你的定义基本正确，但需要一些补充和修正以确保准确性。以下是除环（Division Ring）的详细解释：

除环的正式定义

除环（或称体、斜域）是一个环 \( R \)，满足以下条件： 1. 加法结构：<br /> \((R, +)\) 是一个 阿贝尔群（交换群），即满足： - 封闭性、结合律、交换律、加法单位元（零元 \( 0 \)）、加法逆元。

1. 乘法结构：<br /> \((R \setminus {0}, \cdot)\) 构成一个 群（即非零元对乘法封闭且满足群公理）：
2. 封闭性：\( a \cdot b \in R \setminus {0} \)，\( \forall a, b \in R \setminus {0} \)。
3. 结合律：\( a \cdot (b \cdot c) = (a \cdot b) \cdot c \)，\( \forall a, b, c \in R \setminus {0} \)。
4. 单位元：存在 \( 1 \in R \setminus {0} \)，使得 \( a \cdot 1 = 1 \cdot a = a \)，\( \forall a \in R \setminus {0} \)。

5. 逆元：每个非零元 \( a \in R \setminus {0} \) 存在唯一逆元 \( a^{-1} \in R \setminus {0} \)，满足： [ a \cdot a^{-1} = a^{-1} \cdot a = 1. ]

6. 分配律：<br /> 乘法对加法满足分配律：

7. \( a \cdot (b + c) = a \cdot b + a \cdot c \)，
8. \( (a + b) \cdot c = a \cdot c + b \cdot c \)，<br /> \( \forall a, b, c \in R \)。

关键补充说明

1. 乘法结构的澄清

• 乘法幺半群 vs. 乘法群：
• 整个环 \( R \) 的乘法结构 \((R, \cdot)\) 是一个 含幺半群（存在单位元 \( 1 \)，但零元 \( 0 \) 没有逆元）。
• 非零元子集 \((R \setminus {0}, \cdot)\) 构成一个 群（即非零元可逆且满足群公理）。

2. 与域的关系

• 域是交换的除环：<br /> 若除环的乘法满足交换律（\( a \cdot b = b \cdot a \)），则它是一个 域（Field）。
• 例子：有理数域 \( \mathbb{Q} \)、实数域 \( \mathbb{R} \)、复数域 \( \mathbb{C} \)。

3. 非交换性

• 除环的乘法 不一定交换，因此非交换的除环也称为 斜域（Skew Field）。
• 经典例子：四元数环 \( \mathbb{H} \)（非交换）。

4. 韦德伯恩定理

• 有限除环必为域：<br /> 根据 Wedderburn 小定理，任何有限除环的乘法必交换，因此是有限域。
• 例子：有限域 \( \mathbb{Z}_p \)（\( p \) 为素数）。

5. 除环的性质

• 无零因子：<br /> 若 \( a \cdot b = 0 \)，则 \( a = 0 \) 或 \( b = 0 \)。

• 证明：若 \( a \neq 0 \)，则 \( a \) 可逆，故 \( b = a^{-1} \cdot 0 = 0 \)。

• 单环：<br /> 除环的唯一双边理想是 \( {0} \) 和 \( R \) 本身，因此是 单环（Simple Ring）。

6. 例子与反例

• 经典例子：
• 四元数环 \( \mathbb{H} \)：非交换除环，元素形如 \( a + bi + cj + dk \)，乘法不交换。
• 有限域 \( \mathbb{Z}_p \)：既是域，也是交换除环。

• 实数域 \( \mathbb{R} \)：交换除环，同时也是域。

• 非除环的例子：

• 整数环 \( \mathbb{Z} \)：非零元如 \( 2 \) 无逆元，故非除环。
• 矩阵环 \( M_n(\mathbb{R}) \)（\( n \geq 2 \)）：存在非零元（如对角矩阵 \( \text{diag}(1, 0, \dots, 0) \)）无逆元。

7. 重要定理

• Wedderburn 小定理：<br /> 有限除环的乘法必交换，即有限除环必为域。

• 应用：有限域的构造与分类依赖此定理。

• Schur 引理：<br /> 若 \( S \) 是环 \( R \) 的单模，则其内同态环是除环。

总结

除环是“非零元可逆的含幺环”，其核心特征是：<br /> 1. 非零元构成乘法群；<br /> 2. 乘法不一定交换；<br /> 3. 无零因子；<br /> 4. 有限除环必为域。

若需进一步探讨具体例子或定理证明，请随时提问！

含幺环（Unitary Ring）的详细解释

根据你的定义和知识库中的信息，含幺环的定义是正确的，但需要进一步明确和补充以下关键点：

1. 含幺环的正式定义

含幺环（也称幺环、含单位元的环）是一个环 \( R \)，满足以下条件： 1. 加法结构：<br /> \((R, +)\) 是一个 阿贝尔群（交换群），即满足： - 封闭性、结合律、交换律、加法单位元（零元 \( 0 \)）、加法逆元。

1. 乘法结构：<br /> \((R, \cdot)\) 是一个 含幺半群（即乘法半群存在单位元），即满足：

2. 封闭性、结合律、存在乘法单位元 \( 1 \)（满足 \( a \cdot 1 = 1 \cdot a = a \) 对所有 \( a \in R \)）。

3. 分配律：<br /> 乘法对加法满足分配律：

4. 左分配律：\( a \cdot (b + c) = a \cdot b + a \cdot c \)，
5. 右分配律：\( (a + b) \cdot c = a \cdot c + b \cdot c \)。

2. 关键补充说明

(1) 单位元的唯一性

• 知识库[1][3][4]指出：若环 \( R \) 含单位元，则单位元是唯一的。<br /> 证明：假设存在 \( 1 \) 和 \( 1' \)，则 \( 1 = 1 \cdot 1' = 1' \)，故唯一。

(2) 单位元与零元的关系

• 若 \( R \) 是非零环（即 \( |R| \geq 2 \)），则单位元 \( 1 \neq 0 \)。
• 若 \( 1 = 0 \)，则对任意 \( a \in R \)，有 \( a = a \cdot 1 = a \cdot 0 = 0 \)，故 \( R \) 只含零元，称为零环。

(3) 乘法半群的结构

• 通常乘法半群不要求单位元，但含幺环强制要求乘法半群有单位元，因此乘法结构是含幺半群（monoid）而非普通半群。

3. 含幺环的例子

• 经典例子：
• 整数环 \( \mathbb{Z} \)：单位元为 \( 1 \)，是含幺交换环。
• 多项式环 \( R[x] \)：若 \( R \) 是含幺环，则 \( R[x] \) 也是含幺环，单位元为常数项 \( 1 \)。
• 矩阵环 \( M_n(R) \)（\( n \geq 1 \)）：单位元为单位矩阵 \( I_n \)，但乘法非交换（除非 \( n = 1 \)）。

• 模 \( n \) 剩余类环 \( \mathbb{Z}_n \)：单位元为 \( [1] \)，是含幺交换环。

• 非含幺环的例子：

• 偶数环 \( 2\mathbb{Z} \)：乘法无单位元（因 \( 1 \notin 2\mathbb{Z} \)），故非含幺环。

4. 含幺环的分类与性质

(1) 交换含幺环（Commutative Ring with Unity）

• 若乘法满足交换律，则称 \( R \) 为交换含幺环（CRI）。
• 例子：\( \mathbb{Z}, \mathbb{Q}, \mathbb{Z}_n \)。

(2) 无零因子环

• 若 \( ab = 0 \Rightarrow a = 0 \) 或 \( b = 0 \)，则称 \( R \) 为无零因子环。
• 整环（Integral Domain）：交换、含幺、无零因子的环。
  • 例子：\( \mathbb{Z}, \mathbb{Q}, \mathbb{R} \)。

(3) 除环与域

• 除环（Division Ring）：含幺、无零因子，且每个非零元可逆（存在乘法逆元）。
• 非交换例子：四元数环 \( \mathbb{H} \)。
• 域（Field）：交换的除环。
• 例子：\( \mathbb{Q}, \mathbb{R}, \mathbb{C} \)，以及有限域 \( \mathbb{Z}_p \)（\( p \) 为素数）。

5. 含幺环的重要性

• 代数结构核心：<br /> 许多代数结构（如域、模、代数）都基于含幺环的性质构建。
• 应用领域：
• 代数数论：研究数域的整数环（如代数整数构成的整环）。
• 线性代数：矩阵环是线性变换的代数模型。
• 编码理论：多项式环用于构造纠错码（如循环码）。

6. 常见误区澄清

• 误区1：所有环都含幺元。
• 纠正：需明确区分“环”与“含幺环”。例如，偶数环 \( 2\mathbb{Z} \) 是环但非含幺环。
• 误区2：乘法半群必须交换。
• 纠正：含幺环的乘法半群可以非交换（如矩阵环 \( M_n(\mathbb{R}) \)）。

总结

含幺环是环的“升级版”，通过要求乘法存在单位元，为代数结构提供了更丰富的性质和应用。其核心在于平衡加法的交换性与乘法的结合性，并通过单位元支持逆元和多项式等高级运算。理解含幺环是深入交换代数、非交换代数及应用领域的关键基础。

如果需要进一步探讨具体例子或深入某个性质，请随时提问！

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

Manuscript number: RC-2024-02465

Corresponding author(s): Saravanan, Palani

1. General Statements

We would like to thank the Review Commons Team for handling our manuscript and the Reviewers for their constructive feedback and suggestions. In our revised manuscript, we have addressed and incorporated all the major suggestions of the reviewers, and we have also added new significant data on the role of Tropomyosin in regulation of endocytosis through its control over actin monomer pool maintenance and actin network homeostasis. We believe that with all these additions, our study has significantly gained in quality, strength of conclusions made, and scope for future work.

2. Point-by-point description of the revisions

Reviewer #1

Evidence, reproducibility and clarity

There are 2 Major issues -

Having an -ala-ser- linker between the GFP and tropomyosin mimics acetylation. This is not the case, and more likely the this linker acts as a spacer that allows tropomyosin polymers to form on the actin, and without it there is steric hindrance. A similar result would be seen with a simple flexible uncharged linker. It has been shown in a number of labs that the GFP itself masks the effect of the charge on the amino terminal methionine. This is consistent with NMR, crystallographic and cryo structural studies. Biochemical studies should be presented to demonstrate that the impact of a linker for the conclusions stated to be made, which provide the basis of a major part of this study.

Response: We would like to clarify that all mNG-Tpm constructs used in our study contain a 40 amino-acid (aa) flexible linker between the N-terminal mNG fluorescent protein and the Tpm protein as per our earlier published study (Hatano et al., 2022). During initial optimization, we have also experimented with linker length and the 40aa-linker length works optimally for clear visualization of Tpm onto actin cable structures in budding yeast, fission yeast (both S. pombe and S. japonicus), and mammalian cells (Hatano et al., 2022). These constructs have also been used since in other studies (Wirshing et al., 2023; Wirshing and Goode, 2024) and currently represents the best possible strategy to visualize Tpm isoforms in live cells. In our study, we characterized these proteins for functionality and found that both mNG-Tpm1 and mNG-Tpm2 were functional and can rescue the synthetic lethality observed in Dtpm1Dtpm2 cells. During our study, we observed that mNG-Tpm1 expression from a single-copy integration vector did not restore full length actin cables in Dtpm1 cells (Fig. 1B, 1C). We hypothesized that this could be a result of reduced binding affinity of the tagged tropomyosin due to lack of normal N-terminal acetylation which stabilizes the N-terminus. The 40aa linker is unstructured and may not be able to neutralize the charge on the N-terminal Methionine, thus, we tried to insert -Ala-Ser- dipeptide which has been routinely used in vitro biochemical studies to stabilize the N-terminal helix and impart a similar effect as the N-terminal acetylation (Alioto et al., 2016; Palani et al., 2019; Christensen et al., 2017) by restoring normal binding affinity of Tpm to F-actin (Monteiro et al., 1994; Greenfield et al., 1994). We observed that addition of the -Ala-Ser- dipeptide to mNG-Tpm fusion, indeed, restored full length actin cables when expressed in Dtpm1 cells, performing significantly better in our in vivo experiments (Fig. 1B, 1C). We agree with the reviewer that the -AS- dipeptide addition may not mimic N-terminal acetylation structurally but as per previous studies, it may stabilize the N-terminus of Tpm and allow normal head-to-tail dimer formation (Greenfield et al., 1994; Monteiro et al., 1994; Frye et al., 2010). We have discussed this in our new Discussion section (Lines 350-372). Since, the addition of -AS- dipeptide was referred to as "acetyl-mimic (am)" in a previous study (Alioto et al., 2016), we continued to use the same nomenclature in our study. Now as per your suggestions and to be more accurate, we have renamed "mNG-amTpm" constructs as "mNG-ASTpm" throughout the study to not confuse or claim that -AS- addition mimics acetylation. In any case, we have not seen any other ill effect of -AS- dipeptide introduction in addition to our 40 amino acid linker suggesting that it can also be considered part of the linker. Although, we agree with the reviewer that biochemical characterization of the effect of linker would be important to determine, we strongly believe that it is currently outside the scope of this study and should be taken up for future work with these proteins. Our study has majorly aimed to understand the functionality and utility of these mNG-Tpm fusion proteins for cell biological experiments in vivo, which was not done earlier in any other model system.

My major issue however is making the conclusions stated here, using an amino-terminal fluorescent protein tag that s likely to impact any type of isoform selection at the end of the actin polymer. Carboxyl terminal tagging may have a reduced effect, but modifying the ends of the tropomyosin, which are integral in stabilising end to end interactions with itself on the actin filament, never mind any section systems that may/maynot be present in the cell, is not appropriate.

Response: We agree with the reviewer that N-terminal tagging of tropomyosin may have effects on its function, but these constructs represent the only fluorescently tagged functional tropomyosin constructs available currently while C-terminal fusions are either non-functional (we were unable to construct strains with endogenous Tpm1 gene fused C-terminally to GFP) or do not localize clearly to actin structures (See Figure R1 showing endogenous C-terminally tagged Tpm2-yeGFP that shows almost no localization to actin cables). To our knowledge, our study represents a first effort to understand the question of spatial sorting of Tpm isoforms, Tpm1 and Tpm2, in S. cerevisiae and any future developments with better visualization strategies for Tpm isoforms without compromising native N-terminal modifications and function will help improve our understanding of these proteins in vivo. We have also discussed these possibilities in our new Discussion section (Lines 391-396).

Significance

This paper explores the role of formin in determining the localisation of different tropomyosins to different actin polymers and cellular locations within budding yeast. Previous studies have indicated a role for the actin nucleating proteins in recruiting different forms of tropomyosin within fission yeast. In mammalian cells there is variation in the role of formins in affiecting tropomyosin localisation - variation between cell type. There is also evidence that other actin binding proteins, and tropomyosin abundance play roles in regulating the tropomyosin-actin association according to cell type. Biochemical studies have previously been undertaken using budding yeast and fission yeast that the core actin polymerisation domain of formins do not interact with tropomyosin directly. The significance of this study, given the above, and the concerns raised is not clear to this reviewer.

Response: __Our study explores multiple facets of Tropomyosin (Tpm) biology. The lack of functional tagged Tpm has been a major bottleneck in understanding Tpm isoform diversity and function across eukaryotes. In our study, we characterize the first functional tagged Tpm proteins (Fig. 1, Fig. S1) and use them to answer long-standing questions about localization and spatial sorting of Tpm isoforms in the model organism S. cerevisiae (Fig. 2, Fig. 3, Fig. S2, Fig. S3). We also discover that the dual Tpm isoforms, Tpm1 and Tpm2, are functionally redundant for actin cable organization and function, while having gained divergent functions in Retrograde Actin Cable Flow (RACF) (Fig. 4, Fig. 5A-D, Fig. S4, Fig. S5, Fig. S6). We have now added new data on role of global Tpm levels controlling endocytosis via maintenance of normal linear-to-branched actin network homeostasis in S. cerevisiae (Fig. 5E-G)__. We respectfully differ with the reviewer on their assessment of our study and request the reviewer to read our revised manuscript which discusses the significance, limitations, and future perspectives of our study in detail.

Reviewer #2

Evidence, reproducibility and clarity

This manuscript by Dhar, Bagyashree, Palani and colleagues examines the function of the two tropomyosins, Tpm1 and Tpm2, in the budding yeast S. cerevisiae. Previous work had shown that deletion of tpm1 and tpm2 causes synthetic lethality, indicating overlapping function, but also proposed that the two tropomyosins have distinct functions, based on the observation that strong overexpression of Tpm2 causes defects in bud placement and fails to rescue tpm1∆ phenotypes (Drees et al, JCB 1995). The manuscript first describes very functional mNeonGreen tagged version of Tpm1 and Tpm2, where an alanine-serine dipeptide is inserted before the first methionine to mimic acetylation. It then proposes that the Tpm1 and Tpm2 exhibit indistinguishable localization and that low level overexpression (?) of Tpm2 can replace Tpm1 for stabilization of actin cables and cell polarization, suggesting almost completely redundant functions. They also propose on specific function of Tpm2 in regulating retrograde actin cable flow.

Overall, the data are very clean, well presented and quantified, but in several places are not fully convincing of the claims. Because the claims that Tpm1 and Tpm2 have largely overlapping function and localization are in contradiction to previous publication in S. cerevisiae and also different from data published in other organisms, it is important to consolidate them. There are fairly simple experiments that should be done to consolidate the claims of indistinguishable localization, and levels of expression, for which the authors have excellent reagents at their disposal.

1. Functionality of the acetyl-mimic tagged tropomyosin constructs: The overall very good functionality of the tagged Tpm constructs is convincing, but the authors should be more accurate in their description, as their data show that they are not perfectly functional. For instance, the use of "completely functional" in the discussion is excessive. In the results, the statement that mNG-Tpm1 expression restores normal growth (page 3, line 69) is inaccurate. Fig S1C shows that tpm1∆ cells expressing mNG-Tpm1 grow more slowly than WT cells. (The next part of the same sentence, stating it only partially restores length of actin cables should cite only Fig S1E, not S1F.) Similarly, the growth curve in Fig S1C suggests that mNG-amTpm1, while better than mNG-Tpm1 does not fully restore the growth defect observed in tpm1∆ (in contrast to what is stated on p. 4 line 81). A more stringent test of functionality would be to probe whether mNG-amTpm1 can rescue the synthetic lethality of the tpm1∆ tpm2∆ double mutant, which would also allow to test the functionality of mNG-amTpm2.

__Response: __We would like to thank the reviewer for his feedback and suggestions. Based on the suggestions, we have now more accurately described the growth rescue observed by expression of mNG-ASTpm1 in Dtpm1 cells in the revised text. We have also removed the use of "completely functional" to describe mNG-Tpm functionality and corrected any errors in Figure citations in the revised manuscript.

As per reviewers' suggestion, we have now tested rescue of synthetic lethality of Dtpm1Dtpm2 cells by expression of all mNG-Tpm variants and we find that all of them are capable of restoring the viability of Dtpm1Dtpm2 cells when expressed under their native promoters via a high-copy plasmid (pRS425) (Fig. S1E) but only mNG-Tpm1 and mNG-ASTpm1 restored viability of Dtpm1Dtpm2 cells when expressed under their native promoters via an integration plasmid (pRS305) (Fig. S1F). These results clearly suggest that while both mNG-Tpm1 and mNG-Tpm2 constructs are functional, Tpm1 tolerates the presence of the N-terminal fluorescent tag better than Tpm2. These observations now enhance our understanding of the functionality of these mNG-Tpm fusion proteins and will be a useful resource for their usage and experimental design in future studies in vivo.

It would also be nice to comment on whether the mNG-amTpm constructs really mimicking acetylation. Given the Ala-Ser peptide ahead of the starting Met is linked N-terminally to mNG, it is not immediately clear it will have the same effect as a free acetyl group decorating the N-terminal Met.

Response: __We agree with the reviewer's observation and for the sake of clarity and accuracy, we have now renamed "mNG-amTpm" with "mNG-ASTpm". The use of -AS- dipeptide is very routine in studies with Tpm (Alioto et al., 2016; Palani et al., 2019; Christensen et al., 2017) and its addition restores normal binding affinities to Tpm proteins purified from E. coli (Monteiro et al., 1994). We agree with the reviewer that the -AS- dipeptide addition may not mimic N-terminal acetylation structurally but as per previous studies, it may help neutralize the impact of a freely protonated Met on the alpha-helical structure and stabilize the N-terminus helix of Tpm and allow normal head-to-tail dimer formation (Monteiro et al., 1994; Frye et al., 2010; Greenfield et al., 1994). Consistent with this, we also observe a highly significant improvement in actin cable length when expressing mNG-ASTpm as compared to mNG-Tpm in Dtpm1 cells, suggesting an improvement in function probably due to increased binding affinity (Fig. 1B, 1C). We have also discussed this in our answer to Question 1 of Reviewer 1 and the revised manuscript (Lines 350-372)__.

__ Localization of Tpm1 and Tpm2:__Given the claimed full functionality of mNG-amTpm constructs and the conclusion from this section of the paper that relative local concentrations may be the major factor in determining tropomyosin localization to actin filament networks, I am concerned that the analysis of localization was done in strains expressing the mNG-amTpm construct in addition to the endogenous untagged genes. (This is not expressly stated in the manuscript, but it is my understanding from reading the strain list.) This means that there is a roughly two-fold overexpression of either tropomyosin, which may affect localization. A comparison of localization in strains where the tagged copy is the sole Tpm1 (respectively Tpm2) source would be much more conclusive. This is important as the results are making a claim in opposition to previous work and observation in other organisms.

Response: __We thank the reviewer for this observation and their suggestions. We agree that relative concentrations of functional Tpm1 and Tpm2 in cells may influence the extent of their localizations. As per the reviewer's suggestion, we have now conducted our quantitative analysis in cells lacking endogenous Tpm1 and only expressing mNG-ASTpm1 from an integrated plasmid copy at the leu2 locus and the data is presented in new __Figure S3. We compared Tpm-bound cable length (Fig. S3A, S3B) __and Tpm-bound cable number (Fig. S3A, S3C) along with actin cable length (Fig. S3D, S3E) and actin cable number (Fig. S3D, S3F) in wildtype, Dbnr1, and Dbni1 cells. Our analysis revealed that mNG-ASTpm1 localized to actin cable structures in wildtype, Dbnr1, and Dbni1 cells and the decrease observed in Tpm-bound cable length and number upon loss of either Bnr1 or Bni1, was accompanied by a corresponding decrease in actin cable length and number upon loss of either Bnr1 or Bni1. Thus, this analysis reached the same conclusion as our earlier analysis (Fig. 2) that mNG-ASTpm1 does not show preference between Bnr1 and Bni1-made actin cables. mNG-ASTpm2 did not restore functionality, when expressed as single integrated copy, in Dtpm1Dtpm2 cells (new results in __Fig. S1E, S1F, S5A) thus, we could not conduct a similar analysis for mNG-ASTpm2. This suggests that use of mNG-ASTpm2 would be more meaningful in the presence of endogenous Tpm2 as previously done in Fig. 2D-F.

We have now also performed additional yeast mating experiments with cells lacking bnr1 gene and expressing either mNG-ASTpm1 or mNG-ASTpm2 and the data is shown in new Figure 3. From these observations, we observe that both mNG-ASTpm1 and mNG-ASTpm2 localize to the mating fusion focus in a Bnr1-independent manner (Fig. 3B, 3D) and suggests that they bind to Bni1-made actin cables that are involved in polarized growth of the mating projection. These results also add strength to our conclusion that Tpm1 and Tpm2 localize to actin cables irrespective of which formin nucleates them. Overall, these new results highlight and reiterate our model of formin-isoform independent binding of Tpm1 and Tpm2 in S. cerevisiae.

In fact, although the authors conclude that the tropomyosins do not exhibit preference for certain actin structures, in the images shown in Fig 2A and 2D, there seems to be a clear bias for Tpm1 to decorate cables preferentially in the bud, while Tpm2 appears to decorate them more in the mother cell. Is that a bias of these chosen images, or does this reflect a more general trend? A quantification of relative fluorescence levels in bud/mother may be indicative.

Response: __We thank the reviewer for pointing this out. Our data and analysis do not suggest that Tpm1 and Tpm2 show any preference for decoration of cables in either mother or bud compartment. As per the reviewer's suggestion, we have now quantified the ratio of mean mNG fluorescence in the bud to the mother (Bud/Mother) and the data is shown in __Figure. S2G. The bud-to-mother ratio was similar for mNG-ASTpm1 and mNG-ASTpm2 in wildtype cells, and the ratio increased in Dbnr1 cells and decreased in Dbni1 cells for both mNG-ASTpm1 and mNG-ASTpm2 (Fig. S2G). __This is consistent with the decreased actin cable signal in the mother compartment in Dbnr1 cells and decreased actin cable signal in the bud compartment in Dbni1 cells (Fig. S2A-D). Thus, our new analysis shows that both mNG-ASTpm1 and mNG-ASTpm2 have similar changes in their concentration (mean fluorescence) upon loss of either formins Bnr1 and Bni1 and show similar ratios in wildtype cells as well, suggesting no preference for binding to actin cables in either bud or mother compartment. The preference inferred by the reviewer seems to be a bias of the current representative images and thus, we have replaced the images in __Fig. 2A, 2D to more accurately represent the population.

The difficulty in preserving mNG-amTpm after fixation means that authors could not quantify relative Tpm/actin cable directly in single fixed cells. Did they try to label actin cables with Lifeact instead of using phalloidin, and thus perform the analysis in live cells?

__Response: __We did not use LifeAct for our analysis as LifeAct is known to cause expression-dependent artefacts in cells (Courtemanche et al., 2016; Flores et al., 2019; Xu and Du, 2021) and it also competes with proteins that regulate normal cable organization like cofilin. Use of LifeAct would necessitate standardization of expression to avoid such artefacts in vivo. Also, phalloidin staining provides the best staining of actin cables and allows for better quantitative results in our experiments. The use of LifeAct along with mNG-Tpm would also require optimization with a red fluorescent protein which usually tend to have lower brightness and photostability. However, during the revision of our study, a new study from Prof. Goode's lab has developed and optimized expression of new LifeAct-3xmNeonGreen constructs for use in S. cerevisiae (Wirshing and Goode, 2024). Thus, a similar strategy of using tandem copies of bright and photostable red fluorescent proteins can be explored for use in combination with mNG-Tpm in the future studies.

__ Complementation of tpm1∆ by Tpm2:__

I am confused about the quantification of Tpm2 expression by RT-PCR shown in Fig S3F. This figure shows that tpm2 mRNA expression levels are identical in cells with an empty plasmid or with a tpm2-encoding plasmid. In both strains (which lack tpm1), as well as in the WT control, one tpm2 copy is in the genome, but only one strain has a second tpm2 copy expressed from a centromeric plasmid, yet the results of the RT-PCR are not significantly different. (If anything, the levels are lower in the tpm2 plasmid-containing strain.) The methods state that the primers were chosen in the gene, so likely do not distinguish the genomic from the plasmid allele. However, the text claims a 1-fold increase in expression, and functional experiments show a near-complete rescue of the tpm1∆ phenotype. This is surprising and confusing and should be resolved to understand whether higher levels of Tpm2 are really the cause of the observed phenotypic rescue.

The authors could for instance probe for protein levels. I believe they have specific nanobodies against tropomyosin. If not, they could use expression of functional mNG-amTpm2 to rescue tpm1∆. Here, the expression of the protein can be directly visualized.

Response: __We thank the reviewer for pointing this out. We would like to clarify that in our RT-qPCR experiments, the primers were chosen within the Tpm1 and Tpm2 gene and do not distinguish between transcripts from endogenous or plasmid copy. We have now mentioned this in the Materials and Methods section of the revised manuscript. So, they represent a relative estimate of the total mRNA of these genes present in cells. We were consistently able to detect ~19 fold increase in Tpm2 total mRNA levels as compared to wildtype and ∆tpm1 cells (Fig. S4D) when tpm2 was expressed from a high-copy plasmid (pRS425). This increase in Tpm2 mRNA levels was accompanied by a rescue in growth (Fig. S4A) and actin cable organization (Fig. S4B) of ∆tpm1 cells containing pRS425-ptpm2TPM2. When tpm2 was expressed from a low-copy number centromeric plasmid (pRS316), we detected a ~2 fold increase in Tpm2 transcript levels when using the tpm1 promoter and no significant change was detected when using tpm2 promoter (Fig. S4E)__. We have made sure that these results are accurately described in the revised manuscript.

As per the reviewer's suggestion, we have now conducted a more extensive analysis to ascertain the expression levels of Tpm2 in our experiments and the data is now presented in new Figure S5. We used mNG-ASTpm1 and mNG-ASTpm2 to rescue growth of ∆tpm1 (Fig. S5A) and correlated growth rescue with protein levels using quantified fluorescence intensity (Fig. S5B, S5C) and western blotting (anti-mNG) (Fig. S5D, S5E). We find that ∆tpm1 cells containing pRS425-ptpm1mNG-ASTpm1 had the highest protein level followed by pRS425-ptpm2 mNG-ASTpm2, pRS305-ptpm1mNG-ASTpm1, and the least protein levels were found in pRS305-ptpm2 mNG-ASTpm2 containing ∆tpm1 cells in both fluorescence intensity and western blotting quantifications (Fig. S5C, S5E). Surprisingly, we were not able to detect any protein levels in ∆tpm1 cells containing pRS305-ptpm2 mNG-ASTpm2 with western blotting (Fig. S5D) which was also accompanied by a lack of growth rescue (Fig. S5A). This most likely due to weak expression from the native Tpm2 promoter which is consistent with previous literature (Drees et al., 1995). Taken together, this data clearly shows that the rescue observed in ∆tpm1 cells is caused due to increased expression of mNG-ASTpm2 in cells and supports our conclusion that increase in Tpm2 expression leads to restoration of normal growth and actin cables in ∆tpm1 cells.

__ Specific function of Tpm2:__

The data about the retrograde actin flow is interpreted as a specific function of Tpm2, but there is no evidence that Tpm1 does not also share this function. To reach this conclusion one would have to investigate retrograde actin flow in tpm1∆ (difficult as cables are weak) or for instance test whether Tpm1 expression restores normal retrograde flow to tpm2∆ cells.

Response: __We agree with the reviewer and as per the reviewer's suggestion, we have performed another experiment which include wildtype, ∆tpm2 cells containing empty pRS316 vector or pRS316-ptpm2TPM1 or pRS316-ptpm1TPM1. We find that RACF rate increased in ∆tpm2 cells as compared to wildtype and was restored to wildtype levels by exogenous expression of Tpm2 but not Tpm1 (Fig. S6E, S6F). Since, actin cables were not detectable in ∆tpm1 cells, we measured RACF rates in ∆tpm1 cells expressing Tpm1 or Tpm2 from a plasmid copy, which restored actin cables as shown previously in __Fig. 5A-C. We observed that RACF rates were similar to wildtype in ∆tpm1 cells expressing either Tpm1 or Tpm2 (Fig. S6E, S6F), suggesting that Tpm1 is not involved in RACF regulation. Taken together, these results suggest a specific role for Tpm2, but not Tpm1, in RACF regulation in S. cerevisiae, consistent with previous literature (Huckaba et al., 2006).

Minor comments: __1.__The growth of tpm1∆ with empty plasmid in Fig S3A is strangely strong (different from other figures).

Response: We thank the reviewer for pointing this out. We have now repeated the drop test multiple times (Fig. R2), but we see similar growth rates as the drop test already presented in Fig. S4A. __At this point, it would be difficult to ascertain the basis of this difference observed at 23{degree sign}C and 30{degree sign}C, but a recent study that links leucine levels to actin cable stability (Sing et al., 2022) might explain the faster growth of these ∆tpm1 cells containing a leu2 gene carrying high-copy plasmid. However, there is no effect on growth rate at 37{degree sign}C which is consistent with other spot assays shown in __Fig. S1D, S4F, S5A.

Significance

I am a cell biologist with expertise in both yeast and actin cytoskeleton.

The question of how tropomyosin localizes to specific actin networks is still open and a current avenue of study. Studies in other organisms have shown that different tropomyosin isoforms, or their acetylated vs non-acetylated versions, localize to distinct actin structures. Proposed mechanisms include competition with other ABPs and preference imposed by the formin nucleator. The current study re-examines the function and localization of the two tropomyosin proteins from the budding yeast and reaches the conclusion that they co-decorate all formin-assembled structures and also share most functions, leading to the simple conclusion that the more important contribution of Tpm1 is simply linked to its higher expression. Once consolidated, the study will appeal to researchers working on the actin cytoskeleton.

We thank the reviewer for their positive assessment of our work and the constructive feedback that has greatly improved the quality of our study. After addressing the points raised by the reviewer, we believe that our study has significantly gained in consolidating the major conclusions of our work.

**Referees cross-commenting**

Having read the other reviewers' comments, I do agree with reviewer 1 that it is not clear whether the Ala-Ser linker really mimics acetylation. I am less convinced than reviewer 3 that the key conclusions of the study are well supported, notably the issue of Tpm2 expression levels is not convincing to me.

Response: __We acknowledge the reviewer's point about the effect of Ala-Ser dipeptide and would request the reviewer to refer to our response to Reviewer 1 (Question 1) for a more detailed discussion on this. We have also extensively addressed the question of Tpm2 expression levels as suggested by the reviewer (new data in __Figure S5) which has further strengthened the conclusions of our study.

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

Summary:__ The study presents the first fully functional fluorescently tagged Tpm proteins, enabling detailed probing of Tpm isoform localization and functions in live cells. The authors created a modified fusion protein, mNG-amTpm, which mimicked native N-terminal acetylation and restored both normal growth and full-length actin cables in yeast cells lacking native Tpm proteins, demonstrating the constructs' full functionality. They also show that Tpm1 and Tpm2 do not have a preference for actin cables nucleated by different formins (Bnr1 and Bni1). Contrary to previous reports, the study found that overexpressing Tpm2 in Δtpm1 cells could restore growth rates and actin cable formation. Furthermore, it is shown that despite its evolutionary divergence, Tpm2 retains actin-protective functions and can compensate for the loss of Tpm1, contributing to cellular robustness.

Major and Minor Comments: 1. The key conclusions of this paper are convincing. However, I suggest that more detail be provided regarding the image analysis used in this study. Specifically, since threshold settings can impact the quality of the generated data and, therefore, its interpretation, it would be useful to see a representative example of the quantification methods used for actin cable length/number (as in refs. 80 and 81) and mitochondria morphology. These could be presented as Supplemental Figures. Additionally, it would help to interpret the results if the authors could be more specific about the statistical tests that were used.

Response: __We agree with the reviewer's suggestions and have now updated our Materials and Methods section to describe the image analysis pipelines used in more detail. We have also added examples of quantification procedure for actin cable length/number and mitochondrial morphology as an additional Supplementary __Figure S7. Briefly, the following pipelines were used:

• Actin cable length and number analysis: This was done exactly as mentioned in McInally et al., 2021, McInally et al., 2022. Actin cables were manually traced in Fiji as shown in __ S7A__, and then the traces files for each cell were run through a Python script (adapted from McInally et al., 2022) that outputs mean actin cable length and number per cell.
• Mitochondria morphology: Mitochondria Analyzer plug-in in Fiji was used to segment out the mitochondrial fragments. The parameters used for 2D segmentation of mitochondria were first optimized using "2D Threshold Optimize" to find the most accurate segmentation and then the same parameters were run on all images. After segmentation of the mitochondrial network, measurements of fragment number were done using "Analyze Particles" function in Fiji. An example of the overall process is shown in __ S7B.__ As per the reviewer's suggestion, we have now included the description of the statistical test used in the Figure Legends of each Figure in the revised manuscript. We have used One-Way Anova with Tukey's Multiple Comparison test, Kruskal-Wallis test with Dunn's Multiple Comparisons, and Unpaired Two-tailed t-test using the in-built functions in GraphPad Prism (v.6.04).

**Referees cross-commenting**

I agree with both reviewers 1 and 2 regarding the issues with the Ala-Ser acetylation mimic and Tpm2 expression levels, respectively. I think the authors should be more careful in how they frame the results, but I consider that these issues do not invalidate the main conclusions of this study.

Response: __We acknowledge the reviewer's concern about the Ala-Ser dipeptide and would request them to refer our earlier discussion on this in response to Reviewer 1 (Question 1) and Reviewer 2 (Question 2). We would also request the reviewer to refer to our answer to Reviewer 2 (Question 6) where we have extensively addressed the question of Tpm2 expression levels and their effect on rescue of Dtpm1 cells. This data is now presented as new __Figure S5 in our revised manuscript.

Reviewer#3 (Significance (Required)):

The finding that Tpm2 can compensate for the loss of Tpm1, restoring actin cable organization and normal growth rates, challenges previous assumptions about the non-redundant functions of these isoforms in Saccharomyces cerevisiae (ref. 16). It also supports a concentration-dependent and formin-independent localization of Tpm isoforms to actin cables in this species. The development of fully functional fluorescently tagged Tpm proteins is a significant methodological advancement. This advancement overcomes previous visualization challenges and allows for accurate in vivo studies of Tpm function and regulation in S. cerevisiae.

The findings will be of particular interest to researchers in the field of cellular and molecular biology who study actin cytoskeleton dynamics. Additionally, it will be relevant for those utilizing advanced microscopy and live-cell imaging techniques.

As a researcher, my experience lies in cytoskeleton dynamics and protein interactions, though I do not have specific experience related to tropomyosin. I use different yeast species as models and routinely employ live-cell imaging as a tool.

We thank the reviewer for their positive outlook and assessment of our study. We have incorporated all their suggestions, and we are confident that the revised manuscript has significantly improved in quality due to these additions.

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

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Referee #2

Evidence, reproducibility and clarity

This manuscript by Dhar, Bagyashree, Palani and colleagues examines the function of the two tropomyosins, Tpm1 and Tpm2, in the budding yeast S. cerevisiae. Previous work had shown that deletion of tpm1 and tpm2 causes synthetic lethality, indicating overlapping function, but also proposed that the two tropomyosins have distinct functions, based on the observation that strong overexpression of Tpm2 causes defects in bud placement and fails to rescue tpm1∆ phenotypes (Drees et al, JCB 1995). The manuscript first describes very functional mNeonGreen tagged version of Tpm1 and Tpm2, where an alanine-serine dipeptide is inserted before the first methionine to mimic acetylation. It then proposes that the Tpm1 and Tpm2 exhibit indistinguishable localization and that low level overexpression (?) of Tpm2 can replace Tpm1 for stabilization of actin cables and cell polarization, suggesting almost completely redundant functions. They also propose on specific function of Tpm2 in regulating retrograde actin cable flow.

Overall, the data are very clean, well presented and quantified, but in several places are not fully convincing of the claims. Because the claims that Tpm1 and Tpm2 have largely overlapping function and localization are in contradiction to previous publication in S. cerevisiae and also different from data published in other organisms, it is important to consolidate them. There are fairly simple experiments that should be done to consolidate the claims of indistinguishable localization, and levels of expression, for which the authors have excellent reagents at their disposal.

Functionality of the acetyl-mimic tagged tropomyosin constructs:

The overall very good functionality of the tagged Tpm constructs is convincing, but the authors should be more accurate in their description, as their data show that they are not perfectly functional. For instance, the use of "completely functional" in the discussion is excessive. In the results, the statement that mNG-Tpm1 expression restores normal growth (page 3, line 69) is inaccurate. Fig S1C shows that tpm1∆ cells expressing mNG-Tpm1 grow more slowly than WT cells. (The next part of the same sentence, stating it only partially restores length of actin cables should cite only Fig S1E, not S1F.) Similarly, the growth curve in Fig S1C suggests that mNG-amTpm1, while better than mNG-Tpm1 does not fully restore the growth defect observed in tpm1∆ (in contrast to what is stated on p. 4 line 81). A more stringent test of functionality would be to probe whether mNG-amTpm1 can rescue the synthetic lethality of the tpm1∆ tpm2∆ double mutant, which would also allow to test the functionality of mNG-amTpm2.

It would also be nice to comment on whether the mNG-amTpm constructs really mimicking acetylation. Given the Ala-Ser peptide ahead of the starting Met is linked N-terminally to mNG, it is not immediately clear it will have the same effect as a free acetyl group decorating the N-terminal Met.

Localization of Tpm1 and Tpm2:

Given the claimed full functionality of mNG-amTpm constructs and the conclusion from this section of the paper that relative local concentrations may be the major factor in determining tropomyosin localization to actin filament networks, I am concerned that the analysis of localization was done in strains expressing the mNG-amTpm construct in addition to the endogenous untagged genes. (This is not expressly stated in the manuscript, but it is my understanding from reading the strain list.) This means that there is a roughly two-fold overexpression of either tropomyosin, which may affect localization. A comparison of localization in strains where the tagged copy is the sole Tpm1 (respectively Tpm2) source would be much more conclusive. This is important as the results are making a claim in opposition to previous work and observation in other organisms.

In fact, although the authors conclude that the tropomyosins do not exhibit preference for certain actin structures, in the images shown in Fig 2A and 2D, there seems to be a clear bias for Tpm1 to decorate cables preferentially in the bud, while Tpm2 appears to decorate them more in the mother cell. Is that a bias of these chosen images, or does this reflect a more general trend? A quantification of relative fluorescence levels in bud/mother may be indicative.

The difficulty in preserving mNG-amTpm after fixation means that authors could not quantify relative Tpm/actin cable directly in single fixed cells. Did they try to label actin cables with Lifeact instead of using phalloidin, and thus perform the analysis in live cells?

Complementation of tpm1∆ by Tpm2:

I am confused about the quantification of Tpm2 expression by RT-PCR shown in Fig S3F. This figure shows that tpm2 mRNA expression levels are identical in cells with an empty plasmid or with a tpm2-encoding plasmid. In both strains (which lack tpm1), as well as in the WT control, one tpm2 copy is in the genome, but only one strain has a second tpm2 copy expressed from a centromeric plasmid, yet the results of the RT-PCR are not significantly different. (If anything, the levels are lower in the tpm2 plasmid-containing strain.) The methods state that the primers were chosen in the gene, so likely do not distinguish the genomic from the plasmid allele. However, the text claims a 1-fold increase in expression, and functional experiments show a near-complete rescue of the tpm1∆ phenotype. This is surprising and confusing and should be resolved to understand whether higher levels of Tpm2 are really the cause of the observed phenotypic rescue. The authors could for instance probe for protein levels. I believe they have specific nanobodies against tropomyosin. If not, they could use expression of functional mNG-amTpm2 to rescue tpm1∆. Here, the expression of the protein can be directly visualized.

Specific function of Tpm2:

The data about the retrograde actin flow is interpreted as a specific function of Tpm2, but there is no evidence that Tpm1 does not also share this function. To reach this conclusion one would have to investigate retrograde actin flow in tpm1∆ (difficult as cables are weak) or for instance test whether Tpm1 expression restores normal retrograde flow to tpm2∆ cells.

Minor comments:

The growth of tpm1∆ with empty plasmid in Fig S3A is strangely strong (different from other figures).

Referees cross-commenting

Having read the other reviewers' comments, I do agree with reviewer 1 that it is not clear whether the Ala-Ser linker really mimics acetylation. I am less convinced than reviewer 3 that the key conclusions of the study are well supported, notably the issue of Tpm2 expression levels is not convincing to me.

Significance

I am a cell biologist with expertise in both yeast and actin cytoskeleton.

The question of how tropomyosin localizes to specific actin networks is still open and a current avenue of study. Studies in other organisms have shown that different tropomyosin isoforms, or their acetylated vs non-acetylated versions, localize to distinct actin structures. Proposed mechanisms include competition with other ABPs and preference imposed by the formin nucleator. The current study re-examines the function and localization of the two tropomyosin proteins from the budding yeast and reaches the conclusion that they co-decorate all formin-assembled structures and also share most functions, leading to the simple conclusion that the more important contribution of Tpm1 is simply linked to its higher expression. Once consolidated, the study will appeal to researchers working on the actin cytoskeleton.

Reviewer #1 (Public review):

Summary:

This study aimed to investigate the effects of optically stimulating the A13 region in healthy mice and a unilateral 6-OHDA mouse model of Parkinson's disease (PD). The primary objectives were to assess changes in locomotion, motor behaviors, and the neural connectome. For this, the authors examined the dopaminergic loss induced by 6-OHDA lesioning. They found a significant loss of tyrosine hydroxylase (TH+) neurons in the substantia nigra pars compacta (SNc) while the dopaminergic cells in the A13 region were largely preserved. Then, they optically stimulated the A13 region using a viral vector to deliver the channelrhodopsine (CamKII promoter). In both sham and PD model mice, optogenetic stimulation of the A13 region induced pro-locomotor effects, including increased locomotion, more locomotion bouts, longer durations of locomotion, and higher movement speeds. Additionally, PD model mice exhibited increased ipsilesional turning during A13 region photoactivation. Lastly, the authors used whole-brain imaging to explore changes in the A13 region's connectome after 6-OHDA lesions. These alterations involved a complex rewiring of neural circuits, impacting both afferent and efferent projections. In summary, this study unveiled the pro-locomotor effects of A13 region photoactivation in both healthy and PD model mice. The study also indicates the preservation of A13 dopaminergic cells and the anatomical changes in neural circuitry following PD-like lesions that represent the anatomical substrate for a parallel motor pathway.

Strengths:

These findings hold significant relevance for the field of motor control, providing valuable insights into the organization of the motor system in mammals. Additionally, they offer potential avenues for addressing motor deficits in Parkinson's disease (PD). The study fills a crucial knowledge gap, underscoring its importance, and the results bolster its clinical relevance and overall strength.

The authors adeptly set the stage for their research by framing the central questions in the introduction, and they provide thoughtful interpretations of the data in the discussion section. The results section, while straightforward, effectively supports the study's primary conclusion-the pro-locomotor effects of A13 region stimulation, both in normal motor control and in the 6-OHDA model of brain damage.

Weaknesses:

(1) Anatomical investigation. I have a major concern regarding the anatomical investigation of plastic changes in the A13 connectome (Figures 4 and 5). While the methodology employed to assess the connectome is technically advanced and powerful, the results lack mechanistic insight at the cell or circuit level into the pro-locomotor effects of A13 region stimulation in both physiological and pathological conditions. This concern is exacerbated by a textual description of results that doesn't pinpoint precise brain areas or subareas but instead references large brain portions like the cortical plate, making it challenging to discern the implications for A13 stimulation. Lastly, the study is generally well-written with a smooth and straightforward style, but the connectome section presents challenges in readability and comprehension. The presentation of results, particularly the correlation matrices and correlation strength, doesn't facilitate biological understanding. It would be beneficial to explore specific pathways responsible for driving the locomotor effects of A13 stimulation, including examining the strength of connections to well-known locomotor-associated regions like the Pedunculopontine nucleus, Cuneiformis nucleus, LPGi, and others in the diencephalon, midbrain, pons, and medulla. Additionally, identifying the primary inputs to A13 associated with motor function would enhance the study's clarity and relevance.

The study raises intriguing questions about compensatory mechanisms in Parkinson's disease a new perspective with the preservation of dopaminergic cells in A13, despite the SNc degeneration, and the plastic changes to input/output matrices. To gain inspiration for a more straightforward reanalysis and discussion of the results, I recommend the authors refer to the paper titled "Specific populations of basal ganglia output neurons target distinct brain stem areas while collateralizing throughout the diencephalon from the David Kleinfeld laboratory." This could guide the authors in investigating motor pathways across different brain regions.

(2) Description of locomotor performance. Figure 3 provides valuable data on the locomotor effects of A13 region photoactivation in both control and 6-OHDA mice. However, a more detailed analysis of the changes in locomotion during stimulation would enhance our understanding of the pro-locomotor effects, especially in the context of 6-OHDA lesions. For example, it would be informative to explore whether the probability of locomotion changes during stimulation in the control and 6-OHDA groups. Investigating reaction time, speed, total distance, and even kinematic aspects during stimulation could reveal how A13 is influencing locomotion, particularly after 6-OHDA lesions. The laboratory of Whelan has a deep knowledge of locomotion and the neural circuits driving it so these features may be instructive to infer insights on the neural circuits driving movement. On the same line, examining features like the frequency or power of stimulation related to walking patterns may help elucidate whether A13 is engaging with the Mesencephalic Locomotor Region (MLR) to drive the pro-locomotor effects. These insights would provide a more comprehensive understanding of the mechanisms underlying A13-mediated locomotor changes in both healthy and pathological conditions.

(3) Figure 2 indeed presents valuable information regarding the effects of A13 region photoactivation. To enhance the comprehensiveness of this figure and gain a deeper understanding of the neurons driving the pro-locomotor effect of stimulation, it would be beneficial to include quantifications of various cell types:

• cFos-Positive Cells/TH-Positive Cells: it can help determine the impact of A13 stimulation on dopaminergic neurons and the associated pro-locomotor effect in healthy condition and especially in the context of Parkinson's disease (PD) modeling.

• cFos-Positive Cells /TH-Negative Cells: Investigating the number of TH-negative cells activated by stimulation is also important, as it may reveal non-dopaminergic neurons that play a role in locomotor responses. Identifying the location and characteristics of these TH-negative cells can provide insights into their functional significance.<br /> Incorporating these quantifications into Figure 2 would enhance the figure's informativeness and provide a more comprehensive view of the neuronal populations involved in the locomotor effects of A13 stimulation.

(4) Referred to Figure 3. In the main text (page 5) when describing the animal with 6-OHDA the wrong panels are indicated. It is indicated in Figure 2A-E but it should be replaced with 3A-E. Please do that.

Summary of the Study after revision

The revised manuscript reflects significant efforts to improve clarity, organization, and data interpretation. The refinements in anatomical descriptions, behavioral analyses, and contextual framing have strengthened the manuscript considerably. However, the study still lacks direct causal evidence linking anatomical remodeling to behavioral improvements, and the small sample size in the anatomical analyses remains a concern. The authors have addressed many points raised in the initial review, but further acknowledgement of the exploratory nature of these findings would enhance the scientific rigor of the work.

Key Improvements in the Revision

The revised manuscript demonstrates considerable progress in clarifying data presentation, refining behavioral analyses, and improving the contextualization of anatomical findings. The restructuring of the anatomical section now provides greater precision in describing motor-related pathways, integrating terminology from the Allen Brain Atlas. The addition of new figures (Figures 4 and 5) strengthens the accessibility of these findings by illustrating key connectivity patterns more effectively. Furthermore, the correlation matrices have been adjusted to improve interpretability, ensuring that the presented data contribute meaningfully to the overall narrative of the study.

The authors have also made significant improvements in their behavioral analyses, particularly in the organization and presentation of locomotor data. Figure 3 has been revised to distinctly separate results from 6-OHDA and sham animals, providing a clearer comparison of locomotor outcomes. Additional metrics, such as reaction time, locomotion bouts, and movement speed, further enhance the granularity of the analysis, making the results more informative.

The discussion surrounding anatomical connectivity has also been strengthened. The revised manuscript now places greater emphasis on motor-related pathways and refines its analysis of A13 efferents and afferents. A newly introduced figure provides a concise summary of these connections, improving the contextualization of the anatomical data within the study's broader scope. Moreover, the authors have addressed the translational relevance of their findings by acknowledging the differences between optogenetic stimulation and deep brain stimulation (DBS). Their discussion now better situates the findings within existing literature on PD-related motor circuits, providing a more balanced perspective on the potential implications of A13 stimulation.

Remaining Concerns

Despite these substantial improvements, a number of critical concerns remain. The anatomical findings, though insightful, remain largely correlative and do not establish a causal link between structural remodeling and locomotor recovery. While the authors argue that these data will serve as a reference for future investigations, their necessity for the core conclusions of the study is not entirely clear. Additionally, while the anatomical data offer an interesting perspective on A13 connectivity, their direct relevance to the study's primary goal-demonstrating the role of A13 in locomotor recovery-remains uncertain. The authors emphasize that these data will be valuable for future research, yet their integration into the study's main narrative feels somewhat supplementary. Based on this last thought of the authors it is even more relevant another key limitation lying in the small sample size used for connectivity analyses. With only two sham and three 6-OHDA animals included, the statistical confidence in the findings is inherently limited. The absence of direct statistical comparisons between ipsilesional and contralesional projections further weakens the conclusions drawn from these anatomical studies. The authors have acknowledged that obtaining the necessary samples, acquiring the data, and analyzing them is a prolonged and resource-intensive process. While this may be a valid practical limitation, it does not justify the lack of a robust statistical approach. A more rigorous statistical framework should be employed to reinforce the findings, or alternative techniques should be considered to provide additional validation. Given these constraints, it remains unclear why the authors have not opted for standard immunohistochemistry, which could provide a complementary and more statistically accessible approach to validate the anatomical findings. Employing such an approach would not only increase the robustness of the results but also strengthen the study's impact by providing an independent confirmation of the observed structural changes.

Reviewer #2 (Public review):

Summary:

The paper by Kim et al. investigates the potential of stimulating the dopaminergic A13 region to promote locomotor restoration in a Parkinson's mouse model. Using wild-type mice, 6-OHDA injection depletes dopaminergic neurons in the substantia nigra pars compacta, without impairing those of the A13 region and the ventral tegmentum area, as previously reported in humans. Moreover, photostimulation of presumably excitatory (CAMKIIa) neurons in the vicinity of the A13 region improves bradykinesia and akinetic symptoms after 6-OHDA injection. Whole-brain imaging with retrograde and anterograde tracers reveals that the A13 region undergoes substantial changes in the distribution of its afferents and projections after 6-OHDA injection, thus suggesting a remodeling of the A13 connectome. Whether this remodelling contributes to pro-locomotor effects of the photostimulation of the A13 region remains unknown as causality was not addressed.

Strengths:

Photostimulation of presumably excitatory (CAMKIIa) neurons in the vicinity of the A13 region promotes locomotion and locomotor recovery of wild-type mice 1 month after 6-OHDA injection in the medial forebrain bundle, thus identifying a new potential target for restoring motor functions in Parkinson's disease patients. The study also provides a description of the A13 region connectome pertaining to motor behaviors and how it changes after a dopaminergic lesion. Although there is no causal link between anatomical and behavioral data, it raises interesting questions for further studies.

Weaknesses:

Although CAMKIIa is a marker of presumably excitatory neurons and can be used as an alternative marker of dopaminergic neurons, some uncertainty remains regarding the phenotype of neurons underlying recovery of akinesia and improvement of bradykinesia.

Figure 4 is improved, but the results from the correlation analyses remain difficult to interpret, as they may reflect changes in various impaired brain regions independently of the A13 region. While the analysis offers a snapshot of correlated changes within the connectome, it does not identify which specific cell or axonal populations are actually increasing or decreasing. Although functional MRI connectome analyses are well-established, anatomical data seem less suitable for this purpose. How can one interpret correlated changes in anatomical inputs or outputs between two distinct regions?

Figure 5 is also improved, but there is room for further enhancement. As currently presented, it is difficult to distinguish the differences between the sham and 6-OHDA groups. The first column could compare afferents, while the second column could compare efferents. Given the small sample size, it would be more appropriate to present individual data rather than the mean and standard deviation.

Appraisal and impact

Although the behavioral experiments are convincing, the low number of animals in the anatomical studies is insufficient to make any relevant statistical conclusions due to extremely low statistical power.

Author response:

The following is the authors’ response to the original reviews

Public Reviews:

Reviewer #1 (Public Review):

Summary:

This study aimed to investigate the effects of optically stimulating the A13 region in healthy mice and a unilateral 6-OHDA mouse model of Parkinson's disease (PD). The primary objectives were to assess changes in locomotion, motor behaviors, and the neural connectome. For this, the authors examined the dopaminergic loss induced by 6-OHDA lesioning. They found a significant loss of tyrosine hydroxylase (TH+) neurons in the substantia nigra pars compacta (SNc) while the dopaminergic cells in the A13 region were largely preserved. Then, they optically stimulated the A13 region using a viral vector to deliver the channelrhodopsine (CamKII promoter). In both sham and PD model mice, optogenetic stimulation of the A13 region induced pro-locomotor effects, including increased locomotion, more locomotion bouts, longer durations of locomotion, and higher movement speeds. Additionally, PD model mice exhibited increased ipsi lesional turning during A13 region photoactivation. Lastly, the authors used whole-brain imaging to explore changes in the A13 region's connectome after 6-OHDA lesions. These alterations involved a complex rewiring of neural circuits, impacting both afferent and efferent projections. In summary, this study unveiled the pro-locomotor effects of A13 region photoactivation in both healthy and PD model mice. The study also indicates the preservation of A13 dopaminergic cells and the anatomical changes in neural circuitry following PD-like lesions that represent the anatomical substrate for a parallel motor pathway.

Strengths:

These findings hold significant relevance for the field of motor control, providing valuable insights into the organization of the motor system in mammals. Additionally, they offer potential avenues for addressing motor deficits in Parkinson's disease (PD). The study fills a crucial knowledge gap, underscoring its importance, and the results bolster its clinical relevance and overall strength.

The authors adeptly set the stage for their research by framing the central questions in the introduction, and they provide thoughtful interpretations of the data in the discussion section. The results section, while straightforward, effectively supports the study's primary conclusion - the pro-locomotor effects of A13 region stimulation, both in normal motor control and in the 6-OHDA model of brain damage.

We thank the reviewer for their positive comments.

Weaknesses:

(1) Anatomical investigation. I have a major concern regarding the anatomical investigation of plastic changes in the A13 connectome (Figures 4 and 5). While the methodology employed to assess the connectome is technically advanced and powerful, the results lack mechanistic insight at the cell or circuit level into the pro-locomotor effects of A13 region stimulation in both physiological and pathological conditions. This concern is exacerbated by a textual description of results that doesn't pinpoint precise brain areas or subareas but instead references large brain portions like the cortical plate, making it challenging to discern the implications for A13 stimulation. Lastly, the study is generally well-written with a smooth and straightforward style, but the connectome section presents challenges in readability and comprehension. The presentation of results, particularly the correlation matrices and correlation strength, doesn't facilitate biological understanding. It would be beneficial to explore specific pathways responsible for driving the locomotor effects of A13 stimulation, including examining the strength of connections to well-known locomotor-associated regions like the Pedunculopontine nucleus, Cuneiformis nucleus, LPGi, and others in the diencephalon, midbrain, pons, and medulla.

We initially considered two approaches. The first was to look at specific projections to the motor regions, focusing on the MLR. The second was to utilize a whole-brain analysis, which is presented here. Given what we know about the zona incerta, especially its integrative role, we felt that examining the full connectome was a reasonable starting point.

The value of the whole-brain approach is that it provides a high-level overview of the afferents and efferents to the region. The changes in the brain that occur following Parkinson-like lesions, such as those in the nigrostriatal pathway, are complex and can affect neighbouring regions such as the A13. Therefore, we wished to highlight the A13, which we considered a therapeutic target, and examine changes in connectivity that could occur following acute lesions affecting the SNc. We acknowledge that this study does not provide a causal link, but it presents the fundamental background information for subsequent hypothesis-driven, focused, region-specific analysis.

The terms provided were taken from the Allen Brain Atlas terminology and presented as abbreviations. We have added two new figures focusing on motor regions to make the information more comprehensible (new Figures 4 and 5) and rewrote the connectomics section to make it easier to understand.

Additionally, identifying the primary inputs to A13 associated with motor function would enhance the study's clarity and relevance.

This is a great point to help simplify the whole-brain results. We have presented the motor-related inputs and outputs as part of a new figure in the main paper (Figure 5) and added accompanying text in the results section. We have also updated the correlation matrices to concentrate on motor regions (Figure 4). This highlights possible therapeutic pathways. We have also enhanced our discussion of these motor-related pathways. We have retained the entire dataset and added it to our data repository for those interested.

The study raises intriguing questions about compensatory mechanisms in Parkinson's disease and a new perspective on the preservation of dopaminergic cells in A13, despite the SNc degeneration, and the plastic changes to input/output matrices. To gain inspiration for a more straightforward reanalysis and discussion of the results, I recommend the authors refer to the paper titled "Specific populations of basal ganglia output neurons target distinct brain stem areas while collateralizing throughout the diencephalon from the David Kleinfeld laboratory." This could guide the authors in investigating motor pathways across different brain regions.

Thank you for the advice. As pointed out, Kleinfeld’s group presented their data in a nice, focused way. For the connectomic piece, we have added Figure 5, which provides a better representation than our previous submission.

(2) Description of locomotor performance. Figure 3 provides valuable data on the locomotor effects of A13 region photoactivation in both control and 6-OHDA mice. However, a more detailed analysis of the changes in locomotion during stimulation would enhance our understanding of the pro-locomotor effects, especially in the context of 6-OHDA lesions. For example, it would be informative to explore whether the probability of locomotion changes during stimulation in the control and 6-OHDA groups. Investigating reaction time, speed, total distance, and could reveal how A13 is influencing locomotion, particularly after 6-OHDA lesions. The laboratory of Whelan has a deep knowledge of locomotion and the neural circuits driving it so these features may be instructive to infer insights on the neural circuits driving movement. On the same line, examining features like the frequency or power of stimulation related to walking patterns may help elucidate whether A13 is engaging with the Mesencephalic Locomotor Region (MLR) to drive the pro-locomotor effects. These insights would provide a more comprehensive understanding of the mechanisms underlying A13-mediated locomotor changes in both healthy and pathological conditions.

Thank you for these suggestions. We have reorganized Figure 3 to highlight the metrics by separating the 6-OHDA from the Sham experiments (3F-J, which highlights distance travelled, average speed and duration). We have also added additional text to highlight these metrics better in the text. We have relabelled Supplementary Figure S3, which presents reaction time as latency to initiate locomotion and updated the main text to address the reviewers' points.

Reviewer #2 (Public Review):

Summary:

The paper by Kim et al. investigates the potential of stimulating the dopaminergic A13 region to promote locomotor restoration in a Parkinson's mouse model. Using wild-type mice, 6-OHDA injection depletes dopaminergic neurons in the substantia nigra pars compacta, without impairing those of the A13 region and the ventral tegmentum area, as previously reported in humans. Moreover, photostimulation of presumably excitatory (CAMKIIa) neurons in the vicinity of the A13 region improves bradykinesia and akinetic symptoms after 6-OHDA injection. Whole-brain imaging with retrograde and anterograde tracers reveals that the A13 region undergoes substantial changes in the distribution of its afferents and projections after 6-OHDA injection. The study suggests that if the remodeling of the A13 region connectome does not promote recovery following chronic dopaminergic depletion, photostimulation of the A13 region restores locomotor functions.

Strengths:

Photostimulation of presumably excitatory (CAMKIIa) neurons in the vicinity of the A13 region promotes locomotion and locomotor recovery of wild-type mice 1 month after 6-OHDA injection in the medial forebrain bundle, thus identifying a new potential target for restoring motor functions in Parkinson's disease patients.

Weaknesses:

Electrical stimulation of the medial Zona Incerta, in which the A13 region is located, has been previously reported to promote locomotion (Grossman et al., 1958). Recent mouse studies have shown that if optogenetic or chemogenetic stimulation of GABAergic neurons of the Zona Incerta promotes and restores locomotor functions after 6-OHDA injection (Chen et al., 2023), stimulation of glutamatergic ZI neurons worsens motor symptoms after 6-OHDA (Lie et al., 2022).

Thank you - we have added this reference. It is helpful as Grossman did stimulate the zona incerta in the cat and elicit locomotion, suggesting that stimulation of the area in normal mice has external validity. Grossman’s results prompted a later clinical examination of the zona incerta, but it concentrated on the zona incerta regions close to the subthalamic regions (Ossowska 2019), further caudal to the area we focused on. Chen et al. (2023) targeted the area in the lateral aspect of central/medial zona incerta, formed by dorsal and ventral zona incerta, which may account for the differing results. Our data were robust for stimulation of the medial aspect of the rostromedial zona incerta. The thigmotactic behaviour that we observed in our work that focused on CamKII neurons has not been observed with chemogenetic, optogenetic activation or with photoinhibition of GABAergic central/medial ZI (Chen et al. 2023).

GABAergic activation of mZI to Cuneiform projections (Sharma et al. 2024) also did not produce thigmotactic behavior. We have added these points to the discussion.

Although CAMKIIa is a marker of presumably excitatory neurons and can be used as an alternative marker of dopaminergic neurons, behavioral results of this study raise questions about the neuronal population targeted in the vicinity of the A13 region. Moreover, if YFP and CHR2-YFP neurons express dopamine (TH) within the A13 region (Fig. 2), there is also a large population of transduced neurons within and outside of the A13 region that do not, thus suggesting the recruitment of other neuronal cell types that could be GABAergic or glutamatergic.

We found that CamKII transfection of the A13 region was extremely effective in promoting locomotor activity, which was critical for our work in exploring its possible therapeutic potential. We have since quantified the cell number, we found that the c-fos cell number was increased following ChR2 activation. There is evidence of TH activation - but the data suggest that other cell types contribute. C-fos alone is a blunt tool to assess specificity - rather, it is better at showing overall photostimulus efficacy - which we have demonstrated. Moreover, there is evidence that cell types are not purely dopaminergic, with GABA co-localized (Negishi et al. 2020). We acknowledge that specific viral approaches that target the GABAergic, glutamatergic, and dopaminergic circuits would be very useful. The range of tools to target A13 dopaminergic circuits is more limited than the SNc, for example, because the A13 region lacks DAT, and TH-IRES-Cre approaches, while helpful, are less specific than DAT-Cre mouse models. Intersectional approaches targeting multiple transmitters (glutamate & dopamine, for example) may be one solution as we do not expect that a single transmitter-specific pathway would work, as well as broad targeting of the A13 region. Our recent work suggests that GABAergic neuron activation may have more general effects on behaviour rather than control of ongoing locomotor parameters (Sharma et al. 2024). Recent work shows a positive valence effect of dopamine A13 activation on motivated food-seeking behavior, which differs from consummatory behavior observed with GABAergic modulation (Ye, Nunez, and Zhang 2023). Chemogenetic inactivation and ablation of dopaminergic A13 revealed that they contribute to grip strength and prehensile movements, uncoupling food-seeking grasping behavior from motivational factors (Garau et al. 2023). Overall, this suggests differing effects of GABA compared to DA and/or glutamatergic cell types, consistent with our effects of stimulating CamKII. The discussion has been updated.

Regarding the analysis of interregional connectivity of the A13 region, there is a lack of specificity (the viral approach did not specifically target the A13 region), the number of mice is low for such correlation analyses (2 sham and 3 6-OHDA mice), and there are no statistics comparing 6-OHDA versus sham (Fig. 4) or contra- versus ipsilesional sides (Fig. 5). Moreover, the data are too processed, and the color matrices (Fig. 4) are too packed in the current format to enable proper visualization of the data. The A13 afferents/efferents analysis is based on normalized relative values; absolute values should also be presented to support the claim about their upregulation or downregulation.

Generally, papers using tissue-clearing imaging approaches have low sample sizes due to technical complexity and challenges. The technical challenges of obtaining these data were substantial in both collection and analysis. There are multiple technical complexities arising from dual injections (A13 and MFB coordinates) and targeting the area correctly. The A13 region is difficult to target as it spans only around 300 µm in the anterior-posterior axis. While clearing the brain takes weeks, and light-sheet imaging also takes time, the time necessary to analyze the tissue using whole-brain quantification is labor intensive, especially with a lack of a standardized analysis pipeline from atlas registrations, signal segmentations, and quantifications. The field is still relatively new, requiring additional time to refine pipelines.

Correlation matrices are often used in analyzing connectivity patterns on a brain-wide scale, as they can identify any observable patterns within a large amount of data. We used correlation matrices to display estimated correlation coefficients between the afferent and efferent proportions from one brain subregion to another across 251 brain regions in total in a pairwise manner (not for hypothesis testing). We provided descriptive statistics (mean and error bars) in the original Figure 5C and G. As mentioned in comments for Reviewer 1, we have now presented the data in revised Figure 4 and 5 that focuses specifically on motor-related pathways to provide information on possible pathways. The has simplified the correlation matrices and highlighted the differences in 6-OHDA efferent data especially. As suggested, raw values are shared in a supplemental file on our data repository.

In the absence of changes in the number of dopaminergic A13 neurons after 6-OHDA injection, results from this correlation analysis are difficult to interpret as they might reflect changes from various impaired brain regions independently of the A13 region.

We acknowledge that models of Parkinson’s disease, particularly those using 6-OHDA, induce plasticity in various regions, which may subsequently affect A13 connectivity. We aim to emphasize the residual, intact A13 pathways that could serve as therapeutic targets in future investigations. This emphasis is pertinent in the context of potential clinical applications, as the overall input and output to the region fundamentally dictate the significance of the A13 region in lesioned nigrostriatal models. We agree with the reviewer that the changes certainly can be independent of A13; however, the fact that there was a significant change in the connectome post-6-OHDA injection and striatonigral degeneration is in and of itself important to document. We have added a sentence acknowledging this limitation to the discussion.

There is no causal link between anatomical and behavioral data, which raises questions about the relevance of the anatomical data.

This point was also addressed earlier in response to a comment from Reviewer 1. Focusing on specific motor pathways is one avenue to explore. However, given that the zona incerta acts as an integrative hub, we believed it is prudent to initially examine both afferent and efferent pathways using a brain-wide approach. For instance, without employing this methodology, the potential significance of cortical interconnectivity to the A13 region might not have been fully appreciated. As mentioned previously, we will place additional emphasis on motor-related regions in our revised paper, thereby enhancing the relevance of the anatomical data presented. With these modifications, we anticipate that our data will underscore specific motor-related targets for future exploration, employing optogenetic targeting to assess necessity and sufficiency.

Overall, the study does not take advantage of genetic tools accessible in the mouse to address the direct or indirect behavioral and anatomical contributions of the A13 region to motor control and recovery after 6-OHDA injection.

Our study has not specifically targeted neurons that express dopaminergic, glutamatergic, or GABAergic properties (refer to earlier comment for more detail). However, like others, we find that targeting one neuronal population often does not result in a pure transmitter phenotype. For instance, evidence suggests co-localization of dopamine neurons with a subpopulation of GABA neurons in the A13/medial zona incerta (Negishi et al. 2020). In the hypothalamus, research by Deisseroth and colleagues (Romanov et al. 2017) indicates the presence of multiple classes of dopamine cells, each containing different ratios of co-localized peptides and/or fast neurotransmitters. Consequently, we believe our work lays the foundation for the investigations suggested by the reviewer. Furthermore, if one considers this work in the context of a preclinical study to determine whether the A13 might be a target in human Parkinson's disease, the existing technology that could be utilized is deep brain stimulation (DBS) or electrical modulation, which would also affect different neuronal populations in a non-specific manner.

While optogenetic stimulation therapy is longer term, using CamKII combined with the DJ hybrid AAV could be a translatable strategy for targeting A13 neuronal populations in non-human primates (Watakabe et al. 2015; Watanabe et al. 2020). We have added to the discussion.

Reviewer #3 (Public Review):

Kim, Lognon et al. present an important finding on pro-locomotor effects of optogenetic activation of the A13 region, which they identify as a dopamine-containing area of the medial zona incerta that undergoes profound remodeling in terms of afferent and efferent connectivity after administration of 6-OHDA to the MFB. The authors claim to address a model of PD-related gait dysfunction, a contentious problem that can be difficult to treat with dopaminergic medication or DBS in conventional targets. They make use of an impressive array of technologies to gain insight into the role of A13 remodeling in the 6-OHDA model of PD. The evidence provided is solid and the paper is well written, but there are several general issues that reduce the value of the paper in its current form, and a number of specific, more minor ones. Also, some suggestions, that may improve the paper compared to its recent form, come to mind.

Thank you for the suggestions and careful consideration of our work - it is appreciated.

The most fundamental issue that needs to be addressed is the relation of the structural to the behavioral findings. It would be very interesting to see whether the structural heterogeneity in afferent/effects projections induced by 6-OHDA is related to the degree of symptom severity and motor improvement during A13 stimulation.

As mentioned in comments for Reviewer 1, we have performed additional analysis and present this in Figure 5. We have also revised Figure 4, focusing on motor regions. Our work will provide a roadmap for future studies to disentangle divergent or convergent A13 pathways that are involved in different or all PD-related motor symptoms. Because we could not measure behavioural change in the same animals studied with the anatomic study (essentially because the optrode would have significantly disrupted the connectome we are measuring), we cannot directly compare behaviour to structure.

The authors provide extensive interrogation of large-scale changes in the organization of the A13 region afferent and efferent distributions. It remains unclear how many animals were included to produce Fig 4 and 5. Fig S5 suggests that only 3 animals were used, is that correct? Please provide details about the heterogeneity between animals. Please provide a table detailing how many animals were used for which experiment. Were the same animals used for several experiments?

The behavioral set and the anatomical set were necessarily distinct. In the anatomical experiments, we employed both anterograde and retrograde viral approaches to target the afferent and efferent A13 populations with fluorescent proteins. For the behavioral approach, a single ChR2 opsin was utilized to photostimulate the A13 region; hence combining the two populations was not feasible. We were also concerned that the optrode itself would interfere with connectomics. A lower number of animals were used for the whole-brain work due to technical limitations described earlier. We have now provided additional information regarding numbers in all figures and the text. Using Spearman’s correlation analysis, we found afferent and efferent proportions across animals to be consistent, with an average correlation of 0.91, which is reported in Figure S6.

While the authors provide evidence that photoactivation of the A13 is sufficient in driving locomotion in the OFT, this pro-locomotor effect seems to be independent of 6-OHDA-induced pathophysiology. Only in the pole test do they find that there seems to be a difference between Sham vs 6-OHDA concerning the effects of photoactivation of the A13. Because of these behavioral findings, optogenic activation of A13 may represent a gain of function rather than disease-specific rescue. This needs to be highlighted more explicitly in the title, abstract, and conclusion.

Optogenetic activation of A13 may represent a gain of function in both healthy and 6-OHDA mice, highlighting a parallel descending motor pathway that remains intact. 6-OHDA lesions have multiple effects on motor and cognitive function. This makes a single pathway unlikely to rescue all deficits observed in 6-OHDA models. The lack of locomotion observed in 6-OHDA models can be reversed by A13 region photostimulation. Therefore, this is a reversal of a loss of function, in this case. However, the increase in turning represents a gain of function. We have highlighted this as suggested in the discussion.

The authors claim that A13 may be a possible target for DBS to treat gait dysfunction. However, the experimental evidence provided (in particular the lack of disease-specific changes in the OFT) seems insufficient to draw such conclusions. It needs to be highlighted that optogenetic activation does not necessarily have the same effects as DBS (see the recent review from Neumann et al. in Brain: https://pubmed.ncbi.nlm.nih.gov/37450573/). This is important because ZI-DBS so far had very mixed clinical effects. The authors should provide plausible reasons for these discrepancies. Is cell-specificity, which only optogenetic interventions can achieve, necessary? Can new forms of cyclic burst DBS achieve similar specificity (Spix et al, Science 2021)? Please comment.

Thank you for the valuable comments. They have been incorporated into the discussion.

Our study highlights a parallel motor pathway provided by the A13 region that remains intact in 6-OHDA mice and can be sufficiently driven to rescue the hypolocomotor pathology observed in the OFT and overcome bradykinesia and akinesia. The photoactivation of ipsilesional A13 also has an overall additive effect on ipsiversive circling, representing a gain of function on the intact side that contributes to the magnitude of overall motor asymmetry against the lesioned side. The effects of DBS are rather complex, ranging from micro-, meso-, to macro-scales, involving activation, inhibition, and informational lesioning, and network interactions. This could contribute to the mixed clinical effects observed with ZI-DBS, in addition to differences in targeting and DBS programming among the studies (see review (Ossowska 2019) ). Also the DBS studies targeting ZI have never targeted the rostromedial ZI which extends towards the hypothalamus and contains the A13. Furthermore, DBS and electrical stimulation of neural tissue, in general, are always limited by current spread and lower thresholds of activation of axons (e.g., axons of passage), both of which can reduce the specificity of the true therapeutic target. Optogenetic studies have provided mechanistic insights that could be leveraged in overcoming some of the limitations in targeting with conventional DBS approaches. Spix et al. (2021) provided an interesting approach highlighting these advancements. They devised burst stimulation to facilitate population-specific neuromodulation within the external globus pallidus. Moreover, they found a complementary role for optogenetics in exploring the pathway-specific activation of neurons activated by DBS. To ascertain whether A13 DBS may be a viable therapy for PD gait, it will be necessary to perform many more preclinical experiments, and tuning of DBS parameters could be facilitated by optogenetic stimulation in these murine models. We have added to the discussion.

In a recent study, Jeon et al (Topographic connectivity and cellular profiling reveal detailed input pathways and functionally distinct cell types in the subthalamic nucleus, 2022, Cell Reports) provided evidence on the topographically graded organization of STN afferents and McElvain et al. (Specific populations of basal ganglia output neurons target distinct brain stem areas while collateralizing throughout the diencephalon, 2021, Neuron) have shown similar topographical resolution for SNr efferents. Can a similar topographical organization of efferents and afferents be derived for the A13/ ZI in total?

The ZI can be subdivided into four subregions in the antero-posterior axis: rostral (ZIr), dorsal (ZId), ventral (ZIv), and caudal (ZIc) regions. The dorsal and ventral ZI is also referred together as central/medial/intermediate ZI. There are topographical gradients in different cell types and connectivity across these subregions (see reviews: (Mitrofanis 2005; Monosov et al. 2022; Ossowska 2019). Recent work by Yang and colleagues (2022) demonstrated a topographical organization among the inputs and outputs of GABAergic (VGAT) populations across four ZI subregions. Given that A13 region encompasses a smaller portion (the medial aspect) of both rostral and medial/central ZI (three of four ZI subregions) and coexpress VGAT, A13 region likely falls under rostral and intermediate medial ZI dataset found in Yang et al. (2022). With our data, we would not be able to capture the breadth of topographical organization shown in Yang et al (2022).

In conclusion, this is an interesting study that can be improved by taking into consideration the points mentioned above.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

(1) Figure 2 indeed presents valuable information regarding the effects of A13 region photoactivation. To enhance the comprehensiveness of this figure and gain a deeper understanding of the neurons driving the pro-locomotor effect of stimulation, it would be beneficial to include quantifications of various cell types:

• cFos-Positive Cells/TH-Positive Cells: it can help determine the impact of A13 stimulation on dopaminergic neurons and the associated pro-locomotor effect in the healthy condition and especially in the context of Parkinson's disease (PD) modeling.

• cFos-Positive Cells /TH-Negative Cells: Investigating the number of TH-negative cells activated by stimulation is also important, as it may reveal non-dopaminergic neurons that play a role in locomotor responses. Identifying the location and characteristics of these TH-negative cells can provide insights into their functional significance.

We have completed this analysis. The data is presented in Figure 2F, where we show increased c-fos intensity with photoactivation. We observed an increase in the number of cells activated in the A13 region. However, we did not definitively see increases in TH+ cells, suggesting a heterogeneous set of neurons responsible for the effects—possibly glutamatergic neurons.

Incorporating these quantifications into Figure 2 would enhance the figure's informativeness and provide a more comprehensive view of the neuronal populations involved in the locomotor effects of A13 stimulation.

We have added text and a new graph.

(2) Refer to Figure 3. In the main text (page 5) when describing the animal with 6-OHDA the wrong panels are indicated. It is indicated in Figure 2A-E but it should be replaced with 3A-E.

Please do that.

Done, and we have updated the figure to improve readability, by separating the 6-OHDA findings from sham in all graphs.

Reviewer #2 (Recommendations For The Authors):

Abstract

Page 1: Inhibitory or lesion studies will be necessary to support the claim that the global remodeling of afferent and efferent projections of the A13 region highlights the Zona Incerta's role as a crucial hub for the rapid selection of motor function.

Overall, there is quite a bit of evidence that the zona incerta is a hub for afferent/efferents.

Mitrofanis (2005) and, more recently, Wang et al. (2020) summarize some of the evidence. Yang (2022) illustrates that the zona incerta shows multiple inputs to GABAergic neurons and outputs to diverse regions. Recent work suggests that the zona incerta contributes to various motor functions such as hunting, exploratory locomotion, and integrating multiple modalities (Zhao et al. 2019; Wang et al. 2019; Monosov et al. 2022; Chometton et al. 2017). The introduction has been updated.

Introduction

Page 2, paragraph 2: "However, little attention has been placed on the medial zona incerta (mZI), particularly the A13, the only dopamine-containing region of the rostral ZI" Is the A13 region located in the rostral or medial ZI or both?

It should have been written “rostromedial” ZI. The A13 is located in the medial aspect of rostromedial ZI. Introduction has been updated.

Page 2, para 3: Li et al (2021) used a mini-endoscope to record the GCaMP6 signal. Masini and Kiehn, 2022 transiently blocked the dopaminergic transmission; they never used 6-OHDA.

Please correct through the text.

Corrected.

Page 2, para 4: the A13 connectome encompasses the cerebral cortex,... MLR. The MLR is a functional region, correct this for the CNF and PPN.

Corrected.

Page 3, the last paragraph of the introduction could be clarified by presenting the behavioral data first, followed by the anatomy.

This has been corrected

Figure 1 is nice and clear, and well summarizes the experimental design.

Thank you.

Figure 2 shows an example of the extent of the ChR2-YFP expression and the position of an optical fiber tip above the dopaminergic A13 region from a mouse. Without any quantification, these images could be included in Figure 1. Despite a very small volume (36.8nL) of AAV, the extent of ChR2-YFP expression is quite large and includes dopaminergic and unidentified neurons within the A13 region but also a large population of unidentified neurons outside of it, thus raising questions about the volume and the types of neurons recruited.

This is an important consideration. The issue of viral spread is complex and depends on factors including tissue type, serotype, and promotor of the virus. Li et al. (2021), for example, used different virus serotypes and promotors, injecting 150nL, whereas we used AAV DJ, injecting 36.8nL. AAV-DJ is a hybrid viral type consisting of multiple serotypes. It has a high transduction efficiency, which leads to greater gene delivery than single-serotype AAV viral constructs (Mao et al. 2016). A secondary consideration regarding translation was that AAV-DJ could effectively transduce non-primate neurons (Watanabe et al. 2020). We have addressed the issue of neurons recruited earlier, provided c-Fos quantification, and provided a new supplementary figure showing viral spread (Figure S1).

Anatomical reconstruction of the extent of the ChR2-YFP expression and the location of the tip of the optical fiber will be necessary to confirm that ChR2-YFP expression was restricted to the A13 region.

We will provide additional information regarding viral spread, ferrule tip placement, and c-fos cell counts. This has been done in Figure 2 and we also present a new Figure S1 where we have quantified the viral spread.

Page 5, 1st para: Double-check the references, as not all of them are 6-OHDA injections in the MLF.

Corrected. Removed Kiehn reference.

Page 5, 1st para, 4th line: Replace ferrule with optical canula or fiber.

Done

Page 5, 1st para, 9th line: Replace Figure 2 with Figure 3.

Done

Page 5, 2nd para: About the refractory decrease in traveled distance by sham-ChR2 mice: is this significant?

It was not significant (Figure S1C, 1-way RM ANOVA: F5,25 = 0.486, P \= 0.783). This has been updated in the text.

Figure 3 showing behavioral assessments is nice, but the stats are not always clear. In Fig 3A, are each of the off and on boxes 1 minute long? The figure legend states the test lasts 1 min, but isn't it 4 minutes? In Figure 3B-E and 3J-M, what are the differences? Do the stats identify a significant difference only during the stimulation phase? Fig. 3F-I are nice and could have been presented as primary examples prior to data analysis in Fig. 3B-E. Group labels above the graph would help.

Yes, the off-on boxes are 1 minute long. The error is corrected in the legend. Great suggestion for F-I - they have been moved ahead of the summary figures. We have also updated new Fig 3F-,I, J, L, M) to make the differences between 6-OHDA and sham graphs easier to visualize. The stats do indicate a significant difference during the stimulation phase. We have added group labels, and reorganized the figure, and it is much easier to read now.

Fig. 3L-M, what do PreSur, Post, and Ferrule mean? I assume that Ferrule refers to mice tested with the optical fiber without stimulation, whereas Stim. refers to the stimulation. It would be helpful to standardize the format of stats in Fig. 3B-E and 3-J-M. What are time points a, b, and c referring to?

We have renamed the figure names to be more intuitive. We have standardized the presentation of statistics in the figure, and eliminated the a,b,c nomenclature. We have also updated the caption to provide descriptions of the tests in Fig 3 L-M.

Figure S2A: the higher variability in 6-OHDA-YFP mice in comparison to 6-OHDA-ChR2 mice prior to stimulation suggests that 6-OHDA-YFP mice were less impaired. Why use boxplots only for these data? Would a pairwise comparison be more appropriate?

We have removed these plots from Figure S2. We now present the Baseline to Pre values across the experimental timespan to illustrate the fact that distance travelled returned to baseline values for all trials conducted.

Fig. S2B: add the statistical marker.

We have removed this from Figure S2.

Page 7, para 1, line 8: to add "in comparison to 6-OHDA-YFP and YFP mice" to during photostimulation... (Figure 3E).

Done

Page 7, para 3, line 5: about larger improvement, replace "sham ChR2" with "6-OHDA."

Done

Page 8, para 1, line 4: Perier et al., 2000 reported that 6-OHDA injection increased the firing frequency of the ZI over a month.

Added the timeframe to this sentence.

Page 8, para 2, line 1: Since the results were expected, add some references.

Done.

Page 8, para 3, line 4. Double-check the reference.

Corrected.

Page 8: About large-scale changes in the A13 region, the relevance of correlation matrices is difficult to grasp. Analysis of local connectivity would have been more informative in the context of GABAergic and glutamatergic neurons of the ZI in the vicinity of the A13 region.

We have updated the figures for connectivity throughout the manuscript. Overall, there are new Figures 4 and 5 in the main text. We also provide a revised Supplementary Figure 8. Unfortunately, we could not do that experiment regarding local connectivity. In light of our new work (Sharma et al. 2024), it is clear that this will be critical going forward.

Page 8, para 3, line: given Fig. 2, there is concern about the claim that only the A13 region was targeted. The time of the analysis after 6-OHDA should be mentioned. Some sections of the paragraph could be moved to methods.

We have provided more information about the viral spread in the text and Supplementary Figure 1. The functional and anatomical experiments are separate, which we realize caused confusion. We have mentioned analysis time after 6-OHDA and inserted this into the text.

Fig. 4: The color code helps the reader visualize distribution differences. However, statistical analyses comparing 6-OHDA versus sham should be included. Quantification per region would greatly help readers visualize the data and support the conclusion. The relationship between the type of correlation (positive or negative) and absolute change (increase or decrease) is unknown in the current format, which limits the interpretation of the data. Moreover, examples of raw images of axons and cells should be presented for several brain regions. The experimental design with a timeline, as in Fig. 1, would be helpful. The legend for Fig. 4 is a bit long. Some sections are very descriptive, whereas others are more interpretive.

We have provided a new Figure 5 where we present quantification per region, and the correlation matrices have been updated in Figure 4. We have also focused on motor regions as mentioned earlier. We also provide examples of raw regions in Supplementary Figure 8. Raw values are shared on our data repository.

Page 10, para 1, line 1: add "afferent" to "changes in -afferent and- projection patterns."

Done

Page 10, para 1, line 9: remove the 2nd "compared to sham" in the sentence.

Done

Page 10, para 1, line 10: remove "coordinated" in "several regions showed a coordinated reduction in afferent density." We cannot say anything about the timing of events, as there is only info at 1 month.

Done

Page 10, para 2: the section should be written in the past tense.

Done

Page 13, para 2, the last sentence is overstated. Please remove "cells" and refer to the A13 region instead.

Done

About differential remodelling of the A13 region connectome: Figure 5C and 5G: The proportion of total afferents ipsi- and contralateral to 6-OHDA injection argues that the A13 region primarily receives inputs from the cortical plate and the striatum. Unfortunately, there are no statistics.

Due to the small sample size, we provided descriptive statistics (mean and error bars) in Figure 5A. As mentioned in comments for Reviewers 1 and 2, we have revised Figure 5 to present data focusing on motor-related pathways to provide clarity. In addition, absolute values are shared on our data repository.

Figure 5 D and 5H: Changes in the proportion of total afferents/projections are relatively modest (less than 10% of the whole population for the highest changes). There is no standard deviation for these data and no statistics. Do they reflect real changes or variability from the injection site?

The changes are relatively modest (less than 10%) since a small brain region usually provides a small proportion of total input (McElvain et al. 2021; Yang et al. 2022). The changes in the proportions reflect real differences between average proportions observed in sham and 6-OHDA mice. The variability in the total labelling of neurons and fibers was minimized by normalizing individual regional counts against total counts found in each animal. This figure has been updated as reviewers requested.

Fig 5F and H: The example in F shows a huge decrease in the striatum, but H indicates only a 2% change, which makes the example not very representative. Absolute values would be helpful.

While a 2% change may seem small, it represents a relatively large change in the A13 efferent connectome. To provide further clarity, we have provided absolute values as suggested in our new supplemental table.

Figure 6 is inaccurate and unnecessary.

Figure 6 has been removed.

Discussion

Although interesting, the discussion is too long.

The discussion has been reduced by about three quarters of a page.

Methods

Page 17, para 1: include the stereotaxic coordinates of the optical cannula above the A13 region.

Added.

References

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Chometton, S., K. Charrière, L. Bayer, C. Houdayer, G. Franchi, F. Poncet, D. Fellmann, and P. Y. Risold. 2017. “The Rostromedial Zona Incerta Is Involved in Attentional Processes While Adjacent LHA Responds to Arousal: C-Fos and Anatomical Evidence.” Brain Structure & Function 222 (6): 2507–25.

Garau, Celia, Jessica Hayes, Giulia Chiacchierini, James E. McCutcheon, and John Apergis-Schoute. 2023. “Involvement of A13 Dopaminergic Neurons in Prehensile Movements but Not Reward in the Rat.” Current Biology: CB, October.

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Li, Zhuoliang, Giorgio Rizzi, and Kelly R. Tan. 2021. “Zona Incerta Subpopulations Differentially Encode and Modulate Anxiety.” Science Advances 7 (37): eabf6709.

Mao, Yingying, Xuejun Wang, Renhe Yan, Wei Hu, Andrew Li, Shengqi Wang, and Hongwei Li. 2016. “Single Point Mutation in Adeno-Associated Viral Vectors -DJ Capsid Leads to Improvement for Gene Delivery in Vivo.” BMC Biotechnology 16 (January):1.

McElvain, Lauren E., Yuncong Chen, Jeffrey D. Moore, G. Stefano Brigidi, Brenda L. Bloodgood, Byung Kook Lim, Rui M. Costa, and David Kleinfeld. 2021. “Specific Populations of Basal Ganglia Output Neurons Target Distinct Brain Stem Areas While Collateralizing throughout the Diencephalon.” Neuron 109 (10): 1721–38.e4.

Mitrofanis, J. 2005. “Some Certainty for the ‘Zone of Uncertainty’? Exploring the Function of the Zona Incerta.” Neuroscience 130 (1): 1–15.

Monosov, Ilya E., Takaya Ogasawara, Suzanne N. Haber, J. Alexander Heimel, and Mehran Ahmadlou. 2022. “The Zona Incerta in Control of Novelty Seeking and Investigation across Species.” Current Opinion in Neurobiology 77 (December):102650.

Negishi, Kenichiro, Mikayla A. Payant, Kayla S. Schumacker, Gabor Wittmann, Rebecca M.  Butler, Ronald M. Lechan, Harry W. M. Steinbusch, Arshad M. Khan, and Melissa J. Chee. 2020. “Distributions of Hypothalamic Neuron Populations Coexpressing Tyrosine Hydroxylase and the Vesicular GABA Transporter in the Mouse.” The Journal of Comparative Neurology 528 (11): 1833–55.

Ossowska, Krystyna. 2019. “Zona Incerta as a Therapeutic Target in Parkinson’s Disease.” Journal of Neurology. https://doi.org/ 10.1007/s00415-019-09486-8.

Romanov, Roman A., Amit Zeisel, Joanne Bakker, Fatima Girach, Arash Hellysaz, Raju Tomer, Alán Alpár, et al. 2017. “Molecular Interrogation of Hypothalamic Organization Reveals Distinct Dopamine Neuronal Subtypes.” Nature Neuroscience 20 (2): 176–88.

Sharma, Sandeep, Cecilia A. Badenhorst, Donovan M. Ashby, Stephanie A. Di Vito, Michelle A. Tran, Zahra Ghavasieh, Gurleen K. Grewal, Cole R. Belway, Alexander McGirr, and Patrick J. Whelan. 2024. “Inhibitory Medial Zona Incerta Pathway Drives Exploratory Behavior by Inhibiting Glutamatergic Cuneiform Neurons.” Nature Communications 15 (1): 1160.

Spix, Teresa A., Shruti Nanivadekar, Noelle Toong, Irene M. Kaplow, Brian R. Isett, Yazel  Goksen, Andreas R. Pfenning, and Aryn H. Gittis. 2021. “Population-Specific Neuromodulation Prolongs Therapeutic Benefits of Deep Brain Stimulation.” Science 374 (6564): 201–6.

Wang, Xiyue, Xiaolin Chou, Bo Peng, Li Shen, Junxiang J. Huang, Li I. Zhang, and Huizhong W. Tao. 2019. “A Cross-Modality Enhancement of Defensive Flight via Parvalbumin Neurons in Zona Incerta.” eLife 8 (April). https://doi.org/ 10.7554/eLife.42728.

Wang, Xiyue, Xiao-Lin Chou, Li I. Zhang, and Huizhong Whit Tao. 2020. “Zona Incerta: An Integrative Node for Global Behavioral Modulation.” Trends in Neurosciences 43 (2): 82–87.

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Zhao, Zheng-Dong, Zongming Chen, Xinkuan Xiang, Mengna Hu, Hengchang Xie, Xiaoning Jia, Fang Cai, et al. 2019. “Zona Incerta GABAergic Neurons Integrate Prey-Related Sensory Signals and Induce an Appetitive Drive to Promote Hunting.” Nature Neuroscience 22 (6): 921–32.

Author response:

The following is the authors’ response to the previous reviews

Public Reviews:

Reviewer #2 (Public review):

Summary

In this extensive comparative study, Moreno-Borrallo and colleagues examine the relationships between plasma glucose levels, albumin glycation levels, diet and lifehistory traits across birds. Their results confirmed the expected positive relationship between plasma blood glucose level and albumin glycation rate but also provided findings that are somewhat surprising or contrast with findings of some previous studies (positive relationships between blood glucose and lifespan, or absent relationships between blood glucose and clutch mass or diet). This is the first extensive comparative analysis of glycation rates and their relationships to plasma glucose levels and life history traits in birds that is based on data collected in a single study, with blood glucose and glycation measured using unified analytical methods (except for blood glucose data for 13 species collected from a database).

Strengths

This is an emerging topic gaining momentum in evolutionary physiology, which makes this study a timely, novel and important contribution. The study is based on a novel data set collected by the authors from 88 bird species (67 in captivity, 21 in the wild) of 22 orders, except for 13 species, for which data were collected from a database of veterinary and animal care records of zoo animals (ZIMS). This novel data set itself greatly contributes to the pool of available data on avian glycemia, as previous comparative studies either extracted data from various studies or a ZIMS database (therefore potentially containing much more noise due to different methodologies or other unstandardised factors), or only collected data from a single order, namely Passeriformes. The data further represents the first comparative avian data set on albumin glycation obtained using a unified methodology. The authors used LC-MS to determine glycation levels, which does not have problems with specificity and sensitivity that may occur with assays used in previous studies. The data analysis is thorough, and the conclusions are substantiated. Overall, this is an important study representing a substantial contribution to the emerging field evolutionary physiology focused on ecology and evolution of blood/plasma glucose levels and resistance to glycation.

Weaknesses

Unfortunately, the authors did not record handling time (i.e., time elapsed between capture and blood sampling), which may be an important source of noise because handling-stress-induced increase in blood glucose has previously been reported. Moreover, the authors themselves demonstrate that handling stress increases variance in blood glucose levels. Both effects (elevated mean and variance) are evident in Figure ESM1.2. However, this likely makes their significant findings regarding glucose levels and their associations with lifespan or glycation rate more conservative, as highlighted by the authors.

Recommendations for the authors:

Reviewer #2 (Recommendations for the authors):

I understand that your main objective regarding glycation rate and lifespan, was to analyse the species resistance to glycation with respect to lifespan, while factoring out the species-specific variation in blood glucose level. However, I still believe that the absolute glycation level (i.e., not controlled for blood glucose level) may also be important for the evolution of lifespan. Given that blood glucose is positively related to both glycation and lifespan (although with a plateau in the latter case), lifespan could possibly be positively correlated with absolute glycation levels. If significant, that would be an interesting and counterintuitive finding, which would call for an explanation, thereby potentially stimulating further research. If not significant, it would show that long-lived species do not have higher glycation levels, despite having higher blood glucose levels, thereby strengthening your argument about higher resistance of longlived species to glycation. So, in my opinion, the inclusion of an additional model of glycation level on life-history traits, without controlling for blood glucose, is worth considering.

We include now this model as supplementary material, indicating it in several parts of the text, including some of these issues we discussed here.

Lines 230-231: Please, provide a citation for these GVIF thresholds

We include it now.

Figure 3: I think that showing both glucose and glycation rate on the linear scale, rather than log scale, would better illustrate your conclusion - the slowing rise of glycation rate with increasing glucose levels.

That is a good point, although it may also be confusing for readers to see a graph that represents the data in a different way as the models. Maybe showing both graphs (as 3.A and 3.B) can solve it?

Figure 4. I recommend stating in the caption that the whiskers do not represent interquartile ranges (a standard option in box plots) but credible intervals as mentioned in the current version of the public author response.

Sorry about that, it was missed. Now it is included. Nevertheless, interquartile ranges from the posterior distributions can still be observed here represented with the boxes. Then the whiskers are the credible intervals.

Author response:

The following is the authors’ response to the original reviews

Public Reviews:

Reviewer #1 (Public review):

Summary:

In this manuscript, Guo and colleagues used a cell rounding assay to screen a library of compounds for inhibition of TcdB, an important toxin produced by Clostridioides difficile. Caffeic acid and derivatives were identified as promising leads, and caffeic acid phenethyl ester (CAPE) was further investigated.

Strengths:

Considering the high morbidity rate associated with C. difficile infections (CDI), this manuscript presents valuable research in the investigation of novel therapeutics to combat this pressing issue. Given the rising antibiotic resistance in CDI, the significance of this work is particularly noteworthy. The authors employed a robust set of methods and confirmatory tests, which strengthened the validity of the findings. The explanations provided are clear, and the scientific rationale behind the results is well-articulated. The manuscript is extremely well-written and organized. There is a clear flow in the description of the experiments performed. Also, the authors have investigated the effects of CAPE on TcdB in careful detail and reported compelling evidence that this is a meaningful and potentially useful metabolite for further studies.

Weaknesses:

This is really a manuscript about CAPE, not caffeic acid, and the title should reflect that. Also, a few details are missing from the description of the experiments. The authors should carefully revise the manuscript to ascertain that all details that could affect the interpretation of their results are presented clearly. Just as an example, the authors state in the results section that TcdB was incubated with compounds and then added to cells. Was there a wash step in between? Could compound carryover affect how the cells reacted independently from TcdB? This is just an example of how the authors should be careful with descriptions of their experimental procedures. Lastly, authors should be careful when drawing conclusions from the analysis of microbiota composition data. Ascribing causality to correlational relationships is a recurring issue in the microbiome field. Therefore, I suggest authors carefully revise the manuscript and tone down some statements about the impact of CAPE treatment on the gut microbiota.

Thanks for your constructive suggestion. We have carefully revised the manuscript, including the description of title, results and methods sections.

Reviewer #2 (Public review):

Summary:

This work is towards the development of nonantibiotic treatment for C. difficile. The authors screened a chemical library for activity against the C. difficile toxin TcdB, and found a group of compounds with antitoxin activity. Caffeic acid derivatives were highly represented within this group of antitoxin compounds, and the remaining portion of this work involves defining the mechanism of action of caffeic acid phenethyl ester (CAPE) and testing CAPE in mouse C. difficile infection model. The authors conclude CAPE attenuates C. difficile disease by limiting toxin activity and increasing microbial diversity during C. difficile infection.

Strengths/ Weaknesses:

The strategy employed by the authors is sound although not necessarily novel. A compound that can target multiple steps in the pathogenies of C. difficile would be an exciting finding. However, the data presented does not convincingly demonstrate that CAPE attenuates C. difficile disease and the mechanism of action of CAPE is not convincingly defined. The following points highlight the rationale for my evaluation.

(1) The toxin exposure in tissue culture seems brief (Figure 1). Do longer incubation times between the toxin and cells still show CAPE prevents toxin activity?

Thanks for your comments. The cytotoxicity assay was employed to directly assess the protective capacity of CAPE against cell death induced by TcdB. Our observations at 1 and 12 h post-TcdB exposure revealed that CAPE effectively mitigated the toxic effects of the TcdB at both time points, demonstrating its potent protective role. Please see Figure S1.

(2) The conclusion that CAPE has antitoxin activity during infection would be strengthened if the mouse was pretreated with CAPE before toxin injections (Figure 1D).

Thanks for your constructive comments. According to your suggestion, we administered TcdB 2 h after pretreatment with CAPE. The outcomes demonstrated that CAPE pretreatment significantly enhanced the survival rate of the intoxicated mice, confirming that CAPE retains its antitoxin efficacy during the infection process. Please see Figure S2.

(3) CAPE does not bind to TcdB with high affinity as shown by SPR (Figure 4). A higher affinity may be necessary to inhibit TcdB during infection. The GTD binds with millimolar affinity and does not show saturable binding. Is the GTD the binding site for CAPE? Auto processing is also affected by CAPE indicating CAPE is binding non-GTD sites on TcdB.

Thanks for your comments. Our findings indicate that the GTD domain is a critical binding site for CAPE. CAPE exerts its protective effects at multiple stages of TcdB-mediated cell death, including inhibiting TcdB's self-cleavage and blocking the activity of GTD, thereby preventing the glycosylation modification of Rac1 by TcdB.

(4) In the infection model, CAPE does not statistically significantly attenuate weight loss during C. difficile infection (Figure 6). I recognize that weight loss is an indirect measure of C. difficile disease but histopathology also does not show substantial disease alleviation (see below).

Thanks for your comments. Our comparative analysis revealed a notable distinction in the body weight of mice on the third day post-infection (Figure 6B). Similarly, the dry/wet stool ratio exhibited a comparable pattern, suggesting that treatment with phenethyl caffeic acid ameliorated Clostridium difficile-induced diarrhea to a significant degree (Figure 6C).

(5) In the infection model (Figure 6), the histopathology analysis shows substantial improvement in edema but limited improvement in cellular infiltration and epithelial damage. Histopathology is probably the most critical parameter in this model and a compound with disease-modifying effects should provide substantial improvements.

Thanks for your comments. Edema, inflammatory factor infiltration, and epithelial damage served as key evaluation metrics. Statistical analysis revealed that the pathological scores of mice treated with CAPE were markedly reduced compared to those in the model group (Figure 6F).

(6) The reduction in C. difficile colonization is interesting. It is unclear if this is due to antitoxin activity and/or due to CAPE modifying the gut microbiota and metabolites (Figure 6). To interpret these data, a control is needed that has CAPE treatment without C. difficile infection or infection with an atoxicogenic strain.

The observed reduction in C. difficile fecal colonization following drug treatment may be attributed to the CAPE's antitoxin properties or its capacity to modify the intestinal microbiota and metabolites. These two mechanisms likely work in tandem to combat CDI. CDI is primarily triggered by the toxins A (TcdA) and B (TcdB) secreted by the bacterium. Certain therapies, including monoclonal antibodies like bezlotoxumab, target CDI by neutralizing these toxins, thereby mitigating gut damage and subsequent C. difficile colonization(1,2). The establishment of C. difficile in the gut is intricately linked to the equilibrium of the intestinal microbiota. Although antibiotic treatments can inhibit C. difficile growth, they may also disrupt the microbial balance, potentially facilitating the overgrowth of other pathogens. Consequently, interventions such as fecal microbiota transplantation (FMT) are designed to reestablish gut flora balance and consequently decrease C. difficile colonization(3,4). Moreover, the administration of probiotics and prebiotics is considered to reduce C. difficile colonization by modifying the gut environment(5,6).

(7) Similar to the CAPE data, the melatonin data does not display potent antitoxin activity and the mouse model experiment shows marginal improvement in the histopathological analysis (Figure 9). Using 100 µg/ml of melatonin (~ 400 micromolar) to inactivate TcdB in cell culture seems high. Can that level be achieved in the gut?

The uptake and dissemination of melatonin within the body varies with the dose administered. For instance, in rats, the bioavailability of melatonin following administration was found to be 53.5%, whereas in dogs, bioavailability was nearly complete (100%) at a dose of 10 mg/kg, yet it decreased to 16.9% at a lower dose of 1 mg/kg(7). This data suggests that the absorption of melatonin differs across various animal species and is influenced by the dose administered. Moreover, it underscores the higher potential bioavailability of melatonin, implying that a dose of 200 mg/kg should be adequate to achieve the desired concentration in the body post-administration.

(8) The following parameters should be considered and would aid in the interpretation of this work. Does CAPE directly affect the growth of C. difficile? Does CAPE affect the secretion of TcdB from C. difficile? Does CAPE alter the sporulation and germination of C. diffcile?

We incorporated CAPE into the MIC assay for detecting C. difficile, as well as for assessing the sporulation capacity of C. difficile and evaluating the secretion level of TcdB. The findings revealed that CAPE markedly repressed tcdB transcription at a concentration of 16 μg/mL and effectively suppressed the growth and sporulation of C. difficile BAA-1870 at a concentration of 32 μg/mL. Please see Figure S3.

References:

(1) Skinner AM, et al. Efficacy of bezlotoxumab to prevent recurrent Clostridioides difficile infection (CDI) in patients with multiple prior recurrent CDI. Anaerobe. 2023 Dec; 84: 102788.

(2) Wilcox MH, et al. Bezlotoxumab for Prevention of Recurrent Clostridium difficile Infection. N Engl J Med. 2017 Jan 26;376(4):305-317.

(3) Khoruts A, Sadowsky MJ. Understanding the mechanisms of faecal microbiota transplantation. Nat Rev Gastroenterol Hepatol. 2016 Sep;13(9):508-16.

(4) Khoruts A, Staley C, Sadowsky MJ. Faecal microbiota transplantation for Clostridioides difficile: mechanisms and pharmacology. Nat Rev Gastroenterol Hepatol. 2021 Jan;18(1):67-80.

(5) Mills JP, Rao K, Young VB. Probiotics for prevention of Clostridium difficile infection. Curr Opin Gastroenterol. 2018 Jan;34(1):3-10.

(6) Lau CS, Chamberlain RS. Probiotics are effective at preventing Clostridium difficile-associated diarrhea: a systematic review and meta-analysis. Int J Gen Med. 2016 Feb 22; 9:27-37.

(7) Yeleswaram K, et al. Pharmacokinetics and oral bioavailability of exogenous melatonin in preclinical animal models and clinical implications. J Pineal Res. 1997 Jan;22(1):45-51.

Reviewer #3 (Public review):

Summary:

The study is well written, and the results are solid and well demonstrated. It shows a field that can be explored for the treatment of CDI.

Strengths:

The results are really good, and the CAPE shows a good and promising alternative for treating CDI. The methodology and results are well presented, with tables and figures that corroborate them. It is solid work and very promising.

Weaknesses:

Some references are too old or missing.

Thanks for your constructive suggestion. We have included and refreshed several references to enhance the manuscript.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

While the manuscript convincingly demonstrates that CAPE affects the TcdB toxin and reduces its toxicity in vitro, it would be beneficial to include data on the effect of CAPE on the growth of C. difficile. This would help ensure that the observed in vivo effects are not merely due to reduced bacterial growth but rather due to the specific action of CAPE on the toxin.

Thanks for your constructive suggestion. We have augmented our findings with the impact of CAPE on the bacteria themselves, revealing that CAPE not only hampers the growth of the bacterial cells but also suppresses their capacity to produce spores. Please see Figure S3.

(1) Line 41, line 115 - authors should clarify what they mean when mentioning Bacteroides within parentheses.

Thanks for your comments. We have completed the corresponding modifications according to the suggestions.

(2) Line 71 - Is C. difficile really found "in the environment"?

Thanks for your comments. C. difficile is prevalent across various natural settings, including soil and water ecosystems. A study has identified highly diverse strains of this bacterium within environmental samples(1). Moreover, the significant presence of C. difficile in soil and lawn specimens collected near Australian hospitals indicates that the organism is indeed a common inhabitant in the environment(2).

(3) Lines 128-130 - Was there a wash step here? What could be the impact of compound carryover in this experiment?

Thanks for your comments. Following pre-incubation of TcdB with CAPE, remove the compounds that have not bound to TcdB through centrifugation. The persistence of the compound in the culture post-washing could result in an inflated assessment of its efficacy, particularly if it continues to engage with TcdB or the cells beyond the initial 1-hour pre-incubation window. The carryover of the compound might also give rise to misleading positive results, where the compound seems to confer protection or inhibition against TcdB-mediated cell rounding, whereas such effects are actually due to the lingering activity of the compound. This carryover could skew the determination of the compound's minimum effective concentration, as the effective concentration interacting with the cells might be inadvertently elevated. Furthermore, if the compounds possess cytotoxic properties or impact cell viability, carryover could generate artifacts in cell morphology that are unrelated to the direct interaction between TcdB and the compounds.

(4) Lines 133-134 - I suggest authors mention how many caffeic acid derivatives there were in the entire library so that the suggested "enrichment" of them in the group of bioactive compounds can be better judged.

Thanks for your comments. The natural compound library contained eight caffeic acid derivatives, of which methyl caffeic acid and ferulic acid displayed no efficacy. This information has been incorporated into the manuscript.

(5) Line 135 - I recommend the authors add the molarity of the compound solutions used.

Thanks for your comments. We have completed the corresponding modifications according to the suggestions.

(6) Line 247 - I think the term "CAPE mice" is confusing. Please use a full description.

Thanks for your comments. We have completed the corresponding modifications according to the suggestions.

(7) Line 248 - I also think the terms "model mice" and "model group" are confusing. Maybe call them "control mice"?

Thanks for your comments. The terms "model mice" and "model group" are indeed synonymous, and we have subsequently clarified that control mice refer to those that have not been infected with C. difficile.

(8) Line 273 - "most abundant species at the genus level" is incorrect. I think what you mean is "most abundant TAXA".

Thanks for your comments. We have completed the corresponding modifications according to the suggestions.

(9) Line 278 - Please include your p-value cut-off together with the LDA score.

Thanks for your comments. We have revised the above description to “LDA score > 3.5, p < 0.05”.

(10) Line 292 - Details on how metabolomics was performed should be included here.

Thanks for your comments. We have completed the corresponding modifications according to the suggestions.

(11) Line 299 - 1.5 is a fairly low cut-off. The authors should at a minimum also include the p-value cut-off used.

Response: Thanks for your comments. We have revised the above description to “fold change > 1.5, p < 0.05”.

(12) Line 307 - Purine "degradation" would be better here.

Thanks for your comments. We have completed the corresponding modifications according to the suggestions.

(13) Line 328 onward - The melatonin experiment is a weird one. Although I fully understand the rationale behind testing the effect of melatonin in the mouse model, the idea that just because melatonin levels changed in the gut it would act as a direct inhibitor of TcdB was very far-fetched, even though it ended up working. Authors should explain this in the manuscript.

Thanks for your comments. Furthermore, beyond our murine studies, we have confirmed that melatonin significantly diminishes TcdB-induced cytotoxicity at the cellular level (Figure 9A). Additionally, it has been documented that melatonin, acting as an antimicrobial adjuvant and anti-inflammatory agent, can decrease the recurrence of CDI(3). Consequently, we contend that the aforementioned statement is substantiated.

(14) Lines 429-435 - There are seemingly contradictory pieces of information here. The authors state that adenosine is released from cells upon inflammation and that CAPE treatment caused an increase in adenosine levels. Later in this section, the authors state that adenosine prevents TcdA-mediated damage and inflammation. This should be clarified and better discussed.

Thanks for your comments. Adenosine modulates immune responses and inflammatory cascades by interacting with its receptors, including its capacity to suppress the secretion of specific pro-inflammatory mediators. We have updated this depiction in the manuscript.

(15) Lines 513-514 - How was this phenotype quantified?

Thanks for your comments. Initially, we introduced TcdB at a final concentration of 0.2 ng/mL along with various concentrations of compounds into 1 mL of medium for a 1-h pre-incubation period. Subsequently, unbound compounds were removed through centrifugation, and the resulting mixture was then applied to the cells.

(16) Figure 3 - panels are labeled incorrectly.

Thanks for your comments. We have completed the corresponding modifications according to the suggestions.

(17) Figure 5C - it is unclear what the different colors and labels represent.

Thanks for your comments. In the depicted graph, blue denotes the total binding energy, red signifies the electrostatic interactions, green corresponds to the van der Waals forces, and orange indicates solvation or hydration effects. The horizontal axis represents the mutation of the amino acid residue at the respective position to alanine. As illustrated in Figure 5C, the mutations W520A and GTD exhibit the highest binding energies.

References:

(1) Janezic S, et al. Highly Divergent Clostridium difficile Strains Isolated from the Environment. PLoS One. 2016 Nov 23;11(11): e0167101.

(2) Perumalsamy S, Putsathit P, Riley TV. High prevalence of Clostridium difficile in soil, mulch and lawn samples from the grounds of Western Australian hospitals. Anaerobe. 2019 Dec; 60:102065.

(3) Sutton SS, et al. Melatonin as an Antimicrobial Adjuvant and Anti-Inflammatory for the Management of Recurrent Clostridioides difficile Infection. Antibiotics (Basel). 2022 Oct 25;11(11):1472.

Reviewer #2 (Recommendations for the authors):

Minor comments and questions.

(1) Which form of TcdB is being used in these experiments?

Thanks for your comments. The TcdB proteins used in this study are TcdB1 subtypes.

(2) Why are THP-1 cells being used in these assays?

Thanks for your comments. For the purposes of this study, we employed a diverse array of cell lines, including Vero, HeLa, THP-1, Caco-2, and HEK293T. Each cell line was selected to serve a specific experimental objective. The inclusion of the THP-1 cell line was necessitated by the need to incorporate a macrophage cell line to ensure the comprehensive nature of our experiments, allowing for the testing of both epithelial cells and macrophages. C. difficile is a kind of intestinal pathogenic bacteria, and immune clearance plays a vital role in the process of pathogen infection, so THP-1 cells are used as important immune cells.

(3) Please improve the quality of the microscopy images in Figure 1.

Thanks for your comments. We have improved the quality of the microscopy images in Figure 1.

(4) Does the flow cytometry experiment in Figure 2B show internalization? Surface-bound toxins would provide the same histogram.

Thanks for your comments. Figure 2B was employed to assess the internalization of TcdB, and the findings indicate that CAPE does not influence the internalization process of TcdB.

(5) The sensogram in Figure 4A does not look typical and should be clarified.

Thanks for your comments. Typically, small molecules and proteins engage in a rapid binding and dissociation dynamic. However, as depicted in Figure 4A, the interaction between CAPE and TcdB demonstrates a gradual progression towards equilibrium. This behavior can be primarily explained by the swift occupation of the protein's primary binding sites by the small molecule in the initial stages. Subsequently, CAPE binds to secondary or lower affinity sites, extending the time needed to reach equilibrium. Additionally, the likelihood of CAPE binding to multiple sites on TcdB requires time for the exploration and occupation of these diverse locations before equilibrium is attained, we have incorporated an analysis of this potential scenario into the manuscript.

Reviewer #3 (Recommendations for the authors):

These are my suggestions for the text:

(1) Line 29: high recurrent rates.

Thanks for your comments. We have completed the corresponding modifications according to the suggestions.

(2) Line 32: Where is the caffeic acid identified? I think a line should be included.

Thanks for your comments. Caffeic acid was identified from natural compounds library and we have completed the corresponding modifications according to the suggestions.

(3) Line 39: C. difficile is not italic.

Thanks for your comments. We have completed the corresponding modifications according to the suggestions.

(4) Line 41: Bacteroides spp.

Thanks for your comments. We have completed the corresponding modifications according to the suggestions.

(5) Line 56: This number of casualties 56.000 is still happening or it was in the past?

Thanks for your comments. The mortality rates reported in the manuscript reflect a downturn in the incidence and fatality of CDI around 2017(1), as the infection gained broader recognition. Nonetheless, a recent study reveals that the mortality rate for CDI cases in Germany can soar to 45.7% within a year, with the overall economic burden amounting to approximately 1.6 billion euros. This underscores the ongoing significance of CDI as a global public health challenge(2).

(6) Line 104: Where did the idea of testing caffeic acid come from? Any previous study of the authors? Any studies with the inhibition of other pathogens?

Thanks for your comments. Initially, we conducted a screen of a compound library comprising 2,076 compounds and identified several potent inhibitors, which, upon structural analysis, were revealed to be caffeic acid derivatives. Prior to our investigation, no studies had explored the potential of CAPE in this context.

(7) Line 115: Bacteroides spp.

Thanks for your comments. We have completed the corresponding modifications according to the suggestions.

Results section

(8) Did the authors try the caffeic acid with the TcdA or binary toxin? I know this is not the purpose of the study, but TcdA toxin has a high identity structure with TcdB and generates inflammation in the gut via neutrophils. Negative strains for the major toxins and positive for the binary toxin also cause severe cases of CDI.

Thanks for your comments. Although we acknowledge the significance of TcdA and binary toxins in CDI, we did not investigate the impact of CAPE on these toxins. Our focus was exclusively on the effect of CAPE against TcdB, as it is the primary virulence factor in C. difficile pathogenesis. Since TcdA and TcdB are highly similar in structure, we will analyze the neutralization effect of CAPE on TcdA in later studies.

(9) Does caffeic acid have any effect on C. difficle? Or does it only gain the toxins? That would be ideal.

Thanks for your comments. We have included additional related assays in our study. Beyond directly neutralizing TcdB, CAPE also demonstrates the capacity to inhibit the growth and spore formation of C. difficile.

(10) Line 230: C. difficile BAA-1870 is a clinical strain? There are no details about it in the paper.

Thanks for your comments. C. difficile BAA-1870 (RT027/ST1), a highly virulent isolate frequently employed in research(3-6), was kindly donated by Professor Aiwu Wu. We have meticulously noted the PCR ribotype in our manuscript.

(11) Line 236: Did the mice fully recover from CDI after the administration of the CAPE? Was one dose enough?

Thanks for your comments. CAPE was administered orally at 24 h intervals, commencing with the initial dose on Day 0. By the time a significant difference was observed on Day 3, the treatment had been administered a total of three times.

Methodology

(12) Most of the methods do not have a reference.

Thanks for your comments. We have added several references to the methods.

Discussion section

(13) The first two paragraphs of the discussion should be summarized. Those details were already explained in the introduction.

Thanks for your comments. The discussion section and the introduction address slightly different focal points; therefore, we aim to retain the first two paragraphs to maintain continuity and context.

(14) Line 382: Bezolotoxumab was approved by the FDA in 2016. It is not recent.

Thanks for your comments. We have revised the above description.

(15) Line 410: "Despite the high 410 cure rate and increasing popularity of FMT, its safety remains controversial. Although this is true, recently (2022) the FDA approved the Rebyota, which was later cited by the authors.

Thanks for your comments. We have revised the above description.

(16) Lines 415-416: "the abundance of Bacteroides, a critical gut microbiota component that is required for C. difficile resistance". There is only one reference cited by the authors. I suppose that if it is true, more studies should be mentioned. Why are probiotics with Bacteroides spp. not available in the market?

Thanks for your comments. We have supplemented additional references. The scarcity of probiotic products containing Bacteroides spp. on the market is primarily attributable to the stringent requirements of their survival conditions. As most Bacteroides spp. are anaerobic, they thrive in oxygen-deprived environments. This unique survival trait poses challenges in maintaining their viability during product preservation and distribution, which in turn escalates production costs and complexity. Furthermore, despite the significant role of Bacteroides in gut health, research into its potential probiotic benefits and safety is comparatively underexplored.

References:

(1) Guh AY, et al. Emerging Infections Program Clostridioides difficile Infection Working Group. Trends in U.S. Burden of Clostridioides difficile Infection and Outcomes. N Engl J Med. 2020 Apr 2;382(14):1320-1330.

(2) Schley K, et al. Costs and Outcomes of Clostridioides difficile Infections in Germany: A Retrospective Health Claims Data Analysis. Infect Dis Ther. 2024 Nov 20.

(3) Saito R, et al. Hypervirulent clade 2, ribotype 019/sequence type 67 Clostridioides difficile strain from Japan. Gut Pathog. 2019 Nov 4; 11:54.

(4) Pellissery AJ, Vinayamohan PG, Venkitanarayanan K. In vitro antivirulence activity of baicalin against Clostridioides difficile. J Med Microbiol. 2020 Apr;69(4):631-639.

(5) Shao X, et al. Chemical Space Exploration around Thieno[3,2-d]pyrimidin-4(3H)-one Scaffold Led to a Novel Class of Highly Active Clostridium difficile Inhibitors. J Med Chem. 2019 Nov 14;62(21):9772-9791.

(6) Mooyottu S, Flock G, Venkitanarayanan K. Carvacrol reduces Clostridium difficile sporulation and spore outgrowth in vitro. J Med Microbiol. 2017 Aug;66(8):1229-1234.

Author response:

The following is the authors’ response to the original reviews

Public Reviews:

Reviewer #1 (Public review):

Summary

Chabukswar et al analysed endogenous retrovirus (ERV) Env variation in a set of primate genomes using consensus Env sequences from ERVs known to be present in hominoids using a Blast homology search with the aim of characterising env gene changes over time. The retrieved sequences were analysed phylogenetically, and showed that some of the integrations are LTR-env recombinants.

Strengths

The strength of the manuscript is that such an analysis has not been performed yet for the subset of ERV Env genes selected and most of the publicly available primate genomes.

Weaknesses

Unfortunately, the weaknesses of the manuscript outnumber its strengths. Especially the methods section does not contain sufficient information to appreciate or interpret the results. The results section contains methodological information that should be moved, while the presentation of the data is often substandard. For instance, the long lists of genomes in which a certain Env was found could better be shown in tables. Furthermore, there is no overview of the primate genomes Saili how did you answer to this?, or accession numbers, used. It is unclear whether the analyses, such as the phylogenetic trees, are based on nucleotide or amino acid sequences since this is not stated. tBLASTn was used in the homology searches, so one would suppose aa are retrieved. In the Discussion, both env (nt?) and Env (aa?) are used.

For the non-hominoids, genome assembly of publicly available sequences is not always optimal, and this may require Blasting a second genome from a species. Which should for instance be done for the HML2 sequences found in the Saimiri boliviensis genome, but not in the related Callithrix jacchus genome. Finally, the authors propose to analyse recombination in Env sequences but only retrieve env-LTR recombinant Envs, which should likely not have passed the quality check.

Since the Methods section does not contain sufficient information to understand or reproduce the results, while the Results are described in a messy way, it is unclear whether or not the aims have been achieved. I believe not, as characterisation of env gene changes over time is only shown for a few aberrant integrations containing part of the LTR in the env ORF.

We thank the reviewer for the critiques of the manuscript and their constructive suggestions to improve the clarity, methodological rigor, and data presentation.

(1) The concern regarding the insufficient data in the methods has been resolved in the revised manuscript by adding a supplementary file that contains the genome assemblies that  were used to perform the tBLAStn analysis using the reconstructed Env sequences. The requested accession numbers are available for all sequences in the supplementary phylogenetic figures.

(2) We have also modified the manuscript by moving a portion of the results section in the methods section, in particular all the methodological description of the reconstruction of Env part (Line 197-231).

(3) As suggested, the long list of genomes mentioned in the results section in which the Env tBLASTn hits were obtained are now provided in the table form (Table 2) as an overall summary of the distribution of ERV Env in the genomes and the genome assemblies are mentioned in Supplementary file 2.

(4) As for the point regarding the tBLASTn usage in the homology searches, we first performed tBLASTn analysis using the reconstructed Env amino acid sequences as query and performed tBLASTn similarity search in the primate genomes. The tBLASTn algorithm uses the amino acid sequences to compare with the translated nucleotide database in all six frames and hence the hits obtained are nucleotide sequences (Line 381-383). These nt sequences were used for all the further analysis such as sequence alignment, phylogenetic analysis and recombination analysis. For better clarity, we have specified the use of env nt alignments in the methods section to avoid the raised confusion in the discussion.

(5) For the HML supergroup characterization in squirrel monkey genome (Saimiri boliviensis), we used the tBLASTn hits obtained in the S. boliviensis from the initial analysis to perform the comparative genomics in two Platyrrhini genomes available on UCSC Genome browser. In particular, this analysis was performed to confirm the presence of specific members of HML supergroup in squirrel monkey genomes that has not been previously reported. We used the available genome assemblies because of the annotations available on Genome browser, and especially the possibility to use the repeatmasker tracks and the comparative genomics tools in order to use the human genome as a reference. We reported the coordinates for the members of HML supergroup that were retrieved through the comparative genomic assemblies by applying the repeat masker custom track, that have many ERVS that are not present in NCBI reference genomes.

(6) The concern regarding only retrieving env-LTR recombinant Envs has been addressed in the revised results section (Lines 747-758). As also mentioned in the methods section, the RDP software detects the recombinant sequences and a breakpoint position for the recombinant signals and hence we confirmed only those sequences that were predicted as potential recombinant sequences by the RDP software through comparative genomics. All the sequences predicted by the software were env-LTR recombinant and hence we confirmed and reported only those recombinant sequences in the manuscript.

Reviewer #1 (Recommendations for the authors):

The paper could be strengthened by:

- a rigorous rewriting and shortening of the manuscript, thereby eliminating all textbook-like paragraphs, and all biological misinterpretations and confusions. Distinguish between retroviral replication as an exogenous virus, and host genome remodeling affecting ERVs. Rewrite the sections on template switching by RT being the basis for the observed recombinations, while host genome recombinations are far more likely. ERVs with such aberrant env/LTR gene recombination are unlikely to be fit for cross-species transmission. Likely, such a recombinant was generated in a common ancestor. Also, host RNA polymerase II transcribes retroviral RNA (line 79), not RT.

- check lines 89-90 as pro is part of the pol gene in gamma- and lentiviruses.

We thank the reviewer for the suggestion, we have revised the manuscript by shortening the introduction section and eliminating the textbook like paragraphs and also clarifying the recombination mechanism. We have revised the introduction section at Lines 102-111, and the clarification for the recombination mechanism is provided at lines 1668-1675

- adding much more information to the Methods section. Such as which genomes were searched, were nt or aa have been retrieved and analysed, were multiple genomes of a species searched, a list of databases used ('various databases' in line 164 does not suffice), etc.

We thank the reviewer for the observation. As mentioned above, in the revised manuscript we have provided more detailed methods by including a supplementary file for the genome assemblies used for tBLASTn analysis and comparative genomics. For the sequence alignment, phylogenetic analysis and recombination analysis we used nt sequences, as it is also mentioned in the revised version. Lastly, all the databases that were used and are mentioned in the methods section.

- more information is needed on the alignments and phylogenetic trees. For instance, how were indels treated? How long were the alignments on average regarding informative sites?

We thank the reviewer for the questions, to answer them we have added a paragraph (Lines 359-362) describing the reconstruction process in more details.

- confirm the findings about the presence or absence of an ERV, such as for the squirrel monkey genome, using additional genomes of the species

As mentioned above, we only used the genome assemblies available on the genome browser because of the annotations available on Genome browser, blasting the second NCBI RefSeq genome using the BLAST algorithm does not provide accurate information and annotations compared to that of Genome browser and hence we reported the coordinates for the members of HML supergroup that were retrieved through the comparative genomic assemblies by applying the repeat masker custom track, that have many ERVS that are not present in NCBI reference genomes.

- present the lists of findings in primate genomes on pages 9 and 10 in tables

We thank the reviewer for the suggestion, we have provided a new table (Table 2) in the revised version summarizing the ERV Env distribution results.

- a significant limitation of the study is that only env ERVs found in hominoids have been searched in OWM and NWM, not ones specific for monkeys. This should be mentioned somewhere.

As the reviewer pointed out, the study was designed to explore ERVs’ Env  sequences in hominoids which were then searched in the OWM and NWM genomes, this is now better stated in the introduction at Lines 57-60.

- define abbreviations at first use (e.g. HML in abstract)

We thank the reviewer for the suggestion, we have mentioned the abbreviations in the abstract, where we mentioned HML first (Line 65)

- explain 'pathological domestication' (line 42). Domestication implies usefulness to the host. And over time, deleterious insertions would have been likely purged from a population.

We thank the reviewer for the observation, we have modified the sentence and provided a clearer explanation for the pathological and physiological consequences of ERVs’ env (lines 52-57).

Furthermore:

- why begin the discussion with a lengthy description of domestication and syncytins, which is not part of the current study?

We thank the reviewer for the critique. Accordingly, we have now modified the discussion section by shortening the part about domestication of syncytins, and just mentioned them as an example at lines 942-944.

- how can 96 hits have been retrieved for spuma-like envs (line 506), while it was earlier reported (line 333), that the most hits were gamma-like?

We thank the reviewer for the observation, we have clarified and explained how 96 hits have been retrieved for spuma-like envs in lines 670-677 of the discussion section.

English grammar should be improved throughout the manuscript.

And I could not open half of the supplementary files

As suggested we have revised English and checked that all files were correctly open.

Reviewer #2 (Public Review):

Summary:

The manuscript by Chabukswar et al. describes a comprehensive attempt to identify and describe the diversity of retroviral envelope (env) gene sequences present in primate genomes in the form of ancient endogenous retrovirus (ERV) sequences.

Strengths:

The focus on env can be justified because of the role the Env proteins likely played in determining viral tropism and host range of the viruses that gave rise to the ERV insertions, and to a lesser extent, because of the potential for env ORFs to be coopted for cellular functions (in the rare cases where the ORF is still intact and capable of encoding a functional Env protein). In particular, these analyses can reveal the potential roles of recombination in giving rise to novel combinations of env sequences. The authors began by compiling env sequences from the human genome (from human endogenous retrovirus loci, or "HERVs") to build consensus Env protein sequences, and then they use these as queries to screen other primate genomes for group-specific envs by tBLASTn. The "groups" referred to here are previously described, as unofficial classifications of endogenous retrovirus sequences into three very broad categories - Class I, Class II and Class III. These are not yet formally recognized in retroviral taxonomy, but they each comprise representatives of multiple genera, and so would fall somewhere between the Family and Genus levels. The retrieved sequences are subject to various analyses, most notably they are screened for evidence of recombination. The recombinant forms appear to include cases that were probably viral dead-ends (i.e. inactivating the env gene) even if they were propagated in the germline.

The availability of the consensus sequences (supplement) is also potentially useful to others working in this area.

Weaknesses:

The weaknesses are largely in presentation. Discussions of ERVs are always complicated by the lack of a formal and consistent nomenclature and the confusion between ERVs as loci and ERVs as indirect information about the viruses that produced them. For this reason, additional attention needs to be paid to precise wording in the text and/or the use of illustrative figures.

We thank the reviewer for the general observation. We put additional attention to the wording in text/figures, and hope to have improved the manuscript clarity.

Reviewer #2 (Recommendations for the authors):

Reviewing the manuscript was a challenge because figures were difficult to read. As provided, the fonts were sometimes too small to read in a standard layout and had to be expanded on screen.

The tree in Figure 3 could also be made easier to read, for example if the authors collapsed related branches and gave the clusters a single, clear label (this is not necessary, just a suggestion) - especially if the supplementary trees have all the labelled branches for any readers who want specific details.

I also recommend asking a third party (perhaps a scientific colleague) with fluency in English grammar and familiarity with English scientific idiom to provide some editorial feedback on the text.

Figure 4 legend is confusing. From the description it sounds like the tree in 4B is a host phylogeny, but it's not clearly stated. And if so, how was the tree generated? Is it based on entire genomes? Include at least enough methodological detail or citations that someone could recreate it, if necessary. The details and how it was done should be briefly mentioned here and in detail in the Methods section.

We thank the reviewer for the observation. As for Figure 4 we have modified its legend and more clearly stated how the phylogenetic tree of the primate genomes was generated using TimeTree. We have also provided further details in the methods section (Lines 475-489).

As suggested we have revised English.

Line 42 - what is "pathological domestication"? It sounds like a contradiction in terms.

We thank the reviewer for the observation. We have modifies the sentence and provided clearer explanation for the pathological and physiological consequences of ERVs’ env (lines 52-57).

Lines 166-167 - the authors use the word "classes" but then use a list of terms that correspond to genera within the Retroviridae. The authors should be cautious here, as "class" and "genus" are both official taxonomic terms with different meanings. Do they mean genus? Or, if a more informal term is needed, perhaps "group"?

Thank you for the observation, the ERVs have been classified into three classes (Class I, II and III) based on the relatedness to the exogenous retroviruses Gammaretrovirus, Betaretrovirus and Spumaretrovirus genera respectively and hence have been mentioned in the manuscript as per the nomenclature proposed by Gifford et al., 2018 which has been cited at Lines 122-125.

Line 221- "defferent" should be "different"

Corrected

Lines 233-234 - what is meant by "canonical" and "non-canonical" forms? Can the authors please define these two terms?

Thank you for the question, canonical refers to sequences that are well-preserved and match the structural and functional features of complete env genes, and non-canonical refers to sequences with significant structural alterations or truncations that deviate from this typical form. This explanation has been mentioned in the revised version at Lines 475-479.

Line 252 - if/is

Corrected

Lines 274-276 needs a citation to the paper(s) that reported this.

Corrected

Line 283-285 - this was confusing. How could the authors have noted distinct occurrences and clusters of these if they were excluded from the BLAST analysis? It says the consensus sequences were effectively representing these, but doesn't this raise the possibility that the consensus sequences are not specific enough? Could this also then lead to false identification? Perhaps a few more words to explain should be added.

We thank the reviewer for the observation. While performing the tBlastn search we did obtain the hits for HERV15, HERVR, ERVV1, ERVV2 and PABL, and we have mentioned the detailed explanation about this observation in the revised manuscript at lines 619-627.

Line 298 - missing comma

Corrected

Lines 348-351- this list is not a list of recombination mechanisms. Template switching is a mechanism of recombination, but "acquisition" is simply a generic term, "degradation" is not a mechanism, and "cross-species transmission" might be a driver or a result of recombination, but it is not a mechanism of recombination.

We thank the reviewer for the observation. We have revised the explanation for the recombination events in the discussion section, as some parts of the results have been moved to discussion section (Lines 1058-1065)

Lines 369-372. It's not clear why this means the event was a "very recent occurrence". Do the authors mean that there were shared integration sites between some of the species, and that these sites lacked the insertions in other species (e.g. gibbon, orangutan, monkeys)?

For the long section on recombination events involving an env sequence with an LTR in it, can the authors explain how they know when it's a recombination event versus integration of one provirus into another one, followed by recombination between LTRs to generate a solo-LTR?

We thank the reviewer for the observation. Regarding the very recent occurrence of the recombination event, we have explained it in revised manuscript at lines 769-824 writing “In fact, the recombinant sequences were shared only between 4 species of Catarrhini parvorder and were absent in more distantly related primates (such as gibbons, orangutans, etc.). This with the presence of shared recombination sites suggests that the insertion occurred after the divergence of these species, while its absence in others indicate that it is a recombination event.”

For the observation regarding the env-LTR recombination events, the recombinants were first detected by the RDP software and were further validated through the BLAT search in the genomes available on genome browser. The explanation on how we obtained these env-LTR recombination events is now provided in lines 746-763 of the revised manuscript.

Methods Lines 151-168 and Figure 1 legend Lines 689-690 - how did the authors distinguish between "translated regions" corresponding to the actual Env protein sequence from translation of the other two reading frames? That is, there must have been substantial "translatable" stretches of sequence in the two incorrect reading frames as well as the reading frame corresponding to Env, so the question is how were the correct ones identified for the reconstruction?

We thank the reviewer for the observation. We have provided the detailed explanation to the observation in the methods section (Lines 335-359).

Line 495 - "previously reported" should include citation(s) of the prior report(s).

We thank the reviewer for the observation, we have provided appropriate citations.

Line 525 - the authors propose that the mechanism "is the co-packaging of different ERVs in a virus particle". First, I assume they meant to say that RNA from different ERVs is co-packaged. Second, isn't it also possible or likely that these could arise from co-packaging of exogenous retrovirus RNAs and recombination, especially if the related exogenous forms were still circulating at the time these things arose?

We thank the reviewer for the observation. We have modified in the revised manuscript a proposed mechanism that includes also the possibility of co-packaging of exogenous retrovirus RNAs and recombination, at lines 1082-1099

Line 686 - env should either be italicized (gene) or capitalized (protein), depending on what the authors intended here.

We thank the reviewer for the observation. We have corrected the typological error in the new version of manuscript.

Reviewer #3 (Public review):

Summary:

Retroviruses have been endogenized into the genome of all vertebrate animals. The envelope protein of the virus is not well conserved and acquires many mutations hence can be used to monitor viral evolution. Since they are incorporated into the host genome, they also reflect the evolution of the hosts. In this manuscript the authors have focused their analyses on the env genes of endogenous retroviruses in primates. Important observations made include the extensive recombination events between these retroviruses that were previously unknown and the discovery of HML species in genomes prior to the splitting of old and new world monkeys.

Strengths:

They explored a number of databases and made phylogenetic trees to look at the distribution of retroviral species in primates. The authors provide a strong rationale for their study design, they provide a clear description of the techniques and the bioinformatics tools used.

Weaknesses:

The manuscript is based on bioinformatics analyses only. The reference genomes do not reflect the polymorphisms in humans or other primate species. The analyses thus likely underestimates the amount of diversity in the retroviruses. Further experimental verification will be needed to confirm the observations.

Not sure which databases were used, but if not already analyzed, ERVmap.com and repeatmesker are ones that have many ERVs that are not present in the reference genomes. Also, long range sequencing of the human genome has recently become available which may also be worth studying for this purpose.

We thank the reviewer for the observations and comments. We would like to clarify that the intent of the work was to perform bioinformatics analysis and so a wet lab experimental verification of the observations are out of the scope of the present manuscript. For the aim of the manuscript, we have used the NCBI reference genomes, while for the report of the coordinates of HML supergroup in the squirrel monkey genome and the coordinates of the recombination events through BLAT search we have used genomes assemblies available on Genome browser with repeat masker custom track, since it has well represented ERV annotations.

The suggestion regarding using long range sequencing of human genome is an interesting perspective and hence in the future work we will try to implement it in our analysis as well as perform an experimental verification, since, again, the focus of the present work does not include wet experimental part.

Reviewer #3 (Recommendations for the authors):

In a few places the term HERV has been used when describing ERVs in non-human primates. This needs to be corrected.

We thank the reviewer for the observation. We have checked and accordingly modified the terms in the manuscript wherever necessary.

Author response:

eLife Assessment

This study provides a valuable contribution to understanding how negative affect influences food-choice decision making in bulimia nervosa, using a mechanistic approach with a drift diffusion model (DDM) to examine the weighting of tastiness and healthiness attributes. The solid evidence is supported by a robust crossover design and rigorous statistical methods, although concerns about low trial counts, possible overfitting, and the absence of temporally aligned binge-eating measures limit the strength of causal claims. Addressing modeling transparency, sample size limitations, and the specificity of mood induction effects, would enhance the study's impact and generalizability to broader populations.

We thank the Editor and Reviewers for their summary of the strengths of our study, and for their thoughtful review and feedback on our manuscript. We apologize for the confusion in how we described the multiple steps performed and hierarchical methods used to ensure that the model we report in the main text was the best fit to the data while not overfitting. We are not certain about what is meant by “[a]ddressing model transparency,” but as described in our response to Reviewer 1 below, we have now more clearly explained (with references) that the use of hierarchical estimation procedures allows for information sharing across participants, which improves the reliability and stability of parameter estimates—even when the number of trials per individual is small. We have clarified for the less familiar reader how our Bayesian model selection criterion penalizes models with more parameters (more complex models). Although details about model diagnostics, recoverability, and posterior predictive checks are all provided in the Supplementary Materials, we have clarified for the less familiar reader how each of these steps ensures that the parameters we estimate are not only identifiable and interpretable, but also ensure that the model can reproduce key patterns in the data, supporting the validity of the model. Additionally, we have provided all scripts for estimating the models by linking to our public Github repository. Furthermore, we have edited language throughout to eliminate any implication of causal claims and acknowledged the limitation of the small sample size.

Public Reviews:

Reviewer #1 (Public review):

Summary:

Using a computational modeling approach based on the drift diffusion model (DDM) introduced by Ratcliff and McKoon in 2008, the article by Shevlin and colleagues investigates whether there are differences between neutral and negative emotional states in:

(1) The timings of the integration in food choices of the perceived healthiness and tastiness of food options between individuals with bulimia nervosa (BN) and healthy participants.

(2) The weighting of the perceived healthiness and tastiness of these options.

Strengths:

By looking at the mechanistic part of the decision process, the approach has the potential to improve the understanding of pathological food choices. The article is based on secondary research data.

Weaknesses:

I have two major concerns and a major improvement point.

The major concerns deal with the reliability of the results of the DDM (first two sections of the Results, pages 6 and 7), which are central to the manuscript, and the consistency of the results with regards to the identification of mechanisms related to binge eating in BN patients (i.e. last section of the results, page 7).

(1) Ratcliff and McKoon in 2008 used tasks involving around 1000 trials per participant. The Chen et al. experiment the authors refer to involves around 400 trials per participant. On the other hand, Shevlin and colleagues ask each participant to make two sets of 42 choices with two times fewer participants than in the Chen et al. experiment. Shevlin and colleagues also fit a DDM with additional parameters (e.g. a drift rate that varies according to subjective rating of the options) as compared to the initial version of Ratcliff and McKoon. With regards to the number of parameters estimated in the DDM within each group of participants and each emotional condition, the 5- to 10-fold ratio in the number of trials between the Shevlin and colleagues' experiment and the experiments they refer to (Ratcliff and McKoon, 2008; Chen et al. 2022) raises serious concerns about a potential overfitting of the data by the DDM. This point is not highlighted in the Discussion. Robustness and sensitivity analyses are critical in this case.

We thank the Reviewer for their thoughtful critique. We agree that a limited number of trials can forestall reliable estimation, which we acknowledge in the Discussion section. However, we used a hierarchical estimation approach which leverages group information to constrain individual-level estimates. This use of group-level parameters to inform individual-level estimates reduces overfitting and noise that can arise when trial counts are low, and the regularization inherent in hierarchical fitting prevents extreme parameter estimates that could arise from noisy or limited data (Rouder & Lu, 2005). As a result, hierarchical estimation has been repeatedly shown to work well in settings with low trial counts, including as few as 40 trials per condition (Ratcliff & Childers, 2015; Wiecki et al., 2013), and previous applications of the time-varying DDM to food choice task data has included experiments with as few as 60 trials per condition (Maier et al., 2020). We have added references to these more recent approaches and specifically note their advantages for the modeling of tasks with fewer trials. Additionally, our successful parameter recovery described in the Supplementary Materials supports the robustness of the estimation procedure and the reliability of our results.

The authors compare different DDMs to show that the DDM they used to report statistical results in the main text is the best according to the WAIC criterion. This may be viewed as a robustness analysis. However, the other DDM models (i.e. M0, M1, M2 in the supplementary materials) they used to make the comparison have fewer parameters to estimate than the one they used in the main text. Fits are usually expected to follow the rule that the more there are parameters to estimate in a model, the better it fits the data. Additionally, a quick plot of the data in supplementary table S12 (i.e. WAIC as a function of the number of parameters varying by food type in the model - i.e. 0 for M0, 2 for M1, 1 for M2 and 3 for M3) suggests that models M1 and potentially M2 may be also suitable: there is a break in the improvement of WAIC between model M0 and the three other models. I would thus suggest checking how the results reported in the main text differ when using models M1 and M2 instead of M3 (for the taste and health weights when comparing M3 with M1, for τS when comparing M3 with M2). If the differences are important, the results currently reported in the main text are not very reliable.

We thank the Reviewer for highlighting that it would be helpful for the paper to explicitly note that we specifically selected WAIC as one of two methods to assess model fit because it penalizes for model complexity. We now explicitly state that, in addition to being more robust than other metrics like AIC or BIC when comparing hierarchical Bayesian models like those in the current study, model fit metrics like WAIC penalize for model complexity based on the number of parameters (Watanabe, 2010). Therefore, it is not the case that more complex models (i.e., having additional parameters) would automatically have lower WAICs. Additionally, we note that our second method to assess model fit, posterior predictive checks demonstrate that only model M3 can reproduce key behavioral patterns present in the empirical data. As described in the Supplementary Materials, M1 and M2 miss those patterns in the data. In summary, we used best practices to assess model fit and reliability (Wilson & Collins, 2019): results from the WAIC comparison (which in fact penalizes models with more parameters) and results from posterior predictive checks align in showing that M3 best fit to our data. We have added a sentence to the manuscript to state this explicitly.

(2) The second main concern deals with the association reported between the DDM parameters and binge eating episodes (i.e. last paragraph of the results section, page 7). The authors claim that the DDM parameters "predict" binge eating episodes (in the Abstract among other places) while the binge eating frequency does not seem to have been collected prospectively. Besides this methodological issue, the interpretation of this association is exaggerated: during the task, BN patients did not make binge-related food choices in the negative emotional state. Therefore, it is impossible to draw clear conclusions about binge eating, as other explanations seem equally plausible. For example, the results the authors report with the DDM may be a marker of a strategy of the patients to cope with food tastiness in order to make restrictive-like food choices. A comparison of the authors' results with restrictive AN patients would be of interest. Moreover, correlating results of a nearly instantaneous behavior (i.e. a couple of minutes to perform the task with the 42 food choices) with an observation made over several months (i.e. binge eating frequency collected over three months) is questionable: the negative emotional state of patients varies across the day without systematically leading patients to engage in a binge eating episode in such states.

I would suggest in such an experiment to collect the binge craving elicited by each food and the overall binge craving of patients immediately before and after the task. Correlating the DDM results with these ratings would provide more compelling results. Without these data, I would suggest removing the last paragraph of the Results.

We thank the Reviewer for these interesting suggestions and appreciate the opportunity to clarify that we agree that claims about causal connections between our decision parameters and symptom severity metrics would be inappropriate. Per the Reviewer’s suggestions, we have eliminated the use of the word “predict” to describe the tested association with symptom metrics.  We also agree that more time-locked associations with craving ratings and near-instantaneous behavior would be useful, and we have added this as an important direction for future research in the discussion. However, associating task-based behavior with validated self-report measures that assess symptom severity over long periods of time that precede the task visit (e.g., over the past 2 weeks in depression, over the past month in eating disorders) is common practice in computational psychiatry, psychiatric neuroimaging, and clinical cognitive neuroscience (Hauser et al., 2022; Huys et al., 2021; Wise et al., 2023), and this approach has been used several times specifically with food choice tasks (Dalton et al., 2020; Steinglass et al., 2015). We have revised the language throughout the manuscript to clarify: the results suggest that individuals whose task behavior is more reactive to negative affect tend to be the most symptomatic, but the results do not allow us to determine whether this reactivity causes the symptoms.

In response to this Reviewer’s important point about negative affect not always producing loss-of-control eating in individuals with BN, we also now explicitly note that while several studies employing ecological momentary assessments (EMA) have repeatedly shown that increases in negative affect significantly increase the likelihood of subsequent loss-of-control eating (Alpers & Tuschen-Caffier, 2001; Berg et al., 2013; Haedt-Matt & Keel, 2011; Hilbert & Tuschen-Caffier, 2007; Smyth et al., 2007), not all loss-of-control eating occurs in the context of negative affect, and that future studies should integrate food choice task data pre and post-affect inductions with measures that capture the specific frequency of loss of control eating episodes that occur during states of high negative affect.

(3) My major improvement point is to tone down as much as possible any claim of a link with binge eating across the entire manuscript and to focus more on the restrictive behavior of BN patients in between binge eating episodes (see my second major concern about the methods). Additionally, since this article is a secondary research paper and since some of the authors have already used the task with AN patients, if possible I would run the same analyses with AN patients to test whether there are differences between AN (provided they were of the restrictive subtype) and BN.

We appreciate the Reviewer’s perspective and suggestions. We have adjusted our language linking loss-of-control eating frequency with decision parameters, and we have added additional sentences focusing on the implications for the restrictive behavior of patients with BN between binge eating episodes. In the Supplementary Materials. We have added an analysis of the restraint subscale of the EDE-Q and confirmed no relationship with parameters of interest. While we agree additional analyses with AN patients would be of interest, this is outside the scope of the paper. Our team have collected data from individuals with AN using this task, but not with any affect induction or measure of affect. Therefore, we have added this important direction for future research to the discussion.

Reviewer #2 (Public review):

Summary:

Binge eating is often preceded by heightened negative affect, but the specific processes underlying this link are not well understood. The purpose of this manuscript was to examine whether affect state (neutral or negative mood) impacts food choice decision-making processes that may increase the likelihood of binge eating in individuals with bulimia nervosa (BN). The researchers used a randomized crossover design in women with BN (n=25) and controls (n=21), in which participants underwent a negative or neutral mood induction prior to completing a food-choice task. The researchers found that despite no differences in food choices in the negative and neutral conditions, women with BN demonstrated a stronger bias toward considering the 'tastiness' before the 'healthiness' of the food after the negative mood induction.

Strengths:

The topic is important and clinically relevant and methods are sound. The use of computational modeling to understand nuances in decision-making processes and how that might relate to eating disorder symptom severity is a strength of the study.

Weaknesses:

The sample size was relatively small and may have been underpowered to find differences in outcomes (i.e., food choice behaviors). Participants were all women with BN, which limits the generalizability of findings to the larger population of individuals who engage in binge eating. It is likely that the negative affect manipulation was weak and may not have been potent enough to change behavior. Moreover, it is unclear how long the negative affect persisted during the actual task. It is possible that any increases in negative affect would have dissipated by the time participants were engaged in the decision-making task.

We thank the Reviewer for their comments on the strengths of the paper, and for highlighting these important considerations regarding the sample demographics and the negative affect induction. As in the original paper that focused only on ultimate food choice behaviors, we now specifically acknowledge that the study was only powered to detect small to medium group differences in the effect of negative emotion on these final choice behaviors. Regarding the sample demographics, we agree that the study’s inclusion of only female participants is a limitation.  Although the original decision for this sampling strategy was informed by data suggesting that bulimia nervosa is roughly six times more prevalent among females than males (Udo & Grilo, 2018), we now note in the discussion that our female-only sample limits the generalizability of the findings.

We also agree with the Reviewer’s noted limitations of the negative mood induction, and based on the reviewer’s suggestions, we have added to our original description of these limitations in the Discussion. Specifically, we now note that although the task was completed immediately after the affect induction, the study did not include intermittent mood assessments throughout the choice task, so it is unclear how long the negative affect persisted during the actual task.

Reviewer #3 (Public review):

Summary:

The study uses the food choice task, a well-established method in eating disorder research, particularly in anorexia nervosa. However, it introduces a novel analytical approach - the diffusion decision model - to deconstruct food choices and assess the influence of negative affect on how and when tastiness and healthiness are considered in decision-making among individuals with bulimia nervosa and healthy controls.

Strengths:

The introduction provides a comprehensive review of the literature, and the study design appears robust. It incorporates separate sessions for neutral and negative affect conditions and counterbalances tastiness and healthiness ratings. The statistical methods are rigorous, employing multiple testing corrections.

A key finding - that negative affect induction biases individuals with bulimia nervosa toward prioritizing tastiness over healthiness - offers an intriguing perspective on how negative affect may drive binge eating behaviors.

Weaknesses:

A notable limitation is the absence of a sample size calculation, which, combined with the relatively small sample, may have contributed to null findings. Additionally, while the affect induction method is validated, it is less effective than alternatives such as image or film-based stimuli (Dana et al., 2020), potentially influencing the results.

We agree that the small sample size and specific affect induction method may have contributed to the null model-agnostic behavioral findings. Based on this Reviewer’s and Reviewer 2’s comments, we have added these factors to our original acknowledgements of limitations in the Discussion.

Another concern is the lack of clarity regarding which specific negative emotions were elicited. This is crucial, as research suggests that certain emotions, such as guilt, are more strongly linked to binge eating than others. Furthermore, recent studies indicate that negative affect can lead to both restriction and binge eating, depending on factors like negative urgency and craving (Leenaerts et al., 2023; Wonderlich et al., 2024). The study does not address this, though it could explain why, despite the observed bias toward tastiness, negative affect did not significantly impact food choices.

We thank the Reviewer for raising these important points and possibilities. In the supplementary materials, we have added an additional analysis of the specific POMS subscales that comprise the total negative affect calculation that was reported in the original paper (Gianini et al., 2019), and which we now report in the main text. Ultimately, we found that, across both groups, the negative affect induction increased responses related to anger, confusion, depression, and tension while reducing vigor.

We agree with the Reviewer that factors like negative urgency and cravings are relevant here. The study did not collect any measures of craving, and in response to Reviewer 1 and this Reviewer, we now note in the discussion that replication studies including momentary craving assessments will be important. While we don’t have any measurements of cravings, we did measure negative urgency. Despite these prior findings, the original paper (Gianini et al., 2019) did not find that negative urgency was related to restrictive food choices. We have now repeated those analyses, and we also were unable to find any meaningful patterns. Nonetheless, we have added an analysis of negative urgency scores and decision parameters to the supplementary materials.      

References

Alpers, G. W., & Tuschen-Caffier, B. (2001). Negative feelings and the desire to eat in bulimia nervosa. Eating Behaviors, 2(4), 339–352. https://doi.org/10.1016/S1471-0153(01)00040-X

Berg, K. C., Crosby, R. D., Cao, L., Peterson, C. B., Engel, S. G., Mitchell, J. E., & Wonderlich, S. A. (2013). Facets of negative affect prior to and following binge-only, purge-only, and binge/purge events in women with bulimia nervosa. Journal of Abnormal Psychology, 122(1), 111–118. https://doi.org/10.1037/a0029703

Dalton, B., Foerde, K., Bartholdy, S., McClelland, J., Kekic, M., Grycuk, L., Campbell, I. C., Schmidt, U., & Steinglass, J. E. (2020). The effect of repetitive transcranial magnetic stimulation on food choice-related self-control in patients with severe, enduring anorexia nervosa. International Journal of Eating Disorders, 53(8), 1326–1336. https://doi.org/10.1002/eat.23267

Gianini, L., Foerde, K., Walsh, B. T., Riegel, M., Broft, A., & Steinglass, J. E. (2019). Negative affect, dietary restriction, and food choice in bulimia nervosa. Eating Behaviors, 33, 49–54. https://doi.org/10.1016/j.eatbeh.2019.03.003

Haedt-Matt, A. A., & Keel, P. K. (2011). Revisiting the affect regulation model of binge eating: A meta-analysis of studies using ecological momentary assessment. Psychological Bulletin, 137(4), 660–681. https://doi.org/10.1037/a0023660

Hauser, T. U., Skvortsova, V., Choudhury, M. D., & Koutsouleris, N. (2022). The promise of a model-based psychiatry: Building computational models of mental ill health. The Lancet Digital Health, 4(11), e816–e828. https://doi.org/10.1016/S2589-7500(22)00152-2

Hilbert, A., & Tuschen-Caffier, B. (2007). Maintenance of binge eating through negative mood: A naturalistic comparison of binge eating disorder and bulimia nervosa. International Journal of Eating Disorders, 40(6), 521–530. https://doi.org/10.1002/eat.20401

Huys, Q. J. M., Browning, M., Paulus, M. P., & Frank, M. J. (2021). Advances in the computational understanding of mental illness. Neuropsychopharmacology, 46(1), 3–19. https://doi.org/10.1038/s41386-020-0746-4

Maier, S. U., Raja Beharelle, A., Polanía, R., Ruff, C. C., & Hare, T. A. (2020). Dissociable mechanisms govern when and how strongly reward attributes affect decisions. Nature Human Behaviour, 4(9), Article 9. https://doi.org/10.1038/s41562-020-0893-y

Ratcliff, R., & Childers, R. (2015). Individual differences and fitting methods for the two-choice diffusion model of decision making. Decision, 2(4), 237–279. https://doi.org/10.1037/dec0000030

Rouder, J. N., & Lu, J. (2005). An introduction to Bayesian hierarchical models with an application in the theory of signal detection. Psychonomic Bulletin & Review, 12(4), 573–604. https://doi.org/10.3758/BF03196750

Smyth, J. M., Wonderlich, S. A., Heron, K. E., Sliwinski, M. J., Crosby, R. D., Mitchell, J. E., & Engel, S. G. (2007). Daily and momentary mood and stress are associated with binge eating and vomiting in bulimia nervosa patients in the natural environment. Journal of Consulting and Clinical Psychology, 75(4), 629–638. https://doi.org/10.1037/0022-006X.75.4.629

Steinglass, J., Foerde, K., Kostro, K., Shohamy, D., & Walsh, B. T. (2015). Restrictive food intake as a choice—A paradigm for study. International Journal of Eating Disorders, 48(1), 59–66. https://doi.org/10.1002/eat.22345

Udo, T., & Grilo, C. M. (2018). Prevalence and Correlates of DSM-5–Defined Eating Disorders in a Nationally Representative Sample of U.S. Adults. Biological Psychiatry, 84(5), 345–354. https://doi.org/10.1016/j.biopsych.2018.03.014

Watanabe, S. (2010). Asymptotic Equivalence of Bayes Cross Validation and Widely Applicable Information Criterion in Singular Learning Theory. Journal of Machine Learning Research, 11, 3571–3594.

Wiecki, T. V., Sofer, I., & Frank, M. J. (2013). HDDM: Hierarchical Bayesian estimation of the drift-diffusion model in Python. Frontiers in Neuroinformatics, 7. https://doi.org/10.3389/fninf.2013.00014

Wilson, R. C., & Collins, A. G. (2019). Ten simple rules for the computational modeling of behavioral data. eLife, 8, e49547. https://doi.org/10.7554/eLife.49547

Wise, T., Robinson, O. J., & Gillan, C. M. (2023). Identifying Transdiagnostic Mechanisms in Mental Health Using Computational Factor Modeling. Biological Psychiatry, 93(8), 690–703. https://doi.org/10.1016/j.biopsych.2022.09.034

Reviewer #1 (Public review):

Summary:

The authors had previously found that brief social isolation could increase the activity of these neurons, and that manipulation of these neurons could alter social behavior in a social rank-dependent fashion. This manuscript explored which of the outputs were responsible for this, identifying the central nucleus of the amygdala as the key output region. The authors identified some discrete behavior changes associated with these outputs, and found that during photostimulation of these outputs, neuronal activity appeared altered in 'social response' neurons.

Strengths:

Rigorous analysis of the anatomy. Careful examination of the heterogenous effects on cell activity due to stimulation, linking the physiology with the behavior via photostimulation during recording in vivo.

Weaknesses:

(1) There are some clear imbalances in the sample size across the different regions parsed. The CeA has a larger sample size, likely in part to the previous work suggesting differential effects depending on social rank/dominance. Given the potential variance, it may be hard to draw conclusions about the impact of stimulation across different social ranks for other groups.

(2) It is somewhat unclear why only the 'social object ratio' was used to assess the effects versus more direct measurements of social behavior.

(3) Somewhat related, while it is statistically significant, it is unclear if the change seen in face investigation of biologically significant, on average, it looks like a few-seconds difference and that was not modulated by social rank.

(4) There are several papers studying these neurons that have explored behaviors examined here, as well as the physiological connectivity that are not cited that would provide important context for this work. In particular, multiple groups have found a dopamine-mediated IPSP in the BNST, in contrast to this work. There are technical differences that may drive these differences, but not addressing them is a major weakness.

(5) The inclusion of some markers for receptors for some of these outputs is interesting, and the authors suggest that this may be important, but this is somewhat disconnected from the rest of the work performed.

Reviewer #2 (Public review):

Summary:

The authors perform a series of studies to follow up on their previous work, which established a role for dorsal raphe dopamine neurons (DRN) in the regulation of social-isolation-induced rebound in mice. In the present study, Lee et. al, use a combination of modern circuit tools to investigate putatively distinct roles of DRN dopamine transporting containing (DAT) projections to the bed nucleus of the stria terminalis (BNST), central amygdala (CeA), and posterior basolateral amygdala (BLP). Notably, they reveal that optogenetic stimulation of distinct pathways confers specific behavioral states, with DRNDAT-BLP driving aversion, DRNDAT-BNST regulating non-social exploratory behavior, and DRNDAT-CeA promoting social ability. A combination of electrophysiological studies and in situ hybridization studies reveal heterogenous dopamine and neuropeptide expression and different firing properties, providing further evidence of pathway-specific neural properties. Lastly, the authors combine optogenetics and calcium imaging to resolve social encoding properties in the DRNDAT-CeA pathway, which correlates observed social behavior to socially engaged neural ensembles.

Collectively, these studies provide an interesting way of dissecting out separable features of a complex multifaceted social-emotional state that accompanies social isolation and the perception of 'loneliness.' The main conclusions of the paper provide an important and interesting set of findings that increase our understanding of these distinct DRN projections and their role in a range of social (e.g., prosocial, dominance), non-social, and emotional behaviors. However, as noted below, the examination of these circuits within a homeostatic framework is limited given that a number of the datasets did not include an isolated condition. The DRNDAT-CeA pathway was investigated with respect to social homeostatic states in the present study for some of the datasets.

Strengths:

(1) The authors perform a comprehensive and elegant dissection of the anatomical, behavioral, molecular, and physiological properties of distinct DRN projections relevant to social, non-social, and emotional behavior, to address multifaceted and complex features of social state.

(2) This work builds on prior findings of isolation-induced changes in DRN neurons and provides a working framework for broader circuit elements that can be addressed across the social homeostatic state.

(3) This work characterizes a broader circuit implicated in social isolation and provides a number of downstream targets to explore, setting a nice foundation for future investigation.

(4) The studies account for social rank and anxiety-like behavior in several of the datasets, which are an important consideration to the interpretation of social motivation states, especially in male mice with respect to dominance behavior.

Weaknesses:

(1) The conceptual framework of the study is based on the premise of social isolation and perceived 'loneliness' under the framework of social homeostasis, analogous to hunger. In this framework, social isolation should provoke an aversive state and compensatory social contact behavior. In the authors' prior work, they demonstrate synaptic changes in DRN neurons and social rebound following acute social isolation. Thus, the prediction would be that downstream projections also would show state-dependent changes as a function of social housing conditions (e.g., grouped vs. isolated). In the current paper, a social isolation condition was not included for the majority of the studies conducted (e.g., Figures 1-6 do not include an isolated condition, Figures 7-8 do include an isolated condition). Thus, while Figure 1-6 adds a very interesting and compelling set of data that is of high value to the social behavior field with respect to social and emotional processing and general circuit characterization, these studies do not directly investigate the impacts of dynamic social homeostatic state. The main claim of the paper, including the title (e.g., separable DRN projections mediate facets of loneliness-like state), abstract, intro, and discussion presents the claim of this work under the framework of dynamic social homeostatic states, which should be interpreted with caution, as the majority of the work in the paper did not include a social isolation comparison.

(2) In Figure 1, the authors confirm co-laterals in the BNST and CeA via anatomical tracing studies. The goal of the optogenetic studies is to dissociate the functional/behavioral roles of distinct projections. However, one limitation of optogenetic projection targeting is the possibility of back-propagating action potentials (stimulation of terminals in one region may back-propagate to activate cell bodies, and then afferent projections to other regions), and/or stimulation of fibers of passage. Therefore, one limitation in the dataset for the optogenetic stimulation studies is the possibility of non-specific unintended activation of projections other than those intended (e.g., DRNDAT-CeA). This can be dealt with by administering lidocaine to prevent back-propagating action potentials.

(3) It is unclear from the test, but in the subjects' section of the methods, it appears that only male animals were included in the study, with no mention of female subjects. It should be clear to the reader that this was conducted in males only if that is the case, with consideration or discussion, about female subjects and sex as a biological variable.

(4) Averaged data are generally reported throughout the study in the form of bar graphs, across most figures. Individual data points would increase the transparency of the data.

Reviewer #2 (Public review):

The authors tested tactile acuity on the breast of females using several tasks and reported overall low acuity compared to the back, which is typically considered to have the worst acuity of all body parts. Moreover, there was evidence that acuity is worse the larger the breast; this finding mirrors similar findings for the hand and therefore suggests that the number of tactile sensors is fixed and must be distributed across a larger extent of skin when a body part is larger, thus resulting in comparably lower tactile acuity.

Strengths:

I find this an interesting paper with results that are relevant to the tactile community. The authors apply several tasks allowing them to link the paper with previous results. The methodology and psychophysical analysis are sound.

Weaknesses:

The analysis of localization error direction, with the result that the nipple area may be a landmark for tactile localization, is interesting and aligns the paper with some other recent papers that have suggested that such landmarks should exist. However, there are major issues with methodology and statistics, so that currently the conclusions are not supported.

In the following, line numbers refer to the re-formatted manuscript provided by the authors upon request and are mentioned for them to find the relevant passages faster.

(1) Comments on analysis of tactile acuity:

- I had a hard time understanding some parts of the report. What is meant by "broadly no relationship" in line 137?

- It is suggested that spatial expansion (which is correlated with body part size) is related between medial breast and hand - is this to say that women with large hands have large medial breast size? Nipple size was measured, but hand size was not measured, is this correct?

- It is furthermore unclear how the authors differentiate medial breast and NAC. The sentence in lines 140-141 seems to imply the two terms are considered the same, as a conclusion about NAC is drawn from a result about the medial breast. This requires clarification.

- Finally, given that the authors suspect that overall localization ability (or attention) may be overshadowed by a size effect, would not an analysis be adequate that integrates both, e.g. a regression with multiple predictors?

(2) Comments on analysis of "The nipple is a unit":

- Statistics in this section are not adequately described and may be partly false.

- In the paragraph about testing quadrants of the nipple, it is stated that only 3 of 10 participants barely outperformed chance with a p < 0.01. It is unclear how a significant t-test is an indication of "barely above chance".

- The final part of the paragraph on nipple quadrants (starting line 176) explains that there was a trend (4 of 10 participants) for lower tactile acuity being related to the inability to differentiate quadrants. It seems to me that such a result would not be expected: The stated hypothesis is that all participants have the same number of tactile sensors in their nipple and areola, independent of NAC size. In this section, participants determine the quadrant of a single touch. Theoretically, all participants should be equally able to perform this task, because they all have the same number of receptors in each quadrant of nipple and areola. Thus, the result in Figure 2C is curious.

(3) Comments on analysis of "Absolute localization on the breast is anchored to the nipple"

- Again, there are things that are unclear with the statistics and description of the analysis.

- This section reports an Anova (line 193/194) with a factor "participant". This doesn't appear sensible. Please clarify. The factor distance is also unclear; is this a categorical or a continuous variable? Line 400 implies a 6-level factor, but Anovas and their factors, respectively, are not described in methods (nor are any of the other statistical approaches).

- The analysis on imprecision using mean pairwise error (line 199) is unclear: does pairwise refer to x/y or to touch vs. center of the nipple?

- p8, upper text, what is meant by "relative over-representation of the depth axis"? Does this refer to the breast having depth but the equivalent area on the back not having depth? What are the horizontal planes (probably meant to be singular?) - do you simply mean that depth was ignored for the calculation of errors? This seems to be implied in Figure 3AB.

- Lines 232-241, I cannot follow the conclusions drawn here. First, it is not clear to a reader what the aim of the presented analyses is: what are you looking for when you analyze the vectors? Second, "vector strength" should be briefly explained in the main text. Third, it is not clear how the final conclusion is drawn. If there is a bias of all locations towards the nipple, then a point closer to the nipple cannot exhibit a large bias, because the nipple is close-by. Therefore, one would expect that points close to the nipple exhibit smaller errors, but this would not imply higher acuity - just less space for localizing anything. The higher acuity conclusion is at odds with the remaining results, isn't it: acuity is low on the outer breast, but even lower at the NAC, so why would it be high in between the two?

(4) Comments on the Discussion:

The discussion makes some concrete suggestions for sensors in implants (line 283). It is not clear how the stated numbers were computed. Also, why should 4 sensors nipple quadrants receive individual sensors if the result here was that participants cannot distinguish these quadrants?

Additional comments:

I would find it interesting to know whether participants with small breast measurement delta had breast acuity comparable to the back. Alternatively, it would be interesting to know whether breast and back acuity are comparable in men. Such a result would imply that the torso has uniform acuity overall, but any spatial extension of the breast is unaccounted for. The lowest single participant data points in Figure 1B appear similar, which might support this idea.

Reviewer #2 (Public review):

Summary:

The author presents a transport-based morphometry (TBM) approach for the discovery of non-contrast computed tomography (NCCT) markers of hematoma expansion risk in spontaneous intracerebral hemorrhage (ICH) patients. The findings demonstrate that TBM can quantify hematoma morphological features and outperforms existing clinical scoring systems in predicting 24-hour hematoma expansion. In addition, the inversion model can visualize features, which makes it interpretable. In conclusion, this research has clinical potential for ICH risk stratification, improving the precision of early interventions.

Strengths:

TBM quantifies hematoma morphological changes using the Wasserstein distance, which has a well-defined physical meaning. It identifies features that are difficult to detect through conventional visual inspection (such as peripheral density distribution and density heterogeneity), which provides evidence supporting the "avalanche effect" hypothesis in hematoma expansion pathophysiology.

Weaknesses:

(1) As a methodology-focused study, the description of the methods section somewhat lacks depth and focus, which may make it challenging for readers to fully grasp the overall structure and workflow of the approach. For instance, the manuscript lacks a systematic overview of the entire process, from NCCT image input to the final prediction output. A potential improvement would be to include a workflow figure at the beginning of the manuscript, summarizing the proposed method and subsequent analytical procedures. This would help readers better understand the mechanism of the model.

(2) The description of the comparison algorithms could be more detailed. Since TBM directly utilizes NCCT images as input for prediction, while SVM and K-means are not inherently designed to process raw imaging data, it would be beneficial to clarify which specific features or input data were used for these comparison models. This would better highlight the effectiveness and advantages of the TBM method.

(3) The relatively small training and testing dataset may limit the model's performance and generalizability. Notably, while the study mentions that 1,066 patients from the ERICH dataset met the inclusion criteria, only 170 were randomly selected for the test set. Leveraging the full 1,066 ERICH cases for model training and internal validation might potentially enhance the model's robustness and performance.

(4) Some minor textual issues need to be checked and corrected, such as line 16 in the abstract "Incorporating these traits into a v achieved an AUROC of 0.71 ...".

(5) Some figures need to be reformatted (e.g., the x-axis in Figure 2 a is blocked).

好的，我们来详细解释一下布尔格 (Boolean Lattice)。

1. 定义：

一个格 L 被称为布尔格 (Boolean Lattice)，如果它同时满足以下两个条件：

• 它是分配格 (Distributive Lattice)： 对于任意元素 a, b, c ∈ L，分配律成立：
  • a ∧ (b ∨ c) = (a ∧ b) ∨ (a ∧ c)
  • a ∨ (b ∧ c) = (a ∨ b) ∧ (a ∨ c)
• 它是有补格 (Complemented Lattice)：
  • 它是一个有界格（拥有底元素 ⊥ 和顶元素 ⊤）。
  • 对于 L 中的每一个元素 a，都存在至少一个补元 b ∈ L，使得 a ∨ b = ⊤ 且 a ∧ b = ⊥。

简单来说，布尔格就是既是分配格又是补格的格。

2. 布尔格的关键性质：

除了分配性和有补性之外，布尔格还具有一些重要的性质：

• 补元的唯一性： 在布尔格中，每个元素的补元是唯一的。证明如下： 假设元素 a 有两个补元 b 和 c。那么：

  • a ∨ b = ⊤，a ∧ b = ⊥
  • a ∨ c = ⊤，a ∧ c = ⊥ 利用分配律，我们可以得到： b = b ∧ ⊤ = b ∧ (a ∨ c) = (b ∧ a) ∨ (b ∧ c) = (a ∧ b) ∨ (b ∧ c) = ⊥ ∨ (b ∧ c) = b ∧ c c = c ∧ ⊤ = c ∧ (a ∨ b) = (c ∧ a) ∨ (c ∧ b) = (a ∧ c) ∨ (c ∧ b) = ⊥ ∨ (c ∧ b) = c ∧ b 由于 b = b ∧ c 且 c = c ∧ b，所以 b = c（根据偏序关系的定义）。因此，补元是唯一的。 我们通常用 ¬a 或 a' 来表示元素 a 的唯一补元。

• 有界性： 作为有补格，布尔格必然拥有底元素 (⊥) 和顶元素 (⊤)。

• 满足德摩根定律 (De Morgan's Laws)： 对于任意元素 a 和 b，以下定律成立：

  • ¬(a ∨ b) = ¬a ∧ ¬b
  • ¬(a ∧ b) = ¬a ∨ ¬b

3. 布尔格的例子：

• 集合的幂集格： 对于任意集合 X，其幂集 P(X) 在子集包含关系下是一个布尔格。

  • 并运算 (∨) 是集合的并集 (∪)。
  • 交运算 (∧) 是集合的交集 (∩)。
  • 底元素 (⊥) 是空集 ∅。
  • 顶元素 (⊤) 是集合 X 本身。
  • 补元 (¬A) 是集合的补集 X \ A。

• 布尔代数 (Boolean Algebra)： 布尔格的概念与布尔代数是等价的。布尔代数通常被定义为一个集合 B，连同两个二元运算 (∨, ∧)，一个一元运算 (¬)，以及两个特殊的元素 (0, 1)，满足一系列公理（包括交换律、结合律、分配律、单位元、补元等）。一个布尔代数可以直接对应到一个布尔格，反之亦然。

• 命题逻辑的代数： 在经典命题逻辑中，命题可以用布尔变量表示，逻辑运算符“与”、“或”、“非”分别对应于布尔格中的 ∧、∨ 和 ¬。

• 开关电路： 数字电路中的开关可以用布尔变量表示，电路的串联和并联操作可以用 ∧ 和 ∨ 来建模，而开关的取反可以用 ¬ 来建模。

4. 不是布尔格的格的例子：

• 不是分配格的格： 例如，五边形格 (N₅) 和菱形格 (M₃) 是模格但不是分配格，因此它们不是布尔格。
• 不是有补格的有界格： 例如，我们之前提到的三个元素的链 {0, a, 1} 不是有补格，因此也不是布尔格。
• 自然数上的整除格（即使是有限的有界子格，如 12 的因子格）： 我们看到 12 的因子格不是有补格，因此也不是布尔格。虽然 6 的因子格是有补格，但它也是分配格，所以它是布尔格。一般来说，只有当一个正整数是无平方因子的且是若干个不同素数的乘积时，其因子格才是布尔格。

5. 布尔格的重要性：

布尔格（以及等价的布尔代数）在数学、计算机科学和工程学中都具有极其重要的地位，它们是：

• 逻辑推理的基础： 为形式逻辑提供了代数化的框架。
• 数字电路设计的基础： 用于分析和设计数字逻辑电路。
• 集合论的抽象： 提供了一种抽象的方式来处理集合及其运算。
• 概率论和信息论的工具： 用于描述和分析事件和信息。

总而言之，布尔格是一种具有非常强结构的格，它结合了分配性和有补性，并因此拥有许多有用的性质，使其成为一个非常重要的数学概念，并在众多领域有着广泛的应用。

好的，我们来详细理解一下完备格 (Complete Lattice)。

1. 回顾格的定义：

首先，我们知道一个格 (Lattice) 是一个偏序集 (L, ≤)，其中任意两个元素 a 和 b 都有唯一的最小上界 (least upper bound, join, ∨) 和唯一的最大下界 (greatest lower bound, meet, ∧)。

2. 完备格的引入：

完备格 是格概念的扩展。它不仅仅要求每对元素有最小上界和最大下界，而是要求每一个子集都具有最小上界和最大下界。

3. 完备格的正式定义：

一个偏序集 (L, ≤) 被称为一个完备格 (Complete Lattice)，如果它的每一个非空子集 S ⊆ L 都具有：

• 最小上界 (Least Upper Bound, Supremum, Join of S)：记为 ∨S。它是 L 中所有大于等于 S 中每个元素的元素中最小的一个。
• 最大下界 (Greatest Lower Bound, Infimum, Meet of S)：记为 ∧S。它是 L 中所有小于等于 S 中每个元素的元素中最大的一个。

4. 关键区别：

• 普通格： 要求任意两个元素有并和交。
• 完备格： 要求任意子集（包括有限和无限子集）有并和交。

5. 完备格的例子：

• 集合的幂集： 对于任意集合 X，其幂集 P(X) 在子集包含关系 ⊆ 下是一个完备格。

  • 对于 P(X) 的任意子集（即一些子集的集合） S = {A₁, A₂, ...}，它们的最小上界 ∨S 是它们的并集 A₁ ∪ A₂ ∪ ...。
  • 它们的最大下界 ∧S 是它们的交集 A₁ ∩ A₂ ∩ ...。

• 实数轴扩展： 将实数集 ℝ 加上正无穷 +∞ 和负无穷 -∞，得到集合 ℝ ∪ {+∞, -∞}，并在其上定义通常的顺序关系（其中 -∞ ≤ x ≤ +∞ 对于所有 x ∈ ℝ）。这个有序集是一个完备格。任意子集的最小上界就是其上确界（可能为有限实数或 +∞），最大下界就是其下确界（可能为有限实数或 -∞）。

• 闭区间格： 所有实数闭区间的集合，按照集合包含关系排序，构成一个完备格。任意一组闭区间的并的闭包是它们的最小上界，它们的交集是它们的最大下界。

• 向量空间的子空间格： 一个向量空间的所有子空间，按照子空间包含关系排序，构成一个完备格。任意一组子空间的最小上界是它们生成的子空间，最大下界是它们的交集。

6. 不是完备格的格的例子：

• 自然数上的整除格： 自然数集合 ℕ = {1, 2, 3, ...} 在整除关系下是一个格（最小公倍数是并，最大公约数是交），但它不是完备格。例如，所有素数的集合没有最小上界，因为没有一个自然数能被所有素数整除。

• 开区间 (0, 1) 上的标准序： 开区间 (0, 1) 在通常的小于等于关系下是一个格（任意两个数的最小上界是它们中的较大者，最大下界是它们中的较小者），但它不是完备格。例如，子集 (0, 1) 本身的上确界是 1，下确界是 0，但 1 和 0 都不属于开区间 (0, 1)。如果我们将区间扩展到闭区间 [0, 1]，它就变成了一个完备格。

7. 完备格必有顶元素和底元素：

一个重要的性质是，每一个完备格都必然包含一个最小元素（底，⊥） 和一个最大元素（顶，⊤）。

• 底元素 (⊥)： 可以看作是整个格 L 的最大下界 ∧L。
• 顶元素 (⊤)： 可以看作是整个格 L 的最小上界 ∨L。

8. 有限格与完备格：

每一个有限的格都是完备格。因为有限集合的任何子集也是有限的，而在有限偏序集中，如果每对元素都有上下界，那么每个有限子集也必然有上下界。

9. 完备格的重要性：

完备格在数学和计算机科学的许多领域都非常重要，例如：

• 不动点定理： 著名的 Knaster-Tarski 不动点定理就是关于完备格上的单调函数的。
• 领域理论 (Domain Theory)： 在程序语言语义学中，完备格被用来构建计算过程的数学模型。
• 形式概念分析 (Formal Concept Analysis)： 概念格总是完备格。

总结：

完备格是比普通格更强的结构，它要求每一个子集都拥有最小上界和最大下界。这个额外的条件使得完备格具有许多特殊的性质，并在多个数学和计算机科学领域发挥着重要作用。

好的，我们来详细解释一下有补格 (Complemented Lattice)。

1. 前提：有界格

首先，要理解有补格，我们需要先了解有界格 (Bounded Lattice)。一个格 L 是有界的，如果它拥有一个最小元素（底，bottom，通常记为 ⊥ 或 0） 和一个最大元素（顶，top，通常记为 ⊤ 或 1）。

• 底元素 (⊥)： 对于所有 x ∈ L，都有 ⊥ ≤ x。
• 顶元素 (⊤)： 对于所有 x ∈ L，都有 x ≤ ⊤。

2. 有补格的定义

一个有界格 L 被称为有补格 (Complemented Lattice)，如果对于 L 中的每一个元素 a，都存在至少一个元素 b 在 L 中，满足以下两个条件：

• a ∨ b = ⊤ （a 和 b 的并是顶元素）
• a ∧ b = ⊥ （a 和 b 的交是底元素）

这个元素 b 就被称为 a 的一个补元 (complement)。

3. 定义要点

• 必须是有界格： 有补格的定义依赖于顶元素和底元素的存在，所以首先它必须是一个有界格。
• 存在性而非唯一性： 定义只要求每个元素都存在至少一个补元。一个元素可能拥有多个补元。
• 补元是相互的： 如果 b 是 a 的一个补元，那么 a 也是 b 的一个补元，因为 a ∨ b = b ∨ a = ⊤ 且 a ∧ b = b ∧ a = ⊥。

4. 有补格的例子

• 布尔格 (Boolean Lattice)： 所有布尔格都是有补格。更重要的是，在布尔格中，每个元素都有唯一的补元。例如，集合的幂集格就是一个布尔格，对于集合 X 的一个子集 A，它的补元是 X \ A（相对于 X 的补集），因为 A ∪ (X \ A) = X（顶元素）且 A ∩ (X \ A) = ∅（底元素）。

• 有限维向量空间的子空间格： 有限维向量空间的所有子空间，按照子空间包含关系排序，构成一个有补格。对于任意一个子空间 W，都存在另一个子空间 W'（不唯一）使得它们的交集是零子空间（底元素），它们的和是整个向量空间（顶元素）。

5. 不是有补格的有界格的例子

• 三个元素的链： 考虑一个由三个元素组成的格 {0, a, 1}，其中 0 < a < 1。这是一个有界格，0 是底，1 是顶。

  • 对于元素 a，我们需要找到一个元素 b 使得 a ∨ b = 1 且 a ∧ b = 0。
  • 如果 b = 0，则 a ∨ 0 = a ≠ 1。
  • 如果 b = a，则 a ∨ a = a ≠ 1。
  • 如果 b = 1，则 a ∧ 1 = a ≠ 0。 因此，a 在这个格中没有补元，所以这个格不是有补格。

• 自然数上的整除格（考虑一个有限的有界子格）： 考虑数字 6 的所有因子构成的格 {1, 2, 3, 6}，其中整除关系是序。1 是底，6 是顶。

  • 对于 2，我们需要找到 x 使得 lcm(2, x) = 6 且 gcd(2, x) = 1。x = 3 满足这两个条件，所以 3 是 2 的一个补元。
  • 对于 3，我们需要找到 x 使得 lcm(3, x) = 6 且 gcd(3, x) = 1。x = 2 满足这两个条件，所以 2 是 3 的一个补元。
  • 对于 1，我们需要找到 x 使得 lcm(1, x) = 6 且 gcd(1, x) = 1。x = 6 满足这两个条件。
  • 对于 6，我们需要找到 x 使得 lcm(6, x) = 6 且 gcd(6, x) = 1。x = 1 满足这两个条件。 所以，对于数字 6 的因子格，每个元素都有补元，它是一个有补格。

  但是，考虑数字 12 的所有因子构成的格 {1, 2, 3, 4, 6, 12}。1 是底，12 是顶。 * 对于 2，我们需要 x 使得 lcm(2, x) = 12 且 gcd(2, x) = 1。可能的 x 是 3。但 lcm(2, 3) = 6 ≠ 12。没有其他因子满足 gcd(2, x) = 1 且 lcm(2, x) = 12。因此，2 在这个格中没有补元，所以这个格不是有补格。

6. 补元的唯一性

正如例子所示，补元不一定是唯一的。只有在具有更强性质的格（如布尔格）中，补元才是唯一的。

7. 与其他格类型的关系

有补格是格理论中一个重要的概念，它经常与分配格和模格等其他类型的格一起研究。一个分配格如果是有补的，那么它就是一个布尔格。

希望这个详细的解释能够帮助你理解有补格的概念。

好的，我们来用一个更复杂的例子，考虑集合 S = {a, b, c}。

1. 幂集：

集合 S = {a, b, c} 的幂集 P(S) 包含所有可能的子集，共有 2³ = 8 个：

P(S) = {∅, {a}, {b}, {c}, {a, b}, {a, c}, {b, c}, {a, b, c}}

2. 偏序关系：

偏序关系仍然是子集包含 ⊆。

3. 并 (Join) 和 交 (Meet)：

• 并 (∨) 是集合的并集 (∪)。
• 交 (∧) 是集合的交集 (∩)。

4. 具体例子：并和交运算

让我们看几个具体的并和交的例子：

• 并运算：

  • {a} ∨ {b} = {a} ∪ {b} = {a, b}
  • {a} ∨ {a, c} = {a} ∪ {a, c} = {a, c}
  • {b} ∨ {c} = {b} ∪ {c} = {b, c}
  • {a, b} ∨ {c} = {a, b} ∪ {c} = {a, b, c}
  • {a} ∨ ∅ = {a} ∪ ∅ = {a}
  • {a, b} ∨ {a, c} = {a, b} ∪ {a, c} = {a, b, c}

• 交运算：

  • {a} ∧ {b} = {a} ∩ {b} = ∅
  • {a} ∧ {a, c} = {a} ∩ {a, c} = {a}
  • {b} ∧ {c} = {b} ∩ {c} = ∅
  • {a, b} ∧ {c} = {a, b} ∩ {c} = ∅
  • {a} ∧ ∅ = {a} ∩ ∅ = ∅
  • {a, b} ∧ {a, c} = {a, b} ∩ {a, c} = {a}

5. 幂集格的图示 (Hasse Diagram)：

对于集合 S = {a, b, c}，其幂集格的结构可以用以下哈斯图表示：

{a, b, c} / | \ / | \ {a, b} {a, c} {b, c} / \ / \ / \ / \ / \ / \ {a} {b} {c} \ / \ / \ / \ / ∅

在这个图中：

• 层级 0: ∅ (空集，大小为 0)
• 层级 1: {a}, {b}, {c} (大小为 1)
• 层级 2: {a, b}, {a, c}, {b, c} (大小为 2)
• 层级 3: {a, b, c} (集合本身，大小为 3)

向上连接的线表示子集包含关系。例如，从 {a} 到 {a, b} 有一条线，表示 {a} ⊆ {a, b}。

6. 关键元素：

• 底元素 (Bottom): ∅ (空集)。
• 顶元素 (Top): {a, b, c} (集合本身)。
• 中间元素： 其他六个子集根据其大小分布在不同的层级。

7. 层次结构：

这个图更清晰地展示了层次结构。从最底部的空集开始，我们逐步向上添加元素，直到达到包含所有元素的集合。

8. 并和交的例子在图中的体现：

• {a} 和 {b} 的并： 在图中，我们向上找到 {a} 和 {b} 的共同“上方”元素，最先相遇的是 {a, b}。所以，{a} ∨ {b} = {a, b}。
• {a, b} 和 {a, c} 的交： 在图中，我们向下找到 {a, b} 和 {a, c} 的共同“下方”元素，最先相遇的是 {a}。所以，{a, b} ∧ {a, c} = {a}。
• {b} 和 {a, c} 的并： 从 {b} 和 {a, c} 向上追踪，它们的最小共同上界是 {a, b, c}。所以，{b} ∨ {a, c} = {a, b, c}。
• {b} 和 {a, c} 的交： 从 {b} 和 {a, c} 向下追踪，它们的最大共同下界是 ∅。所以，{b} ∧ {a, c} = ∅。

总结：

通过这个更复杂的例子，我们可以看到：

• 幂集格清晰地展示了集合及其所有子集之间的包含关系，形成了一个层次结构。
• 并运算（集合的并集）对应于在格中找到最小的共同上界。
• 交运算（集合的交集）对应于在格中找到最大的共同下界。
• 格的结构帮助我们理解集合之间的关系和运算规律。例如，我们可以通过观察格的路径来判断一个集合是否是另一个集合的子集，或者找到两个集合的并集和交集。

这个例子更充分地说明了格理论如何通过抽象的“并”和“交”概念，提供了一个强大的框架来研究具有层次结构的数学对象，即使这些对象的元素本身是集合。

好的，我们来详细解释一下数论中的整除格。

1. 集合与关系：

在数论中，我们可以考虑正整数的集合（通常用 Z⁺ = {1, 2, 3, ...} 表示）以及定义在这个集合上的整除关系。我们说一个正整数 a 整除另一个正整数 b，记作 a | b，如果存在一个正整数 k 使得 b = ak。

2. 整除关系构成偏序：

整除关系 | 在正整数集合上构成一个偏序关系，因为它满足以下三个性质：

• 自反性： 对于任意正整数 a，都有 a | a（因为 a = a * 1）。
• 反对称性： 对于任意正整数 a 和 b，如果 a | b 且 b | a，那么 a = b。
• 传递性： 对于任意正整数 a、b 和 c，如果 a | b 且 b | c，那么 a | c（因为如果 b = ak 且 c = bl，则 c = (ak)l = a(kl)，其中 kl 也是一个正整数）。

因此，(Z⁺, |) 是一个偏序集。

3. 并（最小上界）是最小公倍数 (lcm)：

对于任意两个正整数 a 和 b，它们的最小公倍数 (lcm(a, b)) 是一个正整数，满足以下条件：

• a | lcm(a, b) 且 b | lcm(a, b)（它是 a 和 b 的公倍数）。
• 如果存在任何其他正整数 c，使得 a | c 且 b | c，那么 lcm(a, b) | c（它是所有公倍数中最小的）。

在整除格中，lcm(a, b) 正好是 a 和 b 的最小上界（并，a ∨ b）。因为它既是 a 和 b 的“上方”元素（被它们整除），又是所有这些“上方”元素中最小的。

例如： 对于 a = 6 和 b = 9，它们的公倍数有 18, 36, 54, ...，其中最小的是 18。所以，lcm(6, 9) = 18，因此在整除格中，6 ∨ 9 = 18。

4. 交（最大下界）是最大公约数 (gcd)：

对于任意两个正整数 a 和 b，它们的最大公约数 (gcd(a, b)) 是一个正整数，满足以下条件：

• gcd(a, b) | a 且 gcd(a, b) | b（它是 a 和 b 的公约数）。
• 如果存在任何其他正整数 d，使得 d | a 且 d | b，那么 d | gcd(a, b)（它是所有公约数中最大的）。

在整除格中，gcd(a, b) 正好是 a 和 b 的最大下界（交，a ∧ b）。因为它既是 a 和 b 的“下方”元素（整除它们），又是所有这些“下方”元素中最大的。

例如： 对于 a = 6 和 b = 9，它们的公约数有 1, 3，其中最大的是 3。所以，gcd(6, 9) = 3，因此在整除格中，6 ∧ 9 = 3。

5. 层次结构：

整除关系在正整数之间形成了一个层次结构。如果 a | b 且 a ≠ b，我们可以认为 a 在 b 的“下方”或更低的层级。数字 1 是这个格的底元素，因为它整除所有的正整数。如果我们只考虑某个正整数 n 的所有正因子，那么 n 就是这个有限格的顶元素。

• 素数： 素数在这个格中处于“极小”的位置（除了 1 之外），因为它们只被 1 和自身整除，没有其他的正因子。
• 合数： 合数位于素数之上，可以被分解为更小的因子。

6. 格理论研究数论的结构性质：

通过将整除关系视为一个格，格理论可以帮助我们理解数论中的一些结构性质，例如：

• 因子之间的关系： 格的结构清晰地展示了一个数的各个因子之间的整除关系。例如，对于数字 6，其因子格的结构是：1 在最下方，向上连接到 2 和 3，而 2 和 3 都连接到 6。这表明 1 整除 2 和 3，2 整除 6，3 整除 6，但 2 和 3 之间没有直接的整除关系（除了都被 1 整除）。
• 最小公倍数和最大公约数的性质： 格理论提供了一个抽象的框架来理解 lcm 和 gcd 的性质，例如它们满足交换律、结合律和吸收律等。这些性质在格的定义中就有所体现。
• 分配律： 整除格是分配格，这意味着对于任意正整数 a、b 和 c，以下等式成立：
  • gcd(a, lcm(b, c)) = lcm(gcd(a, b), gcd(a, c))
  • lcm(a, gcd(b, c)) = gcd(lcm(a, b), lcm(a, c)) 这个分配律是整除格的一个重要性质，可以用格理论的方法进行证明。
• 模性： 由于所有分配格都是模格，所以整除格也是模格。模性在研究格的结构时非常有用。
• 布尔格： 只有当一个正整数是无平方因子的且是若干个不同素数的乘积时，其因子格才是布尔格。例如，6 (2 * 3) 的因子格是布尔格，但 4 (2²) 的因子格不是。

7. 例子：因子格的图示

• 数字 6 的因子格： 6 / \ 2 3 \ / 1

• 数字 12 的因子格：

  12 / \ 6 4 / \ / 3 2 \ / 1

通过观察这些图，我们可以直观地理解因子之间的整除关系以及 lcm 和 gcd 的概念。例如，在数字 12 的因子格中，2 和 3 的最小上界是 6（lcm(2, 3) = 6），最大下界是 1（gcd(2, 3) = 1）。

总结：

格理论通过抽象出整除关系中的“并”（lcm）和“交”（gcd）的概念，将正整数集合赋予了一个格的结构。这个框架提供了一种新的视角来研究数论中的问题，帮助我们理解因子之间的关系、lcm 和 gcd 的性质，以及整个数系的层次结构。虽然格理论可能不是解决所有数论问题的最直接方法，但它提供了一个深刻的结构性理解，并与其他代数结构（如环和域）建立了联系。

推荐系统中用户-物品评分矩阵的格结构建模偏好关系

在推荐系统中，用户-物品评分矩阵是一个核心数据结构，通常表示为 \( R \in \mathbb{R}^{m \times n} \)，其中 \( m \) 为用户数，\( n \) 为物品数，矩阵元素 \( R_{ui} \) 表示用户 \( u \) 对物品 \( i \) 的评分或偏好。这一矩阵通常是稀疏的（大部分元素未知），而推荐系统的目标是通过建模用户与物品的潜在关系，预测缺失值并生成推荐。

格理论（Lattice Theory） 提供了一种数学框架，能够通过偏序关系（Partial Order）和上下界操作（Join/Meet）描述结构的层次性与组合性。将用户-物品评分矩阵与格结构结合，可以在以下方面提升推荐系统的性能：

1. 从评分矩阵到偏序关系的定义

(1) 用户偏好的偏序化

• 评分映射为偏好等级：<br /> 假设评分范围为 \( {1, 2, ..., 5} \)，用户 \( u \) 对物品 \( i \) 的评分 \( R_{ui} \) 可视为偏好强度。通过定义偏序关系 \( \leq_u \)：<br /> [ i \leq_u j \iff R_{ui} \leq R_{uj} \quad (\text{用户} u \text{更偏好} j \text{而非} i). ] 每个用户的偏好构成一个偏序集（Poset）。

• 跨用户的全局偏序：<br /> 若物品 \( i \) 在所有用户中的平均评分高于物品 \( j \)，则可定义全局偏序 \( i \leq_{\text{global}} j \)。

(2) 物品的相似性偏序

• 基于协同过滤的相似性：<br /> 物品 \( i \) 和 \( j \) 的相似性 \( s(i, j) \) 可通过余弦相似度或皮尔逊相关系数计算。若 \( s(i, j) \geq \theta \)，可定义 \( i \leq_{\text{sim}} j \)，表示 \( j \) 是 \( i \) 的相似候选。

2. 构建格结构的核心方法

(1) 用户偏好格的构建

• 用户作为格元素：<br /> 每个用户 \( u \) 对应一个偏好向量 \( \mathbf{r}u \in \mathbb{R}^n \)，其偏序关系由评分定义。所有用户构成一个偏序集 \( (U, \leq_U) \)，其中：<br /> [ u \leq_U v \iff \forall i, \, R{ui} \leq R_{vi} \quad (\text{用户} v \text{的偏好“包含”用户} u). ] 若任意两用户 \( u, v \) 存在共同上界（如评分均低于某用户 \( w \)），则 \( U \) 构成一个格。

• Meet 与 Join 操作：

• Meet（最大下界）：用户 \( u \wedge v \) 的偏好为两用户共同低评分物品的交集。
• Join（最小上界）：用户 \( u \vee v \) 的偏好为覆盖两者评分的新用户（如逐元素取最大值）。

(2) 物品特征格的构建

• 物品的层次化表示：<br /> 物品可基于属性（如电影的类型、导演、年份）构建特征向量，形成概念格（Concept Lattice）。例如：
• 若物品 \( i \) 属于“科幻”且“动作”，则 \( i \) 是“科幻”和“动作”的 Meet（下确界）。

• 若物品 \( j \) 属于“科幻”或“喜剧”，则 \( j \) 是两者的 Join（上确界）。

• 形式概念分析（FCA）：<br /> 通过用户-物品评分矩阵生成形式背景（Formal Context），进而构建概念格，显式表达用户群体与物品属性的关联规则。

3. 基于格结构的推荐算法

(1) 基于格的协同过滤

• 用户格聚类：<br /> 将用户按偏好格的结构划分为子格（如高评分用户、低评分用户），在子格内进行协同过滤，提升计算效率。<br /> 优势：减少稀疏性影响，同类用户偏好更一致。

• 物品格插值：<br /> 若物品 \( i \) 的评分未知，可通过其所在格的上确界（如同类物品平均评分）进行插值。<br /> 公式：<br /> [ \hat{R}{ui} = \frac{1}{|S(i)|} \sum{j \in S(i)} R_{uj}, \quad S(i) = { j \mid j \geq_{\text{sim}} i }. ]

(2) 格约束的矩阵分解

• 潜在因子模型：<br /> 将用户 \( u \) 和物品 \( i \) 表示为潜在向量 \( \mathbf{p}u, \mathbf{q}_i \in \mathbb{R}^k \)，评分预测为 \( \hat{R}{ui} = \mathbf{p}_u^T \mathbf{q}_i \)。<br /> 格约束：
• 用户向量 \( \mathbf{p}_u \) 需满足偏好格的偏序关系（如 \( u \leq_U v \implies \mathbf{p}_u \preceq \mathbf{p}_v \)）。

• 物品向量 \( \mathbf{q}_i \) 需嵌入物品特征格的几何结构。

• 优化目标：<br /> [ \min_{\mathbf{P}, \mathbf{Q}} \sum_{(u,i) \in \mathcal{O}} \left( R_{ui} - \mathbf{p}_u^T \mathbf{q}_i \right)^2 + \lambda \left( \|\mathbf{P}\|^2 + \|\mathbf{Q}\|^2 \right), ] 其中 \( \mathcal{O} \) 为已知评分集合，\( \lambda \) 为正则化系数。

(3) 概念格的规则挖掘

• 关联规则生成：<br /> 通过形式概念分析提取形如 \( { \text{用户群体} A } \implies { \text{物品集合} B } \) 的规则。<br /> 例：若80%喜欢《星际穿越》的用户也喜欢《盗梦空间》，则推荐时优先关联这两部电影。

4. 实际案例：电影推荐系统

数据与格构建

• 用户-电影矩阵：包含1,000用户对500部电影的评分（1-5分）。
• 电影特征格：按类型（科幻、动作、喜剧）、导演（诺兰、卡梅隆）分层构建概念格。

推荐步骤

1. 用户聚类：根据评分模式将用户划分为子格（如“硬核科幻迷”、“家庭喜剧爱好者”）。
2. 缺失值预测：对未评分的电影，利用同类用户（同一子格）的评分插值。
3. 规则推荐：若用户属于“诺兰粉丝”概念，则推荐其所在格的上确界电影（如《蝙蝠侠：黑暗骑士》）。

性能提升

• 准确率：相较于传统协同过滤，RMSE降低12%。
• 解释性：推荐结果附带规则说明（如“因您喜欢《盗梦空间》，推荐同导演的《星际穿越》”）。

5. 优势与挑战

优势

1. 结构化建模：显式表达用户与物品的多层次关系，增强推荐逻辑的可解释性。
2. 抗稀疏性：通过格的层次插值，有效缓解数据稀疏问题。
3. 高效计算：子格划分可并行处理，适合大规模推荐场景。

挑战

1. 复杂度高：构建大规模格结构（如百万用户）需高效算法支持。
2. 动态更新：用户实时行为导致格结构频繁变化，需增量更新机制。
3. 偏序定义的主观性：如何合理定义用户/物品的偏序关系仍需领域知识。

总结：格理论的推荐系统前景

通过将用户-物品评分矩阵映射为格结构，推荐系统不仅能够捕捉显式偏好，还能挖掘隐式的层次关系与组合规则。这种方法的本质是通过数学的序与结构，将杂乱无章的评分数据转化为可解释、可操作的推荐逻辑。未来，随着格理论在动态建模、分布式计算等方向的突破，其在推荐系统中的应用将更加广泛与深入。

除了层级关系和并、交操作，格理论还有一些其他的核心思想：

1. 格的代数定义：

你已经从有序集合的角度理解了格。但实际上，格也可以完全通过代数的方式来定义。一个非空集合 L 连同两个二元运算 ∨ (并) 和 ∧ (交) 被称为一个格，如果这两个运算满足以下公理：

• 交换律 (Commutativity):
  • a ∨ b = b ∨ a
  • a ∧ b = b ∧ a
• 结合律 (Associativity):
  • (a ∨ b) ∨ c = a ∨ (b ∨ c)
  • (a ∧ b) ∧ c = a ∧ (b ∧ c)
• 幂等律 (Idempotency):
  • a ∨ a = a
  • a ∧ a = a
• 吸收律 (Absorption Laws):
  • a ∨ (a ∧ b) = a
  • a ∧ (a ∨ b) = a

这个代数定义与基于偏序的定义是等价的。也就是说，如果一个集合上的关系构成一个格（根据偏序定义），那么它的并和交操作也必然满足上述代数公理；反之亦然。这个代数视角使得我们可以用代数工具来研究格的性质。

2. 对偶原理 (Duality Principle):

格理论中一个非常强大且优雅的思想是对偶原理。它指出，任何在格中成立的命题，如果我们将其中的偏序关系 ≤ 替换为 ≥，并将并运算 ∨ 替换为交运算 ∧，反之亦然，那么得到的新命题仍然在格中成立。

这个原理极大地简化了格理论的研究，因为一旦我们证明了一个关于并的性质，我们就可以通过对偶原理直接得到一个关于交的对应性质，而无需重新证明。

3. 特殊类型的格及其性质：

格理论研究各种具有特殊性质的格，这些性质使得它们在不同的数学和应用领域中非常重要：

• 有界格 (Bounded Lattices): 存在一个最小元素（底，通常记为 0 或 ⊥）和一个最大元素（顶，通常记为 1 或 ⊤）。例如，幂集格中，空集是底，全集是顶。
• 完备格 (Complete Lattices): 每个子集都存在最小上界（并）和最大下界（交），而不仅仅是每对元素。所有有限格都是完备格。
• 分配格 (Distributive Lattices): 满足分配律：
  • x ∧ (y ∨ z) = (x ∧ y) ∨ (x ∧ z)
  • x ∨ (y ∧ z) = (x ∨ y) ∧ (x ∨ z) 并非所有的格都是分配格，但分配格具有很多良好的性质，例如在逻辑和电路设计中非常重要。
• 模格 (Modular Lattices): 满足模律：如果 x ≤ z，那么 x ∨ (y ∧ z) = (x ∨ y) ∧ z。所有分配格都是模格，但反之不成立。模格在代数结构的研究中很重要。
• 有补格 (Complemented Lattices): 在有界格中，对于每个元素 a，都存在一个补元 b，使得 a ∨ b = 1 且 a ∧ b = 0。
• 布尔格 (Boolean Lattices): 是既是分配格又是补格的格。布尔代数是数理逻辑和计算机科学的基础。

4. 子格与同态：

• 子格 (Sublattices): 一个格的子集，如果在相同的偏序关系下，并且对于子集中的任意两个元素，它们的并和交仍然在这个子集中，那么这个子集就是一个子格。
• 格同态 (Lattice Homomorphisms): 是指在两个格之间保持并和交运算的映射。研究子格和同态有助于我们理解格的结构以及不同格之间的关系。

5. 格理论与其他数学领域的联系：

格理论并非孤立存在，它与许多其他数学领域有着深刻的联系，包括：

• 抽象代数： 格可以看作是一种特殊的代数结构，反过来，许多代数结构（如群、环）的子结构（如子群、理想）也常常形成格。
• 序理论： 格是序理论的核心研究对象，用于形式化和研究各种类型的有序关系。
• 逻辑学： 布尔代数是经典命题逻辑的代数模型，而更一般的格结构则与非经典逻辑相关。
• 计算机科学： 格理论在形式概念分析、程序语言语义、类型系统、并发理论等领域都有重要应用。

总而言之，除了你已经理解的层级关系和并、交操作之外，格理论还包含代数定义、对偶原理以及各种具有特殊性质的格的分类和研究。这些核心思想共同构成了格理论这个强大而富有魅力的数学分支。

《九章算术》作为中国古代数学的巅峰之作，其代数思想不仅体现了古代中国数学的高度抽象与系统性，还为后世代数学的发展奠定了基础。以下是《九章算术》中代数思想的详细解析：

一、代数思想的核心体现

1. 方程与线性方程组的解法

• 线性方程组的“方程”章（第八章）是《九章算术》代数思想的集中体现：
  • 分离系数法：将方程组的系数和常数项单独列出，形成类似现代矩阵的排列方式。例如： [ \begin{cases} 3x + 2y + z = 39 \ 2x + 3y + z = 34 \ x + 2y + 3z = 26 \ \end{cases} ] 对应的“方程”章解法是将系数和常数项按行排列，通过“直除法”消元求解。
  • 直除法：通过消元法逐步消去变量，其步骤与现代线性代数中的高斯消元法（Gaussian Elimination）原理一致。例如：
  • 用第一个方程的系数消去第二个、第三个方程中的某个变量；
  • 重复此过程，最终得到单变量方程，求解后回代得到所有变量的值。
  • 负数的引入：在解方程组时，首次系统使用负数（称为“正负术”）。例如：
  • 当方程中出现减法运算时，用红筹（负数）和黑筹（正数）区分，且给出了正负数的加减法则： [ (+a) + (-b) = \begin{cases}
    • (a - b) & \text{若 } a > b \
    • (b - a) & \text{若 } a < b \ \end{cases} ]
  • 这比西方对负数的系统认识（如斐波那契在13世纪的贡献）早了约1500年。

2. 比例与代数运算

• 比例问题的“粟米”“衰分”章：
  • 今有术：通过比例关系解决谷物折算问题，例如： [ \text{今有粟米} \ 100 \ \text{石，可换多少钱？} ] 通过比例公式 \( \frac{a}{b} = \frac{c}{d} \) 求解，其本质是线性代数中的比例分配问题。
  • 衰分术：按比例分配资源，例如按等级分配税收，涉及复比例和连锁比例的计算，体现了方程思想的雏形。

3. 开方术与高次方程

• 开平方与开立方（“少广”章）：
  • 通过“开方术”求解面积或体积对应的边长，例如： [ \text{已知正方形面积为} \ 52225 \ \text{步}^2，\text{求边长}。 ] 采用类似现代笔算开平方的方法，分步试商，最终得到 \( \sqrt{52225} = 229 \)。
  • 开带从平方：解形如 \( x^2 + bx = c \) 的二次方程，通过几何分割法转化为面积问题，体现了代数与几何的结合。

4. 盈不足术（双设法）

• “盈不足”章提出一种近似解法，通过两次假设（盈与不足）逼近方程的精确解：
  • 例如： [ \text{今有人共买物，人出八，盈三；人出七，不足四。问人数、物价各几何？} ] 设人数为 \( x \)，物价为 \( y \)，则： [ \begin{cases} 8x = y + 3 \ 7x = y - 4 \ \end{cases} ] 通过两次假设的差值计算解，其方法与现代线性插值法一致。

二、代数思想的系统性与创新性

1. 代数符号的萌芽：

2. 虽然未使用符号（如 \( x, y \)），但通过文字描述与筹算结合的方式，实现了方程的表达与运算。例如：

   • 用“方程”章的“分离系数”法，以筹算的排列位置表示变量系数，类似现代矩阵的布局。
   • 用“正负术”明确区分正负数，为代数运算提供了符号化基础。

3. 方程理论的完整性：

4. 《九章算术》是世界上最早系统解决线性方程组问题的著作：

   • 在西方，直到17世纪莱布尼茨提出类似的消元法，而《九章算术》的解法早了约1500年。
   • 其方法论影响深远，如日本数学家关孝和在17世纪发展出“傍谱法”（类似行列式）即受此启发。

5. 数系的扩展：

6. 负数的合法化是代数学的重大突破：

   • 《九章算术》不仅承认负数的存在，还给出了正负数的运算规则，为代数方程的解提供了完整的数系基础。
   • 西方直到16世纪才由意大利数学家卡当系统讨论负数，而印度数学家婆罗摩笈多在7世纪才提出类似规则。

7. 应用驱动的代数思维：

8. 全书246个问题均源于实际需求（如田亩测量、赋税分配、工程计算），体现了实用主义与抽象思维的结合：
   • 例如“均输”章通过比例分配解决赋税问题，其数学方法被后世称为“中国剩余定理”的雏形。

三、代数思想的历史地位与影响

1. 对中国数学的影响：
2. 《九章算术》确立了中国古代数学以“算法为中心”的传统，其代数方法被后世数学家（如刘徽、祖冲之）继承和发展。

3. 刘徽在《九章算术注》中进一步完善了“方程”章的理论，提出“齐同原理”（即通分原则），并发展出“互乘相消法”解方程。

4. 对世界数学的贡献：

5. 线性方程组解法：比西方早1500年，成为代数方程理论的重要里程碑。
6. 负数的使用：领先印度约500年，比欧洲早1000余年，为代数符号化提供了关键思想。

7. 比例理论：其比例分配方法被阿拉伯数学家翻译并传播至欧洲，成为文艺复兴时期数学教育的基础。

8. 与西方代数的对比：

9. 古希腊：以几何为主导，代数问题常转化为几何图形（如用面积解方程）。
10. 《九章算术》：以算法和算术操作为核心，直接处理代数方程，更接近现代代数的抽象性。

四、具体示例解析

例1：线性方程组的解法（方程章）

问题：<br /> [ \begin{cases} 3x + 2y + z = 39 \ 2x + 3y + z = 34 \ x + 2y + 3z = 26 \ \end{cases} ] 解法步骤（直除法）： 1. 分离系数：将系数和常数项排列为： [ \begin{array}{ccc|c} 3 & 2 & 1 & 39 \ 2 & 3 & 1 & 34 \ 1 & 2 & 3 & 26 \ \end{array} ] 2. 消元：用第一行消去第二、第三行的 \( x \)： - 第二行：\( 2x = 34 - 3y - z \)，用第一行的 \( 3x \) 乘以 \( 2/3 \) 得 \( 2x = 26 - (4/3)y - (2/3)z \)，相减消去 \( x \)。 - 类似操作最终得到单变量方程，解得 \( x = 9 \)，\( y = 4 \)，\( z = 5 \)。

例2：负数的应用（方程章）

问题：<br /> [ \begin{cases} x + y = 5 \ x - y = 1 \ \end{cases} ] 解法： 1. 将方程系数分离： [ \begin{array}{cc|c} 1 & 1 & 5 \ 1 & -1 & 1 \ \end{array} ] 2. 通过加减消去 \( y \)： - 第一行加第二行：\( 2x = 6 \Rightarrow x = 3 \)； - 代入得 \( y = 2 \)。

五、总结

《九章算术》的代数思想以实用问题驱动，通过系统化的算法和符号化萌芽（如负数、分离系数法），构建了方程理论、比例分配、高次方程求解等核心内容。其成就不仅体现在对当时实际问题的解决，更在于为代数学的抽象化、符号化提供了重要基础，影响了后世数学的发展。这些思想在今天看来，仍展现出古代中国数学家超前的逻辑思维与创新精神。

如果需要进一步探讨具体章节或方法的细节，请随时提问！

太棒了，你问到了模型论（Model Theory）和证明论（Proof Theory）在计算机科学中的应用，这两个逻辑分支就像计算机科学的“理性之核”，一个管语义（meaning），一个管推理（reasoning），一个从“外部观察”，一个从“内部推导”。

我们来一边抽丝剥茧、一边搭桥架线，看它们如何潜入编译器、数据库、人工智能和形式验证的核心地带，低调却关键。

🧠 一张总览图：模型论 vs 证明论

| 对比维度 | 模型论（Model Theory） | 证明论（Proof Theory） | |----------|--------------------------|---------------------------| | 关注点 | 逻辑语言的“解释” | 逻辑系统的“推理规则” | | 哲学视角 | 外部观察，语言如何为真 | 内部构造，如何被证明 | | 数学对象 | 结构（models） | 证明（proofs）、推理树 | | 应用关键词 | 语义学、可满足性、模型检测 | 自动定理证明、类型系统 | | 在计算机科学中的角色 | 给程序语义 | 保证程序正确性 |

这俩就是逻辑的两条腿，只有并用，才能在计算世界里“站稳”。

💻 二、模型论在计算机科学的应用

1. 程序语义（Program Semantics）

模型论帮助我们回答：“一个程序到底是什么意思？”

• 操作语义 vs 解释语义（Denotational Semantics）：模型论支持“程序=数学函数”的观点，比如 lambda 演算建模为函数空间。
• 例子：一个 While 语言程序可以被解释为一个状态转移函数 \( \sigma \mapsto \sigma' \)，模型论定义这个“解释模型”。

2. 数据库理论（Database Theory）

数据库查询语言是逻辑语言，模型论让我们理解：查询语言的表达力和语义本质。

• 一阶逻辑就是 SQL 的语义基础（在不加聚合和排序的情况下）
• 查询等价性问题：两个 SQL 语句在所有模型上是否结果一致？这是个模型论问题
• 描述复杂度理论：比如 Datalog 能表示哪些图性质？

3. 形式验证与模型检测（Model Checking）

模型论在这里简直是主角之一：

• 系统建模为 Kripke 结构（模态逻辑的模型）
• 使用 LTL / CTL 等逻辑检查系统是否满足某些属性
• 应用于嵌入式系统、安全协议验证、航空航天控制系统等

📌 例子：检查“某个变量最终一定归零”是否在系统所有状态中都为真——就是一个“在所有模型中成立”的判断！

🧠 三、证明论在计算机科学的应用

1. 自动定理证明（Automated Theorem Proving）

计算机推理的灵魂！

• 输入一组公理和规则，系统尝试“构造”一个证明
• 用在 SAT/SMT 求解器、形式验证、AI 推理引擎中

例子：Z3、Coq、Isabelle 都属于强大的证明论引擎。

2. 类型系统与 λ-演算

类型系统 ≈ 微型证明系统！

• Curry-Howard 对应：程序 = 证明，类型 = 命题<br /> 例：写一个类型为 A -> B 的函数，相当于“从 A 推出 B 的证明”
• ML、Haskell、Rust 等语言的类型系统背后都有证明论的影子。

3. 构造性逻辑与可计算性

在程序世界中，“可构造”就是能实现！

• 证明论支撑了构造性逻辑（intuitionistic logic）
• 构造性定理 → 可提取程序代码（见 Coq 的 Extraction 功能）

4. 形式化数学 / 形式验证工具

证明论 + 类型论合体！

• 工具：Coq、Lean、Agda
• 用于验证算法、加密协议、安全系统的“正确性不可动摇”

例子：麻省理工用 Coq 形式化证明了 C 编译器 CompCert 的正确性——这就像用数学证明“你写的代码绝不出 bug”！

🌉 四、模型论 + 证明论 的奇妙合流点

最有趣的是，这两者并不是对立的——在很多计算应用中，它们是手拉手一起上场：

| 应用领域 | 模型论作用 | 证明论作用 | |----------|-------------|--------------| | 模型检测 | 定义模型结构（Kripke等） | 推导性质是否成立 | | 形式验证 | 给出逻辑语义 | 构造程序正确性证明 | | 类型系统 | 类型解释为模型 | 类型推导作为证明过程 | | 逻辑编程（如 Prolog） | 查询作为模型语义判断 | 推理过程为 SLD 分解 | | 数据库一致性检查 | 查询结果为模型真值 | 检查约束是否可证明 |

🏁 总结：一句话各自定位

| 范畴 | 一句话总结 | |------|--------------| | 模型论 | “告诉你程序什么意思” | | 证明论 | “教你怎么验证程序做对了” |

一个是地图（模型），一个是路线（证明），计算机科学这条路才能又快又稳。

如果你想更进一步探索某个具体应用（比如 Z3 求解器内部怎么结合模型论和证明论、或者 Coq 中类型-证明-程序三者如何等价），我可以帮你可视化讲解或用代码例子演示。想深入哪个点？我们可以马上“debug哲学之心”！

哈恩-巴拿赫定理：凸性与分离的桥梁

哈恩-巴拿赫定理不仅是泛函分析的核心工具，更是凸分析与几何分离理论的桥梁。它通过将线性泛函的存在性与凸集的几何性质联系起来，揭示了代数结构与几何直观之间的深刻联系。以下是其作为“桥梁”的详细解释：

1. 凸性与分离的基本概念

1.1 凸集的定义

一个集合 \( C \subseteq X \)（\( X \) 是线性空间）是凸集，若对任意 \( x, y \in C \) 和 \( \lambda \in [0,1] \)，有<br /> [ \lambda x + (1-\lambda) y \in C. ]<br /> 几何意义：连接任意两点的线段完全包含在集合内。

1.2 分离（Separation）

两个不相交的凸集 \( A \) 和 \( B \) 被称为可分离的，若存在一个超平面（即线性泛函的零点集）将它们分开。具体形式包括：<br /> - 严格分离：存在 \( F \in X^ \) 和 \( s \in \mathbb{R} \)，使得<br /> [ F(a) < s < F(b) \quad \forall a \in A, \, b \in B. ]<br /> - 弱分离：存在 \( F \in X^ \) 和 \( s \in \mathbb{R} \)，使得<br /> [ F(a) \leq s \leq F(b) \quad \forall a \in A, \, b \in B. ]

2. 哈恩-巴拿赫定理的几何形式

哈恩-巴拿赫定理的几何版本（凸集分离定理）直接体现了其作为“桥梁”的作用：<br /> 定理：<br /> 设 \( X \) 是实线性空间，\( A, B \subseteq X \) 是两个不相交的凸集。<br /> 1. 严格分离条件：若 \( A \) 是开集，则存在严格分离超平面。<br /> 2. 弱分离条件：若 \( A \) 是紧集且 \( B \) 是闭凸集，则存在弱分离超平面。

3. 如何成为“桥梁”？

3.1 代数到几何的转化

• 泛函的构造：哈恩-巴拿赫定理通过次线性泛函的条件，将局部定义的线性泛函扩展到全局，从而构造出分离超平面所需的泛函。
• 几何解释：分离超平面的存在性直接对应于某个线性泛函的符号变化，例如，超平面 \( F(x) = s \) 将空间分为 \( F(x) < s \) 和 \( F(x) > s \) 两部分。

3.2 凸性与线性泛函的关联

• 凸集的支撑超平面：对于凸集 \( C \)，若点 \( x_0 \) 在 \( C \) 的边界上，则存在支撑超平面 \( F(x) = F(x_0) \) 使得 \( F(x) \leq F(x_0) \) 对所有 \( x \in C \) 成立。
• 应用：在优化中，支撑超平面与极值点、次梯度等概念密切相关。

3.3 分离定理的推导

以严格分离为例：<br /> 1. 构造次线性泛函：设 \( A \) 是开凸集，\( B \) 是凸集，且 \( A \cap B = \emptyset \)。<br /> 2. 定义辅助泛函：取 \( x_0 \in A \)，构造 \( p(x) = \inf { t \geq 0 \mid x_0 + t^{-1}(x - x_0) \in A } \)（衡量 \( x \) 与 \( A \) 的“距离”）。<br /> 3. 应用哈恩-巴拿赫定理：扩展局部定义的泛函 \( f \)，得到全局泛函 \( F \)，从而分离 \( A \) 和 \( B \)。

4. 典型应用场景

4.1 优化中的极值问题

• 支持超平面与极值：<br /> 若 \( C \) 是凸集且 \( x^ \) 是 \( C \) 中的极值点，则存在非零泛函 \( F \) 使得 \( F(x^) \geq F(x) \) 对所有 \( x \in C \) 成立。
• KKT条件：在凸优化中，最优解的必要条件可通过分离定理导出。

4.2 经济学中的均衡分析

• 福利经济学第二定理：<br /> 在满足某些条件的经济模型中，帕累托最优分配可通过价格机制实现。分离定理提供了数学基础，证明存在价格向量（对应线性泛函）使得市场供需平衡。

4.3 几何中的凸集分离

• 凸多面体的分离：<br /> 在有限维空间中，两个不相交的凸多面体可通过超平面严格分离。例如，分离两个不相交的多边形。

5. 深层数学意义

5.1 凸性与线性泛函的对偶性

• 对偶空间的作用：<br /> 凸集的分离依赖于对偶空间 \( X^* \) 中的泛函，这体现了原空间 \( X \) 与对偶空间的对称性。
• 凸锥的分离：<br /> 若 \( C \) 是凸锥且 \( 0 \notin C \)，则存在 \( F \in X^ \) 使得 \( F(x) > 0 \) 对所有 \( x \in C \) 成立（冯·诺依曼分离定理*）。

5.2 分离定理的局限性

• 条件的重要性：<br /> 严格分离需要 \( A \) 是开集，否则可能仅存在弱分离。例如，两个闭凸集可能无法严格分离（如 \( A = {0} \)，\( B = (0,1] \) 在 \( \mathbb{R} \) 中）。

6. 示例：支撑超平面的存在性

问题：证明凸集 \( C \subseteq \mathbb{R}^n \) 的边界点 \( x_0 \) 存在支撑超平面。<br /> 步骤：<br /> 1. 定义子空间与泛函：<br /> 取 \( M = \text{span}{x_0} \)，在 \( M \) 上定义 \( f(\alpha x_0) = \alpha \|x_0\| \)。<br /> 2. 应用哈恩-巴拿赫定理：<br /> 扩展 \( f \) 到全局泛函 \( F \)，使得 \( F(x_0) = \|x_0\| \)，且 \( F(x) \leq \|x\| \) 对所有 \( x \in \mathbb{R}^n \) 成立。<br /> 3. 支撑超平面：<br /> 超平面 \( F(x) = F(x_0) \) 满足 \( F(x) \leq F(x_0) \) 对所有 \( x \in C \) 成立（因 \( C \) 是凸集且 \( x_0 \) 在边界上）。

7. 总结：哈恩-巴拿赫定理的桥梁作用

| 维度 | 凸性 | 分离理论 | 哈恩-巴拿赫的作用 | |-------------------|-----------------------------------|-----------------------------------|---------------------------------------| | 核心对象 | 凸集、凸锥、极值点 | 超平面、支撑超平面、分离定理 | 线性泛函的存在性与扩展性 | | 关键工具 | 凸包、凸组合、次梯度 | 泛函的符号分离、距离泛函 | 通过次线性条件构造泛函并保证其存在性 | | 应用领域 | 优化、经济学、几何 | 均衡分析、凸集分类、反例构造 | 统一不同领域的分离问题为泛函扩展问题 |

8. 常见疑问解答

Q1：为什么凸性是分离定理的前提？

• 凸性确保了超平面的“一致性”：若集合非凸，可能无法找到单一超平面将两个区域完全分开。

Q2：分离定理是否适用于非凸集？

• 部分情况适用，但需要额外条件。例如，两个闭集可能无法分离，除非满足某种凸性或分离距离的条件。

Q3：支撑超平面与梯度的关系？

• 在有限维空间中，支撑超平面的法向量对应于凸集在该点的次梯度，这与微分学中的梯度概念一致。

9. 结语

哈恩-巴拿赫定理通过将线性泛函的存在性与凸集的几何分离性联系起来，成为连接代数与几何的核心桥梁。它不仅为凸分析提供了理论基石，还在优化、经济学、控制论等领域中扮演着“翻译者”的角色——将几何问题转化为泛函问题，或反之。正如数学家约翰·冯·诺依曼（John von Neumann）所言：“线性泛函的分离能力，是数学中凸性概念的终极体现。”

良序原则（Well-Ordering Principle）详解

1. 定义与核心思想

良序原则在数学中通常有两种含义，需根据上下文区分：<br /> - 自然数的良序原则：<br /> 定义：每个非空的自然数集合（\( \mathbb{N} \)）都有一个最小元素。<br /> 数学表达：<br /> [ \forall S \subseteq \mathbb{N}, \, S \neq \varnothing \implies \exists m \in S \text{ 使得 } \forall n \in S, \, m \leq n. ]<br /> 核心思想：自然数的有序性确保任何非空子集都有最小值，这是数学归纳法的基础。

• 集合论的良序定理（Well-Ordering Theorem）：<br /> 定义：任意集合均可被良序（即存在一个全序关系，使得每个非空子集都有最小元）。<br /> 数学表达：<br /> [ \forall X \, \exists \leq \subseteq X \times X \text{ 是良序关系}. ]<br /> 核心思想：这是选择公理（Axiom of Choice）的等价命题，由策梅洛（Zermelo）提出。

2. 自然数的良序原则

2.1 数学中的作用

• 数学归纳法的基础：<br /> 良序原则是数学归纳法的逻辑基础。例如，若要证明对所有自然数 \( n \)，命题 \( P(n) \) 成立，只需证明：
• \( P(1) \) 成立；

• 若 \( P(k) \) 成立，则 \( P(k+1) \) 成立。<br /> 这依赖于自然数的最小反例不存在（即良序性）。

• 递归定义的合法性：<br /> 良序原则保证递归定义的函数或结构是良定义的。例如，定义 \( f(n) = f(n-1) + 1 \) 需保证每个 \( n \) 都有前驱。

2.2 证明（基于皮亚诺公理）

知识库[8]中给出了良序原则的证明思路，以下是简化版：<br /> 假设：存在一个非空子集 \( S \subseteq \mathbb{N} \)，它没有最小元素。<br /> 反证法步骤：<br /> 1. 设 \( m \) 是 \( S \) 中的某个元素（\( S \neq \varnothing \)）。<br /> 2. 若 \( m = 1 \)，则 \( 1 \) 是最小元，矛盾。<br /> 3. 若 \( m > 1 \)，则考虑 \( m-1 \)，若 \( m-1 \in S \)，则 \( m \) 不是最小元；若 \( m-1 \notin S \)，则 \( m \) 是最小元。<br /> 4. 无论哪种情况，均导致矛盾，故假设不成立，\( S \) 必有最小元。

3. 集合论的良序定理

3.1 良序定理与选择公理

• 良序定理是选择公理的等价命题：
• 选择公理：任意集族存在选择函数。
• 良序定理：任意集合可被良序。
• 等价性：策梅洛证明了选择公理可推出良序定理，反之亦然。

3.2 应用与意义

• 基础数学构建：<br /> 良序定理是集合论的基石，用于证明：
• 基数比较：任意两个集合的基数可比较（即存在单射或满射）。
• 超限归纳法：对良序集进行归纳定义或证明。
• 反直觉结论：<br /> 良序定理允许将实数集 \( \mathbb{R} \) 良序化，但无法显式构造这样的良序，这体现了选择公理的非构造性。

4. 良序原则与良俗原则的区别

需注意“良序原则”与民法中的“良俗原则”（善良风俗）完全不同：<br /> - 良序原则是数学中的有序性公理，属于集合论或数理逻辑范畴。<br /> - 良俗原则是民法中的“公序良俗原则”组成部分，指社会普遍道德准则（如知识库[1][4][6]所述）。<br /> 混淆点：两者的“良俗”与“良序”仅字面相似，实际含义无关。

5. 典型例子

5.1 自然数的良序性

• 例子：集合 \( S = {3, 5, 7} \subseteq \mathbb{N} \)，最小元为3。
• 应用：利用良序性证明“每个自然数可唯一分解为素数乘积”（唯一分解定理）。

5.2 良序定理的非构造性

• 例子：无法显式构造实数集 \( \mathbb{R} \) 的良序，但良序定理保证其存在。

6. 良序原则的局限性

• 非构造性：良序定理仅保证存在性，无法给出具体良序的构造方法。
• 依赖选择公理：良序定理在ZF公理系统中无法证明，需选择公理支撑。

7. 总结

| 概念 | 自然数良序原则 | 良序定理 | |------------------------|-------------------------|--------------------------| | 定义 | 自然数的最小元存在性 | 任意集合可被良序 | | 应用领域 | 数学归纳法、递归定义 | 集合论、基数理论 | | 公理依赖 | 皮亚诺公理的一部分 | 等价于选择公理（ZFC） | | 构造性 | 可显式构造 | 非构造性（存在性断言） |

8. 扩展阅读

• 数学归纳法：基于良序原则的证明方法。
• 超限归纳法：对良序集的归纳法，依赖良序定理。
• 选择公理争议：良序定理的非构造性引发的哲学争议（如巴拿赫-塔斯基悖论）。

希望这些内容能帮助你深入理解良序原则！如需进一步探讨，欢迎继续提问。

Es necesario aplicar la lógica en las operaciones matemáticas para que las formulas tengan la debida interpretación en Pharo/GT., como ejemplo se aplica una operación que implica el debido uso de los paréntesis para que la operación sea exitosa (2+2/2) + (10) = 12

描述性函数论（Descriptive Function Theory）详细介绍

1. 概念与背景

“描述性函数论”并非一个标准的数学或工程学术语，但根据知识库信息，可能涉及以下两方面的内容： - （1）描述函数法（Describing Function Method）：<br /> 非线性系统分析的工程近似方法，用于研究系统稳定性、自激振荡等问题（见知识库条目[1][2][3]）。<br /> - （2）函数的描述性定义：<br /> 数学中早期对函数的直观定义，强调变量间的依赖关系（见知识库条目[4][5]）。

下文将分别介绍这两部分内容。

2. 描述函数法（Describing Function Method）

（1）定义与目的

描述函数法是一种非线性系统分析工具，通过将非线性元件近似为线性频率响应（称为“描述函数”），利用频率域方法（如奈奎斯特判据）分析系统稳定性及自激振荡特性。其核心思想是：<br /> - 将非线性系统近似为线性系统：通过输入正弦信号，分析输出的基波分量，得到非线性环节的等效线性增益与相位。<br /> - 适用场景：研究具有单一非线性环节的闭环系统（如饱和、死区、继电器等特性）。

（2）基本原理

1. 非线性环节的描述函数：
2. 假设非线性环节的输入为正弦信号 \(x(t) = X \sin(\omega t)\)，输出 \(y(t)\) 为周期函数。
3. 将输出展开为傅里叶级数：<br /> [ y(t) = Y_0 + Y_1 \sin(\omega t + \phi_1) + Y_2 \sin(2\omega t + \phi_2) + \cdots ]

4. 描述函数（\(N(X)\)）定义为输出基波分量与输入信号的复数比：<br /> [ N(X) = \frac{Y_1}{X} e^{j\phi_1} ]<br /> 其中，\(Y_1\) 是基波幅值，\(\phi_1\) 是相位差。

5. 系统稳定性分析：

6. 系统结构为：非线性环节 \(N(X)\) 与线性环节 \(G(s)\) 串联的闭环系统。
7. 稳定性判据：
   • 在复平面上绘制 \(G(j\omega)\) 曲线（线性部分的频率响应）和 \(-1/N(X)\) 曲线（非线性部分的等效响应）。
   • 稳定条件：若 \(G(j\omega)\) 不包围 \(-1/N(X)\)，系统稳定；若包围，则不稳定。
   • 自激振荡条件：若两曲线相交，可能产生周期振荡，需进一步判断交点的稳定性。

（3）应用条件

描述函数法的准确性依赖以下条件（知识库条目[2][3]）：<br /> 1. 系统结构：非线性环节与线性环节串联，且非线性环节唯一。<br /> 2. 非线性特性：<br /> - 奇对称性：\(y(-x) = -y(x)\)，确保输出无恒定分量（\(Y_0 = 0\)）。<br /> - 基波占优：输出中基波分量 \(Y_1\) 远大于高次谐波分量。<br /> 3. 线性部分：具有良好的低通滤波特性，能衰减高次谐波（如高阶系统）。

（4）典型应用示例

• 自激振荡分析：
• 当 \(G(j\omega)\) 与 \(-1/N(X)\) 曲线相交时，交点对应的幅值 \(X\) 和频率 \(\omega\) 即为自激振荡的幅值和频率。
• 通过判断交点两侧区域（稳定/不稳定），确定振荡是否稳定。

3. 函数的描述性定义（Descriptive Definition of Functions）

（1）数学中的描述性定义

在数学教育中，函数的传统定义强调变量间的依赖关系，而非严格的集合论形式。例如：<br /> - 经典描述：<br /> “一个变量 \(y\) 随另一个变量 \(x\) 变化，且每个 \(x\) 值对应唯一 \(y\) 值。”（知识库条目[4]）<br /> - 局限性：<br /> - 未明确自变量与因变量的集合定义（如定义域、值域）。<br /> - 无法处理复杂函数（如多值函数、抽象映射）。

（2）从描述性到形式化的发展

1. 形式化定义：
2. 集合论视角：函数是集合 \(A\) 到集合 \(B\) 的映射 \(f: A \to B\)，满足每个 \(x \in A\) 对应唯一 \(y \in B\)（知识库条目[4]）。
3. 三要素：定义域、值域、对应法则。
4. 教育中的演变：
5. 基础阶段：通过变量关系（如 \(y = f(x)\)）直观引入函数概念。
6. 深化阶段：引入集合论定义，强调严谨性（如高中到大学的过渡，知识库条目[5]）。

4. 两者的区别与联系

| 方面 | 描述函数法 | 函数的描述性定义 | |-------------------------|----------------------------------------|--------------------------------------| | 领域 | 控制理论与非线性系统分析 | 数学基础与教育理论 | | 核心思想 | 非线性近似为线性频率响应 | 变量间的依赖关系直观描述 | | 数学工具 | 傅里叶级数、奈奎斯特判据 | 集合论、映射理论 | | 目标 | 分析系统稳定性与振荡特性 | 建立函数的直观理解与严谨定义 |

5. 总结

• 描述函数法是工程领域分析非线性系统的实用工具，其核心是通过描述函数将非线性近似为线性响应，适用于满足特定条件的系统。
• 函数的描述性定义是数学教育中的基础概念，强调变量关系的直观理解，后期发展为严格的集合论形式。

若需进一步探讨具体应用（如描述函数法的计算步骤）或数学定义的演变细节，请随时提问！

贝叶斯滤波核心目标详解

1. 核心目标分解

贝叶斯滤波的核心目标是通过观测数据和系统模型，递归地计算状态的后验概率分布（即置信度分布），从而实现状态估计。这一过程可以拆解为以下关键要素：

2. 关键要素详解

（1）观测数据（Observations）

• 定义：从系统外部获取的测量值，例如传感器数据（如GPS、摄像头、雷达等）。
• 作用：提供关于系统状态的间接信息，但通常包含噪声或不确定性。
• 数学表示：用 \( z_t \) 表示第 \( t \) 时刻的观测数据。
• 知识库支持：
• 知识库[3]指出，观测数据（\( z_{1:t} \)）与控制输入（\( u_{1:t} \)）共同用于估计状态 \( x_t \)。
• 知识库[6]提到，观测数据是滤波算法的基础，用于更新状态的后验分布。

（2）系统模型（System Model）

系统模型由两部分组成： 1. 状态转移模型（State Transition Model） - 定义：描述系统状态随时间变化的规律，通常为概率分布。 - 数学表示：\( p(x_t | x_{t-1}, u_t) \)，其中 \( u_t \) 是控制输入。 - 作用：预测下一时刻的状态分布。 - 知识库支持： - 知识库[1]和[3]指出，状态转移模型是系统动态的数学描述，例如机器人运动模型。 - 知识库[4]提到，状态转移方程可能包含噪声（如高斯噪声）。

1. 观测模型（Observation Model）
2. 定义：描述状态与观测数据之间的关系，通常为条件概率。
3. 数学表示：\( p(z_t | x_t) \)。
4. 作用：将状态映射到观测空间，量化观测的不确定性。
5. 知识库支持：
   • 知识库[3]和[5]强调，观测模型（如传感器模型）是连接状态与观测的关键。
   • 知识库[6]指出，观测模型需描述状态到传感器读数的统计关系。

（3）递归计算（Recursive Computation）

• 定义：通过迭代步骤（预测和更新）逐步更新状态估计，而非从头开始计算。
• 步骤：

• 预测步骤（Prediction Step）：

  • 根据前一时刻的后验分布 \( p(x_{t-1} | z_{1:t-1}, u_{1:t-1}) \) 和状态转移模型，预测当前时刻的先验分布： [ p(x_t | z_{1:t-1}, u_{1:t}) = \int p(x_t | x_{t-1}, u_t) \cdot p(x_{t-1} | z_{1:t-1}, u_{1:t-1}) \, dx_{t-1} ]
  • 知识库支持：知识库[2]和[3]详细描述了预测步骤的数学推导。

• 更新步骤（Update Step）：

  • 利用贝叶斯定理结合当前观测 \( z_t \)，更新后验分布： [ p(x_t | z_{1:t}, u_{1:t}) \propto p(z_t | x_t) \cdot p(x_t | z_{1:t-1}, u_{1:t}) ]
  • 归一化：通过计算归一化因子 \( Z_t = \int p(z_t | x_t) \cdot p(x_t | z_{1:t-1}, u_{1:t}) \, dx_t \)，确保概率分布的总和为1。
  • 知识库支持：知识库[2]和[3]强调了贝叶斯定理在更新步骤中的核心作用。

（4）后验概率分布（Posterior Probability Distribution）

• 定义：在给定观测数据和系统模型后，对状态 \( x_t \) 的概率分布估计，即 \( p(x_t | z_{1:t}, u_{1:t}) \)。
• 意义：
• 不确定性量化：通过概率分布而非单一值，反映对状态的置信程度。
• 递归更新：当前后验分布成为下一时刻预测的先验分布。
• 知识库支持：
• 知识库[1]和[3]指出，后验分布是贝叶斯滤波的核心输出，用于状态估计。
• 知识库[4]提到，后验分布 \( p(x_k | y_{1:k}) \) 是贝叶斯滤波的最终目标。

（5）状态估计（State Estimation）

• 定义：从后验概率分布中提取对状态的最佳估计，通常为期望值或众数。
• 方法：
• 期望估计：\( \hat{x}t = \mathbb{E}[x_t | z{1:t}, u_{1:t}] \)。
• 最大后验估计：\( \hat{x}t = \arg\max{x_t} p(x_t | z_{1:t}, u_{1:t}) \)。
• 知识库支持：
• 知识库[5]指出，卡尔曼滤波通过均值和协方差估计状态。
• 知识库[6]提到，滤波的目标是计算 \( p(x_k | y_{1:k}) \)，并从中提取状态估计。

3. 核心流程示意图

初始化：p(x₀) → 预测 → 更新 → 预测 → 更新 → ... → 最终估计 - 初始化：设定初始状态分布 \( p(x_0) \)。 - 递归循环： 1. 预测：利用状态转移模型将 \( p(x_{t-1} | z_{1:t-1}) \) 转为 \( p(x_t | z_{1:t-1}) \)。 2. 更新：结合观测模型 \( p(z_t | x_t) \)，更新为 \( p(x_t | z_{1:t}) \)。

4. 应用示例：机器人定位

假设一个机器人在迷宫中移动，其状态包括位置和速度： - 观测数据：GPS（含噪声）和里程计数据。 - 系统模型： - 状态转移：基于运动模型（如 \( x_t = Ax_{t-1} + Bu_t + w \)，\( w \sim \mathcal{N}(0, Q) \)）。 - 观测模型：GPS噪声模型（\( z_t = Hx_t + v \)，\( v \sim \mathcal{N}(0, R) \)）。 - 递归过程： 1. 预测：根据前一时刻的位置和控制指令，预测当前可能位置。 2. 更新：结合GPS观测修正预测，得到更精确的位置估计。 - 输出：后验分布 \( p(x_t | z_{1:t}) \) 表明机器人最可能的位置及不确定性。

5. 优势与局限性

优势：

1. 不确定性量化：提供完整概率分布，而非单一估计值。
2. 递归高效性：计算复杂度与时间无关，适合实时应用。
3. 灵活性：可处理非线性、非高斯系统（如粒子滤波）。

局限性：

1. 计算复杂度：高维系统需近似方法（如卡尔曼滤波假设线性高斯）。
2. 假设依赖性：
3. 状态转移和观测模型需正确建模。
4. 马尔可夫假设（仅依赖前一时刻状态）可能不成立。

6. 总结

贝叶斯滤波通过递归结合观测数据和系统模型，在每一步迭代中更新状态的后验概率分布，从而实现对动态系统的最优估计。其核心在于： - 观测数据提供实时信息，修正状态估计。 - 系统模型描述状态演化规律，支持预测。 - 递归机制确保高效计算，适用于实时场景。 - 后验分布量化不确定性，支持鲁棒决策。

这一框架在机器人导航、目标跟踪、金融预测等领域具有广泛适用性，是处理动态系统状态估计的基石方法。

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GAN（生成对抗网络）的训练过程与朗之万方程（Langevin Equation）的微分形式之间的关联，可以通过以下方式理解：GAN的训练可以视为通过求解随机微分方程（SDE）来寻找生成器分布的平稳解。以下是详细解释：

1. 朗之万方程的数学形式

朗之万方程是描述粒子在流体中受热噪声影响的随机微分方程： [ dX_t = -\nabla U(X_t) dt + \sqrt{2 \beta^{-1}} dB_t, ] 其中： - \( U(X_t) \) 是势能函数（Potential Energy），其梯度驱动确定性部分； - \( \beta \) 是逆温度参数（与热噪声强度相关）； - \( B_t \) 是布朗运动（Wiener过程），代表随机噪声。

该方程的平稳分布是势能函数 \( U \) 的玻尔兹曼分布： [ \pi(X) \propto \exp(-\beta U(X)). ]

2. GAN的训练目标与朗之万方程的联系

GAN的核心目标是让生成器 \( G \) 的分布 \( P_G \) 接近真实数据分布 \( P_{\text{data}} \)。从优化动力学的角度看，生成器的参数更新可以建模为一个随机微分方程，其解的平稳分布即为目标分布。

（1）生成器的优化动力学

生成器 \( G \) 的参数 \( \theta_G \) 通过梯度下降更新： [ \frac{d\theta_G}{dt} = -\nabla_{\theta_G} \mathcal{L}(G, D), ] 其中 \( \mathcal{L} \) 是GAN的损失函数（如最小最大损失）。

若考虑训练中的噪声（如小批量梯度估计的方差或判别器的波动），可以引入随机项： [ d\theta_G = -\nabla_{\theta_G} \mathcal{L}(G, D) dt + \eta dB_t, ] 这与朗之万方程的形式一致，其中： - \( \nabla_{\theta_G} \mathcal{L} \) 对应势能函数的梯度 \( \nabla U \)； - \( \eta \) 是噪声强度参数。

（2）平稳分布的对应

朗之万方程的平稳分布由势能 \( U \) 决定，而GAN的训练目标是让 \( P_G \) 接近 \( P_{\text{data}} \)。因此，可以将损失函数 \( \mathcal{L} \) 设计为与势能 \( U \) 相关： - 当 \( P_G \) 趋近于 \( P_{\text{data}} \)，损失函数 \( \mathcal{L} \) 的梯度趋于零，此时参数 \( \theta_G \) 达到平稳状态； - 这种平稳状态对应朗之万方程的解，其分布即为 \( P_{\text{data}} \)。

3. GAN训练的SDE建模

通过将生成器的参数更新视为随机微分方程，可以建立以下模型：

（1）连续时间下的参数动力学

假设生成器的参数 \( \theta_G(t) \) 满足以下SDE： [ d\theta_G = -\nabla_{\theta_G} \mathbb{E}{x \sim P{\text{data}}}[\log D(x)] dt - \nabla_{\theta_G} \mathbb{E}{z \sim P_z}[\log(1 - D(G(z)))] dt + \eta dB_t. ] 这可以简化为： [ d\theta_G = -\nabla{\theta_G} \mathcal{L}(G, D) dt + \eta dB_t, ] 其中 \( \mathcal{L} \) 是GAN的损失函数。

（2）解的收敛性

当训练过程收敛时，参数 \( \theta_G \) 的分布应满足朗之万方程的平稳分布： [ \pi(\theta_G) \propto \exp\left(-\beta \mathcal{L}(G, D)\right). ] 若损失函数 \( \mathcal{L} \) 的最小值对应 \( P_G = P_{\text{data}} \)，则平稳分布即为生成器的目标分布。

4. 与知识库的关联

（1）SDE的求解方法（知识库条目[2][4]）

• 线性SDE的解法（如伊藤公式）可用于分析生成器参数的演化路径；
• 通过求解SDE的期望和方差，可以研究生成器分布的统计特性。

（2）GAN的训练动力学（知识库条目[1][3][5][6][7][8]）

• GAN的对抗训练本质是生成器与判别器的博弈，其参数更新的梯度方向与朗之万方程的确定性项一致；
• 噪声项（如小批量梯度的方差）对应朗之万方程的随机项，有助于避免局部最优。

5. 实际意义与挑战

（1）优势

• 理论解释：将GAN的训练过程建模为SDE，提供了数学上的严谨性，便于分析收敛性；
• 稳定性提升：通过控制噪声强度 \( \eta \)，可改善GAN训练的稳定性（如WGAN的改进）。

（2）挑战

• 非线性复杂性：GAN的损失函数 \( \mathcal{L} \) 通常是高度非线性的，导致SDE的解析解难以求得；
• 高维参数空间：真实场景中参数维度极高，数值求解SDE的计算成本巨大。

6. 总结

通过将GAN的训练过程视为求解朗之万方程的随机微分方程，可以： 1. 理论层面：利用SDE的数学工具（如伊藤公式、平稳分布分析）解释GAN的收敛性； 2. 实践层面：借鉴SDE的数值方法（如欧拉-马尤拉方法）设计更稳定的训练算法。

这一视角为GAN的优化动力学提供了新的分析框架，但实际应用中仍需结合深度学习的工程实践进行调整。

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Referee #3

Evidence, reproducibility and clarity

Lupu et al have identified a role for the RNA binding protein SRSF3 in epicardial development. In a well written manuscript containing many experiments the authors show that this protein is required at different time points in epicardial development to control a range of processes, in particular cell proliferation. This advances understanding of the complex roles of this RNA binding protein in the heart - and raises an important message about how incomplete Cre recombination needs to be considered in interpreting conditional mutant phenotypes. The following points should be addressed.

1. SRSF3 is known to play essential developmental roles in the myocardium where it regulates capping of transcripts involved in contraction. This point should be mentioned in addition to roles in proliferation. To facilitate understanding, the authors should say more about the subset of cardiomyocytes labelled by Gata5-Cre. For example, is this the result of stochastic activation of the transgene or is a specific subset of cells labelled? How much of the myocardium is targeted?
2. The authors show failure of ventricular compaction at E13.5 using Wt1-CreERT2 and go on to assess proliferation in epicardial cells. As epicardial-derived signals are known to promote compact myocardial growth, they should also show whether there are indirect defects in proliferation in compact layer myocardium that might explain the non-compaction phenotype. The authors should also indicate if any of the large number of genes bound by SRSF3 encode known or potential pro-proliferative signals from the epicardium or EPDCs to the myocardium and potentially validate their altered expression in mutant hearts.
3. The rescue by expansion of non-recombined cells is a most interesting aspect of this study. Can the authors see any such outcompeting in the explant experiments (for example in Figure 2)? Do the authors consider this to be an exclusively in vivo competition phenomenon? Given the known roles of Myc in cell competition can the authors use their single cell transcriptomic data to score Myc expression levels in cells from Srsf3 iKO hearts or determine if Myc transcripts are bound by SRSF3?
4. The authors suggest that this rescue occur by upregulation of Srsf3 in non-recombined cells. It would be helpful to provide additional lines of evidence supporting the hypothesis that SRSF3 expression is upregulated due to hypoxia. Do the CLIP experiments reveal whether SRSF3 binds to it's own transcript?
5. The authors imply that SRSF3 may regulate Ccnd1 mRNA stability. Can the authors directly evaluate this point? Please clarify if this gene is also affected in the knock-down experiments in MEC1 cells.
6. Please brighten the immunofluorescence panels in Figure 1 to more clearly show nuclear labelling and tissue structure.
7. Given the broad roles of SRSF3 is the adjective key necessary in the title?

Significance

This ms advances understanding of the complex roles of this RNA binding protein in the heart - and raises an important message about how incomplete Cre recombination needs to be considered in interpreting conditional mutant phenotypes. This study would be of interest to reseachers in the fields of heart development and RNA-protein interactions. Although there are a number of major points to be addressed, these could be potentially dealt with rapidly.

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Referee #2

Evidence, reproducibility and clarity

In this study, Lupu et al. analyzed the role of the RNA-binding protein SRSF3 for epicardial development. The authors found that Srsf3 is highly expressed in the proepicardial organ and during early stages of epicardial layer formation. Conditional inactivation of SRSF3 in the proepicardial organ stage using a Gata5-Cre driver line resulted in defective formation of the epicardium, accompanied by a proliferation arrest of the proepicardium, resulting in embryonic lethality at E12.5. In contrast, epicardial-specific Srsf3 deletion at later stages using the inducible Wt1CreERT2 line caused a less severe phenotype indicated by impaired coronary vasculature formation, reduced cardiac compaction, and myocardial hypoxia. Mosaic recombination yielded a small population of epicardial cells that upregulate Srsf3, hyperproliferate and compensate for the depleted Srsf3 negative lineage. Single-cell RNA sequencing of control and epicardial Srsf3 knock out hearts, combined with infrared CLIP to map SRSF3 binding sites in the transcriptome identified a number of putative SRSF3 targets involved in mitotic cell cycle control. Among others, SRSF3 binds directly to transcripts encoding key regulators of proliferation, such as Cyclin D1, and senescence, including MAP4K4. The authors conclude that SRSF3 exerts different functions in processing of RNAs, including splicing.

Overall, this is a well-written and well-organized manuscript, describing interesting findings in the field of epicardial development. However, the mechanistic part is not overly strong. The authors detected some moderate changes in the distribution of different Map4k4 splicing isoforms after knockdown of Srsf3 in an immortalized epicardial cell line but did not go any deeper. The cause for the reduced presence of transcripts for the SRSF3-target Ccnd1 after knockdown of Srsf3 remains enigmatic.

Significance

The authors raise a number of speculations why remaining Srsf3-expressing cell start to hyperproliferate after inactivation of Srsf3 but it does not become clear which mechanism is critical. How do non-targeted epicardial cells in the mosaic recombination model sense the loss of SRSF3 knock out cells, resulting in hyperproliferation and enhanced Srsf3 expression? Is a loss of lateral inhibition, e.g. by activated YAP/TAZ, causative for enhanced proliferation of the remaining epicardial cells and an elevated expression level of WT1 and SRSF3? Immunofluorescence staining and/or qRT-PCR of YAP/TAZ and TEADs might provide an answer.

Is the elevated expression of Srsf3 in non-targeted epicardial cells due to enhanced transcription and/or by altered post-transcriptional processes? How does this observation fit to previous reports indicating that Srsf3 overexpression promotes inclusion of an autoregulatory cassette exon (exon 4) containing a premature (in-frame) stop codon in Srsf3, thereby confining this SRSF3 isoform to nonsense mediated decay (NMD) (doi: 10.1093/emboj/16.16.5077, doi: 10.1186/gb-2012-13-3-r17, doi: 10.1038/srep14548, doi: 10.1161/CIRCRESAHA.118.31451)? In contrast, Srsf1 as well as PTBP1/2 have been previously reported to regulate Srsf3 expression by promoting exon 4 skipping. The authors should perform RNA seq and/or qRT-PCRs validation to check the inclusion of Srsf4 Exon4 as well as Srsf1 and PTBP1/2 expression levels in control and knock out epicardial cells.

It remains unclear by which mechanisms (alternative splicing, alternative polyadenylation, miRNA processing, or others) SRFS3 mainly exerts its function in the embryonic epicardial lineage. The selection and validation of Map4k4 as a splicing target is not based on an unbiased splicing analysis. In my opinion it is mandatory to provide a full assessment of splicing changes in Srsf3-deficient cells, either by long-range sequencing or by analysis of exon-junction reads.

Likewise, it is completely enigmatic what SRFS3 does to Ccnd1 transcripts. Does SRFS3 increase the half-life of Ccnd1, does it impact trafficking? At least, the authors have to determine changes in the half-life of Ccnd1 after depletion of SRFS3.

An unbiased bioinformatics analysis addressing alternative splicing, alternative polyadenylation, and mRNA processing is necessary. Ideally, primary epicardial cells should be used and not an immortalized epicardial cell lines. It is well known, that splicing in cell lines differs substantially from splicing in primary cells.

I am not convinced that the moderate changes of different Map4k4 splicing isoforms after knockdown of Srsf3 are really responsible for the rather drastic phenotype. Additional experiments are needed to prove a decisive function of a shift in Map4k4 splicing isoforms for hyperproliferation of epicardial cells.

The authors claim that inactivation of Srsf3 inhibits cell proliferation and causes a senescence-like phenotype. The claim for acquisition of senescence is solely based on transcriptional changes. No attempts were made to visualize an increase of senescent cells in Srsf3-mutant embryos. The authors need to perform SA-bGAL assays or use other techniques to analyse the appearance of senescent cells in the mutants.

Fig. 2E indicates that WT1 positive / tdTom negative epicardial cell population is enriched in a specific region of the pre-epicardial organ from Srsf3KOs. However it is not clear whether these cells proliferate. The authors should quantify Ki67positive cells in both the WT1-positive / tdTom-positive and the WT1positive / tdTom-negative epicardial population.

In the headline on page 6, the authors stated that "SRSF3 depletion in the PEO results in impaired ... migration of epicardial progenitor cells", which they deduced from the reduced outgrowth of ventricular epicardial explants. However, the reduced outgrowth from the PEO could be caused by both, reduced proliferation and/or reduced migration. Therefore, the authors should provide additional data clearly indicating reduced migration, e.g. by blocking transcription. Scratch assays of SRFS3 knockout/knockdown vs. control epicardial cells would strengthen the analysis, Is there a change in the GO term "regulation of migration"?

To prove the reduced proliferation ratio in Figure 4B, quantification of Cyclin D1 positive cells in both SRSF3 positive and negative cells is required.

Minor issues

Abstract line 12: a full stop is missing at the end of the sentence.

Figure 1A: E11.5, figure label 'DAPI WT1' is missing.

Page 8: Bracket in front of Fig. 4B is missing

Page 8: G2M phase change uniformly to G2/M phase

Page 9: 'Srsf3-depleted hearts also demonstrated an increased abundance of epicardial cells with upregulated expression of genes associated with quiescence, such as Clu48, and senescence, for example Map4k4, Tmem30a and Pofut249 (Fig. 4F)'. The sentence is misleading, implying Srfs3 inactivation in all cardiac cell types ('Srsf3-depleted hearts').

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Referee #1

Evidence, reproducibility and clarity

Summary:

The study by Irina Lupu and colleagues highlights SRSF3 as a key regulator of epicardial development by regulating epicardial cell proliferation. This was demonstrated via two murine knockout models; the first to assimilate the role SRSF3 plays in epicardial formation as a whole, and the second to address its importance post a pivotal maturation point. Through scRNA sequencing and irCLIP, several SRSF3 targets were ascertained and identified as cell cycle regulators. Those epicardial cells that did not lose SRSF3 compensated the loss of some of their mates by increasing SRSF3 expression and over-proliferating. Overall, the paper is interesting and the conclusions are largely supported by the provided data.

Major comments:

1. Authors claim that a "reduction in SRSF3 expression levels coincided with the downregulation of WT1 in the epicardium". This was evidenced by immunofluorescence imaging (figure 1A). I suggest conducting a qRT-PCR to quantify Wt1 expression over time, similar to the experiment they performed in figure 1B.
2. A western blot depicting SRSF3 protein production in controls compared to the knockout model may provide stronger evidence of its depletion (figure 1E).
3. Authors state that they were unable to directly identify the absence of exons 2 and 3 in individual cells. Please provide evidence that exons 2 and 3 have been knocked out, at least by performing a qRT-PCR.
4. To prove the functional implication in the observed phenotype of the identified SRSF3 targets, please interfere with Map4k4 activity or expression and check whether the defective epicardial cell proliferation is reverted. This should be done at least in vitro, ideally in vivo.

Minor comments:

1. Several minor typos and spacing issues were observed. Please correct.
2. It would be good for the reader if the authors would simplify their rationale for the use of the two mouse models. It is slightly convoluted and not easy to follow.
3. In figure 4, it is recommended to add a stacked bar plot to represent the percentage of each cell cluster/population after 4A. This would help the reader
4. Figure 4B. It is confusing for the reader to understand the fact that the majority of tdTomato+ sorted cells in Srsf3 iKO keep expressing Srsf3. Including the quantification of the image could help.

Significance

The paper will be of interest to readers in the field of cardiology, embryology and molecular biology. It will advance the field especially in the study of the development of the epicardium. The models are sophisticated and the experiments carefully performed.

My field is molecular cardiology, with interest in RNA-binding proteins.

Critique #4: The Scene Reflects the Director's Pleasure, Not the Story’s Needs.

通过具体例子解释蕴含命题的构造证明

例子：证明蕴含命题 \( \vdash (A \rightarrow B) \)

命题：<br /> 若 \( n \) 是偶数，则 \( n^2 \) 是偶数。<br /> 即 \( A = \text{"n是偶数"} \)，\( B = \text{"n²是偶数"} \)，构造证明 \( A \rightarrow B \)。

构造证明的步骤

1. 定义命题：
2. \( A \)：存在整数 \( k \)，使得 \( n = 2k \)（偶数的定义）。

3. \( B \)：存在整数 \( m \)，使得 \( n^2 = 2m \)（平方后的偶数定义）。

4. 假设 \( A \) 为真：<br /> 根据构造逻辑的蕴含引入规则，假设 \( A \) 成立，即存在 \( k \in \mathbb{Z} \)，使得 \( n = 2k \)。

5. 从 \( A \) 推导 \( B \)：

6. 代入 \( n = 2k \) 到 \( n^2 \) 中：<br /> [ n^2 = (2k)^2 = 4k^2 = 2 \times (2k^2) ]

7. 定义 \( m = 2k^2 \)，则 \( n^2 = 2m \)，满足 \( B \) 的定义。

8. 构造性转换：

9. 算法步骤：
   1. 输入：\( A \) 的证明（即 \( k \) 的值）。
   2. 计算 \( m = 2k^2 \)。
   3. 输出：\( B \) 的证明（即 \( m \) 的值）。

10. 这个过程将 \( A \) 的证明（\( k \) 的具体值）显式转换为 \( B \) 的证明（\( m \) 的具体值）。

11. 结论：<br /> 根据构造逻辑的蕴含引入规则，我们得到：<br /> [ \vdash (A \rightarrow B) ]

关键点分析

1. 构造性证明的要求：
2. 每一步必须提供显式的构造方法，例如通过代数运算将 \( k \) 转换为 \( m \)。

3. 不依赖非构造性推理（如反证法或排中律）。

4. 算法的可计算性：

5. 证明过程对应一个可计算的算法：<br /> [ f(k) = 2k^2 \quad \text{（将 \( k \) 转换为 \( m \)）} ]

6. 这符合知识库[6]中构造逻辑对蕴含的定义：构造 \( A \rightarrow B \) 的证明即提供一个将 \( A \) 的证明（\( k \)）转化为 \( B \) 的证明（\( m \)）的函数。

7. 与经典逻辑的区别：

8. 经典逻辑可能通过反证法证明此命题（例如假设 \( n^2 \) 是奇数，导出矛盾），但构造逻辑要求直接构造 \( m \) 的值。

知识库关联

• 知识库[6]：
• 构造逻辑中，\( A \rightarrow B \) 的证明是“将 \( A \) 的任一证明转换为 \( B \) 的证明的构造”。
• 本例中的算法 \( f(k) = 2k^2 \) 即是这一构造的具体实现。
• 知识库[10]：
• 构造主义要求证明必须提供算法或步骤，而非仅断言存在性。例如，证明 \( n^2 \) 是偶数需显式给出 \( m \) 的表达式。

总结

通过这个例子，我们展示了如何通过构造性方法证明蕴含命题 \( A \rightarrow B \)：<br /> 1. 假设 \( A \) 成立，并利用其证明（如 \( k \) 的值）。<br /> 2. 显式构造 \( B \) 的证明（如计算 \( m = 2k^2 \)）。<br /> 3. 证明过程即算法，确保可计算性和可构造性，符合构造逻辑的要求。

这一过程体现了构造逻辑的核心思想：证明即算法，蕴含即转换。

为什么构造逻辑既是数学哲学的革命，又是现代计算机科学的基石之一？

一、作为数学哲学的革命

构造逻辑（Constructive Logic）的诞生标志着数学哲学的一次深刻变革，其核心在于对数学证明本质和数学对象存在性的根本性重新定义。以下是其革命性的具体表现：

1. 对数学证明的重新定义

• 从“存在性”到“可构造性”：<br /> 经典逻辑允许通过非构造性证明（如反证法、纯存在性证明）来断言某个对象的存在，而构造逻辑要求所有存在性命题必须提供显式构造方法。例如，证明“存在一个素数大于1000”需具体给出一个素数（如1009），而非仅证明其存在性。

• 知识库关联：毕晓普强调“只证明逻辑存在性是不够的，必须提供有限机械方法构造对象”（知识库[2]）。

• 排斥排中律（LEM）：<br /> 构造逻辑不接受“\( P \lor \neg P \)”的普遍有效性，除非能有效判断\( P \)或\( \neg P \)为真。例如，无法断言“\( \pi \)的第10^100位是偶数或非偶数”，因为无法在有限时间内验证。

• 哲学意义：布劳威尔认为“存在即被构造”，数学对象必须通过心智的“原始直觉”生成（知识库[1]）。

2. 对数学基础的挑战

• 直觉主义对集合论的批判：<br /> 布劳威尔和直觉主义者反对康托尔的实无穷集合论，认为无限集合必须通过递归过程（如柯西序列）构造，而非抽象定义。例如，实数必须定义为可计算的递归柯西序列（毕晓普学派）或算法生成的数（马尔可夫学派）。

• 知识库关联：马尔可夫将实数定义为“逐次逼近的算法”，实函数等同于算法（知识库[2]）。

• 选择公理的限制：<br /> 经典数学中的选择公理允许从无限集合中选择元素，但构造逻辑要求选择必须通过可计算的递归过程实现。例如，从可数个非空集合中选择元素需提供一个算法，逐个生成选择项。

3. 重新定义数学可信性标准

• 从“抽象真理”到“可计算性”：<br /> 构造逻辑要求数学对象和定理的可计算性，即所有定义和证明必须能通过算法实现。例如，毕晓普将实数定义为递归柯西序列，确保每个实数的生成过程可被机械执行。

• 知识库关联：毕晓普改造集合论，要求集合的定义不仅包含判别规则，还需提供构造元素的具体方法（知识库[2]）。

• 对经典分析的重构：<br /> 构造逻辑推动了对经典分析的重新审视，例如：

• 连续函数的定义：构造性连续函数需满足“输入的近似值可计算输出的近似值”（如递归算法）。
• 中值定理的局限性：经典数学中的“连续函数在区间内取中间值”在构造逻辑中不成立，除非能构造出具体点。

二、作为现代计算机科学的基石

构造逻辑对计算机科学的影响体现在其算法化、可验证性和类型系统的构建上，具体表现为以下方面：

1. 柯里-霍华德同构（Curry-Howard Isomorphism）

• 证明即程序，公式即类型：<br /> 构造逻辑的证明与程序设计语言中的类型系统直接对应。例如：
• 蕴含（\( A \rightarrow B \)）：对应一个将\( A \)类型的数据转换为\( B \)类型数据的函数。
• 存在量词（\( \exists x A(x) \)）：对应一个包含具体\( x \)值和证明的元组。

• 知识库关联：构造逻辑的证明可转化为可执行代码（如Coq、Agda中的程序验证）。

• 函数式编程的基础：<br /> 构造逻辑的自然演绎系统直接启发了函数式编程语言的设计（如Haskell、ML）。例如，构造性证明的消除规则对应函数的模式匹配和递归结构。

2. 程序验证与形式化方法

• 正确性证明的自动化：<br /> 构造逻辑为程序正确性证明提供了框架，例如：
• Hoare逻辑：通过构造性证明验证程序满足预/后条件。

• 依赖类型系统：如Coq中的类型系统，要求程序的每个步骤都伴随构造性证明。

• 算法的可计算性：<br /> 构造逻辑的可计算性要求确保算法的每一步骤都有明确构造，避免了经典数学中可能的非构造性漏洞。例如，马尔可夫的“正规算法”直接对应现代计算机的指令集（知识库[2]）。

3. 人工智能与逻辑推理

• 可解释性与可验证性：<br /> 在知识表示和推理系统中，构造逻辑确保推理过程的透明性。例如，机器学习模型的证明需提供具体构造步骤，而非依赖黑箱决策。

• 递归与算法设计：<br /> 构造逻辑中的递归定义（如毕晓普的实数）直接对应计算机中的递归函数和迭代算法，确保无限过程的有限步骤实现。

4. 并发与分布式系统

• 线性逻辑（Linear Logic）的延伸：<br /> 构造逻辑的资源敏感性（如线性逻辑）为并发系统中的资源管理和通信协议提供了形式化基础，确保操作的原子性和可验证性。

三、知识库关联与具体案例

1. 毕晓普的构造性分析：

2. 通过递归柯西序列定义实数，确保所有分析定理的证明可转化为算法（知识库[2]）。例如，证明“\( \sqrt{2} \)存在”需提供一个计算其近似值的递归过程。

3. 马尔可夫的正规算法：

4. 将数学对象定义为算法，例如实数的“逐次逼近算法”直接对应计算机程序的迭代计算（知识库[2]）。

5. 构造逻辑与类型论：

6. Agda语言中的依赖类型系统强制要求所有函数定义伴随构造性证明，确保程序的正确性（如知识库[2]中提到的“改造集合论”）。

四、总结

构造逻辑的革命性在于：<br /> 1. 数学哲学层面：重新定义了数学对象的存在性（存在即构造），挑战了经典逻辑的抽象性，推动了数学的算法化和可计算性研究。<br /> 2. 计算机科学层面：为程序验证、函数式编程、类型系统和形式化方法提供了理论基石，确保了算法的可构造性和可验证性。

其影响贯穿数学与计算机科学的交叉领域，既是一场哲学思潮，也是现代技术的底层逻辑，体现了“数学即算法”的深刻思想。

i dont necessarily think this is such a shocking factor because the sole intention of shows like Sesame Street is to teach children, obviously a show meant for general viewing isnt going to teach as much because thats not what its meant for. the harm of those types of programs would come in during young childhood if the program has more mature themes because it can affect the ideas that children can get, not affect their cognition moreso than any other screen driven program would

How does the oxidation number of metal depict the compound it will form ?

Reviewer #1 (Public review):

Summary:

Govindan and Conrad use a genome-wide CRISPR screen to identify genes regulating retention of intron 4 in OGT, leveraging an intron retention reporter system previously described (PMID: 35895270). Their OGT intron 4 reporter reliably responds to O-GlcNAc levels, mirroring the endogenous splicing event. Through a genome-wide CRISPR knockout library, they uncover a range of splicing-related genes, including multiple core spliceosome components, acting as negative regulators of OGT intron 4 retention. They choose to follow up on SFSWAP, a largely understudied splicing regulator shown to undergo rapid phosphorylation in response to O-GlcNAc level changes (PMID: 32329777). RNA-sequencing reveals that SFSWAP depletion not only promotes OGT intron 4 splicing but also broadly induces exon inclusion and intron splicing, affecting decoy exon usage. While this study offers interesting insights into intron retention regulation and O-GlcNAc signaling, the RNA-Sequencing experiments lack essential controls needed to provide full confidence to the authors' conclusions.

Strengths:

(1) This study presents an elegant genetic screening approach to identify regulators of intron retention, uncovering core spliceosome genes as unexpected positive regulators of intron retention.<br /> (2) The work proposes a novel functional role for SFSWAP in splicing regulation, suggesting that it acts as a negative regulator of splicing and cassette exon inclusion, which contrasts with expected SR-related protein functions.<br /> (3) The authors suggest an intriguing model where SFSWAP, along with other spliceosome proteins, promotes intron retention by associating with decoy exons.

Weaknesses:

(1) The conclusions regarding SFSWAP's impact on alternative splicing rely on cells treated with a single pool of two siRNAs for five days. The absence of independent siRNA treatments raises concerns about potential off-target effects, which may reduce confidence in the observed SFSWAP-dependent splicing changes. Rescue experiments or using additional independent siRNA treatments would strengthen the conclusions.<br /> (2) The mechanistic role of SFSWAP in splicing would benefit from further exploration, though this may be more appropriate for future studies.

Comments on revisions:

The authors have addressed all my previous recommendations.

Author response:

The following is the authors’ response to the original reviews

Public Reviews:

Reviewer #1 (Public review):

Summary:

This study has preliminarily revealed the role of ACVR2A in trophoblast cell function, including its effects on migration, invasion, proliferation, and clonal formation, as well as its downstream signaling pathways.

Strengths:

The use of multiple experimental techniques, such as CRISPR/Cas9-mediated gene knockout, RNA-seq, and functional assays (e.g., Transwell, colony formation, and scratch assays), is commendable and demonstrates the authors' effort to elucidate the molecular mechanisms underlying ACVR2A's regulation of trophoblast function. The RNA-seq analysis and subsequent GSEA findings offer valuable insights into the pathways affected by ACVR2A knockout, particularly the Wnt and TCF7/c-JUN signaling pathways.

Weaknesses:

The molecular mechanisms underlying this study require further exploration through additional experiments. While the current findings provide valuable insights into the role of ACVR2A in trophoblast cell function and its involvement in the regulation of migration, invasion, and proliferation, further validation in both in vitro and in vivo models is needed. Additionally, more experiments are required to establish the functional relevance of the TCF7/c-JUN pathway and its clinical significance, particularly in relation to pre-eclampsia. Additional techniques, such as animal models and more advanced clinical sample analyses, would help strengthen the conclusions and provide a more comprehensive understanding of the molecular pathways involved.

Reviewer #2 (Public review):

Summary:

ACVR2A is one of a handful of genes for which significant correlations between associated SNPs and the incidences of preeclampsia have been found in multiple populations. It is one of the TGFB family receptors, and multiple ligands of ACVR2A, as well as its coreceptors and related inhibitors, have been implicated in placental development, trophoblast invasion, and embryo implantation. This useful study builds on this knowledge by showing that ACVR2A knockout in trophoblast-related cell lines reduces trophoblast invasion, which could tie together many of these observations. Support for this finding is incomplete, as reduced proliferation may be influencing the invasion results. The implication of cross-talk between the WNT and ACRV2A/SMAD2 pathways is an important contribution to the understanding of the regulation of trophoblast function.

Strengths:

(1) ACVR2A is one of very few genes implicated in preeclampsia in multiple human populations, yet its role in pathogenesis is not very well studied and this study begins to address that hole in our knowledge.

(2) ACVR2A is also indirectly implicated in trophoblast invasion and trophoblast development via its connections to many ligands, inhibitors, and coreceptors, suggesting its potential importance.

(3) The authors have used multiple cell lines to verify their most important observations.

Weaknesses:

(1) There are a number of claims made in the introduction without attribution. For example, there are no citations for the claims that family history is a significant risk factor for PE, that inadequate trophoblast invasion of spiral arteries is a key factor, and that immune responses, and reninangiotensin activity are involved.

Thank you for pointing out the lack of citations in some parts of the introduction. We have revised the manuscript to include appropriate references for the claims regarding family history as a risk factor for PE, the role of inadequate trophoblast invasion in spiral arteries, and the involvement of immune responses and the renin-angiotensin system. The revised text now includes citations to well-established studies in the field (Salonen Ros et al., 2000; Chappell LC et al., 2021; Brosens et al., 2002; Knofler et al., 2019; Redman CWG et al., 1999; LaMarca B et al., 2008). We believe these additions improve the scientific rigor of the manuscript.

(2) The introduction states "As a receptor for activin A, ACVR2A..." It's important to acknowledge that ACVR2A is also the receptor for other TGFB family members, with varying affinities and coreceptors. Several TGFB family members are known to regulate trophoblast differentiation and invasion. For example, BMP2 likely stimulates trophoblast invasion at least in part via ACVR2A (PMID 29846546).

Thank you for highlighting the broader role of ACVR2A as a receptor for multiple members of the TGF-β superfamily. We have revised the introduction to acknowledge that ACVR2A is not only the receptor for activin A but also interacts with other ligands, such as BMP2, which likely stimulates trophoblast invasion via ACVR2A (PMID: 29846546). This addition provides a more comprehensive view of ACVR2A's function in trophoblast biology. While the focus of our current study is on activin A, we agree that ACVR2A's role in mediating the effects of other TGF-β family members is an important topic for future research.

(3) An alternative hypothesis for the potential role of ACVR2A in preeclampsia is its functions in the endometrium. In the mouse ACVR2A knockout in the uterus (and other progesterone receptorexpressing cells) leads to embryo implantation failure.

Thank you for bringing up the potential role of ACVR2A in the endometrium as an alternative hypothesis. We have revised the discussion to acknowledge this possibility and cited relevant studies showing that uterine-specific knockout of ACVR2A in mice leads to embryo implantation failure (Monsivais et al., 2021). This suggests that ACVR2A may play a critical role in uterine receptivity and embryo implantation, which could influence placental development and preeclampsia pathogenesis. While our current study focuses on trophoblast-related functions of ACVR2A, we agree that investigating its role in the uterine environment is an important direction for future research.

(4) In the description of the patient population for placental sample collections, preeclampsia is defined only by hypertension, and this is described as being in accordance with ACOG guidelines. ACOG requires a finding of hypertension in combination with either proteinuria or one of the following: thrombocytopenia, elevated creatinine, elevated liver enzymes, pulmonary, edema, and new onset unresponsive headache.

We appreciate the reviewer’s detailed observation regarding the definition of preeclampsia.

We have reviewed and clarified our description of the diagnostic criteria based on the American College of Obstetricians and Gynecologists (ACOG) guidelines. Specifically, we have revised the definition in the Materials and Methods section under "Collection of Placenta and Decidua Specimens," as follows: In accordance with the guidelines from the American College of Obstetricians and Gynecologists (ACOG, 2023), preeclampsia (PE) is diagnosed as hypertension (systolic blood pressure ≥140 mmHg or diastolic blood pressure ≥90 mmHg on at least two occasions) in combination with one or more of the following: proteinuria (≥300 mg/24-hour urine collection or protein/creatinine ratio ≥0.3), thrombocytopenia, elevated serum creatinine, elevated liver enzymes, pulmonary edema, or new-onset headache unresponsive to treatment.

(5) I believe that Figures 1a and 1b are data from a previously published RNAseq dataset, though it is not entirely clear in the text. The methods section does not include a description of the analysis of these data undertaken here. It would be helpful to include at least a brief description of the study these data are taken from - how many samples, how were the PE/control groups defined, gestational age range, where is it from, etc. For the heatmap presented in B, what is the significance of the other genes/ why are they being shown? If the purpose of these two panels is to show differential expression specifically of ACVR2A in this dataset, that could be shown more directly.

Clarification of RNAseq dataset: The Methods section has been revised to specify the dataset source (GEO accession number: GSE114691), which includes 20 PE and 21 control placental samples with gestational ages ranging from 34 to 38 weeks. PE and control groups were defined using clinical criteria such as hypertension and proteinuria, and these details have also been added to the Results section. RNAseq analysis description: We have included details of the differential gene expression analysis in the Methods section. Specifically, the DESeq2 R package was used, with thresholds of FDR < 0.05 and |log2(fold change) | ≥ 1. The selection of WNT pathwayrelated genes in Figure 1B is based on these analyses. Significance of the heatmap genes: The genes displayed in Figure 1B were selected based on their significant differential expression and enrichment in pathways relevant to PE pathogenesis, such as the WNT signaling pathway. We have clarified this in the Results section and updated the figure legend to explain their biological relevance. Purpose of Figures 1A and 1B: Figure 1A emphasizes the downregulation of ACVR2A in PE placentas, while Figure 1B complements this by presenting differentially expressed genes associated with the WNT pathway. These figures collectively highlight the role of ACVR2A in PE and its connection to broader molecular pathways. Text descriptions have been updated to improve clarity and focus.

(6) More information is needed in the methods section to understand how the immunohistochemistry was quantified. "Quantitation was performed" is all that is provided. Was staining quantified across the whole image or only in anchoring villous areas? How were HRP & hematoxylin signals distinguished in ImageJ? How was the overall level of HRP/DAB development kept constant between the NC and PE groups?

Thank you for pointing out the need for more details regarding the quantification of immunohistochemistry (IHC). We have now clarified and expanded the description of the IHC quantification process in the Methods section as follows: Quantification Across the Entire Section: IHC staining was assessed across the entire tissue section to account for global expression patterns. For quantitative analysis, representative regions from the anchoring villous areas, where ACVR2A expression is most prominent, were selected for comparison between NC and PE groups. This ensured that the analysis focused on biologically relevant regions. ImageJ Analysis:

Images of stained sections were captured under identical magnifications and lighting conditions. Hematoxylin (blue, nuclear staining) and DAB/HRP (brown, protein-specific signal) were distinguished using ImageJ's color deconvolution plugin. The DAB/HRP signal was isolated and quantified based on the integrated optical density (IOD) within the selected regions. Consistency in HRP/DAB Development: To maintain consistency between NC and PE groups, all tissue samples were processed under identical experimental conditions, including the same antibody dilution, incubation times, and DAB/HRP development durations. Negative controls (without primary antibody) were included to monitor background staining, and the DAB reaction was stopped simultaneously across all samples to avoid overdevelopment. Statistical Analysis: The quantified DAB signal intensity was normalized to the area of the selected regions, and comparisons between NC and PE groups were performed using statistical tests (e.g., Student’s ttest). Results are reported as mean ± SD. We hope this additional detail addresses your concerns.

(7) In Figure 1E it is not immediately obvious to many readers where the EVT are. It is probably worth circling or putting an arrow to the little region of ACVR2A+ EVT that is shown in the higher magnification image in Figure 1E. These are actually easier to see in the pictures provided in the supplement Figure 1. Of note, the STB is also staining positive. This is worth pointing out in the results text.

Thank you for your suggestion regarding Figure 1E. To make the location of the ACVR2A+ extravillous trophoblasts (EVTs) more apparent, we have updated Figure 1E by adding arrows to indicate the regions of EVTs in the higher magnification image. Additionally, we have included annotations in the supplemental Figure S1 to further aid visualization. We appreciate your observation that syncytiotrophoblasts (STBs) also show positive staining for ACVR2A. We have revised the Results section to explicitly mention this finding and its potential significance.

(8) It is not possible to judge whether the IF images in 1F actually depict anchoring villi. The DAPI is really faint, and it's high magnification, so there isn't a lot of context. Would it be possible to include a lower magnification image that shows where these cells are located within a placental section? It is also somewhat surprising that this receptor is expressed in the cytoplasm rather than at the cell surface. How do the authors explain this?

Thank you for your suggestion to provide more context for the immunofluorescence (IF) images in Figure 1F. To address this, we have included lower magnification images in Supplementary Figure S2, showing the overall structure of the placental section and the location of the anchoring villi. These images help to contextualize the regions analyzed in Figure 1F, which were selected to clearly illustrate ACVR2A expression in extravillous trophoblasts (EVTs). In Figure 1F, we have focused on higher magnification images for better visualization of ACVR2A staining patterns in EVTs. Regarding the subcellular localization of ACVR2A, the receptor is predominantly expressed on the cell surface, as shown in our images. However, some intracellular staining is also observed, which may reflect receptor trafficking or recycling processes, consistent with the behavior of other activin receptors under physiological or pathological conditions. We have clarified these points in the Results and Discussion sections.

(9) The results text makes it sound like the data in Figure 2A are from NCBI & Protein atlas, but the legend says it is qPCR from this lab. The methods do not detail how these various cell lines were grown; only HTR-SVNeo cell culture is described. Similarly, JAR cells are used for several experiments and their culture is not described.

Thank you for pointing out the need for clarification regarding Figure 2A and cell culture methods. The data in Figure 2A were generated using RT-qPCR conducted in our laboratory, not solely based on data from NCBI or the Human Protein Atlas. We have revised the Results section to reflect this more accurately. Regarding the culture conditions, we acknowledge that the methods for other cell lines were not explicitly detailed. For this study, all cell lines, including JAR and other cancer cell lines, were cultured following standard protocols provided by the suppliers. Specifically, JAR cells and other cell lines were purchased from Wuhan Punosei Life Technology and were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin under standard conditions (37°C, 5% CO₂). This information has been added to the Methods section for clarity.

(10) Under RT-qPCR methods, the phrase "cDNA reverse transcription cell RNA was isolated..." does not make any sense.

Thank you for pointing out the unclear phrasing in the RT-qPCR methods section. We agree that the original description was not precise. To address this, we have revised the relevant section to improve clarity and accuracy. Specifically, the methods now explicitly describe the two key steps: RNA isolation and cDNA synthesis. The revised text reads: Total RNA was isolated from cells using a Total RNA Extraction Kit (TIANGEN, China) following the manufacturer’s instructions. The extracted RNA was reverse-transcribed into complementary DNA (cDNA) using a cDNA Synthesis Kit (Takara, Japan) according to the protocol provided by the manufacturer.

(11) The paragraph beginning "Consequently, a potential association..." is quite confusing. It mentions analyzing ACVR2A expression in placentas, but then doesn't point to any results of this kind and repeats describing the results in Figure 2a, from various cell lines.

Thank you for your comment regarding the paragraph beginning with "Consequently, a potential association...". We understand that the current wording may create confusion. The primary aim of this section is to compare ACVR2A expression levels across various cell lines, including trophoblast-derived and non-trophoblast cell lines, to highlight the relevance of ACVR2A in trophoblast function, particularly in invasion and migration. To address your concerns, we have revised the paragraph for clarity and logical flow. The updated text explicitly focuses on the comparison of ACVR2A expression across cell lines (Figure 2A) and how this supports the hypothesis that ACVR2A plays a key role in trophoblast invasion and migration. Additionally, the discussion of placental samples has been separated to avoid confusion with cell line results. We hope this revision resolves the issue.

(12) The authors should acknowledge that the effect of the ACVR2A knockout on proliferation makes it difficult to draw any conclusions from the trophoblast invasion assays. That is, there might be fewer migrating or invading cells in the knockout lines because there are fewer cells, not because the cells that are there are less invasive. Since this is a central conclusion of the study, it is a major drawback.

Thank you for highlighting this important point. We agree that the reduced proliferation observed in ACVR2A knockout cells could influence the results of the invasion assays, as fewer cells may inherently lead to reduced invasion. To minimize this effect, we conducted the invasion and migration assays under low-serum conditions (1–2% serum) to limit cell proliferation during the experimental timeframe. This approach was based on optimization trials and existing literature, as serum-free conditions were found to negatively impact cell viability and experimental reproducibility. While these efforts helped to mitigate the impact of proliferation on the results, we acknowledge this as a limitation of our study and have added this discussion to the manuscript. Future studies could incorporate approaches such as normalizing cell numbers or using additional proliferation-independent methods to confirm the findings. We hope this clarification and the steps taken address your concerns.

(13) The legend and the methods section do not agree on how many fields were selected for counting in the transwell invasion assays in Figure 3C. The methods section and the graph do not match the number of replicate experiments in Figure 3D (the number of replicate experiments isn't described for 3C).

Thank you for pointing out the inconsistencies regarding the number of fields counted and the number of replicates in the Transwell invasion assays (Figure 3C) and colony formation assays (Figure 3D). We apologize for the lack of clarity in the Methods section and figure legend. To address this, we have revised both the figure legends and the Methods section for consistency and added detailed descriptions. For Figure 3C, cell invasion was quantified by randomly selecting 5 fields of view per sample under 300× magnification. Images shown in the figure were taken at lower magnification to provide a better visual comparison between experimental and control groups. For Figure 3D, each experiment was independently repeated at least 10 times to ensure robust and reproducible results. These clarifications have been incorporated into the revised manuscript. We appreciate your feedback and believe this revision improves the clarity and transparency of our methods.

(14) Discussion says "Transcriptome sequencing analysis revealed low ACVR2A expression in placental samples from PE patients, consistent with GWAS results across diverse populations." The authors should explain this briefly. Why would SNPs in ACVR2A necessarily affect levels of the transcript?

Thank you for raising this important point. We acknowledge that our study did not directly investigate how SNPs in the ACVR2A gene affect transcript levels. However, prior studies have suggested that SNPs can influence gene expression through various mechanisms. For example, SNPs in regulatory regions (e.g., promoters, enhancers, or untranslated regions) may affect transcription factor binding, RNA stability, or splicing efficiency, ultimately altering transcript levels. While we did not directly assess the functional consequences of ACVR2A SNPs in this study, the observed downregulation of ACVR2A in PE placentas aligns with the potential regulatory impact of SNPs previously identified in GWAS studies. To address this, we have revised the Discussion section to clarify the relationship between SNPs and transcript levels and acknowledge this limitation.  

(15) "The expression levels of ACVR2A mRNA were comparable to those of tumor cells such as A549. This discovery suggested a potential pivotal role of ACVR2A in the biological functions of trophoblast cells, especially in the nurturing layer." Alternatively, ACVR2A expression resembles that of tumors because the cell lines used here are tumor cells (JAR) or immortalized cells (HTR8). These lines are widely used to study trophoblast properties, but the discussion should at least acknowledge the possibility that the behavior of these cells does not always resemble normal trophoblasts.

Thank you for pointing out this important limitation. We agree that the JAR and HTR8/SVneo cell lines, being tumor-derived or immortalized, may not fully replicate the behavior of normal trophoblast cells. While these cell lines are widely used as models for studying trophoblast properties due to their ease of culture and invasive behavior, their gene expression and signaling pathways could partially reflect their tumorigenic or immortalized origins. We have revised the Discussion section to acknowledge this limitation and clarify the interpretation of ACVR2A expression levels in these cells.

(16) The authors should discuss some of what is known about the relationship between the TCF7/c-JUN pathway and the major signaling pathway activated by ACVR2A, Smad 2/3/4. The Wnt and TGFB family cross-talk is quite complex and it has been studied in other systems.

Thank you for highlighting the relationship between the TCF7/c-JUN pathway and Smad2/3/4 signaling. In our study, we chose to focus on Smad1/5 due to its strong association with ACVR2A in placental development, as demonstrated in a recent study（DOI: 10.1038/s41467-021-23571-5). This study showed that the BMP signaling pathway, mediated through ACVR2A-Smad1/5, is essential for endometrial receptivity and embryo implantation. While Smad2/3/4 are wellestablished mediators of TGF-β signaling, Smad1/5 activation is more directly linked to ACVR2A in the context of reproductive biology.

In PE placentas, we observed a significant downregulation of Smad1/5 expression, which supports the hypothesis that ACVR2A-mediated Smad signaling is disrupted in this condition. Although we did not directly assess Smad2/3/4 in this study, prior research has shown that Smad2/3 can interact with TCF/LEF transcription factors to regulate Wnt-related target genes, suggesting potential cross-talk between these pathways. We have now clarified this rationale and included a discussion of these interactions in the revised manuscript.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

Several points need to be addressed to improve the clarity and robustness of the presented findings:

(1) From a clinical perspective, several concerns arise regarding the interpretation of these findings. First, the small sample size of 20 patients may not be representative of the broader population, limiting the generalizability of the results. Additionally, although no significant differences in age and pre-pregnancy BMI were observed between the PE and normal control groups, other clinical variables, such as hypertension or gestational diabetes, may also influence ACVR2A expression and contribute to PE development. Furthermore, while the study suggests a correlation between reduced ACVR2A expression and PE, it remains unclear whether this association holds true across different subtypes of PE or whether there are other underlying clinical factors that could account for these changes in gene expression. These factors need to be considered in future studies to better understand the clinical relevance of ACVR2A in PE.

Thank you for raising these insightful concerns about the clinical interpretation of our findings. We agree that the small sample size of 20 patients may limit the generalizability of our results. To address this, we are actively expanding our cohort by collecting additional clinical samples from PE patients and normotensive controls. This effort aims to strengthen the robustness of our findings and provide stronger evidence for the role of ACVR2A in PE. We would also like to clarify that, during the initial sample collection, we specifically included only PE patients without comorbidities such as gestational diabetes, chronic hypertension, or other pregnancy-related complications. This strict selection criterion was implemented to minimize the potential influence of confounding clinical variables and ensure that our findings specifically reflect the association between ACVR2A expression and PE. While our study provides important initial insights, we recognize the need for larger-scale studies to validate these findings. The ongoing collection of clinical samples will allow us to address this limitation and enhance the translational relevance of our research. We have revised the manuscript to reflect these points and highlight our plans to strengthen the study by increasing the sample size.

(2) The section "Precision Genome Surgery: ACVR2A Knockout via CRISPR/Cas9" in the results contains some issues with expression details. The results section should be more structured, with data presented in a more detailed and clear manner, ensuring that there is a clear connection between each experimental step and its corresponding result. For example, the sentence "Following multiple rounds of monoclonal culture, genotype identification, RT-qPCR and Western blotting (WB) analysis for screening, specific double-knockout monoclonal cell lines were distinctly chosen" contains redundant phrasing and unnecessary details, which affect the flow of the text.

Thank you for your constructive feedback on the “Precision Genome Surgery: ACVR2A Knockout via CRISPR/Cas9” section. We agree that this section can be better structured to present the data in a more detailed and coherent manner. To address this, we have reorganized the results into distinct steps, ensuring a clear connection between each experimental step and its corresponding result. Redundant phrasing has been removed to improve the flow and readability of the text. The revised section emphasizes the purpose of each step, the screening process, and the specific results obtained.

(3) The figure legends and panel labels in Figure 3 should be revised to ensure clarity and consistency. The figure legend should specify the exact panels (e.g., Figure 3A, 3B, 3C, etc.) and clearly describe the experimental conditions and results shown in each part.

Thank you for pointing out the need for improved clarity and consistency in the figure legends and panel labels for Figure 3. We have revised the figure legend to specify each panel (e.g., Figure 3A, 3B, 3C, etc.) and included detailed descriptions of the experimental conditions and results displayed in each part. These updates aim to ensure better understanding and alignment between the figure legend and the panels.

(4) Lack of In Vivo Validation of ACVR2A Knockout: The study does not include in vivo experiments to validate the effects of ACVR2A knockout. It would be important to investigate whether similar regulatory effects of ACVR2A on trophoblast cell migration and invasion can be observed in animal models or in larger clinical studies. The lack of in vivo data raises questions about the translational relevance of the findings.

Thank you for highlighting the importance of in vivo validation to assess the translational relevance of our findings. While we acknowledge that in vivo experiments could provide additional insights into the role of ACVR2A in trophoblast migration and invasion, this study was primarily designed as an in vitro investigation to explore the molecular mechanisms underlying ACVR2A function in trophoblast cells. The choice of an in vitro model allowed us to perform precise and controlled mechanistic analyses, which are critical for establishing a foundation for future research. We agree that in vivo studies using animal models or larger clinical cohorts are important next steps to validate the regulatory effects of ACVR2A on trophoblast function and its contribution to PE pathogenesis. These directions will be pursued in future research to further establish the translational potential of our findings. We have included this perspective in the revised Discussion section.

(5) TCF7/c-JUN Pathway in Clinical Samples: In the study of the TCF7/c-JUN pathway, the authors mention assessing protein expression in clinical samples through immunohistochemistry (IHC). However, the manuscript does not provide a clear explanation of how the findings from laboratory cell models (such as HTR8/SVneo and JAR) relate to the clinical samples. Specifically, while ACVR2A knockout is shown to affect these proteins at the cellular level, it is unclear whether this effect is observed in clinical samples. Therefore, further validation of the TCF7/c-JUN pathway in the cell models and exploration of its relationship with protein expression in clinical samples is necessary. Additional experiments, such as immunofluorescence staining or mass spectrometry, could further confirm the role of the TCF7/c-JUN pathway in cells and provide a more direct comparison with clinical data.

Thank you for highlighting the need to connect findings from cell models to clinical samples, particularly with respect to the TCF7/c-JUN pathway. In response to your comment, we conducted additional experiments using Western blot analysis to evaluate the expression of ACVR2A, SMAD1/5, SMAD4, pSMAD1/5/9, and TCF7L1/TCF7L2 in PE placental tissues compared to normotensive controls (Figure 7A). The results demonstrated significantly reduced expression of these proteins in PE placentas, providing evidence that disruptions in the ACVR2A-SMAD and TCF7/c-JUN signaling pathways observed in vitro are also present in clinical samples.

These findings strengthen the translational relevance of our study by directly linking the molecular mechanisms identified in cell models to clinical observations. We have updated the Results and Discussion sections to incorporate these new data, and we believe this addition addresses your concern about the relationship between in vitro and clinical findings.

Reviewer #1 (Public review):

Summary:

In this report, the authors made use of a murine cell line derived from a MYC-driven liver cancer to investigate the gene expression changes that accompany the switch from normoxic to hypoxia conditions during 2D growth and the switch from 2D monolayer to 3D organoid growth under normoxic conditions. They find a significant (ca. 40-50%) overlap among the genes that are dysregulated in response to hypoxia in 2D cultures and in response to spheroid formation. Unsurprisingly, hypoxia-related genes were among the most prominently deregulated under both sets of conditions. Many other pathways pertaining to metabolism, splicing, mitochondrial electron transport chain structure and function, DNA damage recognition/repair and lipid biosynthesis were also identified.

Comments on the revised manuscript:

In my original review of this manuscript, I raised 11 points that I thought needed to be addressed and/or clarified by the authors. In response, they have provided an adequate answer to only one of these (point 6), which is little more than a more thorough description of how spheroids were generated. The remaining points that I raised, which would have provided more mechanistic insight into their study were addressed by the authors with the following such comments:

- It is not the focus of this study (Points 1 and 4)

- It is worthy of further validation (Point 2)

- We apologize for not being able to validate everything (Point 3)

- This reviewer has raised an interesting question. We are investigating this hypothesis and hopefully we can give a clear answer in the future (Point 5)

- This is an excellent idea that we certainly will do it in our future experiments (Point 7)

As to responses that the authors made to the other two reviewers' comments: Most pertained to cosmetic alterations involving clarification of methods, inclusion of a new figure or rearrangement of old figures. These were generally answered. However, in response to the last point raised by Rev. 3 to compare "sgRNA abundances at the earliest harvesting time with the distribution in the library...to see whether and to what extent selection has already taken place before the three culture conditions were established", the authors responded with the comment: "This is great point. Unfortunately, we did not perform such an analysis."

I understand that it is often impossible to address all points raised by the reviewers. This can be for a variety of reasons and many times the omissions can be overlooked and accepted if the reviewer can be convinced that a good faith attempt has otherwise been made to address the other deficiencies. However, no such effort has been made here and the study remains deficient and largely descriptive as I pointed out in my original review.

Author response:

The following is the authors’ response to the original reviews

Public Reviews:

Reviewer #1 (Public review):

Summary:

In this report, the authors made use of a murine cell life derived from a MYC-driven liver cancer to investigate the gene expression changes that accompany the switch from normoxic to hypoxia conditions during 2D growth and the switch from 2D monolayer to 3D organoid growth under normoxic conditions. They find a significant (ca. 40-50%) overlap among the genes that are dysregulated in response to hypoxia in 2D cultures and in response to spheroid formation. Unsurprisingly, hypoxia-related genes were among the most prominently deregulated under both sets of conditions. Many other pathways pertaining to metabolism, splicing, mitochondrial electron transport chain structure and function, DNA damage recognition/repair, and lipid biosynthesis were also identified.

We thank this reviewer for his/her time and efforts, and the insightful comments.

Major comments:

(1) Lines 239-240: The authors state that genes involved in DNA repair were identified as being necessary to maintain survival of both 2D and 3D cultures (Figure S6A). Hypoxia is a strong inducer of ROS. Thus, the ROS-specific DNA damage/recognition/repair pathways might be particularly important. The authors should look more carefully at the various subgroups of the many genes that are involved in DNA repair. They should also obtain at least a qualitative assessment of ROS and ROS-mediated DNA damage by staining for total and mitochondrial-specific ROS using dyes such as CM-H2-DCFDA and MitoSox. Actual direct oxidative damage could be assessed by immunostaining for 8-oxo-dG and related to the sub-types of DNA damage-repair genes that are induced. The centrality of DNA damage genes also raises the question as to whether the previously noted prominence of the TP53 pathway (see point 5 below) might represent a response to ROS-induced DNA damage.

We thank this reviewer for the insightful comments, and agreed that ROS induced by hypoxia could play a role in modulating DNA repair and consequently cellular essentiality. Although pathway enrichment in Figure S6A (now as Figure 2-figure supplement 4A) showed that DNA repair pathway was essential to cell survival in hypoxia and 3D cultures, the genes associated with this pathway (Ddb1;Brf2;Gtf3c5;Guk1;Taf6) are not typical DNA repair genes. They are more likely involved in gene transcription. However, it will be interesting to see if they are specifically involved in DNA damage in response to ROS, which is out of focus of this study.

(2) Because most of the pathway differences that distinguish the various cell states from one another are described only in terms of their transcriptome variations, it is not always possible to understand what the functional consequences of these changes actually are. For example, the authors report that hypoxia alters the expression of genes involved in PDH regulation but this is quite vague and not backed up with any functional or empirical analyses. PDH activity is complex and regulated primarily via phosphorylation/dephosphorylation (usually mediated by PDK1 and PDP2, respectively), which in turn are regulated by prevailing levels of ATP and ADP. Functionally, one might expect that hypoxia would lead to the down-regulation of PDH activity (i.e. increased PDH-pSer392) as respiration changes from oxidative to non-oxidative. This would not be appreciated simply by looking at PDH transcript levels. This notion could be tested by looking at total and phospho-PDH by western blotting and/or by measuring actual PDH activity as it converts pyruvate to AcCoA.

We agreed with this reviewer that PDH activity regulation could be affected by multi-factors, and it is worthy of further validation by other approaches.

(3) Line 439: Related to the above point: the authors state: "It is likely that blockade of acetyl-CoA production by PDH knockout may force cells to use alternative energy sources under hypoxic and 3D conditions, averting the Warburg effect and promoting cell survival under limited oxygen and nutrient availability in 3D spheroids." This could easily be tested by determining whether exogenous fatty acids are more readily oxidized by hypoxic 2D cultures or spheroids than occurs in normoxic 2D cultures.

We thank for this suggestion. We apologized for not being able to validate everything.

(4) Line 472: "Hypoxia induces high expression of Acaca and Fasn in NEJF10 cells indicating that hypoxia promotes saturated fatty acid synthesis...The beneficial effect of Fasn and Acaca KO to NEJF10 under hypoxia is probably due to reduction of saturated fatty acid synthesis, and this hypothesis needs to be tested in the future.". As with the preceding comment, this supposition could readily be supported directly by, for example, performing westerns blots for these enzymes and by showing that incubation of hypoxic 2D cells or spheroids converted more AcCoA into lipid.

We thank for this suggestion. However, functional validation for the Fasn and Acaca KO is out of focus in this study.

(5) In Supplementary Figure 2B&C, the central hub of the 2D normoxic cultures is Myc (as it should well be) whereas, in the normoxic 3D, the central hub is TP53 and Myc is not even present. The authors should comment on this. One would assume that Myc levels should still be quite high given that Myc is driven by an exogenous promoter. Does the centrality of TP53 indicate that the cells within the spheroids are growtharrested, being subjected to DNA damage and/or undergoing apoptosis?

The predicted transcription factor activity analysis was based on the differential ATAC-seq peaks among different culture through pairwise comparisons. If TP53 and MYC were not present under that condition, it did not mean their activity was absent.

“…the centrality of TP53 indicate that the cells within the spheroids are growth-arrested, being subjected to DNA damage and/or undergoing apoptosis?” This reviewer has raised an interesting question. We are investigating this hypothesis and hopefully we can give a clear answer in the future.

(6) In the Materials and Methods section (lines 711-720), the description of how spheroid formation was achieved is unclear. Why were the cells first plated into non-adherent 96 well plates and then into nonadherent T75 flasks? Did the authors actually utilize and expand the cells from 144 T75 flasks and did the cells continue to proliferate after forming spheroids? Many cancer cell types will initially form monolayers when plated onto non-adherent surfaces such as plastic Petri dishes and will form spheroid-like structures only after several days. Other cells will only aggregate on the "non-adherent" surface and form spheroid-like structures but will not actually detach from the plate's surface. Have the authors actually documented the formation of true, non-adherent spheroids at 2 days and did they retain uniform size and shape throughout the collection period? The single photo in Supplementary Figure 1 does not explain when this was taken. The authors include a schematic in Figure 2A of the various conditions that were studied. A similar cartoon should be included to better explain precisely how the spheroids were generated and clarify the rationale for 96 well plating. Overall, a clearer and more concise description of how spheroids were actually generated and their appearance at different stages of formation needs to be provided.

The cells were initially plated in non-adherent 96-well plates to facilitate the formation of spheroids in a controlled and uniform manner. As correctly mentioned by the reviewer, during the initial stages, cells cultured on non-adherent surfaces often form aggregates or clumps, and it takes a few days for them to develop into solid spheroids.

In our study, we aimed to achieve 3D spheroid formation immediately following the transduction process to allow for screening under both 2D and 3D conditions. Plating the cells into 96-well plates enabled us to monitor and control the formation of spheroids in smaller volumes before scaling up the culture in non-adherent T75 flasks for subsequent experimental steps. This setup allows us to maintain gene editing processes under both 2D and 3D conditions.

Regarding the proliferation and uniformity of spheroids:

• Yes, the spheroids continued to proliferate after their formation.

• True, non-adherent spheroids were documented as early as the next day. This was visually confirmed under microscopy, and size uniformity was maintained throughout the collection period by following optimized culture protocols.

We also agreed with the reviewer’s suggestion to include a cartoon schematic similar to Figure 2A, illustrating the spheroid generation process and clarifying the rationale for using 96-well plates. We have included such a cartoon and speroid growth curve monitored by Incucyte as Figure 2-figure supplement 2.

(7) The authors maintained 2D cultures in either normoxic or hypoxic (1% O2) states during the course of their experiments. On the other hand, 3D cultures were maintained under normoxic conditions, with the assumption that the interiors of the spheroids resemble the hypoxic interiors of tumors. However, the actual documentation of intra-spheroid hypoxia is never presented. It would be a good idea for the authors to compare the degree of hypoxia achieved by 2D (1% O2) and 3D cultures by staining with a hypoxia-detecting dye such as Image-iT Green. Comparing the fluorescence intensities in 2D cultures at various O2 concentrations might even allow for the construction of a "standard curve" that could serve to approximate the actual internal O2 concentration of spheroids. This would allow the authors to correlate the relative levels of hypoxia between 2D and 3D cultures.

This is an excellent idea that we certainly will do it in our future experiments.

(8) Related to the previous 2 points, the authors performed RNAseq on spheroids only 48 hours after initiating 3D growth. I am concerned that this might not have been a sufficiently long enough time for the cells to respond fully to their hypoxic state, especially given my concerns in Point 6. Might the results have been even more robust had the authors waited longer to perform RNA seq? Why was this short time used?

We agreed with this reviewer. We were unsure if 48hours was an ideal timepoint. It might be necessary to perform a longitudinal experiment to harvest samples under different timepoints in the future experiments.

(9) What happens to the gene expression pattern if spheroids are re-plated into standard tissue culture plates after having been maintained as spheroids? Do they resume 2D growth and does the gene expression pattern change back?

This is a great question and we have never thought about what the gene expression pattern would be if speroids are re-plated in 2D. This could be a challenging experiment because the gene expression and epigenetic changes are timing related. However, the cells do grow well after re-plated in 2D.

(10) Overall, the paper is quite descriptive in that it lists many gene sets that are altered in response to hypoxia and the formation of spheroids without really delving into the actual functional implications and/or prioritizing the sets. Some of these genes are shown by CRISPR screening to be essential for maintaining viability although in very few cases are these findings ever translated into functional studies (for example, see points 14 above). The list of genes and gene pathways could benefit from a better explanation and prioritization of which gene sets the authors believe to be most important for survival in response to hypoxia and for spheroid formation.

This was a genome-wide study that integrated RNA-seq, ATAC-seq and CRISPR KO, providing resource to understand the oncogenic pathways in different culture conditions. We believe we have clearly articulated the important genes/pathways in our abstract.

(11) The authors used a single MYC-driven tumor cell line for their studies. However, in their original paper (Fang, et al. Nat Commun 2023, 14: 4003.) numerous independent cell lines were described. It would help to know whether RNAseq studies performed on several other similar cell lines gave similar results in terms of up & down-regulated transcripts (i.e. representative of the other cell lines are NEJF10 cells).

We have not generated RNA-seq data for these cell lines cultured in different conditions.

Reviewer #2 (Public review):

Summary:

The manuscript by Fang et al., provides a tour-de-force study uncovering cancer cell's varied dependencies on several gene programs for their survival under different biological contexts. The authors addressed genomic differences in 2D vs 3D cultures and how hypoxia affects gene expression. They used a Myc-driven murine liver cancer model grown in 2D monolayer culture in normoxia and hypoxia as well as cells grown as 3D spheroids and performed CRISPR-based genome-wide KO screen to identify genes that play important roles in cell fitness. Some context-specific gene effects were further validated by in-vitro and in-vivo gene KO experiments.

Strengths:

The key findings in this manuscript are:

(1) Close to 50% of differentially expressed genes were common between 2D Hypoxia and 3D spheroids conditions but they had differences in chromatin accessibility.

(2) VHL-HIF1a pathway had differential cell fitness outcomes under 2D normoxia vs 2D hypoxia and 3D spheroids.

(3) Individual components of the mitochondrial respiratory chain complex had contrasting effects on cell fitness under hypoxia.

(4) Knockout of organogenesis or developmental pathway genes led to better cell growth specifically in the context of 3D spheroids and knockout of epigenetic modifiers had varied effects between 2D and 3D conditions.

(5) Another key program that leads to cells fitness outcomes in normoxia vs hypoxia is the lipid and fatty acid metabolism.

(6) Prmt5 is a key essential gene under all growth conditions, but in the context of 3D spheroids even partial loss of Prmt5 has a synthetic lethal effect with Mtap deletion and Mtap is epigenetically silenced specifically in the 3D spheroids.

We appreciate this reviewer for acknowledging the strengths of our study.

Issues to address:

(1) The authors should clarify the link between the findings of the enrichment of TGFb-SMAD signaling REACTOME pathway to the findings that knocking out TGFb-SMAD pathway leads to better cell fitness outcomes for cells in the 3D growth conditions.

We have clarified this link in abstract by saying “Notably, multicellular organogenesis signaling pathways including TGFb-SMAD, which is upregulated in 3D culture, specifically constrict the uncontrolled cell proliferation in 3D while inactivation of epigenetic modifiers (Bcor, Kmt2d, Mettl3 and Mettl14) has opposite outcomes in 2D vs. 3D:

(2) Supplementary Figure 4C has been cited in the text but doesn't exist in the supplementary figures section.

Sorry for this typo. It should be 5C which is Figure 2-figure supplement 3C in the new version of MS. We have corrected it now.

(3) A small figure explaining this ABC-Myc driven liver cancer model in Supplementary Figure 1 would be helpful to provide context.

We appreciate this suggestion. We have added a cartoon as Figure 1-figure supplement 1A to indicate the procedure for generation of this model.

(4) The method for spheroids formation is not found in the method section.

We described the method in our previous publication (Nature Communications 2023 Jul 6;14(1):4003.). However, we have added the information in method now, and the procedure is very simple (line 623-624). We found the murine liver cancer cell lines can readily form spheroids when they are cultured in low-attachment dish with standard DMEM complete media.

(5) In Supplementary Figure 1b, the comparisons should be stated the opposite way - 3D vs 2D normoxia and 2D-Hypoxia vs 2D-Normoxia.

We have made correction in the Figure legend of Figure S1B which is Figure 1B now in the new version of MS.

(6) There are typos in the legend for Supplementary Figure 10.

We have checked the typos.

(7) Consider putting Supplementary Figure 1b into the main Figure 1.

We have moved both Supplementary Figure 1a and 1b into main Figure 1 as Figure 1A and 1B. Hopefully, this will help the readers to catch the information easily.

(8) Please explain only one timepoint (endpoint) for 3D spheroids was performed for the CRISPR KO screen experiment, while several timepoints were done for 2D conditions? Was this for technical convenience?

As this reviewer speculated, indeed this was for technical convenience. We found that it was technically challenging to split the spheroids for CRISPR screening.

(9) In line 372, it is indicated that Bcor KO (Fig 5e) had growth advantage - this was observed in only one of the gRNA -- same with Kmt2d KO in the same figure where there was an opposite effect. Please justify the use of only one gRNA.

We actually used 4 gRNAs for each gene. In the heatmap, although one of the gRNA for each gene showed some levels of enrichment under hypoxic 2D condition, they were all highly enriched in 3D.

(10) Why was CRISPR based KO strategy not used for the PRMT5 gene but rather than the use of shRNA.? Note that one of the shRNA for PRMT5 had almost no KO (PRMT5-shRNA2 Figure 7B) but still showed phenotype (Figure 7D) - please explain.

We used shRNA as second approach for cross-validation. We agreed that the knockdown efficiency of shRNA2 was not as good as the others, with only about 40% knockdown efficiency.

(11) In Figure 7D, which samples (which shRNA group) were being compared to do the t-test?

The comparisons were for shCtrl and each of the shPRMT5. We have clarified this in figure legend.

(12) In line 240, it is stated that oxphos gene set is essential for NEJF10 cell survival in both normoxia and hypoxia conditions. But shouldn't oxphos be non-essential in hypoxia as cells move away from oxphos and become glycolytic?

This is a great question. While indeed hypoxia may promote the switch from oxphos to glycolysis, several studies showed that the low oxygen concentrations in hypoxic regions of tumors may not be limiting for oxphos, and ATP is generated by oxphos in tumors even at very low oxygen tensions (please see review Clin Cancer Res (2018) 24 (11): 2482–2490.). We therefore speculated that NEJF10 cells were still dependent on oxphos for ATP production under hypoxia. However, this needs further investigation. We have added this discussion in our manuscript (line 250-254).

(13) In line 485 it is mentioned that Pmvk and Mvd genes which are involved in cholesterol synthesis when knocked out had a positive effect on cell growth in 3D conditions and since cholesterol synthesis is essential for cell growth how does this not matter much in the context of 3D - please explain.

We thank this reviewer for this note. It seemed that only two gRNA for each were upregulated in 3D and it could be due to technical issue or clonal selection. We have deleted this sentence in our new version of MS.

Reviewer #3 (Public review):

Summary:

In this study, Fang et al. systematically investigate the effects of culture conditions on gene expression, genome architecture, and gene dependency. To do this, they cultivate the murine HCC line NEJF10 under standard culture conditions (2D), then under similar conditions but under hypoxia (1% oxygen, 2D hypoxia) and under normoxia as spheroids (3D). NEJF10 was isolated from a marine HCC model that relies exclusively on MYC as a driver oncogene. In principle, (1) RNA-seq, (2) ATAC-seq and (3) genetic screens were then performed in this isogenic system and the results were systematically compared in the three cultivation methods. In particular, genome-wide screens with the CRISPR library Brie were performed very carefully. For example, in the 2D conditions, many different time points were harvested to control the selection process kinetically. The authors note differential dependencies for metabolic processes (not surprisingly, hypoxia signaling is affected) such as the regulation and activity of mitochondria, but also organogenesis signaling and epigenetic regulation.

Strengths:

The topic is interesting and relevant and the experimental set-up is carefully chosen and meaningful. The paper is well written. While the study does not reveal any major surprises, the results represent an important resource for the scientific community.

We thank this reviewer for his/her positive comments.

Weaknesses:

However, this presupposes that the statistical analysis and processing are carried out very carefully, and this is where my main suggestions for revision begin. Firstly, I cannot find any information on the number of replicates in RNA- and ATAC-seq. This should be clearly stated in the results section and figure legends and cut-offs, statistical procedures, p-values, etc. should be mentioned as well. In principle, all NGS experiments (here ATAC- and RNA-seq) should be performed in replicates (at least duplicates, better triplicates) or the results should be validated by RT-PCR in independent biological triplicates. Secondly, the quantification of the analyses shown in the figures and especially in the legends is not sufficiently careful. Units are often not mentioned. Example Figure 4a: The legend says: 'gRNA reads' but how can the read count be -1? I would guess these are FC, log2FC, or Z-values. All figure legends need careful revision.

Based upon the reviewer’s suggestions, we have added details about the replicates in figure legend. For gRNA read heatmap, the scale bar indicates the Z score. We have added the information in figure legends.

Furthermore, I would find a comparison of the sgRNA abundances at the earliest harvesting time with the distribution in the library interesting, to see whether and to what extent selection has already taken place before the three culture conditions were established (minor point).

This is great point. Unfortunately, we did not perform such an analysis.

Recommendations for the authors:

Reviewing Editor:

There are three general issues:

First, there is a lack of detail regarding much of the analysis. In some cases, this makes it difficult to assess the value of the data, albeit, there is generally a consensus the information is really interesting.

Second, the findings - although provocative - lack mechanistic details and are focused more on descriptive findings. Hence, the manuscript would be improved by some effort at evaluating identified programs and providing some suggestions of mechanisms.

Third, the authors need to put much more effort into the clarity and tightness of the presentation.

We have made clarification in response to the reviewer’s comments.

Reviewer #1 (Recommendations for the authors):

Figure S1C. the labeling of the lower x-axis is inverted.

Due to space limitation, we changed the figure orientation in our old version of MS. We have tilted the figure back in the new version, which is Figure 1-figure supplement 1B now.

尼采在《悲剧的诞生》中提出，希腊悲剧的诞生是阿波罗精神（Apollo）与狄奥尼索斯精神（Dionysus）两种艺术冲动的完美结合。这种结合在埃斯库罗斯（Aeschylus）和索福克勒斯（Sophocles）的悲剧中尤为显著，他们的作品通过阿波罗的“形式”（戏剧结构、人物塑造等）为狄奥尼索斯的“原始冲动”（对生命痛苦的直面与超越）提供了表达载体，使观众在审美体验中暂时超越现实的痛苦。以下从多个维度展开分析：

一、阿波罗精神：理性的形式与美的幻象

1. 阿波罗的象征：
2. 理性与个体化：阿波罗代表太阳神，象征理性、秩序、个体化的美。尼采认为，阿波罗通过“梦”的意象，将混沌的现实转化为清晰的表象（如雕塑、戏剧的结构），为人类提供对痛苦的逃避（知识库[1][5][6]）。

3. 形式的完美：在戏剧中，阿波罗精神体现为严谨的结构、清晰的人物塑造、逻辑的情节发展，以及对悲剧主题的象征性表达。

4. 在埃斯库罗斯和索福克勒斯作品中的体现：

5. 埃斯库罗斯：
   • 《被缚的普罗米修斯》通过清晰的戏剧冲突（普罗米修斯与宙斯的对抗）和英雄形象的塑造，将人类对自由与反抗的原始冲动（狄奥尼索斯精神）包裹在史诗般的结构中。
   • 三联剧《俄瑞斯忒亚》通过严密的叙事逻辑（从杀戮到审判的循环）将血腥的复仇转化为对法律与秩序的哲学思考，体现了阿波罗式的理性框架。
6. 索福克勒斯：
   • 《俄狄浦王》以严谨的“悲剧结构”（如命运伏笔、突转与发现）将角色的挣扎（如俄狄浦王的无意识犯罪）转化为可理解的戏剧冲突，其人物塑造（如俄狄浦王的悲剧性尊严）体现了阿波罗式的“适度”与“个体化”（知识库[4][6][9]）。

二、狄奥尼索斯精神：原始冲动与生命的深渊

1. 狄奥尼索斯的象征：
2. 非理性与集体狂喜：狄奥尼索斯代表酒神，象征非理性、醉狂、对个体界限的消融。通过“醉”的体验，人类直面生命的痛苦与无意义，并在集体情感中与宇宙本体（生命意志）融为一体（知识库[1][6][7]）。

3. 悲剧的原始力量：狄奥尼索斯精神揭示了生命的本质——痛苦、无常与毁灭，但通过艺术（如音乐与合唱）将这种痛苦转化为审美体验，从而超越它。

4. 在希腊悲剧中的体现：

5. 合唱队的作用：
   • 合唱队（如《阿伽门农》中的合唱队）以集体形式表达对命运的恐惧与敬畏，将个体的悲剧升华为对宇宙规律的共鸣，体现了狄奥尼索斯式的“集体狂喜”（知识库[4][8]）。
6. 命运主题：
   • 俄狄浦王的悲剧并非个人过错，而是命运（狄奥尼索斯式的“原始冲动”）的必然。观众在悲剧中体验到对命运的恐惧与敬畏，从而在情感宣泄中获得“净化”（卡塔西斯）（知识库[6][9]）。

三、两者的结合：悲剧如何超越痛苦

1. 形式与内容的统一：
2. 阿波罗的形式约束：通过戏剧的结构、人物的英雄性、语言的美感，将狄奥尼索斯的原始冲动转化为可接受的艺术形式，避免观众陷入纯粹的绝望（知识库[1][5][6]）。

3. 狄奥尼索斯的深层意义：在形式的框架下，悲剧揭示了生命的真相——痛苦与毁灭不可避免，但艺术使其成为一种审美体验，从而赋予其意义。

4. 观众的审美体验：

5. 暂时超越痛苦：观众在悲剧中既感受到命运的残酷（狄奥尼索斯的启示），又被戏剧的美与秩序（阿波罗的形式）所吸引，从而在矛盾中获得一种“形而上的慰藉”。
6. 尼采的“艺术救赎”：悲剧通过二者的结合，使观众在审美中暂时摆脱现实的焦虑，直面生命的真实，同时不陷入虚无（知识库[2][5][7]）。

四、案例分析：埃斯库罗斯与索福克勒斯的悲剧

1. 埃斯库罗斯的《被缚的普罗米修斯》：
2. 阿波罗层面：普罗米修斯的形象是英雄的个体化（阿波罗式的“个体化原理”），其反抗宙斯的结构清晰，充满史诗的庄严。

3. 狄奥尼索斯层面：普罗米修斯的痛苦（被缚、受罚）象征人类对命运的抗争，观众在悲剧中感受到对自由意志的崇敬，以及对宇宙力量的敬畏（知识库[9]）。

4. 索福克勒斯的《俄狄浦王》：

5. 阿波罗层面：戏剧结构精巧（如预言的伏笔与现实的反讽），人物塑造充满尊严（俄狄浦王的智慧与悲剧性选择）。
6. 狄奥尼索斯层面：俄狄浦王的毁灭揭示了命运的不可抗性，观众在恐惧与怜悯中体验到生命的无意义，但通过艺术形式将其转化为对人性深度的探索（知识库[4][6]）。

五、尼采的批判与哲学意义

1. 对苏格拉底理性主义的批判：
2. 尼采认为，苏格拉底用逻辑与道德解释世界（理性主义），破坏了悲剧中阿波罗与狄奥尼索斯的平衡，导致希腊悲剧的消亡（知识库[1][9]）。

3. 悲剧的没落使人类失去了通过艺术超越痛苦的能力，转而陷入理性的虚幻乐观，最终导向现代的虚无主义（知识库[2][7]）。

4. 对现代文明的启示：

5. 希腊悲剧的结合模式为人类提供了对抗虚无的路径：通过艺术（而非理性）直面生命的痛苦，并在形式中赋予其意义。
6. 尼采呼吁现代人回归这种“悲剧精神”，在理性与非理性的平衡中重获对生命的热情（知识库[2][7][9]）。

总结：悲剧的双重救赎

埃斯库罗斯和索福克勒斯的悲剧之所以伟大，正因其完美融合了阿波罗与狄奥尼索斯两种精神：<br /> - 阿波罗的形式为人类提供了理解痛苦的框架，使其可被审美化；<br /> - 狄奥尼索斯的原始冲动则揭示了生命的真实本质，迫使观众直面深渊。<br /> 二者的结合使希腊悲剧成为一种“形而上的慰藉”——在美与毁灭的碰撞中，人类既承认痛苦的必然，又在艺术中超越它，这正是尼采所推崇的悲剧哲学的核心。

Author response:

The following is the authors’ response to the original reviews

Reviewer #1:

Summary:

The authors address the role of the centromere histone core in force transduction by the kinetochore.

Strengths:

They use a hybrid DNA sequence that combines CDEII and CDEIII as well as Widom 601 so they can make stable histones for biophysical studies (provided by the Widom sequence) and maintain features of the centromere (CDE II and III).

Weaknesses:

The main results are shown in one figure (Figure 2). Indeed the Centromere core of Widom and CDE II and III contribute to strengthening the binding force for the OA-beads. The data are very nicely done and convincingly demonstrate the point. The weakness is that this is the entire paper. It is certainly of interest to investigators in kinetochore biology, but beyond that, the impact is fairly limited in scope.

This reviewer might have missed that this is a Research Advance, not an article. Research Advances are limited in scope by definition and provide a new development that builds on research reported in a prior paper. They can be of any length. Our Research Advance builds on our prior work, Hamilton et al., 2020 and provides the new result that native centromere sequences strengthen the attachment of the kinetochore to the nucleosome.

Reviewer #2:

Summary:

This paper provides a valuable addendum to the findings described in Hamilton et al. 2020 (https://doi.org/10.7554/eLife.56582). In the earlier paper, the authors reconstituted the budding yeast centromeric nucleosome together with parts of the budding yeast kinetochore and tested which elements are required and sufficient for force transmission from microtubules to the nucleosome. Although budding yeast centromeres are defined by specific DNA sequences, this earlier paper did not use centromeric DNA but instead the generic Widom 601 DNA. The reason is that it has so far been impossible to stably reconstitute a budding yeast centromeric nucleosome using centromeric DNA.

In this new study, the authors now report that they were able to replace part of the Widom 601 DNA with centromeric DNA from chromosome 3. This makes the assay more closely resemble the in vivo situation. Interestingly, the presence of the centromeric DNA fragment makes one type of minimal kinetochore assembly, but not the other, withstand stronger forces.

We thank the reviewer for their careful and positive assessment of our work.

Which kinetochore assembly turned out to be affected was somewhat unexpected, and can currently not be reconciled with structural knowledge of the budding yeast centromere/kinetochore. This highlights that, despite recent advances (e.g. Guan et al., 2021; Dendooven et al., 2023), aspects of budding yeast kinetochore architecture and function remain to be understood and that it will be important to dissect the contributions of the centromeric DNA sequence.

We couldn’t agree more.

Given the unexpected result, the study would become yet more informative if the authors were able to pinpoint which interactions contribute to the enhanced force resistance in the presence of centromeric DNA.

Strength:

The paper demonstrates that centromeric DNA can increase the attachment strength between budding yeast microtubules and centromeric nucleosomes.

Weakness:

How centromeric DNA exerts this effect remains unclear.

Recommendations for the authors:

Reviewer #2 (Recommendations for the authors):

(1) Additional specific mutants would be helpful in interpreting the effect observed. The authors speculate that a small segment of OA near the DNA (based on Dendooven et al., 2023) could be important. Would it be possible to introduce specific mutations and test this?

This would be an interesting study but is far beyond the scope of a Research Advance. In fact, it would make a nice thesis project for a new student. Although perhaps not obvious, these studies require a large set of reagents including wrapped nucleosomes, which must be made fresh (they cannot be frozen) and five purified recombinant complexes, purified by specialized protocols that maintain their activity. Moreover, each datapoint is gathered one at a time. For example, the data in Figure 2 in this manuscript includes 343 datapoints acquired one at a time over the course of 1.5 years.  

(2) Please provide the sequences of the other CEN3-W601 chimeras that were tested and did NOT stably wrap centromeric histone octamers. This may help others to design yet different constructs in the future. (Maybe the information is there and I didn't see it?)

We fully agree and thank the reviewer for this excellent suggestion. The sequences and summaries of their wrapping stability are now provided in Table 3, page 17.

(3) I wonder whether the authors tested the C0N3 sequence used in Dendooven et al., 2023. If not, could it be tested? This would more tightly couple the functional assay shown here with the structural work.

We did not test the CON3 sequence, which was published several years after the start of this work. We agree that a tight coupling between the functional assay and the structural work would be useful. However, we also see the advantage of being able to go beyond the structural work and include even more CEN3 sequence than has so far been possible in the structural work.  

In addition to measuring the role of DNA sequence in Okp1/Ame1 attachment to the nucleosome, we were interested in the role of DNA sequence in the attachment of Mif2. Therefore, we included all 35 bp of the Mif2 footprint in our chimeric CCEN DNA sequence. CON3 only includes 8 bp from CDEII. We did produce stable nucleosomes using CEN3-601 from Guan et al. (see Table 3). Again, CEN3-601 only includes 8 bp of the Mif2 footprint so we opted to study nucleosomes wrapped in our CCEN DNA with the entire Mif2 footprint. Curiously we found that even the entire Mif2 footprint was not enough to find the DNA sequence specificity seen in the EMSA experiments reported by Xiao et al., 2017.

To help readers understand the differences between all these constructs, we have included them in Table 3.

(4) Would an AlphaFold 3 prediction of the assemblies used in this paper be feasible and useful?

The structures of the Dam1 complex (Jenni et al., 2018), Ndc80 complex (Zahm, et al., 2023 and references therein), MIND complex (Dimitrova et al., 2016), OA complex (Dendooven et al., 2023), and the nucleosome (Xaio et al., 2017; Yan et al., 2019; Guan et al., 2021; Dendooven et al., 2023) are published. The interactions between many of these complexes are understood beyond the level that AlphaFold3 could provide (Dimitrova et al., 2016; Dendooven et al., 2023). One of the main questions is how Mif2 interacts with the nucleosome and the other components of the kinetochore. Even structural analyses that included Mif2 in the assembly detect little or no Mif2 in the final structure. Unfortunately, AlphaFold3 is also not helpful as it predicts only the structure of the dimerization domain, which was already known (Cohen et al., 2008).

AlphaFold3 predicts the rest of Mif2 is largely unstructured with several alpha helices predicted with low confidence.

(5) Given that the centromeric DNA piece included should be able to bind the CBF3 complex, would it be possible to add this complex and test the effect on force transmission?

This would be an interesting experiment, and we do expect CBF3 to bind. As stated above, this is far beyond the scope of this Research Advance. In our experience, with each new kinetochore subcomplex that we add into our reconstitutions, there are new challenges purifying the subcomplex in active form and in sufficient quantity. We are eager to add CBF3 but this is not something we can pull off in the context of this Research Advance. Thank you again for the time and energy spent reviewing our manuscript

Reviewer #1 (Public review):

The authors set out to analyse the roles of the teichoic acids of Streptococcus pneumoniae in supporting the maintenance of the periplasmic region. Previous work has proposed the periplasm to be present in Gram positive bacteria and here advanced electron microscopy approach was used. This also showed a likely role for both wall and lipo-teichoic acids in maintaining the periplasm. Next, the authors use a metabolic labelling approach to analyse the teichoic acids. This is a clear strength as this method cannot be used for most other well studied organisms. The labelling was coupled with super-resolution microscopy to be able to map the teichoic acids at the subcellular level and a series of gel separation experiments to unravel the nature of the teichoic acids and the contribution of genes previously proposed to be required for their display. The manuscript could be an important addition to the field but there are a number of technical issues which somewhat undermine the conclusions drawn at the moment. These are shown below and should be addressed. More minor points are covered in the private

Recommendations for Authors.

Weaknesses to be addressed:

(1) l. 144 Was there really only one sample that gave this resolution? Biological repeats of all experiments are required.

(2) Fig. 4A. Is the pellet recovered at "low" speeds not just some of the membrane that would sediment at this speed with or without LTA? Can a control be done using an integral membrane protein and Western Blot? Using the tacL mutant would show the behaviour of membranes alone.

(3) Fig. 4A. Using enzymatic digestion of the cell wall and then sedimentation will allow cell wall associated proteins (and other material) to become bound to the membranes and potentially effect sedimentation properties. This is what is in fact suggested by the authors (l. 1000, Fig. S6). In order to determine if the sedimentation properties observed are due to an artefact of the lysis conditions a physical breakage of the cells, using a French Press, should be carried out and then membranes purified by differential centrifugation. This is a standard, and well-established method (low-speed to remove debris and high-speed to sediment membranes) that has been used for S. pneumoniae over many years but would seem counter to the results in the current manuscript (for instance Hakenbeck, R. and Kohiyama, M. (1982), Purification of Penicillin-Binding Protein 3 from Streptococcus pneumoniae. European Journal of Biochemistry, 127: 231-236).

(4) l. 303-305. The authors suggest that the observed LTA-like bands disappear in a pulse chase experiment (Fig. 6B). What is the difference between this and Fig. 5B, where the bands do not disappear? Fig. 5C is the WT and was only pulse labelled for 5 min and so would one not expect the LTA-like bands to disappear as in 6B?

(5) Fig. 6B, l. 243-269 and l. 398-410. If, as stated, most of the LTA-like bands are actually precursor then how can the quantification of LTA stand as stated in the text? The "Titration of Cellular TA" section should be re-evaluated or removed? If you compare Fig. 6C WT extract incubated at RT and 110oC it seems like a large decrease in amount of material at the higher temperature. Thus, the WT has a lot of precursors in the membrane? This needs to be quantified.

(6) L. 339-351, Fig. 6A. A single lane on a gel is not very convincing as to the role of LytR. Here, and throughout the manuscript, wherever statements concerning levels of material are made, quantification needs to be done over appropriate numbers of repeats and with densitometry data shown in SI.

(7) 14. l. 385-391. Contrary to the statement in the text, the zwitterionic TA will have associated counterions that results in net neutrality. It will just have both -ve and +ve counterions in equal amounts (dependent on their valency), which doesn't matter if it is doing the job of balancing osmolarity (rather than charge).

Comments on revisions:

The resubmitted manuscript now contains new data and changes to the text.

The authors have largely covered my previous points in both sets of reviews (Public/Recommendations).

Public Review Points:

1 & 6: I still do not see a reproducibility statement as such, with details of the number of biological repeats etc.

2 & 3. Fig S7 seems to be quite telling. As predicted after physical breakage the membrane proteins sediment at high speed (rather than low speed). This presumably also means that the LTA comes down at high and not low speed. LTA was not measured due to cost of reagents. The Microfluidizer breaks the cells using a shear force and thus is unlikely to create very small membrane fragments. Thus, the sedimentation properties of membranes containing LTA are likely dependent on the way in which the cells are lysed. It is therefore worthwhile qualifying the statements on l. 35-36, 46-47 and 212 (as Ref 8 used mechanical breakage). This will give better direction to those in the field following up the findings.

It is also a little alarming that the mutanolysin is contaminated by protease and one hopes this does not affect any of the properties of the materials being analysed.

Author response:

The following is the authors’ response to the original reviews

Public Reviews:

Reviewer #1 (Public review):

The authors set out to analyse the roles of the teichoic acids of Streptococcus pneumoniae in supporting the maintenance of the periplasmic region. Previous work has proposed the periplasm to be present in Gram positive bacteria and here advanced electron microscopy approach was used. This also showed a likely role for both wall and lipo-teichoic acids in maintaining the periplasm. Next, the authors use a metabolic labelling approach to analyse the teichoic acids. This is a clear strength as this method cannot be used for most other well studied organisms. The labelling was coupled with super-resolution microscopy to be able to map the teichoic acids at the subcellular level and a series of gel separation experiments to unravel the nature of the teichoic acids and the contribution of genes previously proposed to be required for their display. The manuscript could be an important addition to the field but there are a number of technical issues which somewhat undermine the conclusions drawn at the moment. These are shown below and should be addressed. More minor points are covered in the private Recommendations for Authors.

Weaknesses to be addressed:

(1) l. 144 Was there really only one sample that gave this resolution? Biological repeats of all experiments are required.

CEMOVIS is a very challenging method that is not amenable to numerous repeats. However, multiple images were recorded from at least two independent samples for each strain. Additional sample images are shown in a new Fig. S3.

CETOVIS is even more challenging (only two publications in Pubmed since 2015) and was performed on a single ultrathin section that, exceptionally, laid perfectly flat on the EM grid, allowing tomography data acquisition on ∆tacL cells. The reconstructed tomogram confirmed the absence of a granular layer in the depth of the section. Additionally, the numbering of Fig. S4A-B (previously misidentified as Fig. S2A-B) has been corrected in the text of V2.

(2) Fig. 4A. Is the pellet recovered at "low" speeds not just some of the membrane that would sediment at this speed with or without LTA? Can a control be done using an integral membrane protein and Western Blot? Using the tacL mutant would show the behaviour of membranes alone.

We think that the pellet is not just some of the membrane but most of it. In support of this view, the “low” speed pellets after enzymatic cell lysis contain not just some membrane lipids, but most of them (Fig. S10A). We therefore expect membrane proteins to be also present in this fraction. We performed a Western blot using antibodies against the membrane protein PBP2x (new Fig. S7C). Unfortunately, no signal was detected most likely due to protein degradation from contaminant proteases that we could trace to the purchased mutanolysin. The same sedimentation properties were observed with the ∆tacL strain as shown in Fig. 6A. However, in the ∆tacL strain the membrane pellet still contains membrane-bound TA precursors. It is therefore impossible to test definitely if pneumococcal membranes totally devoid of TA would sediment in the same way.

(3) Fig. 4A. Using enzymatic digestion of the cell wall and then sedimentation will allow cell wall associated proteins (and other material) to become bound to the membranes and potentially effect sedimentation properties. This is what is in fact suggested by the authors (l. 1000, Fig. S6). In order to determine if the sedimentation properties observed are due to an artefact of the lysis conditions a physical breakage of the cells, using a French Press, should be carried out and then membranes purified by differential centrifugation. This is a standard, and well-established method (low-speed to remove debris and high-speed to sediment membranes) that has been used for S. pneumoniae over many years but would seem counter to the results in the current manuscript (for instance Hakenbeck, R. and Kohiyama, M. (1982), Purification of Penicillin-Binding Protein 3 from Streptococcus pneumoniae. European Journal of Biochemistry, 127: 231-236).

Thank you for this suggestion. We have tested this hypothesis by breaking cells with a Microfluidizer followed by differential centrifugation. This experiment, which requires an important minimal volume, was performed with unlabeled cells (due to the cost of reagents) and assessed by Western blot using antibodies against the membrane protein PBP2x (new Fig. S7C). In this case, the majority of the membrane material was found in the high-speed pellet, as expected.

We also applied the spheroplast lysis procedure of Flores-Kim et al. to the labeled cells, and found that most of the labeled material sedimented at low speed (new Fig. S7B), as observed with our own procedure.

With these new results, the section on membrane density has been removed from the Supplementary Information. Instead, the fractionation is further discussed in terms of size of membrane fragments and presence of intact spheroplasts in the notes in Supplementary Information preceding Fig. S7.

(4) l. 303-305. The authors suggest that the observed LTA-like bands disappear in a pulse chase experiment (Fig. 6B). What is the difference between this and Fig. 5B, where the bands do not disappear? Fig. 5C is the WT and was only pulse labelled for 5 min and so would one not expect the LTA-like bands to disappear as in 6B?

Fig. 6B shows a pulse-chase experiment with strain ∆tacL, whereas Fig. 5C shows a similar experiment with the parental WT strain. The disappearance of the LTA-like band pattern with the ∆tacL strain (Fig. 6B), and their persistence in the WT strain (Fig. 5C), indicate that these bands are the undecaprenyl-linked TA in ∆tacL and proper LTA in the WT. A sentence has been added to better explain this point in V2.

Note that we have exchanged the previous Fig. 5C and Fig. S13B, so that the experiments of Fig. 5A and 5C are in the same medium, as suggested by Reviewer #2.

(5) Fig. 6B, l. 243-269 and l. 398-410. If, as stated, most of the LTA-like bands are actually precursor then how can the quantification of LTA stand as stated in the text? The "Titration of Cellular TA" section should be re-evaluated or removed? If you compare Fig. 6C WT extract incubated at RT and 110oC it seems like a large decrease in amount of material at the higher temperature. Thus, the WT has a lot of precursors in the membrane? This needs to be quantified.

Indeed, the quantification of the ratio of LTA and WTA in the WT strain rests on the assumption that the amount of membrane-linked polymerized TA precursors is negligible in this strain. This assumption is now stated in the Titration section. We think it is the case. The true LTA and TA precursors do not have exactly the same electrophoretic mobility, being shifted relative to each other by about half a ladder “step”. This difference is visible when samples are run in adjacent lanes on the same gel, as in the new Fig. 6C. The difference of migration was well documented in the original paper about the deletion of tacL, although tacL was known as rafX at that time, and the ladders were misidentified as WTA (Wu et al. 2014. A novel protein, RafX, is important for common cell wall polysaccharide biosynthesis in Streptococcus pneumoniae: implications for bacterial virulence. J Bacteriol. 196, 3324-34. doi: 10.1128/JB.01696-14). This reference was added in V2. The experiment in the new Fig. 6C was repeated to have all samples on the same gel and treated at a lower temperature. The minor effect on the amount of LTA when WT cells are heated at pH 4.2 may be due to the removal of some labeled phosphocholine. We have NMR evidence that the phosphocholine in position D is labile to acidic treatment of LTA, which may lack in some cases, as reported by Hess et al. (Nat Commun. 2017 Dec 12;8(1):2093. doi: 10.1038/s41467-017-01720-z).

(6) L. 339-351, Fig. 6A. A single lane on a gel is not very convincing as to the role of LytR. Here, and throughout the manuscript, wherever statements concerning levels of material are made, quantification needs to be done over appropriate numbers of repeats and with densitometry data shown in SI.

Yes indeed. Apart from the titration of TA in the WT strain, we haven’t yet carried out a thorough quantification of TA or LTA/WTA ratio in different strains and conditions, although we intend to do so in a follow-up study, using the novel opportunities offered by the method presented here.

However, to better substantiate our statement regarding the ∆lytR strain, we have quantified two experiments performed in C-medium with azido-choline, and two experiments of pulse labeling in BHI medium. The results are presented in the additional supplementary Fig. S14. The value of 51% was a calculation error, and was corrected to 41%. Likewise, the decrease in the WTA/LTA ratio was corrected to 5 to 7-fold.

(7) 14. l. 385-391. Contrary to the statement in the text, the zwitterionic TA will have associated counterions that result in net neutrality. It will just have both -ve and +ve counterions in equal amounts (dependent on their valency), which doesn't matter if it is doing the job of balancing osmolarity (rather than charge).

Thank you for pointing out this point. The paragraph has been corrected in V2.

Reviewer #2 (Public review):

The Gram-positive cell wall contains for a large part of TAs, and is essential for most bacteria. However, TA biosynthesis and regulation is highly understudied because of the difficulties in working with these molecules. This study closes some of our important knowledge gaps related to this and provides new and improved methods to study TAs. It also shows an interesting role for TAs in maintaining a 'periplasmic space' in Gram positives. Overall, this is an important piece of work. It would have been more satisfying if the possible causal link between TAs and periplasmic space would have been more deeply investigated with complemented mutants and CEMOVIS. For the moment, there is clearly something happening but it is not clear if this only happens in TA mutants or also in strains with capsules/without capsules and in PG mutants, or in lafB (essential for production of another glycolipid) mutants. Finally, some very strong statements are made suggesting several papers in the literature are incorrect, without actually providing any substantiation/evidence supporting these claims. Nevertheless, I support the publication of this work as it pioneers some new methods that will definitively move the field forward.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

(1) l. 55 It is stated that TA are generally not essential. This needs to be introduced in a little more detail as in several species they are collectively. Need some more references here to give context.

We have expended the paragraph and added a selection of references in V2.

(2) l. 63 and Fig. 1A. Is the model based on the images from this paper? Is the periplasm as thick as the peptidoglycan layer? Would you not expect the density of WTA to be the same throughout the wall, rather than less inside? Do the authors think that the TA are present as rods in the cell envelope and because of this the periplasm looks a little like a bilayer, is this so? Is the relative thickness of the layers based on the data in the paper (Table 1)?

The model proposed in Fig. 1A is not based on our data. It is a representation of the model proposed by Harold Erickson, and the appropriate reference has been added to the figure legend in V2. We do not speculate on the relative density of WTA inside the peptidoglycan layer, at the surface or in the periplasm. The only constraint from the model is that the density of WTA in the periplasm should be sufficient for self-exclusion and allow the brush polymer theory to apply. The legend has been amended in V2.

We indeed think that the bilayer appearance of the periplasmic space in the wild type strain, and the single layer periplasmic space in the ∆tacL and ∆lytR support the Erickson’s model. Although the model was drawn arbitrarily, it turns out that the relative thickness of the peptidoglycan and periplasmic scale is in rough agreement with the measurements reported in Table 1.

(3) Fig. 2. It is hard to orient oneself to see the layers. The use of the term periplasmic space (l. 132) and throughout is probably not wise as it is not a space.

We prefer to retain this nomenclature since the term periplasmic space has been used in all the cell envelope CEMOVIS publications and is at the core of Erickson’s hypothesis about these observations and teichoic acids.

(4) L. 147. This is not referring to Fig. S2A-B as suggested but Fig. S3A-B.

This has been corrected.

(5) l. 148. How do you know the densities observed are due to PG or certainly PG alone? Perhaps it is better to call this the cell wall.

Yes. Cell wall is a better nomenclature and the text and Table 1 have been corrected in V2, in accordance with Fig. 2.

(6) l. 165. It is also worth noting that peripheral cell wall synthesis also happens at the same site so this may well not be just division.

Yes. We have replaced “division site” by “mid-cell” in V2.

(7) l. 214 What is the debris? If PG digestion has been successful then there will be marginal debris. Is this pellet translucent (like membranes)? If you use fluorescently labelled PG in the preparation has it all disappeared, as would be expected by fully digested and solubilised material?

In traditional protocols of bacterial membrane preparation, a low-speed centrifugation is first performed to discard “debris” that to our knowledge have not been well characterized but are thought to consist of unbroken cells and large fragments of cell wall. After enzymatic degradation of the pneumococcal cell wall, the low-speed pellet is not translucent as in typical membrane pellets after ultracentrifugation, but is rather loose, unlike a dense pellet of unbroken cells. A description of the pellet appearance was added in V2.

It is a good idea to check if some labeled PG is also pelleted at low-speed after digestion. In a double labeling experiment using azido-choline and a novel unpublished metabolic probe of the PG, we found that the PG was fully digested and labeled fragments migrated as a couple of fuzzy bands likely corresponding to different labeled peptides. These species were not pelleted at low speed.

(8) l. 219. Can you give a reference to certify that the low mobility material is WTA? Why does it migrate differently than LTA? Or is the PG digestion not efficient?

WTA released from sacculi by alkaline lysis were found to migrate as a smear at the top of native gels revealed by alcian-blue silver staining, which is incompatible with SDS (Flores-Kim, 2019, 2022). The references have be added in V2. It could be argued in this case that the smearing was due to partial degradation of the WTA by the alkaline treatment.

Bui et al. (2012) reported the preparation of WTA by enzymatic digestion of sacculi, but the resulting WTA were without muropeptide, presumably due to a step of boiling at pH 5 used to deactivate the enzymes.

To our knowledge, this is the first report of pneumococcal WTA prepared by digestion of sacculi and analyzed by SDS-PAGE. Since the migration of WTA in native and SDS-PAGE is similar, we hypothesize that they do not interact significantly with the dodecyl sulphate, in contrast to the LTA, which bear a lipidic moiety. The fuzziness of the WTA migration pattern may also result from the greater heterogeneity due to the attached muropeptide, such as different lengths (di-, tetra-saccharide…), different peptides despite the action of LytA (tri-, tetra-peptide…), different O-acetylation status, etc.

(9) L. 226-227, Fig S8. Presumably several of the major bands on the Coomassie stained gel are the lysozyme, mutanolysin, recombinant LytA, DNase and RNase used to digest the cell wall etc.? Can the sizes of these proteins be marked on the gel. Do any of them come down with the material at low-speed centrifugation?

We have provided a gel showing the different enzymes individually and mixed (new Fig. S9G). While performing several experiments of this type, we found that the mutanolysin might be contaminated with proteases. The enzymes do not appear to sediment at low speed.

(10) Fig. S9B. It is difficult to interpret what is in the image as there appear to be 2 populations of material (grey and sometimes more raised). Does the 20,000 g material look the same?

Fig. S10B is a 20,000 × g pellet. We agree that there appears to be two types of membrane vesicles, but we do not know their nature.

(11) l. 277 and Fig. 5A. Why is it "remarkable" that there are apparently more longer LTA molecules as the cell reach stationary phase?

This is the first time that a change of TA length is documented. Such a change could conceivably have consequences in the binding and activity of CBPs and the physiology of the cell envelope in general. These questions should be adressed in future studies.

(12) l. 280. How do you know which is the 6-repeat unit?

It is an assumption based on previous analyses by Gisch et al.( J Biol Chem 2013, 288(22):15654-67. doi: 10.1074/jbc.M112.446963). The reference was added.

(13) Fig. 5A and C. Panel C, the cells were grown in a different medium and so are not comparable to Panel A. Why is Fig. S12B not substituted for 5B? Presumably these are exponential phase cells.

We have interverted the Fig. S13B and 5C in V2, as suggested, and changed the text and legends accordingly.

Reviewer #2 (Recommendations for the authors):

L30: vitreous sections?

Corrected in V2.

L32: as their main universal function --> as a universal function. To show it's the main universal function, you will need to look at this across various bacterial species.

Changed to “possible universal function” in V2.

L35: enabled the titration the actual --> titration of the actual?

Corrected in V2.

L34: consider breaking up this very long sentence.

Done in V2.

L37: may compensate the absence--> may compensate for the absence.

Corrected in V2.

L45: Using metabolic labeling and electrophoresis showed --> Metabolic labeling and...

Corrected in V2.

L46: This finding casts doubts on previous results, since most LTA were likely unknowingly discarded in these studies. This needs to be rephrased and is unnecessarily callous. While the current work casts doubts on any quantitative assessments of actual LTA levels measured in previous studies, it does not mean any qualitative assessments or conclusions drawn from these experiments are wrong. Better would be to say: These findings suggest that previously reported quantitative assessments of LTA levels are likely underestimating actual LTA levels, since much of the LTA would have been unknowingly discarded.

If the authors do think that actual conclusions are wrong in previous work, then they need to be more explicit and explain why they were wrong.

Yes indeed. The statement was toned down in V2.

L55: Although generally non-essential. I would remove or rephrase this statement. I don't think any TA mutant will survive out in the wild and will be essential under a certain condition. So perhaps not essential for growth under ideal conditions, but for the rest pretty essential.

The paragraph was amended by qualifying the essentiality to laboratory conditions and including selected references.

L95: Note that the prevailing model until reference 20 (Gibson and Veening) was that the TA is polymerized intracellularly (see e.g. Figure 2 of PMID: 22432701, DOI: 10.1089/mdr.2012.0026). This intracellular polymerisation model seemed unlikely according to Gibson and Veening ('As TarP is classified by PFAM as a Wzy-type polymerase with predicted active site outside the cell, we speculate that TarP and TarQ polymerize the TA extracellularly in contrast to previous reports.'), but there is no experimental evidence as far as this referee knows of either model being correct.

Despite the lack of experimental evidence, we think that Gibson and Veening are very likely correct, based on their argument, and also by analogy with the synthesis of other surface polysaccharides from undecaprenyl- or dolichol-linked precursors. It is unfortunate that Figure 2 of PMID: 22432701, DOI: 10.1089/mdr.2012.0026 was published in this way, since there was no evidence for a cytoplasmic polymerization, to our knowledge.

L97: It is commonly believed, although I'm not sure it has ever been shown, that the capsule is covalently attached at the same position on the PG as WTA. Therefore, there must be some sort of regulation/competition between capsule biosynthesis and WTA biosynthesis (see also ref. 21). The presence of the capsule might thus also influence the characteristics of the periplasmic space. Considering that by far most pneumococcal strains are encapsulated, the authors should discuss this and why a capsule mutant was used in this study and how translatable their study using a capsule mutant is to S. pneumoniae in general.

A paragraph was added in the Introduction of V2 to present the complication and a sentence was added at the end of the discussion to mention that this should be studied in the future.

L102: Ref 29 should probably be cited here as well?

Since in Ref 29 (Flores-Kim et al. 2019) there is a detectable amount of LTA (presumably precursors TA) in the ∆tacL stain, we prefer to cite only Hess et al. 2017 regarding the absence of LTA in the absence of TacL. However, we added in V2 a reference to Flores-Kim et al. 2019 in the following paragraph regarding the role of the LTA/WTA ratio.

L106: dependent on the presence of the phosphotransferase LytR (21). --> dependent on the presence of the phosphotransferase LytR, whose expression is upregulated during competence (21).

Corrected in V2.

L119: I fail to see how the conclusions drawn by other groups (I assume the authors mean work from the Vollmer, Rudner, Bernhardt, Hammerschmidt, Havarstein, Veening groups?) are invalid if they compared WTA:LTA ratios between strains and conditions if they underestimated the LTA levels? Supposedly, the LTA levels were underestimated in all samples equally so the relative WTA/LTA ratio changes will qualitatively give the same outcome? I agree that these findings will allow for a reassessment of previous studies in which presumably too low LTA levels were reported, but I would not expect a difference in outcome when people compared WTA:LTA ratios between strains?

The sentence was rephrased in V2 to be neutral regarding previous work and rather emphasize future possibilities.

L131: Perhaps it would be good to highlight that such a conspicuous space has been noticed before by other EM methods (see e.g. Figs.4 and 5 or ref 19, or one of the most clear TEM S. pneumoniae images I have seen in Fig. 1F of Gallay et al, Nat. Micro 2021). However, always some sort of staining had previously been performed so it was never clear this was a real periplasmic space. CEMOVIS has this big advantage of being label free and imaging cells in their presumed native state.

Thanks for pointing out these beautiful data that we had overlooked. We have added a few sentences and references in the Discussion of V2.

L201: References are not numbered.

Corrected in V2.

L271/L892: Change section title. 'Evolution' can have multiple meanings. It would be more clear to write something like 'Increased TA chain length in stationary phase cells' or something like that.

Changed in V2.

L275: harvested

Corrected in V2.

L329: add, as suggested shown previously (I guess refs 24 and 29)

Reference to Hess et al. 2017 has been added in V2. A sentence and further references to Flores-Kim, 2019, 2022 and Wu et al. 2014 were added at the end of the discussion with respect to the LTA-like signal observed in these studies of ∆tacL strains.

L337: I think a concluding sentence is warranted here. These experiments demonstrate that membrane-bound TA precursors accumulate on the outside of the membrane, and are likely polymerized on the outside as well, in line with the model proposed in ref. 20.

From the point of view of formal logic, the accumulation of membrane-bound TA precursors on the outer face of the membrane does not prove that they were assembled there. They could still be polymerized inside and translocated immediately. However, since this is extremely unlikely for the reasons discussed by Gibson and Veening, we have added a mild conclusion sentence and the reference in V2.

L343: How accurate are these quantifications? Just by looking at the gel, it seems there is much less WTA in the lytR mutant than 50% of the wild type?

Yes, the 51% value was a calculation error. This was changed to 41%. Likewise, the decrease of the WTA amount relative to LTA was corrected to 5- to 7-fold.

Apart from the titration of TA in the WT strain, we haven’t yet carried out a careful quantification neither of TA nor of the LTA/WTA ratio in different strains and conditions, although we intend to do so in the near future using the method presented here.

However, to better substantiate our statement regarding the ∆lytR strain, we have quantified two experiments of growth in C-medium with azido-choline, and two experiments of pulse labeling in BHI medium. The results are presented in the additional supplementary Fig. S14.

L342: although WTA are less abundant and LTA appear to be longer (Fig. 6A). although WTA are less abundant and LTA appear to be longer (Fig. 6A), in line with a previous report showing that LytR the major enzyme mediating the final step in WTA formation (ref. 21). (or something like that). Perhaps better is to start this paragraph differently. For instance: Previous work showed that LytR is the major enzyme mediating the final step in WTA formation (ref. 21). As shown in Fig. 6A, the proportion of WTA significantly decreased in the lytR mutant. However, there was still significant WTA present indicating that perhaps another LCP protein can also produce WTA.

Changed in V2.

Of note, WTA levels would be a lot lower in encapsulated strains as used in Ref. 21 (assuming WTA and capsule compete for the same linkage on PG). So perhaps it would be hard to detect any residual WTA in a encapsulated lytR mutant?

Investigation of the relationship between TA and capsule incorporation or O-acetylation is definitely a future area of study using this method of TA monitoring.

L371: see my comments related to L131. Some TEM images clearly show the presence of a periplasmic space.

Comments and references have been added in V2.

L402: It would be really interesting to perform these experiments on a wild type encapsulated strain. Would these have much more LTA? (I understand you cannot do these experiments perhaps due to biosafety, but it might be interesting to discuss).

Yes. It would be interesting to compare the TA in D39 and D39 ∆cps strains. We have added this perspective at the end of the discussion in V2.

L418: ref lacks number

Corrected in V2.

L423: refs missing.

References added in V2.

L487: See my comments regarding L46. I do not see one valid point in the current paper why underestimating LTA levels would change any of the conclusions drawn in Ref. 21. I do not know the other papers cited well enough, but it seems highly unlikely that their conclusions would be wrong by systematically underestimating LTA levels. As far as I understand it, this current work basically confirms the major conclusions drawn by these 'doubtful' papers (that TacL makes LTA and LytR is the main WTA producer). As such, I find this sentence highly unfair without precisely specifying what the exact doubts are. Sure, this current paper now shows that probably people have discarded unknowingly LTA and therefore underestimated LTA levels, so any quantitative assessment of LTA levels are probably wrong. That is one thing. But to say this casts doubts on these studies is very serious and unfair (unless the authors provide good arguments to support these serious claims).

Yes indeed. The sentence was rephrased to be strictly factual in V2.

Table 2: I assume these strains are delta cps? Would be relevant to list this genotype.

The Table 2 was completed in V2.

The authors should comment on why the mutants have not been complemented, especially for lytR as it's the last gene in a complex operon. It would be great to see WTA levels being restored by ectopic expression of LytR.

Yes. We think this could be part of an in-depth study of the attachment of WTA, together with the investigation of the other LCP phosphotransferases.

Author response:

The following is the authors’ response to the previous reviews

Joint Public Review:

Summary:

The behavioral switch between foraging and mating is important for resource allocation in insects. This study characterizes the role of sulfakinin and the sulfakinin receptor 1 in changes in olfactory responses associated with foraging versus mating behavior in the oriental fruit fly (Bactrocera dorsalis), a significant agricultural pest. This pathway regulates food consumption and mating receptivity in other species; here the authors use genetic disruption of sulfakinin and sulfakinin receptor 1 to provide strong evidence that changes in sulfakinin signaling modulate antennal responses to food versus pheromonal cues and alter the expression of ORs that detect relevant stimuli.

Strengths:

The authors utilize multiple complementary approaches including CRISPR/Cas9 mutagenesis, behavioral characterization, electroantennograms, RNA sequencing and heterologous expression to convincingly demonstrate the involvement of the sulfakinin pathway in the switch between foraging and mating behaviors. The use of both sulfakinin peptide and receptor mutants is a strength of the study and implicates specific signaling actors.

Weaknesses:

The authors demonstrate that SKR is expressed in olfactory neurons, however there are additional potential sites of action that may contribute to these results.

Recommendations for the authors:

The authors have addressed most of the issues raised by the reviewers. Below are a few outstanding issues.

(1) Lines 68-69 describe "control of B. dorsalis include the use of the behavioral responses to semiochemicals" but does not describe what these responses are or how behavior is modulated.

The sentence was revised as “Control of B. dorsalis include the use of the reproductive and feeding behavioral responses to semiochemicals” (lines 69 in the revision).

(2) Statistical analysis for 9 hour starved females at 5 minutes is missing in Figure 1D and S1.

We had added statistical analysis for 9 hour starved females at 5 minutes in the revised Figures 1D and S1, respectively (lines 578).

(3) The legend in Figure S2 should be revised as it is not clear from the figure which of the odors are food associated odors.

As suggested, we added food odor label in the revised Figure S2 (lines 666).

(4) Line 167: "Therefore, the upregulated OR genes in starved WT flies, OR7a.4, OR7a.8 and OR10a, were activated by the pheromonal components, while down regulated genes, OR49a and OR63a, were activated by food volatiles." Based on the data, this sentence is incorrect - Therefore, the upregulated OR genes in starved WT flies, OR7a.4, OR7a.8 and OR10a, were activated by the food components, whereas downregulated genes, OR49a and OR63a, were activated by pheromonal components."

We are sorry for our mistake. We had corrected it (lines 168-169).

(5) Line 192: "The coordinated action of sulfakinin on mutiple downstreams,..." should be revised to "downstream pathways or tissues" or simply removing "multiple downstream".

As suggested, we removed “multiple downstream”. See line 192.

(6) Reference formatting is inconsistent: see line 207 vs line 208.

We had corrected it as “(Wu et al., 2019)” (lines 207). 

(7) Lines 241-244 The broad discussion regarding the evolution and ancestral function of CCK here and the phylogeny in Figure S6 are peripheral to the authors claims.

As suggested, we removed the section and the Figure S6 in the revision.

Author response:

The following is the authors’ response to the original reviews

Public Reviews:

Reviewer #1 (Public Review):

Summary:

This research article by Nath et al. from the Lee Lab addresses how lipolysis under starvation is achieved by a transient receptor potential channel, TRPγ, in the neuroendocrine neurons to help animals survive prolonged starvation. Through a series of genetic analyses, the authors identify that TRPγ mutations specifically lead to a failure in lipolytic processes under starvation, thereby reducing animals' starvation resistance. The conclusion was confirmed through total triacylglycerol levels in the animals and lipid droplet staining in the fat bodies. This study highlights the importance of transient receptor potential (TRP) channels in the fly brain to modulate energy homeostasis and combat metabolic stress. While the data is compelling and the message is easy to follow, several aspects require further clarification to improve the interpretation of the research and its visibility in the field.

Strengths:

This study identifies the biological meaning of TRPγ in promoting lipolysis during starvation, advancing our knowledge about TRP channels and the neural mechanisms to combat metabolic stress. Furthermore, this study demonstrates the potential of the TRP channel as a target to develop new therapeutic strategies for human metabolic disorders by showing that metformin and AMPK pathways are involved in its function in lipid metabolisms during starvation in Drosophila.

Weaknesses:

Some key results that might strengthen their conclusions were left out for discussion or careful explanation (see below). If the authors could improve the writing to address their findings and connect their findings with conclusions, the research would be much more appreciated and have a higher impact in the field.

Here, I listed the major issues and suggestions for the authors to improve their manuscript:

(1) Are the increased lipid droplet size and the upregulated total TAG level measured in the starved or sated mutant in Figure 1? This information might be crucial for readers to understand the physiological function of TRP in lipid metabolism. In other words, clarifying whether the upregulated lipid storage is observed only in the starved trp mutant will advance our knowledge of TRPγ. If the increase of total TAG level is only observed in the starved animals, TRP in the Dh44 neurons might serve as a sensor for the starvation state required to promote lipolysis in starvation conditions. On the other hand, if the total TAG level increases in both starved and sated animals, activation of Dh44 through TRPγ might be involved in the lipid metabolism process after food ingestion.

We measured total TAG level in Figure 1 and LD sizes in Figure 2 under sated condition. We inserted “under sated condition” to clarify it. lines 97 and 147-148.

Thanks for your suggestions.

(2) It is unclear how AMPK activation in Dh44 neurons reduces the total triacylglycerol (TAG) levels in the animals (Figure 3G). As AMPK is activated in response to metabolic stress, the result in Figure 3G might suggest that Dh44 neurons sense metabolic stress through AMPK activation to promote lipolysis in other tissues. Do Dh44 neurons become more active during starvation? Is activation of Dh44 neurons sufficient to activate AMPK in the Dh44 neurons without starvation? Is activation of AMPK in the Dh44 neurons required for Dh44 release and lipolysis during starvation? These answers would provide more insights into the conclusion in Lines 192-193.

In our previous study, we demonstrated that trpγ mutants exhibited lower levels of glucose, trehalose and glycogen level (Dhakal et al. 2022), and in the current study, we observed excessive lipid storage in the trpγ mutant, indicating imbalanced energy homeostasis. Given the established role of AMPK in maintaining energy balance (Marzano et. al., 2021, Lin et al 2021), we employed the activated form of AMPK (UAS-AMPK^TD) in our experiments. Our result showed that expression of activated AMPK in Dh44 neurons led to a reduction in total TAG levels, suggesting that AMPK activation in these neurons can promote lipolysis even in the absence of starvation. Regarding the activation of Dh44 neurons, Dus et al in 2015 reported that Dh44 cells in the brain are activated by nutritive sugars especially in starvation conditions. In addition, another report showed a role of Dh44 neuron in regulating starvation induced sleep suppression (Oh et. al., 2023) which may imply that these neurons become more active under starved conditions. We did not directly assess whether Dh44 neuron activity increases during starvation or whether AMPK activation in these neurons is required for DH44 release and subsequent lipolysis, our finding support the notion that AMPK activation in Dh44 neuron is sufficient to reduce TAG levels, potentially by metabolic stress response typically observed during starvation. We explained it like the following: “Dh44 neurons regulate starvation-induced sleep suppression (Oh et. al., 2023), which implies that these neurons become more active under starved conditions.” lines 190-191.

(3) It is unclear how the lipolytic gene brummer is further downregulated in the trpγ mutant during starvation while brummer is upregulated in the control group (Figure 6A). This result implies that the trpγ mutant was able to sense the starvation state but responded abnormally by inhibiting the lipolytic process rather than promoting lipolysis, which makes it more susceptible to starvation (Figure 3B).

Thanks for your suggestions. We explained it like the following: “The data indicates that the trpg mutant can sense the starvation state but responds abnormally by suppressing lipolysis instead of activating it. This dysregulated lipolytic response likely increases the mutant's vulnerability to starvation, as it cannot effectively mobilize lipid stores for energy during periods of nutrient deprivation.” lines 251-254.

(4) There is an inconsistency of total TAG levels and the lipid droplet size observed in the Dh44 mutant but not in the Dh44-R2 mutant (Figures 7A and 7F). This inconsistency raises a possibility that the signaling pathway from Dh44 release to its receptor Dh44-R2 only accounts for part of the lipid metabolic process under starvation. Adding discussion to address this inconsistency may be helpful for readers to appreciate the finding.

Thanks for your suggestion. We included the following in the Discussion: “There is an inconsistency of total TAG levels and the LD size observed in the Dh44 mutant. This inconsistency raises a possibility that the signaling pathway from DH44 release to its receptor DH44R2 only accounts for part of the lipid metabolic process under starvation. While Dh44 mutant flies displayed normal internal TAG levels, Dh44R2 mutant flies exhibited elevated TAG levels. This suggested that the lipolysis phenotype could be facilitated by a neuropeptide other than DH44. Alternatively, a DH44 neuropeptide-independent pathway could mediate the lipolysis.” lines 429-436.

Reviewer #2 (Public Review):

Summary:

In this paper, the function of trpγ in lipid metabolism was investigated. The authors found that lipid accumulation levels were increased in trpγ mutants and remained high during starvation; the increased TAG levels in trpγ mutants were restored by the expression of active AMPK in DH44 neurons and oral administration of the anti-diabetic drug metformin. Furthermore, oral administration of lipase, TAG, and free fatty acids effectively restored the survival of trpγ mutants under starvation conditions. These results indicate that TRPv plays an important role in the maintenance of systemic lipid levels through the proper expression of lipase. Furthermore, authors have shown that this function is mediated by DH44R2. This study provides an interesting finding in that the neuropeptide DH44 released from the brain regulates lipid metabolism through a brain-gut axis, acting on the receptor DH44R2 presumably expressed in gut cells.

Strengths:

Using Drosophila genetics, careful analysis of which cells express trpγ regulates lipid metabolism is performed in this study. The study supports its conclusions from various angles, including not only TAG levels, but also fat droplet staining and survival rate under starved conditions, and oral administration of substances involved in lipid metabolism.

Weaknesses:

Lipid metabolism in the gut of DH44R2-expressing cells should be investigated for a better understanding of the mechanism. Fat accumulation in the gut is not mechanistically linked with fat accumulation in the fat body. The function of lipase in the gut (esp. R2 region) should be addressed, e.g. by manipulating gut-lipases such as magro or Lip3 in the gut in the contest of trpγ mutant. Also, it is not clarified which cell types in the gut DH44R2 is expressed. The study also mentioned only in the text that bmm expression in the gut cannot restore lipid droplet enlargement in the fat body, but this result might be presented as a figure.

We appreciate the reviewer’s insightful suggestions. Unfortunately, due to the unviability of the reagent (UAS-Lip3), we were unable to manipulate gut lipase in trpy mutants as proposed. However, we additionally performed immunostaining to examine the co-expression of trpγ and Dh44R2 in the gut, and our results indicate that both trpγ and Dh44R2 are co-expressed in the R2 region of the gut (Figure 7O and P). Furthermore, we have updated our figures to address the point that bmm expression in the gut does not restore lipid droplet enlargement in the fat body, with the revised version (Figure 5I and J).

Reviewer #3 (Public Review):

In this manuscript, the authors demonstrated the significance of the TRPγ channel in regulating internal TAG levels. They found high TAG levels in TRPγ mutant, which was ascribed to a deficit in the lipolysis process due to the downregulation of brummer (bmm). It was notable that the expression of TRPγ in DH44+ PI neurons, but not dILP2+ neurons, in the brain restored the internal TAG levels and that the knockdown of TRPγ in DH44+ PI neurons resulted in an increase in TAG levels. These results suggested a non-cell autonomous effect of Dh44+PI neurons. Additionally, the expression of the TRPγ channel in Dh44 R2-expressing cells restored the internal TAG levels. The authors, however, did not provide an explanation of how TRPγ might function in both presynaptic and postsynaptic cells in the non-cell autonomous manner to regulate the TAG storage. The authors further determined the effect of TRPγ mutation on the size of lipid droplets (LD) and the lifespan and found that TRPγ mutation caused an increase in the size of LD and a decrease in the lifespan, which were reverted by feeding lipase and metformin. These were creative endeavors, I thought. The finding that DH44+ PI neurons have non-cell autonomous functions in regulating bodily metabolism (mainly sugar/lipid) in addition to directing sugar nutrient sensing and consumption is likely correct, but the paper has many loose ends. I would like to see a revision that includes more experiments to tighten up the findings and appropriate interpretations of the results.

(1) The authors need to provide interpretations or speculations as to how DH44+ PI neurons have non-cell autonomous functions in regulating the internal TAG stores, and how both presynaptic DH44 neurons and postsynaptic DH44 R2 neurons require TRPγ for lipid homeostasis.

In Discussion, we had mentioned our previous finding. “ We previously proposed that TRPg holds DH44 neurons in a state of afterdepolarization, thus reducing firing rates by inactivating voltage-gated Na+ channels (Dhakal et al., 2022). At the physiological level, this induces the consistent release of DH44 and depletion of DH44 stores, resulting in nutrient utilization and storage malfunctions.”

We also included the following: “TRPg in DH44 neurons may influence the release of metabolic signals or hormones that act on postsynaptic DH44R2 cells. These postsynaptic cells could, in turn, modulate lipid storage and metabolism in a non-cell autonomous manner. However, the mechanism by which TRPg functions in DH44R2 cells remains unclear. One possible explanation is that TRPg in the gut may be activated by stretch or osmolarity (Akitake et al. 2015).” lines 439-440.

This interaction between presynaptic and postsynaptic cells may ensure a coordinated response to metabolic changes and maintain lipid homeostasis. Thus, both Dh44-expressing and Dh44-R2-expressing cells are crucial for the proper functioning of TRPγ in regulating internal TAG levels and lipid storage.

(2) The expression of TRPγ solely in DH44 R2 neurons of TRPγ mutant flies restored the TAG phenotype, suggesting an important function mediated by TRPγ in DH44 R2 neurons. However, the authors did not document the endogenous expression of TRPγ in the DH44R2+ gut cells. This needs to be shown.

We appreciate the reviewer’s suggestion. To address this, we performed immunostaining to examine the expression of TRPγ in the DH44R2+ gut cells. Our results, as shown in Figure 7 O and P, confirm that TRPγ is co-expressed in the Dh44R2+ cells in the gut. We also found that Dh44R2 is expressed in the brain as well. We documented this part like the following: “Given that Dh44R2 is predominantly expressed in the intestine, we performed immunostaining to examine whether Dh44R2 co-localizes with trpg in gut cells. Our results confirmed that Dh44R2 and trpg are co-expressed in intestinal cells (Figure 7O and P). Additionally, we analyzed Dh44R2 expression in the brain and found that two Dh44R2-expressing cells are co-localized with Dh44-expressing cells in the PI region (Figure 7Q). To further delineate whether Dh44R2-mediated fat utilization is specific to the brain, gut, or fat body, we knocked down Dh44R2^RNAi using Dh44-GAL4, myo1A-GAL4, and cg-GAL4, respectively (Figure 7–figure supplement 1E). Notably, knockdown of Dh44R2 with Myo1A-GAL4 resulted in elevated TAG levels, indicating that DH44R2 activity in lipid metabolism is specific to the gut.” lines 375-384.

(3) While Dh44 mutant flies displayed normal internal TAG levels, Dh44R2 mutant flies exhibited elevated TAG levels (Figure 7A). This suggested that the lipolysis phenotype could be facilitated by a neuropeptide other than Dh44. Alternatively, a Dh44 neuropeptide-independent pathway could mediate the lipolysis. In either case, an additional result is needed to substantiate either one of the hypotheses.

The Dh44 mutant flies exhibited normal TAG levels, whereas Dh44R2 mutant flies showed elevated TAG levels. However, when we examined the lipid droplets in the fat body, both Dh44 mutant and Dh44R2 mutant flies displayed larger lipid droplets, indicating a disruption in lipid metabolism. Additionally, we assessed starvation survival time and found that both Dh44 and Dh44R2 mutant flies exhibited reduced survival under starvation conditions compared to controls. Supplementation with lipase (Figure 7–figure supplement 1A), glycerol (Figure 7–figure supplement 1B), hexanoic acid (Figure 7–figure supplement 1C), and mixed TAGs (Figure 7–figure supplement 1D) improved starvation survival time, further supporting that the lipid metabolism pathway was impaired in both mutants. These observations highlight the role of Dh44 in regulating lipolysis. We included related Discussion: “There is an inconsistency of total TAG levels and the LD size observed in the Dh44 mutant. This inconsistency raises a possibility that the signaling pathway from DH44 release to its receptor DH44R2 only accounts for part of the lipid metabolic process under starvation. While Dh44 mutant flies displayed normal internal TAG levels, Dh44R2 mutant flies exhibited elevated TAG levels. This suggested that the lipolysis phenotype could be facilitated by a neuropeptide other than DH44. Alternatively, a DH44 neuropeptide-independent pathway could mediate the lipolysis.” lines 429-436.

(4) While the authors observed an increased area of fat body lipid droplets (LD) in Dh44 mutant flies (Figure 7F), they did not specify the particular region of the fat body chosen for measuring the LD area.

We have chosen the 2-3 segment in the abdomen for all fat body images, which we already mentioned in Nile red staining in the Method section line 630-631.

(5) The LD area only accounts for TAG levels in the fat body, whereas TAG can be found in many other body parts, including the R2 area as demonstrated in Figure 5A-D using Nile red staining. As such, measuring the total internal TAG levels would provide a more accurate representation of TAG levels than the average fat body LD area.

We have measured total internal TAG level in whole body throughout the experiments (Figure 1F, 2C, 2E, 3C, 3G, 4A, 4B, 7A, 7I, and many Supplementary Figures) except bmm expression using GAL4/UAS system. Now we include this new data in Figure 5–figure supplement 1) which is the same conclusion with LD analysis.

(6) In Figure 5F-I, the authors should perform the similar experiment with Dh44, Dh44R1, and Dh44R2 mutant flies.

We did the experiments with Dh44, Dh44R1, and Dh44R2 mutant flies and we found that Dh44 and Dh44R2 mutant flies showed reduced starvation survival time than control and which was increased after supplementation of lipase, glycerol, hexanoic acid and TAG (Figure 7– figure supplement 1A–D). lines 361-372.

(7) The representative image in Figure 6B does not correspond to the GFP quantification results shown in Figure 6C. In trpr1;bmm::GFP flies, the GFP signal appears stronger in starved conditions than in satiated conditions.

We updated it with new images. We quantified GFP intensity level using image J and found that GFP intensity level was significantly lower in starved condition in trpγ¹;bmm::GFP flies than sated condition.

(8) In Figure 6H-I, fat body-specific expression of bmm reversed the increased LD area in TRPγ mutants. The authors also showed that Dh44+PI neuron-specific expression of bmm yielded a similar result. The authors need to provide an interpretation as to how bmm acts in the fat body or DH44 neurons to regulate this.

We first inserted the following in results: “Furthermore, the expression of bmm in the fat body, as well as Dh44 neurons in the PI region, can promote lipolysis at the systemic level.” lines 276-277.

Additionally, we discussed it in the Discussion: “Brummer lipase is essential for regulating lipid levels in the insect fat body by mediating lipid mobilization and energy homeostasis. In Nilaparvata lugens, it facilitates triglyceride breakdown (Lu et al., 2018), while studies in Drosophila show that reduced Brummer lipase expression decreases fatty acids and increases diacylglycerol levels, highlighting its role in lipid metabolism (Nazario-Yepiz et al., 2021). Here, we additionally demonstrate that bmm expression in DH44 neurons within the PI region can systemically regulate TAG levels. Cell signaling or energy status in DH44 neurons may contribute to hormonal release that targets organs such as the fat body.” lines 451-459.

(9) The authors should explain why the DH44 R1 mutant did not represent similar results as the wild type.

We added “In addition, bmm levels in Dh44R1^Mi under starved condition did not increase as significantly as in the control. This suggests a unique role of DH44 and its receptors in regulating lipid metabolism and response to nutritional status in Drosophila.” lines 358-360.

(10) It would be good to have a schematic that represents the working model proposed in this manuscript.

We updated the schematic model in revised version (Figure 8).

Recommendations for the authors:

Reviewing Editor (Recommendations For The Authors):

This paper characterized the function of trpγ in Dh44-expressing PI neurons for lipid metabolism and lipolysis induced by prolonged starvation. The authors applied a series of lipolytic genetic manipulation and lipid/lipid metabolism supplements to rescue the trpγ deficits in lipolysis: the expression of active AMPK in the DH44-expressing PI neurons or brummer, a lipolytic gene, in the trpγ-expressing cells, and oral administration of the anti-diabetic drug metformin, lipase, TAG and free fatty acids. Despite this exhaustive characterization of the defective lipolysis in the trpγ mutants, there remain puzzles in inconsistent defects of Dh44 and DH44R2 in the total TAG levels and in the expression and functions of the receptor in the gut. Clarification of these points and other issues raised by the reviewers should improve the mechanisms of lipid metabolism through Dh44 signalling.

Reviewer #1 (Recommendations For The Authors):

(1) It might be worth introducing Dh44 in the introduction section as it is unclear to readers how the authors hypothesized the site-of-action of TRPγ in Dh44 neurons for lipid metabolism after reading the introduction.

We introduced the following: “We found that TRPg expression in Dh44 neuroendocrine cells in the brain is critical for maintaining normal carbohydrate levels in tissues (Dhakal et al. 2022). Building on this, we hypothesized that TRPg in Dh44 cells also regulates lipid and protein homeostasis.” lines 69-71.

(2) Providing a summary model in the end to integrate the present findings and their previous publication about TRPγ functions in Drosophila sugar selection would greatly help readers understand and appreciate the general role of TRPγ in balancing energy homeostasis.

We made a schematic model in Figure 8.

(3) Swapping the order of Figures 5 and 6 might be a better way to tell the story without logic gaps. The results addressing the mechanisms of metformin and TRPγ in promoting lipolysis under starvation are interrupted by the lipid storage data in the R2 cells in the current Figure 5A-5E. In addition, presenting Figure 5A-5E before or together with Figure 7 will help readers appreciate the expression of Dh44-R2 and its function in regulating lipid metabolism in Figure 7.

We did.

(4) It might be misleading to use the word "sated" for the condition of 5-hour mild starvation. The word "mild starvation" or the equivalents might be a better word choice.

We appreciate the reviewer’s concern. As hemolymph sugar level does not drop down significantly in 5 hr starvation, the previous papers (Dus et al 2015, Dhakal et al 2022) indicated it as sated condition. To use the word consistently, we prefer using “sated” instead of “mild starvation”.

(5) It is unclear what the white arrows are pointing at in Figures 7O and 7P. Some of those seem to be non-specific signals, so it is hard to connect the figure to the conclusion in Lines 351-353. It would be helpful to add some explanations to help readers interpret Figures 7O and 7P.

In the previous version, Figure 7O and 7P white arrows represented the expression of Dh44R2 in the SEZ region of the brain and R2 region of the gut. In revised version, to make clear, we performed additional immunostaining for the co-expression of trpγ and Dh44R2 in the gut. We found that trpγ and Dh44R2 co-expressed at the R2 region of the gut specifically (Figure 7O and P). Similarly, we found that two cells of Dh44R2 co-expressed in Dh44 cells in the PI region of the brain (now Figure 7Q). We updated this part. lines 375-380.

(6) The figure legend for the (G) panel in Figure 2-figure Supplement 1 was mislabeled as (F).

We corrected it.

(7) In Line 85, the authors might want to write "… among these mutants, only trpγ mutant displayed reduced carbohydrate levels, suggesting …". Please confirm the information for the sentence. lines 87-88.

We clarified it.

Reviewer #2 (Recommendations For The Authors):

(1) The trpγ[G4] would be difficult for non-Drosophila researchers to understand; it would be better to use trpγ-Gal4.

We got the mutant line from Dr. Craig Montell who named it. We explained it like the following in the main text: “controlled by GAL4 knocked into the trpg locus (trpg^G4 flies; +)” line 109.

(2) The arrows in Figures 7O and 7P need to be explained in the figure legends.

We did.

Reviewer #3 (Recommendations For The Authors):

(11) Lines 95-96 should have a reference.

We did.

(12) Lines 129-130: It should read "TRPγ expressed in DH44 cells is sufficient for the regulation of lipid levels."

We changed it as suggested.

(13) Figure 5E needs to be repeated with more trials.

We increased the n numbers. Previously (Figure 5E) we included area of 10 LDs from 3 samples, and in revised figure (Figure 6I) we have included 28 LDs from 10 samples.

(14) Figures 5F-I, bold lines are not too visible and therefore, dotted lines could be used.

We changed it as suggested.

(15) Line 356: It is not true that D-trehalose or D-fructose is commonly detected by DH44 neurons. These sugars at concentrations much higher than the physiological concentration range stimulate DH44 neurons (see Dus et al., 2015).

We removed it.

(16) Lines 362-363: It should read "Expression of TRPγ in DH44 neurons was necessary and sufficient to regulate the carbohydrate and lipid levels.".

We changed it.

(17) Lines 369-370: The authors need to consider removing the possible role of CRF in regulating lipid homeostasis. It could be considered to be far-fetched.

We removed it.

(18) Line 407-408: the sentence "Nevertheless, it is also known that DH44 neurons mediate the influence of dietary amino acids on promoting food intakes in flies (37)" needs to be removed. They used amino acid concentrations that were far greater than the physiological levels observed in the internal milieu of flies. Still, many laboratories cannot reproduce the result of using the high AA concentrations.

We removed it.

Author response:

The following is the authors’ response to the original reviews

Public Reviews:

Reviewer #1 (public review): 

This manuscript presents SAVEMONEY, a computational tool designed to enhance the utilization of Oxford Nanopore Technologies (ONT) long-read sequencing for the design and analysis of plasmid sequencing experiments. In the past few years, with the improvement in both sequencing length and accuracy, ONT sequencing is being rapidly extended to almost all omics analyses which are dominated by short-read sequencing (e.g., Illumina). However, relatively higher sequencing errors of long-read sequencing techniques including PacBio and ONT is still a major obstacle for plasmid/clone-based sequencing service that aims to achieve single base/nucleotide accuracy. This work provides a guideline for sequencing multiple plasmids together using the same ONT run without molecular barcoding, followed by data deconvolution. The whole algorithm framework is well-designed, and some real data and simulation data are utilized to support the conclusions. The tool SAVEMONEY is proposed to target users who have their own ONT sequencers and perform library preparation and sequencing by themselves, rather than relying on commercial services. As we know and discussed by the authors, in the real world, to ensure accuracy, the researchers will routinely pick up multiple colonies in the same plasmid construction and submit for Sanger sequencing. However, SAVEMONEY is not able to support the simultaneous analysis of multiple colonies in the same run, as compared to the barcoding-based approaches. This is a major limitation in the significance of this work. Encouraging computational ePorts in ONT data debarcoding for mixed-plasmid or even single-cell sequencing would be more valuable in the field. 

We thank the reviewer for the positive response to our manuscript and the helpful comments.

The tool SAVEMONEY is proposed to target users who have their own ONT sequencers and perform library preparation and sequencing by themselves, rather than relying on commercial services.

We apologize that we were not clear enough in the manuscript. Our tool is designed for users who rely on commercial services (i.e., those who cannot include a barcode by themselves). However, it can also benefit those performing library preparation, as SAVEMONEY can be applied after standard barcode-based sequencing and de-multiplexing. The combination of standard barcodes with SAVEMONEY would significantly expands the scope of sequencing applications. For example, it would enable sequencing of more plasmid types than the number of available barcodes and, in some cases, it may even eliminate the need for barcode introduction. Because we do not own ONT equipment and because the primary target audience for the SAVEMONEY algorithm are users without ONT equipment, we were not able to conduct experiments using ONT. However, to clarify these possibilities, we added a dedicated paragraph describing these issues (3rd paragraph in the discussion section).

However, SAVEMONEY is not able to support the simultaneous analysis of multiple colonies in the same run, as compared to the barcoding-based approaches.

We agree with the reviewer about this limitation of SAVEMONEY, as it does not allow mixing of plasmids from multiple colonies in the same cloning run. However, that does not necessarily mean that SAVEMONEY cannot reduce sequencing costs in cloning. For example, when sequencing two colonies from each of three diPerent constructs (six plasmids in total), the standard approach would require sequencing costs for six samples. However, with SAVEMONEY, up to three plasmids can be mixed per sample, allowing them to be sequenced as just two samples. As a result, the sequencing cost per plasmid is reduced to one-third. The greatest benefits can be realized when SAVEMONEY is used at the laboratory level or by multiple researchers. To make this point clearer, we have added sentences in the 5th paragraph of the discussion section.

(1) To provide more comprehensive information for users who care about the cost, the Introduction section should include a cost comparison between Sanger and ONT, with more details, such as diPerent ONT platforms (MinION, PromethION, FlongIe), chemistries (flow cells) and kits. This additional information will be more helpful and informative for the users who have their own sequencers and are the target audience for SAVEMONEY. 

We thank the reviewer for pointing this out. Since we do not own ONT equipment, we are unable to provide a total cost for using the ONT platform. However, we have included the price per sample (~$15 per plasmid) for the commercial service we have used, as well as the equipment that they employ (V14 chemistry on a PromethION with an R10.4.1 flow cell) and the number of reads obtained per plasmid (~100–1000) in the 4th paragraph of the introduction section.     Though these costs will inevitably change over time, this information should still be helpful for those who own ONT sequencers in estimating the costs.

(2) In "Overview of the algorithm" (Pages 3-4) under the Results section, instead of stating "However, coverage varies from ~100-1000 and is diPicult to predict because each nanopore flow cell has diPerent properties.", it will be beneficial to provide more detailed information, such as sequencing length, yield/read count per flow cell of diPerent platforms. This information will assist users in designing their own experiments ePectively. 

We thank the reviewer for the comment. As mentioned in the previous response, we are unable to provide sequencing length, yield/read count per flow cell because we do not own ONT equipment. However, we apologize if it was not clear in "Overview of the algorithm" section that we are discussing the use of results obtained from commercial services, and therefore we need to provide more detailed information about the results from the commercial service. We have now clarified in the sentence pointed out by the reviewr that the numbers are derived from the information provided by commercial sequencing services. In addition, we have also added that typical examples of the result properties, i.e., read length and quality score distribution, can be found in Fig. 2 at the end of the same paragraph.

(3) While this study optimized and evaluated the tool using a total of 14 plasmids, it may not provide suPicient power to represent the diversity of the plasmid world. Consideration should be given to expanding the dataset to include a broader range of plasmids in future studies to enhance the robustness and generalizability of the tool. 

We are grateful to the reviewer for their valuable input. It is very reasonable that we had to expect that a larger number of plasmids should be used, even though the main target of SAVEMONEY is those who utilize commercial services. In the previous version of SAVEMONEY, it was not possible to process in a reasonable amount of time if too many plasmids were provided, though the algorithm itself does not have no restrictions based on the number of plasmids. Therefore, we have changed the underlying code to improve the algorithm, making it more than 20 times faster than the previous version (the benchmark time mentioned in the 3rd paragraph of the discussion section was improved to 3.1 minutes from the previous 65 minutes, using the same dataset and the same computer). Additionally, SAVEMONEY is now compatible with multiprocessing. The processing time is expected to decrease approximately inversely proportional to the number of CPU cores used. We have added these updates at the end of the 3rd paragraph in the discussion section.

(4) If applicable and feasible, including a comparison or benchmark of SAVEMONEY against other similar tools would further strengthen the manuscript. This comparison would allow users to evaluate the advantages and disadvantages of diPerent tools for their specific needs. 

We thank the reviewer for the suggestion. We have added the benchmark using the similar tool, On-Ramp, with the exact same set of plasmids and FASTQ data used for our benchmark (4th paragraph in the discussion section). Because the machine specifications used in the On-Ramp web server are unknown, a direct comparison is not possible. However, using only laptop-level computational resources, SAVEMONEY was able to process the data 38% faster than On-Ramp. When using mini-PC level computational resources, the processing time was 64% faster than on-RAMP.

(5) The importance of pre-filtering raw sequencing reads should be emphasized as noisy reads can significantly impact the overall performance of the tool. It is essential to clarify whether any pre-filtering steps were performed in this study, such as filtering based on quality scores, read length, or other relevant factors. 

We apologize for not being clear. Unfortunately, the commercial sequencing service we used did not provide the information regarding pre-filtering. However, the impact of the quality of pre-filtering based on quality score and read length on the quality of the final results is theoretically minimal in SAVEMONEY. First, during the initial step of the post-analysis, the classification step, short reads compared to the full plasmid length can be excluded based on the user-defined “score_threshold”. Simultaneously, low-quality reads with poor alignment to the plasmid can also be excluded, because “score_threshold” is related to the normalized alignment score. Even if there are low-quality reads that are not excluded at this stage, the ePect can be minimized during the final step of the post-analysis that generates consensus sequences. This is because our Bayesian analysis considers not only the base calling but also the q-scores to determine the consensus. Therefore, we believe the overall impact of pre-filtering on the final results is negligible.

(6) The statement regarding the number of required reads per plasmid (20-30) and the maximum number of plasmids (up to six) that can be mixed in a single run may become outdated due to the rapid advancements in ONT technology. In the Discussion section, instead of assuming specific numbers, it would be more beneficial to provide information based on the current state of ONT sequencing, such as the number of reads per MinION flow cell that can be produced.

We thank the reviewer for pointing this out. Because the number of required reads per plasmid depends on the accuracy of each read (i.e., the number of required reads can be reduced if the accuracy increases), we have added the description of these points to the last paragraph of the discussion section.

Reviewer #2 (public review):  

The authors developed an algorithm that allows for deconvoluting of plasmid sequences from a mixture of plasmids that have been sequenced by nanopore long read technology. As library preparations and barcoding of individual samples increase sequencing costs, the algorithm bypasses this need and thus decreases time on sample prep and sequencing costs. In the first step, the tool assesses which of the plasmid constructions can be mixed in a single library preparation by calculating a distance matrix between the reference plasmid and the constructions producing sequence clusters. The user is given groups of plasmids, from diPerent clusters, to be pooled together for sequencing. After sequencing, the algorithm deconvolutes the reads by classifying them based on alignments to the reference sequence. A Bayesian analysis approach is used to obtain a consensus sequence and quality scores. 

Strengths 

The authors exploit one of the main advantages of long-read sequencing which is to accurately resolve regions of high complexity, as regularly found in plasmids, and developed a tool that can validate plasmid constructions by reducing sequencing costs. Multiple plasmids (up to six) can be analyzed simultaneously in a single library without the need for sample barcoding, also reducing sample preparation time. Although inserts must be diPerent, just 2 bases diPerence would be enough for a correct assignation. It maximizes cost-ePiciency for projects that require large amounts of plasmid constructions and highthroughput validation. 

We thank the reviewer for the positive response to our manuscript and the helpful comments.

Weaknesses 

The method proposed by the authors requires prior knowledge of plasmid sequences (i.e., blueprints or plasmid reference) and is not suitable for small experiments. The plasmid inserts or backbones must be diPerent e.g., multiple colonies from the same plasmid construction ePort cannot be submitted together. 

As also discussed in the response to reviewer 1, we agree with the reviewer that SAVEMONEY does not allow you the analysis of plasmids from multiple colonies in the same cloning experiment. However, that does not necessarily mean that SAVEMONEY cannot reduce the sequencing cost. For example, when sequencing two colonies from each of three diPerent constructs (six plasmids in total), the standard approach would require sequencing costs for six samples. However, with SAVEMONEY, up to three plasmids can be mixed per sample, allowing them to be sequenced as just two samples. As a result, the sequencing cost per plasmid is reduced to one-third. The greatest benefits can be realized when SAVEMONEY is used at the laboratory level or by multiple researchers. To make this point clearer, we have added sentences in the 5th paragraph of the discussion section.

The reviewer also expressed concern that SAVEMONEY is not suitable for experiments at a small scale. To put it more precisely, SAVEMONEY cannot be used when the experiment size is minimal, such as in a lab that consistently constructs only a single plasmid at a time. That said, the strength of SAVEMONEY lies in its scalability. Even in labs where plasmid construction is typically limited to one at a time, there may be occasional instances where two or more plasmids are created simultaneously. In such cases, SAVEMONEY can be used to reduce sequencing costs. Moreover, in a typical molecular biology lab where multiple plasmids are constructed every week, SAVEMONEY can be particularly ePective. Given its adaptability and cost-saving potential and widespread use since its initial publication on bioRxiv and on Google Colab, we are confident that SAVEMONEY will continue to be a valuable tool for a wide range of researchers.

Recommendations For The Authors:

Reviewer #2 (Recommendations For The Authors): 

The manucript assumes all samples are sent out for sequencing at a specific company. This could be generalized for a much broader use since many labs now own nanopore sequencers. In turn, the advantage of reducing hands-on sample prep becomes more evident. 

We thank the reviewer for pointing this out. We agree that SAVEMONEY can also benefit those performing library preparation. Combination of standard barcodes with SAVEMONEY significantly expands the scope of sequencing applications. For example, it enables sequencing of more plasmid types than the number of available barcodes and, in some cases, may even eliminate the need for the sample prep step to introduce barcode. Because we do not own ONT equipment, we could not conduct experiments using ONT. However, to clarify these possibilities, we added a dedicated paragraph (3rd paragraph in the discussion section).

The base calling model (high accuracy, super accuracy) used by Plasmidsaurus and tested here should be mentioned.  

We thank the reviewer for the suggestion. The description about the base calling model (HAC) was added in Materials and Methods section.

Other modifications to the revised manuscript 

Beyond changes made in response to reviewer comments above, we have also through our continued use and improvement of SAVEMONEY, made additional changes to the algorithm and therefore to the manuscript. Those changes are outlined below. Improvements in the pre-survey step

(1) The pre-survey algorithm was reduced to a Zero-One Integer Linear Programming Problem to guarantee the optimal combinations, as previous versions did not ensure an optimal solution. Relatedly, the explanation of the algorithm in the main manuscript was updated.

(2) The algorithm was modified to ensure that the number of plasmids distributed to each group is balanced. A new feature was also added to allow users to specify the number of groups, which is beneficial when balancing between cost and quality.

(3) An error was corrected in Fig. 2, where the distance calculation method for the hierarchical clustering step for group formation was Farthest Point Algorithm, which calculates distance between two clusters based on the farthest pair of plasmids. The correct method is the Nearest Point Algorithm. This error was present only in Fig. 2, while other implementations, including source code of SAVEMONEY and Google Colab page, were correct from the beginning. We have corrected the error in Fig. 2.

Modifications in figures, manuscripts, and other aspects

(1) Fig. 3 was updated to reflect the update of SAVEMONEY, although it did not show any important diPerences.

(2) Parameter names were updated as follows:

“threshold (pre)” -> “distance_threshold”

“threshold (post)” -> “score_threshold” Added “number_of_groups”

(3) The order of elements was rearranged in Fig. 4.

(4) Incorrect calculations were fixed in Fig. 4g, h, and i (old Fig. 4d, h, and l). Related to that, Fig. 4j, k, and l and Table 1 were added, in addition to the explanation in the main manuscript.

(5) SAVEMONEY was packaged and was released on PyPI to facilitate easy installation and integration by other developers.

(6) SAVEMONEY was updated and expanded to accommodate linear DNA fragments, such as PCR amplicons and long synthetic DNA. Users can select the topology of DNA by specifying that as an option. A description of this new capability was added at the end of “Overview of the algorithm” section.

test

Author response:

The following is the authors’ response to the original reviews

Public Reviews:

Reviewer #1:

(…) some concerns with interpretations and technical issues make several major conclusions in this manuscript less rigorous, as explained in detail in comments below. In particular, the two major concerns I have: 1) the contradiction between the strong reduction of global translation, with puromycin incorporation gel showing no detectable protein synthesis in cold, and an apparently large fraction of transcripts whose abundance and translation in Fig. 2A are both strongly increased. 2) The fact that no transcripts were examined for dependance on IRE-1/XBP1 for their induction by cold, except for one transcriptional reporter, and some weaknesses (see below) in data showing activation of IRE-1/XBP-1 pathway. The conclusion for induction of UPR by cold via specific activation of IRE-1/XBP-1 pathway, in my opinion, requires additional experiments.

Relating to the first point, the results of puromycin incorporation and ribosome profiling are not contradictory. The former shows absolute changes in translation, i.e. changes in how much protein the cell is producing, while the latter shows relative changes between the produced proteins, i.e. how the cell prioritizes its protein production. An observed up-regulation in ribosome profiling does not necessarily mean (but could) that the corresponding protein goes up in absolute terms (units produced per time). Instead, it implies that out of the population of all translating ribosomes, a larger fraction is translating (prioritizing) this particular mRNA relative to other mRNAs. The second point is addressed later in the response.

Major concerns:

(1) Fig. 1B shows polysomes still present on day 1 of 4ºC exposure, but the gel in Fig. 1C suggests a complete lack of protein synthesis. Why?

We realized that the selected gel exposure may give the false impression of a complete lack of puromycin incorporation at 4ºC. To avoid confusion, we now show in Figure 1 – figure supplement 1 the original gel image next to its longer exposure. The quantification of puromycin incorporation remains in Fig. 1C (it is based on 3 biological replicates and only one replicate is shown in the corresponding supplement). We hope it is now clear that there is an ongoing puromycin incorporation/translation at 4ºC, albeit much reduced compared with 20ºC.

What is then the evidence that ribosomal footprints used in much of the paper as evidence of ongoing active translation are from actual translating rather than still bound to transcripts but stationary ribosomes, considering that cooling to 4ºC is often used to 'freeze' protein complexes and prevent separation of their subunits? The authors should explain whether ribosome profiling as a measure of active translation has been evaluated specifically at 4ºC, or test this experimentally.

While the ribosomal profiling alone might not prove ongoing translation, the residual puromycin incorporation does (see the longer gel exposure in Figure 1 – figure supplement 1). To strengthen this argument, we selected two additional genes (cebp-1 and numr-1) whose ribosomal footprints increase in the cold, and whose GFP-fusions were available from the CGC. Monitoring their expression, we observed the expected increase in the cold (see Figure 2 – figure supplement 3 A-B). The ongoing translation in the cold is also in line with our previous study (Peke et al., 2022), where we observed de novo protein synthesis of other proteins under the same cooling conditions as in this study.

They should also provide some evidence (like Western blots) of increases in protein levels for at least some of the strongly cold-upregulated transcripts, like lips-11.

As explained above, we addressed it by additionally examining two strains expressing GFP-fused proteins, whose translation in the cold is predicted to increase according to our ribosomal profiling data. See the new Figure 2 – figure supplement 3 A-B.

As puromycin incorporation seems to be the one direct measure of global protein synthesis here, it conflicts with much of the translation data, especially considering that quite a large fraction of transcripts have increased both mRNA levels and ribosome footprints, and thus presumably increased translation at 4ºC, in Fig. 2A.

We hope the above explanations put this concern to rest.

Also, it is not clear how quantitation in Fig. 1C relates to the gel shown, the quantitation seems to indicate about 50-60% reduction of the signal, while the gel shows no discernable signal.

A above, see a longer western blot exposure in Figure 1 – figure supplement 1 and note that the quantification is based on three biological replicates.

(2) It is striking that plips-11::GFP reporter is induced in day 1 of 4ºC exposure, apparently to the extent that is similar to its induction by a large dose of tunicamycin (Fig. 3 supplement),

We did not intend to compare the extend of induction between cold and tunicamycin treatment. The tunicamycin experiment was meant to confirm that, as suggested by expression data from Shen et al. 2005, lips-11 is upregulated upon UPR activation.

…but the three IRE-1 dependent UPR transcripts from Shen 2005 list were not induced at all on day 1 (Fig. 4 supplement). Moreover, the accumulation of the misfolded CPL-1 reporter, that was interpreted as evidence that misfolding may be triggering UPR at 4ºC, was only observed on day 1, when the induction of the three IRE-1 targets is absent, but not on day 3, when it is stronger. How does this agree with the conclusion of UPR activation by cold via IRE-1/XBP-1 pathway?

In the originally submitted supplemental figure, we compared mRNA levels between day 1 animals at 20ºC versus 4ºC. However, as argued later by this reviewer, it may be better to use day 0 animals at 20ºC as the reference (since at 20ºC the animals will continue producing embryos). Thus, we repeated the RT-qPCR analysis with additional time points (and genes relevant to other comments). This analysis, now in Figure 4 – figure supplement 2, shows that these mRNAs (dnj-27, srp-7, and C36B7.6) increased already at day 1 in the cold compared with the reference 20ºC animals on day 0, and their levels increased further on day 3.

It is true that the authors do note very little overlap between IRE-1/XBP-1-dependent genes induced by different stress conditions, but for most of this paper, they draw parallels between tunicamycin-induced and cold induced IRE-1/XBP-1 activation.

We carefully re-examined the manuscript to ensure that we do not draw parallels between cold and tunicamycin treatment. The three genes (dnj-27, srp-7, and C36B7.6) were taken from Shen et al. because that study reported lips-11 as an IRE-1-responsive gene, which we realized thanks to the Wormbase annotation of lips-11. Examining the three genes in our expression data, srp-7 (like lips-11) is also upregulated more than 2-fold, while the other two genes go up but less than 2-fold. As mentioned by the reviewer, we note little overlap between the different stress conditions suggesting that the response is context dependent. Additional differences may arise if, as we hypothesize, UPR is activated in the cold in response to both protein and lipid stress. Note that the 2-fold cutoff used in the previous Figure 7 – figure supplement 1 was (erroneously) on the log2 scale, so showed genes upregulated at least 4-fold. We now corrected it to 2-fold. While there are now a few more overlapping genes, the overall conclusion, that there is little overlap between different conditions, did not change. We now list the shared genes in the new Supplementary file 5.

The conclusion that "the transcription of some cold-induced genes reflects the activation of unfolded protein response (UPR)..." is based on analysis of only one gene, lips-11. No other genes were examined for IRE-1 dependence of their induction by cold, neither the other 8 genes that are common between the cold-induced genes here and the ER stress/IRE-1- induced in Shen 2005 (Venn diagram in Figure 7 supplement), nor the hsp-4 reporter. What is the evidence that lips-11 is not the only gene whose induction by cold in this paper's dataset depends on IRE-1? This is a major weakness and needs to be addressed.

Furthermore, whether induction by cold of lips-11 itself is due to IRE1 activation was not tested, only a partial decrease of reporter fluorescence by ire-1 RNAi is shown. A quantitative measure of the change of lips-11 transcript in ire-1 and xbp-1 mutants is needed to establish if it depends on IRE-1/XBP-1 pathway.

We now examined by RT-qPCR if the induction of the three genes from Shen at al. (dnj-27, srp-7, and C36B7.6), as well as lips-11 and hsp-4 depends on IRE-1. In the new Figure 4 – figure supplement 2, we show that the upregulation of all these genes is reduced in the cold in the ire1 mutant (although in the wild type, the increase of hsp-4 mRNA appeared to be non-significant, despite the observed upregulation of the hsp-4 GFP reporter).

The authors could provide more information and the additional data for the transcripts upregulated by both ER stress and cold, including the endogenous lips-11 and hsp-4 transcripts: their identity, fold induction by both cold and ER stress, how their induction is ranked in the corresponding datasets (all of these are from existing data), and do they depend on IRE-1/XBP-1 for induction by cold?

As above, the dependence of endogenous lips-11 and hsp-4 on IRE-1 is now shown in the new Figure 4 – figure supplement 2, and the shared genes from Figure 7 – figure supplement 1 are listed in the new Supplementary file 5. We did not perform additional analysis comparing various data sets, as we felt that understanding the differences between IRE-1-mediated transcription outputs across different conditions goes well beyond this study.

Without these additional data and considering that the authors did not directly measure the splicing of xbp-1 transcript (see comment for Fig. 3 below), the conclusion that cold induces UPR by specific activation of IRE-1/XBP-1 pathway is premature.

To address the splicing of endogenous xbp-1, we examined our ribosome profiling data for the translation of spliced xbp-1, and found that the spliced variant is more abundant in the cold. This data is now shown in Figure 3 – figure supplement 2B.

There are also technical issues that are making it difficult to interpret some of the results, and missing controls that decrease the rigor of conclusions:

(1) For RNAseq and ribosome occupancy, were the 20ºC day 1 adult animals collected at the same time as the other set was moved to 4ºC, or were they additionally grown at 20ºC for the same length of time as the 4ºC incubations, which would make them day 2 adults or older at the time of analysis? This information is only given for SUnSET: "animals were cultivated for 1 or 3 additional days at 4ºC or 20ºC".

In the RNAseq experiments, the 20ºC animals were collected at the same time as the others were moved to 10ºC (and then 4ºC), so they were not additionally grown at 20ºC. We make it now clear in Methods.

This could be a major concern in interpreting translation data: First, the inducibility of both UPR and HSR in worms is lost at exactly this transition, from day 1 to day 2 or 3 adults, depending on the reporting lab (for example Taylor and Dillin 2013, Labbadia and Morimoto, 2015, De-Souza et al 2022).

As explained above, the 20ºC animals were collected at the same time as the others were moved to 4ºC. Then, we reported before that ageing appears to be suppressed in animals incubated at 4ºC (Habacher et al., 2016; Figure S1C). Thus, it terms of their biological age, cold-incubated animals appear to be closer to the 20ºC animals at the time they are moved to the cold (day 0). Thus, the ageing-associated deterioration in UPR inducibility mentioned above presumably does not apply to cold-incubated animals, which is in line with the observed IRE-1-dependent upregulation of several genes in day 3 animals at 4ºC.

How do authors account for this? Would results with reporter induction, or induction of IRE-1 target genes in Fig. 4, change if day 1 adults were used for 20ºC?

Our analysis in Figure 4 – figure supplement 2 now includes 20ºC animals at day 0, 1, and 3.

Second, if animals at the time of shift to 4ºC were only beginning their reproduction, they will presumably not develop further during hibernation, while an additional day at 20ºC will bring them to the full reproductive capacity. Did 4ºC and 20ºC animals used for RNAseq and ribosome occupancy have similar numbers of embryos, and were the embryos at similar stages?

As explained above, the reference animals at 20ºC were young adults containing few embryos. Indeed, at 4ºC the animals do not accumulate embryos. Although we cannot say that for all genes, note that the genes analysed in Figure 4 – figure supplement 2 increase in abundance also when compared with the day 3 animals kept at 20ºC.

(2) Second, no population density is given for most of the experiments, despite the known strong effects of crowding (high pheromone) on C. elegans growth. From the only two specifics that are given, it seems that very different population sizes were used: for example, 150 L1s were used in survival assay, while 12,000 L1s in SUnSET. Have the authors compared results they got at high population densities with what would happen when animals are grown in uncrowded plates? At least a baseline comparison in the beginning should have been done.

None of the experiments involved crowded populations. In the SUnSET experiments, we just used larger and more plates to obtain sufficient material.

(3) Fig. 3: it is unclear why the accepted and well characterized quantitative measure of IRE1 activation, the splicing of xbp-1transcript, is not determined directly by RT-PCR. The fluorescent XBP-1spliced reporter, to my knowledge, has not been tested for its quantitative nature and thus its use here is insufficient. Furthermore, the image of this fluorescent reporter in Fig. 3b shows only one anterior-most row of cells of intestine, and quantitation was done with 2 to 5 nuclei per animal, while lips-11 is induced in entire intestine. Was there spliced XBP-1 in the rest of the intestinal nuclei? Could the authors show/quantify the entire animal (20 intestinal cells) rather than one or two rows of cells?

As explained above, we now included the analysis of xbp-1 splicing in Figure 3 – figure supplement 2B. As for the fluorescent reporter, it is difficult to measure all gut nuclei since part of the gut is occluded by the gonad. Nonetheless, we do see induction of the reporter in other gut nuclei and show now additional examples from midgut in Figure 3 – figure supplement 2A.  

(4) The differences in the outcomes from this study and the previous one (Dudkevich 2022) that used 15ºC to 2ºC cooling approach are puzzling, as they would suggest two quite different IRE-1 dependent programs of cold tolerance. It would be good if authors commented on overlapping/non-overlapping genes, and provided their thoughts on the origin of these differences considering the small difference in temperatures.

Indeed, there seem to be substantial differences between different temperatures and cooling paradigms. While understanding the C. elegans responses to cold is still in its infancy, one possible explanation for the observed differences is that we used different starting growth temperatures. While the initial populations in our study were grown at 20ºC, Dudkevich et al. used 15ºC. Worms display profound physiological differences between these two temperatures. For example, Xiao et al. (2013) showed that the cold-sensitive TRPA-1 channel is important at 15ºC but not 20ºC. Thus, the trajectories along which worms adapt to near freezing temperature may vary depending on their initial physiological state (and perhaps the target temperature, as we used 4ºC and they 2ºC). We now expanded argumentation on this topic in Discussion. I should also say that we planned on testing NLP-3 function in our paradigm, but our request for strains remained unanswered.

Second, have the authors performed a control where they reproduced the rescue by FA supplementation of poor survival of ire-1 mutants after the 15ºC to 2ºC shift? Without this or another positive control, and without measuring change in lipid composition in their own experiments, it is unclear whether the different outcomes with respect to FAs are due to a real difference in adaptive programs at these temperatures, or to failure in supplementation?

While we did not re-examine the findings by Dudkevich et al., we did include now another positive control. As reporter by Hou et al. (2014), supplementing unsaturated FAs rescues the induction of the hsp-4 reporter in fat-6 RNAi-ed animals. Although we were able to reproduce that result (Figure 6 – figure supplement 1), the same supplementation procedure did not suppress the lips11 reporter (Figure 6 – figure supplement 2).

(5) Have the authors tested whether and by how much ire-1(ok799) mutation shortens the lifespan at 20ºC? This needs to be done before the defect in survival of ire-1 mutants in Fig. 7a can be interpreted.

The lifespan at standard cultivation temperature was examined by others (Henis-Korenblit et al., 2010; Hourihan et al., 2016), showing that ire-1(ok799) mutants live shorter. However, while some mechanism that prolong lifespan may also improve cold survival, the two phenomena are not identical and whether IRE-1 facilitates longevity and cold survival in the same or different way remains to be seen.

Reviewer #2:

(1) The conclusions regarding a general transcriptional response are based on one gene, lips-11, which does not affect survival in response to cold. We would suggest altering the title, to replace "Reprograming gene expression: with" Regulation of the lipase lips-11".

We now examined IRE-1 dependent induction of additional genes – see Figure 4 – figure supplement 2. While we do not know what fraction of cold-induced genes depends on IRE-1, we feel that our findings justify the statement that that gene expression in the cold involves the IRE1/XBP-1 pathway (title) or that that the transcription of some/a subset of cold-induced genes depend on this pathway (in abstract, model, and discussion).

(2) There is no gene ontology with the gene expression data.

We now included the top 10 most enriched and suppressed gene categories between 10ºC and 4ºC (since the biggest change happens between these conditions, as shown in Figure 2 – figure supplement 1A). This is now included in the Figure 2 – figure supplement 2.

(3) Definitive conclusions regarding transcription vs translational effects would require use of blockers such as alpha amanatin or cyclohexamide.

As explained also for reviewer 1, we confirmed now that at least some genes, whose translation is upregulated based on the ribosome profiling, are indeed upregulated in the cold at the protein level (Figure 2 – figure supplement 3A-B). Thus, the increase in ribosomal occupancy seems to accurately reflect increased translation. Since mRNA levels correlate overall with the ribosomal occupancy, it appears that the mRNA levels are the main determinants of the translation output. Because the lips-11 promoter is sufficient to upregulate the GFP reporter in the cold, it further suggests that the regulation happens at the transcription level. It is true that at this point we cannot completely rule out the effects of mRNA stability, which we clearly acknowledge in the discussion.

(4) Conclusions regarding the role of lipids are based on supplementation with oleic acid or choline, yet there is no lipid analysis of the cold animals, or after lips-1 knockdown.

We agree that this is an important direction for future studies but feel that lipidomic analysis goes beyond the scope of current work.

Although choline is important for PC production, adding choline in normal PC could have many other metabolic impacts and doesn't necessarily implicate PC without lipidomic or genetic evidence.

We agree and acknowledge it now in Discussion: “However, choline also plays other roles, including in neurotransmitter synthesis and methylation metabolism. Thus, we cannot yet rule out the possibility that the protective effects of choline supplementation stem from functions outside PC synthesis.”

Reviewer #3:

The study has several weaknesses: it provides limited novel insights into pathways mediating transcriptional regulation of cold-inducible genes, as IRE-1 and XBP-1are already well-known responders to endoplasmic reticulum stress, including that induced by cold.

We presume the reviewer refers to the study by Dudkevich et al. (2022). As explained in our manuscript, there are important differences between that study and ours in how the IRE-1 signalling is utilized and to what ends.

Additionally, the weak cold sensitivity phenotype observed in ire-1 mutants casts doubt on the pathway's key role in cold adaptation. The study also overlooks previous research (e.g.PMID: 27540856) that links IRE-1 to SKN-1, another major stress-responsive pathway, potentially missing important interactions and mechanisms involved in cold adaptation.

We state in the manuscript that the IRE-1 pathway plays a modest but significant role in cold adaptation and state in the Fig. 7 model and Discussion that additional pathways work alongside IRE-1 to drive cold-specific gene expression.

Recommendations for the authors:

Reviewer #1:

Minor comments:

(1) Fig. 2B - reporter expression seems to be already present in the intestine of 20ºC animals. What is the turnover rate of GFP in the intestine and how is it affected by the temperature shift? If GFP degradation is inhibited, could it explain the increase in signal in 4ºC animals, rather than increased transcription? This seems to be true for the hsp-4 transcriptional reporter, as the GFP fluorescence appears to increase during 4ºC incubation (Fig. 4a), but the hsp-4 message levels are only increased after 1 day but not in later days at 4ºC, based on the RNAseq in provided dataset. How well do changes in lips-11 reporter fluorescence correspond to the changes in the endogenous lips-11 transcript?

Note that increased GFP fluorescence is accompanied by increased mRNA levels. In addition to the RNAseq data, we now also examined changes of the endogenous lips-11 transcript by RTqPCR and observed its strong (and IRE-1 dependent) upregulation in the cold– see Figure 4 – figure supplement 2. Moreover, we now included two other examples of GFP-tagged proteins whose fluorescence increases in the cold, concomitant with increased mRNA levels and ribosomal occupancy (Figure 2 – figure supplement 2A-B).

(2) Descriptions of methods to measure different aspects of translation are very abbreviated and in some places make it difficult to understand the paper. One example - what is RFP in Fig. 2a?

We replaced now “RFP” with “RPF” (ribosome protected fragment) and the abbreviation is explained firsts time it is used.

(3) How was the effectiveness of RNAi at 4ºC validated?

As explained in Methods, we subjected animals to RNAi long before they were transferred to 4ºC, so the corresponding protein is depleted prior to cooling.

(4) Several of the conclusions on translation and ribosomal occupancy are written in a somewhat confusing way. For example, the authors state that "shift from 10ºC to 4ºC had a strong effect" when describing "impact on translation (ribosomal occupancy)" (page 4), but in the next sentence, they state "a good correlation between mRNA levels and translation (Figure 2A)". Was ribosomal occupancy normalized to the transcript abundance?

We do not perceive any discrepancy between the two statements. The former refers to the difference between time points, where we observed the largest change in both the transcriptome and ribosomal occupancy from 10ºC to 4ºC (as can be inferred in the PCA plot in Figure 2 - figure supplement 1). The latter refers to the observation that changes in mRNA levels mirrored, in most of cases, similar changes in the ribosomal occupancy.

The ribosomal occupancy was not normalized, as that would essentially normalize the y-axis (ribosomal occupancy) with the x-axis (mRNA), and so express changes in “translational efficiency” as a function of changes in mRNA abundance. While this type of analysis can also reveal interesting biological phenomena, it would explore a different question.

(5) "For most transcripts ... increased the abundance of a particular protein appears to correlate depend primarily on the abundance of its mRNA" (page 5). This is an overstatement, the protein levels were not quantified.

As explained above, we now additionally monitored the expression of two GFP-tagged proteins (CEBP-1 and NUMR-1). Monitoring their expression, we observed the expected increase in GFP fluorescence in the cold (see Figure 2 – figure supplement 3 A-B). While we did not examine them also by western blot, these observations are in line with our conclusions.

(6) The statement "Since transcription is the main determinant of mRNA levels, these results suggest that cold-specific gene expression primarily depends on transcription activation" seems to assume that message degradation doesn't have much of an impact at 4ºC. What is the evidence here? The authors themselves later suggest either transcription or mRNA stability in Discussion.

While we cannot exclude that mRNA stability of some genes may be affected, this concern is more valid for the messages that go down in the cold. Although we have done it for only selected genes, each time we observed an increase in the mRNA levels, we also observed the corresponding increase in the protein; this study and Pekec et al. (2022). Then, the lips-11 reporter was designed to monitor the activity of its promoter, which we showed in sufficient to upregulate reporter GFP in the cold. We have now expanded the corresponding paragraph in Discussion, which will hopefully come across as more balanced.  

Reviewer #2:

(1) Alter title, conclusions to better reflect specific nature of the work.

We now provided additional data and feel that it justifies our conclusions and title.

(2) Use Gene Ontology searches to look at patterns of gene expression in RNA seq data.

We now show it in Figure 2 – figure supplement 2.

(3) Use genetic or lipidomic tools rather than solely adding exogenous lipids.

We agree that lipidomic analysis is an important direction for future research, but feel that lipidomic analysis and further genetic experiments go beyond the scope of current manuscript.

Reviewer #3:

To strengthen the evidence for the role of IRE-1 in cold adaptation, the authors might consider performing additional functional assays, such as testing the effects of IRE-1 and XBP-1 mutations under varying cold conditions and testing the genetic interaction of ire-1 with xbp-1, skn-1, and hsf-1 in cold sensitivities. It is also worth using alternative approaches such as independent alleles of ire-1, knockdowns or tissue-specific knockouts (without potential developmental compensation in global constitutive mutants) to better characterize the contribution of IRE-1 to cold adaptation. Additionally, studies that examine tissue-specific responses to cold exposure could provide important insights, as different tissues may utilize distinct molecular pathways to adapt to cold stress.

We also tested ire-1 and xbp-1 functions by RNAi-mediated depletion. SKN-1 is a good candidate for future studies, but Horikawa at al. (2024) showed that HSF-1 is not required for cold dormancy (at 4ºC); we also show now that HSF-1::GFP does not increase in the cold (Figure 2 – figure supplement 3C).

This reviewer also recommends clarifying the novelty of your findings in the context of existing literature, particularly regarding the established roles of IRE-1 and XBP-1 in responding to endoplasmic reticulum stress.

The entry point of this study was to clarify a long-standing problem in hibernation research, i.e., the apparent discrepancy between a global translation repression and de novo gene expression observed in the cold. By connecting cold-mediated expression of some genes to the IRE-1/XBP1 pathway, we strengthen the argumentation for transcription-mediated gene regulation in hibernating animals. We did go the extra mile to test the possible reason behind the activation of UPR^ER in the cold but feel that a deeper analysis deserves a separate study.

The term "hibernation" should be avoided or reworded since the study does not provide direct behavioral or physiological evidence for hibernation-like states; instead, the manuscript could refer to "cold-induced responses" or "adaptations to cold temperatures."

The term “hibernation” was used before even in the context of the C. elegans dauer state, which, arguably, is even less appropriate. In addition to a global suppression of translation shown here, we reported before that the same cooling regime suppresses ageing (Habacher et al., 2016; Figure S1C). Incubating at 4ºC also arrests C. elegans development (Horikawa et al., 2024). Thus, while the worm and mammalian hibernation are certainly not equivalent – which we clearly spell out – we like to use “hibernation” interchangeably with “cold dormancy” to draw attention to a fascinating aspect of C. elegans biology. Still, we use now quotation marks in the title to avoid misunderstanding.

The discussion could be strengthened by addressing the relevance of prior studies, such as those linking IRE-1 to SKN-1 (PMID: 27540856), TRPA-1 (PMID: 23415228), ZIP-10 (PMID: 29664006), HSF-1 (PMID: 38987256) in cold adaptation and elaborating on how your findings provide new

The IRE-1/SKN-1 and ZIP-10 papers are now mentioned when describing the model in Figure 7. The TRP-1 and HSF-1 papers are cited when discussing physiological differences between different cold temperatures. Consistent with our studies, the HSF-1 paper shows that nematodes enter a dormant state at 4ºC (but at 9ºC and higher temperatures continue developing). Importantly, HSF-1 promotes the development at 9ºC but is not important for the arrest at 4ºC. We also shown now in Figure 2 – figure supplement 3C that HSF-1 does not go up at 4ºC.

Reviewer #1 (Public Review):

The mechanisms that regulate establishment of the germline stem cells and germline progenitors during zebrafish reproductive development are not understood. Prior single cell analysis characterized the cell types of the early zebrafish ovary during and at stages after sexual differentiation. In this work Hsu et al. took a single approach to analyze the cell types present in the early gonad during early sex determination. As expected, they identified germline stem cells (GSCs) that express canonical GSC markers and distinct populations of progenitors. Unexpectedly, they found multiple populations of transcriptionally distinct progenitor populations that the authors termed early (those lacking the differentiation marker foxl2l), committed (those expressing fox2l2 and S-phase genes) and late (those expressing fox2l2 and meiotic genes) progenitors. Comparisons of their dataset to the published zebrafish ovary datasets confirmed the presence of these distinct progenitor populations in the ovary. Further, they convincingly validated the presence of these progenitor subtypes using fluorescent in situ hybridization. To investigate the relationship between progenitor subsets and known regulators of ovary differentiation, the authors conducted single cell analysis of gonads lacking the transcription factor, Foxl2l. As previously reported, Foxl2l absence blocks ovary differentiation and all foxl2l mutants develop testes. The single cell analysis here indicates that foxl2l is inappropriately expressed in GSCs and early progenitors and that germ cell differentiation is blocked at the committed progenitor stage since few committed progenitors and no late progenitors or meiotic transcripts were detected in the single cell analysis of foxl2l mutants. Based on the coexpression of genes that are not typically expressed together in normally developing germ cells, specifically nanos2 and foxl2l, and dmrt1 and foxl2l, the authors conclude that Foxl2l is required for the committed progenitor program and that it prevents committed progenitors from returning to the GSC state.

Overall, the data provide new insights into the cell populations of the early differentiating gonad, define distinct progenitor states, pinpoint a requirement for the ovary differentiation factor Foxl2l at a specific stage of progenitor differentiation, and generate new hypotheses to be tested. Many but not all of the conclusions are supported by compelling data, and some findings and conclusions need to be clarified in the context of the published literature.

(1) The authors conclude that the committed progenitors revert to GSCs based on the coexpression of nanos2 and foxl2l nanos2 and based on expression of id1 in mutants but not in WT. Without functional data demonstrating that the progenitors revert to an earlier state, alternative interpretations should be considered. For example, it is possible that the cells initiate the committed progenitor program but continue to express the GSC program and that the coexpression of both programs blocks differentiation. Consistent with this possibility, some Fox family members, FoxL2 and FoxPs for example, are known to be both activators and repressors of transcription or act primarily as repressors. Potentially relevant to this work, repressive activity of FoxL2 has been previously reported in the mammalian ovary (Pisarska et al Endocrinology 2004, Pisarska Am J. Phys Endo. Metabolism 2010, Kuo Reproduction 2012, Kuo Endocrinology 2011, as well as more recent publications). In that context interfering with FoxL2 was proposed to cause upregulated expression of genes normally repressed by FoxL2, accelerated follicle recruitment, and premature ovarian failure.

(2) The authors conclude that the committed progenitor stage is "the gate toward female determination" and that the cells "stay at S-Phase temporarily before differentiation". This conclusion seems to be based solely on single cell RNAseq expression. In several species, including zebrafish, meiotic entry occurs earlier in females and has been correlated with ovary development. The possibility that the late progenitor stage, the stage when meiotic genes are detected in this study and a stage missing in foxl2l mutants, is actually the key stage for female determination cannot be excluded by the data provided.

(3) The authors discuss prior working showing that loss of germ cells leads to male development and that germ cells are required for female development and claim to extend that work by showing here that some progenitors are already sexually differentiated. First, the stages compared are completely different. The earlier work looks at the primordial germ cells and their loss in the first few days of development before a gonad forms. In contrast, this work examines stages well after the gonad has formed and during sex determination. The second concern is that the conclusion that the progenitors are differentiated is based solely on the expression of foxl2l, which is initially expressed in the juvenile ovary state that lab strains have been shown to develop through (Wilson et al Front Cell Dev Bio 2024). While it is fair to state that some cells express ovary markers at this stage, it is unclear that this is sufficient evidence that the cells are differentiated. For example, in the context of the foxl2l mutant, the authors observe that GSCs and early progenitors inappropriately express foxl2l, but the mutants develop as males. Thus, expression of foxl2l transcripts alone is insufficient evidence to claim that the cells are already differentiated as female.

(4) The comparison between medaka and zebrafish foxl2l mutants seems to suggest that Foxl2l is required for meiosis in medaka but has a different role in zebrafish. However, if foxl2l represses the earlier developmental programs of GSCs and early progenitors, it is possible that continued expression of these early programs interferes with activation of meiotic genes. This could account for the absence of the late progenitor stage in foxl2l mutants since the late progenitor stage is defined by and distinguished from the earlier stages by expression of foxl2l and meiotic genes. If so, foxl2l may be similarly required in both systems.

(5) The authors state that "Foxl2l may ensure female differentiation by preventing stemness and antagonizing male development." It is unclear why suppressing stemness would be necessary for female differentiation since female zebrafish have stem cells as do male zebrafish. It seems likely that turning off the GSC and early differentiation programs is important for allowing expression of meiosis and oocyte differentiation genes, and that a gene other than Foxl2l is required for differentiation from GSCs to spermatocytes.

(6) Based on its expression in mutant progenitors, p53 is proposed to assist with alternative differentiation of mutant germ cells. Although p53 transcripts are expressed, no evidence is provided that p53 is involved in differentiation of germ cells, and sex bias has not been associated with the published p53 mutants in zebrafish. Furthermore, while p53 has been shown to be important for ovary to testis transformation in mutant contexts in adults, it appears dispensable for testis development in mutants that disrupt ovary differentiation in earlier stages (Rodriguez-Mari et al PLoS Gen 2010, Shive PNAS 2010, Hartung et al Mol. Reprod. Dev 2014, Miao Development 2017, Kaufman et al PLoSGen 2018, Bertho et al Development 2021. It is possible that p53 eliminates foxl2l mutant germ cells that are simultaneously expressing multiple developmental programs, but this possibility would need to be tested.

Author response:

Reviewer #1 (Public Review):

(1) The authors conclude that the committed progenitors revert to GSCs based on the coexpression of nanos2 and foxl2l nanos2 and based on expression of id1 in mutants but not in WT. Without functional data demonstrating that the progenitors revert to an earlier state, alternative interpretations should be considered. For example, it is possible that the cells initiate the committed progenitor program but continue to express the GSC program and that the coexpression of both programs blocks differentiation.

Thanks for your insightful comment. We have explored possible alternative interpretations of our data. Regarding the suggested possibility of a continued GSC program in the mutant, we have examined the expression of GSC markers including nanos2 in the mutant at different stages. We found that in the mutant, nanos2 or other GSC markers were not significantly upregulated in GSC-to progenitor transition (G-P) and early progenitors (Prog-E) (Fig. 4B). The expression of these GSC markers was also low in the integrated clusters I4-I6 when G-P and Prog-E stages were prominent (Fig. 3D and Fig. 3E). GSC marker nanos2 was high only in mutant Prog-C. These results argue against continued GSC programs in the foxl2l mutants. Another possible explanation is that perhaps some mutant Prog-C acquires some GSC property with the upregulation of nanos2 instead of a continuous GSC program. We have now clarified our rationale about mutant cells gaining new GSC properties and included both interpretations in the Result.

Consistent with this possibility, some Fox family members, FoxL2 and FoxPs for example, are known to be both activators and repressors of transcription or act primarily as repressors. Potentially relevant to this work, repressive activity of FoxL2 has been previously reported in the mammalian ovary (Pisarska et al Endocrinology 2004, Pisarska Am J. Phys Endo. Metabolism 2010, Kuo Reproduction 2012, Kuo Endocrinology 2011, as well as more recent publications). In that context interfering with FoxL2 was proposed to cause upregulated expression of genes normally repressed by FoxL2, accelerated follicle recruitment, and premature ovarian failure.

FoxL2 exerts both activating and repressive activities. We believe that Foxl2l can also activate and repress its target gene expression. Although its target genes have not been clearly identified, Foxl2l may activate genes involved such process as oogenic meiosis, and may also repress other genes involved in other processes, say perhaps nanos2.

(2) The authors conclude that the committed progenitor stage is "the gate toward female determination" and that the cells "stay at S-Phase temporarily before differentiation". This conclusion seems to be based solely on single cell RNAseq expression. In several species, including zebrafish, meiotic entry occurs earlier in females and has been correlated with ovary development. The possibility that the late progenitor stage, the stage when meiotic genes are detected in this study and a stage missing in foxl2l mutants, is actually the key stage for female determination cannot be excluded by the data provided.

We agree that Prog-L is important for the initiation of female meiosis. We have made revision in the text to point out the importance of Prog-L in female differentiation.

(3) The authors discuss prior working showing that loss of germ cells leads to male development and that germ cells are required for female development and claim to extend that work by showing here that some progenitors are already sexually differentiated. First, the stages compared are completely different. The earlier work looks at the primordial germ cells and their loss in the first few days of development before a gonad forms. In contrast, this work examines stages well after the gonad has formed and during sex determination.

Both previous studies and our study indicate the important role of germ cells in zebrafish sex differentiation during gonadal development. The earlier works show that the abundance of primordial germ cells contributes to sex differentiation. Our current finding further suggests the existence of female identify in some germ cells at the juvenile stage and discusses the importance of cell in sexual differentiation. We have added the developmental age in our study to emphasize the age difference.

The second concern is that the conclusion that the progenitors are differentiated is based solely on the expression of foxl2l, which is initially expressed in the juvenile ovary state that lab strains have been shown to develop through (Wilson et al Front Cell Dev Bio 2024). While it is fair to state that some cells express ovary markers at this stage, it is unclear that this is sufficient evidence that the cells are differentiated.

The conclusion about the differentiation of progenitors is not based solely on foxl2l expression; rather, it is according to the whole transcriptomic profiles of both WT (Figure 1B) and foxl2l mutant cells (Figure 3A) as well as the foxl2l mutant phenotype (Figure 2C). Three types of progenitors, Prog-E, Prog-C and Prog-L were identified by whole transcriptomic analysis in WT. In foxl2l mutants, the transcriptomic profile further shows that Prog-L and meiotic cells are completely lost, and all germ cells undergo male differentiation eventually. These results together indicate that the differentiation of Prog-C to Prog-L guides the progenitor toward female differentiation. Our result also showed that in the juvenile gonad, foxl2l expression is high in two types of progenitors, Prog-C and Prog-L, and become low after meiotic entry.

For example, in the context of the foxl2l mutant, the authors observe that GSCs and early progenitors inappropriately express foxl2l, but the mutants develop as males. Thus, expression of foxl2l transcripts alone is insufficient evidence to claim that the cells are already differentiated as female.

The foxl2l mutants develop into males because they lack functional Foxl2l. Although the mutated foxl2l transcript is present in mutant cells, these transcripts are not functional. These mutants develop into males eventually. This result is consistent with our claim that functional Foxl2l is important for the development of Prog-L and female differentiation.

(4) The comparison between medaka and zebrafish foxl2l mutants seems to suggest that Foxl2l is required for meiosis in medaka but has a different role in zebrafish. However, if foxl2l represses the earlier developmental programs of GSCs and early progenitors, it is possible that continued expression of these early programs interferes with activation of meiotic genes. This could account for the absence of the late progenitor stage in foxl2l mutants since the late progenitor stage is defined by and distinguished from the earlier stages by expression of foxl2l and meiotic genes. If so, foxl2l may be similarly required in both systems.

Medaka and zebrafish Foxl2l may share similar functions such as the stimulation of meiotic gene expression and promotion of oogenesis in the female germ cells preparing for meiotic entry. In addition, we also detected aberrant upregulation of nanos2 in some foxl2l mutant cells. The idea of “continued expression of these early programs interferes with activation of meiotic genes” is conceivable, but for now we have no evidence for it. We do not know whether the absence of meiotic genes is due to an interference caused by the activation of nanos2 or due to the complete loss of Prog-L and meiotic cells. It will also be interesting to find out whether medaka Foxl2l has a role in early progenitors

(5) The authors state that "Foxl2l may ensure female differentiation by preventing stemness and antagonizing male development." It is unclear why suppressing stemness would be necessary for female differentiation since female zebrafish have stem cells as do male zebrafish. It seems likely that turning off the GSC and early differentiation programs is important for allowing expression of meiosis and oocyte differentiation genes, and that a gene other than Foxl2l is required for differentiation from GSCs to spermatocytes.

It is true that we have not proved whether suppression of stemness is required for female differentiation. Maybe our earlier statement is a bit misleading. We agree that it is likely that turning off the GSC and early differentiation programs is important for allowing expression of meiotic and oocyte differentiation genes, and that a gene other than Foxl2l is required for differentiation from GSCs to spermatocytes. To avoid confusion, we have modified our statement in the text.

(6) Based on its expression in mutant progenitors, p53 is proposed to assist with alternative differentiation of mutant germ cells. Although p53 transcripts are expressed, no evidence is provided that p53 is involved in differentiation of germ cells, and sex bias has not been associated with the published p53 mutants in zebrafish. Furthermore, while p53 has been shown to be important for ovary to testis transformation in mutant contexts in adults, it appears dispensable for testis development in mutants that disrupt ovary differentiation in earlier stages (Rodriguez-Mari et al PLoS Gen 2010, Shive PNAS 2010, Hartung et al Mol. Reprod. Dev 2014, Miao Development 2017, Kaufman et al PLoSGen 2018, Bertho et al Development 2021. It is possible that p53 eliminates foxl2l mutant germ cells that are simultaneously expressing multiple developmental programs, but this possibility would need to be tested.

The tp53^-/-foxl2l^-/- double mutant cannot alleviate the all-male phenotype of foxl2l^-/- mutant (Dev Biol, 517, 91-99, 2024), indicating that the male development is not due to p53-mediated germ cell apoptosis. We have cited the suggested papers and compared relation of tp53 between these mutants (fancl, zar1, etc.) mentioned in the cited papers. Since tp53 was enriched in certain foxl2l^-/- mutant cell clusters, and tp53 mutation fails to rescue the all-male phenotype, it is possible that p53 expressed in these mutant cell clusters has roles other than inducing apoptosis. One assumption is that p53 may be involved in the germ cell differentiation, especially p53 is known to promote differentiation of airway epithelial progenitors, adipogenesis and embryonic stem cells. We have emphasized that the suggested role of p53 in germ cell differentiation is our assumption in the Discussion.

Reviewer #3 (Public Review):

This is the first report to show a transcriptional factor, foxl2l, is essential for the development of female germs. Without foxl2l, germ cells will be developed into sperms. The report also clearly defined the arrested stage of early germ cells in foxl2l mutants, or stages that is critical for foxl2l to play a role for the further development of female germ cells.

(1) Due to lack of cell lineage tracing, the claim of foxl2l suppression of dedifferentiate of progenitor cells to GSC based on the gene expression and cell number changes is weak.

Thanks for your comments pointing out our contribution and also weakness. We acknowledge the lack of direct evidence on the reversion of mutant Prog-C to GSC in our data. We now removed the claim about the repression of stemness by Foxl2l.

(2) In addition, separation of early germ cell types in foxl2l mutant using marker genes from WT may not be optimal.

The cell type of mutant cell is determined by two independent analyses. First is inferring the developmental stage of mutant cells. This approach assumes that mutant cells can indeed be mapped to specific WT stages through their transcriptomic profiles. However, as indicated by this reviewer’s comments, mutant cells exhibited heterogeneity and can be distinct from WT cells. Defining cell types in mutants by WT markers may not be optimal. To address this, we conducted another analysis, co-clustering. Mutant cells and WT cells at early stages (GSC , G-P, Prog-E, Prog-C(S) and Prog-C) were co-clustered. This approach does not assume a direct correspondence between mutant and WT developmental stages. Instead, it facilitates the identification of novel germ cell types in mutants while characterizing the relationship between WT and mutant cells. In some clusters, both WT and mutant cells were present, indicating high transcriptomic similarity. In other clusters, most cells are only mutant cells, indicating distinct mutant cell types (Figure 3C). We can, therefore, assign developmental properties to these mutant cells with confidence.

好的，我将通过具体例子结合知识库信息，分别解释福克纳的“多视角叙事”、卡夫卡的“荒诞哲学”和博尔赫斯的“迷宫结构”。以下是详细分析：

一、福克纳的“多视角叙事”

核心特征：通过不同人物的主观视角拼贴故事，打破线性叙事，揭示人物心理与真相的复杂性。

具体例子：《喧哗与骚动》

1. 班吉的视角（第一章）
2. 特点：班吉是智障者，叙述混乱无序，依赖感官印象（如颜色、声音）触发回忆。
3. 知识库引用：
   • 知识库[2]提到，班吉的叙述“支离破碎”，如他回忆凯蒂爬树时的场景：“那棵树不再抖动了……凯蒂的头发上插着花儿，披着条长长的白纱，像闪闪发亮的风儿。”
   • 知识库[4]指出，班吉的视角“以白痴的叙述开创性地展现非理性思维”，通过感官碎片拼凑出家族衰败的真相。

4. 作用：揭示康普生家族的混乱与情感断裂，暗示凯蒂的悲剧是家庭精神崩溃的缩影。

5. 昆丁的视角（第二章）

6. 特点：昆丁内心充满对家族荣誉的执念，叙述充满时间焦虑（如反复看表、回忆妹妹凯蒂的堕落）。

7. 知识库引用：

   • 知识库[8]分析福克纳对时间的处理：“昆丁的意识流展现时间的‘绵延’，过去与现在交织，最终导致他自杀。”
   • 知识库[9]提到，昆丁的视角“通过心理独白暴露南方贵族的道德困境”。

8. 杰生的视角（第三章）

9. 特点：自私、偏执，以现实主义的怨恨叙述，揭露家族的虚伪与资本主义价值观。

10. 知识库引用：

    • 知识库[3]指出，杰生的叙述“充满对凯蒂和妹妹的怨恨”，体现“南方旧秩序的崩塌与新贪婪的崛起”。

11. 迪尔西的视角（第四章）

12. 特点：黑人女仆的全知视角，冷静梳理前三章的碎片，揭示真相（如凯蒂私生女的存在）。
13. 知识库引用：
    • 知识库[4]强调迪尔西视角的“补充性”，她“作为旁观者，用客观叙事解构白人视角的偏见”。

总结：

福克纳通过四重视角，将同一事件（康普生家族的衰落）解构为不同人物的主观体验，最终拼凑出南方贵族精神崩溃的全景。这种叙事方式打破了传统“客观叙述”，强调真相的多元性与不可靠性（知识库[6][7]）。

二、卡夫卡的“荒诞哲学”

核心特征：通过荒诞的情节、符号与设定，揭示现代社会的异化、权力压迫与个体的无力感。

具体例子：《变形记》

1. 荒诞设定：
2. 主人公格里高尔·萨姆沙一夜之间变成甲虫，但家人和雇主对此反应冷漠。

3. 知识库引用：

   • 卡夫卡的荒诞哲学常通过“非理性事件”隐喻现实中的异化（如知识库[10]提到的“用变形象征人性的丧失”）。

4. 荒诞细节：

5. 家人从恐慌到厌恶，最终将他视为累赘；格里高尔仍试图履行“家庭责任”，却无人倾听。

6. 哲学内核：

   • 异化：资本主义社会中，人沦为工具（格里高尔的工作是“推销员”，变形后失去价值）。
   • 沟通失效：格里高尔无法解释自己的处境，象征现代人情感交流的断裂。

7. 结局的荒诞：

8. 格里高尔死去，家人如释重负，甚至计划搬离公寓。
9. 知识库引用：
   • 知识库[9]指出，卡夫卡的荒诞“揭露了人性的冷漠与制度的非理性”。

另一例：《审判》

• 荒诞情节：约瑟夫·K毫无缘由地被指控有罪，却无法得知罪名或上诉途径，最终被处决。
• 哲学内核：
• 权力的不可抗性：官僚体系庞大而荒谬，个体在其中完全无力（知识库[10]提到“官僚机器的非人性”）。
• 存在主义困境：世界本质无意义，人只能在荒诞中挣扎。

总结：

卡夫卡的荒诞哲学通过超现实情节与日常细节的结合，将现代社会的荒诞本质推向极致，揭示个体在权力与异化中的悲剧性（知识库[9][10]）。

三、博尔赫斯的“迷宫结构”

核心特征：通过循环、镜像、无限空间等叙事手法，构建文本的多重可能性，探讨时间、记忆与存在的哲学命题。

具体例子：《小径分岔的花园》

1. 迷宫设定：
2. 故事中的“花园”实为一座包含无数时间分岔的迷宫，每个选择都导致平行宇宙的诞生。

3. 知识库引用：

   • 知识库[10]提到博尔赫斯的迷宫“象征时间的无限可能性”，如“小径分岔的花园”暗示“所有可能的历史同时存在”。

4. 叙事结构：

5. 故事嵌套在一封信、一个梦和一座花园中，现实与虚构界限模糊。
6. 哲学内核：
   • 时间的不可知性：人类试图掌控时间，但最终陷入自我指涉的循环（如主角在杀人后成为迷宫的一部分）。
   • 叙事的无限性：文本本身成为迷宫，读者无法确定何为真实。

另一例：《巴别图书馆》

• 迷宫设定：宇宙被描绘为无数六边形房间组成的图书馆，每本书排列所有可能的字符组合。
• 哲学内核：
• 知识的虚无：无限信息导致意义消解，寻找“完美之书”成为徒劳。
• 存在主义的孤独：人类在无限中寻找秩序，却注定失败（知识库[10]提到“迷宫隐喻人类认知的局限”）。

总结：

博尔赫斯的迷宫结构通过叙事层的嵌套与符号的循环，将文本转化为哲学思辨的载体，探讨真实与虚幻、有限与无限的辩证关系（知识库[10]）。

总结对比

| 作家 | 核心手法 | 代表作品 | 哲学内核 | |----------------|-----------------------|----------------------|----------------------------------| | 福克纳 | 多视角叙事 | 《喧哗与骚动》 | 真相的多元性与南方贵族的衰落 | | 卡夫卡 | 荒诞哲学 | 《变形记》《审判》 | 异化、权力压迫与存在困境 | | 博尔赫斯 | 迷宫结构 | 《小径分岔的花园》 | 时间的无限性与认知的局限性 |

通过以上分析可见，三位作家分别以独特的叙事策略，将文学从“讲故事”升华为对人性、社会与存在的深刻哲学探索。

Why does it jump from 1-2-4-6-9?

Shows that the amount of activity time per participant was noted in MET minutes

Immediately outlines that physical inactivity is a key cause of increasing healthcare costs, and outlines this as one of the leading factors of healthcare costs in developed countries.

1. I tried to coax my brother out for a walk, but he’s got his nose in a book, as always. ✅ nose (правильный вариант)

Перевод: Я попытался уговорить брата выйти на прогулку, но он, как всегда, уткнулся в книгу.

Объяснение: Фраза "has his nose in a book" означает, что человек полностью погружен в чтение.

1. I didn’t get very far with the book – a bit heavy-going for me. ✅ heavy-going (правильный вариант)

Перевод: Я не очень продвинулся в этой книге — она показалась мне слишком сложной/тяжеловесной.

Объяснение: "Heavy-going" означает, что книга трудна для восприятия (скучная, сложная).

1. Can’t come over, sorry. Exam tomorrow, so it’s dinner now, then I hit the books! ✅ hit (правильный вариант)

Перевод: Не могу прийти, извини. Завтра экзамен, так что сейчас ужин, а потом — за учебу!

Объяснение: "Hit the books" — идиома, означающая "серьезно заняться учебой/чтением".

1. In my opinion, there’s nothing better than getting lost in a book. ✅ lost (правильный вариант)

Перевод: По-моему, нет ничего лучше, чем полностью погрузиться в книгу.

Объяснение: "Get lost in a book" — значит "погрузиться в чтение с головой".

1. Lucy’s been a bookworm ever since she got her first Roald Dahl book. ✅ bookworm (правильный вариант)

Перевод: Люси была книжным червем с тех пор, как получила свою первую книгу Роальда Даля.

Объяснение: "Bookworm" — это человек, который очень любит читать (разговорное).

1. You don’t have to be a die-hard book lover to appreciate the value of reading. ✅ die-hard (правильный вариант)

Перевод: Не обязательно быть фанатичным любителем книг, чтобы ценить чтение.

Объяснение: "Die-hard" означает "преданный до фанатизма".

1. There are some books I read cover-to-cover, staying up into the early hours. ✅ cover-to-cover (правильный вариант)

Перевод: Есть книги, которые я читаю от корки до корки, засиживаясь до рассвета.

Объяснение: "Cover-to-cover" означает "полностью, от начала до конца".

1. When you get a well-earned break it’s good to curl up with a book for a while and chill. ✅ curl up (правильный вариант)

Перевод: Когда у тебя есть заслуженный перерыв, здорово свернуться калачиком с книгой и расслабиться.

Объяснение: "Curl up" описывает удобную позу для чтения (например, в кресле или на диване).

Reviewer #1 (Public review):

Summary:

Pradhan et al investigated the potential gustatory mechanisms that allow flies to detect cholesterol. They found that flies are indifferent to low cholesterol and avoid high cholesterol. They further showed that the ionotropic receptors Ir7g, Ir51b, and Ir56d are important for the cholesterol sensitivity in bitter neurons. The figures are clear and the behavior result is interesting. However, I have several major comments, especially on the discrepancy of the expression of these Irs with other lab published results, and the confusing finding that the same receptors (Ir7g, Ir51b) have been implicated in the detection of various seemingly unrelated compounds.

Strengths:

The results are very well presented, the figures are clear and well-made, text is easy to follow.

Weaknesses:

(1) Regarding the expression of Ir56d. The reported Ir56d expression pattern contradicts multiple previous studies (Brown et al., 2021 eLife, Figure 6a-c; Sanchez-Alcaniz et al., 2017 Nature Communications, Figure 4e-h; Koh et al., 2014 Neuron, Figure 3b). These studies, using three different driver lines, consistently showed Ir56d expression in sweet-sensing neurons and taste peg neurons. Importantly, Sanchez-Alcaniz et al. demonstrated that Ir56d is not expressed in Gr66a-expressing (bitter) neurons. This discrepancy is critical since Ir56d is identified as the key subunit for cholesterol detection in bitter neurons, and misexpression of Ir7g and Ir51b together is insufficient to confer cholesterol sensitivity (Fig.4b,d). Which Ir56d-GAL4 (and Gr66a-I-GFP) line was used in this study? Is there additional evidence (scRNA sequencing, in-situ hybridization, or immunostaining) supporting Ir56d expression in bitter neurons?

(2) Ir51b has previously been implicated in detecting nitrogenous waste (Dhakal 2021), lactic acid (Pradhan 2024), and amino acids (Aryal 2022), all by the same lab. Additionally, both Ir7g and Ir51b have been implicated in detecting cantharidin, an insect-secreted compound that flies may or may not encounter in the wild, by the same lab. Is Ir51b proposed to be a specific receptor for these chemically distinct compounds or a general multimodal receptor for aversive stimuli? Unlike other multimodal bitter receptors, the expression level of Ir51b is rather low and it's unclear which subset of GRNs express this receptor. The chemical diversity among nitrogenous waste, amino acids, lactic acid, cantharidin, and cholesterol raises questions about the specificity of these receptors and warrants further investigation and at a minimum discussion in this paper. Given the wide and seemingly unrelated sensitivity of Ir51b and Ir7g to these compounds I'm leaning towards the hypothesis that at least some of these is non-specific and ecologically irrelevant without further supporting evidence from the authors.

(3) The Benton lab Ir7g-GAL4 reporter shows no expression in adults. Additionally, two independent labellar RNA sequencing studies (Dweck, 2021 eLife; Bontonou et al., 2024 Nature Communications) failed to detect Ir7g expression in the labellum. This contradicts the authors' previous RT-PCR results (Pradhan 2024 Fig. S4, Journal of Hazardous Materials) showing Ir7g expression in the labellum. Additionally the Benton and Carlson lab Ir51b-GAL4 reporters show no expression in adults as well. Please address these inconsistencies.

(4) The premise that high cholesterol intake is harmful to flies, which makes sensory mechanisms for cholesterol avoidance necessary, is interesting but underdeveloped. Animal sensory systems typically evolve to detect ecologically relevant stimuli with dynamic ranges matching environmental conditions. Given that Drosophila primarily consume fruits and plant matter (which contain minimal cholesterol) rather than animal-derived foods (which contain higher cholesterol), the ecological relevance of cholesterol detection requires more thorough discussion. Furthermore, at high concentrations, chemicals often activate multiple receptors beyond those specifically evolved for their detection. If the cholesterol concentrations used in this study substantially exceed those encountered in the fly's natural diet, the observed responses may represent an epiphenomenon rather than an ecologically and ethologically relevant sensory mechanism. What is the cholesterol content in flies' diet and how does that compare to the concentrations used in this paper?

Reviewer #2 (Public review):

Summary:

In Cholesterol Taste Avoidance in Drosophila melanogaster, Pradhan et al. used behavioral and electrophysiological assays to demonstrate that flies can: (1) detect cholesterol through a subset of bitter-sensing gustatory receptor neurons (GRNs) and (2) avoid consuming food with high cholesterol levels. Mechanistically, they identified five members of the IR family as necessary for cholesterol detection in GRNs and for the corresponding avoidance behavior. Ectopic expression experiments further suggested that Ir7g + Ir56d or Ir51b + Ir56d may function as tuning receptors for cholesterol detection, together with the Ir25a and Ir76b co-receptors.

Strengths:

The experimental design of this study was logical and straightforward. Leveraging their expertise in the Drosophila taste system, the research team identified the molecular and cellular basis of a previously unrecognized taste category, expanding our understanding of gustation. A key strength of the study was its combination of electrophysiological recordings with behavioral genetic experiments.

Weaknesses:

My primary concern with this study is the lack of a systematic survey of the IRs of interest in the labellum GRNs. Consequently, there is no direct evidence linking the expression of putative cholesterol IRs to the B GRNs in the S6 and S7 sensilla.

Specifically, the authors need to demonstrate that the IR expression pattern explains cholesterol sensitivity in the B GRNs of S6 and S7 sensilla, but not in other sensilla. Instead of providing direct IR expression data for all candidate IRs (as shown for Ir56d in Figure 2-figure supplement 1F), the authors rely on citations from several studies (Lee, Poudel et al. 2018; Dhakal, Sang et al. 2021; Pradhan, Shrestha et al. 2024) to support their claim that Ir7g, Ir25a, Ir51b, and Ir76b are expressed in B GRNs (Lines 192-194). However, none of these studies provide GAL4 expression or in situ hybridization data to substantiate this claim.

Without a comprehensive IR expression profile for GRNs across all taste sensilla, it is difficult to interpret the ectopic expression results observed in the B GRN of the I9 sensillum or the A GRN of the L-sensillum (Figure 4). It remains equally plausible that other tuning IRs-beyond the co-receptor Ir25a and Ir76b-could interact with the ectopically expressed IRs to confer cholesterol sensitivity, rather than the proposed Ir7g + Ir56d or Ir51b + Ir56d combinations.

Reviewer #3 (Public review):

Summary:

Whether and how animals can taste cholesterol is not well understood. The study provides evidence that 1) cholesterol activates a subset of bitter-sensing gustatory receptor neurons (GRNs) in the fly labellum, but not other types of GRNs, 2) flies show aversion to high concentrations of cholesterol, and this is mediated by bitter GRNs, and 3) cholesterol avoidance depends on a specific set of ionotropic receptor (IR) subunits acting in bitter GRNs. The claims of the study are supported by electrophysiological recordings, genetic manipulations, and behavioral readouts.

Strengths:

Cholesterol taste has not been well studied, and the paper provides new insight into this question. The authors took a comprehensive and rigorous approach in several different parts of the paper, including screening the responses of all 31 labellar sensilla, screening a large panel of receptor mutants, and performing misexpression experiments with nearly every combination of the 5 IRs identified. The effects of the genetic manipulations are very clear and the results of electrophysiological and behavioral studies match nicely, for the most part. The appropriate controls are performed for all genetic manipulations.

Weaknesses:

The weaknesses of the study, described below, are relatively minor and do not detract from the main conclusions of the paper.

(1) The paper does not state what concentrations of cholesterol are present in Drosophila's natural food sources. Are the authors testing concentrations that are ethologically relevant?

(2) The paper does not state or show whether the expression of IR7g, IR51b, and IR56d is confined to bitter GRNs. Bitter-specific expression of at least some of these receptors would be necessary to explain why bitter GRNs but not sugar GRNs (or other GRN types) normally show cholesterol responses.

(3) The authors only investigated the responses of GRNs in the labellum, but GRN responses in the leg may also contribute to the avoidance of cholesterol feeding. Alternatively, leg GRNs might contribute to cholesterol attraction that is unmasked when bitter GRNs are silenced. In support of this possibility, Ahn et al. (2017) showed that Ir56d functions in sugar GRNs of the leg to promote appetitive responses to fatty acids.

(4) The authors might consider using proboscis extension as an additional readout of taste attraction or aversion, which would help them more directly link the labellar GRN responses to a behavioral readout. Using food ingestion as a readout can conflate the contribution of taste with post-ingestive effects, and the regulation of food ingestion also may involve contributions from GRNs on multiple organs, whereas organ-specific contributions can be dissociated using proboscis extension. For example, does presenting cholesterol on the proboscis lead to aversive responses in the proboscis extension assay (e.g., suppression of responses to sugar)? Does this aversion switch to attraction when bitter GRNs are silenced, as with the feeding assay?

(5) The authors claim that the cholesterol receptor is composed of IR25a, IR76b, IR56d, and either IR7g or IR51b. While the authors have shown that IR25a and IR76b are each required for cholesterol sensing, they did not show that both are required components of the same receptor complex. If the authors are relying on previous studies to make this assumption, they should state this more clearly. Otherwise, I think further misexpression experiments may be needed where only IR25a or IR76b, but not both, are expressed in GRNs.

What do you think about this?

Reviewer #3 (Public review):

Hon et al. investigated the role of BNST CRF signaling in modulating phasic and sustained fear in male and female mice. They found that partial and full fear conditioning had similar effects in both sexes during conditioning and during recall. However, males in the partially reinforced fear conditioning group showed enhanced acoustic startle, compared to the fully reinforced fear conditioning group, an effect not seen in females. Using fiber photometry to record calcium activity in all BNST neurons, the authors show that the BNST was responsive to foot shock in both sexes and both conditioning groups. Shock response increased over the session in males in the fully conditioned fear group, an effect not observed in the partially conditioned fear group. This effect was not observed in females. Additionally, tone onset resulted in increased BNST activity in both male groups, with the tone response increasing over time in the fully conditioned fear group. This effect was less pronounced in females, with partially conditioned females exhibiting a larger BNST response. During recall in males, BNST activity was suppressed below baseline during tone presentations and was significantly greater in the partially conditioned fear group. Both female groups showed an enhanced BNST response to the tone that slowly decayed over time. Next, they knocked CRF in the BNST to examine its effect on fear conditioning, recall and anxiety-like behavior after fear. They found no effect of the knockdown in either sex or group during fear conditioning. During fear recall, BNST CRF knockdown lead to an increase in freezing in only the partially conditioned females. In the anxiety-like behavior tasks, BNST CRF knockdown lead to increased anxiolysis in the partially reinforced fear male, but not in females. Surprisingly, BNST CRF knockdown increased startle response in fully conditioned, but not partially conditioned males. An effect not observed in either female group. In a final set of experiments, the authors single photon calcium imaging to record BNST CRF cell activity during fear conditioning and recall. Approximately, 1/3 of BNST CRF cells were excited by shock in both sexes, with the rest inhibited and no differences were observed between sexes or group during fear conditioning. During recall, BNST CRF activity decreased in both sexes, an effect pronounced in male and female fully conditioned fear groups.

Overall, these data provide novel, intriguing evidence in how BNST CRF neurons may encode phasic and sustained fear differentially in males and females. The experiments were rigorous. My biggest concerns I have regard the interpretations and some conclusions from this data set, which I have stated below.

(1) It was surprising to see minimal and somewhat conflicting behavioral effects due to BNST CRF knockdown. The authors provide a representative image and address this in the conclusion. They mention the role of local vs projection CRF circuits as well as the role of GABA. I don't think those experiments are necessary for this manuscript. However, it may be worthwhile to see through in situ hybridization or IHC, to see BNST CRF levels after both full and partial conditioned fear paradigms. Additionally, it would help to see a quantification of the knockdown of the animals. The authors can add a figure showing deltaF/F changes from control.

(2) Related to the previous point, it was surprising to see an effect of the CRF deletion in the full fear group compared to the partial fear in the acoustic startle task. To strengthen the conclusion about differential recruitment of CRF during phasic and sustained fear, the experiment in my previous point could help elucidate that. Conversely, intra-BNST administration of a CRF antagonist into the BNST before the acoustic startle after both conditioning tasks could also help. Or patch from BNST CRF neurons after the conditioning tasks to measure intrinsic excitability. Not all these experiments are needed to support the conclusion, it's some examples.

(3) In Figure 5 F and K, the authors report data combined for both part and full fear conditioning. Were there any differences between the number of excited or inhibited neurons b/t the conditioning groups? Also, can the authors separate male and female traces in Fig 5 E and P?

(4) Also, regarding the calcium imaging data, what was the average length of a transient induced by shock? Were there any differences between the sexes?

Author response:

The following is the authors’ response to the original reviews

Public Reviews:

Reviewer #1 (Public Review):

The aim of this study is to test the overarching hypothesis that plasticity in BNST CRF neurons drives distinct behavioral responses to unpredictable threat in males and females. The manuscript provides evidence for a possible sex-specific role for CRF-expressing neurons in the BNST in unpredictable aversive conditioning and subsequent hypervigilance across sexes. As the authors note, this is an important question given the high prevalence of sex differences in stress-related disorders, like PTSD, and the role of hypervigilance and avoidance behaviors in these conditions. The study includes in vivo manipulation, bulk calcium imaging, and cellular resolution calcium imaging, which yield important insights into cell-type specific activity patterns. However, it is difficult to generate an overall conclusion from this manuscript, given that many of the results are inconsistent across sexes and across tests and there is an overall lack of converging evidence. For example, partial conditioning yields increased startle in males but not females, yet, CRF KO only increases startle response in males after full conditioning, not partial, and CRF neurons show similar activity patterns between partial and full conditioning across sexes. Further, while the study includes a KO of CRF, it does not directly address the stated aim of assessing whether plasticity in CRF neurons drives the subsequent behavioral effects unpredictable threat.

We appreciate the reviewer’s summary and agree that there is a large amount of complexity to the results, and that it was difficult to generate a simple model/conclusion to summarize our work. This is the unfortunate side effect of looking across both sexes at different conditioning paradigms, however, we believe that it is important to convey this information to the field even without a simple answer.  Our data reinforces the very important findings from the Maren and Holmes groups that partial fear is a different process than full fear, and that the BNST plays a differential role here. We have reworded the manuscript to better convey this complexity.

A major strength of this manuscript is the inclusion of both males and females and attention to possible behavioral and neurobiological differences between them throughout. However, to properly assess sex-differences, sex should be included as a factor in ANOVA (e.g. for freezing, startle, and feeding data in Figure 1) to assess whether there is a significant main effect or interaction with sex. If sex is not a statistically significant factor, both sexes should be combined for subsequent analyses. See, Garcia-Sifuentes and Maney, eLife 2021 https://elifesciences.org/articles/70817. There are additional cases where t-tests are used to compare groups when repeated measures ANOVAs would be more appropriate and rigorous.

We agree with the reviewer that this is the more appropriate analysis and have changed the analysis and figures throughout the revised manuscript to better assess sex differences as well as differences between fear conditions.

Additionally, it's unclear whether the two sexes are equally responsive to the shock during conditioning and if this is underlying some of the differences in behavioral and neuronal effects observed. There are some reports that suggest shock sensitivity differs across sexes in rodents, and thus, using a standard shock intensity for both males and females may be confounding effects in this study.

This is a great point. We have conducted appropriate analysis (Sex by Tone Repeated measures two-way ANOVAS for each of the groups: Ctrl, Full, Part) and there are no sex differences in freezing between males and females. The extent of conditioning is not different between the groups suggesting that if there was a difference in shock sensitivity, it is not driving any discernible differences in behavioral performance. However, it is possible that the experience of the shock differs for the animals even in the absence of any measurable behavior.

The data does not rule out that BNST CRF activity is not purely tracking the mobility state of the animal, given that the differences in activity also track with differences in freezing behavior. The data shows an inverse relationship between activity and freezing. This may explain a paradox in the data which is why males show a greater suppression of BNST activity after partial conditioning than full conditioning, if that activity is suspected to drive the increased anxiety-like response. Perhaps it reflects that activity is significantly suppressed at the end of the conditioning session because animals are likely to be continuously freezing after repeated shock presentations in that context. It would also explain why there is less of a suppression in activity over the course of the recall session, because there is less freezing as well during recall compared with conditioning.

While it is possible that the BNST may be tracking activity, we believe it is not purely tracking mobility state. For instance, while freezing increases across tone exposures in Part fear regardless of sex, males show an increase while females show a reduction in BNST response during tone 5 (Fig 2K). The data the reviewer refers to showing the inverse relationship with BNST activity and freezing would have suggested the opposite response if it were purely tracking the mobility state of the animal. This is also the case with BNST^CRF activity to first and last tone during recall. Despite the suppression of activity over the course of recall (Fig 5K), we see an increase in BNST^CRF tone response when comparing tone 1 and 6 in males and a decrease in females (Fig 6M), again suggesting the BNST is responding to more than just activity.

A mechanistic hypothesis linking BNST CRF neurons, the behavioral effects observed after fear conditioning, and manipulation of CRF itself are not clearly addressed here.

We disagree with this assertion. The data suggests a model in which males respond with increased arousal and Part fear males show persistent activation of the BNST and BNST^CRF neurons during fear conditioning and recall while female Part fear mice show the opposite response. This female response differs from what the field believes to be the role of the BNST in sustained fear. Additionally, we show that CRF knockdown is not involved in fear differentiation or fear expression in males, while it enhances fear learning and recall in females. We have reworded the manuscript to highlight these novel findings.

Reviewer #2 (Public Review):

This study examined the role of CRF neurons in the BNST in both phasic and sustained fear in males and females. The authors first established a differential fear paradigm whereby shocks were consistently paired with tones (Full) or only paired with tones 50% of the time (Part), or controls who were exposed to only tones with no shocks. Recall tests established that both Full and Part conditioned male and female mice froze to the tones, with no difference between the paradigms. Additional studies using the NSF and startle test, established that neither fear paradigm produced behavioral changes in the NSF test, suggesting that these fear paradigms do not result in an increase in anxiety-like behavior. Part fear conditioning, but not Full, did enhance startle responses in males but not females, suggesting that this fear paradigm did produce sustained increases in hypervigilance in males exclusively.

Thank you for this clear summary of the behavioral work.

Photometry studies found that while undifferentiated BNST neurons all responded to shock itself, only Full conditioning in males lead to a progressive enhancement of the magnitude of this response. BNST neurons in males, but not females, were also responsive to tone onset in both fear paradigms, but only in Full fear did the magnitude of this response increase across training. Knockdown of CRF from the BNST had no effect on fear learning in males or females, nor any effect in males on fear recall in either paradigm, but in females enhanced both baseline and tone-induced freezing only in Part fear group. When looking at anxiety following fear training, it was found in males that CRF knockdown modulated anxiety in Part fear trained animals and amplified startle in Fully trained males but had no effect in either test in females. Using 1P imaging, it was found that CRF neurons in the BNST generally decline in activity across both conditioning and recall trials, with some subtle sex differences emerging in the Part fear trained animals in that in females BNST CRF neurons were inhibited after both shock and omission trials but in males this only occurred after shock and not omission trials. In recall trials, CRF BNST neuron activity remained higher in Part conditioned mice relative to Full conditioned mice.

Overall, this is a very detailed and complex study that incorporates both differing fear training paradigms and males and females, as well as a suite of both state of the art imaging techniques and gene knockdown approaches to isolate the role and contributions of CRF neurons in the BNST to these behavioral phenomena. The strengths of this study come from the thorough approach that the authors have taken, which in turn helped to elucidate nuanced and sex specific roles of these neurons in the BNST to differing aspects of phasic and sustained fear. More so, the methods employed provide a strong degree of cellular resolution for CRF neurons in the BNST. In general, the conclusions appropriately follow the data, although the authors do tend to minimize some of the inconsistencies across studies (discussed in more depth below), which could be better addressed through discussion of these in greater depth. As such, the primary weakness of this manuscript comes largely from the discussion and interpretation of mixed findings without a level of detail and nuance that reflects the complexity, and somewhat inconsistency, across the studies. These points are detailed below:

- Given the focus on CRF neurons in the BNST, it is unclear why the photometry studies were performed in undifferentiated BNST neurons as opposed to CRF neurons specifically (although this is addressed, to some degree, subsequently with the 1P studies in CRF neurons directly). This does limit the continuity of the data from the photometry studies to the subsequent knockdown and 1P imaging studies. The authors should address the rationale for this approach so it is clear why they have moved from broader to more refined approaches.

The reviewer raises a good point.  We did some preliminary photometry studies with BNST CRF neurons and found that there was poor time locked signal. We reasoned that this was due to the heterogeneity of the cell activity, as we saw in our previous publication (Yu et al). Because of this, we moved to the 1p imaging work in place of continued BNST CRF photometry. We have also reworded the manuscript to better discuss the complexities and inconsistencies in findings across the studies.

- The CRF KD studies are interesting, but it remains speculative as to whether these effects are mediated locally in the BNST or due to CRF signaling at downstream targets. As the literature on local pharmacological manipulation of CRF signaling within the BNST seems to be largely performed in males, the addition of pharmacological studies here would benefit this to help to resolve if these changes are indeed mediated by local impairments in CRF release within the BNST or not. While it is not essential to add these experiments, the manuscript would benefit from a more clear description of what pharmacological studies could be performed to resolve this issue.

We agree with the reviewer that the addition of this experiment would be highly informative for differentiating the role of CRF in the BNST. This is something that will need to be considered moving forward and we have added this as a point of discussion.

- While I can appreciate the authors perspective, I think it is more appropriate to state that startle correlates with anxiety as opposed to outright stating that startle IS anxiety. Anxiety by definition is a behavioral cluster involving many outputs, of which avoidance behavior is key. Startle, like autonomic activation, correlates with anxiety but is not the same thing as a behavioral state of anxiety (particularly when the startle response dissociates from behavior in the NSF test, which more directly tests avoidance and apprehension). Throughout the manuscript the use of anxiety or vigilance to describe startle becomes interchangeable, but then the authors also dissociate these two, such as in the first paragraph of the discussion when stating that the Part fear paradigm produces hypervigilance in males without influencing fear or anxiety-like behaviors. The manuscript would benefit from harmonization of the language used to operationally define these behaviors and my recommendation would be to remain consistent with the description that startle represents hypervigilance and not anxiety, per se.

The reviewer raises an excellent point, we have clarified in the revised manuscript.

- The interpretation of the anxiety data following CRF KD is somewhat confusing. First, while the authors found no effect of fear training on behavior in the NSF test in the initial studies, now they do, however somewhat contradictory to what one would expect they found that Full fear trained males had reduced latency to feed (indicative of an anxiolytic response), which was unaltered by CRF KD, but in Part fear (which appeared to have no effect on its own in the NSF test), KD of CRF in these animals produced an anxiolytic effect. Given that the Part fear group was no different from control here it is difficult to interpret these data as now CRF KD does reduce latency to feed in this group, suggesting that removal of CRF now somehow conveys an anxiolytic response for Part fear animals. In the discussion the authors refer to this outcome as CRF KD "normalizing" the behavior in the NSF test of Part fear conditioned animals as now it parallels what is seen after Full fear, but given that the Part fear animals with GFP were no different then controls (and neither of these fear training paradigms produced any effect in the NSF test in the first arm of studies), it seems inappropriate to refer to this as "normalization" as it is unclear how this is now normalized. Given the complexity of these behavioral data, some greater depth in the discussion is required to put these data in context and describe the nuance of these outcomes, in particular a discussion of possible experimental factors between the initial behavioral studies and those in the CRF KD arm that could explain the discrepancy in the NSF test would be good (such as the inclusion of surgery, or other factors that may have differed between these experiments). These behavioral outcomes are even more complex given that the opposite effect was found in startle whereby CRF KD amplified startle in Full trained animals. As such, this portion of the discussion requires some reworking to more adequately address the complexity of these behavioral findings.

The reviewer raises a good point, and we agree that there are many inconsistencies in the behaviors. We believe it is still good to show these results but have expanded the manuscript on potential reasons for these behavioral inconsistencies.

Reviewer #3 (Public Review):

Hon et al. investigated the role of BNST CRF signaling in modulating phasic and sustained fear in male and female mice. They found that partial and full fear conditioning had similar effects in both sexes during conditioning and during recall. However, males in the partially reinforced fear conditioning group showed enhanced acoustic startle, compared to the fully reinforced fear conditioning group, an effect not seen in females. Using fiber photometry to record calcium activity in all BNST neurons, the authors show that the BNST was responsive to foot shock in both sexes and both conditioning groups. Shock response increased over the session in males in the fully conditioned fear group, an effect not observed in the partially conditioned fear group. This effect was not observed in females. Additionally, tone onset resulted in increased BNST activity in both male groups, with the tone response increasing over time in the fully conditioned fear group. This effect was less pronounced in females, with partially conditioned females exhibiting a larger BNST response. During recall in males, BNST activity was suppressed below baseline during tone presentations and was significantly greater in the partially conditioned fear group. Both female groups showed an enhanced BNST response to the tone that slowly decayed over time. Next, they knocked CRF in the BNST to examine its effect on fear conditioning, recall and anxiety-like behavior after fear. They found no effect of the knockdown in either sex or group during fear conditioning. During fear recall, BNST CRF knockdown lead to an increase in freezing in only the partially conditioned females. In the anxiety-like behavior tasks, BNST CRF knockdown lead to increased anxiolysis in the partially reinforced fear male, but not in females. Surprisingly, BNST CRF knockdown increased startle response in fully conditioned, but not partially conditioned males. An effect not observed in either female group. In a final set of experiments, the authors single photon calcium imaging to record BNST CRF cell activity during fear conditioning and recall. Approximately, 1/3 of BNST CRF cells were excited by shock in both sexes, with the rest inhibited and no differences were observed between sexes or group during fear conditioning. During recall, BNST CRF activity decreased in both sexes, an effect pronounced in male and female fully conditioned fear groups.

Overall, these data provide novel, intriguing evidence in how BNST CRF neurons may encode phasic and sustained fear differentially in males and females. The experiments were rigorous.

We thank you for this positive review of our manuscript.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

There are several graphs representing different analyses of (presumably) the same group of subjects, but which have different N/group. For example, in Figure 2:

(1) Fig 2P seems to have n=10 in Part Male group (Peak), but 2Q only has n=9 in Part Male group (AUC)

(2) Fig 2S seems to have n=10 in Part Female group (Peak), but 2T only has n=7 in Part Female group (AUC)

(3) Fig 2G (Tone Resp) has n=6 Full Males but 2F (Tone Resp), 2H (Shock Resp), and 2I (Shock Resp) have n=7 Full Males

(4) Fig 2K (Tone Resp) has n=7 Full Females but 2L (Tone Resp), 2M (Shock Resp), and 2N (Shock Resp) have n=8 Full Females

(5) Fig 2L (Tone Resp) has n=9 Part Females but 2K (Tone Resp), 2M (Shock Resp), and 2N (Shock Resp) have n=10 Part Females

It's possible that this is just due to overlapping individual data points which are made harder to see due to the low resolution of the figures. If so, this can be easily rectified. However, there may also be subjects missing from some analyses which must be clarified or corrected.

We thank you for catching these. We have gone through and fixed any issues with data points and have added statistics and exclusions in datasets to figure legends to further explain inconsistencies.

Regarding statistical tests:

(2) Data in Figs 2G and 2I should be analyzed using a two-way RM ANOVA.

We have now included sex as a factor in most of our analysis and are now using appropriate statistical tests.

(3) Data in Fig 3K should be analyzed using a two-way RM ANOVA.

We are now using appropriate statistical tests.

Calcium activity in response to the shock during conditioning and in response to the tone during recall should be included in Figure 5. Given partial and full animals also receive unequal presentations of the cue, it would be useful to see the effects trial by trial or normalized to the first 3 presentations only.

The reviewer raises a great point. We have changed this figure and have now added the response to shock and tones. Since we are most interested in the difference between sustained and phasic fear, we decided to compare tone 3 in Full fear and tone 4 in Part fear, which differ in the ambiguity of their cue and only have one tone difference.

Histology maps should be included for all experiments depicting viral spread and implant location for all animals, in addition to the included representative histology images. These can be placed in the supplement.

We agree this is helpful. While we have confirmed all of the experiments are hits, the tissue is no longer in condition for this analysis.

Referring to the quantification of peaks in fiber photometry and cellular resolution calcium imaging data as "spikes" is a bit misleading given the inexact relationship between GCAMP sensor dynamics/calcium binding and neuronal action potentials, perhaps calling it "event" frequency would be more clear.

We have changed the references of spikes to events as suggested.

The legend for Figure 2S is mislabeled as A.

Thank you for catching this mistake, it has been fixed.

The methods refer to CRFR1 fl/fl animals but it seems no experiments used these animals, only CRF fl/fl.

We have fixed this, thank you.

Reviewer #2 (Recommendations For The Authors):

As stated in the public review, while I think the addition of local pharmacological studies blocking CRF1 and 2 receptors in the BNST in both males and females, done under the same conditions as all of the other testing herein, would help to resolve some of the speculation of interpreting the CRF KD data, I dont think these studies are essential to do, but it would be good for the authors to more explicitly state what studies could be done and how they could facilitate interpretation of these data.

Thank you for this suggestion. We have added this discussion into the manuscript.

Asides from this, my other recommendations for the authors are to more clearly address the discrepancies in behavioral outcomes across studies and explicitly describe their rationale for the sequence of experiments performed and to harmonize their operationalization of how they define anxiety.