niche have been identified, each with a distinct function - some promoting stemness, while others regulate differentiation. Expression of Dpp and Gbb alone does not substantiate the claim that these tumor cells have acquired the niche-like property. To support their assertion that these tumors mimic the niche, the authors should provide additional evidence showing that these tumor cells also express other niche-associated markers. Alternatively, they could revise the manuscript title to more accurately reflect their findings.

In the Method section, the authors need to provide details on how dpp-lacZ expression levels were quantified and normalized.

Author response:

Reviewer #1 (Public review):

Summary:

This preprint from Shaowei Zhao and colleagues presents results that suggest tumorous germline stem cells (GSCs) in the Drosophila ovary mimic the ovarian stem cell niche and inhibit the differentiation of neighboring non-mutant GSC-like cells. The authors use FRT-mediated clonal analysis driven by a germline-specific gene (nos-Gal4, UASp-flp) to induce GSC-like cells mutant for bam or bam's cofactor bgcn. Bam-mutant or bgcn-mutant germ cells produce tumors in the stem cell compartment (the germarium) of the ovary (Figure 1). These tumors contain non-mutant cells - termed SGC for single-germ cells. 75% of SGCs do not exhibit signs of differentiation (as assessed by bamP-GFP) (Figure 2). The authors demonstrate that block in differentiation in SGC is a result of suppression of bam expression (Figure 2). They present data suggesting that in 73% of SGCs, BMP signaling is low (assessed by dad-lacZ) (Figure 3) and proliferation is less in SGCs vs GSCs. They present genetic evidence that mutations in BMP pathway receptors and transcription factors suppress some of the non-autonomous effects exhibited by SGCs within bam-mutant tumors (Figure 4). They show data that bam-mutant cells secrete Dpp, but this data is not compelling (see below) (Figure 5). They provide genetic data that loss of BMP ligands (dpp and gbb) suppresses the appearance of SGCs in bam-mutant tumors (Figure 6). Taken together, their data support a model in which bam-mutant GSC-like cells produce BMPs that act on nonmutant cells (i.e., SGCs) to prevent their differentiation, similar to what is seen in the ovarian stem cell niche. 

Strengths:

(1) Use of an excellent and established model for tumorous cells in a stem cell microenvironment.

(2) Powerful genetics allow them to test various factors in the tumorous vs nontumorous cells.

(3) Appropriate use of quantification and statistics.

We greatly appreciate these comments.

Weaknesses:

(1) What is the frequency of SGCs in nos>flp; bam-mutant tumors? For example, are they seen in every germarium, or in some germaria, etc, or in a few germaria?

This is a great question. Because the SGC phenotype depends on the presence of germline tumor clones, our quantification was restricted to germaria that contained them.These quantification data ("SGCs and/or germline cysts per germarium with germline clones") will be presented in the revised Figure 1.

(2) Does the breakdown in clonality vary when they induce hs-flp clones in adults as opposed to in larvae/pupae?

Our initial attempts to induce ovarian hs-flp germline clones by heat-shocking adult flies were unsuccessful, with very few clones being observed. Therefore, we shifted our approach to an earlier developmental stage. Successful induction was achieved by subjecting late-L3/early-pupal animals to a twice-daily heatshock at 37°C for 6 consecutive days (2 hours per session with a 6-hour interval, see Lines 325-329) (Zhao et al., 2018).

(3) Approximately 20-25% of SGCs are bam+, dad-LacZ+. Firstly, how do the authors explain this? Secondly, of the 70-75% of SGCs that have no/low BMP signaling, the authors should perform additional characterization using markers that are expressed in GSCs (i.e., Sex lethal and nanos).

These 20-25% of SGCs are bamP-GFP⁺ dad-lacZ-, not bam⁺ dad-lacZ⁺ (see Figure 2C and 3D). They would be cystoblast-like cells that may have initiated a differentiation program toward forming germline cysts (see Lines 109-117). The 70-75% of SGCs that have low BMP signaling exhibit GSC-like properties, including: 1) dot-like spectrosomes; 2) dad-lacZ positivity; 3) absence of bamP-GFP expression. While additional markers would be beneficial, we think that this combination of properties is sufficient to classify these cells as GSC-like. 

(4) All experiments except Figure 1I (where a single germarium with no quantification) were performed with nos-Gal4, UASp-flp. Have the authors performed any of the phenotypic characterizations (i.e., figures other than Figure 1) with hs-flp?

Yes, we initially identified the SGC phenotype through hs-flp-mediated mosaic analysis of bam or bgcn mutant in ovaries. However, as noted in our response to Weakness (2), this approach was very labor-intensive. Therefore, we switched to using the more convenient nos::flp system for subsequent experiments. To our observation, there was no difference in the SGC phenotype between these two approaches, confirming that the nos::flp system is a valid and more practical alternative for its study. 

(5) Does the number of SGCs change with the age of the female? The experiments were all performed in 14-day-old adult females. What happens when they look at a young female (like 2-day-old). I assume that the nos>flp is working in larval and pupal stages, and so the phenotype should be present in young females. Why did the authors choose this later age? For example, is the phenotype more robust in older females? Or do you see more SGCs at later time points?

These are very good questions. Such time-course analysis data will be provided in revised Figure 1. The SGC phenotype depends on the presence of bam or bgcn mutant germline clones. Germaria from 14-day-old flies contained bigger and more such clones than those from younger flies. This age-dependent increase in clone size and frequency significantly enhanced the efficiency of our quantification (see Lines 129-131). 

(6) Can the authors distinguish one copy of GFP versus 2 copies of GFP in germ cells of the ovary? This is not possible in the Drosophila testis. I ask because this could impact the clonal analyses diagrammed in Figure 4A and 4G and in 6A and B. Additionally, in most of the figures, the GFP is saturated, so it is not possible to discern one vs two copies of GFP.

We greatly appreciate this comment. It was also difficult for us to distinguish 1 and 2 copies of GFP in the Drosophila ovary. In Figure 4A-F, to resolve this problem, we used a triplecolor system, in which red germ cells (RFP^+/+ GFP^-/-) are bam mutant, yellow germ cells (RFP^+/- GFP^+/-) are wild-type, and green germ cells (RFP^-/- GFP^+/+) are punt or med mutant. In Figure 4G-J, we quantified the SGC phenotype only in black germ cells (GFP^-/-), which are wild-type (control) or mad mutant.  In Figure 6, we quantified the SGC phenotype only in green germ cells (both GFP^+/+ and GFP^+/-), all of which are wild-type.

(7) More evidence is needed to support the claim of elevated Dpp levels in bam or bgcn mutant tumors. The current results with the dpp-lacZ enhancer trap in Figure 5A, B are not convincing. First, why is the dpp-lacZ so much brighter in the mosaic analysis (A) than in the no-clone analysis (B)? It is expected that the level of dpplacZ in cap cells should be invariant between ovaries, and yet LacZ is very faint in Figure 5B. I think that if the settings in A matched those in B, the apparent expression of dpp-lacZ in the tumor would be much lower and likely not statistically significant. Second, they should use RNA in situ hybridization with a sensitive technique like hybridization chain reactions (HCR) - an approach that has worked well in numerous Drosophila tissues, including the ovary.

We appreciate this critical comment. The settings of immunofluorescent staining and confocal parameters in Figure 5A were the same as those in 5B. To our observation, the level of dpp-lacZ in cap cells was variable across germaria, even within the same ovary, as quantified in Figure 5C. We will provide RNA in situ hybridization data to further strengthen the conclusion that bam or bgcn mutant germline tumors secret BMP ligands.  

(8) In Figure 6, the authors report results obtained with the bamBG allele. Do they obtain similar data with another bam allele (i.e., bamdelta86)?

No. Given that bam^BG was functionally indistinguishable from bam^Δ86 in inducing the SGC phenotype (compare Figure 6F, I with Figure 6-figure supplement 3C), we believe that repeating these experiments with bam^Δ86 would be redundant and would not alter the key conclusion of our study. Thanks for the understanding!

Reviewer #2 (Public review):

While the study by Zhang et al. provides valuable insights into how germline tumors can non-autonomously suppress the differentiation of neighboring wild-type germline stem cells (GSCs), several conceptual and technical issues limit the strength of the conclusions.

Major points:

(1) Naming of SGCs is confusing. In line 68, the authors state that "many wild-type germ cells located outside the niche retained a GSC-like single-germ-cell (SGC) morphology." However, bam or bgcn mutant GSCs are also referred to as "SGCs," which creates confusion when reading the text and interpreting the figures. The authors should clarify the terminology used to distinguish between wild-type SGCs and tumor (bam/bgcn mutant) SGCs, and apply consistent naming throughout the manuscript and figure legends.

We apologize for any confusion. In our manuscript, the term "SGC" is reserved specifically for wild-type germ cells that maintain a GSC-like morphology outside the niche. bam or bgcn mutant germ cells are referred to as GSC-like tumor cells (Lines 87-88), not SGCs.

(a) The same confusion appears in Figure 2. It is unclear whether the analyzed SGCs are wild-type or bam mutant cells. If the SGCs analyzed are Bam mutants, then the lack of Bam expression and failure to differentiate would be expected and not informative. However, if the SGCs are wild-type GSCs located outside the niche, then the observation would suggest that Bam expression is silenced in these wildtype cells, which is a significant finding. The authors should clarify the genotype of the SGCs analyzed in Figure 2C, as this information is not currently provided.

The SGCs analyzed in Figure 2A-C are wild-type, GSC-like cells located outside the niche. They were generated using the same genetic strategy depicted in Figures 1C and 1E (with the schematic in Figure 1B). The complete genotypes for all experiments are available in Source data 1. 

(b) In Figures 4B and 4E, the analysis of SGC composition is confusing. In the control germaria (bam mutant mosaic), the authors label GFP⁺ SGCs as "wild-type," which makes interpretation unclear. Note, this is completely different from their earlier definition shown in line 68.

The strategy to generate SGCs in Figure 4B-F (with the schematic in Figure 4A) is completely different from that in Figure 1C-F, H, and I (with the schematic in Figure 1B). In Figure 4B-F, we needed to distinguish punt^-/- (or med^-/-) with punt^+/- (or med^+/-) germ cells. As noted in our response to Reviewer #1’s Weakness (6), it was difficult for us to distinguish 1 and 2 copies of GFP in the Drosophila ovary. Therefore, we chose to use the triple-color system to distinguish these germ cells in Figure 4B-F (see genotypes in Source data 1). 

(c) Additionally, bam⁺/⁻ GSCs (the first bar in Figure 4E) should appear GFP⁺ and Red⁺ (i.e., yellow). It would be helpful if the authors could indicate these bam⁺/⁻ germ cells directly in the image and clarify the corresponding color representation in the main text. In Figure 2A, although a color code is shown, the legend does not explain it clearly, nor does it specify the identity of bam⁺/⁻ cells alone. Figure 4F has the same issue, and in this graph, the color does not match Figure 4A.

The color-to-genotype relationships for the schematics in Figures 2A and 4E are provided in Figures 1B and 4A, respectively. Due to the high density of germ cells, it is impractical to label each genotype directly in the images. In contrast to Figure 4E, the colors in Figure 4F do not represent genotypes; instead, blue denotes the percentage of SGCs, and red denotes the percentage of germline cysts, as indicated below the bar chart. 

(2) The frequencies of bam or bgcn mutant mosaic germaria carrying [wild-type] SGCs or wild-type germ cell cysts with branched fusomes, as well as the average number of wild-type SGCs per germarium and the number of days after heat shock for the representative images, are not provided when Figure 1 is first introduced. Since this is the first time the authors describe these phenotypes, including these details is essential. Without this information, it is difficult for readers to follow and evaluate the presented observations.

Thanks for this constructive suggestion. We will include such quantification data in the revised manuscript.

(3) Without the information mentioned in point 2, it causes problems when reading through the section regarding [wild-type] SGCs induced by impairment of differentiation or dedifferentiation. In lines 90-97, the authors use the presence of midbodies between cystocytes as a criterion to determine whether the wild-type GSCs surrounded by tumor GSCs arise through dedifferentiation. However, the cited study (Mathieu et al., 2022) reports that midbodies can be detected between two germ cells within a cyst carrying a branched fusome upon USP8 loss.

Unlike wild-type cystocytes, which undergo incomplete cytokinesis and lack midbodies, those with USP8 loss undergo complete cell division, with the presence of midbodies (white arrow, Figure 1F’ from Mathieu et al., 2022) as a marker of the late cytokinesis stage (Mathieu et al., 2022). 

(a) Are wild-type germ cell cysts with branched fusomes present in the bam mutant mosaic germaria? What is the proportion of germaria containing wild-type SGCs versus those containing wild-type germ cell cysts with branched fusomes?

(b) If all bam mutant mosaic germaria carry only wild-type GSCs outside the niche and no germaria contain wild-type germ cell cysts with branched fusomes, then examining midbodies as an indicator of dedifferentiation may not be appropriate.

We greatly appreciate this critical comment. bam mutant mosaic germaria indeed contained wild-type germline cysts, as evidenced by an SGC frequency of ~70%, rather than 100% (see Figures 2H, 4F, 4J, 6F, 6I, and Figure 6-figure supplement 3C). Since the SGC phenotype depends on the presence of bam or bgcn mutant germline tumors, we quantified it as “the percentage of SGCs relative to the total number of SGCs and germline cysts that are surrounded by germline tumors” (see Lines 124-129). Quantifying the SGC phenotype as "the percentage of germaria with SGCs" would be imprecise. This is because the presence and number of SGCs were highly variable among germaria with bam mutant germline clones, and a small number of germaria entirely lacked these clones. We will provide the data of "SGCs and/or germline cysts per germarium with germline clones" in revised Figure 1.

(c) If, however, some germaria do contain wild-type germ cell cysts with branched fusomes, the authors should provide representative images and quantify their proportion.

Such representative germaria are shown in Figure 2G, 3B, 3C, 6D, 6E, and 6H. The percentage of germline cysts can be calculated by “100% - SGC%”.

(d) In line 95, although the authors state that 50 germ cell cysts were analyzed for the presence of midbodies, it would be more informative to specify how many germaria these cysts were derived from and how many biological replicates were examined.

As noted in our response to points a) and b) above, the germ cells surrounded by germline tumors, rather than germarial numbers, are more precise for analyzing the phenotype. For this experiment, we examined >50 such germline cysts via confocal microscopy. As the analysis was performed on a defined cellular population, this sample size should be sufficient to support our conclusion. 

(4) Note that both bam mutant GSCs and wild-type SGCs can undergo division to generate midbodies (double cells), as shown in Figure 4H. Therefore, the current description of the midbody analysis is confusing. The authors should clarify which cell types were examined and explain how midbodies were interpreted in distinguishing between cell division and differentiation.

We assayed for the presence of midbodies or not specifically within the germline cysts surrounded by bam mutant tumors, not within the tumors themselves (Lines 94-95). As detailed in Lines 88-97, the absence of midbodies was used as a key criterion to exclude the possibility of dedifferentiation.  

(5) The data in Figure 5 showing Dpp expression in bam mutant tumorous GSCs are not convincing. The Dpp-lacZ signal appears broadly distributed throughout the germarium, including in escort cells. To support the claim more clearly, the authors should present corresponding images for Figures 5D and 5E, in which dpp expression was knocked down in the germ cells of bam or bgcn mutant mosaic germaria. Showing these images would help clarify the localization and specificity of Dpp-lacZ expression relative to the tumorous GSCs.

We greatly appreciate this comment. RNA in situ hybridization data will be provided to further strengthen the conclusion that bam or bgcn mutant germline tumors secret BMP ligands.

(6) While Figure 6 provides genetic evidence that bam mutant tumorous GSCs produce Dpp to inhibit the differentiation of wild-type SGCs, it should be noted that these analyses were performed in a dpp⁺/⁻ background. To strengthen the conclusion, the authors should include appropriate controls showing [dpp⁺/⁻; bam⁺/⁻] SGCs and [dpp⁺/⁻; bam⁺/⁻] germ cell cysts without heat shock (as referenced in Figures 6F and 6I).

Schematic cartoons in Figure 6A and 6B demonstrate that these analyses were performed in a dpp^+/- background. Figure 6-figure supplement 1 indicates that dpp^+/- or gbb^+/- does not affect GSC maintenance, germ cell differentiation, and female fly fertility. Figure 6C is the control for 6D and 6E, and 6G is the control for 6H, with quantification in 6F and 6I.  We used nos::flp, not the heat shock method, to induce germline clones in these experiments (see genotypes in Source data 1).

(7) Previous studies have reported that bam mutant germ cells cause blunted escort cell protrusions (e.g., Kirilly et al., Development, 2011), which are known to contribute to germ cell differentiation (e.g., Chen et al., Frontiers in Cell and Developmental Biology, 2022). The authors should include these findings in the Discussion to provide a broader context and to acknowledge how alterations in escort cell morphology may further influence differentiation defects in their model.

Thanks for teaching us! Such discussion will be included in the revised manuscript.

(8) Since fusome morphology is an important readout of SGCs vs differentiation. All the clonal analysis should have fusome staining.

SGC is readily distinguishable from multi-cellular germline cyst based on morphology. In some clonal analysis experiments, fusome staining was not feasible due to technical limitations such as channel saturation or antibody incompatibility. Thanks for the understanding! 

(9) Figure arrangement. It is somewhat difficult to identify the figure panels cited in the text due to the current panel arrangement.

The figure panels were arranged to optimize space while ensuring that related panels are grouped in close proximity for logical comparison. We would be happy to consider any specific suggestions for an alternative layout that could improve clarity. Thanks!

(10) The number of biological replicates and germaria analyzed should be clearly stated somewhere in the manuscript-ideally in the Methods section or figure legends. Providing this information is essential for assessing data reliability and reproducibility.

Thanks for this constructive suggestion. Such information will be included in figure legends in the revised manuscript.

Reviewer #3 (Public review):

Summary:

Zhang et al. investigated how germline tumors influence the development of neighboring wild-type (WT) germline stem cells (GSC) in the Drosophila ovary. They report that germline tumors inhibit the differentiation of neighboring WT GSCs by arresting them in an undifferentiated state, resulting from reduced expression of the differentiation-promoting factor Bam. They find that these tumor cells produce low levels of the niche-associated signaling molecules Dpp and Gbb, which suppress bam expression and consequently inhibit the differentiation of neighboring WT GSCs non-cell-autonomously. Based on these findings, the authors propose that germline tumors mimic the niche to suppress the differentiation of the neighboring stem cells.

Strengths:

This study addresses an important biological question concerning the interaction between germline tumor cells and WT germline stem cells in the Drosophila ovary. If the findings are substantiated, they could provide valuable insights applicable to other stem cell systems.

We greatly appreciate these comments.

Weaknesses:

Previous work from Xie's lab demonstrated that bam and bgcn mutant GSCs can outcompete WT GSCs for niche occupancy. Furthermore, a large body of literature has established that the interactions between escort cells (ECs) and GSC daughters are essential for proper and timely germline differentiation (the differentiation niche). Disruption of these interactions leads to arrest of germline cell differentiation in a status with weak BMP signaling activation and low bam expression, a phenotype virtually identical to what is reported here. Thus, it remains unclear whether the observed phenotype reflects "direct inhibition by tumor cells" or "arrested differentiation due to the loss of the differentiation niche". Because most data were collected at a very late stage (more than 10 days after clonal induction), when tumor cells already dominate the germarium, this question cannot be solved. To distinguish between these two possibilities, the authors could conduct a time-course analysis to examine the onset of the WT GSC-like singlegerm-cell (SGC) phenotype and determine whether early-stage tumor clones with a few tumor cells can suppress the differentiation of neighboring WT GSCs with only a few tumor cells present. If tumor cells indeed produce Dpp and Gbb (as proposed here) to inhibit the differentiation of neighboring germline cells, a small cluster or probably even a single tumor cell generated at an early stage might prevent the differentiation of their neighboring germ cells.

Thanks for this critical comment. Such time-course analysis data will be provided in revised Figure 1.

The key evidence supporting the claim that tumor cells produce Gpp and Gbb comes from Figures 5 and 6, which suggest that tumor-derived dpp and gbb are required for this inhibition. However, interpretation of these data requires caution. In Figure 5, the authors use dpp-lacZ to support the claim that dpp is upregulated in tumor cells (Figure 5A and 5B). However, the background expression in somatic cells (ECs and pre-follicular cells) differs noticeably between these panels. In Figure 5A, dpp-lacZ expression in somatic cells in 5A is clearly higher than in 5B, and the expression level in tumor cells appears comparable to that in somatic cells (dpplacZ single channel). Similarly, in Figure 5B, dpp-lacZ expression in germline cells is also comparable to that in somatic cells. Providing clear evidence of upregulated dpp and gbb expression in tumor cells (for example, through single-molecular RNA in situ) would be essential.

We greatly appreciate this critical comment. In our data, the expression of dpp-lacZ in cap cells was variable across germaria, even within the same ovary, as quantified in Figure 5C. The images in Figures 5A and 5B were selected as representative examples of positive signaling. To directly address the reviewer's point and strengthen our conclusion, we will perform RNA in situ hybridization data in the revised manuscript to visualize the expression of BMP ligands within the bam or bgcn mutant germline tumor cells.

Most tumor data present in this study were collected from the bam[86] null allele, whereas the data in Figure 6 were derived from a weaker bam[BG] allele. This bam[BG] allele is not molecularly defined and shows some genetic interaction with dpp mutants. As shown in Figure 6E, removal of dpp from homozygous bam[BG] mutant leads to germline differentiation (evidenced by a branched fusome connecting several cystocytes, located at the right side of the white arrowhead). In Figure 6D, fusome is likely present in some GFP-negative bam[BG]/bam[BG] cells. To strengthen their claim that the tumor produces Dpp and Gbb to inhibit WT germline cell differentiation, the authors should repeat these experiments using the bam[86] null allele.

Although a structure resembling a "branched fusome" is visible in Figure 6E (right of the white arrowhead), it is an artifact resulting from the cytoplasm of GFP-positive follicle cells, which also stain for α-Spectrin, projecting between germ cells of different clones (see the merged image). In both our previous (Zhang et al., 2023) and current studies, bam^BG was functionally indistinguishable from bam^Δ86 in its ability to block GSC differentiation and induce the SGC phenotype (compare Figure 6F, I with Figure 6-figure supplement 3C). Given this, we believe that repeating the extensive experiments in Figure 6 with the bam^Δ86 allele would be scientifically redundant and would not change the key conclusion of our study. We thank the reviewer for their consideration.

It is well established that the stem niche provides multiple functional supports for maintaining resident stem cells, including physical anchorage and signaling regulation. In Drosophila, several signaling molecules produced by the niche have been identified, each with a distinct function - some promoting stemness, while others regulate differentiation. Expression of Dpp and Gbb alone does not substantiate the claim that these tumor cells have acquired the niche-like property. To support their assertion that these tumors mimic the niche, the authors should provide additional evidence showing that these tumor cells also express other niche-associated markers. Alternatively, they could revise the manuscript title to more accurately reflect their findings.

Dpp and Gbb are the key niche signals from cap cells for maintaining GSC stemness. Our work demonstrates that germline tumors can specifically mimic this signaling function, not the full suite of cap cell properties, to create a non-cell-autonomous differentiation block. The current title “Tumors mimic the niche to inhibit neighboring stem cell differentiation” reflects this precise concept: a partial, functional mimicry of the niche's most relevant activity in this context. We feel it is an appropriate and compelling summary of our main conclusion.

In the Method section, the authors need to provide details on how dpp-lacZ expression levels were quantified and normalized.

Thanks for this suggestion. Such information will be included in the revised manuscript.

Reading this section personally hit very close to home for me. I also have ADHD, and have a tendency to hyperfocus when it comes to art as I can spend hours on a single painting, but then struggle to focus for more than 30 minutes on a homework assignment. I find it very true that people tend to "mask" their disabilities, as almost everyone I know has something they hide in order to fit in. I as well often hide that fact that homework often takes longer for me due to my ADHD. Knowing this, this is why I strongly think it's important we as a society work to becoming more inclusive in all aspects of life!

Universal Design makes sense because it moves the work from the disabled person to the builder. When spaces include ramps, elevators, and clear labels (including braille), many different people benefit at the same time. It treats access as part of the plan, not a special fix. The hard part is balancing needs when space and budgets are tight, but a fair goal is to offer equal, visible paths so no one is sent to a “back door.”

This paragraph describes a situation that I can feel in person in real life. Nowadays, some platforms provide personalized mode for the way that the platforms work, which ensure to a very great extent that people do not feel like they are "disabled" when using internet. I saw a video of a blind computer programmer before, and this paragraph recalled my memory about that programmer. She uses Alt-text and listen to all the code she types to make a program run successfully, which made me feel the help that accessible design brings to us.

This feels parallel to a common criticism I see applied to social media and other digital platforms. The criticism is that its made for white and/or rich and/or male people. In most cases, I don't think its intentional, but it is true that the least oppressed groups get catered to the most. This is relevant here because many things passively cater toward those who are not disabled the most.

describing the goal of extending a healthy life as long as possible before a sudden decline near the end, rather than experiencing a long period of illness.

the action of excavating something, especially an archaeological site.

Mistake (and unclear what is really meant)

eLife Assessment

This manuscript presents significant and important work that advances single-molecule imaging technology of transcription with simultaneous analysis of several parameters. However, currently, the evidence is incomplete and requires further quantitation/description of the technologies used, further controls, and additional analysis of the data by other methods.

Reviewer #1 (Public review):

Summary:

This study investigates the effects of transcriptional activation on chromatin dynamics and mobility. Using a breast cancer model, the authors examine the effects of estrogen receptor-a (ERa) stimulation and the resulting transcriptional activation on chromatin behavior at ERa-dependent loci during three distinct phases: unstimulated, acute stimulation, and chronic stimulation. Through live DNA and RNA imaging, the authors claim that ERa-dependent target genes display distinct bursting dynamics during periods of acute versus chronic simulation, accompanied by an overall increase in chromatin mobility. Notably, they claim that ERa-dependent loci display increased mobility during the non-bursting phase compared to the bursting phase. The study also attempts to explore the role of condensates in mediating these transcriptional and chromatin mobility changes using a single-molecule tracking assay to identify a unique population of low diffusion-coefficient molecules that appears upon E2 stimulation and is sensitive to 1,6-hexanediol.

Strengths:

While the study develops interesting tools that have the potential to provide useful insights into the relationship between transcriptional state, genomic locus mobility, and condensate formation, several major claims lack key supportive evidence, and the methods are inadequately established and described.

Weaknesses:

(1) The use of 1,6 hexanediol experiments is not suitable for drawing conclusions in live cell experiments, as this assay is now widely recognized to be plagued with artifacts and inadequate as a test for condensate formation. 1,6 hexanediol perturbs all hydrophobic interactions and has effects ranging from perturbing kinase and phosphatase activities (Düster et al, J. Biol. Chem., 2021), immobilizing and condensing chromatin in living cells (Itoh et al., Life Sci. Alliance 2021), disrupting nuclear pore complexes (Ribbeck et al., EMBO 2002), nuclear transport (Barrientos et al., Nucleus, 2023), and does not disrupt charge-mediated phase separation (Zheng et al., EMBO, 2025). There is also a discussion on these effects in a recent article: Current practices in the study of biomolecular condensates: a community comment, Alberti, Nat. Comm., 2025.

(2) The chromatin mobility is analyzed using displacement, and the differences are typically less than 50 nm. There is no discussion on the precision of this measurement and what these small differences may mean. No control loci are assessed to see if this effect is specific to the genes of interest or global.

(3) The SMT analysis is performed using Mean Square Displacement fitting of short single trajectories, which is error-prone, and no analysis is performed on the localization precision or error in estimation of the key parameters. Potential artifacts from this analysis are reflected in the distribution of alpha and diffusion coefficients that are presented in this paper, which include physically impossible values on which major claims rest.

(4) No experiment is performed to directly connect foci/cluster/condensation formation of ER at the genes of interest. Given these points alone, it is impossible to assess whether any of the claims made in the current manuscript are correct.

Reviewer #2 (Public review):

Summary:

The authors use a combination of state-of-the-art live-cell imaging techniques to track transcriptional bursting, DNA mobility, and single-molecule tracking to discern biophysical behaviours of chromatin and condensate formation in response to ER𝛼 activation. Surprisingly, the authors find that loci in estradiol-stimulated cells display enhanced mobility during the non-bursting phase. The authors attribute the reduced mobility of the loci during transcriptional bursts to condensate formation of ER𝛼 on enhancers regulating the bursting gene. Inhibition of transcription with flavopiridol shifts the loci and ER𝛼 to a non-confined state. These findings open the door to performing more complex multi-color live-cell imaging assays to fully interrogate the role of transcription factor condensates, DNA mobility, and subnuclear localization in the regulation of transcriptional bursting kinetics, and should be of great benefit to researchers studying mechanisms of gene regulation.

Strengths:

The authors presented a series of advanced multi-color live cell imaging assays used to correlate changes in DNA mobility with transcriptional bursting of a gene. By using such a defined temporal trigger associated with the addition of estroldiol to cells, the authors were also able to elegantly characterize changes in the diffusive properties of different classes of ER𝛼 during the acute (early, <2 hours) and chronic (late, >2 hours) phases of estrogen-responsive gene activation. Interestingly, one particular class of ER𝛼 that changed between acute and chronic phases was also responsive to 1,6-hexanediol treatment, suggesting that the authors are assaying ER𝛼 behaviours related to condensate formation. The authors also examined how the proximity of the NRIP1 gene to interchromatin granules impacted transcriptional bursting kinetics. There was no correlation of DNA mobility nor transcription bursting associated with localization to interchromatin granules, suggesting that other higher-order, architectural associations are regulating these processes. The imaging data were also supported by genomic GRO-seq and ChIP-seq assays showing changes in genomic occupancy of a number of transcription factors, including ER𝛼, during the pre-acute, acute, and chronic phases.

Weaknesses:

Although there are a number of compelling strengths to support the author's interpretation of the data, the paper is written in a way that lacks clarity and detail on a number of technical components. This lack of details, in particular related to how endogenous tagging of DNA, ER𝛼, and interchromatin granules (e.g. SC35) potentially impacts transcriptional bursting, makes it difficult for the reader to sufficiently judge any potential limitations of these complex engineered cell lines. Another potential weakness is the lack of any experiments directly measuring ER𝛼 diffusive properties in close proximity to the bursting gene. It is noted that this type of experiment examining transcription factor binding on a bursting gene is very technically challenging, given the different timescales of measurement of bursting (seconds-minutes) versus ER𝛼 diffusion (sub-seconds). However, these types of experiments would go a long way to supporting the authors' conclusions regarding how changes in DNA mobility and transcription bursting may be directly related to ER𝛼 condensate formation on enhancers.

Reviewer #3 (Public review):

Summary:

In this manuscript, the authors explore dynamic chromosomal mobility and transcriptional bursting events in mammalian cells, particularly focusing on ERα-dependent gene activation. The authors investigate how the physical movement of DNA loci changes during different phases of gene transcription (bursting vs. non-bursting, acute vs. chronic stimulation). Using advanced live-cell imaging techniques, including SMT of ERα and dual DNA/RNA visualization, the study reveals a multi-state model of DNA mobility linked to the formation of transcription factor condensates. The authors conclude that differential DNA kinetics serve as a reliable indicator for detecting condensate formation during gene activation, offering new insights into the mechanisms regulating gene expression within the nucleus.

Strengths:

The authors have done substantial work, and a major strength of the manuscript is being able to image both DNA and RNA from the same gene, as well as the TF that acts on that gene. This multi-pronged approach leads to complementary insights into transcription bursting mechanisms.

Weaknesses:

A major weakness of the manuscript is the lack of appropriate controls that support the specificity of the effects observed. The exclusive focus on condensates as the underlying mechanism to explain their data is also a bit limiting.

This guy is a sloppy slopper

This article discuss the social model that suggest ideas that stem around the belief/ideas around disabilities fully stem from societal attudies. Entailing that the model of disability is structured more by societal barriers rather then mental/physical health.

This source shows why alt-text matters: it should be short, concrete, and task-focused. Useful alt-text names the subject, the action, the setting, and any visible text in the image. A simple policy would help: when someone uploads an image, the tool asks for alt-text with a small prompt. AI can suggest a draft, but a human should review. This makes posts more accessible and also more professional.

This article introduces a female who is working on using AI technology to help people with disabilities. It first introduces this female's diploma and working experiences, and then tells us the main working focus of this female. She's trying to cultivate accessible computing research lab and design studio to help people understand the world of disabilities and help disabilities to live a normal life. From this article, I can see the importance of accessible designs.

It really made me stop and think about what “normal vision” even means. If some women can see more colours than most people, then the typical human colour range is just one version of reality but our “normal” might actually be a limitation.

A disability is something ultimately deemed by society. Society's structure and standards are what shape/determine what a disability is. The concept of a disability has also changed from "madness" to a state of of medical illness over the years.

I personally like this version of the definition of disability, and the disability is a general meaning for the society. In this case, the people with disability will not feel offended anymore.

this continues to highlight how ethics, particularly within the realm of health practice and promotion and policy, needs to be constantly evolving, to keep up to date with contemporary issues, and new understandings

takes into consideration the equity imbalance between different groups of people - what population will benefit the most and which form of distribution would lead to the greatest health outcome for the most people?

During COVID - restriction public movement and enforcing lockdown may have been considered restricting the human rights of freedom/liberty in many places; however, the threat of infection was very high and already shown to cause loss of life. The lockdowns aimed to be a preventative measure to reduce the amount of disease transmission, also taking pressure off the health care system treating already sick patients

It is important to note that although it is normal and respectable for individuals and groups to hold different personal and societal values - all values do not have the same moral status, and it may become more important to discuss the ethics values for issues that become complex.

It is also important to consider the values of other individuals and groups, to provide one with a greater understanding of different issues, and enable a more understanding society

As there is a difference between ethical, social and personal values, certain situations can lead to conflict between the values of different groups. e.g. an ethical value may encourage the general population to help someone in an accident, but an individual's personal values may disagree with that

Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

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Reply to the reviewers

We thank all the reviewers for their comments and suggestions, which has helped in revising the manuscript for a broader audience. Some of the experiments that was suggested by the reviewers has been performed and included in the revised manuscript. The response to reviewers is provided below their comments.

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

MprF proteins exist in many bacteria to synthesize aminoacyl phospholipids that have diverse biological functions, e.g. in the defense against small cationic peptides. They integrate two functions, the aminoacylation of lipids, i.e. the transfer of Lys, Arg or Ala from tRNAs to the head group, and the flipping of these modified lipids to the membrane outer leaflet. The authors present structures of MprF from Pseudomonas aeruginosa and describe these structures in great detail. As MprF enzymes confer antibiotic resistance and are therefore highly important, studying them is significant and interesting. Consequently, their structures have been substantially characterized in recent years, including the publication of the dimeric full-length MpfR from Rhizobium (Song et al., 2021).

While the structural work appears to be solid and carried out well on the technical part, one big criticism is how the data are presented in the manuscript, how they are analyzed and how they are put into relation to previous work. As structures of Mpfr from Rhizobium have been published, it is not required and rather distracting to explain the methodological details and the structure of Pseudomonas MprF in such great detail. Instead, the manuscript would benefit very strongly from reaching the interesting and novel parts, the comparison with the previous structures, as early as possible. Overall, the manuscript should be substantially shortened to not divert the reader's attention away from the novel parts by drowning them in miniscule description of the structural features such as secondary structure elements or lipid molecule positions where it remains completely unclear what their relevance is to the story and the message of the paper. Finally, during this revision, care should be taken to improve the language and maybe involve a native speaker in doing so.

It is true that we have described the experimental details of PaMprF in detail including the constructs. We had reconstructed the map of dimeric PaMprF in 2020 but with the publication of the homologues structures (Song et al 2021 and the unpublished Rhizobium etli structure), we had to make sure the PaMprF dimer is not an artefact. Hence, our attempts to rule out this with different constructs and extensive testing with various detergents. Thus, we would like to keep this in the manuscript. We realise the importance of focusing on novel/interesting parts and have reshuffled sections (comparing structures and validating the dimer interface) followed by description of modelling of lipid molecules.

Even more importantly, since the authors observe a dimer interface which strongly deviates from the previously presented arrangement of another species, the most important thing would be to properly characterize this interface and experimentally validate it, both of which has not been done sufficiently. When also taking into account that there were significant differences in the arrangement of the dimer between their structures in GDN and nanodisc, and that in the GDN structure, the cholesterol backbone of GDN appears to be involved in the interface (there should not be any cholesterol in native bacterial membranes!), there is a realistic chance that the observed dimer is an artefact. If the authors cannot convincingly rule out this possibility, all their conclusions are meaningless.

The trials with cholesterol hemisuccinate stems more of out of curiosity (we are aware that no cholesterol is present in bacterial membranes). We had started the initial analysis of PaMprF with DDM and by itself it was largely monomeric (unpublished observation and supported by recent publication of PaMprF in DDM – Hankins et al 2025). When we observed that GDN was essential for the stability of the dimer (and not even LMNG), we asked if a combination of CHS with DDM will keep the dimer intact, which didn’t work and GDN was found to be important. The use of CHS for prokaryotic membrane protein studies has now been reported in few different systems and a recent one includes – Caliseki et al., 2025. We would like to keep the observation with CHS in the manuscript, and we have moved this figure to Appendix Fig. S3C.

In addition, in a recent report on MgtA, a magnesium transporter (Zeinert et al., 2025), it was observed that DDM/LMNG resulted in monomeric enzyme, while GDN resulted in dimeric enzyme albeit, the dimer interface was in the soluble domain. We have added this reference and observation of MgtA in the discussion (page 13, lines 407-411).

We like to think that the milder GDN tends to keep the membrane proteins or oligomers of membrane proteins more stable but further studies on multiple labile membrane protein systems will be required to substantiate this.

Hence, while I think that the data presented here would be worth publishing. However, a major drawback is that the authors do not sufficiently analyse, characterise and validate the dimer interface and fail to show that the dimer is biologically relevant.

Further major points: - The authors always jump between their structures in detergent and nanodisc during all the descriptions, which makes following the story even more difficult. Please first describe one of the structures and then (briefly) discuss relevant similarities and differences afterwards.

The flow and description of the structures is now modified and the figures have now been rearranged to make it easier to follow. The panel in figure 2 describing the overlay of the GDN and nanodisc is now moved to Appendix Fig. S2B. Thus, figure 2 has only description of salient features of the structures (the interacting residues between the membrane and soluble domain) and the terminal helix.

• The difference in dimerization between Pseudomonas and Rhizobium is the most interesting and surprising feature (if true) of the new structures. However, it is not really presented as such. The authors should put more emphasis on making clear that this is a complete rotation of the monomers with respect to each other (by how many degrees?) and they should visualize it even more clearly in Figure 4 (and label the figure so that it is possible to understand it without having to read the text or the legend first).

We thought the colouring of the TM helices should make the difference in interface more obvious (the N and C-terminal TM helices in different colours). Now, we have also labelled the TM helices, so that it is easier to follow (this was also shown in panel E). The rotation is ~180° and this is now mentioned in the figure legend.

• P. 10: The authors insinuate that only one of the dimer interfaces, either Pseudomonas or Rhizobium could be real, but disregard the possibility that both might be the biologically relevant interfaces of the respective species and that there might have been a switch of interfaces during evolution. They should also mention and discuss this possibility.

We didn’t imply that one of the interfaces is real but clearly mentioned that it could also be different conformational state (page 7, lines 226-228). In the revised version, we have included a multiple sequence alignment (we had not included in the initial draft as it had been presented in several previous publications). The MSA (Appendix Fig. S6) reveals that neither of the interfaces are highly conserved.

• Fig. 5G: The authors claim that the higher molecular band that appears in the mutant is a "dimer with aberrant migration" of >250 kDa as opposed to the expected 150 kDa. They should explain how they came to this conclusion and how they can be sure that the band does not correspond to a higher oligomer (trimer or tetramer). They could show, by extraction and purification scheme similar to the wildtype using first LMNG and then GDN, followed by at least a preliminary EM analysis, that the crosslinked mutant MprF is indeed a dimer, or use other biophysical methods to do the same, otherwise this experiment does not show much. Furthermore, they should also include a cysteine mutant in the part of Pseudomonas MprF that would be involved in a Rhizobium-like interface in their crosslinking experiments to check whether they could also stabilize dimers in this case.

The band of the double mutant after crosslinking (or even without crosslinking) migrates at higher molecular weight than that expected for a dimer, and could potentially be a higher molecular band that a dimer. We also note that in the previous publication by Song et al 2021, the crosslinking of RtMprF also resulted in a higher molecular weight band (shown also by Western blot).

We now substantiate the dimer of PaMprF with different approaches. We employed blue-native gel and also SDS-PAGE of the purified protein. This clearly shows that the higher molecular band after crosslinking is a dimer (Figure 4B and Fig. EV4D). In particular, in the BN-PAGE, the treatment of mutants with crosslinkers revealed a dimeric band even in the presence of SDS. Further, we have performed cryoEM analysis of the mutants - H386C/F389C and H566C. The images, classes and reconstruction show that the enzyme forms a dimer similar to the WT. Interestingly, we also observe in H566C mutant in nanodisc, a small population that has similar architecture to the Rhizobium-like interface (classes shown in Fig. EV7 and Appendix Fig. S5). This prompted us to look closely at other datasets and it is clear that during the process of reconstitution in nanodisc, we observe both kinds of dimer interface but the PaMprF dimer is predominant. We also observe higher order oligomers (tetramer) in GDN but as only few views are visible, a reconstruction could not be obtained (Appendix Fig. S5). In addition, we also introduced two cysteines on the Rhizobium-like interface and no crosslinking on the membranes were observed (Figure 4B). But it is possible that these chosen mutants are not accessible to the crosslinker. Thus, we conclude that the oligomers of PaMprF is sensitive to nature of detergents and labile.

• As the question whether the observed interface is real or an artefact is very central to the value of the structural data and the drawn conclusions from it, the authors should make more effort to analyze and try to validate the interface. First, an analysis of interface properties (buried surface area, nature of the interactions, conservation) should be performed for the interface as observed in the Pseudomonas structure but also for a (hypothetical) Rhizobium-like interface of two Pseudomonas monomers (such a model of a dimer should be easily obtainable by AlphaFold using the available Rhizobium structures as models). Then, experimental methods such as FRET or crosslinking-MS would allow to draw more solid conclusions on the distances between potential interface residues. While these experiments are a certain effort, the question whether the dimer interface is real is so central to the paper that it would be worthwhile to make this effort.

We have included the interface area and nature of interactions in the revised manuscript (page 7, lines 221-223).

We attempted AlphaFold for predicting the dimeric structure of PaMprF (and included RtMprF also). Some of the attempts from the predictions is summarised in figure 1.

The prediction of monomer is of high confidence but the oligomer (here dimer) is of low confidence (from ipTM values). Even the prediction for Rhizobium enzyme has low confidence, and gives a complete different architecture (and in some trials with lipids, it gives an inverted or non-physiological dimer). Only when the monomer of PaMprF with lipids and tRNA was given as input (requested by reviewer 2 and described below), it predicts oligomeric structure with some confidence but rest were not informative.

• As it seems that detergents might disrupt or modify the dimer interface, it might be an alternative to solubilize the protein in a more native environment by polymer-stabilized nanodiscs using DIBMA or similar molecules.

We have tried to use SMALPs for extraction of PaMprF. We were able to solubilise but unable to enrich the enzyme sufficient for structural studies currently and will require further optimisation.

• Since parts of the Discussion are mostly repetitions of the Results part and other parts of the Discussion also contain a large extend of structure analysis one would usually rather expect in the Results part instead of the Discussion, the authors should consider condensing both to a combined (and overall much shorter) Results & Discussion section.

We have rewritten much of the discussion section and removed any repetition from the results sections. We would prefer to keep the results and discussion separate.

Minor points: - Explain abbreviations the first time they appear in the text, e.g. TTH

This is now expanded in the first instance

• Figure labels are very minimalistic. This should be improved, e.g. by putting labels to important structural features that appear in the text, otherwise the figures are not an adequate support for the text.

The font size for the labels have been increased.

• Figure 5: Label where the different oligomers run on the gels

Labelled.

Reviewer #1 (Significance (Required)):

While the structural work appears to be solid and carried out well on the technical part, one big criticism is how the data are presented in the manuscript, how they are analyzed and how they are put into relation to previous work. As structures of Mpfr from Rhizobium have been published, it is not required and rather distracting to explain the methodological details and the structure of Pseudomonas MprF in such great detail. Instead, the manuscript would benefit very strongly from reaching the interesting and novel parts, the comparison with the previous structures, as early as possible. Overall, the manuscript should be substantially shortened to not divert the reader's attention away from the novel parts by drowning them in miniscule description of the structural features such as secondary structure elements or lipid molecule positions where it remains completely unclear what their relevance is to the story and the message of the paper. Finally, during this revision, care should be taken to improve the language and maybe involve a native speaker in doing so.

Even more importantly, since the authors observe a dimer interface which strongly deviates from the previously presented arrangement of another species, the most important thing would be to properly characterize this interface and experimentally validate it, both of which has not been done sufficiently. When also taking into account that there were significant differences in the arrangement of the dimer between their structures in GDN and nanodisc, and that in the GDN structure, the cholesterol backbone of GDN appears to be involved in the interface (there should not be any cholesterol in native bacterial membranes!), there is a realistic chance that the observed dimer is an artefact. If the authors cannot convincingly rule out this possibility, all their conclusions are meaningless.

Hence, while I think that the data presented here would be worth publishing. However, a major drawback is that the authors do not sufficiently analyse, characterise and validate the dimer interface and fail to show that the dimer is biologically relevant.

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

Shaileshanand J. et al., reported the structures of Multiple Peptide Resistance Factor, MprF, which is a bi-functional enzyme in bacteria responsible for aminoacylation of lipid head groups. The authors purified MprF from Pseudomonas aeruginosa in GDN micelles and nanodiscs, and by applying cryo-EM single particle method, they successfully reached near-atomic resolution, and built corresponding atomic models. By applying structural analysis as well as biochemistry methods, the authors demonstrated dimeric formation of MprF, exhibited the dynamic nature of the catalytic domain of this enzyme, and proposed a possible model on tRNA binding and aminoacylation.

Major comments 1. In abstract, the authors stated 'Several lipid-like densities are observed in the cryoEM maps, which might indicate the path taken by the lipids and the coupling function of the two functional domains. Thus, the structure of a well characterised PaMprF lays a platform for understanding the mechanism of amino acid transfer to a lipid head group and subsequent flipping across the leaflet that changes the property of the membrane.' Firstly, those lipid-like densities were demonstrated in Fig 3A, since densities of lipids of purified membrane proteins often exist within regions of relatively low local resolution, or low quality, I think more detailed description on how the authors defined which part of the density belongs to lipid and how they acquired the modeling of some of the lipids is required. And the authors modeled phosphatidylglycerol into the GDN MprF, I would require additional experiment, for instance, mass spectrometry over the purified sample, to demonstrate the existence of this specific lipid with the sample. Secondly, regarding the last sentence in the abstract, how these structures lay a platform for further understanding was poorly discussed in both result section and discussion section, since the authors clearly stated 'This cavity perhaps provides a path for holding lipids...', then the statement in the next sentence 'Taken together... the vicinity to the cavities described above indicates the possible path taken by the lipids to enter and exit the enzyme' does not have a reliable evidence to support this conclusion, I would suggest the authors move these statements into discussion section, and elaborate more over this issue since it is an important part in the abstract, or make a more solid proof using other approaches, such as molecular dynamics simulation, to make these statements solid in the result section.

The membranes of E. coli have predominantly phosphatidyl ethanolamine (PE) and phosphatidyl glycerol (PG) as the next abundant lipid with cardiolipin though smaller in number, plays an important role in functioning of many membrane proteins. In our map, the non-protein density are unambiguous and they can be observed as long density reflective of acyl chains (note that GDN used in purification has no acyl chain) and hence attributed these densities to lipids (Fig. EV4E/F and Figure 5A). Only in few of these densities, head group could be modelled and the identity of the lipid as PG at the dimer interface is based on the requirement of negatively charged lipids for oligomerisation of membrane proteins in general (for example – KcsA tetramer formation requires PG, Marius et al., 2005; Valiyaveetil et al., 2002;2004). It is true that the lipid densities are at the peripheral regions of the map but here only acyl chains have been modelled. Within the membrane domain, one reasonably ordered lipid is observed and by analogy with R. tropici structure, it is possible to build a modified-PG (in PaMprF here ala-PG). However, the density of the head group is not unambiguous (unlike lysine in the R. tropici, whose density stands out) and hence we have modelled it as PG alone. In the methods (page 20, lines 649-650), the identification and modelling of lipid densities is described.

We agree that mass spectrometry analysis of purified lipids will be useful but it will not be able to tell the position of the lipid in the map (model) and for this we still require a map at higher resolution with better ordered lipids. We have recently built/developed the workflow for native MS and we plan to initiate analysis of PaMprF in the near future, which will provide details for the lipid purified with the enzyme.

We had initiated molecular dynamics simulation during the review process, and we had included tRNA molecules (shorter version) as we felt the connection between tRNA binding and lipid modification was important. This would have also explained the path taken by lipids (performed by Hankins et al., 2025 in their publication). However, this is likely to require more work (and computing resources) and both mass spectrometry and molecular dynamics will be part of the future work.

We have rewritten the discussion and changed the last line of the abstract to the following

“From the structures, the binding modes of tRNA and lipid transport can be postulated and the mobile secondary structural elements in the synthase domain might play a mechanistic role”.

(in the abstract, lines 24-26).

Fig 2B, it seems the H566 sidechains were overlapping in the zoom-in figure of distance measurement between H566 residues, to clarify this, authors should either present another figure with rotation, to better demonstrate their relative locations, or swap this zoom-in figure with another figure with rotations. Also, could the authors briefly commenting on why they chose H566 for distance measurement specifically?

The side chain of residue H566 in the nanodisc model face towards each other at the interface, hence this residue was chosen to shown the proximity.

Related to previous comment, I see one additional green square in Fig. 2A and an additional green square in Fig. 2B, without any zoom-in images provided on these regions. Besides, they're focusing on two different domains with same color, any particular reason why they're there? If so, please provide the information in figure legends.

The green squares in panels 2A and 2B are the regions that have been zoomed in panels 2D and 2E showing the interactions of the TTH. This is now made clear in the legend as well as in the figure.

Related to previous comment, authors should also provide distance measurement over electrostatic interaction sites in Fig. 2A, since distance plays as an important factor in these forces.

The electrostatic interactions have been included.

For Fig. 2C, since in Fig. 1, the authors have already indicated the differences between reconstruction of the GDN and nanodisc datasets, this information provided here seems to be a bit abundant, I suggest either move this panel to Fig. 1, to make a visualization on both electron densities as well as atomic models, or move this panel to supplementary figures.

We thank the reviewer for the suggestion. The panel, figure 2C is moved to Appendix Fig. S2B.

Fig. 3B, some of the spheres of the lipids were also marked as red, any particular reason why they're red? Do they indicate they're phosphate heads? If so, could the authors provide evidences how they define these orientations of the lipid heads? If not, any particular reason why they're red?

Although, there are non-protein densities (i.e., density beyond noise that remain after modelling of protein residues and found individually) have been modelled as lipids (In Fig. EV4E, these additional densities are shown). Except for few, all these densities have been modelled only as acyl chain. The lipids modelled with head group and phosphate (that have oxygen) and the fit of the density are shown in both figure 3A and EV4F. Hence, the red (oxygen) is seen in the space filling model of lipids (the density for few lipids are shown, also in the response to the comment below).

Fig. 3C, the fitted model of lipid and its corresponding density should be added to Fig. S4, to give more detailed view on the quality of the fitting.

The figure 3 has now been reorganised and the new figure (fig. 5) has only 3 panels. We have provided an enlarged view of the lipids in the membrane domain along with unmodelled densities in 3A. In addition, in fig. EV4F, fit of the lipid to density (select lipids) are shown.

Fig. 4D and 4E, could the authors also indicate the RMSD values when comparing the differences of RtMprF, PaMprF, ReMprF, this information would be helpful to understand how big of a difference within these three models.

The RMSD values of the structural comparison is given in the text.

Fig. 6E, the coloring used for CCA-Ala were similar to the blue part of soluble domain, could the authors change the coloring a bit? Also, for Fig. 6F, I would suggest the authors provide a prediction model, such as using AlphaFold3, of this tRNA interaction site, to further validate this proposed model.

The colour of the CCA part is changed in the revised figure. Following the suggestion of the reviewer, we used AlphaFold3 to predict the complex formation of PaMprF with tRNA (or shorter version) (Figure 2). As mentioned above in response to reviewer 1, the prediction of dimeric enzyme was of low confidence and this is also reflected when a combination of tRNA, lipids and enzyme sequence are given. Instead of full-length tRNA, if only the CCA end is provided, then the prediction program does position this in the postulated cavity. Only with the monomeric enzyme and tRNA does one get a reasonable model. With respect to the proposed model in 6F, currently we don’t have any evidence and this remains a postulate. In the revised manuscript, we have replaced this with conservation figure, which we thought is more relevant.

In Supplementary Figures S1 and S3, the angular distribution of maps exhibited preferred orientation to certain extent, 3D FSC estimation should also be supplied for these maps, as an indication of whether the reconstructed densities were affected or not.

We have included the 3DFSC plots for all the data sets (including the new ones in figures EV1, 2, 5, 6, 7). It is evident that the nanodisc datasets in general are slightly anisotropic.

For Fig S3B, could the authors switch to another image with better contrast?

This is now replaced with an image to show the particles.

Minor comments 1. Fig. 2E and 2F, distance measurement should also be supplied to these two panels.

We have now included the distance measurement in both the panels, which are now Fig. 2D and 2E.

Fig. 5D, since in Fig. 4F and 4G already mentioned the skeleton of GDN, this modeling part should be presented before exhibit it in dimer interface, the authors should rearrange the sequence over these three panels.

The figures in the revised manuscript has been rearranged. Figure 5 (now figure 4) has been modified to include the biochemical analysis (crosslinking studies) and the panel 5D has been removed.

In Supplementary Figure S3, which density was shown for the PaMprF local resolution estimation result? Authors should provide this information as two maps were shown in this figure.

The local resolution is for C2 symmetrised map and this is now mentioned in the panel.

CROSS-REFEREE COMMENTS Both Reviewer #1 and #3 made comments over technical issue, their evaluation over functional aspects of this protein is what I was lacking over my comments, also, their evaluation of the biological narrative, relevance toward previous research is also more insightful. Finally, they offer valuable suggestions on how to adjust the article to make it more readable, and better describing the biological story which I would suggest the authors to pay attention to.

Reviewer #2 (Significance (Required)):

Significance The authors mainly focused on the structure of MprF in Pseudomonas aeruginosa, this protein is essential for the resistance to cationic antimicrobial peptides. A combination of structural and biochemical analysis provided evidences to the dimeric formation to this enzyme, and the analysis over differences of purified proteins using GDN and nanodisc was particular interesting, which provide new insight regarding the flexible nature of this enzyme, and potentially could be beneficial to the membrane protein community, as it demonstrates the differences in detergent/nanodisc of choice could affect the assembly of the protein of interest. Still, some of the statements in the manuscript, for instance, the assignment of lipids was over-claimed and could be benefited from additional approaches to support the issue. I would suggest some refinement in the discussion section as well as some of the figures.

My expertise: cryo-EM single particle analysis; cryo-ET; sub-tomo averaging; cryo-FIB;

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

Jha and Vinothkumar characterize the cryoEM structure of the alanyl-phosphatidylglycerol producing multiple peptide resistance factor (MprF) of Pseudomonas aeruginosa. MprF proteins mediate the transfer of amino acids from aminoacyl-tRNAs to negatively charged phospholipids resulting in reduced membrane interactions with cationic antimicrobial peptides (produced by the host and competing microorganisms). The phospholipid modifications involve in most cases the transfer of lysine or alanine to phosphatidylglycerol. MprF proteins are membrane proteins consisting of a soluble and hydrophobic domain. Multiple functional studies have shown that the soluble domain of MprF mediates the aminoacylation of phosphatidylglycerol, while the hydrophobic domain mediates the "flipping" of aminoacylated phospholipids across the membrane, a process that is crucial to repulse or prevent the interaction of antimicrobial peptides encountered at the outer leaflet of bacterial membranes. Aside from its role in conferring antimicrobial peptide resistance, other roles of MprF have been described including more physiological roles such as improving growth under acidic conditions. Interestingly, MprF proteins are also found in Gram-negative bacteria which are already protected by an additional membrane that includes LPS. However, in Pseudomonas aeruginosa, MprF confers phenotypes that are similar to those observed in Gram-positive bacteria. Importantly, crystal structures of the soluble domain have led to important insights into aminoacyl phospholipid synthesis and recent studies on the cryoEM structure of Rhizobium tropici have confirmed functional and preliminary structural studies with other MprF proteins. The cryoEM structure from R. tropici confirmed the dimeric structure of MprF and supported a role of the hydrophobic domain in flipping lysyl-phosphatidylglycerol across the membrane. A comparison of the structures of lysyl-phosphatidylglycerol with alanyl-phosphatidylglycerol producing MprFs could reveal new insights into the mechanism of transferring aminoacyl-phospholipids from the soluble domain to the hydrophobic domain and translocation of alanyl- vs lysyl-phosphatidylglycerol across the membrane.

Major concerns

1. The study by Jha and Vinothkumar provides the cryoEM structure of an alanyl-phosphatidylglycerol producing MprF protein which is in principle an important milestone in gaining a better understanding of the mechanism of aminoacyl-phospholipid synthesis and flipping, including the potentially different requirements of accommodating different aminoacyl -tRNAs and aminoacyl-phospholipid species. However, this is not addressed. The authors present a "distinct architecture" compared to the structure of R. tropici- MprF, without providing functional insights and the focus of the study shifts to the role of detergents in determining MprF structures via cryoEM. Thus, after fundamental discoveries have been made with crystal structures of the soluble domain and cryoEM structure of R. tropici, this study -while valuable as a resource- seems to offer only an incremental advance in understanding the mode of action of MprF and the potential different requirements for transferring alanyl-phosphatidylglycerol to the hydrophobic domain and flipping across the membrane. The reader is left with the finding of a distinct architecture with no further explanation or hypothesis.

We thank the reviewer for his/her comments. It is true that the crystal structures of soluble domains of MprF (from 3 species) and the cryoEM structures are now available (two Rhizobium species). However, the cryoEM maps that we have obtained has several salient features including the distinct dimeric interface and the position of the C-terminal helix of the soluble domain. This in particular is important. In the previous study, Hebecker et al 2011 had reported that the terminal helix of PaMprF was important for the activity and the construct without the TM domain can also function in modifying the lipids. The full-length cryoEM map of PaMprF in GDN now provides an idea how this occurs, with the terminal helix buried at the interface. Further, the proposed tRNA binding site (from Hebecker et al 2015, lysine amide bound structure) face other in the dimeric architecture of R. tropici and it is not clear how the full-length tRNA will bind without disrupting the dimer. In contrast, the dimer architecture observed for PaMprF has the tRNA binding site facing away and they can bind to the enzyme without any constraints. We think the mobile/dynamic elements (or secondary structure) of the synthase domain play a major role in interaction with substrates and mechanism. The current structures provide some evidence for this and form the basis of future studies. Instead of cartoon description, we have now included a conservation plot of the molecule in explaining the possible mechanism along with the surface representation in figure 6.

Differences to R.tropici MprF and other studies are difficult to follow as only a topological map of the Pseudomonas MprF is provided and conserved amino acids that have been shown to be crucial in mediating synthesis and flipping are not highlighted in the text or in the figures, specifically addressed, or discussed. Conserved amino acids in the presented cryoEM structure could provide important mechanistic insights and could address substrate specificity/requirements for aminoacyl phospholipid synthesis, transfer to the hydrophobic domain and flipping.

The conservation of residues across MprF homologues have been presented in previous published articles and hence, initially we had not included in the manuscript. We have now included multiple sequence alignment of select homologues of MprF highlighting conserved residues (Appendix Fig. S6) as well a figure (Fig. 6F) colouring the molecule with conservation scores with CONSURF. In figure 6F, zoomed in version, we highlight the many of the conserved residues in the synthase domain as they play a role in substrate selectivity.

Authors characterize an alanyl-phosphatidylglycerol producing MprF but do not detect the lipid in the cryoEM structure. Thus, the potential path taken by alanyl-phosphatidylglycerol remains unclear. Authors model the detected lipids as phosphatidylglycerol, which may be an interesting finding as it would indicate that MprF is generally capable of flipping phospholipids (this is however not discussed). While it is plausible that MprF flippases may be able to flip phosphatidyglycerol it could have a different path and structural requirements. It is also difficult to follow what the suggested pathway of flipping is in the Pseudomonas-MprF flippase (compared to R.tropici). Authors could provide a similar overview figure as in Song et al. and indicate what the potential differences are.

We modelled phosphatidylglycerol as the lipid as the current density doesn’t allow to model ala-PG ambiguously though it is found in the same position as the lys-PG in the R. tropici maps. The recent in-vitro assay by Hankins et al 2025 shows that PaMprF is able to flip wide range of lipids and we would also like to point out that PG from outer leaflet can be flipped, whose headgroup can be modified at the inner leaflet and flipped back. As shown by Song et al 2021 and Hebecker et al 2011, the specificity for the substrates is in the synthase domain (by mutagenesis and swapping). We don’t think there will be any difference between the lys-PG and Ala-PG path but in our opinion the positional relation between the soluble and membrane domain is the most important and has remained the focus of the manuscript along with the dimeric architecture. The figure 6 in the manuscript is descriptive of this and provides a summary of the structural observation from the presented structures.

Minor concerns

• Page 13: the following sentence should be rephrased: "Among the missing links in the current cryoEM maps is the lack of well-ordered density for lipid molecules on the inner leaflet closer to the re-entrant helices but it is reasonable to assume from the cluster of positive charge that there will be lipid molecules and are dynamic. "

This is has been rephrased.

• Page 4: Klein et al do not show that the Pseudomonas aeruginosa MprF mediates flipping

Corrected to reflect only the modification of lipid and not flipping.

Reviewer #3 (Significance (Required)):

General assessment: see review

Advance: Minor

Audience: Specialized

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

Referee #3

Evidence, reproducibility and clarity

Jha and Vinothkumar characterize the cryoEM structure of the alanyl-phosphatidylglycerol producing multiple peptide resistance factor (MprF) of Pseudomonas aeruginosa. MprF proteins mediate the transfer of amino acids from aminoacyl-tRNAs to negatively charged phospholipids resulting in reduced membrane interactions with cationic antimicrobial peptides (produced by the host and competing microorganisms). The phospholipid modifications involve in most cases the transfer of lysine or alanine to phosphatidylglycerol. MprF proteins are membrane proteins consisting of a soluble and hydrophobic domain. Multiple functional studies have shown that the soluble domain of MprF mediates the aminoacylation of phosphatidylglycerol, while the hydrophobic domain mediates the "flipping" of aminoacylated phospholipids across the membrane, a process that is crucial to repulse or prevent the interaction of antimicrobial peptides encountered at the outer leaflet of bacterial membranes. Aside from its role in conferring antimicrobial peptide resistance, other roles of MprF have been described including more physiological roles such as improving growth under acidic conditions. Interestingly, MprF proteins are also found in Gram-negative bacteria which are already protected by an additional membrane that includes LPS. However, in Pseudomonas aeruginosa, MprF confers phenotypes that are similar to those observed in Gram-positive bacteria. Importantly, crystal structures of the soluble domain have led to important insights into aminoacyl phospholipid synthesis and recent studies on the cryoEM structure of Rhizobium tropici have confirmed functional and preliminary structural studies with other MprF proteins. The cryoEM structure from R. tropici confirmed the dimeric structure of MprF and supported a role of the hydrophobic domain in flipping lysyl-phosphatidylglycerol across the membrane. A comparison of the structures of lysyl-phosphatidylglycerol with alanyl-phosphatidylglycerol producing MprFs could reveal new insights into the mechanism of transferring aminoacyl-phospholipids from the soluble domain to the hydrophobic domain and translocation of alanyl- vs lysyl-phosphatidylglycerol across the membrane.

Major concerns:

1. The study by Jha and Vinothkumar provides the cryoEM structure of an alanyl-phosphatidylglycerol producing MprF protein which is in principle an important milestone in gaining a better understanding of the mechanism of aminoacyl-phospholipid synthesis and flipping, including the potentially different requirements of accommodating different aminoacyl -tRNAs and aminoacyl-phospholipid species. However, this is not addressed. The authors present a "distinct architecture" compared to the structure of R. tropici- MprF, without providing functional insights and the focus of the study shifts to the role of detergents in determining MprF structures via cryoEM. Thus, after fundamental discoveries have been made with crystal structures of the soluble domain and cryoEM structure of R. tropici, this study -while valuable as a resource- seems to offer only an incremental advance in understanding the mode of action of MprF and the potential different requirements for transferring alanyl-phosphatidylglycerol to the hydrophobic domain and flipping across the membrane. The reader is left with the finding of a distinct architecture with no further explanation or hypothesis.

2. Differences to R.tropici MprF and other studies are difficult to follow as only a topological map of the Pseudomonas MprF is provided and conserved amino acids that have been shown to be crucial in mediating synthesis and flipping are not highlighted in the text or in the figures, specifically addressed, or discussed. Conserved amino acids in the presented cryoEM structure could provide important mechanistic insights and could address substrate specificity/requirements for aminoacyl phospholipid synthesis, transfer to the hydrophobic domain and flipping.

3. Authors characterize an alanyl-phosphatidylglycerol producing MprF but do not detect the lipid in the cryoEM structure. Thus, the potential path taken by alanyl-phosphatidylglycerol remains unclear. Authors model the detected lipids as phosphatidylglycerol, which may be an interesting finding as it would indicate that MprF is generally capable of flipping phospholipids (this is however not discussed). While it is plausible that MprF flippases may be able to flip phosphatidyglycerol it could have a different path and structural requirements. It is also difficult to follow what the suggested pathway of flipping is in the Pseudomonas-MprF flippase (compared to R.tropici). Authors could provide a similar overview figure as in Song et al. and indicate what the potential differences are.

Minor concerns:

1. Page 13: the following sentence should be rephrased: "Among the missing links in the current cryoEM maps is the lack of well-ordered density for lipid molecules on the inner leaflet closer to the re-entrant helices but it is reasonable to assume from the cluster of positive charge that there will be lipid molecules and are dynamic. "

2. Page 4: Klein et al do not show that the Pseudomonas aeruginosa MprF mediates flipping

Significance

General assessment:

The study by Jha and Vinothkumar provides the cryoEM structure of an alanyl-phosphatidylglycerol producing MprF protein which is in principle an important milestone in gaining a better understanding of the mechanism of aminoacyl-phospholipid synthesis and flipping, including the potentially different requirements of accommodating different aminoacyl -tRNAs and aminoacyl-phospholipid species. However, this is not addressed. The authors present a "distinct architecture" compared to the structure of R. tropici- MprF, without providing functional insights and the focus of the study shifts to the role of detergents in determining MprF structures via cryoEM. Thus, after fundamental discoveries have been made with crystal structures of the soluble domain and cryoEM structure of R. tropici, this study -while valuable as a resource- seems to offer only an incremental advance in understanding the mode of action of MprF and the potential different requirements for transferring alanyl-phosphatidylglycerol to the hydrophobic domain and flipping across the membrane

Advance: Minor

Audience: Specialized

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

Referee #2

Evidence, reproducibility and clarity

Shaileshanand J. et al., reported the structures of Multiple Peptide Resistance Factor, MprF, which is a bi-functional enzyme in bacteria responsible for aminoacylation of lipid head groups. The authors purified MprF from Pseudomonas aeruginosa in GDN micelles and nanodiscs, and by applying cryo-EM single particle method, they successfully reached near-atomic resolution, and built corresponding atomic models. By applying structural analysis as well as biochemistry methods, the authors demonstrated dimeric formation of MprF, exhibited the dynamic nature of the catalytic domain of this enzyme, and proposed a possible model on tRNA binding and aminoacylation.

Major comments:

1. In abstract, the authors stated 'Several lipid-like densities are observed in the cryoEM maps, which might indicate the path taken by the lipids and the coupling function of the two functional domains. Thus, the structure of a well characterised PaMprF lays a platform for understanding the mechanism of amino acid transfer to a lipid head group and subsequent flipping across the leaflet that changes the property of the membrane.' Firstly, those lipid-like densities were demonstrated in Fig 3A, since densities of lipids of purified membrane proteins often exist within regions of relatively low local resolution, or low quality, I think more detailed description on how the authors defined which part of the density belongs to lipid and how they acquired the modeling of some of the lipids is required. And the authors modeled phosphatidylglycerol into the GDN MprF, I would require additional experiment, for instance, mass spectrometry over the purified sample, to demonstrate the existence of this specific lipid with the sample. Secondly, regarding the last sentence in the abstract, how these structures lay a platform for further understanding was poorly discussed in both result section and discussion section, since the authors clearly stated 'This cavity perhaps provides a path for holding lipids...', then the statement in the next sentence 'Taken together... the vicinity to the cavities described above indicates the possible path taken by the lipids to enter and exit the enzyme' does not have a reliable evidence to support this conclusion, I would suggest the authors move these statements into discussion section, and elaborate more over this issue since it is an important part in the abstract, or make a more solid proof using other approaches, such as molecular dynamics simulation, to make these statements solid in the result section.

2. Fig 2B, it seems the H566 sidechains were overlapping in the zoom-in figure of distance measurement between H566 residues, to clarify this, authors should either present another figure with rotation, to better demonstrate their relative locations, or swap this zoom-in figure with another figure with rotations. Also, could the authors briefly commenting on why they chose H566 for distance measurement specifically?

3. Related to previous comment, I see one additional green square in Fig. 2A and an additional green square in Fig. 2B, without any zoom-in images provided on these regions. Besides, they're focusing on two different domains with same color, any particular reason why they're there? If so, please provide the information in figure legends.

4. Related to previous comment, authors should also provide distance measurement over electrostatic interaction sites in Fig. 2A, since distance plays as an important factor in these forces.

5. For Fig. 2C, since in Fig. 1, the authors have already indicated the differences between reconstruction of the GDN and nanodisc datasets, this information provided here seems to be a bit abundant, I suggest either move this panel to Fig. 1, to make a visualization on both electron densities as well as atomic models, or move this panel to supplementary figures.

6. Fig. 3B, some of the spheres of the lipids were also marked as red, any particular reason why they're red? Do they indicate they're phosphate heads? If so, could the authors provide evidences how they define these orientations of the lipid heads? If not, any particular reason why they're red?

7. Fig. 3C, the fitted model of lipid and its corresponding density should be added to Fig. S4, to give more detailed view on the quality of the fitting.

8. Fig. 4D and 4E, could the authors also indicate the RMSD values when comparing the differences of RtMprF, PaMprF, ReMprF, this information would be helpful to understand how big of a difference within these three models.

9. Fig. 6E, the coloring used for CCA-Ala were similar to the blue part of soluble domain, could the authors change the coloring a bit? Also, for Fig. 6F, I would suggest the authors provide a prediction model, such as using AlphaFold3, of this tRNA interaction site, to further validate this proposed model.

10. In Supplementary Figures S1 and S3, the angular distribution of maps exhibited preferred orientation to certain extent, 3D FSC estimation should also be supplied for these maps, as an indication of whether the reconstructed densities were affected or not.

11. For Fig S3B, could the authors switch to another image with better contrast?

Minor comments:

1. Fig. 2E and 2F, distance measurement should also be supplied to these two panels.

2. Fig. 5D, since in Fig. 4F and 4G already mentioned the skeleton of GDN, this modeling part should be presented before exhibit it in dimer interface, the authors should rearrange the sequence over these three panels.

3. In Supplementary Figure S3, which density was shown for the PaMprF local resolution estimation result? Authors should provide this information as two maps were shown in this figure.

CROSS-REFEREE COMMENTS

Both Reviewer #1 and #3 made comments over technical issue, their evaluation over functional aspects of this protein is what I was lacking over my comments, also, their evaluation of the biological narrative, relevance toward previous research is also more insightful. Finally, they offer valuable suggestions on how to adjust the article to make it more readable, and better describing the biological story which I would suggest the authors to pay attention to.

Significance

Significance

The authors mainly focused on the structure of MprF in Pseudomonas aeruginosa, this protein is essential for the resistance to cationic antimicrobial peptides. A combination of structural and biochemical analysis provided evidences to the dimeric formation to this enzyme, and the analysis over differences of purified proteins using GDN and nanodisc was particular interesting, which provide new insight regarding the flexible nature of this enzyme, and potentially could be beneficial to the membrane protein community, as it demonstrates the differences in detergent/nanodisc of choice could affect the assembly of the protein of interest. Still, some of the statements in the manuscript, for instance, the assignment of lipids was over-claimed and could be benefited from additional approaches to support the issue. I would suggest some refinement in the discussion section as well as some of the figures.

My expertise: cryo-EM single particle analysis; cryo-ET; sub-tomo averaging; cryo-FIB;

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Referee #1

Evidence, reproducibility and clarity

MprF proteins exist in many bacteria to synthesize aminoacyl phospholipids that have diverse biological functions, e.g. in the defense against small cationic peptides. They integrate two functions, the aminoacylation of lipids, i.e. the transfer of Lys, Arg or Ala from tRNAs to the head group, and the flipping of these modified lipids to the membrane outer leaflet. The authors present structures of MprF from Pseudomonas aeruginosa and describe these structures in great detail. As MprF enzymes confer antibiotic resistance and are therefore highly important, studying them is significant and interesting. Consequently, their structures have been substantially characterized in recent years, including the publication of the dimeric full-length MpfR from Rhizobium (Song et al., 2021).

• While the structural work appears to be solid and carried out well on the technical part, one big criticism is how the data are presented in the manuscript, how they are analyzed and how they are put into relation to previous work. As structures of Mpfr from Rhizobium have been published, it is not required and rather distracting to explain the methodological details and the structure of Pseudomonas MprF in such great detail. Instead, the manuscript would benefit very strongly from reaching the interesting and novel parts, the comparison with the previous structures, as early as possible. Overall, the manuscript should be substantially shortened to not divert the reader's attention away from the novel parts by drowning them in miniscule description of the structural features such as secondary structure elements or lipid molecule positions where it remains completely unclear what their relevance is to the story and the message of the paper. Finally, during this revision, care should be taken to improve the language and maybe involve a native speaker in doing so.

• Even more importantly, since the authors observe a dimer interface which strongly deviates from the previously presented arrangement of another species, the most important thing would be to properly characterize this interface and experimentally validate it, both of which has not been done sufficiently. When also taking into account that there were significant differences in the arrangement of the dimer between their structures in GDN and nanodisc, and that in the GDN structure, the cholesterol backbone of GDN appears to be involved in the interface (there should not be any cholesterol in native bacterial membranes!), there is a realistic chance that the observed dimer is an artefact. If the authors cannot convincingly rule out this possibility, all their conclusions are meaningless.

• Hence, while I think that the data presented here would be worth publishing. However, a major drawback is that the authors do not sufficiently analyse, characterise and validate the dimer interface and fail to show that the dimer is biologically relevant.

Major points:

• The authors always jump between their structures in detergent and nanodisc during all the descriptions, which makes following the story even more difficult. Please first describe one of the structures and then (briefly) discuss relevant similarities and differences afterwards.

• The difference in dimerization between Pseudomonas and Rhizobium is the most interesting and surprising feature (if true) of the new structures. However, it is not really presented as such. The authors should put more emphasis on making clear that this is a complete rotation of the monomers with respect to each other (by how many degrees?) and they should visualize it even more clearly in Figure 4 (and label the figure so that it is possible to understand it without having to read the text or the legend first).

• P. 10: The authors insinuate that only one of the dimer interfaces, either Pseudomonas or Rhizobium could be real, but disregard the possibility that both might be the biologically relevant interfaces of the respective species and that there might have been a switch of interfaces during evolution. They should also mention and discuss this possibility.

• Fig. 5G: The authors claim that the higher molecular band that appears in the mutant is a "dimer with aberrant migration" of >250 kDa as opposed to the expected 150 kDa. They should explain how they came to this conclusion and how they can be sure that the band does not correspond to a higher oligomer (trimer or tetramer). They could show, by extraction and purification scheme similar to the wildtype using first LMNG and then GDN, followed by at least a preliminary EM analysis, that the crosslinked mutant MprF is indeed a dimer, or use other biophysical methods to do the same, otherwise this experiment does not show much. Furthermore, they should also include a cysteine mutant in the part of Pseudomonas MprF that would be involved in a Rhizobium-like interface in their crosslinking experiments to check whether they could also stabilize dimers in this case.

• As the question whether the observed interface is real or an artefact is very central to the value of the structural data and the drawn conclusions from it, the authors should make more effort to analyze and try to validate the interface. First, an analysis of interface properties (buried surface area, nature of the interactions, conservation) should be performed for the interface as observed in the Pseudomonas structure but also for a (hypothetical) Rhizobium-like interface of two Pseudomonas monomers (such a model of a dimer should be easily obtainable by AlphaFold using the available Rhizobium structures as models). Then, experimental methods such as FRET or crosslinking-MS would allow to draw more solid conclusions on the distances between potential interface residues. While these experiments are a certain effort, the question whether the dimer interface is real is so central to the paper that it would be worthwhile to make this effort.

• As it seems that detergents might disrupt or modify the dimer interface, it might be an alternative to solubilize the protein in a more native environment by polymer-stabilized nanodiscs using DIBMA or similar molecules.

• Since parts of the Discussion are mostly repetitions of the Results part and other parts of the Discussion also contain a large extend of structure analysis one would usually rather expect in the Results part instead of the Discussion, the authors should consider condensing both to a combined (and overall much shorter) Results & Discussion section.

Minor points:

• Explain abbreviations the first time they appear in the text, e.g. TTH

• Figure labels are very minimalistic. This should be improved, e.g. by putting labels to important structural features that appear in the text, otherwise the figures are not an adequate support for the text.

• Figure 5: Label where the different oligomers run on the gels

Significance

While the structural work appears to be solid and carried out well on the technical part, one big criticism is how the data are presented in the manuscript, how they are analyzed and how they are put into relation to previous work. As structures of Mpfr from Rhizobium have been published, it is not required and rather distracting to explain the methodological details and the structure of Pseudomonas MprF in such great detail. Instead, the manuscript would benefit very strongly from reaching the interesting and novel parts, the comparison with the previous structures, as early as possible. Overall, the manuscript should be substantially shortened to not divert the reader's attention away from the novel parts by drowning them in miniscule description of the structural features such as secondary structure elements or lipid molecule positions where it remains completely unclear what their relevance is to the story and the message of the paper. Finally, during this revision, care should be taken to improve the language and maybe involve a native speaker in doing so.

• Even more importantly, since the authors observe a dimer interface which strongly deviates from the previously presented arrangement of another species, the most important thing would be to properly characterize this interface and experimentally validate it, both of which has not been done sufficiently. When also taking into account that there were significant differences in the arrangement of the dimer between their structures in GDN and nanodisc, and that in the GDN structure, the cholesterol backbone of GDN appears to be involved in the interface (there should not be any cholesterol in native bacterial membranes!), there is a realistic chance that the observed dimer is an artefact. If the authors cannot convincingly rule out this possibility, all their conclusions are meaningless.

• Hence, while I think that the data presented here would be worth publishing. However, a major drawback is that the authors do not sufficiently analyse, characterise and validate the dimer interface and fail to show that the dimer is biologically relevant.

viewing something as a voyeur makes the observation seem more credible, viewing something as an audience member makes the observation seem less credible

Highlights the tension between technological advancement and human flourishing

a grassroot resistance against over-technologizing education.

rapid innovation can compromise human reflection and social cohesion.

Modern technological acceleration can hinder these communal values.

technology often segregates perspectives through algorithm echo chambers.

digital acceleration and pandemic isolation amplified self-centered behaviors.

Modern tech can undermine mutual care by promoting isolation and individualism.

This reflects a core Socratic method-examining the present to imagine possible better futures.

highlights both the promise and pressure of technology; Socratic reflection would ask weather reliance on tech distracts from deeper educational goals.

connects to hope, method, and critique.

This phrase captures the Socratic ideal—knowledge grounded in uncertainty.

This mirrors the dialectic method—questioning, observing contradictions, and reflecting toward moral clarity.

A key ethical insight—hope is not escapism, but moral resistance, akin to Socratic self-knowledge in adversity.

A warning about over-reliance on technology, consistent with your argument that tech utopias threaten democratic self-examination.

this idea suggests relying on intellectual complacency can limit imagination.

the author signals that utopian thought can be both creative and dangerous, echoing topic about the threat of technological utopias to democracy.

Emphasizes human and democratic priorities over technological determinism; reflects Socratic principles of examined life.

school closures show impact on technology-driven crises on social institutions, relevant to discussions of technological utopias and democracy.

illustrates societal fragility and need for reflection, echoing Socratic self-examination.

Final focus reinforces the importance of reflection, care, and community in technological societies.

The word "hope" connects Ernst Bloch's philosophy, framing futurity as a moral and imaginative act rather than prediction.

This subsetting of raw data is incorrect. Listwise deletion of records prior to setting the survey design object will problematize variance estimation.

This is what I was looking for in the earlier primary models section regarding stratum-specific estimates. Why break it up this way?

The approach you use here is called a linear combination of coefficients (or LINCOM) method. It's an appropriate method and properly implemented here.

For future reference, though, you could just re-implement your initial cox models while altering the reference level of the modifier in order to obtain stratum specific estimates because the beta-coefficient is the stratum-specific estimate for the given reference level of the modifier.

I highly encourage you to explore ggplot() as your primary visualization modality, given its flexibility in generating high quality graphics. The plot function here can work, but the graphics produced in your initial markdown HTML were difficult to parse visually.

I don't fully understand the rationale for this approach and why you consider it "robust." This seems needlessly complicated.

The svykm() function can accommodate a singular categorical predictor like your depression and food security indicators.

Indeed, if you wanted, you could generate a 4-level categorical predictor of the interaction between depression and food insecurity.

For models 3a and 3b I cannot identify how you generate stratum-specific estimates of your main effect as reported in Table 2 of your main submission.

This makes me wonder whether you accurately represent the quantities reported in Table 2...

The potentially problematic result of this practice is that each of your models will use a different sample size...

This code section seems unnecessarily complicated given the tools available from the tableone or gtsummary or the course-specific svyTable1 packages, usage of which would be less prone to user-error.

This subsetting procedure is correct, but the object df was initially improperly subset per line 439 of Section 1 (Overview).

I was unable to re-run this file because of this assumption.

In practice, your file size is not so large that re-running the import + data preparation protocols would be time-prohibitive. For the sake of file management, if you wish to create two separate files (one for data importing/cleaning and one for analysis) then that is workable. But iteratively saving and relying upon new data sets complicates reproducibility.

Again, importing data directly from the CDC FTP will obviate the need to set relative/absolute directory pathways.

If weights are used in analyses, they should be WTMEC2YR.

You are HIGHLY encouraged to import mortality data directly from the CDC FTP web portal to facilitate reproducibility.

For example, if you were to submit this file as supplementary material for publication, your code would not be reproducible because mortality files are imported from a local directory.

This code is incorrect.

NHANES supplies R code for importing mortality files (see "Sample R program").

NOTE that the R import file contains instructions for importing NHIS mortality data and NHANES mortality data. Follow instructions for NHANES. This code is also adapted in the course text (see "Link mortality data to NHANES").

Discover how web design drives business growth, boosting brand credibility, enhancing user experience, increasing conversions, and improving SEO.

refers to the thought preservation through contextual change

This article highlights how people, such as famous influencers, create unrealistic body standards through editing and Photoshop. Through these standards, it can harm people's mental health since they have a thought to compare themselves to others. These comparisons are very damaging, and they're causing people to resort to surgery to compete with the comparisons.

I agree with this statement. A prototype doesn't have to be the final version of your solution, it is just the first step to getting there. I used to view this differently; I thought that a prototype was the final stage of the design process and needed very few changes as it was closest to the actual product. However, I have realised that a prototype helps you test out your ideas and use it to make a better version in the future.

I like how this section frames prototyping as a process of decision-making rather than just building for the sake of it. I agree that choosing what to prototype depends a lot on the context of it. Time, resources, and risk are all things that need to be accounted for. This made me realize that smart prototyping is rarely just about creativity, but strategy too.

Annotation (Stronger Alternative): The author says marketing is too complex for humans and that automation is the only answer. That point makes sense, but it feels a little one sided. A stronger version of this argument would mention that while automation helps with speed and data, human judgment still matters for creativity and ethics. Mixing both human and machine input would make the point more balanced and believable.

Annotation (Validity): The explanation of semi supervised, and transfer learning is logically structured, moving from defining each concept to explaining how they work. However, the conclusion that transfer learning is “effective at leveraging existing knowledge” could use more direct support. The author assumes effectiveness without showing clear evidence or examples beyond image analysis, so the argument would be stronger if they explained why or how it works well in that field.

Annotation (Soundness): Most of the explanation here is accurate and well grounded in established reinforcement learning concepts. However, saying the “greedy algorithm produces reasonable results” is a bit weak and vague. It’s true only in limited situations where rewards don’t change much or exploration isn’t needed, but in most real world cases, a purely greedy approach performs poorly. The author should clarify the conditions under which the greedy algorithm is actually effective to make the statement sounder and more precise.

Annotation (Ambiguity or Misleading Language): The phrase “Unbeknownst to the consumer” makes it sound like people have no idea that algorithms are behind their searches or ads. That might be a bit vague and exaggerated since most people know the internet uses some kind of automation. The author could be clearer if they mean that people don’t know how much automation is involved instead of saying they don’t know at all.

don't use hyperlinks since the links change daily

on work cited page ONLY add sources you cited from

format

It is important to acknowledge the shortcomings of MLA formatting in order to better understand its uses.

add "qtd. in" Incase of an indirect source preferrably find direct source

when author is unknown replace it with the source title

not citing a source is taking credit for someone's work

mark citations to avoid plagiarism

add citations to credit the authors and inform readers where the information is coming from

It is important to have a good set of priorities when citing sources. It is important to have less of the correct information, rather than more of the wrong information.

This sentence made me wonder why in text citations are in parentheses, so upon a google search "In-text citations are in parentheses to provide immediate, non-disruptive source information to the reader."

This is unclear from the reader's perspective, likely because of the compound noun "platform mechanics references" -- break it up: "Student references to reddit or subreddit mechanics demonstrate..."

Transition is hard to follow from one idea to the next. The flow of content and expressions needs to be more methodical and progressive with throughlines that bind together content, expression, or the argumentative progression of the chapter.

Consider revising the structure of this sentence to make it more economical and clearly constructed.

Add priority to the computational analysis of metadiscourse across the CUNY Reddit Hub, chronicled in the two most recent blog post entries. Pull from those feed items to write paragraph-level content framing the two posts on metadiscourse, situated within the literature of public-sphere theory in digital spaces, arguing for these subreddit communities as constitutive of local networked publics for their reflexive circulation of discourse, among other factors discussed in Warner's "Publics and Counterpublics" paper.

I find this very interesting because a criticism that is often levied at fairy tales is their enforcing of patriarchal values. This lesson thought by the fairy tales still in a way assumes patriarchal society but it doesn't treat finding a husband as the end goal but as something that can have it's own concerns. This isn't princess marries prince charming because he showed up. It is more like princess thinks about if prince charming is gonna murder her instead of naively following him.

This shows that fairy tales are not an a made up story but rather a reflection of reality. The term happy ending refers to the hope that people have in overcoming hardships. This shows growth from suffering rather than growth from avoiding it. Story telling makes the truth easier to except by adding imagery and magic.

This explains the life of farm workers and what they go through, these stories and facts are being told in order to help the workers receive healthier working conditions.

This is another case thats being described, a boy known as Felipe, was born with no limbs or legs, his mother was exposed to pesticides without knowing the harm they did to her, during her pregnancy.

I believe they share the stories of farm workers, who suffered the consequences of pesticides. For example, Johnnie, who suffered with cancer.

This explains the unfair treatment of black people had in America and calls for recognition of their human rights and dignity.

It talks about how the books that were read opened their eyes to history and gave them insights towards black history and before slavery in America.

This starts off with how Malcom X's life was, such as changing his name during prison, and promoted his ideas with the nation of islam. He changed his beliefs to support unity in civil rights during a trip. Sadly he was assassinated soon after.

good question. they overlap a bit but they serve different purposes.

command = intent

event = outcome

command pattern is about explicit control of an action. you create a Command object, pass it to an executor, and call execute() when you want. you know who asked for it, when it ran, and what the outcome was. it’s about invoking behavior in a controlled and traceable way.

event-driven architecture is about reactions to things that already happened. an event is a statement of fact: “user.created”. it doesn’t command anyone to do something; it just signals that something occurred. listeners may respond or ignore it, you don’t control that directly.

so:

• use commands when you want deterministic, transactional actions with well-defined ownership and lifecycle (e.g. workflows, CQRS “write” side, retryable jobs).
• use events when you want decoupled, asynchronous fan-out or notifications (e.g. “send welcome email” after user created).

many systems use both. commands cause events. example: CreateUserCommand → executed → emits UserCreatedEvent.

Here’s how it works:

Each service in your system gets a sidecar proxy (like Envoy) deployed next to it. This proxy intercepts all inbound and outbound traffic for that service.

These proxies handle network-level logic: retries, timeouts, encryption (mTLS), rate limiting, circuit breaking, etc.

A control plane (in Istio or Linkerd) manages all these proxies. It pushes configurations dynamically — so you can, for example, shift 10% of traffic to a new version or enforce mTLS across all services without touching the app code.

Because all traffic goes through these proxies, you get automatic observability — metrics, tracing, and logs across your entire service graph.

Kẻ tấn công có thể dễ dàng thiết lập một cuộc tấn công ET ở những nơi công cộng khi mạng sử dụng Wi-Fi mở.

Thực tế cho thấy, nghiên cứu của Norton cho biết có đến 68% người dùng Wi-Fi công cộng đã trở thành nạn nhân của tội phạm mạng, chủ yếu do mất cắp dữ liệu nhạy cảm . Con số này phản ánh rõ mức độ nguy hiểm thực tế của các cuộc tấn công như Evil Twin Ngoài ra, cảnh sát liên bang Úc cáo buộc người đàn ông này đã tạo ra mạng wifi "song sinh độc ác" - mô phỏng các mạng wifi hợp pháp - để lừa người dùng nhập thông tin cá nhân của họ.

Essentially meaning watching an idea start with a few people and eventually develops into something bigger (which leads to unexpected big changes).

Though the evolution of technologies today have obviously been heavily influenced by its origins, there is always something that needs to be new and exciting, never before seen. Could be a reach, but this could be a great example of how things that are constantly evolving were not created for certain human practices, and that our generations are now greedy, always seeking to be better and to invent more technologically advanced things (an example of this in the automobile context would be self-driving cars; just get a chauffeur at that point. Another example in the context of internet is AI - shows how lazy we are when it comes to research. Google wasn't enough, nor was ChatGPT in its original simplicity of assisting with research, AI has evolved to be so efficient, it has started to bleed into robotics.) Essentially, some things are better left simple.

Supports his earlier claim that "Networking has had an effect on the world more profound than that of the more spectacular and expensive Apollo moon missions"

Change to:

it provide prefigurative evidence

Make sure to highlight the institutional shortcomings that preceded the pandemic and were perhaps exacerbated by it more dramatically, making these faultlines hypervisible upon breakdown of university facilities and services during the early days of the pandemic in Spring 2020.

Summary: "Economic relationships are voluntary and uncoerced contractual relationships that are typically mutually beneficial. Such relationships make exploitation a philosophically interesting concept and give exploitation complaints their distinctive moral bite."

"This engagement supports the attempt, in Chapter 7, to sketch what a non-exploitative, democratically emancipated economy might look like. The unifying thread throughout is the critique of capital as monetary title to alien purposiveness." Might be a neat thing to look at for my story.

For my story, I should look into different perspectives of why the system is upheld (though this book and others). Liberal, republican, socialist, etc.

Summary: recent critics of capatalism argue abt the economic inequality of it and "violating basic precepts of rational justification." This is connected to surplus extraction under capitalism. It's marketed as a system of freedom and equality, but remains neither of those things though the following points:

1.) Unjust Abuse of power. 2.)Exploitation is a dividend of servitude, specifically "having to respond to the extractive ends and dispositions of the powerful." Money for example 3.) Structural Domination is a useful and coherent notion. 4.) The Capitalist has global variety. Like Colonial Imperialism, and liberal imperialism (what's happening nowadays.

Summary: Capitalism is basically a more modern form of fuedalism, where "might makes right" and whatever the lord says goes. This was abolished in the 18th and 19th centuries and was replaced with contracts, and ownership more in the relationship to things, capital.

Annotation Comment: This passage caught my attention because of how dramatically Miner emphasizes something that to most, seems completely normal, such as brushing our teeth and dentist visits. By presenting these everyday Western hygienic habits in terms of anthropology, Miner forces readers to see their own culture as if it were strange or out of the ordinary. It’s a clever way to highlight how ethnocentrism affects how we describe and evaluate differing cultures. Main Takeaway: Rituals are not restricted to ‘primitive’ or preindustrial societies. In modern societies like the United States, normalized routines such as brushing teeth function as cultural rituals that express shared values, in this case, being hygiene, beauty, and societal acceptance. Rituals exist in every society and reveal the values that a culture holds as most valuable.

I agree that implicit inputs such as location tracking raise ethical concerns, especially for people in vulnerable situations. I think this section does a great job of denoting how invisible data collection can have real life consequences. This made me acknowledge how "user input" is not always a voluntary thing... and that the design decisions regarding data collection are also decisions about safety and justice.

I honestly don't even really understand why its a question of whether or not states should be banning companies from creating these apps. I understand how it could be difficult to monitor companies that are overseas. From my understanding at least I thought the US government could still ban people in the US from using these websites .

My mouth physically dropped when reading that these sites are brining in millions of dollars a year. I also was confused about the fact that it says a new federal law was made regarding posting nonconsensual photos which is confusing me because hasn't it always been against the law to post nude pictures of anyone even with concent, if that wasn't a law before that's even more shocking.

I was not expecting this at all when first reading and choosing this article. I think the worries about parents posting pictures of their children is one things but this is a whole different level of crazy. My initial reaction to hearing there are apps that can do this is who the heck is making these apps and what's wrong with them. This just adds a whole new level to the debate about posting children.

This is way too terse and punchy. It's also borderline incoherent without any strong verbs, like a child was taking notes. Revise this and update the dissertation writing style guide to reflect the lessons learn with this stylistic edit.

Use the first person narrator "I used ___ to identify correlations in ___ "

The government would give basic needs to the people they forced out, basically setting up the internment camps.

All government departments had to help with the internment process providing supplies and services.

This section says the new order doesn’t cancel older wartime orders. It just adds more power for the military to act on the West Coast.

The military could use soldiers and police to make sure people followed these orders.

FDR allows the military to mark certain areas as “military zones.” Anyone could be forced to leave those zones if the military said so.

Tấn công MITM sử dụng ARP Poisoning là kỹ thuật được sử dụng phổ biến nhất để thực hiện các cuộc tấn công MITM và điều này là do tính bảo mật kém của giao thức ARP và cũng vì đây là cách đơn giản nhất để thực hiện cuộc tấn công

Telemedicine has the potential to improve access for populations historically excluded from high-quality healthcare, but attention must be paid to the context in which it is implemented such that it does not worsen health disparities by exacerbating inequities in access or by introducing inequities in quality.

Innovations such as telemedicine greatly improved healthcare delivery during the COVID-19 pandemic and beyond. It enabled individuals in quarantine, those living in remote areas, or those unable to physically visit their doctors to connect with healthcare providers, exchange health information, and receive treatment remotely. However, in other contexts, health inequities deepened for populations lacking the financial resources to access electronic devices or the digital literacy needed to navigate new technologies.

The diagram in the article illustrates healthcare as a complex adaptive system and highlights how individuals interact with the process of accessing healthcare throughout their lifespan. It depicts the care-seeking process and the nuances of healthcare delivery, which are influenced by a variety of factors; including human, structural, environmental, and sociopolitical elements.

leaders must commit to interventions that seek to repair, remediate, restructure, and remove structures that confer disadvantage in care delivery alongside the implementation of innovation

A concrete action that a nurse leader can take to address disparities should be inspired by the R4P framework—Remove, Repair, Remediate, Restructure, and Provide. For example, the nurse leader can use feedback loops to assess the nature of disparities specific to a particular community and respond in a way that aligns with the community’s unique needs and context. They may collaborate with assistance programs to provide electronic devices, offer access to free or reduced-cost internet, and organize teaching sessions to help individuals learn how to use these technologies effectively.

I believe there is a crossover here where the technology can help build content skills that students can carryover to everyday lives. I also believe that there are many cases where the technology itself that we use in the classroom can carry over into everyday life. Our district is primarily Google driven and many of the tools we use like sheets and calendars that extend learning are useful in life outside of the classroom.

The part about making sure all the tea was destroyed shows how determined they were. They didn’t want anyone using it or selling it. It makes sense that Britain reacted so strongly afterward with the Coercive Acts.

Hewes makes it sound like everything was calm and organized, not like a wild riot. That makes me think they wanted people to see the Tea Party as a proud, controlled protest instead of random destruction.

This part shows how serious they were about what they were doing. They all took personal risks for what they believed in, which really shows the early revolutionary spirit—standing up for freedom even if it meant getting in trouble.

It’s interesting that everyone kept quiet afterward. It reminds me of how some protest groups today stay anonymous to protect themselves, kind of like the Sons of Liberty did later.

It’s kind of surprising that the British ships didn’t try to stop them. Maybe the soldiers didn’t want to start a fight, or maybe they didn’t expect the colonists to actually go through with destroying all the tea.

I wonder why they decided to dress up as Native Americans. Was it just to stay anonymous, or did they do it to make a point about being “American” and different from the British?

Griffin’s Wharf was the harbor area in Boston where the Tea Party took place. The fact that British warships were right there shows how much control the British military had over the city even before the war started.

Governor Thomas Hutchinson was a loyal British official in Massachusetts. He refused to send the tea ships back, which basically caused the Tea Party to happen. It shows how government leaders weren’t listening to the people’s frustration anymore.

This happened just before the American Revolution. At that time, colonists were really angry about the Tea Act and other taxes that they thought were unfair. The Boston Tea Party became a big moment where people openly protested British control.

A tomahawk is a small axe that Native Americans originally used. The men at the Boston Tea Party carried fake ones and dressed like “Indians” to hide who they were and to show they were breaking away from British identity.

The word “rebels” here means the American colonists who went against British rule. The British used it as a negative label to make them sound like troublemakers instead of people standing up for their rights.

Problematic subletting? The preferred approach would be to recategorize as censored and perform a cause-specific cox analysis.

I have validated your code here using two different methods, so good job.

However, your code is difficult to parse and unnecessarily complicated. Compared to your SAP, where you relied on the predict() function to generate estimates, here you employ a manual linear combination of coefficients (LINCOM) method. This is ideal because you do not need to specify a reference grid to obtain final estimates, a feature which you don't take full advantage of.

Included in my code review is a sample code file that takes your code and reduces it to the necessary components, which might prove easier to read.

This looks like problematic subsetting, you are list-wise deleting records before generating the survey design object.

Given the descriptive nature of the KM plot it would be preferable to implement the analysis on the un-imputed analytic data set.

However, here it doesn't matter assuming the data were subset to complete case observations on the exposure and outcome already.

You should implement descriptive statistics on the unimputed analytic data set, not the imputed data set.

I highly recommend importing mortality files directly from the CDC FTP web portal. Otherwise your code will not be self-contained when submitted for publication.

My students have many different interests and abilities and it is awesome when I can find a way for a student to show me they are competent with the material without having to use traditional methods. It can also be a pain to deal with varied methods for no reason so having the tech be actually doing something I couldn't do easier a different way is a great criteria.

It is always a struggle with students to keep them focused on learning. I like how it differentiates between time on the technology and time the time focusing on the assignment.

Research on this text: Almost all endurance athletes train at high altitudes. The Olympic 1500 meter runner Josh Kerr went to UNM and he occasionally trains in New Mexico, because of its high altitude and low humidity.

Lactic acid == Lactate is a byproduct of muscle metabolism that builds up when there is not enough oxygen in the blood stream. Physiopedia - Lactate Threshold

Research on this text: A lot of the athletes at the 1968 Olympic games in Mexico city did complain about the high elevation affecting there performance. Here is article supporting this statement: Elevation, Records, and Controversy

VO2max == The maximum rate in which you body can use oxygen.

Harvard Health - What is VO2max?

I would highly recommend importing mortality data directly from the CDC FTP website.

As submitted, your code here is not self-contained, which it should be assuming it were submitted as supplemental materials for publication.

If I am reading this correctly, you are subsetting the raw data, which is problematic.

Where does this come from? Also it was not mentioned in the methods section or in the results/supplementary materials, so I assume you don't intend to do anything with it?

I encourage you to use theme_classic() or to explore the ggthemes::theme_stata() function, which both generate more professional looking figures.

I have validated your code using two separate methods and arrived at the same results, good work.

However, your method here is unnecessarily complex. Compared to your SAP code, this time around you attempted to estimate the HR using a linear-combination of coefficients approach (whereas you used the predict() function last time).

When employing this manual LINCOM approach, the reference grid of the adjustment covariates is irrelevant because you do not have any multiplicative interaction terms. In my code review I am including a simpler approach, which might be easier for external readers to parse.

If we initialize an array and attempt to return it, the array will be located on the stack frame of the function and will not be safely accessible by the user once it is returned outside of the function's stack frame.

Changes can be seen if we are dereferencing dest, such as when we set dest = src. When we are not dereferencing dest, such as dest++, we are only modifying the local pointer in order to work with its contents, and such modifications cannot be seen by the caller.

This is also a fact. It's a count of how many days the government has been shut down for and can be verified by several sources. You'd get the same answer wherever you look.

This is factual information. If you looked up what the SNAP program is online, you'd get the same explanation of how the program works.

Criticism is opinion. It's their judgement about how the money is being spent. Some people are in support of Trump building the new ballroom at the White House, and others strongly oppose it.

This is a fact that came from a research group. It's based on numbers and thorough analysis.

This is an opinion put out by congresswoman Rosa DeLauro, who comes from my home state of Connecticut. It shows clearly how she feels, not a proven fact. She has been very publicly outspoken about how she opposes many of Trump's initiatives.

Fact, SNAP is a huge program that's used by millions of Americans, and this is a more specific statistic that can easily be looked up.

This is an opinion. It's a more dramatic way of saying there's no money, not a clear number, fact, or statistic.

This is a factual claim that's linked to a government source. It can be validated through USDA announcements.

We need to think critically about how that shapes our understanding of the past and other cultures.

This shows that we can feed people and protect the planet at the same time, as long as we use a balanced mix of farming methods.

This is how white supremacy lives on

Now when people say "Marxism" it's a buzzword against anyone who want equal rights.

For many tribes, the fish symbolizes good luck and prosperity. It can also symbolize fertility and the cycle of life and death. In the Cherokee story, this symbol of a fish makes sense in the context of the story. The woman was struck with a fish and was able to multiply. This instance represents prosperity and fertility which correlates with why the Cherokee tribe symbolizes the fish in that way.

Is Coyote is just a name or a different meaning for something. In this work, Coyote is not referred to as, "the" Coyote but instead just Coyote like it is a name. It is interesting because of the context of the story.

Why was the Bald Eagle the chief of animals? The Bald Eagle is now our countries most treasured bird. It is significant and interesting that in the Salinan Indian creation story the Bald Eagle is the chief of animals and now it is our countries bird. The Bald Eagle was chosen as our national bird in 1782 because of its symbolism of strength and freedom. It sounds like the Salinan peoples associated the Bald Eagle with the same feelings of strength and freedom that we do now.

The first contact with the Cherokee people and Europeans happened in 1540. The Cherokee people were led by one man, a chief. During the 1830's, the tribe was forcibly moved and migrated on a long journey (The Trail of Tears). Many people during this migration did not make it and died on their journey. The forcible migration was a result of the Indian Removal Act which was authorized by the president to allow lands by the Mississippi in exchange for Indian lands.

Ethnology- the study of the characteristics of various peoples and the differences and relationships between them.

The Salinan tribe were first in contact with Europeans in the year 1602. They were located in California by the Santa Maria river snd mountains. The Salinan tribe also relied on the river and streams for food like salmon and mussels. They also foraged for berries and nuts.

During the Columbian Exchange in the 1490's, many new ideas and ideology were brought about by both the Native Americans and the Europeans. From both sides, some new information was integrated into old ways of thinking but some information was also disregarded because both peoples believed they were right and set in their ways. Not only new ideas were spread during the Columbian Exchange but resources as well as deadly diseases were spread.

The Cherokee tribe also thought the world was flat just like people long ago. The Cherokee people thought the earth was floating in the ocean being held up but it is interesting how they say that the sun goes down and then returns on its upper side to the starting place to start again. The Cherokee tribe had no idea that the moon and sun were two different things. Aristotle proved the earths roundness long before the Cherokee tribe but they did not have knowledge of this and believed in something else entirely.

It is very important to regard the close connection that both the Cherokee and Salinan tribe had with animals. They believed that animals were symbols and represented an idea and purpose in their own lives. Animals were represented as symbols and signs for many different things and they regarded them as such. Since these tribes had such close and personal relationships with animals and the idea of them, the animals were treated with so much more respect.

When this happens, will the world ever come again? The Cherokee tribe thought that the earth was suspended floating in the water and then at one point, it would simply sink. From this ideology, I am curious as to if the Cherokee tribe believed/believes that there would be a new world after them or if there was a different world before them?

News of the king’s death shows how connected colonists were to Britain at this time.

just a description of what the virus is and why it spreads.

The authors also reference the ADAPPT model (De Leeuw & Chang, 2024), which views bilingual language systems as constantly changing rather than reaching a final stable state.

The findings support a Dynamic Systems Theory (DST) approach: language systems evolve in complex, nonlinear ways, influenced by both internal (cross-language interaction) and external (social and emotional) factors.

Using the SLM-revised (Flege & Bohn, 2021), the authors explain this through the Category Precision Hypothesis: speakers with more precise L1 categories can more easily form new, accurate L2 ones.

This suggests that both languages can develop robust, native-like categories simultaneously, depending on factors like exposure, experience, and L2 learning age.

Traditional theories predict that better L2 pronunciation causes L1 attrition, but this study found the opposite: bilinguals who produced nativelike L2 vowels also maintained nativelike L1 vowels.

The authors suggest that future research should investigate other cognitive factors (e.g., memory, grammatical sensitivity) and sociolinguistic influences.

Sound discrimination aptitude (a measure of phonetic ability) did not significantly predict production accuracy.

This implies that external factors, like exposure, input quality, and language prestige, may play a larger role than cognitive aptitude in shaping bilingual pronunciation.

The study also discusses how perception and production might relate, but concludes that more research, especially on vowel perception, is needed to clarify this connection.

Results showed cross-language interactions affected vowels differently: ・For /ɪ/ and /u-ʊ/, bilinguals showed deviations from monolingual norms, but the direction of change differed by group. ・The vowel /a-æ/ was produced natively in both languages, suggesting that clear perceptual distance between L1 and L2 sounds helps bilinguals maintain separate categories.

These results align with the Speech Learning Model (SLM), which states that more distinct L1-L2 sounds are easier to separate and learn accurately.

frequent urge to go to the bathroom without being able to go