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congealed into those same bodies.

English Explanation:

The phrase "congealed into those same bodies" refers to a process where a substance changes from a fluid or gaseous state into a solid or more stable form. In this context, it suggests that various elements or materials that were once in a more fluid state have now solidified or formed a conforming shape, which could represent literal bodies (like physical objects) or metaphorical ones (like identity, culture, or other constructs). The use of the term "those same bodies" implies that there is a continuity or connection to previously mentioned entities or forms, indicating a transformation that is significant in its context.

Chinese Explanation:

“凝固成那些相同的身体”这个短语描述了一种物质从流体或气体状态转变为固体或更稳定形态的过程。在这个背景下，它暗示着曾经处于更流动状态的各种元素或材料现在已经固化或形成了一个符合形状的状态，这可能代表字面上的身体（如物理对象）或隐喻上的身体（如身份、文化或其他构建）。使用“那些相同的身体”这个短语，暗示了与之前提到的实体或形式之间的延续性或联系，这表明在其背景下的转变是重要的。

peppered freely through the sea;

English Explanation:

The phrase "peppered freely through the sea" suggests a scattering or distribution of something throughout the sea. The use of the word "peppered" implies that these elements are spread out in a way that might resemble how pepper is sprinkled over food—non-uniformly and in different quantities. This imagery evokes a sense of randomness and diversity, indicating that whatever is being described (perhaps objects, creatures, or particles) is not concentrated in one area but rather dispersed widely across the marine environment. The phrase conveys a vivid picture of variety and abundance in the sea, encouraging readers to imagine a dynamic and enriched aquatic setting.

Chinese Explanation:

“peppered freely through the sea”这个短语暗示某种东西在海洋中散布或分布。使用“peppered”这个词意味着这些元素以一种可能类似于将胡椒撒在食物上的方式被散布——不均匀地、数量不同。这种意象唤起了一种随机性和多样性的感觉，表明所描述的事物（或许是物体、生物或颗粒）并不是集中在一个区域，而是广泛分散在海洋环境中。这个短语传达了一幅丰富多彩的海洋画面，鼓励读者想象一个充满活力和丰富的水下世界。

become bundled up.

English Explanation

The phrase "become bundled up" typically means to wrap oneself in layers of clothing for warmth. It suggests that someone is dressing in a way that protects them from cold weather, often involving multiple pieces of clothing such as jackets, scarves, hats, and gloves. The imagery conveys a sense of preparation for cold conditions, emphasizing comfort and protection from the elements.

In a broader context, "bundled up" can also refer to being grouped together, whether representing people, objects, or ideas. When referring to people, it may indicate a sense of camaraderie or being close-knit, while in the context of objects or ideas, it can suggest a collection or assembly.

Chinese Explanation

“become bundled up”这个短语通常意味着穿上多层衣物以保持温暖。它暗示着某人在寒冷的天气中穿着，以保护自己，通常涉及多件衣服，如夹克、围巾、帽子和手套。这个形象传达了一种为寒冷天气做好准备的感觉，强调了舒适和保护免受恶劣天气的影响。

在更广泛的背景下，“bundled up”也可以指将某些东西聚集在一起，无论是代表人、物品还是思想。当指人时，它可能表示一种友情或紧密团结的感觉，而在物品或思想的上下文中，它可以表示一种集合或汇编。

criss-crossed

The term "criss-crossed" typically describes a pattern or movement where lines or paths intersect with each other in a crisscross fashion. This can refer to physical objects, such as strings or roads, that cross over one another at various angles, creating an intricate arrangement. In a broader sense, it can also describe complex relationships, interactions, or ideas that intertwine, overlap, or influence each other.

English Explanation

1. Pattern: "Criss-crossed" can refer to a geometric or visual pattern where lines cross each other. For example, a piece of fabric may have a criss-cross pattern that gives it a unique texture.

2. Movement: It can also describe movement itself. For example, if someone is walking in a garden where the paths criss-cross, it means they are navigating through paths that intersect in various directions.

3. Metaphorical Use: Beyond the physical, it can have metaphorical implications, such as relationships or narratives that intertwine, representing complexity in interactions or connections among different entities.

4. Examples:

5. In a sewing design, yarn can be criss-crossed to create visual aesthetics.
6. In traffic patterns, roads can criss-cross each other, leading to more intricate navigation.

中文解释

“交错”这个词通常描述的是一种图案或运动，其中线条或路径以交错的方式相互交叉。这可以指代物理对象，比如绳子或道路，它们在不同的角度交叉，从而形成复杂的排列。更广泛地说，它也可以描述复杂的关系、互动或思想，这些关系、互动或思想相互交织、重叠或影响彼此。

1. 图案：“交错”可以指一种几何或视觉图案，其中线条相互交叉。例如，一块布料可能具有交错的图案，使其具有独特的纹理。

2. 运动：它也可以描述运动本身。例如，如果某人在一个花园里走路，而小路是交错的，这意味着他们正在穿越相互交叉的路径。

3. 比喻用法：除了物理意义外，它还可以具有比喻意义，例如关系或叙述交织在一起，代表不同实体之间互动或连接的复杂性。

4. 例子：

5. 在缝纫设计中，纱线可以交错以创造视觉美感。
6. 在交通模式中，道路可能会交错在一起，导致更复杂的导航。

This comprehensive understanding highlights both the literal and metaphorical uses of "criss-crossed" in various contexts.

salient

English Explanation

The term "salient" functions as an adjective and has several meanings depending on the context in which it is used. Generally, "salient" refers to something that stands out or is prominent; it can denote the most noticeable or important aspects of something.

1. In General Use: When describing a feature, characteristic, or point, "salient" indicates that it attracts attention or is particularly significant. For instance, you might say, "The salient features of the proposal include its cost-effectiveness and sustainability."

2. In Geography: In geographical terms, a "salient" can refer to a landform that juts out from the main body of land or territory, such as a peninsula.

3. In Psychology: The term can also relate to the way certain stimuli stand out in perception, making them more likely to be noticed or remembered.

In summary, "salient" emphasizes something that is conspicuous or noteworthy and is used in various fields to highlight key points or features.

Chinese Explanation

“salient” 这个词作为形容词，依使用的语境有几种含义。一般来说，“salient”指的是某物突出的、显著的；它可以表示某事物中最显眼或最重要的方面。

1. 一般用法：当描述某个特征、特性或观点时，“salient”表示引起注意或特别重要的。例如，可以说：“该提案的显著特征包括其成本效益和可持续性。”

2. 地理学上的用法：在地理学中，“salient”可以指突出的地形，例如从主体陆地或领土突出的半岛。

3. 心理学上的用法：该词也可以与某些刺激在感知中如何突出来有关，使这些刺激更容易被注意或记住。

总之，“salient” 强调的是突出的或值得注意的事物，在各个领域中用来突出关键点或特征。

predominate

Certainly! Let’s break down the excerpt “predominate” in both English and Chinese.

English Explanation:

The term "predominate" is a verb that means to be the strongest or main element in a particular context or situation. It implies that something is prevailing or has a greater influence, presence, or importance over other elements. For example, in a discussion about natural resources, water might predominate as the most crucial resource, meaning it's more essential or abundant compared to others.

In usage: - In a sentence: "In the debate about climate change, scientific facts predominate over opinion." - This means that scientific facts have a more significant impact or influence in that debate than personal opinions.

Chinese Explanation:

“Predominate” 是一个动词，意思是在特定的环境或情况中，某个元素是最强大或主要的。这个词暗示某种事物占主导地位，或者它在其他元素中具有更大的影响力、存在感或重要性。例如，在谈论自然资源时，水可能是最重要的资源，这意味着它比其他资源更为基础或丰富。

在用法上： - 在一句话中：“在关于气候变化的辩论中，科学事实占主导地位，而不是个人观点。” - 这意味着在那场辩论中，科学事实比个人观点更具影响力。

Summary:

So, "predominate" indicates a state of dominance or superiority among various options or arguments, highlighting the most significant or influential aspect in a given scenario.

中文总结：

“Predominate” 指在不同选择或论点中占据主导或优越地位，强调在特定情境中最重要或最具影响力的方面。

by the random jostling of smaller particles.

English Explanation:

The excerpt "by the random jostling of smaller particles" refers to a phenomenon in which smaller particles are constantly moving and colliding with each other in an unpredictable or chaotic manner. This random movement causes the particles to push against one another, creating a kind of agitation or disturbance in their environment. The term "jostling" suggests a physical bumping or shaking, which can lead to various effects, such as the spreading of particles, mixing of substances, or even the transfer of energy between the particles. This concept is often relevant in fields like physics, chemistry, and material science, where the behavior of particles plays a crucial role in understanding larger systems and reactions.

Chinese Explanation:

这段摘录“由较小粒子的随机挤动”指的是一种现象，其中较小的粒子不断地以不可预测或混乱的方式移动并相互碰撞。这种随机运动导致粒子互相推挤，形成一种激动或扰动的状态。术语“挤动”暗示着物理上的碰撞或摇动，这可能会导致多种效应，例如粒子的扩散、物质的混合，甚至粒子之间能量的转移。这个概念在物理学、化学和材料科学等领域中往往是相关的，因为粒子的行为在理解更大系统和反应中起着至关重要的作用。

manifesto

English Explanation

A "manifesto" is a public declaration of intentions, motives, or views of an individual or group. Manifestos are often used in political contexts, as they outline the beliefs and goals of a political party or movement. They can also be found in artistic contexts, where they express the philosophy or direction of a particular art movement or style.

Typically, a manifesto is a written document that aims to inform, persuade, or mobilize the audience. It may articulate the principles and values that the authors stand for and call for action or change. Famous examples of manifestos include "The Communist Manifesto" by Karl Marx and Friedrich Engels, and the "Surrealist Manifesto" by André Breton, each representing significant social or artistic movements.

The key characteristics of manifestos include:

1. Clarity: The ideas should be presented clearly to ensure the audience understands the intentions.
2. Passion: A manifesto often reflects the emotional commitment of the authors towards their beliefs.
3. Call to Action: Many manifestos conclude with a call for change, urging the audience to take specific steps in response to the ideas presented.

In summary, a manifesto is an influential tool used to articulate and advocate for a set of beliefs, encouraging others to join in a cause or movement.

Chinese Explanation

“宣言”是个人或团体的意图、动机或观点的公开声明。宣言常用于政治环境中，因为它们概述了政党或运动的信念和目标。宣言在艺术背景下也能找到其身影，表达特定艺术运动或风格的哲学或方向。

通常，宣言是一份书面文件，旨在告知、说服或动员受众。它可能会阐明作者所信奉的原则和价值观，并呼吁采取行动或改变。著名的宣言例子包括卡尔·马克思和弗里德里希·恩格斯的《共产党宣言》，以及安德烈·布勒东的《超现实主义宣言》，它们分别代表了重要的社会或艺术运动。

宣言的主要特点包括：

1. 清晰性：思想应该清晰地表达，以确保受众理解意图。
2. 激情：宣言通常反映了作者对其信念的情感承诺。
3. 行动号召：许多宣言以呼吁改变结束，敦促受众根据所提出的观点采取具体措施。

总之，宣言是一个有影响力的工具，用于阐明和倡导一套信念，鼓励他人加入某一事业或运动。

be crammed into one chapter.

English Explanation:

The phrase "be crammed into one chapter" suggests that a large amount of information, ideas, or content is being forced or packed into a single chapter of a book or a document. This could imply that the chapter may feel overwhelming or overloaded because it contains too much material, making it difficult for the reader to absorb or understand everything clearly. The use of "crammed" indicates a lack of space or a sense of rush, where the chapter does not have the luxury of sufficient depth or detail that might be found in a more spread-out treatment of the subject.

Chinese Translation:

“被塞进一章”这个短语暗示着大量的信息、观点或内容被强行或紧凑地放入一本书或文件的单独一章中。这可能意味着这一章会感到令人难以承受或超负荷，因为它包含了过多的材料，这使得读者很难清晰地吸收或理解所有内容。“塞”这个词传达了一种空间不足或匆忙的感觉，暗示着这一章没有足够的深度或细节，而这些在对主题的更为分散的处理当中可能会出现。

ferry

English Explanation

The term "ferry" primarily refers to a type of boat or vessel used to transport people, vehicles, or goods across a body of water. Ferries are commonly operated on shorter routes where a bridge or tunnel may not exist, providing a crucial link between two points that are separated by water.

There are various types of ferries: 1. Passenger Ferries: These are designed to carry people from one location to another and may offer amenities like seating and restrooms. 2. Vehicle Ferries: These ferries can transport cars, trucks, and sometimes larger vehicles. Passengers can often stay in their vehicles during the crossing. 3. Cargo Ferries: Specialized vessels that are intended to carry goods, freight, and various types of cargo.

Ferries usually run on a set schedule and can operate on rivers, lakes, or seas. They are often a vital mode of transportation in areas with many islands or where bridges are impractical. The operation of ferries is an essential part of logistics and public transport in many regions.

Chinese Explanation (中文解释)

“渡船”主要指一种用于在水域之间运输人、车辆或货物的船只或船舶。渡船通常在较短的航线上运营，在一些不通桥或隧道的地方提供了一条重要的连接线路。

渡船有多种类型： 1. 客渡船：这些渡船专为将人从一个地方运输到另一个地方设计，通常会提供座位和卫生间等设施。 2. 车辆渡船：这类渡船可以运输汽车、卡车，有时也能容纳较大的车辆。乘客通常可以在过渡期间呆在自己的车辆内。 3. 货物渡船：专门设计用于运输货物、货运和各种类型的货物的船舶。

渡船通常按固定时间表运行，可以在河流、湖泊或海洋上运营。在有许多岛屿或桥梁不切实际的地方，渡船常常是重要的交通方式。渡船的运营在许多地区的物流和公共交通中扮演着重要角色。

cellular uniformity

Explanation in English:

Cellular Uniformity refers to the consistency or similarity in the structure and function of cells within a specific tissue, organism, or system. In biological contexts, cellular uniformity implies that the cells share similar characteristics, such as size, shape, and function. This is important for the proper functioning of tissues and organs.

Key Points:

1. Definition: Cellular uniformity means that the cells look and behave alike within a certain group.

2. Importance:

3. It can indicate a healthy state of tissue, where cells are functioning well together.

4. Lack of uniformity may suggest pathological conditions, such as cancer, where cells can be very dissimilar (heterogeneous).

5. Examples:

6. In muscle tissue, uniformity is essential to ensure that muscle contractions occur in a coordinated manner.
7. In skin tissues, uniformity helps maintain protective barriers and functions.

In summary, cellular uniformity is crucial for maintaining the overall integrity and functionality of biological systems.

Explanation in Chinese (中文解释):

细胞均匀性是指在特定组织、个体或系统中细胞的结构和功能的一致性或相似性。在生物学背景下，细胞均匀性意味着细胞在形态、大小和功能等特征上具有相似性。这对组织和器官的正常功能至关重要。

关键点:

1. 定义：细胞均匀性意味着在某个群体内，细胞的外观和行为相似。

2. 重要性：

3. 这可以表明组织的健康状态，细胞之间能够良好合作。

4. 缺乏均匀性可能表明病理状况，例如癌症，其中细胞可能非常不同（异质性）。

5. 示例：

6. 在肌肉组织中，均匀性对确保肌肉收缩协调进行是必要的。
7. 在皮肤组织中，均匀性有助于维持保护屏障和功能。

总之，细胞均匀性对于维持生物系统的整体完整性和功能至关重要。

scrupulously

English Explanation:

The term "scrupulously" is an adverb that derives from the adjective "scrupulous." It conveys a sense of meticulousness and conscientiousness in actions or behavior. When someone acts scrupulously, they do so with great attention to detail, moral integrity, and a commitment to doing what is right—even when it might be easier to cut corners or take shortcuts.

For example, a scrupulous accountant would ensure that all financial records are accurate and comply with legal standards, without omitting any details for convenience. Similarly, a person who is scrupulous in their personal relationships would treat others with fairness and respect, adhering to ethical principles.

In summary, being scrupulous implies a dedication to ethical standards and a careful, thorough approach in whatever one does.

Chinese Explanation (中文解释):

“scrupulously”是一个副词，源自形容词“scrupulous”。这个词表达了一种在行为或举止中极为细致和尽责的态度。当一个人以scrupulously的方式行事时，他们会高度关注细节，遵循道德原则，并承诺做正确的事情——即使这样做可能比省略某些程序或作捷径更为困难。

例如，一位尽责的会计师会确保所有财务记录都准确无误，并符合法律标准，而不是为了方便而忽略某些细节。类似地，在个人关系中尽责的人会公平地对待他人，遵循伦理原则。

总之，scrupulous的含义是对道德标准的奉献以及在所做的一切中采取谨慎、彻底的方法。

contrivances

English Explanation:

The term "contrivances" refers to devices, inventions, or means that are created or devised for a specific purpose. It can imply cleverness or ingenuity in the way something is constructed or arranged. This word can also carry a slightly negative connotation when it suggests that something is artificial, contrived, or overly complicated—essentially, a solution that might not seem natural or straightforward.

In literature or discussions, "contrivances" are often used to describe plot devices or mechanisms that are used to advance a narrative but may feel forced or unrealistic. For example, in a story, if a character suddenly finds a convenient tool to solve a problem right when needed, that might be considered a contrivance.

Chinese Explanation:

“Contrivances”这个词指的是为特定目的而创造或设计的设备、发明或手段。它可以暗示在构造或安排某事时的聪明才智。这个词在某些情况下也可能带有轻微的贬义，暗示某物是人工制作的、精心设计的，或者过于复杂——本质上是指一种可能不自然或不简单的解决方案。

在文学或讨论中，“contrivances”常用来描述推动叙事的情节装置或机制，但这些装置可能会感觉被强加或不现实。例如，在一个故事中，如果一个角色恰好在需要的时候找到了一个方便的工具来解决问题，这种情况可能被视为一种“contrivance”。

lineages

English Explanation

The term "lineages" refers to the sequences of lines or lines of descent from a common ancestor. In biological contexts, it describes the evolutionary history and relationships among various species, often indicated through phylogenetic trees. In human contexts, lineages can denote family trees, heritage, or the transmission of cultural practices and traditions. Each lineage can represent a distinct group that shares a common ancestor, showcasing how traits, genes, or cultural elements are passed down through generations.

Chinese Explanation

“血统”一词指的是从共同祖先出来的血缘关系或谱系。在生物学中，它描述的是不同物种之间的进化历史和关系，通常通过系统发生树来表示。在人类文化中，血统可以表示家谱、遗产或文化实践和传统的传承。每个血统可以代表一个独特的群体，分享着共同的祖先，展示了特征、基因或文化元素如何在世代之间传承。

well-tempered

English Explanation

The term "well-tempered" refers to a state of being balanced, composed, or stable, often used to describe a person’s temperament or personality. A well-tempered individual is typically calm, reasonable, and even-keeled, showing emotional stability in various situations. The concept can also extend to subjects like music or instruments, where "well-tempered" describes a tuning system that ensures harmonious sound across different keys, allowing for a wide range of musical expression.

In general, the phrase implies a level of moderation and thoughtful consideration, suggesting a positive and adaptable approach to both personal interactions and creative endeavors.

Chinese Explanation

“well-tempered” 这个词指的是一种平衡、镇定或稳定的状态，常用来形容一个人的性情或个性。一个性格温和的人通常表现得冷静、理智，情绪稳定，能够在各种情况下保持平和。这一概念也可以应用于音乐或乐器上，“well-tempered”描述的是一种调音系统，确保在不同的音调中能够产生和谐美妙的声音，从而允许多样的音乐表现。

总的来说，这个短语暗示着一种适度和深思熟虑的态度，表示在个人交往和创造性工作中积极和适应性强的方式。

in the roster of cell divisions

English Explanation

The phrase "in the roster of cell divisions" refers to the list or catalog of various types of cell division processes that occur within biological systems. Cell division is a fundamental biological process through which a single cell divides to form two or more daughter cells. There are different types of cell division, primarily mitosis and meiosis, which serve various purposes in growth, development, and reproduction of organisms.

• Mitosis is the process by which a single cell divides to produce two genetically identical daughter cells, essential for growth, repair, and asexual reproduction.
• Meiosis, on the other hand, is a type of cell division that reduces the chromosome number by half, leading to the formation of gametes (sperm and eggs) for sexual reproduction.

In a broader sense, the "roster" implies a comprehensive overview or categorization of these processes, highlighting their significance in cellular and organismal functions.

Chinese Explanation

“在细胞分裂的名单上”这个短语指的是生物系统中发生的各种细胞分裂过程的列表或目录。细胞分裂是一个基本的生物过程，通过这个过程，一个细胞分裂成两个或更多的子细胞。细胞分裂有不同的类型，主要包括有丝分裂和减数分裂，它们在生物的生长、发育和繁殖中发挥各自的作用。

• 有丝分裂是指一个细胞分裂产生两个基因相同的子细胞的过程，这一过程对于生长、修复和无性繁殖至关重要。
• 减数分裂则是通过将染色体数量减少一半的细胞分裂类型，导致配子（精子和卵子）的形成，以用作有性繁殖。

从更广泛的角度来看，“名单”意味着对这些过程的全面概述或分类，突出它们在细胞和有机体功能中的重要性。

clutter

English Explanation

Clutter refers to a collection of things lying about in an untidy state. It can encompass a wide range of items, from physical objects like clothes, papers, and furniture to digital files and information that are disorganized or overwhelming. Clutter can occur in various environments, such as homes, workplaces, and even in digital spaces like computers and smartphones.

Clutter often has negative connotations. It can lead to stress, decreased productivity, and difficulty in finding important items. In psychology, clutter can represent mental chaos, affecting one's mood and overall quality of life. Many people engage in decluttering processes to create a more organized and peaceful living or working environment.

In a more abstract sense, clutter can also refer to a lack of clarity in ideas or information, where the essential points may be obscured by excessive details or distractions.

Chinese Explanation

杂乱是指杂乱无序地堆积在一起的物品。它可以包括各种物品，从衣物、文件和家具等实物到组织不当或令人不堪重负的数字文件和信息。杂乱可以发生在各种环境中，例如家中、工作场所，甚至在计算机和智能手机等数字空间中。

杂乱通常带有负面含义。它可能会导致压力、降低工作效率和找不到重要物品。在心理学上，杂乱可以代表精神上的混乱，影响一个人的情绪和整体生活质量。许多人参与清理活动，以创造一个更有序和宁静的生活或工作环境。

在更抽象的意义上，杂乱也可以指思想或信息的不清晰，其中关键点可能因为过多的细节或干扰而模糊不清。

ploughshares

English Explanation

The term "ploughshares" refers to the components of a plow that are designed to cut into the soil. In agriculture, a plow is a tool used to turn over and aerate the soil, making it suitable for planting crops. The "shares" specifically are the sharp, metal parts that penetrate the earth and slice through the dirt. The term is often highlighted in discussions around the concepts of peace and war, particularly due to its biblical association with the phrase "beating swords into ploughshares," which signifies transforming weapons of war into tools for peaceful agriculture and productivity. This symbolizes a shift from conflict to cooperation and sustenance.

中文解释

"ploughshares"（犁铧）指的是犁的构件，专门设计用于切入土壤。在农业中，犁是一种用来翻耕和通气土壤的工具，使土壤适合种植作物。"shares"特别指的是穿透地面并切割泥土的锋利金属部分。这个术语经常在和平与战争的讨论中被强调，特别因其与“把剑打造成犁铧”的圣经短语相关联，象征着将战争武器转化为和平农业和生产工具。这象征着从冲突转向合作与生存的变化。

herring

English Explanation

The term "herring" refers to a type of small, schooling fish that belongs to the Clupeidae family. These fish are typically found in the North Atlantic Ocean and are known for their silvery bodies and distinctive lateral line. Herrings play an important role in marine ecosystems as a food source for larger fish, birds, and marine mammals. They are also economically significant for humans and are commonly harvested for consumption, often found in various culinary forms such as pickled, smoked, or canned.

In culinary contexts, herring is appreciated for its rich taste and is a staple in many northern European cuisines. Herrings can be prepared in various ways, leading to a wide range of traditional dishes.

中文解释

“鲱鱼”是指一种小型集群鱼类，属于鲱形科（Clupeidae）。这些鱼通常生存于北大西洋，以其银白色的身体和显著的侧线而闻名。鲱鱼在海洋生态系统中扮演着重要角色，是更大鱼类、鸟类和海洋哺乳动物的重要食物来源。对人类来说，它们也具有经济重要性，通常被捕捞供人食用，常见的烹饪形式有腌制、熏制或罐装。

在烹饪上下文中，鲱鱼因其丰富的味道而受到喜爱，是许多北欧国家料理中的主食。鲱鱼可通过多种方式进行准备，导致了各种传统菜肴的出现。

inkling

English Explanation

The term "inkling" refers to a slight knowledge or vague idea about something. It is often used to describe an intuition or a hint that a person has, which is not fully formed or confirmed. For instance, if someone has an inkling about a possible outcome of an event, they have a general sense or an initial feeling about it, but they do not have definite evidence or a detailed understanding.

In literature or casual conversation, "inkling" can also imply a beginning of understanding or awareness regarding a situation. It suggests that while the person may not have complete information, there is some underlying sense or suspicion that hints at what may be true.

Chinese Explanation

“inkling”这个词指的是对某事的一种轻微的了解或模糊的概念。它通常用来描述一个人拥有的直觉或暗示，这种直觉或暗示并没有形成或确认得很清楚。例如，如果某人对事件可能的结果有一种“inkling”，那么他们对这一结果有一种大致的感觉或初步的想法，但并没有确凿的证据或详细的理解。

在文学或日常对话中，“inkling”还可以暗示对某种情况的初步理解或意识。它表明，尽管这个人可能没有完整的信息，但他们对真相有某种潜在的感觉或怀疑。

straggly

English Explanation

The term "straggly" is an adjective used to describe something that appears messy, untidy, or uneven in appearance. It often refers to things that grow or spread out in an irregular or disorganized manner. For example, "straggly hair" might describe hair that hangs loosely, with some strands out of place or unkempt. Similarly, "straggly plants" could refer to plants that are not well-maintained, with uneven growth or an appearance of neglect.

The word can evoke images of disarray or lack of care, often implying that something has grown or developed in a haphazard fashion. Straggly can also be used metaphorically to describe abstract concepts, such as a "straggly argument" that lacks coherence or clarity.

Chinese Explanation

“straggly” 是一个形容词，用来描述某物显得凌乱、杂乱或不均匀的外观。这个词常常用来形容以不规则或无序的方式生长或扩散的事物。例如，“straggly hair”（凌乱的头发）可能指的是头发松散地垂着，某些发 strands 不在适当的位置或显得不整齐。类似地，“straggly plants”（杂乱的植物）可能指的是那些保养不善的植物，呈现出不均匀的生长或被忽视的外观。

这个词可能唤起一种杂乱或缺乏关心的视觉印象，通常暗示某物以一种随意的方式成长或发展。 “straggly” 也可以在比喻意义上使用，形容某个抽象概念，例如“straggly argument”（不连贯的论点），缺乏一致性或清晰性。

This shedding of parts

English Explanation

The phrase "this shedding of parts" suggests a process in which certain components or elements are being released, discarded, or lost. The term "shedding" often implies a natural or gradual process of losing something that is no longer needed or beneficial. Context can significantly alter the meaning of this phrase. For instance:

1. Biological Context: In nature, animals shed skin, fur, or feathers as a way to grow or adapt. Similarly, plants might shed leaves during winter as a survival mechanism.

2. Metaphorical Context: In social or psychological terms, "shedding" might refer to letting go of past experiences, habits, or beliefs that are no longer helpful. This can be part of personal development or transformation.

3. Industrial or Technical Context: In manufacturing or technology, the phrase might refer to components from a machine being removed for maintenance or updates, suggesting a process of refinement or improvement.

In summary, "this shedding of parts" indicates a deliberate or natural process of removal, which may lead to growth or improvement, depending on the specific situation being described.

中文解释

“这一部分的脱落”这个短语暗示着某些成分或元素正在被释放、丢弃或失去。“脱落”一词通常意味着一种自然或渐进的过程，其中失去某些东西是因为它们不再需要或有益。上下文可以显著改变这句话的意思。比如说：

1. 生物学背景：在自然界中，动物会脱皮、掉毛或掉羽毛，以便生长或适应环境。同样，植物在冬天可能会掉落树叶，作为一种生存机制。

2. 隐喻背景：在社交或心理方面，“脱落”可能指的是放下过去的经历、习惯或信念，这些东西不再有帮助。这可以是个人发展或转型的一部分。

3. 工业或技术背景：在制造或技术领域，这个短语可能指的是机器的某些组件被移除，以便进行维护或更新，这暗示着一种精炼或改进的过程。

总之，“这一部分的脱落”指的是一种有意或自然的去除过程，根据所描述的具体情况，这可能导致成长或改进。

amorphous

English Explanation

The term "amorphous" refers to a state of matter that lacks a defined structure or shape. In the context of materials science, it typically describes solids that do not have a long-range order or crystalline structure. Instead, the atoms or molecules in an amorphous substance are arranged in a random, irregular manner.

Common examples of amorphous materials include glass, gels, and some types of plastics. Unlike crystalline solids, which have a well-organized arrangement of particles that repeat throughout the material, amorphous solids do not exhibit this degree of order, which can affect their physical properties, such as melting point, viscosity, and hardness.

In other fields, such as biology, "amorphous" can also refer to the absence of a definite form or structure in biological entities, such as certain cells or tissues.

Chinese Explanation

“非晶体的”这个术语指的是一种没有明确结构或形状的物质状态。在材料科学中，它通常描述的是缺乏长程有序或晶体结构的固体。相反，非晶物质中的原子或分子以随机、不规则的方式排列。

非晶材料的常见例子包括玻璃、凝胶和某些类型的塑料。与具有高度有序排列的粒子的晶体固体不同，非晶固体并没有这种程度的有序，这会影响它们的物理性质，如熔点、粘度和硬度。

在其他领域，如生物学，“非晶体的”也可以指生物实体中缺乏明确形式或结构的情况，例如某些细胞或组织。

straggling

Explanation in English:

The term "straggling" generally refers to the act of moving slowly or being scattered in a disorganized manner. It often implies that someone or something is lagging behind or not keeping pace with others. This can be used in various contexts, such as in a group of people walking together where one may fall behind, or in the context of plants growing in a non-uniform way, where some plants may be spread out instead of growing closely together.

In a broader sense, "straggling" can also refer to a lack of organization or cohesion, whether in physical spaces or abstract situations, like thoughts or ideas that are not aligned.

Explanation in Chinese:

“straggling”这个词通常指的是移动缓慢或以无组织的方式散落。这一般意味着某人或某物落后于其他人或未能与他人步调一致。这个词可以用在各种语境中，比如一群人一起走的时候，有一个人可能落在后面；或者在植物生长方面，一些植物可能生长得散乱，而不是紧密生长在一起。

更广泛地说，“straggling”也可以指缺乏组织性或凝聚力，无论是在物理空间中还是抽象的情境中，比如思想或观点没有一致性。

the immortal germ line.

Explanation in English:

The phrase "the immortal germ line" refers to a crucial concept in biology and genetics, particularly in the study of reproduction and heredity. The "germ line" is the sequence of cells, such as sperm and egg cells, that contribute to the genetic material passed from one generation to the next. These cells are involved in reproduction and are responsible for ensuring that genetic information is transmitted.

The term "immortal" in this context indicates that the germ line can perpetuate itself indefinitely through successive generations. While somatic (body) cells age and die, germ line cells can theoretically continue to reproduce without losing their genetic integrity over time. This concept is essential in understanding how certain traits and genetic characteristics can be maintained within a population over long periods, even as individual organisms age and perish.

In summary, "the immortal germ line" highlights the idea that the fundamental genetic material is preserved and passed down through generations, enabling the continuation of life and the evolution of species.

Explanation in Chinese (翻译):

“永恒的生殖系”这个短语指的是生物学和遗传学中的一个重要概念，尤其是在研究生殖和遗传时。“生殖系”是指那些提供遗传物质、并从一代传递到下一代的细胞序列，比如精子和卵子。这些细胞参与生殖，负责确保遗传信息的传递。

在这个上下文中，“永恒”的含义是指生殖系可以通过连续的世代无限地自我延续。虽然体细胞（身体细胞）会衰老和死亡，但生殖系细胞从理论上来说可以不断繁殖，而不会随着时间的推移而丧失其遗传完整性。这个概念对于理解某些特征和遗传特性如何在一个种群中维持很重要，因为即使个体生物衰老和死亡，这些特征仍然可以通过生殖系得以保留。

总之，“永恒的生殖系”强调了基本遗传物质如何被保存并传递给下一代，使生命能够延续以及物种的进化得以实现。

tentacles

English Explanation

Tentacles are long, flexible, and often elongated appendages that are typically found on certain animals, such as octopuses, squids, jellyfish, and some species of mollusks. They serve various functions depending on the organism:

1. Movement: In many animals, such as octopuses, tentacles are used for locomotion by pushing against the water.

2. Sensing: Tentacles can be equipped with sensory organs to help the animal detect changes in their environment, including the presence of food or predators.

3. Capturing Prey: Many marine creatures use their tentacles to catch and hold onto prey. For example, jellyfish use their tentacles to sting and immobilize their prey.

4. Manipulation: Tentacles can be quite dexterous, allowing animals to manipulate objects, such as tools or prey.

The term "tentacle" can also be used metaphorically in literature and culture to describe something that reaches out or influences various aspects of a situation, much like how a tentacle reaches out in different directions.

中文解释

触手是一些动物（如章鱼、鱿鱼、水母以及某些软体动物）特有的长而灵活的附肢。它们在不同生物中具有多种功能：

1. 运动：在许多动物中，如章鱼，触手用于在水中移动，通过推水来前进。

2. 感知：触手上配有感官器官，可以帮助动物探测环境变化，包括食物或捕食者的存在。

3. 捕捉猎物：许多海洋生物使用触手来抓住和固定猎物。例如，水母使用触手来刺伤和使猎物失去活动能力。

4. 操作：触手非常灵活，允许动物像操纵工具或猎物一样操控物体。

“触手”这个词在文学和文化中也可以隐喻性地使用，形容某些事物向外延伸或影响某种情况的各个方面，就像触手向不同方向伸展一样。

in the fabric of membranes;

English Explanation

The phrase "in the fabric of membranes" refers to the structural composition and arrangement of membranes, which are thin layers that separate different environments or components within biological systems. Membranes play a crucial role in living organisms, as they help maintain the integrity of cells and organelles by acting as barriers that regulate the movement of substances in and out.

• Fabric: This term implies that membranes are composed of various elements that are woven together to create a functional structure. Much like fabric in textiles, biological membranes are made of different molecules, primarily lipids and proteins. The specific arrangement and types of these molecules contribute to the properties of the membrane, such as fluidity and permeability.

• Membranes: These are essential biological structures. The most well-known type is the cell membrane, which surrounds each cell and provides a protective barrier. Additionally, membranes can be found around organelles such as the nucleus and mitochondria, playing a key role in cellular functions.

Overall, the phrase suggests a focus on the intricate and interconnected nature of these membranes and their importance in biological processes.

Chinese Explanation

“在膜的结构中”这个短语指的是膜的结构组成和排列，膜是将生物系统中的不同环境或成分分开的薄层。膜在生物体中扮演着至关重要的角色，因为它们通过作为屏障来调节物质的进出，从而维持细胞和细胞器的完整性。

• 结构（Fabric）: 这个术语暗示膜是由各种元素编织在一起形成的功能性结构。就像纺织品中的织物一样，生物膜由不同的分子组成，主要是脂质和蛋白质。这些分子的特定排列和类型决定了膜的性质，如流动性和通透性。

• 膜（Membranes）: 这是一种重要的生物结构。最著名的类型是细胞膜，包围每个细胞并提供保护性屏障。此外，膜还可以在细胞器周围找到，例如细胞核和线粒体，在细胞功能中发挥关键作用。

总体而言，这个短语强调了这些膜的复杂和相互关联的特性以及它们在生物过程中重要性。

cavalier

English Explanation:

The term "cavalier" has several meanings depending on the context in which it is used.

1. Historical Context: Originally, "cavalier" referred to a supporter of the king during the English Civil War in the 17th century. These individuals were loyal to King Charles I and opposed the Parliamentarians, also known as the Roundheads. The term has since taken on a more general connotation of someone who is a knight or a horseman.

2. General Usage: In contemporary language, "cavalier" can also describe a person who shows a lack of concern or seriousness about something. For example, if someone is described as having a "cavalier attitude" towards safety concerns, it means they are indifferent or dismissive about the importance of safety.

3. Personality Trait: As an adjective, it can be used to characterize someone as being dismissive, showing a carefree or nonchalant attitude and sometimes implying arrogance or disregard for consequences.

In summary, "cavalier" can refer to historical figures associated with royalist sentiments, or it can describe a person's attitude that is carefree and perhaps disrespectful towards important matters.

Chinese Explanation:

“骑兵”（cavalier）这个词根据使用的上下文有几种不同的含义。

1. 历史背景：最初，“骑兵”指的是17世纪英国内战中支持国王的人。这些人是查理一世的忠实支持者，反对议会派，也被称为“圆头党”。这个词后来演变为更广泛的含义，指某个骑士或骑马的人。

2. 一般用法：在当代语言中，“骑兵”可以用来形容某个人对某事缺乏关注或严肃态度。例如，如果某人被描述为对安全问题有“骑士态度”，则指他对安全的重要性表现得无所谓或不屑一顾。

3. 性格特征：作为形容词，它可以用来描述某人表现出轻率、无忧无虑的态度，有时暗示傲慢或对后果的不在乎。

总之，“骑兵”既可以指与王室情感有关的历史人物，也可以描述一个人对重要事务抱有无所谓甚至不尊重的态度。

feathers and fangs

English Explanation

The phrase "feathers and fangs" symbolizes two contrasting natural elements that can represent different aspects of life or character.

1. Feathers: They are often associated with birds and can signify lightness, freedom, and grace. Feathers may also represent beauty and the ability to rise above challenges, akin to the idea of flying above adversity. Additionally, in various cultures, feathers can be seen as symbols of spirituality or connection to the divine.

2. Fangs: In contrast, fangs are sharp teeth commonly found in predators and are associated with power, danger, and aggression. They symbolize instincts related to survival, strength, and the more primal aspects of an individual or a situation.

Combining these two elements in one phrase indicates a duality or a balance between gentle, graceful qualities (like those associated with feathers) and fierce, powerful attributes (like those represented by fangs). This combination may illustrate the complexity of nature or human characteristics, suggesting that a person or situation may embody both gentleness and strength.

Chinese Explanation

短语“羽毛和獠牙”象征着两种对比鲜明的自然元素，可以代表生活或性格的不同方面。

1. 羽毛：通常与鸟类相关，象征着轻盈、自由和优雅。羽毛也可能代表美丽和超越挑战的能力，就像在逆境中翱翔的想法一样。此外，在各种文化中，羽毛可以被视为灵性或与神圣的连接的象征。

2. 獠牙：相比之下，獠牙是猛禽常见的尖锐牙齿，与力量、危险和攻击性相关。它们象征着与生存、力量和更原始的个体或情境相关的本能。

将这两个元素结合在一个短语中，表明了一种双重性或在温和优雅的品质（如羽毛所代表的）与强烈有力的特性（如獠牙所体现的）之间的平衡。这种结合可能展现了自然或人类特征的复杂性，暗示一个人或一种情况可能同时具备温柔和力量的特质。

hereafter.

The term "hereafter" generally refers to the future or a time that follows the present. It can imply a sense of continuity or the subsequent events that will occur after the current moment. In legal and formal writings, "hereafter" is often used to indicate terms, conditions, or definitions that will be explained later in the document.

English Explanation:

1. Temporal reference: "Hereafter" denotes a point in time that is beyond the current moment. It signifies that whatever is being discussed will take place or be relevant in the future.

2. Usage in context: In legal documents or formal texts, "hereafter" may precede a definition or description that will apply further along in the text, ensuring clarity for the reader.

3. Examples: A sentence like "The party hereafter referred to as 'the buyer' shall be responsible for payment" means that any future mention of "the buyer" will refer to the party previously identified.

Chinese Explanation:

1. 时间参考: “hereafter” 指的是超出当前时刻的一个时间点。它表示所讨论的事项将在未来发生或相关。

2. 上下文中的用法: 在法律文书或正式文本中，“hereafter” 可能出现在一个定义或描述的开头，以便明确在文本的后面会有适用的条款或条件。

3. 示例: 句子“在此之后所称的‘买方’应负责付款” 意味着所有未来提到的“买方”将指代之前确定的那个方。

In summary, "hereafter" serves as a reference point for what will follow in future discussions or legal definitions, both in English and Chinese contexts.

总结来说， "hereafter" 作为对未来讨论或法律定义的参考点，无论在英语还是中文环境中都有类似的用法和意义。

jockeying

English Explanation

The term "jockeying" has several meanings depending on the context in which it is used. Primarily, it refers to the act of competing or maneuvering for advantage, often in a strategic or cunning manner. It can be related to various fields such as sports, business, or politics.

1. In Horse Racing: The most literal interpretation of "jockeying" pertains to horse racing, where "jockeys" are individuals who ride horses during races. The term captures the notion of jockeys maneuvering their horses strategically to gain a competitive edge over others.

2. In Politics/Business: "Jockeying" can also refer to the strategies employed by individuals or groups to position themselves favorably, secure power, influence decisions, or outmaneuver opponents. This could involve negotiating, forming alliances, or strategically placing oneself in a favorable light.

3. General Use: In broader usage, "jockeying" can describe any scenario where individuals are trying to gain a favorable position or advantage in a given situation, such as during competitions, debates, or negotiations.

In summary, "jockeying" embodies the ideas of strategy, competition, and maneuvering to achieve a goal.

Chinese Explanation

"jockeying" 这个词的意思根据上下文不同而有所变化。主要来说，它指的是为了获得优势而进行的竞争或操控，通常是以一种策略性或巧妙的方式进行的。这一术语可以与体育、商业或政治等多个领域相关联。

1. 在马术比赛中： "jockeying" 的最直接的解释与赛马有关，其中 "jockeys" 是指在比赛中骑马的人。这个词反映了骑手在比赛中战略性地操纵马匹，以便在竞争中取得优势的概念。

2. 在政治/商业中： "jockeying" 也可以指个人或团体为了在权力争夺、影响决策或超越对手而采取的策略。这可能涉及谈判、结成联盟，或将自己置于有利的光环中。

3. 普遍使用：在更广泛的用法中，"jockeying" 可以描述任何情况下，个人试图在特定情境中获得有利位置或优势的情形，比如在竞争、辩论或谈判中。

总之，"jockeying" 体现了策略、竞争和操控以达成目标的概念。

jostling

Sure! Let's break down the term "jostling" both in English and Chinese.

English Explanation:

The term "jostling" typically refers to the act of bumping, pushing, or shoving against someone or something, often in a crowded or competitive environment. It implies a physical interaction where individuals may be trying to get ahead or make their presence known, often resulting in a sense of chaos or disorder.

For example, in a busy subway station, you might experience jostling as people move quickly to get on and off trains, brushing against each other in the process. The word can also have metaphorical connotations, such as in a competitive situation where different parties vie for position, attention, or resources, like in a political campaign or a marketplace.

Chinese Explanation:

“Jostling”这个词通常指的是在拥挤或竞争环境中，人与人或人与物之间的撞击、推搡或挤压。这意味着一种身体上的互动，个体可能试图争取前进或让自己被注意到，通常会导致一种混乱或无序的感觉。

例如，在一个繁忙的地铁站，你可能会经历“jostling”，因为人们快速行走，上下列车的过程中相互碰撞。这个词也可以有隐喻上的含义，例如在竞争场合，不同的参与者争夺位置、关注或资源，比如在政治竞选或市场中。

Summary:

In summary, "jostling" captures both the physical act of pushing and the competitive dynamics present in various scenarios, signifying a struggle for space or advantage.

综上所述，“jostling”既体现了推搡的身体行为，也体现了各种场景中存在的竞争动态，意味着为了空间或优势而进行的斗争。

broad-fronted budding,

English Explanation:

The term "broad-fronted budding" likely refers to a specific kind of reproductive strategy or growth pattern in certain plants or organisms. In biological contexts, "bud" typically denotes a young, undeveloped shoot or flower that is in the process of maturing and growing.

• Broad-fronted: This phrase can suggest a wide or expansive approach to growth or development. It implies that the budding occurs at a broader scale, which could be related to the formation of multiple buds or a wide area from which buds are formed.

Overall, "broad-fronted budding" may indicate a method or characteristic of growth that allows for more extensive expansion or proliferation of the parent organism, potentially increasing its reproductive success and survival.

Chinese Explanation:

“宽面芽”这个术语可能指的是某些植物或生物特定的繁殖策略或生长模式。在生物学语境中，“芽”通常指的是年轻的、未发育成熟的枝条或花朵，正处于成熟和生长的过程中。

• 宽面：这个短语可以表示一种宽广或广泛的生长或发展的方式。它暗示芽的形成是在更大范围内进行的，这可能与多个芽的形成或芽形成的宽区域有关。

总的来说，“宽面芽”可能指的是一种生长方法或特征，能够使母体生物的扩展或繁殖更加广泛，从而潜在地增加其繁殖成功率和生存率。

budded off

English Explanation

The term "budded off" refers to a process typically seen in biology, particularly in the context of asexual reproduction in organisms such as plants or certain fungi.

In this process, a new individual organism (often referred to as a "bud") forms as an outgrowth from the parent organism. This bud eventually detaches and becomes independent. For example, in plants like succulents, a new plant will develop from a bud that grows on the main plant. Once the bud has matured sufficiently, it separates from the parent plant, resulting in two distinct plants: the original parent and the new budded-off plant.

This method of reproduction allows for rapid population increase and can lead to genetically identical offspring, as the new organism is essentially a clone of the parent.

Chinese Explanation

“budded off” 这个术语通常用于生物学，特别是用于描述有机体（如植物或某些真菌）无性繁殖的过程。

在这个过程中，一个新的个体（通常称为“芽”）作为父母有机体的一个生长部分形成。这种芽最终会分离出来，变得独立。例如，在多肉植物中，一个新植物会从主植物的芽上发育而来。一旦芽成熟到足够的程度，它就会脱离父母植物，从而形成两个不同的植物：原来的父母植物和新的“budded off”植物。

这种繁殖方式可以快速增加种群数量，并可能导致基因相同的后代，因为新有机体本质上是父母的克隆。

vessel

Certainly! The term "vessel" can have multiple meanings depending on the context in which it is used. Below, I will explain its various meanings in both English and Chinese.

English Explanation:

1. General Definition: A vessel is a container that holds liquid or substances. This can refer to various types of containers, such as jars, bottles, or any other object designed to hold something.

2. In Biology: In a biological context, a vessel refers to a tubular structure in the body that carries fluids. For example, blood vessels transport blood throughout the body, helping to distribute nutrients and remove waste.

3. In Maritime Terms: A vessel may also refer to a ship or boat. This usage emphasizes the transportation aspect of vessels, especially in marine contexts.

4. In Legal Terms: In legal contexts, "vessels" may refer to ships or other floating conveyances as recognized by law, often subject to maritime laws and regulations.

Chinese Explanation (中文解释):

1. 一般定义：在一般情况下，"vessel"（容器）指的是一个用来盛装液体或物质的容器。这可以包括各种类型的容器，比如罐子、瓶子或其他设计用来盛装东西的物体。

2. 在生物学中：在生物学的语境中，"vessel"（血管）指的是体内一种管状结构，主要用于运输液体。例如，血管负责将血液运送遍布全身，帮助分配营养和排除废物。

3. 在海事术语中：在海事术语中，"vessel"（船舶）可以指船或小艇。这个词强调了运输的方面，特别是在海洋相关的语境中。

4. 在法律术语中：在法律语境中，"vessels"（船舶）可以指法律认可的船只或其他漂浮交通工具，通常受海事法律和法规的约束。

Summary:

In summary, "vessel" is a versatile term that can refer to containers, biological structures, maritime vehicles, or legally recognized ships depending on the context.

总结来说，"vessel" 是一个多用途的术语，根据上下文可以指容器、生物结构、海事交通工具或法律认可的船只。

To be sure, packs may bud off daughter packs.

Certainly! Let's break down the excerpt:

English Explanation

The phrase "To be sure, packs may bud off daughter packs" suggests that there is a process by which larger groups (called "packs") can give rise to smaller, separate groups (called "daughter packs"). The term "bud off" is borrowed from biological language, where it typically describes how new offspring can grow out from a parent organism. In this context, it implies that these packs could potentially split or branch out to form new entities.

This concept can be applied to various contexts, such as biological systems, social groups, or organizational structures where a main group can generate smaller, independent units. The word "may" indicates that this is a possibility rather than a certainty, suggesting that not all packs will necessarily produce daughter packs.

Chinese Explanation

这个短语“可以肯定的是，群体可能会分裂出子群体”暗示了一个过程，其中较大的群体（称为“群体”）能够衍生出较小的、独立的群体（称为“子群体”）。术语“分裂出”借用了生物学的语言，通常描述新后代是如何从父母生物体上生长出来的。在这个上下文中，它意味着这些群体可能会分裂或分支出形成新的实体。

这个概念可以应用于各种情境，例如生物系统、社交团体或组织结构，其中一个主要群体可以产生更小的独立单位。词语“可能”表明这是一个可能性，而不是肯定的事情，这暗示并不是所有的群体都会产生子群体。

If you have any further questions or need clarification, feel free to ask!

the same unpartisan meiosis

English Explanation

The phrase "the same unpartisan meiosis" can be broken down into two parts for a clearer understanding:

1. Unpartisan: This term typically refers to a stance that is neutral and does not favor any particular party or group. In a broader context, it implies an unbiased perspective or approach that avoids political affiliations or preferences.

2. Meiosis: In biological terms, meiosis is a type of cell division that results in the production of gametes (sperm and eggs) with half the number of chromosomes, allowing for genetic diversity through sexual reproduction. It can also mean a rhetorical figure of speech that intentionally understates or downplays a situation for effect.

Combining these two concepts, "the same unpartisan meiosis" could imply a neutral or unbiased approach to a process (possibly decision-making, discussion, or analysis) that downplays or reduces the complexity of an issue. The phrase may suggest that an objective viewpoint is being maintained throughout, and that this process avoids taking sides or showing favoritism.

Chinese Explanation

短语“the same unpartisan meiosis”可以拆分为两个部分，以便更清晰地理解：

1. Unpartisan（中立的）：这个词通常指的是一种中立的立场，不偏向任何特定的政党或团体。更广泛的说，它暗示一种公正的视角或方法，避免政治的偏见或喜好。

2. Meiosis（减数分裂）：在生物学中，减数分裂是一种细胞分裂方式，产生拥有一半染色体数量的生殖细胞（精子和卵子），通过有性生殖促进遗传多样性。它也可以指一种修辞手法，故意低估或淡化某一情境，以达到特定效果。

结合这两个概念，“the same unpartisan meiosis”可能暗示了一种对某个过程（可能是决策、讨论或分析）采取的中立或无偏见的方法，该方法淡化或减少了问题的复杂性。该短语可能表明在整个过程中保持了客观视角，并且此过程避免了偏袒某一方或表现出偏向性。

paltry

Explanation in English

The term "paltry" is an adjective used to describe something that is considered to be insignificant, trivial, or of little worth. It often conveys the idea that the amount or quality of something is so low that it does not meet expectations or is not worthy of attention. "Paltry" can be used to refer to amounts of money, efforts, acknowledgments, or even contributions that are considered inadequate or meager. For example, if someone receives a paltry sum of money for their hard work, it suggests that the payment is disappointingly small relative to the effort put in.

Examples: 1. "Despite her hard work, she received a paltry paycheck at the end of the month." 2. "The amount of food provided at the event was paltry; many guests left still hungry."

Chinese Translation

“paltry” 是一个形容词，用来描述被认为微不足道、琐碎或价值不高的事物。这个词常常表达出某事物的数量或质量低到不能满足期望，或者不值得关注的含义。 “paltry” 可以用来形容金钱、努力、认可，甚至是被认为不足够或微薄的贡献。例如，如果某人因为自己的辛勤工作而得到一笔 paltry 的报酬，这意味着这笔报酬相较于付出的努力来说非常之小，让人失望。

例子： 1. “尽管她努力工作，但她月末收到的薪水却是微不足道的。” 2. “活动提供的食物数量很少，许多客人离开时仍感到饿。”

In summary, "paltry" indicates a sense of inadequacy or disappointment regarding the value of something.

rudimentary coherence

English Explanation

The term "rudimentary coherence" can be broken down into two parts: "rudimentary" and "coherence."

1. Rudimentary: This word refers to something that is basic or elementary. It suggests that the subject in question is at a fundamental level, lacking in sophistication or completeness. In many contexts, "rudimentary" indicates the foundational aspects of something before it is developed into a more complex or refined state.

2. Coherence: Coherence refers to the quality of being logical, consistent, and understandable. It usually implies that various elements or ideas fit together in a way that makes sense, promoting clarity and a unified understanding of a topic.

When combined, "rudimentary coherence" implies a basic level of logical connection or consistency among parts or ideas. It suggests that while there may be some form of organization or clarity, it is limited and may not be fully developed. This phrase could be used in various contexts, such as writing, arguments, or theories, where the structure is present but not sophisticated or detailed.

Chinese Explanation

“rudimentary coherence” 这个术语可以分为两个部分：“rudimentary”和“coherence”。

1. rudimentary（基础的，初步的）: 这个词指的是某事物的基本或初级状态。它暗示相关主题处于基本层次，缺乏复杂性或完整性。在许多上下文中，“rudimentary”表示某种事物在深入发展成更复杂或精致状态之前的基础方面。

2. coherence（连贯性）: 连贯性指的是逻辑性、一致性和易理解的特质。它通常意味着各种元素或观点以一种合理的方式结合在一起，从而促进了对主题的清晰和统一的理解。

将“rudimentary”和“coherence”结合在一起，"rudimentary coherence" 暗示出在各部分或思想之间存在一种基本的逻辑连接或一致性。它表明尽管可能有某种形式的组织或清晰度，但这种清晰度是有限的，可能并不完全发达。这个短语可以在各种上下文中使用，比如写作、论证或理论，其中结构存在但并不复杂或详尽。

wishy-washy

English Explanation

The term "wishy-washy" is an informal adjective used to describe someone or something that is indecisive, weak, or lacking in clarity and firmness. When a person is called wishy-washy, it means they have trouble making decisions or commitments, often resulting in vague or ambiguous opinions or actions. This term can also refer to ideas, plans, or statements that lack decisiveness or conviction, presenting a diluted or uncertain stance on a matter. It's often perceived negatively, suggesting a lack of strength or determination.

Contextual Use: - "Her wishy-washy attitude during the meeting frustrated her colleagues, who sought clear direction." - "The proposal was too wishy-washy to gain approval; it needed a stronger, more definite plan."

中文解释

“wishy-washy”是一个非正式的形容词，常用来形容一个人或事物优柔寡断、软弱或缺乏清晰和坚定。被称为“wishy-washy”的人往往在做决策或承诺时感到困难，导致模糊或含糊的观点或行动。这个词也可以用来指那些缺乏决断性或信念的思想、计划或陈述，对于某个问题的立场显得稀薄或不确定。这个词通常被看作是贬义，暗示缺乏力量或决心。

使用上下文： - “她在会议中的优柔寡断让同事们感到沮丧，他们希望能有明确的方向。” - “提案太过优柔寡断，无法获得批准；需要一个更强烈、更明确的计划。”

facile

English Explanation:

The word "facile" is an adjective that has a few different meanings depending on the context in which it is used.

1. Easy or Smooth: The most common use of "facile" refers to something that is easily achieved or accomplished. For example, a facile solution to a problem is one that is straightforward and does not require much effort to understand or implement.

2. Superficial or Lacking Depth: In some contexts, "facile" can have a negative connotation, suggesting that something is too simplistic or does not delve deeply into the complexities of a situation. For instance, a facile argument might oversimplify an issue without acknowledging its nuances.

3. Skillful or Fluent: Additionally, "facile" can describe someone who is very skilled at a particular task or is able to express themselves fluently, often with ease. For example, a facile writer is one who manages to present their ideas clearly and eloquently.

Overall, "facile" can imply ease in a positive sense (efficiency and skill) or in a negative sense (superficiality or oversimplification).

Chinese Explanation:

"facile" 是一个形容词，它的含义根据上下文的不同而有所不同。

1. 容易或顺利: "facile" 最常见的用法是指某事很容易实现或完成。例如，对一个问题的简单解决方案称为 "facile" 方案，表示这个解决方案直接明了，理解或实施并不需要太多努力。

2. 肤浅或缺乏深度: 在某些情况下，"facile" 可能带有负面含义，表示某事过于简单化，没有深入探讨情况的复杂性。例如，肤浅的论点可能会忽略问题的细微差别。

3. 熟练或流利: 此外，"facile" 还可以用来形容某人在特定任务中非常熟练，或能够流利地表达自己，通常显得轻松自如。例如，"facile" 的作家能够清晰而优雅地呈现他们的想法。

总的来说，"facile" 可以在积极的意义上（高效和技能）或消极的意义上（肤浅或过于简单化）来暗示轻松。

it clears up a tiresome controversy over the level at which natural selection acts.

English Explanation:

The excerpt discusses a resolution to a disagreement that has been ongoing and possibly frustrating regarding the specific level or scale at which natural selection operates. In evolutionary biology, natural selection is a process where organisms better adapted to their environment tend to survive and produce more offspring. However, there has been some debate among scientists and researchers about whether natural selection primarily affects individuals, groups, populations, or even species as a whole.

The phrase "it clears up a tiresome controversy" suggests that this resolution is significant enough to alleviate a considerable amount of confusion or dispute within the scientific community. The use of the word "tiresome" implies that this controversy has been long-standing and possibly distracting from more productive research or conversation.

Chinese Explanation (中文解释):

这段摘录讨论了一个令人厌倦的争议的解决，这个争议是关于自然选择作用的具体层次或规模。在进化生物学中，自然选择是一个过程，在这个过程中，更适应环境的生物往往生存得更好并且繁殖更多后代。然而，科学家和研究人员之间对此的辩论，一直围绕着自然选择是主要作用于个体、群体、种群，还是甚至整个物种。

“它澄清了一个令人厌倦的争议”这句话表明这个解决方案的重要性足以减轻科学界内部相当程度的困惑或争论。使用“令人厌倦”这个词暗示这个争议已经持续了很久，并可能使得人们无法更有效地进行研究或交流。

Summary:

The excerpt signifies a significant breakthrough in resolving a complex debate about natural selection's scope in evolutionary processes, aiming to clarify misunderstandings in the scientific discourse, which has been persistent and taxing.

这段摘录表明在关于自然选择作用范围的复杂辩论中的一个重大突破，旨在澄清科学讨论中的误解，而这个争论一直存在并且令人疲惫。

They are cast in different, complementary and in many respects equally important roles,

English Explanation:

This excerpt indicates that there are multiple individuals or groups being discussed, and each of them has distinct roles that contribute to a larger purpose or function. The term "complementary" suggests that these roles work together harmoniously, enhancing each other's effectiveness. Additionally, the phrase "equally important" emphasizes that no single role is considered more significant than the others; rather, all roles are vital for the success of the whole.

In essence, this statement highlights the idea of teamwork and collaboration, where diverse contributions are valued and necessary for achieving common goals.

Chinese Explanation:

这段摘录表明，讨论中涉及多个个体或群体，每一个都有其独特的角色，这些角色共同推动一个更大的目的或功能。词语“互补”暗示这些角色能够和谐地协同工作，提升彼此的有效性。此外，“同样重要”这一短语凸显了没有单一角色被视为比其他角色更重要；相反，所有角色对整体的成功都是至关重要的。

从本质上说，这句话强调了团队合作与协作的理念，表明多样化的贡献被重视，并对实现共同目标是必不可少的。

Gene and individual organism are not rivals for the same starring role in the Darwinian drama.

Certainly! Let’s break down the excerpt and explain it in both English and Chinese.

English Explanation

The excerpt suggests that genes and individual organisms do not compete with each other for prominence in the context of evolutionary theory, particularly in Darwinian evolution.

1. Gene: Refers to the basic unit of heredity in living organisms. Genes are segments of DNA that contain the instructions for building proteins, which are essential for the structure, function, and regulation of the body’s cells.

2. Individual Organism: This refers to a single living entity (e.g., a human, animal, or plant) that encompasses various systems and functions within its biology.

3. Darwinian Drama: This phrase alludes to the process of evolution and natural selection, as proposed by Charles Darwin. Within this framework, evolution is viewed as a complex narrative where survival and reproduction are key themes.

The statement implies that both genes and individual organisms play important but different roles in the process of evolution. Genes are responsible for passing traits from one generation to the next, while individual organisms are the entities that experience those traits in their environments. Rather than one being more important than the other, they complement each other in the overall process of evolutionary change.

中文解释

这段摘录表明，基因和个体生物在达尔文进化理论的背景下并不为同一个主角竞争。

1. 基因：指的是生物体遗传的基本单位。基因是DNA的片段，包含构建蛋白质的指令，而蛋白质对生物细胞的结构、功能和调节至关重要。

2. 个体生物：指的是单一的生物实体（例如，人体、动物或植物），其生物学包括各种系统和功能。

3. 达尔文戏剧：这个短语暗指进化和自然选择的过程，即查尔斯·达尔文所提出的理论。在这个框架下，进化被视为一个复杂的叙事，其中生存和繁殖是关键主题。

这段话的意思是，基因和个体生物在进化过程中扮演着重要但不同的角色。基因负责将特征从一代传递到下一代，而个体生物则是在环境中体验这些特征的实体。基因和个体生物并不是彼此竞争的重要性，而是相辅相成，共同参与了进化变化的整体过程。

form lineages of identical copies

English Explanation

The phrase "form lineages of identical copies" refers to the process of creating sequences or chains of copies that are all identical to one another. In biological or evolutionary terms, a lineage usually describes a sequence of organisms or entities that share a common ancestor. When we say "identical copies," we imply that these copies are exact replicas of each other, without any variations.

In the context of genetics, for instance, one might consider how genetic material can replicate itself, leading to a lineage of genetically identical cells. In a broader sense, this phrase can be applied to various fields, such as technology (like photocopying documents) or programming (creating instances of a particular class in software).

Understanding this concept is crucial because it relates to the principles of replication, evolution, and the transmission of traits or information through consecutive generations or copies.

Chinese Explanation

“形成相同复制品的谱系”这个短语指的是创建一系列全都相同的复制品的过程。在生物学或进化论的术语中，谱系通常描述了一系列共享共同祖先的生物或实体。当我们说“相同的复制品”时，意味着这些复制品是彼此完全相同的，没有任何变化。

例如，在遗传学的背景下，可以考虑遗传物质如何自我复制，从而导致一系列遗传上相同的细胞。在更广泛的方面，这个短语也可以应用于各种领域，比如技术（例如复印文件）或编程（创建特定类的实例）。

理解这个概念是至关重要的，因为它与复制、进化和通过连续世代或复制传递特征或信息的原则相关。

teems with

English Explanation:

The phrase "teems with" is an idiomatic expression in English that means to be full of or to be overflowing with a large quantity of something. It often conveys a sense of abundance, liveliness, or bustling activity. For example, one might say, "The garden teems with flowers in the spring," indicating that during this season, the garden is filled with a vibrant array of flowers, possibly to the point of overflowing. The term can be used in various contexts, including nature, activities, people, or ideas, and often evokes a vivid image of richness and diversity.

Chinese Explanation:

“teems with” 是一个英语习语，意思是充满了或盛满了大量的某物。这个短语通常传达出一种丰盛、生机勃勃或活动繁忙的感觉。比如，人们可能会说：“春天时花园里充满了花”，这表示在这个季节，花园里开满了生机勃勃的花朵，甚至可能多到溢出的程度。这个表达可以用在不同的语境中，包括自然、活动、人物或想法，常常描绘出丰富多彩的生动形象。

administers an aggression-arousing drug

Sure! Let's break down the excerpt "administers an aggression-arousing drug" thoroughly in both English and Chinese.

English Explanation:

• Administers: This term means to give or apply something, often in a medical context. In this case, it refers to the act of providing a drug to someone.
• An aggression-arousing drug: This phrase describes a type of drug that has the potential to increase or provoke aggressive behavior in individuals. "Aggression-arousing" implies that the drug influences the brain or body's chemistry in a way that heightens feelings of anger or hostility.

Putting it all together, the excerpt indicates that someone (likely a medical professional or researcher) is giving a specific type of drug that is designed or known to increase aggressive behavior in the person receiving it. This may be relevant in a psychological or pharmacological study or treatment context.

Chinese Explanation (中文解释):

• Administers（给予/施用）：这个词通常在医学语境中使用，意思是给予或施用某种东西。在此情况下，它指的是向某人提供一种药物。
• An aggression-arousing drug（激发攻击性的药物）：这个短语描述了一种药物，其可能性是增加或激发个人的攻击行为。“激发攻击性”意味着这种药物影响大脑或身体的化学反应，以加重愤怒或敌对感。

综合来看，这段摘录表明某人（可能是医疗专业人员或研究人员）正在给予一种特定类型的药物，这种药物被设计或已知能够增加接受者的攻击性行为。这在心理学或药理学的研究或治疗中可能是相关的。

Summary (总结):

In both English and Chinese, "administers an aggression-arousing drug" refers to the action of giving a drug that increases aggressive behavior, typically in a clinical, research, or experimental setting.

under the sway of a dope-peddling caterpillar

English Explanation

The phrase "under the sway of a dope-peddling caterpillar" employs metaphorical language to create an image that can be interpreted in various ways.

1. "Under the sway of": This expression means to be influenced or controlled by someone or something. It suggests a lack of autonomy, implying that the subject (which is not explicitly mentioned) is being influenced in a significant way.

2. "A dope-peddling caterpillar":

3. "Dope-peddling": This term refers to selling illegal drugs or substances. In a broader sense, it can denote someone who promotes harmful or destructive influences.
4. "Caterpillar": This image can evoke various interpretations. A caterpillar can symbolize transformation, as it eventually becomes a butterfly. However, in this context, it may represent something small and seemingly innocuous that has a significant negative impact or influence.

Overall, the excerpt suggests a scenario where an individual or group is being heavily influenced by a seemingly minor but detrimental element (the caterpillar), which is associated with harmful activities (dope-peddling). This can reflect themes of manipulation, addiction, or being led astray by harmful influences.

Chinese Explanation

"在一个贩卖毒品的毛毛虫的影响下"这个短语使用隐喻语言，创造出可以用多种方式解读的形象。

1. "在......的影响下"：这个表达意味着受到某人或某事的影响或控制。它暗示着缺乏自主权，意味着主题（尽管没有明说）在重要方面受到影响。

2. "贩卖毒品的毛毛虫"：

3. "贩卖毒品的"：这个术语指的是贩卖非法药物或物质。从更广泛的意义来看，它可以指代促进有害或毁灭性影响的人。
4. "毛毛虫"：这个形象可以激发多种解读。毛毛虫可以象征转变，因为它最终会变成蝴蝶。然而，在此语境中，它可能代表某种微小且看似无害但对人产生重大负面影响或影响力的东西。

总体而言，这段摘录暗示一个场景，其中一个个人或群体受到一种看似微小但有害元素（毛毛虫）强烈影响，而这种元素与有害活动（贩卖毒品）相关。这可以反映操控、上瘾或被有害影响误导的主题。

Except, significantly, the caterpillar responsible for drugging it.

English Explanation

The excerpt "Except, significantly, the caterpillar responsible for drugging it." suggests that there is a notable exception or important point regarding a caterpillar. This caterpillar plays a critical role because it is responsible for drugging another organism or entity, although the excerpt does not specify what that organism is. The use of the word "significantly" emphasizes the importance of this caterpillar's actions, indicating that understanding its role is crucial to the broader context of the discussion.

Essentially, the excerpt implies a situation where everything mentioned is generally unaffected or removed from a particular issue, except for this one caterpillar, which has a significant and impactful role that may influence the circumstances involved.

Chinese Explanation

这段摘录“Except, significantly, the caterpillar responsible for drugging it.”暗示着有一个显著的例外，即一只毛毛虫。这个毛毛虫扮演着重要的角色，因为它负责使另一个生物或实体中毒，尽管摘录并没有具体说明那个生物是什么。“Significantly”这个词的使用强调了这只毛毛虫的行为的重要性，表明理解它的角色对于讨论的更广泛背景至关重要。

简而言之，这段摘录意味着提到的事情通常都没有受到影响，或与特定问题无关，除了这一只毛毛虫。这只毛毛虫具有重要且深远的影响，可能会影响相关的情况。

potion

Certainly! Let's delve into the term "potion."

English Explanation:

A "potion" typically refers to a liquid substance that is often infused with magical or medicinal properties. It is commonly found in literature, folklore, and role-playing games where potions are created for various effects, such as healing, transformation, or granting special abilities.

Historically, potions have been associated with alchemy and the practices of ancient healers who believed that certain mixtures could bring about changes to the body or mind. Ingredients might include herbs, minerals, and magical elements. In modern contexts, the term can also be used metaphorically to describe any concoction or mixture that has significant effects on a person's condition or situation.

Usage in Literature and Media: - In fantasy novels, a hero might drink a potion to gain strength or speed. - In games, players often collect or craft potions to heal or enhance their character's abilities.

Chinese Translation and Explanation:

“药水”是一个常指含有魔法或药物特性的液体物质的术语。在文学、民俗和角色扮演游戏中，药水经常被用作各种效果的创造，例如治疗、变形或赋予特殊能力。

历史上，药水与炼金术和古代治疗师的实践有关，这些治疗师相信某些混合物可以带来身体或心理上的变化。药水的成分可能包括草药、矿物和魔法元素。在现代语境中，这个术语也可以比喻地用来描述任何对一个人的状况或情况产生重大影响的混合物。

在文学和媒体中的使用： - 在幻想小说中，英雄可能会喝一剂药水以获得力量或速度。 - 在游戏中，玩家通常会收集或制作药水，以治愈或增强角色的能力。

This comprehensive overview should provide a clear understanding of what "potion" signifies both in English and Chinese contexts.

cast an altogether more subtle spell.

English Explanation:

The phrase "cast an altogether more subtle spell" suggests the act of creating a kind of influence or effect that is nuanced and delicate, rather than overt or obvious. To "cast a spell" often carries a magical connotation, meaning to enchant or captivate someone. When described as "altogether more subtle," it implies that this influence is not heavy-handed or easily recognized; rather, it operates in a refined, understated manner. The use of "altogether" emphasizes that the level of subtlety is significant, indicating that this spell is distinctively different from other, more obvious forms of enchantment.

Chinese Translation:

这个短语“施放一种完全更为微妙的魔法”意味着创造一种影响力或效果，它是细致而微妙的，而不是直白或明显的。 “施放魔法”通常带有魔幻的含义，意味着迷住或吸引某人。当描述为“完全更为微妙”时，这暗示这种影响并不是粗暴或容易被识别的，而是以一种精致、低调的方式运作。“完全”（altogether）的使用强调这种微妙的程度是显著的，表明这种魔法与其他更明显的迷人形式有明显的不同。

nozzles

Certainly! Let's explore the term "nozzles" in depth.

English Explanation

A nozzle is a device designed to control the direction or characteristics of a fluid (liquid or gas) as it exits an enclosed chamber or pipe. Nozzles are commonly used in various applications, including:

1. Engineering and Manufacturing: In these fields, nozzles are used in machining processes to direct cutting fluids and coolants.

2. Aerospace: In rocket engines, nozzles are crucial for directing the high-speed exhaust gases produced during combustion, which helps propel the rocket forward.

3. Agriculture: Spraying systems utilize nozzles to disperse pesticides, fertilizers, or irrigation water over crops, ensuring an even distribution.

4. Automotive: Fuel injectors in vehicles use nozzles to atomize fuel into the engine’s combustion chamber, promoting efficient combustion.

The design of a nozzle can greatly affect the flow rate, pressure, and velocity of the fluid passing through it. There are different types of nozzles, such as convergent, divergent, and convergent-divergent types, each designed for specific functions.

中文解释 (Chinese Explanation)

喷嘴 是一种装置，旨在控制流体（液体或气体）在其从封闭腔体或管道中排出时的方向或特性。喷嘴在各种应用中被广泛使用，包括：

1. 工程与制造：在这些领域中，喷嘴用于加工过程中，导向切削液和冷却剂。

2. 航空航天：在火箭发动机中，喷嘴对于引导在燃烧过程中产生的高速废气至关重要，这有助于推动火箭前进。

3. 农业：喷雾系统利用喷嘴将杀虫剂、肥料或灌溉水均匀地分散在作物上，确保分布均匀。

4. 汽车：车辆中的燃油喷射器使用喷嘴将燃油雾化成进入发动机燃烧室，促进高效燃烧。

喷嘴的设计可以极大地影响通过它的流体的流量、压力和速度。喷嘴有不同类型，如收敛喷嘴、发散喷嘴和收敛-发散喷嘴，分别针对特定功能设计。

If you have more specific aspects of "nozzles" you would like to explore or have questions about, feel free to ask!

exude seductive nectar.

English Explanation

The phrase "exude seductive nectar" can be broken down into two main parts for better understanding:

1. Exude: This verb means to release or give off a substance gradually. It implies a natural emanation, where something flows out effortlessly. In this context, it suggests that something is being produced or discharged in a way that is noticeable and appealing.

2. Seductive Nectar: The word "seductive" indicates that whatever is being described has a quality that attracts or entices others, often in a captivating or charming manner. "Nectar" is a sweet liquid produced by flowers, which is often associated with bees and other pollinators. It is typically considered delicious and appealing.

Putting it together, the phrase suggests that something is releasing a sweet, enticing substance that draws others in, much like how flowers attract pollinators with their nectar. This expression could be used metaphorically to describe a situation, feeling, or object that captivates attention or evokes desire.

Chinese Explanation

短语“散发魅惑的花蜜”可以分为两个主要部分进行详细理解：

1. 散发 (exude): 这个动词的意思是逐渐释放或排放某种物质。它暗示了一种自然的散发，表示某物在不费力的情况下流出。在这个上下文中，它暗示某种东西正在以一种显而易见和令人愉悦的方式被产生或排出。

2. 魅惑的花蜜 (seductive nectar): “魅惑的”这个词表示所描述的事物具有吸引或诱惑他人的特质，通常以一种迷人或诱人的方式。“花蜜”是花朵产生的一种甜液体，通常与蜜蜂和其他授粉者相关。它通常被认为是美味和诱人的。

综上所述，这个短语意味着某种东西正在散发一种甜美而诱人的物质，这种物质吸引他人，就像花朵用其花蜜吸引授粉者一样。这个表达可以比喻地用于描述一种情况、感觉或对象，吸引注意力或唤起渴望。

telescopic spouts

English Explanation

Telescopic spouts are specialized components often found in various types of containers, such as bottles or dispensing systems. These spouts are designed to extend and retract, much like a telescope, providing flexibility in the pouring or dispensing process.

Functionality: The main purpose of telescopic spouts is to enable the user to control the flow of a liquid or other substances with greater precision. When the spout is extended, it allows for a longer reach, making it easier to pour into containers with a greater distance or reach over obstacles. When it's retracted, the spout is more compact, making storage more convenient and reducing the risk of spills.

Applications: Telescopic spouts are commonly used in various industries, including: - Household products: Such as shampoo bottles or liquid soap dispensers. - Industrial applications: In factories for dispensing oils or chemicals. - Food and beverage: For sauces, dressings, or any liquid that requires controlled pouring.

In essence, telescopic spouts enhance usability and efficiency by allowing users to manage how much and where they pour substances without creating messes.

中文解释

伸缩喷嘴是一种专业组件，通常见于各种类型的容器中，如瓶子或分配系统。这些喷嘴的设计使其能够像望远镜一样伸缩，从而在倒出或分配过程中提供灵活性。

功能性：伸缩喷嘴的主要目的是使用户能够更精确地控制液体或其他物质的流动。当喷嘴伸展时，可以达到更长的距离，方便在距离较远的容器中倒入液体或跨越障碍物倒出。当喷嘴缩回时，它更加紧凑，方便存放，并减少溢出的风险。

应用：伸缩喷嘴通常用于多个行业，包括： - 家居产品：如洗发水瓶或液体肥皂分配器。 - 工业应用：在工厂中用于分配油或化学品。 - 食品和饮料：用于酱料、调味品或任何需要控制倒出量的液体。

总之，伸缩喷嘴通过让用户更好地管理倒出物质的方式和目标，增强了使用的便利性和效率，避免了混乱的局面。

summoning

English Explanation:

The term "summoning" generally refers to the act of calling upon or bringing forth someone or something, often with a sense of authority or urgency. In various contexts, it can mean:

1. Legal Context: In legal terms, "summoning" may refer to an official notification or order to appear in court. A summons is often issued by a court to require an individual to respond to a legal proceeding.

2. Spiritual or Mystical Context: In spiritual or mystical practices, "summoning" can refer to invoking or calling upon spirits, deities, or other supernatural entities, typically for a specific purpose, such as seeking guidance, protection, or performing a ritual.

3. General Use: More broadly, summoning can refer to any situation where someone or something is called upon, such as summoning courage in a challenging situation or summoning help when needed.

In summary, summoning involves calling forth or bringing into presence someone or something, with a range of applications depending on the context in which it is used.

Chinese Explanation:

“召唤”这一术语通常指的是呼唤或引出某人或某物的行为，通常带有权威性或紧迫感。在不同的语境中，它可以有以下几种含义：

1. 法律语境： 在法律术语中，“召唤”可能指的是正式的通知或命令，要求某人出庭。传票通常由法院发出，要求个人对法律程序作出回应。

2. 灵性或神秘语境： 在灵性或神秘的实践中，“召唤”可能指的是引导或呼唤灵魂、神明或其他超自然存在，通常是为了特定的目的，如寻求指导、保护或进行仪式。

3. 一般用法： 更广泛地说，召唤可以指任何需要呼唤某人或某物的情况，例如在困难时刻召唤勇气，或在需要时召唤帮助。

总之，召唤涉及呼唤或引入某人或某物的行为，根据所用语境的不同，这一行为有着不同的应用。

bristle

Certainly! Here's a thorough explanation of the word "bristle" in both English and Chinese.

English Explanation

Bristle can be understood in several contexts:

1. Noun:
2. In its noun form, "bristle" typically refers to a short, stiff hair or fiber. For example, many animals have bristles on their bodies that can serve various purposes, including protection or sensory perception. Bristles are commonly found on the fur of pigs or the coat of some other animals.

3. Additionally, in the context of cleaning tools, like brushes, bristles refer to the stiff hairs or fibers that make up the brush and help in scrubbing or cleaning surfaces.

4. Verb:

5. As a verb, "to bristle" means to react defensively or with aggression, often in response to a perceived threat or insult. When someone "bristles," they may become tense or show signs of irritation or anger. For example, you might say, "She bristled at the critique," meaning she reacted negatively or defensively to the criticism.

In summary, "bristle" can refer to both physical hair-like structures in animals or brushes, as well as a metaphorical reaction to conflict or provocation.

Chinese Explanation

bristle（刚毛） 这个词可以从多个方面来理解：

1. 名词：
2. 在名词形式中，“bristle”通常指的是短而硬的毛发或纤维。例如，许多动物的身体上都有刚毛，能够起到各种用途，包括保护作用或感知功能。刚毛通常可以在猪的毛发或其他某些动物身上找到。

3. 此外，在清洁工具的背景下，如刷子，刚毛指的是构成刷子的硬毛或纤维，能够帮助擦洗或清洁表面。

4. 动词：

5. 作为动词时，“to bristle”意味着在面对感觉到的威胁或侮辱时采取防御性或具有攻击性的反应。当某人“bristles”时，他们可能会变得紧张，或表现出不满或愤怒的迹象。例如，你可以说：“她对批评感到愤怒”，意思是她对批评的反应是负面或者防御性。

总之，“bristle”可以指动物或刷子上的物理毛发结构，也可以比喻人们在冲突或挑衅时的反应。

pillagers

The term "pillagers" refers to individuals or groups who engage in pillaging, which is the act of raiding and plundering, often in a violent manner. Historically, pillagers are often associated with armies or marauding bands that invade towns or settlements to steal valuables, loot resources, and cause destruction. The act of pillaging is typically characterized by the use of force and violence, and those who pillage may be motivated by greed, the desire for power, or the need for resources.

Pillaging has been a common practice throughout history, particularly during times of war or social unrest when law and order may break down. Examples of pillaging can be found in numerous historical contexts, such as during the Viking invasions, the conquests of various empires, and more recently in the context of civil wars or revolutions.

In a broader sense, "pillagers" can also refer to anyone who takes advantage of others' misfortunes for their own benefit, whether through legal or illegal means.

“掠夺者”这个词是指那些进行掠夺的人或团体，掠夺是指以暴力方式进行突袭和抢劫的行为。历史上，掠夺者通常与军队或强盗团伙有关，他们侵入城镇或定居点，盗窃贵重物品、掠夺资源并造成破坏。掠夺的行为通常以使用武力和暴力为特征，掠夺者可能出于贪婪、权力欲或对资源的需求而进行掠夺。

掠夺在历史上是一种常见的行为，尤其是在战争或社会动荡时期，法律和秩序可能会崩溃。掠夺的例子在众多历史背景中都可以找到，例如在维京人的入侵、各个帝国的征服，以及更近期的内战或革命。

从更广泛的意义上说，“掠夺者”也可以指任何在他人不幸中利用他人以获取自身利益的人，无论是通过合法还是非法的手段。

caterpillar

English Explanation:

The term "caterpillar" refers to the larval stage of certain insects, particularly members of the order Lepidoptera, which includes butterflies and moths. Caterpillars are known for their distinct elongated bodies, which are often cylindrical and segmented. They typically have a number of prolegs, which help them to hold onto surfaces and aid in locomotion.

Caterpillars primarily feed on leaves and are critical to the ecosystem as herbivores. They play a significant role in plant population control and serve as a food source for many predators, including birds and small mammals.

In the life cycle of a butterfly or moth, the caterpillar stage comes after the egg stage and precedes the pupal stage (chrysalis or cocoon), before ultimately transforming into an adult insect. This transformation involves a process called metamorphosis, where the caterpillar undergoes significant physical changes.

Caterpillars also have various adaptations for survival, such as camouflage to blend in with their surroundings to avoid predators, the ability to produce defensive chemicals, and in some species, bright coloration to signal toxicity.

Chinese Explanation:

“毛虫”是指某些昆虫的幼虫阶段，特别是鳞翅目（包括蝴蝶和蛾）的成员。毛虫以其独特的细长身体而闻名，通常呈圆柱形并且分段。它们通常有多个附肢，这些附肢称为“虚肢”，帮助它们抓住表面并促进移动。

毛虫主要以叶子为食，并且作为食草动物对生态系统至关重要。它们在植物种群控制中扮演着重要角色，并为许多捕食者（包括鸟类和小型哺乳动物）提供食物来源。

在蝴蝶或蛾的生命周期中，毛虫阶段是在卵阶段之后，并在蛹阶段（蛹或茧）之前，最终转变为成虫。这一转变涉及称为变态的过程，在这一过程中，毛虫经历重大的生理变化。

毛虫也具备各种生存适应能力，例如伪装以与环境融为一体，以避开捕食者，产生防御性化学物质的能力，以及在某些物种中，鲜艳的色彩用以信号警告其有毒性。

a sitting target for freeloaders.

English Explanation

The phrase "a sitting target for freeloaders" describes a situation or person that is easy for others to take advantage of, particularly those who seek to obtain something without giving anything in return.

• "sitting target": This expression comes from hunting, where a "sitting target" is an easy target to hit because it isn't moving. In a metaphorical sense, it implies something or someone that is vulnerable or unprotected, making it straightforward for others to exploit or attack.

• "freeloaders": This term refers to individuals who take advantage of others' generosity or resources without contributing anything of equal value themselves. Freeloaders often rely on the goodwill or resources of others to meet their needs without any reciprocation.

In combination, the phrase suggests that there is an individual or situation that is particularly susceptible to being exploited by those who want to benefit without putting in effort or resources of their own.

中文解释

短语“a sitting target for freeloaders”描述了一个人或一种情况，容易被他人利用，特别是那些寻求获取某种东西而不付出任何回报的人。

• “sitting target”（静态目标）：这个表达来源于狩猎，在狩猎中，"sitting target"是指一个静止不动的猎物，很容易被击中。从隐喻的角度来看，它意味着某物或某人处于脆弱或没有保护的状态，使得他人可以轻易地利用或攻击。

• “freeloaders”（搭便车者）：这个术语指的是那些利用他人慷慨或资源，而自己并没有付出同等价值的人。搭便车者通常依赖他人的善意或资源来满足自己的需求，而不作任何回报。

综上所述，这个短语暗示有某个个人或情况特别容易受到那些想要在不付出努力或资源的情况下获利的人的剥削。

a central hoard

English Explanation

The term "central hoard" refers to a collection or accumulation of valuable items, resources, or artifacts that are stored in a central location, typically for the purpose of protection, preservation, or controlled distribution. This concept is often used in various contexts:

1. Archaeology: In archaeological studies, a central hoard might refer to a cache of ancient artifacts or treasures found together, suggesting that they were significant during the time they were buried or hidden.

2. Economics: In economic terms, a central hoard can refer to a reserve of financial assets or commodities maintained by a central authority, such as a government or a central bank, often to stabilize the economy.

3. Gaming/Literature: In the context of games or storytelling, a central hoard could symbolize a treasure trove that plays a critical role in the plot, often representing wealth, power, or the culmination of a quest.

Overall, the idea of a "central hoard" emphasizes the strategic importance of centralized accumulation, whether it be for security, economic reasons, or narrative significance.

Chinese Explanation

“中央宝藏”这个词指的是在一个中心位置存放的有价值物品、资源或文物的集合或聚积，通常目的是为了保护、保存或控制分配。这个概念常常在不同的语境中出现：

1. 考古学：在考古研究中，“中央宝藏”可能指的是一起发现的古代文物或珍宝，暗示这些物品在它们被埋藏或藏匿的时期是重要的。

2. 经济学：在经济学中，中央宝藏可以指由中央权威（例如政府或中央银行）维护的一种金融资产或商品的储备，通常用于稳定经济。

3. 游戏/文学：在游戏或故事叙述的背景中，中央宝藏可以象征着在情节中发挥重要作用的财富，通常代表着财富、权力或一场任务的高潮。

总的来说，“中央宝藏”这个概念强调了集中积累的战略重要性，无论是出于安全、经济原因，还是叙事的意义。

a wide catchment area

Explanation in English:

The phrase "a wide catchment area" refers to a broad geographical zone or region from which an organization, service, or institution attracts its participants, customers, or clients. The term "catchment area" is often used in contexts such as education, healthcare, or public services, where it indicates the area that a school, hospital, or service serves.

When we say "wide," it implies that the area is extensive, covering a large population or a significant geographical distance. For instance, a school with a wide catchment area may draw students from various nearby towns or neighborhoods, rather than being limited to the immediate vicinity.

In summary, "a wide catchment area" suggests a large and diverse region that contributes to the user base or clientele of a service or organization.

Chinese Translation:

“广泛的服务区域”这个短语指的是一个广泛的地理区域，在这个区域内，某个组织、服务或机构吸引其参与者、客户或顾客。“服务区域”这个术语通常用于教育、医疗或公共服务等场合，它表示某个学校、医院或服务所覆盖的区域。

当我们说“广泛”时，意味着该区域较为广阔，涵盖了大量的人口或广泛的地理距离。例如，一所具有广泛服务区域的学校可能会吸引来自不同城镇或社区的学生，而不仅限于周围的邻近地区。

总之，“广泛的服务区域”暗示着一个大而多样化的区域，这个区域对某个服务或组织的用户群体或客户群体有着重要的贡献。

specialist hangers-on

English Explanation

The term "specialist hangers-on" refers to individuals who align themselves with a particular expert, professional, or specialist in a specific field, often without having substantial expertise or credentials themselves. These "hangers-on" may be drawn to the expert for various reasons, including the desire to gain knowledge, influence, or status by association. They may try to stay close to the specialist to benefit from their knowledge or reputation, but they often do not contribute significantly or may even depend on the specialist for their opinions or decisions.

The term can carry a negative connotation, implying that these individuals are somewhat parasitic, relying on the expertise of someone else rather than developing their own skills or knowledge. The idea is that they lack the commitment or ability to achieve success in their own right, instead opting to extend their relevance and presence through their association with experts.

Chinese Explanation

“专家依附者”这个词指的是那些与某个在特定领域中拥有专业知识或技能的专家或专业人士保持联系，但自己并没有实质性专业知识或资历的人。这些“依附者”可能出于各种原因与专家建立关系，包括希望通过这种联系获取知识、影响力或地位。他们可能试图靠近专家，以便从他们的知识或声誉中获益，但通常没有显著的贡献，甚至可能依赖专家的观点或决策。

这个词语可能带有一种负面含义，暗示这些人有些像寄生虫，依赖别人的专业知识，而不是发展自己的技能或知识。这个概念的核心是，他们缺乏独立取得成功的能力或决心，而选择通过与专家的关系来延续自己的相关性和存在感。

menagerie

Certainly! The term "menagerie" refers to a collection of live animals, often kept for public display or entertainment. Historically, menageries were typically associated with traveling exhibitions that showcased exotic animals. These collections could include a variety of species, such as lions, tigers, bears, elephants, and other wildlife, both domestic and exotic.

In modern contexts, the term can sometimes be used metaphorically to describe a diverse or varied group of people or things. For instance, one might refer to a gathering of different personalities, ideas, or objects as a "menagerie" to highlight the diversity present.

Here’s a thorough explanation in Chinese:

在中文中，“动物园” (menagerie) 是指一群活的动物，通常是为了公众展示或娱乐而保存的。历史上，动物园通常与旅行展览有关，这些展览展示异国情调的动物。这些动物的集合可能包括各种物种，例如狮子、老虎、熊、大象以及其他野生动物，无论是家养的还是外来的。

在现代语境中，这个词有时也可以比喻地用来形容一组多样化或各异的人或事物。例如，人们可以称一个聚集着不同个性、想法或物件的场合为“动物园”，以突出其中的多样性。

In both English and Chinese, "menagerie" captures the essence of diversity and variety, whether in a literal sense referring to animals or in a metaphorical sense referring to different people or ideas.

formidable indeed

English Explanation:

The phrase "formidable indeed" is an expression used to convey a sense of admiration, respect, or fear towards someone or something that is impressively powerful, strong, or capable.

• "Formidable": This word means inspiring fear or respect through being impressively large, powerful, intense, or capable. For example, a formidable opponent in a competition would be someone who is exceptionally skilled or challenging to overcome.

• "Indeed": This word is used for emphasis, affirming what has just been stated. It suggests that what follows adds weight or importance to the previous statement.

When combined, "formidable indeed" emphasizes the extent to which something or someone is formidable. It might be used in contexts ranging from describing a challenging task, a respected leader, a significant obstacle, or an impressive achievement. The phrase underscores the seriousness and the level of challenge presented by the subject in question.

Chinese Translation:

短语 "formidable indeed" 的意思是用来表达对某人或某事的钦佩、尊重或敬畏之情，通常是因为它令人印象深刻、强大或有能力。

• "formidable"（可怕的）：这个词意味着因其令人敬畏的体积、力量、强度或能力而引起恐惧或尊重。例如，在竞争中，"formidable opponent"（强大的对手）指的是技能异常出众或挑战性极大的对手。

• "indeed"（确实）：这个词用于强调，确认刚刚陈述的内容。它表明接下来要说的内容对之前的陈述增加了分量或重要性。

将二者结合时，"formidable indeed" 就强调了某人或某事的可怕程度。可以用在描述一项具有挑战性的任务、一个受人尊敬的领导者、一个重要的障碍或一项令人印象深刻的成就等不同的上下文中。这个短语突出了所述对象所带来的严重性和所面临的挑战程度。

matricide

English Explanation of "Matricide"

The term "matricide" refers to the act of killing one's mother. The word is derived from the Latin roots "mater," meaning mother, and "cide," meaning the act of killing. Matricide can occur in various contexts, including psychological, social, and legal frameworks.

In psychological terms, matricide may be associated with complex family dynamics, where underlying issues such as trauma, abuse, or mental illness can lead to extreme actions. Socially, matricide is often viewed with significant moral and ethical weight, typically eliciting shock and horror in society at large due to the natural bond that exists between a mother and child.

Legally, matricide is treated as a serious crime and can carry severe penalties. The motivations and circumstances surrounding the act are often examined in court to determine the perpetrator's mental state and intent. Overall, the concept of matricide raises profound questions about familial relationships, morality, and the human psyche.

中文解释“弑母”

“弑母”一词指的是杀死自己的母亲。这个词源于拉丁语词根“mater”，意为母亲，和“cide”，意为杀戮的行为。弑母可以发生在不同的情境中，包括心理、社会和法律框架。

在心理学上，弑母可能与复杂的家庭动态有关，其中潜在的问题，如创伤、虐待或心理疾病，可能导致极端行为。在社会层面上，弑母通常被视为一个具有重大道德和伦理分量的事件，通常会引发公众的震惊与恐惧，因为母子之间存在自然的纽带。

在法律上，弑母被视为一种严重罪行，可能会带来严厉的惩罚。围绕这一行为的动机和情境常常在法庭上进行审查，以确定犯罪者的心理状态和意图。总体而言，弑母这一概念引发了有关家庭关系、道德和人类心理的深刻问题。

turns her against her own mother.

English Explanation

The excerpt "turns her against her own mother" indicates a situation in which an individual influences or manipulates someone to feel hostile or resentful towards their mother. This can occur due to various reasons, such as:

1. Manipulation by Others: Someone outside the mother-daughter relationship (e.g., a friend or relative) may provide negative information or opinions about the mother, causing the daughter to question her mother's intentions or feelings.

2. Conflicts of Interest: The individual might present a conflict that aligns with their interests, leading the daughter to perceive her mother as an opponent rather than a supporter.

3. Emotional Distress: The daughter may already have unresolved issues with her mother, and the influence of another person exacerbates these feelings, creating further division between them.

The overall effect is a deterioration of trust and affection in the mother-daughter relationship, which can lead to long-lasting emotional consequences.

Chinese Explanation

这段摘录“让她对自己的母亲产生反感”表明了一种情境，个体影响或操纵某人，使其对母亲感到敌意或怨恨。这种情况可能由于以下几种原因而发生：

1. 他人的操控：母女关系以外的某个人（例如朋友或亲戚）可能提供有关母亲的负面信息或观点，导致女儿质疑母亲的意图或情感。

2. 利益冲突：这位个体可能会提出一个与其利益一致的冲突，使女儿将母亲视为对立者，而不是支持者。

3. 情感困扰：女儿可能已经与母亲存在未解决的问题，而另一个人的影响加剧了这些情感，导致她们之间的关系进一步恶化。

整体效果是母女关系中的信任和亲密感的下降，这可能导致长期的情感后果。

woos her from deeply ingrained duties

English Explanation

The excerpt "woos her from deeply ingrained duties" implies a scenario where someone (likely a man) is trying to attract or win the affection of a woman who is tied to her long-standing responsibilities or obligations. The phrase "woos her" indicates a romantic pursuit, suggesting that the man is making efforts to charm or entice her. The term "deeply ingrained duties" suggests that these responsibilities are not just casual tasks but are deeply rooted in her life, possibly linked to tradition, family, or societal expectations. The idea here reflects a tension between personal desire and the weight of obligations, suggesting that the romance may challenge or conflict with her existing commitments. Ultimately, this excerpt captures the complexity of human relationships where love, duty, and personal aspirations intersect.

Chinese Explanation

这段摘录“将她从根深蒂固的责任中吸引出来”暗示了一个场景，在这个场景中，有人（可能是一个男人）试图吸引或赢得一个女性的芳心，而这个女性与她长期以来的责任或义务紧密相连。“将她吸引过来”表示了一种浪漫的追求，暗示这个男人在努力去魅惑或引诱她。“根深蒂固的责任”这一表述说明这些责任并非随意的任务，而是在她的生活中深深扎根，可能与传统、家庭或社会期望有关。这里的意思体现了个人欲望与责任重量之间的紧张关系，暗示这段浪漫可能会挑战或与她现有的承诺发生冲突。最终，这段摘录捕捉了人际关系的复杂性，在这里，爱情、责任和个人愿望交织在一起。

grabs die reins of her muscles,

Sure! Let's break down the excerpt "grabs die reins of her muscles" in both English and Chinese.

English Explanation:

The phrase "grabs die reins of her muscles" uses a metaphorical expression. Here are the components of the phrase:

1. "grabs" - This verb indicates a strong, assertive action. It suggests an active effort to take control or hold onto something.

2. "the reins" - This phrase typically refers to the straps used to control a horse. Metaphorically, "reins" symbolize control or mastery over something.

3. "of her muscles" - This part specifies what the "reins" are controlling: her muscles. The phrase suggests a connection between thought (or willpower) and the physical body.

When combined, the whole expression can imply that someone is taking control of their physical capabilities or is trying to exert discipline over their body. It conveys a sense of empowerment and control over one's strength or abilities.

Chinese Explanation:

这句“grabs die reins of her muscles”中的表达使用了隐喻。我们来拆解这一短语：

1. “grabs”（抓住） - 这个动词表示一种强有力的行为。它暗示着积极地努力控制或握住某样东西。

2. “the reins”（缰绳） - 这个短语通常指的是用于控制马匹的带子。在隐喻上，“缰绳”象征着对某事的控制或掌握。

3. “of her muscles”（她的肌肉） - 这部分具体说明了“缰绳”控制的是什么：她的肌肉。这个短语暗示了思想（或意志力）与身体之间的联系。

结合在一起，这整个表达可以暗示某人正在控制自己的生理能力或试图对自己的身体施加纪律。它传达了一种对个人力量或能力的掌控感和自信感。

In conclusion, this excerpt is rich in meaning, suggesting ways in which an individual may harness their strength or body through willpower and control.

总结来说，这段摘录意味深长，暗示个人可能通过意志力和控制来掌握自己的力量或身体。

It floods die brain of

The excerpt "It floods die brain of" appears to be a fragment and may have typographical errors. A likely intended phrase could be "It floods the brain of," which suggests a concept of overwhelming the mind or a significant influence on thought processes.

Explanation in English:

This phrase implies that something (likely an emotion, thought, or stimulus) is overwhelming or saturating someone's brain. The term "floods" indicates an excessive amount or intensity, akin to how a flood overwhelms an area with water. In this context, "the brain" refers to the cognitive processes associated with thinking, feeling, and reacting.

For example, one might say, "It floods the brain of a person with ideas," meaning that a rush of ideas is overwhelming for that individual, possibly leading to creativity or confusion. Such language is often used in discussions about cognitive overload, emotional experiences, or the impact of external stimuli.

Explanation in Chinese (中文解释):

这个短语“它淹没了……的大脑”暗示着某种东西（可能是情感、思想或刺激）正在淹没或充满某人的大脑。“淹没”这个词表示一种过量或强烈的状态，就像洪水淹没一个地方一样。在这种情况下，“大脑”指的是与思考、感觉和反应有关的认知过程。

例如，人们可能会说“它淹没了某人脑海中的想法”，这意味着大量的想法对那个人来说是压倒性的，可能导致创造力或混乱。这种语言通常用于讨论认知过载、情感体验或外部刺激的影响。

Since the original excerpt includes a possible typo ("die" instead of "the"), this analysis assumes a correction for clarity. If you have a specific context in mind or if you're looking for something different, please provide more details, and I would be happy to refine the explanation!

insidious

English Explanation

The term "insidious" refers to something that progresses in a gradual and subtle way but with harmful effects. It often denotes situations, actions, or traits that may appear harmless or even benign at first but ultimately lead to negative consequences. The term is frequently used to describe diseases, schemes, or behaviors that are deceptive and may not be immediately obvious to those affected. For instance, an "insidious disease" may slowly damage a person's health without showing clear symptoms until it is too late to treat effectively. In a broader context, insidious can also refer to malicious intent that is masked by a front of friendliness or normalcy, making it difficult for individuals to recognize the danger until it is too late.

Chinese Explanation (中文解释)

“insidious” 这个词指的是一种逐渐和微妙的发展方式，却带来有害的影响。它通常表示某些情况、行为或特质，起初可能看起来无害或轻微，但最终会导致负面的后果。这个词常常用于描述疾病、阴谋或行为，这些事情具有欺骗性，可能不会立即显现出对受影响者的危害。例如，“隐匿的疾病”（insidious disease）可能在没有明显症状的情况下慢慢损害一个人的健康，直到为时已晚，无法有效治疗。在更广泛的上下文中，insidious 也可以指被友好或正常的表象所掩盖的恶意意图，使得人们很难在危机来临之前识别出危险。

If you would like more information or examples involving the term "insidious," please let me know!

attuned

English Explanation

The term "attuned" is derived from the verb "attune," which means to bring into harmony or to adjust to a particular condition or awareness. When someone is described as "attuned," it suggests that they are in sync with their environment, emotions, or the feelings of others. This state of being attuned often involves a heightened sensitivity or awareness; for example, a person who is attuned to their own emotions may be very aware of their feelings and how these affect their behavior. Similarly, someone who is attuned to others may be skilled in understanding and responding to the emotions and needs of those around them.

In interpersonal contexts, being attuned can enhance communication and relationships, as it allows individuals to connect on a deeper level. It can also refer to being in tune with broader aspects, like cultural or societal norms, suggesting a level of understanding that goes beyond superficial awareness.

Chinese Explanation

“attuned”这个词来源于动词“attune”，意思是使和谐或调整到特定的状态或意识。当一个人被形容为“attuned”时，这表示他们与周围环境、情感或他人的感受保持一致。这种状态通常涉及一种更高的敏感性或意识；例如，能够了解自己情感的人可能对自己的感受非常敏感，并且意识到这些感受如何影响他们的行为。类似地，能够理解他人的人可能擅长于理解和回应他人的情感和需求。

在人际关系中，处于“attuned”状态可以增强沟通和关系，因为这使得个人能够在更深层次上连接。它也可以指与更广泛的方面（例如文化或社会规范）保持一致，暗示着一种超越肤浅意识的理解水平。

behest

English Explanation

The term "behest" refers to an authoritative order or command. It signifies a strong expression of will or desire, often coming from someone in a position of power. When someone acts "at the behest of" another, it means they are doing something because that person has requested or directed them to do so.

For example, if a manager asks an employee to complete a specific task "at their behest," it implies that the employee is obliged to follow the manager's request due to their higher authority. The word carries connotations of obligation and compliance, suggesting that the action is a response to an external authority rather than an independent choice.

Chinese Explanation

“behest” 这个词指的是一种权威的命令或指示。它表达了强烈的意志或愿望，通常来源于有权力的人。当某人“在别人的命令下”行动时，这意味着他们是在应他人的请求或指示进行某项工作。

例如，如果一位经理让一名员工“在其命令下”完成特定任务，这意味着员工由于经理的权威，需要遵从其要求。这个词带有义务和服从的含义，暗示这种行为是对外部权威的回应，而不是独立的选择。

caste

The term "caste" refers to a social stratification system found primarily in India and some other South Asian societies. It denotes a hierarchical division of society based on heredity, occupation, and social status. The caste system categorizes people into different groups, typically referred to as "castes," which are often associated with specific professions and social roles. The rigid nature of the caste system historically restricted social mobility and dictated many aspects of life, including marriage, social interactions, and economic opportunities.

The caste system is traditionally divided into four main categories, known as "varnas":

1. Brahmins: The priestly class responsible for religious rituals and spiritual knowledge.
2. Kshatriyas: The warrior class, responsible for protection and governance.
3. Vaishyas: The merchant class, involved in agriculture and trade.
4. Shudras: The working class, performing various labor tasks.

In addition to these broad categories, there are thousands of sub-castes, or "jatis," that reflect more specific social roles and regional variations.

In modern times, the caste system has been legally abolished in India, but its effects and influence remain significant in some areas, impacting social interactions and community dynamics. Efforts to promote equality and address caste-based discrimination continue to be central to social reform movements.

中文解释：

“种姓”一词指的是一种主要存在于印度和一些南亚社会的社会分层制度。它表示社会根据血缘、职业和社会地位进行的等级划分。种姓制度将人们划分为不同的组别，通常被称为“种姓”，这些种姓通常与特定的职业和社会角色相关联。种姓制度的严格性历史上限制了社会流动，并决定了生活的许多方面，包括婚姻、社会交往和经济机会。

种姓制度传统上分为四个主要类别，称为“瓦尔那”（varnas）：

1. 婆罗门（Brahmins）：负责宗教仪式和精神知识的祭司阶层。
2. 刹帝利（Kshatriyas）：负责保护和治理的战士阶层。
3. 商人（Vaishyas）：参与农业和贸易的商人阶层。
4. 首陀罗（Shudras）：执行各种劳动任务的工人阶层。

除了这些大类别外，还有成千上万的亚种姓（jatis），反映了更加具体的社会角色和地区差异。

在现代，印度已法定废除种姓制度，但其影响在某些地区依然显著，影响着社会交往和社区动态。促进平等和解决基于种姓的歧视的努力仍然是社会改革运动的重要组成部分。

The Insect Societies is Monomorium santschii.

English Explanation

The excerpt you provided refers to an insect species known as "Monomorium santschii." This species belongs to a larger group classified as "Insect Societies," which indicates that it may be a type of social insect. Social insects, such as ants, bees, and termites, live in complex communities organized by a social structure.

"Monomorium" is the genus name, which groups together species that share certain characteristics, and "santschii" is the specific species name that identifies this particular type of ant within the genus.

The term "Insect Societies" suggests that this ant species likely exhibits social behaviors, living in colonies or communities where individuals cooperate in tasks like foraging, caring for the young, and defending their nest. This organization is a significant aspect of their ecological role and vastly influences their survival and reproductive strategies.

Chinese Explanation

您提供的摘录提到了一种昆虫物种，名为“Monomorium santschii”。该物种属于更大的类别，称为“昆虫社会”，这表明它可能是一种社会性昆虫。社会性昆虫，例如蚂蚁、蜜蜂和白蚁，生活在根据社会结构组织的复杂社区中。

“Monomorium”是属名，它将共享某些特征的物种归为一类，而“santschii”是物种名称，用于辨识该属中的特定类型的蚂蚁。

“昆虫社会”这一术语表明，这种蚂蚁物种可能表现出社会行为，生活在群体或社区中，个体之间协作进行觅食、抚育幼虫和保护巢穴等任务。这种组织结构是它们生态角色的重要方面，对它们的生存和繁殖策略有着深远的影响。

coerce a stand-in.

English Explanation:

The phrase "coerce a stand-in" can be broken down into two components: "coerce" and "stand-in."

1. Coerce: This verb means to compel someone to do something by using force or threats. It implies a lack of voluntary choice in the matter, suggesting that the person being coerced is being pressured or manipulated into a situation or action against their will.

2. Stand-in: This term generally refers to a substitute or replacement for someone else. In various contexts, such as theater, film, or even everyday situations, a "stand-in" is someone who takes the place of another person temporarily. For example, a stand-in might replace an actor during rehearsals or when the principal actor is unavailable.

Putting it together, the phrase "coerce a stand-in" implies forcing or pressuring a substitute or a temporary replacement into a situation or to perform a specific action that they might not want to do. This could be relevant in contexts where someone is taking the place of another, and they are being pressured into performing duties or actions under duress.

中文解释：

短语“coerce a stand-in”可以拆分为两个部分：“coerce”（强迫）和“stand-in”（替代者）。

1. Coerce（强迫）：这个动词的意思是通过使用武力或威胁来迫使某人做某事。它暗示着在这一过程中缺乏自愿的选择，表明被强迫的人在一定程度上受到压力或操控，而不得不进入某种情况或采取某种行动。

2. Stand-in（替代者）：这个术语通常指的是替代或替换某人的人。在各种情境中，比如戏剧、电影，甚至日常生活中，“stand-in”是指暂时替代另一个人的人。例如，在排练中，替代者可能会在主要演员缺席时代替其出场。

结合在一起，短语“coerce a stand-in”意味着强迫或施压一个替代者进入某种情况或执行特定的行动，而这种行动他们可能并不愿意去做。这可能与某人在替代他人时受压迫去执行某些职责或行动的情景相关。

Odiers become queens who fly out to seek pastures new and royal heads yet unsevered

English Explanation:

This excerpt seems to evoke a metaphorical or poetic image. The term "Odiers" might refer to a specific group or character type—potentially those who are noble or significant in their society. When it states that they "become queens," it indicates a transformation or elevation in status, suggesting that these individuals are taking on a position of power and authority.

The phrase "fly out to seek pastures new" indicates a desire for exploration or the pursuit of new opportunities. The use of "fly" implies freedom and aspiration, suggesting that these queens—or perhaps women being elevated to royalty—are not constrained by their current circumstances and are actively seeking new horizons or environments that could provide growth or better circumstances.

The mention of "royal heads yet unsevered" further enriches the imagery. It suggests that there are still potential leaders or figures of authority who have not yet been metaphorically "cut off" or removed from their positions. This could imply a transitional phase, where new leaders are being sought, but the current ones have not yet been displaced or affected.

Overall, the excerpt conveys themes of empowerment, exploration, and the fluidity of power dynamics in leadership.

Chinese Explanation:

这段摘录似乎传达了一个隐喻或诗意的形象。“Odiers”可能指的是某个特定的群体或角色类型——可能是社会中显贵或重要的人。当它说他们“变成女王”时，暗示了一种转变或地位的提升，表明这些人正在承担一种权力和权威的角色。

“飞出去寻找新的牧场”这一短语表示渴望探索或追求新的机会。“飞”这个词暗示了自由和抱负，表明这些女王——或者说被提升为王室的女性——不受现状的限制，正在积极寻求新的地平线或能够提供成长或更好环境的地方。

提到“尚未被割首的王室头颅”进一步丰富了这个意象。这暗示着还有潜在的领导者或权威人物尚未在象征上被“切断”或从其位置上移除。这可以暗示一个过渡阶段，新领导者正在寻找中，但现任的还没有被取代或受到影响。

总体而言，这段文字传达了 empowerment（赋权）、探索和领导权力动态流动性等主题。

pastures

English Explanation

The term "pastures" refers to land used for grazing livestock. These areas are typically covered with grass or other vegetation, providing a natural source of food for animals such as cattle, sheep, and goats. Pastures can vary in size and quality, depending on various factors including soil type, climate, and management practices.

In agricultural practices, pastures are essential for sustainable livestock farming, as they allow animals to feed in a natural environment, which can lead to healthier livestock and less dependence on artificial feeds. Properly managed pastures contribute to soil health and biodiversity, and they can also play a role in carbon sequestration, helping to mitigate climate change.

The management of pastures includes practices such as rotational grazing, where livestock are moved between different pasture areas to prevent overgrazing, allowing vegetation to recover and maintain its productivity.

中文解释

“草地”一词指的是用于放牧牲畜的土地。这些区域通常覆盖着草或其他植物，为牛、羊和山羊等动物提供自然的食物来源。草地的大小和质量可以根据土壤类型、气候和管理方式等各种因素而有所不同。

在农业实践中，草地对于可持续的畜牧业至关重要，因为它们使动物能够在自然环境中进食，这可以导致更健康的牲畜，并减少对人工饲料的依赖。妥善管理的草地有助于土壤健康和生物多样性，而且也可以在碳封存方面发挥作用，有助于缓解气候变化。

草地的管理包括轮流放牧等实践，其中牲畜在不同的草地区域之间移动，以防止过度放牧，使植被得以恢复并保持其生产力。

artfully macabre understatement,

English Explanation:

The phrase "artfully macabre understatement" combines three distinct ideas:

1. Artfully: This word suggests that something is done with skill, creativity, and aesthetic appreciation. It implies a level of sophistication or intentionality in the way something is presented or created.

2. Macabre: This term relates to the grim or ghastly aspects of death and the supernatural. It often evokes feelings of horror or fascination, dealing with themes that are dark, eerie, or unsettling. In art and literature, "macabre" can denote works that explore the more sinister or morbid elements of the human experience.

3. Understatement: This refers to a figure of speech or a way of expressing something that deliberately downplays or minimizes the severity or significance of a situation or emotion. It contrasts with exaggeration and can create a subtle impact, allowing the audience to grasp the underlying meaning beneath the surface.

Putting it all together, "artfully macabre understatement" describes a creative expression or artistic approach that skillfully conveys dark or grim themes in a way that is subtle and restrained. It suggests an intentional choice to not overtly emphasize horror or death but instead to imply it through understatement, allowing for deeper engagement and interpretation by the audience.

Chinese Explanation:

短语“艺术性地阴森低调”结合了三个不同的概念：

1. 艺术性地（Artfully）：这个词意味着某事是以技巧、创造力和审美欣赏的方式完成的。它暗示着在某事的呈现或创作过程中有一定的复杂性或意图。

2. 阴森（Macabre）：这个词与死亡和超自然的阴暗或可怕方面有关。它通常唤起恐怖或迷恋的感觉，涉及黑暗、怪异或令人不安的主题。在艺术和文学中，“阴森”可以指探讨人类经历中更阴险或病态元素的作品。

3. 低调（Understatement）：这指的是一种修辞手法或表达方式，故意淡化或减轻情境或情感的严重性或重要性。它与夸张形成对比，可以创造一种微妙的影响，使观众能在表面之下领会潜在的意义。

将这些概念结合在一起，“艺术性地阴森低调”形容了一种创造性的表达或艺术方法，这种方式巧妙地以低调的方式传达黑暗或阴沉的主题。它暗示了故意选择不夸张地强调恐怖或死亡，而是通过低调来暗示，从而让观众更深入地参与和解读。

propensity

English Explanation

The term "propensity" refers to a natural tendency or inclination towards a particular behavior, action, or disposition. It implies that an individual or group has a predisposition to act in certain ways based on their characteristics, experiences, or environment. For example, someone might have a propensity for kindness if they frequently display compassionate behavior towards others. In psychological or sociological contexts, propensity can be used to study patterns of behavior and predict future actions.

In addition, "propensity" can be involved in various fields such as economics, where it might describe consumer behavior, or in medicine, where it could relate to an individual's likelihood of developing certain health conditions based on genetic or lifestyle factors.

中文解释

“倾向”这个词指的是一种自然的倾向或对特定行为、行动或性格的倾向。它意味着个体或群体根据其特征、经历或环境有某种行为上的倾向。例如，如果一个人经常对他人表现出同情行为，那么可以说他具有善良的倾向。在心理学或社会学的背景下，“倾向”可以用来研究行为模式并预测未来的行为。

此外，“倾向”还可以应用于各种领域，例如经济学中，它可能描述消费者行为，或者在医学中，它可能与个体基于遗传或生活方式因素开发某些健康状况的可能性有关。

to dispose of

Explanation in English:

The phrase "to dispose of" generally means to get rid of something, to eliminate, or to transfer ownership of something. It can refer to different contexts, such as disposing of waste (throwing it away), disposing of property (selling or giving it away), or disposing of assets (liquidating them for cash).

In legal terms, "to dispose of" can also refer to the act of settling or resolving a matter, such as disposing of a case in court. The phrase often implies a finality to the action taken, meaning that once something has been disposed of, it is no longer in one's possession or ownership.

Explanation in Chinese:

"to dispose of" 这个短语一般指的是处理某物，消除或转让某物的所有权。它可以在不同的上下文中使用，比如处理废物（扔掉），处理财产（出售或赠送）或处理资产（将其变现为现金）。

在法律术语中，"to dispose of" 还可以指解决或结束某个事务的行为，例如在法庭上解决案件。这个短语通常暗示所采取的行为具有最终性，意味着一旦某物被处理掉后，就不再属于某人或在某人的拥有之内。

Insect 'cuckoos' defy listing;

English Explanation

The phrase "Insect 'cuckoos' defy listing" refers to a group of insects that are metaphorically compared to cuckoo birds, which are known for their unique reproductive strategy of laying eggs in the nests of other birds. In this context, "cuckoos" likely denotes insects that exhibit behavior or characteristics that resemble this parasitic strategy, possibly by laying their eggs in or stealing resources from the nests or habitats of other insect species.

The term "defy listing" implies that these insects might be resisting classification or naming in a formal or scientific context. This could be due to their unique behaviors, which make them difficult to categorize, or perhaps because they are poorly understood or recognized in the scientific community. The excerpt suggests a challenge in documenting these insects, whether due to their elusive nature, hybrid characteristics, or emerging behaviors that do not fit into existing taxonomic frameworks.

Chinese Explanation

这段话中的“昆虫‘布谷鸟’挑战分类”指的是一类昆虫，这些昆虫在某种程度上与布谷鸟相类比，布谷鸟以其独特的繁殖策略著称，即将卵子产在其他鸟类的巢中。在这个背景下，“布谷鸟”可能是指那些表现出类似这种寄生行为的昆虫，可能是通过在其他昆虫的巢穴中产卵或偷取资源。

“挑战分类”一词意味着这些昆虫可能正在抵抗正式或科学分类的过程。这可能是因为它们的独特行为使得它们难以被归类，或者是因为科学界对它们的理解或认知较少。这段摘录暗示在记录这些昆虫方面存在挑战，这可能是由于它们难以捉摸的特性、混合特征或不符合现有分类框架的行为所致。

aptly

Explanation in English:

The word "aptly" is an adverb that means in a manner that is appropriate or suitable. It is used to describe actions, words, or behaviors that fit well with a particular context or situation. When something is done aptly, it is done in a way that is particularly fitting or relevant, enhancing communication or understanding.

For example: - If someone calls a very funny movie "hilarious," they are apt to describe it accurately, as "hilarious" is a word that aptly conveys the movie's humor. - In a discussion about environmental issues, using the term “sustainable” to describe practices that do not deplete resources would be an aptly chosen word.

The word derives from "apt," which has connotations of being suitable or appropriate. Therefore, using "aptly" implies that the subject has been cleverly and thoughtfully expressed in the right context.

Explanation in Chinese:

“aptly” 是一个副词，意思是以适当或合适的方式来做事情。它用来描述与特定情境或情况非常契合的行为、言辞或举止。当某件事情以“aptly”的方式进行时，它就以恰当的方式完成，增强了沟通或理解的效果。

例如： - 如果有人把一部非常搞笑的电影称为“搞笑”，那么这个描述就恰如其分，因为“搞笑”这个词恰当地传达了电影的幽默。 - 在讨论环境问题时，使用“可持续性”这个词来描述不会耗尽资源的做法就是一个合适的用词。

“aptly”源于“apt”，这个词有适合或恰当的含义。因此，使用“aptly”表明主题在正确的上下文中巧妙而深思熟虑地表达。

vertebrates

Certainly! Let's break down the term "vertebrates" and explain it in detail in both English and Chinese.

English Explanation:

Vertebrates are a subphylum of animals within the phylum Chordata. They are characterized by having a backbone or spinal column, which is formed from a series of vertebrae. This group includes a wide range of animals such as:

1. Mammals (e.g., humans, tigers, whales)
2. Birds (e.g., eagles, penguins, sparrows)
3. Reptiles (e.g., snakes, lizards, turtles)
4. Amphibians (e.g., frogs, salamanders)
5. Fish (e.g., sharks, salmon, goldfish)

Vertebrates are distinguished from invertebrates, which are animals that do not have a backbone. The backbone provides structural support, allows for greater movement, and protects the spinal cord, which is part of the nervous system. The evolutionary development of vertebrates has led to diverse adaptations that allow them to thrive in various environments, from deep oceans to high mountains.

Chinese Explanation:

脊椎动物是动物界的一个亚门，属于脊索动物门（Chordata）。它们的特征是具有脊柱或脊椎，这一结构由一系列脊椎骨组成。该组包括多种动物，如：

1. 哺乳动物（例如：人类、老虎、鲸鱼）
2. 鸟类（例如：鹰、企鹅、麻雀）
3. 爬行动物（例如：蛇、蜥蜴、乌龟）
4. 两栖动物（例如：青蛙、蝾螈）
5. 鱼类（例如：鲨鱼、三文鱼、金鱼）

脊椎动物与无脊椎动物相区分，无脊椎动物是指没有脊柱的动物。脊柱提供结构支持，允许更大的运动能力，并保护脊髓，脊髓是神经系统的一部分。脊椎动物的进化过程导致了多样的适应，使它们能够在从深海到高山等各种环境中生存。

Summary:

In summary, "vertebrates" refers to a major group of animals characterized by the presence of a backbone, which plays a crucial role in their structure and function. This group includes mammals, birds, reptiles, amphibians, and fish, all of which exhibit a wide range of adaptations to their environments.

总结一下，“脊椎动物”指的是以脊柱为特征的一个主要动物群体，脊柱在它们的结构和功能中起着关键作用。该群体包括哺乳动物、鸟类、爬行动物、两栖动物和鱼类，所有这些动物都有多样的适应能力以适应其环境。

besotted

English Explanation

The term "besotted" is an adjective used to describe someone who is so infatuated or deeply in love with someone else that it impairs their judgment or reason. When a person is besotted, they often exhibit extreme affection or devotion, and they may overlook flaws or negative traits in the object of their affection. The term carries a connotation of excessive or irrational love, suggesting that the person is almost blinded by their feelings. It can also imply a sense of obsession, where one’s emotions dominate their thoughts and actions.

Example Usage: - "Ever since John met Sarah, he has been completely besotted with her, making him forget all his commitments."

中文解释

“besotted”是一个形容词，用来描述一个人对另一个人非常迷恋或深深相爱的状态，以至于影响了他们的判断力或理智。当一个人“besotted”时，他们通常表现出极度的爱慕或专情，可能会忽视他们所爱之人身上的缺点或负面特质。这个词带有过度或不理性爱情的含义，暗示这个人几乎被自己的情感所蒙蔽。它还可以暗示一种痴迷的感觉，一个人的情感主导了他们的思想和行为。

举例说明： - “自从约翰遇见莎拉后，他对她完全着迷，以至于忘记了所有的责任。”

Note de synthèse : L'organisation des temps de l'enfant

CCTE Session#1 - audition 2 : "Organisation des temps des enfants" https://www.youtube.com/watch?v=g35R5sCToQo

Cette note de synthèse s'appuie sur les échanges de la session "Organisation des temps des enfants", mettant en lumière les perspectives d'Éric Charbonnier (analyste et expert en éducation à l'OCDE) et de Stéphanie Constant (maîtresse de conférence en psychologie du développement et de l'éducation).

Elle aborde les thèmes cruciaux des rythmes de vie des enfants, de l'équilibre entre temps scolaire, périscolaire et extrascolaire, et des enjeux liés à leur bien-être et à l'équité.

Thèmes centraux et idées clés

1. La définition et l'importance du temps de loisir pour l'enfant

• Stéphanie Constant insiste sur la nécessité de redéfinir le loisir non pas comme un simple "hors scolaire", mais comme un "temps à soi" pour l'enfant, un temps "libéré des contraintes" scolaires et domestiques.

Elle souligne que ce temps est souvent "organisé, planifié, bien rempli à l'avance par les adultes", ce qui limite la liberté et l'autonomie de l'enfant.

• Le jeu comme apprentissage informel : Le jeu est présenté comme un vecteur essentiel d'apprentissages informels, où l'enfant construit son cadre, prend des décisions et des initiatives. "Quand les enfants jouent ou quand ils sont qu'ils font des activités on va dire de loisirs non structurés plus autonome c'est porteur d'énormément d'apprentissage informel en fait".
• Les limites de l'approche éducative permanente : Une éducation permanente, même dans le temps de loisir, peut être "contreproductive justement pour le développement pour les apprentissages des enfants et bien sûr pour leur santé". Il est crucial de trouver un équilibre entre activités structurées (visant le développement de compétences spécifiques) et activités non structurées, plus libres.

2. L'équilibre entre temps scolaire et périscolaire : Le cas français

Éric Charbonnier et Stéphanie Constant abordent l'articulation complexe entre le temps scolaire et le temps périscolaire, en particulier en France.

• La journée scolaire française : La France se distingue par "la journée d'école la plus longue de tous les pays de l'OCDE" pour les enfants de 3 à 11 ans. Cette intensité nécessite que le temps périscolaire serve à "recharger les batteries", "souffler", "s'amuser" et développer des compétences non travaillées à l'école.
• Objectifs du système éducatif : Au-delà des fondamentaux (lire, compter, écrire), l'école doit permettre aux enfants de "développer tout un ensemble de compétences qui vont leur permettre de s'épanouir", comme la coopération, la confiance en soi, la motivation.
• Le débat sur les 4 jours/4 jours et demi : Ce débat récurrent en France est jugé "spécifique" et souvent influencé par des "décisions des adultes sur l'intérêt des enfants", notamment le budget des municipalités.

Bien que les études ne montrent pas de différences significatives en termes de résultats scolaires entre les deux systèmes, cela s'explique par un manque d'exploitation qualitative des matinées supplémentaires. "Ce n'est pas uniquement le nombre d'heures qui assure la réussite éducative". * Les devoirs à la maison : L'aide aux devoirs est jugée plus efficace pendant le temps scolaire et par les enseignants, afin de libérer le temps périscolaire pour des activités non scolaires.

3. Les inégalités et les solutions innovantes

La question des inégalités est transversale aux discussions, qu'il s'agisse de l'accès aux loisirs ou de la réussite scolaire.

• Inégalités d'accès aux loisirs : Les dispositifs d'accueil extrascolaire sont souvent vécus comme "contraignants" par les enfants, qui n'y vont "rarement par choix". Les professionnels, malgré leurs intentions, tendent à privilégier les activités structurées dans une "perspective éducative", même pendant les vacances.
• L'émancipation adolescente : À l'adolescence, on observe un désengagement des activités structurées (clubs sportifs, culturels) en raison de la "supervision adulte". Il est crucial de proposer des alternatives pour éviter l'isolement ou les pratiques à risque.
• Dispositifs pour réduire les inégalités :La "boîte à jouer" (Playbox) : Un dispositif peu coûteux et peu utilisé, basé sur des objets de récupération, qui favorise l'imagination, dépasse les stéréotypes de genre et les catégories d'âge, encourage la mixité et la solidarité.
• Les terrains d'aventure : Des espaces extérieurs en construction perpétuelle, souvent implantés dans des quartiers défavorisés, permettant aux enfants de bricoler, d'utiliser des outils et de développer une "pédagogie du risque". Ils sont accessibles et favorisent le lien social. "Les terrains d'aventure ça mériterait d'être plus connu et surtout d'être développé".
• Le rôle des parents : Même une courte interaction (10-20 minutes) comme la lecture ou le jeu a un "impact considérable" sur le développement des enfants. Il est essentiel d'encourager les parents sans les culpabiliser, en valorisant diverses formes d'interactions.

4. L'adaptation au changement climatique et les rythmes internationaux

La discussion s'élargit aux défis environnementaux et aux comparaisons internationales des systèmes éducatifs.

• Infrastructures scolaires : L'adaptation des temps de l'enfant au changement climatique implique de "repenser nos établissements et repenser comment on peut les moderniser", notamment face aux fortes chaleurs.

• Comparaison des vacances scolaires : La France a plus de vacances scolaires que la moyenne de l'OCDE, mais ce sont surtout les "vacances intermédiaires" qui sont plus longues (8 semaines, un "record du monde" selon Éric Charbonnier), et non les vacances d'été.

• Modèles éducatifs internationaux : Les pays modèles (Finlande, Estonie, Australie, Royaume-Uni) allient "qualité, équité sociale et bien-être".

Ils se distinguent par des enseignants mieux préparés pédagogiquement, des classes moins chargées et des rythmes plus adaptés. Le programme scolaire ne doit pas "dicter les décisions" et le travail des personnels.

• Santé mentale des jeunes : La santé mentale est une "préoccupation internationale" croissante, exacerbée par des événements comme la pandémie de COVID-19.

Il est nécessaire de collecter davantage de données et de mettre en place des dispositifs pour améliorer le bien-être psychologique des jeunes.

5. Recommandations et perspectives

• Approche qualitative et budgétisation : Toute réforme des rythmes doit être pensée en termes de "qualité d'éducation" et être "budgétisée" pour éviter les retours en arrière.

• Flexibilité et adaptation territoriale : Les rythmes doivent être flexibles et adaptés aux spécificités territoriales et saisonnières.

• Remettre l'enfant au cœur : Les "pédagogies alternatives" qui placent l'enfant "au cœur des apprentissages" en respectant ses rythmes biologiques et son plaisir d'apprendre, comme les "écoles forestières", sont des pistes à explorer.

• Continuité scolaire-loisirs : Développer la coordination entre les acteurs éducatifs (PEDT) et des initiatives simples comme les pédibus pour combattre les inégalités d'accès aux activités extrascolaires.

• Expérimentation et évaluation : Les réformes devraient passer par des phases d'"expérimentation et d'évaluation" pour assurer leur pérennité et éviter les revirements politiques fréquents.

Différencier les réflexions "en fonction des niveaux d'éducation et de l'âge des enfants" est également crucial.

• En conclusion, la réflexion sur les temps de l'enfant en France est complexe et doit dépasser les seuls considérations horaires ou budgétaires pour se centrer sur le bien-être global, l'autonomie et l'équité des opportunités pour chaque enfant.

Note d'information détaillée : Le bien-être et les droits des enfants

CCTE Session#1 - audition 1 : "Bien-être et droits des enfants" https://www.youtube.com/watch?v=E0_tjkDN4Ug

Ce document synthétise les thèmes principaux, les idées essentielles et les faits marquants issus des extraits de la session d'audition "Bien-être et droits des enfants" de la Convention Citoyenne sur les Temps de l'Enfant (CCTE), ainsi que de l'intervention de la Ministre de l'Éducation Nationale.

Il intègre des citations directes pour illustrer les points clés.

Introduction et Cadre de la Convention

• La Convention Citoyenne sur les Temps de l'Enfant (CCTE) est une initiative lancée par le Premier ministre, demandant au Conseil économique, social et environnemental (CE) de "structurer au mieux les temps de la vie quotidienne des enfants pour favoriser leurs apprentissages, leur développement et préserver leur santé" (Ministre Borne).

Ce processus vise à engager les citoyens dans une réflexion sur un sujet à la fois "intime et de société", en complémentarité avec le Parlement, afin de faire émerger une "intelligence collective libérée des logiques partisanes" (Ministre Borne).

Les discussions doivent se concentrer sur l'ensemble des temps de l'enfant, y compris le temps périscolaire et extrascolaire, la nécessité de repenser le contenu et la qualité de ces temps, et non seulement leur durée.

La première session d'audition a réuni trois experts :

• Grégoire Borst, Professeur de psychologie du développement et de neurosciences cognitives de l'éducation, directeur du laboratoire psychologie du développement et de l'éducation de l'enfant. Ses travaux portent notamment sur les inégalités sociales et éducatives.
• Sophie Marinopoulos, Psychologue et psychanalyste spécialiste de l'enfance et de la famille, fondatrice de l'association "Les Pâtes au Beurre", axée sur le bien-être relationnel.
• Éric Delemar, Défenseur des Enfants, adjoint à la Défenseure des droits, dont la mission est de défendre et faire connaître les droits des enfants.

Thème 1 : Santé et Bien-être des Enfants et Adolescents

• Constats alarmants : La santé mentale des enfants et adolescents, particulièrement celle des adolescents, s'est "considérablement dégradée" au cours des dix dernières années. L'adolescence est une période de risque accrue en raison de la sensibilité du cerveau au stress et à l'anxiété.

• Augmentation des symptômes dépressifs : "On était autour de 15 % de la population adolescente qui présentait des symptômes dépressifs avant Covid, après Covid on est autour de 35 %" (Grégoire Borst). Il y a des différences marquées selon le sexe, avec "deux fois plus de symptômes dépressifs chez les femmes que chez les garçons" (Grégoire Borst).

• Impact du COVID-19 : La pandémie a eu un "impact direct [et] un facteur de risque aggravant pour la santé mentale de ces adolescents", principalement dû à l'"abandon en partie des relations sociales" et la fermeture des établissements scolaires (Grégoire Borst).
• Inégalités sociales : La dégradation de la santé mentale et les impacts de la fermeture des écoles pendant le COVID ont touché "beaucoup plus les enfants de milieux sociaux défavorisés que les enfants de milieux sociofavorisés" (Grégoire Borst).
• Sédentarité : La santé physique est également impactée par une "explosion de la sédentarité chez les enfants et chez les adolescents". Le périmètre de déplacement des enfants a considérablement diminué en 20 ans, passant de "entre 5 et 9 km autour du foyer à moins de 300 m" (Grégoire Borst), posant un "vrai problème de santé publique" (Grégoire Borst).
• Causes profondes et contexte : Sophie Marinopoulos souligne que la situation n'était pas optimale avant le COVID : "on n'était pas très en forme avant le Covid hein... on a une modernité qui ne va pas très bien avec le temps de l'enfance et aussi le temps des parents" (Sophie Marinopoulos).

Le bien-être est fondamentalement un "bien-être relationnel" (Sophie Marinopoulos).

Éric Delemar rappelle que dès 2018, le réseau européen des défenseurs des enfants alertait déjà sur les difficultés de santé mentale chez les pré-adolescents et adolescents.

La France, par le Défenseur des droits, avait déjà observé et fait des recommandations sur l'état de la pédopsychiatrie (baisse du nombre de pédopsychiatres, difficultés de la santé scolaire, etc.).

Rôle des enfants dans la prise de parole : Les enfants eux-mêmes ont exprimé leurs préoccupations : "nous pendant le Covid on nous a demandé on s'est inquiété pour la vie de nos grands-parents on on nous a dit qu'on était des réservoirs à Covid à angoisse et qu'on allait peut-être être responsable de la maladie de nos grands-parents on s'est inquiété pour le salaire et le travail des noss de nos parents qui s'est inquiété pour nous ?" (Éric Delemar, rapport 2021 co-réalisé avec Claire Hédon).

Thème 2 : Rythmes Biologiques et Temps de l'Enfant

• Manque d'adaptation de l'école : Il est clair que l'école "ne prend pas suffisamment en compte les problématiques les besoins physiologiques des enfants en premier lieu le sommeil" (Grégoire Borst).

Le sommeil est un "catalyseur extrêmement important y compris de la santé physique et de la santé mentale" (Grégoire Borst).

Déficit de sommeil généralisé : La population française souffre d'un déficit global de sommeil, particulièrement les enfants et surtout les adolescents, qui représentent "la population la plus vulnérable en terme de déficit de sommeil" (Grégoire Borst).

Une dette de sommeil prend "3 à 4 mois pour récupérer" (Grégoire Borst).

Recommandations concrètes :

• Sensibilisation des parents : Mettre en place un "vrai parcours de parentalité" dès la maternité pour informer les parents sur le développement biologique, affectif, cognitif et physiologique de l'enfant, notamment l'importance du sommeil (Grégoire Borst). Ce déficit de sommeil est plus important dans les milieux défavorisés.
• Sieste à l'école maternelle : Permettre aux enfants de maternelle de faire "une sieste d'au moins 2 heures" est un "facteur de réduction des inégalités" (Grégoire Borst).
• Décalage des horaires scolaires pour les adolescents : "Décaler la première heure de cours à 9h30 point" (Grégoire Borst).

Cette mesure, connue depuis 20 ans, est essentielle car "physiologiquement au moment où ils rentrent dans la puberté il y a un décalage du rythme de leur sommeil" (Grégoire Borst).

Ce décalage permettrait d'améliorer la santé mentale et les apprentissages, car le sommeil est crucial pour la mémorisation et la neuroplasticité. Cette réforme "coûte 0 €" (Grégoire Borst).

• Importance de la relation et du jeu : Sophie Marinopoulos insiste sur le "temps de la relation [qui] ne doit jamais être oublié" (Sophie Marinopoulos).

Il est essentiel pour les bébés de retrouver leurs parents au réveil et de vivre des séparations apaisées.

L'éducation à la séparation est vitale pour la sécurité interne de l'enfant.

Le jeu est fondamental : "quand il joue il construit sa vie interne il construit sa valeur il construit sa dignité il construit l'amour de lui-même il apprend à donner à recevoir il apprend l'échec il apprend la frustration il apprend à sublimer" (Sophie Marinopoulos).

• Droit au loisir et à l'ennui : Éric Delemar met en avant l'Article 31 de la Convention internationale des droits de l'enfant, qui garantit le droit au loisir et à la culture. "le jeu pour les enfants c'est un peu le travail pour les adultes" (Éric Delemar).

Il souligne le besoin de temps de repos et du "droit à l'ennui", citant l'exemple de Newton.

La société actuelle a du mal à tolérer l'ennui chez les enfants, voulant qu'ils soient "autonomes tout de suite tout de suite tout de suite" (Éric Delemar).

Thème 3 : Droits de l'Enfant, Protection et Écrans Lutte contre les violences : La protection contre les violences faites aux enfants est à l'origine du droit international des enfants.

"Sans doute que notre société irait mieux si on s'était mieux occupé de certains adultes quand ils avaient été enfants" (Éric Delemar).

Le concept d'"intérêt supérieur de l'enfant" est l'indissociabilité et l'interdépendance des droits (justice, loisir, protection, etc.) (Éric Delemar).

Malgré les efforts, les moyens ne sont pas "à la hauteur des enjeux des dégradations" (Éric Delemar). "les enfants ne sont pas des petitêt être humain ne veut pas dire petit droit et c'est pas parce qu'ils font 50 cm ou 50 kg de moins qu'on aura qu'on a le droit d'utiliser la force pour se faire obéir" (Éric Delemar).

Les "bonnes claques" ont des conséquences avérées sur le développement cérébral et la santé mentale.

Troubles de l'attention et carences relationnelles : La violence chez les enfants est souvent liée à des "troubles relationnels précoces" et un manque de "ressources internes" pour faire autrement que de passer à l'acte (Sophie Marinopoulos, citant Maurice Berger). L'accès aux mots est une "liberté énorme".

Les troubles de l'attention sont un "grand sujet de santé mentale" (Sophie Marinopoulos), car un enfant inattentif ne "se nourrit [pas] de ce qu'il regarde" et ne construit pas sa vie intérieure. La santé mentale n'est pas seulement le domaine des spécialistes, mais la "santé de nos relations" (Sophie Marinopoulos).

Impact des écrans : Les écrans sont un sujet de grande préoccupation pour les parents, qui se sentent "totalement dépassés" (Sophie Marinopoulos).

• L'écran n'est pas un parent : L'écran "veut l'enfant tout le temps il le veut tout à lui toute la journée le plus possible" (Sophie Marinopoulos), créant une addiction.
• Rapport "Les enfants et les écrans à la recherche du temps perdu" : Ce rapport de 29 recommandations préconise une approche multifactorielle, au-delà de la simple interdiction.
• Avant 3 ans : Pas d'exposition aux écrans, car cela a un "impact négatif sur le sommeil" de l'enfant (Grégoire Borst).
• Entre 3 et 6 ans : Pas d'écran seul, nécessite un accompagnement et une limitation du temps.
• Inégalités sociales face aux écrans : Les familles défavorisées, monoparentales ou avec des horaires décalés, sont plus susceptibles d'exposer leurs enfants aux écrans par manque d'alternatives de garde. Il ne faut pas "culpabiliser" ces familles, mais proposer des "alternatives dans la société" (Grégoire Borst).
• Alternatives et éducation : Il est nécessaire de "peupler l'espace public d'alternative aux écrans" (Grégoire Borst), par exemple en proposant des "wagons jeu" dans les trains (Grégoire Borst). L'éducation au numérique, notamment à l'école, est une "absolue nécessité" pour développer la pensée critique des enfants.
• Le rôle de la société adulte : La Ministre Borne souligne que "le temps de l'enfant est absorbé par le temps des écrans" et que "dès 11 ans la moitié des jeunes dort 2 heures de moins que nécessaire" (Ministre Borne).

Elle annonce la généralisation de la pause numérique au collège et milite pour une décision européenne interdisant l'accès aux réseaux sociaux aux moins de 15 ans.

Elle insiste sur le rôle des adultes : "si nous-même on passe notre temps sur nos écrans c'est sûr qu'on donne pas un bon exemple à notre jeunesse" (Ministre Borne).

La société doit encourager davantage d'interactions et d'échanges réels.

Conclusion et Perspectives

Changements de paradigme nécessaires :

• Lieux d'accueil petite enfance : Ne pas les considérer comme de simples "lieux de garde", mais comme des "lieux d'éveil" (Éric Delemar) où l'on peut jouer, être à l'extérieur, prendre des risques.

• Soutien à la parentalité : Doit être universel ("pour tous les parents") et non seulement destiné aux parents "défaillants" (Éric Delemar), avec des moyens adéquats pour les structures d'accueil et les écoles maternelles.

• Prise en compte de la parole des enfants : L'Article 12 de la CIDE est crucial. Il faut "donner le courage [aux enfants] de prendre la parole, de s'assurer qu'ils ne seront pas moqués humilié" (Éric Delemar).

• Remettre l'enfance au cœur des préoccupations : Accepter "l'enfance de nos enfants" (Sophie Marinopoulos), dans une société qui tend au "no kids". C'est un travail continu pour la "paix" interne et externe, en nourrissant les enfants pour qu'ils aient les "ressources internes" et ne recourent pas à la violence.

• Articulation des temps et inégalités : Grégoire Borst insiste sur la nécessité de "réfléchir en tout cas dans vos réflexions d'avoir toujours en tête la question de comment on articule les différents temps de l'enfant" (Grégoire Borst) (scolaire, périscolaire, extrascolaire).

L'absence de coordination entre ces temps est un "facteur d'inégalité scolaire extrêmement important" (Grégoire Borst).

Il faut des "politiques publiques qui permettent effectivement de façon de de réfléchir de façon globale à comment on réduit les inégalités dès les 1000 premiers jours" (Grégoire Borst).

La Ministre Borne réitère l'importance de la liberté des conventionnaires à "douter, de questionner, d'explorer, libre de bousculer les idées préconçues de formuler des propositions neuves" (Ministre Borne) et leur assure que leur travail sera pris en compte par le gouvernement.

La convention doit permettre de faire émerger des "points de convergence, des orientations claires, des solutions concrètes" (Ministre Borne) pour repenser et améliorer la vie des enfants en France.

the management thing seems so off and yet the part of me that wants to object to it is so right there with them about the experience of dealing with a colleague using it

https://dl.acm.org/doi/10.1145/3706598.3713564

The Meta AI app includes a Discover feed, a place to share and explore how others are using AI.

https://about.fb.com/news/2025/04/introducing-meta-ai-app-new-way-access-ai-assistant/

Tipo "El rincón del vago"?

https://reddit.com/r/typewriters/comments/1lrkpuy/whats_inside_your_typewriter/

"The Hermes 3000 Mk2 I picked up last Saturday had a purple Polish pencil (still sharp), a marble, a drawing pin, and a well-mouldy green Smartie."

also: - broken glass<br /> - ball bearings<br /> - receipts<br /> - picture of a cat - typewriter servicing receipt - eraser shavings - multiple safety razor blades<br /> - paper clips - disintegrated foam

I can understand both sides of this scenario. I understand that the professor just wants to help. He wants to give people back an ability they either once had or didn’t even have to begin with. On the other side I completely understand her perspective. I have a close friend that is deaf and has been majority of her life. She is very active in the deaf community and enjoys being a part of it. I think if she was given this opportunity to “fix” her hearing that she wouldn’t do it. She believes that she isn’t just “deaf” but that it has helped shape and make her who she is as a person. She talks about how sometimes it is really peaceful to just shut everything in the world out and be by herself to think. When she talked about how people see someone being “broken” if they are deaf, I understood what she was saying. Sometimes people automatically assume that if you have a disability there is something wrong with you and that you should feel bad. My son is autistic and I have had multiple people ask me how I handle it or how I’m “fixing” him. There is nothing to fix, everything about him is what makes him who he is. If he wasn’t autistic, he wouldn’t have the personality that he has. It isn’t something that is going to hold him back in life or something that he needs to change and fix.

Cocoon

Semiotic: The semiotic approach, also known as semiology, is a field of study that focuses on signs and how meaning is created and communicated through them.

Very true, even if I am Ecuadorian. All of the people from South American countries( Mexico, Peru, chile, Argentina etc relatively understand each other because of broadly similar conceptual map.

Colour in pictures of your favourite Disney characters on a rainy day.

How transitions are important in a speech or essay.

Make sure to assess your supporting points.

What your main point means.

Without an outline you'll lose credibility

This is a prime example that elaborates on why social justice is a nations effort and not just for social workers. There is maltreatment and discrimination amongst all forms of healthcare and political policies.

Should there be only one definition? Having one definition that everyone abides by deletes room for interpretation and adverse meanings.

This is extremely important in any field of work, not just social work. All forms of work should be to serve the people and give them the best life possible. However, that is not the society we live in today. Social workers may not discriminate but what if officers do? What if judges do?

How can one expect social workers alone to end discrimination, oppression, poverty, and other forms of injustice? These are thing that have been passed down generations and have been ongoing for so long. I believe this is a nations effort to be made and not just for social workers to aim to do. Policies have to change, position of power shifts has to change, and so much more. However, will there truly ever be an "end"?

interesting story

key elements ook bruikbaar voor vraagarticulatie / participatieve governance

zie ook: https://www.mediatechdemocracy.com/from-recommendations-to-implementation?utm_source=substack&utm_medium=email

david van reybrouck in actie! maar ook inspirerend voor tgl dingen/100 vragen

Apart from the formatting characters, this should be easy to do—the simplest OCR to ever implement.

mean x SQUARED

population variance

Version 2 of this preprint has been peer-reviewed and recommended by Peer Community in Genomics.<br /> See the peer reviews and the recommendation.

Moet aangepast worden

eher streichen

hier Fachbegriff predictive maintenance schon einführen

Sehr detaillierte Gliederung, aber schon recht umfangreich. Ggf. einige Aspekte nicht unter eigenen Überschriften verarbeiten, sondern in anderen Abschnitten mitdiskutieren.

nicht für die Praxis, sondern nur in Lernzusammenhängen

Was ist "neu"? Kannst du vielleicht auch weglassen.

eLife Assessment

This useful manuscript addresses some key molecular mechanisms on the neuroprotective roles of soluble TREM2 in neurodegenerative diseases. The study will advance our understanding of TREM2 mutations, particularly on the damaging effect of known TREM2 mutations, and also provides solid evidence why soluble TREM2 can antagonize Aβ aggregation.

Reviewer #1 (Public review):

In this manuscript, Saeb et al reported the mechanistic roles of the flexible stalk domain in sTREM2 function using molecular dynamics simulations. They have reported some interesting molecular bases explaining why sTREM2 shows protective effects during AD, such as partial extracellular stalk domain promoting binding preference and stabilities of sTREM2 with its ligand even in the presence of known AD-risk mutation, R47H. Furthermore, they found that the stalk domain itself acts as the site for ligand binding by providing an "expanded surface", known as 'Expanded Surface 2' together with the Ig-like domain. Also, they observed no difference in the binding free energy of phosphatidyl-serine with wild TREM2-Ig and mutant TREM2-Ig, which is a bit inconsistent with the previous report with experiment studies by Journal of Biological Chemistry 293, (2018), Alzheimer's and Dementia 17, 475-488 (2021), Cell 160, 1061-1071 (2015).

Reviewer #2 (Public review):

Significance:

TREM2 is an immunomodulatory receptor expressed on myeloid cells and microglia in the brain. TREM2 consists of a single immunoglobular (Ig) domain that leads into a flexible stalk, transmembrane helix, and short cytoplasmic tail. Extracellular proteases can cleave TREM2 in its stalk and produce a soluble TREM2 (sTREM2). TREM2 is genetically linked to Alzheimer's disease (AD), with the strongest association coming from an R47H variant in the Ig domain. Despite intense interest, the full TREM2 ligand repertoire remains elusive, and it is unclear what function sTREM2 may play in the brain. The central goal of this paper is to assess the ligand-binding role of the flexible stalk that is generated during the shedding of TREM2. To do this, the authors simulate the behavior of constructs with and without stalk. However, it is not clear why the authors chose to use the isolated Ig domain as a surrogate for full-length TREM2. Additionally, experimental binding evidence that is misrepresented by the authors contradicts the proposed role of the stalk.

Summary and strengths:

The authors carry out MD simulations of WT and R47H TREM2 with and without the flexible stalk. Simulations are carried out for apo TREM2 and for TREM2 in complex with various lipids. They compare results using just the Ig domain to results including the flexible stalk that is retained following cleavage to generate sTREM2. The computational methods are well-described and should be reproducible. The long simulations are a strength, as exemplified in Figure 2A where a CDR2 transition happens at ~400-600 ns. The stalk has not been resolved in structural studies, but the simulations suggest the intriguing and readily testable hypothesis that the stalk interacts with the Ig domain and thereby contributes to the stability of the Ig domain and to ligand binding. I suspect biochemists interested in TREM2 will make testing this hypothesis a high priority.

Comments on latest version:

The authors have addressed my critiques and carried out additional simulations, as requested. I would upgrade my assessment of the evidence to "solid."

Author response:

The following is the authors’ response to the original reviews

Public Review:

Review #1 (Public review):

Also, they observed no difference in the binding free energy of phosphatidyl-serine with wild TREM2-Ig and mutant TREM2-Ig, which is a bit inconsistent with the previous report with experiment studies by Journal of Biological Chemistry 293, (2018), Alzheimer's and Dementia 17, 475-488 (2021), Cell 160, 1061-1071 (2015).

We agree with the reviewer that our results do not fully recapitulate experimental findings and directly note this in the body of our work, particularly given the known limitations of free energy calculations in MD simulations, as outlined in the Limitations section. Our claim is that the loss-of-function effects of the R47H variant extend beyond decreased binding affinities which are likely due to variable binding patterns. We have also re-analyzed and highlighted statistically significant differences in interaction entropies. Ultimately, our claim is that mutational effects extend beyond experimentally confirmed differences in binding affinities.

Perhaps the authors made significant efforts to run a number of simulations for multiple models, which is nearly 17 microseconds in total; none of the simulations has been repeated independently at least a couple of times, which makes me uncomfortable to consider this finding technically true. Most of the important conclusions that authors claimed, including the opposite results from previous research, have been made on the single run, which raises the question of whether this observation can be reproduced if the simulation has been repeated independently. Although the authors stated the sampling number and length of MD simulations in the current manuscript as a limitation of this study, it must be carefully considered before concluding rather than based on a single run.

To address this comment, we have added numerous replicates to our simulations of WT and R47H (s)TREM2 without lipids and substantially increased the total simulation time. Each pure protein system now has six total microsecond-long technical replicates. The addition of replicates strengthens the validity of the work and allows us to make stronger novel conclusions than with one simulation alone, particularly for claims regarding the CDR2 loop and sTREM2 stalk.  In our models with phospholipids, running multiple independent biological replicates of the same system offers a more rigorous methodology than simply repeating simulations of the same docked model. This strategy allows us to sample several distinct starting configurations, thereby minimizing biases introduced by docking algorithms and single-model reliance.

sTREM2 shows a neuroprotective effect in AD, even with the mutations with R47H, as evidenced by authors based on their simulation. sTREM2 is known to bind Aβ within the AD and reduce Aβ aggregation, whereas R47H mutant increases Aβ aggregation. I wonder why the authors did not consider Aβ as a ligand for their simulation studies. As a reader in this field, I would prefer to know the protective mechanism of sTREM2 in Aβ aggregation influenced by the stalk domain.

Our initial approach for this study used Aβ as a ligand rather than phospholipids. However, we noted the difficulties in simulating Aβ, particularly in choosing relevant Aβ structures and oligomeric states (n-mers). We believe that phospholipids represent an equally pertinent ligand for TREM2, given its critical role in lipid sensing and metabolism. Furthermore, there is growing recognition in the AD research community of the need to move beyond Aβ and focus on other understudied pathological mechanisms.

In a similar manner, why only one mutation is considered "R47H" for the study? There are more server mutations reported to disrupt tethering between these CDRs, such as T66M. Although this "T66M" is not associated with AD, I guess the stalk domain protective mechanism would not be biased among different diseases. Therefore, it would be interesting to see whether the findings are true for this T66M.

In most previous studies, the mechanism for CDR destabilization by mutant was explored, like the change of secondary structures and residue-wise interloop interaction pattern. While this is not considered in this manuscript, neither detailed residue-wise interaction that changed by mutant or important for 'ligand binding" or "stalk domain".

These are both excellent points that deserve extensive investigation, although we note that our paper does include significant protein-protein and protein-ligand interaction mapping that encompasses both the CDR2 loop and stalk, analyses which were not performed in any previous papers. In a separate paper, we explored more detailed residue-wise interactions for the CDR2 loop (Lietzke et al., Alzheimer’s and Dementia, 2025). While R47H is the most common and prolific mutation in literature, an extensive catalog of other mutations is important to explore. To this end, we are currently preparing a separate publication that will explore a larger mutational library and include more detailed sTREM2 analyses. 

The comparison between the wild and mutant and other different complex structures must be determined by particular statistical calculations to state the observed difference between different structures is significant. Since autocorrelation is one of the major concerns for MD simulation data for predicting statistical differences, authors can consider bootstrap calculations for predicting statistical significance.

The addition of numerous replicates across systems negates potential effects from autocorrelation and allows us to include standard deviations to critically assess the validity of our claims.

Review #2 (Public review):

The authors state that reported differences in ligand binding between the TREM2 and sTREM2 remain unexplained, and the authors cite two lines of evidence. The first line of evidence, which is true, is that there are differences between lipid binding assays and lipid signaling assays. However, signaling assays do not directly measure binding. Secondly, the authors cite Kober et al 2021 as evidence that sTREM2 and TREM2 showed different affinities for Abeta1-42 in a direct binding assay. Unfortunately, when Kober et al measured the binding of sTREM2 and Ig-TREM2 to Abeta they reported statistically identical affinities (Kd = 3.8 {plus minus} 2.9 µM vs 5.1 {plus minus} 3.7 µM) and concluded that the stalk did not contribute measurably to Abeta binding.

We appreciate the reviewer’s insight and acknowledge the need to clarify our interpretation of Kober et al. (2021). We have adjusted how we cite Kober et al. and reframed the first paragraph in the second results section.

In line with these findings, our energy calculations reveal that sTREM2 exhibits weaker—but still not statistically significant—binding affinities for phospholipids compared to TREM2. These results suggest that while overall binding affinity might be similar, differences in binding patterns or specific lipid interactions could still contribute to functional differences observed between TREM2 and sTREM2.

The authors appear to take simulations of the Ig domain (without any stalk) as a surrogate for the full-length, membrane-bound TREM2. They compare the Ig domain to a sTREM2 model that includes the stalk. While it is fully plausible that the stalk could interact with and stabilize the Ig domain, the authors need to demonstrate why the full-length TREM2 could not interact with its own stalk and why the isolated Ig domain is a suitable surrogate for this state.

We believe that this is a major limitation of all computational work of TREM2 to-date, and of experimental work which only presents the Ig-like domain. This is extensively discussed in the limitations section of our paper and treated carefully throughout the text. We are currently working toward a separate manuscript that will represent the first biologically relevant model of full-length TREM2 in a membrane and will rigorously assess the current paradigm of using the Ig-like domain as an experimental surrogate for TREM2.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

(1) Perhaps the authors made significant efforts to run a number of simulations for multiple models, which is nearly 17 microseconds in total; none of the simulations has been repeated independently at least a couple of times, which makes me uncomfortable to consider this finding technically true. Most of the important conclusions that authors claimed, including the opposite results from previous research, have been made on the single run, which raises the question of whether this observation can be reproduced if the simulation has been repeated independently. Although the authors stated the sampling number and length of MD simulations in the current manuscript as a limitation of this study, it must be carefully considered before concluding rather than based on a single run.

To address this comment, we have added numerous replicates to our simulations of WT and R47H (s)TREM2 without lipids and substantially increased the total simulation time. Each pure protein system now has six total microsecond-long technical replicates. The addition of replicates strengthens the validity of the work and allows us to make stronger novel conclusions than with one simulation alone, particularly for claims regarding the CDR2 loop and sTREM2 stalk.  In our models with phospholipids, running multiple independent biological replicates of the same system offers a more rigorous methodology than simply repeating simulations of the same docked model. This strategy allows us to sample several distinct starting configurations, thereby minimizing biases introduced by docking algorithms and single-model reliance. 

(2) sTREM2 shows a neuroprotective effect in AD, even with the mutations with R47H, as evidenced by authors based on their simulation. sTREM2 is known to bind Aβ within the AD and reduce Aβ aggregation, whereas R47H mutant increases Aβ aggregation. I wonder why the authors did not consider Aβ as a ligand for their simulation studies. As a reader in this field, I would prefer to know the protective mechanism of sTREM2 in Aβ aggregation influenced by the stalk domain.

Our initial approach for this study used Aβ as a ligand rather than phospholipids. However, we noted the difficulties in simulating Aβ, particularly in choosing relevant Aβ structures and oligomeric states (n-mers). We believe that phospholipids represent an equally pertinent ligand for TREM2, given its critical role in lipid sensing and metabolism. Furthermore, there is growing recognition in the AD research community of the need to move beyond Aβ and focus on other understudied pathological mechanisms.

(3) In a similar manner, why only one mutation is considered "R47H" for the study? There are more server mutations reported to disrupt tethering between these CDRs, such as T66M. Although this "T66M" is not associated with AD, I guess the stalk domain protective mechanism would not be biased among different diseases. Therefore, it would be interesting to see whether the findings are true for this T66M.

(4) In most previous studies, the mechanism for CDR destabilization by mutant was explored, like the change of secondary structures and residue-wise interloop interaction pattern. While this is not considered in this manuscript, neither detailed residue-wise interaction that changed by mutant or important for 'ligand binding" or "stalk domain".

These are both excellent points that deserve extensive investigation, although we note that our paper does include significant protein-protein and protein-ligand interaction mapping that encompasses both the CDR2 loop and stalk, analyses which were not performed in any previous papers. In a separate paper, we explored more detailed residue-wise interactions for the CDR2 loop (Lietzke et al., Alzheimer’s and Dementia, 2025). While R47H is the most common and prolific mutation in literature, an extensive catalog of other mutations is important to explore. To this end, we are currently preparing a separate publication that will explore a larger mutational library and include more detailed sTREM2 analyses.

(5) The comparison between the wild and mutant and other different complex structures must be determined by particular statistical calculations to state the observed difference between different structures is significant. Since autocorrelation is one of the major concerns for MD simulation data for predicting statistical differences, authors can consider bootstrap calculations for predicting statistical significance.

The addition of numerous replicates across systems negates potential effects from autocorrelation and allows us to include standard deviations to critically assess the validity of our claims.

Reviewer #2 (Recommendations for the authors):

Major points:

(1) I encourage the authors to review Figure 5D and the text of section 2.7 from Kober et al 2021, which argued that "(t)he identical (within error) binding affinities indicated that the TREM2 Ig domain composes the majority (if not entirety) of the mAβ42 binding surface."

We appreciate the reviewer’s insight and acknowledge the need to clarify our interpretation of Kober et al. (2021). We have adjusted how we cite Kober et al and reframed the first paragraph in the second results section.

(2) The abstract and text need extensive revision to address the major concerns, which jeopardize the biological premise and significance of the work.

We have made changes to the abstract and text to reflect concerns and revisions.

(3) The title and abstract should change to reflect the contents of the paper. The authors do not directly measure lipid binding, nor are any of the computations done in a membrane environment. The authors do not measure anything in the brain.

We have modified the title to better reflect the content of the paper. The paper measures lipid binding in the form of free energy calculations and interaction maps.

Minor points:

(1) How does the conservation of the TREM2 stalk compare to the Ig domain as they relate to the TREM2 family?

While this study may inspire further exploration of other TREM receptors, we do not believe that our results extend to other TREM family members because of relatively low homology.

(2) Please show the locations of the glycosylation sites on a model in Figure 1 and discuss their potential contribution to the ligand binding surfaces.

N-linked glycosylation points are now noted on the sequence map of Figure 1 and updated in the text.

(3) There is an isoform of TREM2 that produces a secreted product that is similar to the sTREM2 produced by proteolysis. The authors should comment as to whether their findings would apply to secreted TREM2.

We have addressed this with a new line in the ‘Ideas and Speculation’ section.

(4) This sentence on p. 2, line 73 references a review, not a study:

This has been corrected.

(5) "Yet, one study suggested effective TREM2 stimulation by PLs may require co-presentation with other molecules, potentially reflecting the nature of lipoprotein endocytosis30"

This has been corrected.

(6) Is "inclusive" on line 88 a typo for inconclusive?

This has been corrected.

(7) "Further, there is a strong correlation between the levels of sTREM2 in the cerebrospinal fluid and that of Tau, however correlation with Aβ is inclusive"

This has been corrected.

eLife Assessment

This study convincingly demonstrates that odors evoke a feeding response in Drosophila, mediated by gustatory receptors and observed as a proboscis extension. The evidence is comprehensive, encompassing behavior, functional imaging and electrophysiology. This important results on the molecular and cellular basis of multimodal integration across olfaction and gustation will be of interest for the study of chemosensation, sensory biology, and animal behavior.

Reviewer #1 (Public review):

Summary:

Odor- and taste-sensing are mediated by two different systems, the olfactory and gustatory systems, and have different behavioral roles. In this study, Wei et al. challenge this dichotomy by showing that odors can activate gustatory receptor neurons (GRNs) in Drosophila to promote feeding responses, including the proboscis extension response (PER) that was previously thought to be driven only by taste. While previous studies suggested that odors can promote PER to appetitive tastants, Wei et al. go further to show that odors alone cause PER, this effect is mediated through sweet-sensing GRNs, and sugar receptors are required. The study also shows that odor detection by bitter-sensing GRNs suppresses PER. The authors' conclusions are supported by behavioral assays, calcium imaging, electrophysiological recordings, and genetic manipulations. The observation that both attractive and aversive odors promote PER leaves an open question as to why this effect is adaptive. Overall, the study sheds new light on chemosensation and multimodal integration by showing that odor and taste detection converge at the level of sensory neurons, a finding that is interesting and surprising while also being supported by another recent study (Dweck & Carlson, Sci Advances 2023).

Strengths:

(1) The main finding that odors alone can promote PER by activating sweet-sensing GRNs is interesting and novel.

(2) The study uses video tracking of the proboscis to quantify PER rather than manual scoring, which is typically used in the field. The tracking method is less subjective and provides a higher-resolution readout of the behavior.

(3) The study uses calcium imaging and electrophysiology to show that odors activate GRNs. These represent complementary techniques that measure activity at different parts of the GRN (axons versus dendrites, respectively) and strengthen the evidence for this conclusion.

(4) Genetic manipulations show that odor-evoked PER is primarily driven by sugar GRNs and sugar receptors rather than olfactory neurons. This is a major finding that distinguishes this work from previous studies of odor effects on PER and feeding (e.g., Reisenman & Scott, 2019; Shiraiwa, 2008) that assumed or demonstrated that odors were acting through olfactory neurons.

Weaknesses/Limitations:

(1) Many of the odor effects on behavior or neuronal responses were only observed at very high concentrations. Most effects seemed to require concentrations of at least 10^-2 (0.01 v/v), which is at the high end of the concentration range used in olfactory studies (e.g., Hallem et al., 2004), and most experiments in the paper used a far higher concentration of 0.5 v/v. It is unclear whether these are concentrations that would be naturally encountered by flies. In addition, it is difficult to compare the concentrations used for electrophysiology and behavior given that they are presented in solution versus volatile form.

(2) The timecourse of GRN activation by odors seems quite prolonged (and possibly delayed, depending on the exact timing of odor onset to the fly), and this timecourse is not directly compared with activation by tastes to determine whether it is a property of the calcium sensor or a real difference.

(3) While the overall effect of different conditions is tested using appropriate statistical methods, post-hoc tests are not always used to determine which specific groups are different from each other (e.g., which odors and concentrations elicit significant PER compared to air or mineral oil controls in Fig. 1; which odors show impaired responses without olfactory organs in Fig. 2A).

Discrepancies with previous studies:

These discrepancies are important to note but should not necessarily be considered "weaknesses" of the present study.

(1) It is not entirely clear why PER to odors alone has not been previously reported, especially as this study shows that it is a broad effect evoked by many different odors. Previous studies (Oh et al., 2021; Reisenman & Scott, 2019; Shiraiwa, 2008) tested the effect of odors on PER and only observed enhancement of PER to sugar rather than odor-evoked PER; some of these studies explicitly show no effect of odor alone or odor with low sugar concentration. In the Response to Reviewers, the authors propose that genetic background may explain discrepancies, but this is not discussed much in the paper itself. Differences in behavioral quantification (automated vs. manual scoring, quantification of PER duration versus probability) may also contribute.

(2) The calcium imaging data showing that sugar GRNs respond to a broad set of odors contrasts with results from Dweck & Carlson (Sci Adv, 2023) who recorded sugar neurons with electrophysiology and observed responses to organic acids, but not other odors. This discrepancy is mentioned in the Discussion but the underlying reason is not clear.

Reviewer #3 (Public review):

Summary:

Using flies, Kazama et al. combined behavioral analysis, electrophysiological recordings, and calcium imaging experiments to elucidate how odors activate gustatory receptor neurons (GRNs) and elicit a proboscis extension response, which is interpreted as a feeding response.

The authors used DeepLabCut v2.0 to estimate the extension of the proboscis, which represents an unbiased and more precise method for describing this behavior compared to manual scoring.

They demonstrated that the probability of eliciting a proboscis extension increases with higher odor concentrations. The most robust response occurs at a 0.5 v/v concentration, which, despite being diluted in the air stream, remains a relatively high concentration. Although the probability of response is not particularly high it is higher than control stimuli. Notably, flies respond with a proboscis extension to both odors that are considered positive and those regarded as negative.

The authors used various transgenic lines to show that the response is mediated by GRNs. Specifically, inhibiting Gr5a reduces the response, while inhibiting Gr66a increases it in fed flies. Additionally, they find that odors induce a strong positive response in both types of GRNs, which is abolished when the labella of the proboscis are covered. This response was also confirmed through electrophysiological tip recordings.

Finally, the authors demonstrated that the response increases when two stimuli of different modalities, such as sucrose and odors, are presented together, suggesting clear multimodal integration

Strengths:

The integration of various techniques, which collectively supports the robustness of the results.<br /> The assessment of electrophysiological recordings in intact animals, preserving natural physiological conditions.

Weaknesses:

Only highly concentrated odours are capable of evoking positive responses and, even then, the proportion remains relatively low.

The authors have incorporated my suggestions.

Author response:

The following is the authors’ response to the original reviews

Public Review:

Reviewer #1 (Public review): 

Summary: 

Odor- and taste-sensing are mediated by two different systems, the olfactory and gustatory systems, and have different behavioral roles. In this study, Wei et al. challenge this dichotomy by showing that odors can activate gustatory receptor neurons (GRNs) in Drosophila to promote feeding responses, including the proboscis extension response (PER) that was previously thought to be driven only by taste. While previous studies suggested that odors can promote PER to appetitive tastants, Wei et al. go further to show that odors alone cause PER, this effect is mediated through sweet-sensing GRNs, and sugar receptors are required. The study also shows that odor detection by bitter-sensing GRNs suppresses PER. The authors' conclusions are supported by behavioral assays, calcium imaging, electrophysiological recordings, and genetic manipulations. The observation that both attractive and aversive odors promote PER leaves an open question as to why this effect is adaptive. Overall, the study sheds new light on chemosensation and multimodal integration by showing that odor and taste detection converge at the level of sensory neurons, a finding that is interesting and surprising while also being supported by another recent study (Dweck & Carlson, Sci Advances 2023).

Strengths: 

(1) The main finding that odors alone can promote PER by activating sweet-sensing GRNs is interesting and novel.

(2) The study uses video tracking of the proboscis to quantify PER rather than manual scoring, which is typically used in the field. The tracking method is less subjective and provides a higherresolution readout of the behavior.

(3) The study uses calcium imaging and electrophysiology to show that odors activate GRNs. These represent complementary techniques that measure activity at different parts of the GRN (axons versus dendrites, respectively) and strengthen the evidence for this conclusion. 

(4) Genetic manipulations show that odor-evoked PER is primarily driven by sugar GRNs and sugar receptors rather than olfactory neurons. This is a major finding that distinguishes this work from previous studies of odor effects on PER and feeding (e.g., Reisenman & Scott, 2019; Shiraiwa, 2008) that assumed or demonstrated that odors were acting through olfactory neurons.

We appreciate the reviewer’s positive assessment of the novelty and significance of our work.

Weaknesses/Limitations: 

(1) The authors may want to discuss why PER to odors alone has not been previously reported, especially as they argue that this is a broad effect evoked by many different odors. Previous studies testing the effect of odors on PER only observed odor enhancement of PER to sugar (Oh et al., 2021; Reisenman & Scott, 2019; Shiraiwa, 2008) and some of these studies explicitly show no effect of odor alone or odor with low sugar concentration; regardless, the authors likely would have noticed if PER to odor alone had occurred. Readers of this paper may also be aware of unpublished studies failing to observe an effect of PER on odor alone (including studies performed by this reviewer and unrelated work by other colleagues in the field), which of course the authors are not expected to directly address but may further motivate the authors to provide possible explanations.

We appreciate the reviewer’s comment. We believe that the difference in genotype is likely the largest reason behind this point. This is because the strength varied widely across genotypes and was quite weak in some strains including commonly used w[1118] empty Gal4 and w[1118] empty spit Gal4 as shown in Figure1- figure supplement 3 (Figure S3 in original submission). However, given that we observed odor-evoked PER in various genotypes (many in main Figures and three in Figure1- figure supplement 3 including Drosophila simulans), the data illustrate that it is a general phenomenon in Drosophila. Indeed, although Oh et al. (2021) did not emphasize it in the text, their Fig. 1E showed that yeast odor evoked PER at a probability of 20%, which is much higher than the rate of spontaneous PER in many genotypes. Therefore, this literature may represent another support for the presence of odor-evoked PER. We have expanded our text in the Discussion to describe these issues.

Another possibility is our use of DeepLabcut to quantitatively track the kinematics of proboscis movement, which may have facilitated the detection of PER.

(2) Many of the odor effects on behavior or neuronal responses were only observed at very high concentrations. Most effects seemed to require concentrations of at least 10-2 (0.01 v/v), which is at the high end of the concentration range used in olfactory studies (e.g., Hallem et al., 2004), and most experiments in the paper used a far higher concentration of 0.5 v/v. It is unclear whether these are concentrations that would be naturally encountered by flies.

We acknowledge that the concentrations used are on the higher side, suggesting that GRNs may need to be stimulated with relatively concentrated odors to induce PER. Although it is difficult to determine the naturalistic range of odor concentration, it is at least widely reported that olfactory neurons including olfactory receptor neurons and projection neurons do not saturate, and exhibit odor identity-dependent responses at the concentration of 10^-2 where odor-evoked PER can be observed. Furthermore, we have shown in Figure 6 that low concentration (10^-4) of banana odor, ethyl butyrate, and 4-methycyclohexanol all significantly increased the rate of odor-taste multisensory PER even in olfactory organs-removed flies, suggesting that low concentration odors can influence feeding behavior via GRNs in a natural context where odors and tastants coexist at food sites. Finally, we note that odors were further diluted by a factor of 0.375 by mixing the odor stream with the main air stream before being applied to the flies as described in Methods.

(3) The calcium imaging data showing that sugar GRNs respond to a broad set of odors contrasts with results from Dweck & Carlson (Sci Adv, 2023) who recorded sugar neurons with electrophysiology and observed responses to organic acids, but not other odors. This discrepancy is not discussed.  

As the reviewer points out, Dweck and Carlson (Sci Adv, 2023) reported using single sensillum electrophysiology (base recording) that sugar GRNs only respond to organic acids whereas we found using calcium imaging from a group of axons and single sensillum electrophysiology (tip recording) that these GRNs respond to a wide variety of odors. Given that we observed odor responses using two methods, the discrepancy is likely due to the differences in genotype examined. We now have discussed this point in the text.

(4) Related to point #1, it would be useful to see a quantification of the percent of flies or trials showing PER for the key experiments in the paper, as this is the standard metric used in most studies and would help readers compare PER in this study to other studies. This is especially important for cases where the authors are claiming that odor-evoked PER is modulated in the same way as previously shown for sugar (e.g., the effect of starvation in Figure S4).

For starved flies, we would like to remind the reviewer that the percentage of trials showing PER is reported in Fig. 1E, which shows a similar trend as the integrated PER duration. For fed flies, we have analyzed the percentage of PER and added the result to Figure 2-figure supplement 1C (Figure S4 in original submission).

(5) Given the novelty of the finding that odors activate sugar GRNs, it would be useful to show more examples of GCaMP traces (or overlaid traces for all flies/trials) in Figure 3. Only one example trace is shown, and the boxplots do not give us a sense of the reliability or time course of the response. A related issue is that the GRNs appear to be persistently activated long after the odor is removed, which does not occur with tastes. Why should that occur? Does the time course of GRN activation align with the time course of PER, and do different odors show differences in the latency of GRN activation that correspond with differences in the latency of PER (Figure S1A)?

Following the reviewer’s suggestion, we now report GCaMP responses for all the trials in all the flies (both Gr5a>GCaMP and Gr66a>GCaMP flies), where the time course and trial-to-trial/animal-toanimal variability of calcium responses can be observed (Figure 3-figure supplement 2).

Regarding the second point, we recorded responses to both sucrose and odors in some flies and found that calcium responses of GRNs are long-lasting not only to odors but also to sucrose, as shown in Author response image 1. This may be due in part to the properties of GCaMP6s and slower decay of intracellular calcium concentration as compared to spikes.

Author response image 1.

Example calcium responses to sucrose and odor (MCH) in the same fly (normalized by the respective peak responses to better illustrate the time course of responses). Sucrose (blue) and odor (orange) concentrations are 100 mM, and 10^-1 respectively. Odor stimulation begins at 5 s and lasts for 2 s. Sucrose was also applied at the same timing for the same duration although there was a limitation in controlling the precise timing and duration of tastant application. Because of this limitation, we did not quantify the off time constant of two responses.

To address whether the time course of GRN activation aligns with the time course of PER, and whether different odors evoke different latencies of GRN activation that correspond to latencies of PER, we plotted the time course of GRN responses and PER, and further compared the response latencies across odors and across two types of responses in Gr5a>GCaMP6s flies. As shown in Author response image 2, no significant differences were found in response latency between the six odors for PER and odor responses. Furthermore, Pearson correlation between GRN response latencies and PER latencies was not significant (r = 0.09, p = 0.872).

Author response image 2.

(A) PER duration in each second in Gr5a-Gal4>UAS-GCaMP6s flies. The black lines indicate the mean and the shaded areas indicate standard error of the mean. n = 25 flies. (B) Time course of calcium responses (ΔF/F) to nine odors in Gr5a GRNs. n = 5 flies. (C) Latency to the first odor-evoked PER in Gr5a-Gal4>UAS-GCaMP6s flies. Green bar indicates the odor application period. p = 0.67, one-way ANOVA. Box plots indicate the median (orange line), mean (black dot), quartiles (box), and 5-95% range (bar). Dots are outliers. (D) Latency of calcium responses (10% of rise to peak time) in Gr5a GRNs. Green bar indicates the odor application period. p = 0.32, one-way ANOVA. Box plots indicate the median (orange line), mean (black dot), quartiles (box), and 5-95% range (bar). Dots are outliers.

(6) Several controls are missing, and in some cases, experimental and control groups are not directly compared. In general, Gal4/UAS experiments should include comparisons to both the Gal4/+ and UAS/+ controls, at least in cases where control responses vary substantially, which appears to be the case for this study. These controls are often missing, e.g. the Gal4/+ controls are not shown in Figure 2C-G and the UAS/+ controls are not shown in Figure 2J-L (also, the legend for the latter panels should be revised to clarify what the "control" flies are). For the experiments in Figure S5, the data are not directly compared to any control group. For several other experiments, the control and experimental groups are plotted in separate graphs (e.g., Figure 2C-G), and they would be easier to visually compare if they were together. In addition, for each experiment, the authors should denote which comparisons are statistically significant rather than just reporting an overall p-value in the legend (e.g., Figure 2H-L).

We thank the reviewer for the input. We have conducted additional experiments for four Gal4/+controls in Figure 2 and added detailed information about control flies in the figure legend (Figure 2C-F).

For the RNAi flies shown in Figure 2 and Figure 2-figure supplement 3, we used the recommended controls suggested by the VDRC. These control flies were crossed with tubulin-Gal4 lines to include both Gal4 and UAS control backgrounds.

Regarding Figure S5 in original submission (current Figure 2-figure supplement 2), we now present the results of statistical tests which revealed that PER to certain odors is statistically significantly stronger than that to the solvent control (mineral oil) for both wing-removed and wing-leg-removed flies.

For Figure 2C-F, we now plot the results for experimental and control groups side by side in each figure.

Regarding the results of statistical tests, we have provided more information in the legend and also prepared a summary table (supplemental table). 

(7) Additional controls would be useful in supporting the conclusions. For the Kir experiments, how do we know that Kir is effective, especially in cases where odor-evoked PER was not impaired (e.g., Orco/Kir)? The authors could perform controls testing odor aversion, for example. For the Gr5a mutant, few details are provided on the nature of the control line used and whether it is in the same genetic background as the mutant. Regardless, it would be important to verify that the Gr5a mutant retains a normal sense of smell and shows normal levels of PER to stimuli other than sugar, ruling out more general deficits. Finally, as the method of using DeepLabCut tracking to quantify PER was newly developed, it is important to show the accuracy and specificity of detecting PER events compared to manual scoring.  

A previous study (Sato, 2023, Front Mol Neurosci) showed that the avoidance to 100 μM 2methylthiazoline was abolished, and the avoidance to 1 mM 2MT was partially impaired in Orco>Kir2.1 flies. However, because Orco-Gal4 does not label all the ORNs and we have more concrete results on flies in which all the olfactory organs are removed as well as specific GRNs and Gr are manipulated, we decided to remove the data for Orco>kir2.1 flies and have updated the text and Figure 2 accordingly.

For the Gr5a mutant and its control, we have added detailed information about the genotype in the figure legend and in the Methods. We have used the exact same lines as reported in Dahanukar et al. (2007) by obtaining the lines from Dr. Dahanukar. Dahanukar et al. has already carefully examined that Gr5a mutant loses responses only to certain types of sugars (e.g. it even retains normal responses to some other sugars), demonstrating that Gr5a mutants do not exhibit general deficits.

As for the PER scoring method, we manually scored PER duration and compared the results with those obtained using DeepLabCut in wild type flies for the representative data. The two results were similar (no statistical difference). We have reported the result in Figure1-figure supplement 1C.

(8) The authors' explanation of why both attractive and aversive odors promote PER (lines 249-259) did not seem convincing. The explanation discusses the different roles of smell and taste but does not address the core question of why it would be adaptive for an aversive odor, which flies naturally avoid, to promote feeding behavior.  

We have extended our explanation in the Discussion by adding the following possibility: “Enhancing PER to aversive odors might also be adaptive as animals often need to carry out the final check by tasting a trace amount of potentially dangerous substances to confirm that those should not be further consumed.”

Reviewer #2 (Public review): 

Summary: 

A gustatory receptor and neuron enhances an olfactory behavioral response, proboscis extension. This manuscript clearly establishes a novel mechanism by which a gustatory receptor and neuron evokes an olfactory-driven behavioral response. The study expands recent observations by Dweck and Carlson (2023) that suggest new and remarkable properties among GRNs in Drosophila. Here, the authors articulate a clear instance of a novel neural and behavioral mechanism for gustatory receptors in an olfactory response.

Strengths: 

The systematic and logical use of genetic manipulation, imaging and physiology, and behavioral analysis makes a clear case that gustatory neurons are bona fide olfactory neurons with respect to proboscis extension behavior.

Weaknesses: 

No weaknesses were identified by this reviewer.  

We appreciate the reviewer’s recognition of the novelty and significance of our work.

Reviewer #3 (Public review): 

Summary: 

Using flies, Kazama et al. combined behavioral analysis, electrophysiological recordings, and calcium imaging experiments to elucidate how odors activate gustatory receptor neurons (GRNs) and elicit a proboscis extension response, which is interpreted as a feeding response. 

The authors used DeepLabCut v2.0 to estimate the extension of the proboscis, which represents an unbiased and more precise method for describing this behavior compared to manual scoring.

They demonstrated that the probability of eliciting a proboscis extension increases with higher odor concentrations. The most robust response occurs at a 0.5 v/v concentration, which, despite being diluted in the air stream, remains a relatively high concentration. Although the probability of response is not particularly high it is higher than control stimuli. Notably, flies respond with a proboscis extension to both odors that are considered positive and those regarded as negative.

The authors used various transgenic lines to show that the response is mediated by GRNs.

Specifically, inhibiting Gr5a reduces the response, while inhibiting Gr66a increases it in fed flies. Additionally, they find that odors induce a strong positive response in both types of GRNs, which is abolished when the labella of the proboscis are covered. This response was also confirmed through electrophysiological tip recordings.

Finally, the authors demonstrated that the response increases when two stimuli of different modalities, such as sucrose and odors, are presented together, suggesting clear multimodal integration.

Strengths: 

The integration of various techniques, that collectively support the robustness of the results.

The assessment of electrophysiological recordings in intact animals, preserving natural physiological conditions.

We appreciate the reviewer’s recognition of the novelty and significance of our work.

Weaknesses: 

The behavioral response is observed in only a small proportion of animals.  

We acknowledge that the probability of odor-evoked PER is lower compared to sucrose-evoked PER, which is close to 100 % depending on the concentration. To further quantify which proportion of animals exhibit odor-evoked PER, we now report this number besides the probability of PER for each odor shown in Fig. 1E. We found that, in wild type Dickinson flies, 73% and 68 % of flies exhibited PER to at least one odor presented at the concentration of 0.5 and 0.1.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors): 

Minor comments/suggestions: 

- Define "MO" in Figure 1D.  

We have defined it as mineral oil in the figure legend.

- Clarify how peak response was calculated for GCaMP traces (is it just the single highest frame per trial?).

We extended the description in the Methods as follows: “The peak stimulus response was quantified by averaging ΔF/F across five frames at the peak, followed by averaging across three trials for each stimulus. Odor stimulation began at frame 11, and the frames used for peak quantification were 12 to 16.” We made sure that information about the image acquisition frame rate was provided earlier in the text.

- Clarify how the labellum was covered in Figure 3 and show that this does not affect the fly's ability to do PER (e.g., test PER to sugar stimulation on tarsus) - otherwise one might think that gluing the labella could affect PER.

In Figure 3, only calcium responses were recorded, and PER was not recorded simultaneously from the same flies. To ensure stable recording from GRN axons in the SEZ, we kept the fly’s proboscis in an extended position as gently as possible using a strip of parafilm. In some of the imaging experiments, we covered the labellum with UV curable glue, whose purpose was not to fix the labellum in an extended position but to prevent the odors from interacting with GRNs on the labellum. We have added a text in the Methods to explain how we covered the labellum.

- Clarify how the coefficients for the linear equation were chosen in Figure 3G.  

We used linear regression (implemented in Python using scikit-learn) to model the relationship between neural activity and behavior, aiming to predict the PER duration based on the calcium responses of two GRN types, Gr5a and Gr66a. The coefficients were estimated using the LinearRegression function. We added this description to the Methods. 

- Typo in "L-type", Figure 4A.  

We appreciate the reviewer for pointing out this error and have corrected it.

- Clarify over what time period ephys recordings were averaged to obtain average responses.

We have modified the description in the Methods as follows: “The average firing rate was quantified by using the spikes generated between 200 and 700 ms after the stimulus contact following the convention to avoid the contamination of motion artifact (Dahanukar and Benton, 2023; Delventhal et al., 2014; Hiroi et al., 2002).

- The data and statistics indicate that MCH does not enhance feeding in Figure 6G, so the text in lines 207-208 is not accurate.

We have modified the text as follows: “A similar result was observed with ethyl butyrate, and a slight, although not significant, increase was also observed with 4-methylcyclohexanol (Figure 6G).”

- P-value for Figure S9 correlation is not reported.  

We appreciate the reviewer for pointing this out. The p-value is 0.00044, and we have added it to the figure legend (current Figure 5-figure supplement 1).

Reviewer #2 (Recommendations for the authors): 

Honestly, I have no recommendations for improvement. The manuscript is extremely well-written and logical. The experiments are persuasive. A lapidary piece of work.

We appreciate the reviewer for the positive assessment of our work.

Reviewer #3 (Recommendations for the authors): 

- I suggest explaining the rationale for selecting a 4-second interval, beginning 1 second after the onset of stimulation.

Integrated PER duration was defined as the sum of PER duration over 4 s starting 1 s after the odor onset. This definition was set based on the following data.

(1) We used a photoionization detector (PID) to measure the actual time that the odor reaches the position of a tethered fly, which was approximately 1.1 seconds after the odor valve was opened. Therefore, we began analyzing PER responses 1 second after the odor onset (valve opening) to align with the actual timing of stimulation.

(2) As shown in Fig.1D and 1F, the majority of PER occurred within 4 s after the odor arrival.

We have now added the above rationale in the Methods.

- I could not find the statistical analysis for Figures 1E and 1G. If these figures are descriptive, I suggest the authors revise the sentences: 'Unexpectedly, we found that the odors alone evoked repetitive PER without an application of a tastant (Figures 1D-1G, and Movie S1). Different odors evoked PER with different probability (Figure 1E), latency (Figure S1A), and duration (Figures 1F, 1G, and S2)'.

We have added the results of statistical analysis to the figure legend.

- In Figure 2, the authors performed a Scheirer-Ray-Hare test, which, to my knowledge, is a nonparametric test for comparing responses across more than two groups with two factors. If this is the case, please provide the p-values for both factors and their interaction

We now show the p-values for both factors, odor and group as well as their interaction in the supplementary table. 

- In line 83, I suggest the authors avoid claiming that 'these data show the olfactory system modulates but is not required for odor-evoked PER,' as they are inhibiting most, but not all olfactory receptor neurons. In this regard, is it possible to measure the olfactory response to odors in these flies?  

We thank the reviewer for the comment. Because Orco-Gal4 does not label all the ORNs and because we have more concrete results on flies in which all the olfactory organs are removed as well as specific GRNs and Gr are manipulated, we decided to remove the data for Orco>kir2.1 flies and have updated the text and Figure 2 accordingly.

- In Figure 2, I wonder if there are differences in the contribution of various receptors in detecting different odors. A more detailed statistical analysis might help address this question.

Although it might be possible to infer the contribution of different gustatory receptors by constructing a quantitative model to predict PER, it is a bit tricky because the activity of individual GRNs and not Grs are manipulated in Figure 2 except for Gr5a. The idea could be tested in the future by more systematically manipulating many Grs that are encoded in the fly genome.

- For Figures 2J-L, please clarify which group serves as the control.  

We have added this information to the legend. 

- In Figure 3, I recommend including an air control in panels D and F to better appreciate the magnitude of the response under these conditions.

The responses to all three controls, air, mineral oil and water, were almost zero. As the other reviewer suggested to present trial-to-trial variability as well, we now show responses to all the controls in all the trials in all the animals tested in Figure 3-figure supplement 2.

- I had difficulty understanding Figure 3G. Could the authors provide a more detailed explanation of the model?

We used linear regression (implemented in Python using scikit-learn) to model the relationship between neural activity and behavior, aiming to predict the PER duration based on the calcium responses of two GRN types, Gr5a and Gr66a. The weights for GRNs were estimated using the LinearRegression function. The weight for Gr5a and Gr66a was positive and negative, respectively, indicating that Gr5a contributes to enhance whereas Gr66a contributes to reduce PER.

To evaluate the model performance, we calculated the coefficient of determination (R²), which was 0.81, meaning the model explained 81% of the variance in the PER data.

The scatter plot in Fig. 3G shows a tight relationship between the predicted PER duration (y-axis) plotted against the actual PER duration (x-axis), demonstrating a strong predictive power of the model.

We added the details to the Methods.

- In Figure S4a, the reported p-value is 0.88, which seems to be a typo, as the text indicates that PER is enhanced in a starved state.

Thank you for pointing this out. We have modified the figure legend to describe that PER was enhanced in a starved state only for the experiments conducted with odors at 10^-1 concentration (current Figure 2-figure supplement 1).

eLife Assessment

This important study provides convincing evidence that the Kinesin protein family member KIF7 regulates the development of the cerebral cortex and its connectivity and the specificity of Sonic Hedgehog signaling by controlling the details of Gli repressor vs activator functions. This study provides important new insights into general aspects of cortical development.

Reviewer #1 (Public review):

Summary:

This is an interesting follow-up to a paper published in Human Molecular Genetics reporting novel roles in corticogenesis of the Kif7 motor protein that can regulate the activator as well as the repressor functions of the Gli transcription factors in Shh signalling. This new work investigates how a null mutation in the Kif7 gene affects the formation of corticofugal and thalamocortical axon tracts and the migration of cortical interneurons. It demonstrates that Kif7 null mutant embryos present with ventriculomegaly and heterotopias as observed in patients carrying KIF7 mutations. The Kif7 mutation also disrupts the connectivity between cortex and thalamus and leads to an abnormal projection of thalamocortical axons. Moreover, cortical interneurons show migratory defects that are mirrored in cortical slices treated with the Shh inhibitor cyclopamine suggesting that the Kif7 mutation results in a down-regulation of Shh signalling. Interestingly, these defects are much less severe at later stages of corticogenesis.

Strengths/weaknesses:

The findings of this manuscript are clearly presented and are based on detailed analyses. Using a compelling set of experiments, especially the live imaging to monitor interneuron migration, the authors convincingly investigate Kif7's roles and their results support their major claims. The migratory defects in interneurons and the potential role of Shh signalling present novel findings and provide some mechanistic insights but rescue experiments would further support Kif7's role in interneuron migration. Similarly, the mechanism underlying the misprojection which has previously been reported in other cilia mutants remains unexplored. Taken together, this manuscript makes novel contributions to our understanding of the role of primary cilia in forebrain development and to the aetiology of the neural symptons in ciliopathy patients.

Comments on revisions:

The authors addressed most of the points I raised in my original review. However, I am not convinced by the figures the authors present on Shh protein expression. The "bright tiny dots" of Shh protein in the cortex are not visible on the images in Figure 7. I wonder whether the authors could present higher magnification and/or black and white images with increased contrast.

Reviewer #2 (Public review):

Summary:

This study investigates the role of KIF7, a ciliary kinesin involved in the Sonic Hedgehog (SHH) signaling pathway, in cortical development using Kif7 knockout mice. The researchers examined embryonic cortex development (mainly at E14.5), focusing on structural changes and neuronal migration abnormalities.

Strengths:

(1) The phenotype observed is interesting, and the findings provide neurodevelopmental insight into some of the symptoms and malformations seen in patients with KIF7 mutations.

(2) The authors assess several features of cortical development, including structural changes in layers of the developing cortex, connectivity of the cortex with thalamus, as well as migration of cINs from CGE and MGE to cortex.

Weaknesses:

(1) The Kif7 null does have phenotype differences from individual mutations seen in patients. It would be interesting to add more thoughts about how the null differs from these mutants in ciliary structure and SHH signaling via the cilium.

(2) The description of altered cortex development at E14.5 is perhaps rather descriptive. It would be useful to assess more closely the changes occurring in different cell types and stages. For this it seems very important to have a time course of cortical development and how the structural organization changes over time. This would be easy to assess with the addition of serial sections from the same mice. It might also be interesting to see how SHH signaling is altered in different cortical cell types over time with a SHH signaling reporter mouse.

(3) Abnormal neurodevelopmental phenotypes have been widely reported in the absence of other key genes affecting primary cilia function (Willaredt et al., J Neurosci 2008; Guo et al., Nat Commun 2015). It would be interesting to have more discussion of how the Kif7 null phenotype compares to some of these other mutants.

(4) The authors see alterations in cIN migration to the cortex and observe distinct differences in the pattern of expression of Cxcl12 as well as suggest cell intrinsic differences within cIN in their ability to migrate. The slice culture experiments though make it a little difficult to interpret the cell intrinsic effects on cIN of loss of Kif7, as the differences in Cxcl12 patterns still exist presumably in the slice cultures. It would be useful to assess their motility in an assay where they were isolated, as well as assess transcriptional changes in cINs in vivo lacking KIF7 for expression patterns that may affect motility or other aspects of migration.

Comments on revisions:

The authors have made significant and thoughtful responses as well as experimental additions to the authors comments. Their efforts are appreciated and the manuscript is much improved.

Author response:

The following is the authors’ response to the original reviews

Reviewer #1 (Public review):

Summary:

This is an interesting follow-up to a paper published in Human Molecular Genetics reporting novel roles in corticogenesis of the Kif7 motor protein that can regulate the activator as well as the repressor functions of the Gli transcription factors in Shh signalling. This new work investigates how a null mutation in the Kif7 gene affects the formation of corticofugal and thalamocortical axon tracts and the migration of cortical interneurons. It demonstrates that the Kif7 null mutant embryos present with ventriculomegaly and heterotopias as observed in patients carrying KIF7 mutations. The Kif7 mutation also disrupts the connectivity between the cortex and thalamus and leads to an abnormal projection of thalamocortical axons. Moreover, cortical interneurons show migratory defects that are mirrored in cortical slices treated with the Shh inhibitor cyclopamine suggesting that the Kif7 mutation results in a down-regulation of Shh signalling. Interestingly, these defects are much less severe at later stages of corticogenesis.

Strengths/weaknesses:

The findings of this manuscript are clearly presented and are based on detailed analyses. Using a compelling set of experiments, especially the live imaging to monitor interneuron migration, the authors convincingly investigate Kif7's roles and their results support their major claims. The migratory defects in interneurons and the potential role of Shh signalling present novel findings and provide some mechanistic insights but rescue experiments would further support Kif7's role in interneuron migration. Similarly, the mechanism underlying the misprojection which has previously been reported in other cilia mutants remains unexplored. Taken together, this manuscript makes novel contributions to our understanding of the role of primary cilia in forebrain development and to the aetiology of neural symptoms in ciliopathy patients.

We again thank Reviewer 1 for her/his positive assessment of our article. We have addressed several weaknesses identified by the reviewer, supplementing the initial results with new data, and correcting or clarifying the text where necessary. Our detailed responses to the reviewer’s recommendations appear at the end of each comment.

Reviewer #1 (Recommendations for the authors):

(1) The authors report remarkable phenotypic changes in E14.5 embryos in the projection patterns of corticofugal/thalamocortical axons and in interneuron migration, but some of those phenotypes appear much less severe at E16.5. This might be indicative of a delay in development. Does the migration of interneurons to more dorsal regions correspond to an extended Cxcl12 expression? Do interneuorons still show migratory defects at E16.5? To address a potential delay, the authors could, if feasible, repeat Tbr2/Tomato and L1 or neurofilament stainings in E18.5 embryos?

The question of a possible developmental delay in Kif7 -/- embryos is important. To document this topic, we have extended our study initially focused on embryonic stage E14.5 to earlier (E12.5) and later (E16.5, E18.5/P0) developmental stages. We added new data on E12.5 (Fig. 1, Fig. 3, Fig. S4) and E18.5 (Fig. 3, Fig. 4) embryos in the main figures, and considerably extended the data on E16.5 embryos (Fig. 1, Fig. 3). The legends of figures and the text of the result section (p5-p6) have been modified accordingly. We now describe developmental defects in Kif7 -/- embryos, which are not simple developmental delays. The sequences of thalamic axon development and cIN migration are representative of this complexity.

Thalamic axons: the pioneer projection is misrouted to the amygdala at E14.5 (Fig. 4B) whereas most Kif7 -/- thalamic axons extend to the cortex at E16.5, with a slight delay compared to WT axons (Fig. 4D). At E18.5, the Kif7 -/- thalamo-cortical projection appears rather normal in the rostral forebrain but is drastically reduced in the median and caudal forebrain (Fig. 4E). This strong decrease is confirmed by neurofilament staining performed at E18.5 which identifies a major loss of corticofugal and thalamo-cortical projections in Kif7 -/- brains (Fig. 4F). 

Migrating cIN: During normal development, CXCL12 maintains cIN in their tangential pathways as they start to colonize the cortical wall (E13.5/E14.5). Then CXCL12 drops in the SVZ (Tiveron et al., 2006; Caronia-Brown and Grove, 2011) allowing wild type cIN to invade the cortical plate (Stumm et al., 2003; Li et al., 2008; Atkins et al., 2023). In Kif7 -/- embryos, CXCL12 is never expressed in the SVZ of the dorsal cortex. Therefore Kif7 -/- cIN migrate radially in the dorsal cortex instead of tangentially. We have improved our text in the result section to clarify this transient defect (p8-9).

(2) Figure 1D: The overview of the Gsh2 and Tbr2 stainings does not allow us to see details of the PSPB. The lines indicating the position of the PSPB are not helpful either. Higher magnifications are required to see whether there are subtle differences at these boundaries as observed for other cilia mutants.

We thank the reviewer for her/his question that allowed us to identify a mild default of patterning at the PSB, illustrated by high magnification pictures in the Fig. 1D and described in the result section (p5). This subtle defect of PSB patterning is consistent with previous observations in Kif7 -/- embryos (Putoux et al, 2019) and appears milder than the PSB defect in hypomorphic Gli3 Pdn mutants (PSB shifted dorsally and less well defined as illustrated in Kuschel et al, 2003 and Magnani et al., 2010).

(3) Figure 3: The authors report an interesting mis-projection of thalamocortical axons towards the amygdala. A very similar pattern has been described in Gli3 hypomorphic Pdn mutants (Magnani et al., 2010), in Rfx3, and in Inpp5e null mutant embryos (Magnani et al., 2015). These papers lend further support that this Kif7 phenotype is Gli3 dependent and should be cited in the manuscript. Moreover, the mechanism(s) underlying this mis-projection remain unexplored. Is this phenotype rescued in the previously reported Kif7/ Gli3D699 double mutants? Is there an abnormal expression of axon guidance molecules?

We deeply thank the reviewer for drawing our attention to the abnormal projection of thalamic axons to the amygdala described in the Gli3 Pdn mutant and in two ciliary mutants, Rfx3 -/- and Lnpp5e -/-. We cite these two papers (Magnani et al., 2010, 2015) in the revised manuscript (p7). In the Gli3 Pdn mutant, transplantation experiments show that a patterning defect of the ventral telencephalon (VT) underlies the mis-projection of the thalamus to the amygdala (Magnani et al, 2010). In the Rfx3 ciliary mutant, two possible mechanisms are proposed: pre-thalamus patterning defect and ectopic Netrin and Slit1 expression in the VT (Magnani et al, 2015). We do agree that understanding the mechanism of the thalamic misprojection in the Kif7 mutant would be of great interest. However, given the complexity of the putative mechanisms described in the Gli3 Pdn and Rfx3 mutants, we believe that this question deserves further investigation in a future study. Finally, the possibility that the thalamic projection defect observed in Kif7 -/- embryos could be rescued in Kif7/Gli3699 (double mutants in which Gli3R is overexpressed in the dorsal and ventral forebrain) is very unlikely. Our two main arguments are:

(1) Magnani et al (2015) did not rescue the TCA pathfinding defect in the Rfx3 -/- ciliary mutant when they overexpressed GLI3-R (see TCA description in the Rfx3/ Gli3699 double mutant, last paragraph of the result section). The authors concluded “This finding could be explained by a requirement for Gli activator and not Gli repressor function in VT {ventral telencephalon} patterning and indeed, Gli3 western blots showed that the levels of Gli3R are not altered in the VT of Rfx3 -/- embryos”.

(2) The GLI3-R/Gli3-FL ratio is decreased in the cortex of the Kif7 -/- embryos (dorsal telencephalon) as expected, whereas it is very low in the MGE of WT embryos (ventral telencephalon) and remains unaltered in the Kif7 -/- embryos (Fig. 2B).  

Similarly, the analysis of Kif7 -/- cIN migratory defects leads us to conclude that Kif7 ablation impairs Gli activation function rather than Gli repressor function in the VT where cIN are generated.

(4) Figure 4: The authors should discuss the difference between Tbr2 and Cxcl12 expression which does not extend into the dorsal-most cortical SVZ.

We observed that the transient CXCL12 expression is lacking in the SVZ of the dorsal cortex of Kif7 -/- embryos at E14.5, in a region where TBR2 cells abnormally reach the cortical surface and intermingle with post-mitotic cells. A sentence in our previous version (lines 233-234) could suggest a link between the abnormal location of TBR2 expressing cells and the lack of CXCL12 expression. Having found no data in the literature to explain the absence of CXCL12 expression in the brain by an abnormal cellular environment or by a defect in transcription factor expression, we do not want to further elaborate on differences and similarities between TBR2 and CXCL12 expression patterns in the Kif7 -/- brain. We have modified our text accordingly in the result section of the revised manuscript (p8-9). 

(5) Figure 5: The authors convincingly describe migratory defects of interneurons. The treatment with Shh agonist and antagonist provides some mechanistic insights but genetic or pharmacological rescue experiments would lend further support. For example, they could treat Kif7 mutant sections with Shh agonists or analyse Kif7/Gli3D699 double mutants.

We thank the reviewer for her/his positive assessment of our analysis of the cIN migration. Unfortunately, the rescue experiments proposed by the reviewer should not help to further support our conclusions. First, Kif7 ablation in cIN prevents the processing of any SHH signal in the transcriptional pathway. Second, increasing GLI3R by crossing Kif7 -/- animals with Gli3D699 mice could possibly rescue the alterations of layering in the dorsal cortex where the GLI3R/GLI-FL ratio is strongly decreased and the SHH pathway activated. Such a rescue had been previously described for corpus callosum defects (Putoux et al., 2019). However, because cIN are generated in the ventral forebrain where SHH signaling predominantly activates the formation of GLI-A and where Kif7 ablation does not alter the GLI3 ratio, GLI3R re-introduction in the basal forebrain should rather increase the migratory defects of Kif7 -/- cIN instead of producing a rescue. To further support our conclusion, we analyzed the migratory behavior of Kif7 -/- cIN in a WT cortical environment. The results illustrated in the Fig. 6A and described in page 9 of the result section confirm that the migration defects of Kif7 -/-  cIN are reminiscent of an inhibition but not an activation of the  transcriptional SHH pathway (same phenotype as in Kif3a ciliary mutants described in Baudoin et al, 2012).

(6) Figure 6: The authors describe the Shh mRNA and protein expression with relevance to interneuron migration. In contrast to the in situ hybridisation, the immunofluorescence analysis is not very convincing and requires further controls. The authors should at least show a no primary antibody control and, if available, could include a staining on Shh mutants. These additional controls are important as Shh protein expression in the developing cortex is highly controversial and a recent paper describes a different pattern (Manuel et al., 2022: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3001563#). Moreover, it remains unclear whether the Shh protein expression is uniform within the cortex or follows lateral to medial or ventricular to pial gradients. A more thorough description and corresponding figures would be helpful. 

Manuel et al. (2022) used the SHH KO (generated by Chiang et al., 1996) that develops a long proboscis to validate the rabbit anti-SHH antibodies (from Genetech) used in their study. They show a lack of SHH signal in the SHH KO. However, it is difficult to identify the cortex in this mouse line and the authors did not specify which part of the SHH protein was used to generate antibodies. We wished to use the SHH KO generated by Chiang and backcrossed on a C57B/6 line (Rash and Grove, 2007) that develops a layered neocortex at E17.5. However,

(1) the SHH KO was obtained by replacing exon2 with a PGK-neo cassette and could express a 101 aa truncated protein comprising the N-ter part of the protein, and

(2) the antibody we used, is a polyclonal N-ter antibody that targets the active SHH protein (Cys25-Gly198 part of SHH protein used as immunogen to produce the antibody). We thus thought that this labeling experiment will not give information on the specificity of the antibody, some epitopes being able to recognize the truncated protein produced in the SHH KO.

To overcome the lack of a good mutant mice to validate the SHH N-ter antibodies, we analyzed the SHH immunostaining pattern at E12.5 and compared the expression profile with previously published SHH mRNA expression patterns. The border of the third ventricle and the ZLI were strongly immunostained by SHH-Nter antibodies and these regions were shown to express SHH mRNA at E12.5-E13.5 (Kicker et al. 2004, Loulier et al., 2005, Sahara et al., 2007 and Fig. 7B1). In brain sections at E14.5, only the choroid plexus was strongly labeled and some structures showed diffused labeling. We analyzed the distribution of SHH mRNAs in the cortex using a highly sensitive technique (RNAscop) at E14.5 and showed that very few cortical cells expressed SHH mRNA and at very low level. Anti-SHH-Nter antibodies immunostained numerous bright dots throughout the cortical neuropile, which is not surprising for a diffusible factor like SHH. However, the labeling was not homogeneous and showed a ventricle to pial gradient at E12.5 and aligned distributions in the different cortical layers at E14.5. We have described the expression pattern in more detail and modified the Fig. S4 by adding an image of immunostaining performed without SHH N-ter antibody.  

(7) Figure S1: The Gli3 Western blot needs to be quantified. As the authors only show one control and one mutant sample, it remains unclear how representative this blot is. In addition to Gli3R and Gli3FL, the authors should also determine the ratio of both isoforms. Are there also differences in the MGE?

We now produce results of Gli3 western blots in the cortex and MGE of several E14.5 Kif7 KO (n=4) and WT (n=4) embryos. The GLI3R/GLI3FL ratio has been determined in the cortex and in the MGE of WT and mutant embryos. Results are illustrated in the Fig. 2. 

Minor points:

The authors should carefully amend the literature on Gli genes and forebrain development. For example:

(1) Line 85: Add Hasenpusch-Theil et al., 2018.

We added this reference.

(2) Line 141: Remove Magnani et al., 2010 (they characterized hypomorphic Gli3 Pdn mutants) and replace with Kuschel et al., 2003.

Since our revised figure 2 illustrates GLI3 western blots and compare GLI3R/GLI3FL ratios in the cortex and MGE of WT and Kif7-/- embryos, we no longer cite these papers in the result section.

(3) Line 380: Replace reference with Theil, 2005.

We have replaced Magnani et al, 2014 by Theil 2005 in the sentence.

(4) Line 414: Rallu et al is not an appropriate reference for this as this manuscript does not investigate the expression of a single cortical marker in Shh/Gli3 double mutants.

We removed the reference Rallu et al. in the sentence.

(5) Reference in line 355: do not use Vancouver style.

We apologize for the mistake that was corrected.

(6) Spelling: Line 447 it should read "choroid plexus"

We again apologize for the mistake that has been corrected.

Reviewer #2 (Public review):

Summary:

This study investigates the role of KIF7, a ciliary kinesin involved in the Sonic Hedgehog (SHH) signaling pathway, in cortical development using Kif7 knockout mice. The researchers examined embryonic cortex development (mainly at E14.5), focusing on structural changes and neuronal migration abnormalities.

Strengths:

(1) The phenotype observed is interesting, and the findings provide neurodevelopmental insight into some of the symptoms and malformations seen in patients with KIF7 mutations.<br /> (2) The authors assess several features of cortical development, including structural changes in layers of the developing cortex, connectivity of the cortex with the thalamus, as well as migration of cINs from CGE and MGE to the cortex.

We greatly thank Reviewer 2 for her/his positive assessment of our work that characterize the neurodevelopmental defects induced by KIF7 ablation. We have deeply reorganized and implemented data in the figures to show changes occurring in different cortical cell types and at different stages. We have moreover corrected and clarified the text where necessary. Our detailed responses to the reviewer’s recommendations appear at the end of each comment.

Weaknesses:

(1) The Kif7 null does have phenotype differences from individual mutations seen in patients. It would be interesting to add more thoughts about how the null differs from these mutants in ciliary structure and SHH signaling via the cilium.

We are grateful to the Reviewer for recalling that Kif7 ablation alters SHH signaling within primary cilium and has a strong effect on ciliary structure. In the revised version of the manuscript, we discuss data from the literature that describe these alterations in human (Putoux et al, 2011) and in murine KIF7 depleted cells (He et al, 2015; Cheung et al., 2009; Lai et al., 2021) (discussion p13).

(2) The description of altered cortex development at E14.5 is perhaps rather descriptive. It would be useful to assess more closely the changes occurring in different cell types and stages. For this it seems very important to have a time course of cortical development and how the structural organization changes over time. This would be easy to assess with the addition of serial sections from the same. It might also be interesting to see how SHH signaling is altered in different cortical cell types over time with a SHH signaling reporter mouse.

We thank the Reviewer for her/his request that helped us to improve our description of developmental defaults in the Kif7 -/- cortex.  In the revised manuscript, we have expanded our study initially focused on embryonic stage E14.5 to earlier (E12.5) and later (E16.5, E18.5 /P0) developmental stages. Instead of focusing on median forebrain sections, we have expanded our observations to rostral and caudal sections. Altogether, these new observations allow us to describe more precisely the complex developmental defects in the Kif7 -/- cortex over time, in specific cortical regions (dorsal versus lateral cortex, and rostral versus caudal levels). Figures 1, 3, 4, and S4 have been deeply edited to present new data on E12.5 (Fig. 1, Fig. 3, Fig. S4), E16.5 (Fig. 1, Fig. 3) and E18.5 (Fig. 3, Fig. 4) embryos. We have modified the legends and text in the result section (p5-6) accordingly. We agree with the Reviewer that deciphering how SHH signaling is altered in the different cortical cells over time should be highly interesting and relevant. Nevertheless, we anticipate complex analyses and consider that they should be retained for future studies.

(3) Abnormal neurodevelopmental phenotypes have been widely reported in the absence of other key genes affecting primary cilia function (Willaredt et al., J Neurosci 2008; Guo et al., Nat Commun 2015). It would be interesting to have more discussion of how the Kif7 null phenotype compares to some of these other mutants.

We agree with this Reviewer concern. In the revised manuscript, we discuss our results with regard to previous observations in other ciliary mutants. The murine cobblestone mutant described in Willaredt et al. (2008) indeed shows defects similar to those we describe in the Kif7 -/- mouse. We thank again the Reviewer for her/his helpful comment that allowed us to strengthen and better interpret our results. Guo et al (2015) did not conduct a study of ciliary mutants. Nevertheless, their characterization of cortical developmental defects following invalidation of genes involved in human ciliopathies identified cell autonomous defects in cortical progenitors and in differentiating cortical neurons, which corroborate our observations (p.15)

(4) The authors see alterations in cIN migration to the cortex and observe distinct differences in the pattern of expression of Cxcl12 as well as suggest cell-intrinsic differences within cIN in their ability to migrate. The slice culture experiments though make it a little difficult to interpret the cell intrinsic effects on cIN of loss of Kif7, as the differences in Cxcl12 patterns still exist presumably in the slice cultures. It would be useful to assess their motility in an assay where they were isolated, as well as assess transcriptional changes in cINs in vivo lacking KIF7 for expression patterns that may affect motility or other aspects of migration.

To circumvent the difference in the expression profile of CXCL12 in the dorsal cortex of WT and Kif7 -/- embryos on the migratory behavior of cIN, we compared the trajectories and dynamics of WT and Kif7 -/- cIN imaged in the lateral cortex where CXCL12 expression appears similar in WT and Kif7 -/- brains.

We moreover followed the reviewer recommendation and analyzed the migratory behavior of Kif7 -/- cIN that migrate as isolated cells on a dissociated substrate of WT cortical cells. We sincerely thank the reviewer for her/his suggestion as the results revealed an interesting and relevant ciliary phenotype in migrating Kif7 -/- cIN. This additional experiment confirms that Kif7 -/- cIN exhibit the same migratory defects as those initially characterized in the Kif3a -/-  ciliary mutant.  The new results are illustrated in the Fig. 6A and described in the result section (p9). We agree with the reviewer that the analysis of transcriptional changes that could affect Kif7 -/- cIN motility and migration would be very interesting to study, but this study is beyond the scope of the present article.

eLife Assessment

This study reports important new insights into the roles of a long noncoding RNA, lnc-FANCI-2, in the progression of cervical cancer induced by a type of human papillomavirus. Through a blend of cell biological, biochemical, and genetic analyses of RNA and protein expression, protein-protein interaction, cell signaling, and cell morphology, the authors provide convincing evidence that lnc-FANCI-2 affects cervical cancer outcome by regulating the RAS signaling pathway. These findings will be of interest to scientists in the fields of cervical cancer, long noncoding RNA, and cell signaling.

Reviewer #1 (Public review):

Summary:

The authors attempted to dissect the function of a long non-coding RNA, lnc-FANCI-2, in cervical cancer. They profiled lnc-FANCI-2 in different cell lines and tissues, generated knockout cell lines, and characterized the gene using multiple assays.

Strengths:

A large body of experimental data has been presented and can serve as a useful resource for the scientific community, including transcriptomics and proteomics datasets. The reported results also span different parts of the regulatory network and open up multiple avenues for future research.

Weaknesses:

The write-up is somewhat unfocused and lacks deep mechanistic insights in some places.

Comments on revisions:

The manuscript is much improved. I am satisfied with the authors' responses.

Reviewer #3 (Public review):

Summary:

A long noncoding RNA, lnc-FANCI-2, was reported to be regulated by HPV E7 oncoprotein and a cell transcription factor, YY1 by this group. The current study focuses on the function of lnc-FANCI-2 in HPV-16 positive cervical cancer is to intrinsically regulate RAS signaling, thereby facilitating our further understanding additional cellular alterations during HPV oncogenesis. Authors used the advanced technical approaches such as KO, transcriptome and (IRPCRP) and LC- MS/MS analyses in the current study and concluded that KO Inc-FANCI-2 significantly increase RAS signaling, especially phosphorylation of Akt and Erk1/2.

Strengths:

(1) HPV E6E7 are required for full immortalization and maintenance of malignant phenotype of cervical cancer, but they are NOT sufficient for full transformation and tumorigenesis. This study helps further the understanding of other cellular alterations in HPV oncogenesis.<br /> (2) lnc-FANCI-2 is upregulated in cervical lesion progression from CIN1, CIN2-3 to cervical cancer, cancer cell lines and HPV transduced cell lines.<br /> (3) Viral E7 of high-risk HPVs and host transcription factor YY1 are two major factors promoting lnc-FANCI-2 expression.<br /> (4) Proteomic profiling of cytosolic and secreted proteins showed inhibition of MCAM, PODXL2 and ECM1 and increased levels of ADAM8 and TIMP2 in KO cells.<br /> (5) RNA-seq analyses revealed that KO cells exhibited significantly increased RAS signaling but decreased IFN pathways.<br /> (6) Increased phosphorylated Akt and Erk1/2, IGFBP3, MCAM, VIM, and CCND2 (cyclin D2) and decreased RAC3 were observed in KO cells.

Comments on revisions:

The revised manuscript has been significantly improved. The authors addressed all my concerns.

Author response:

The following is the authors’ response to the previous reviews

Public Reviews:

Reviewer #1 (Public review):

Summary:

The authors attempted to dissect the function of a long non-coding RNA, lnc-FANCI-2, in cervical cancer. They profiled lnc-FANCI-2 in different cell lines and tissues, generated knockout cell lines, and characterized the gene using multiple assays.

Strengths:

A large body of experimental data has been presented and can serve as a useful resource for the scientific community, including transcriptomics and proteomics datasets. The reported results also span different parts of the regulatory network and open up multiple avenues for future research.

Thanks for your positive comments on the strengths.

Weaknesses:

The write-up is somewhat unfocused and lacks deep mechanistic insights in some places.

As the lnc-FANCI-2 as a novel lncRNA had never been explored for any functional study, our report found that it regulates RAS signaling. Thus, this report focuses on lnc-FANCI-2 and RAS signaling pathway but also includes some important screening data, which are important for our readers to understand how we could reach the RAS signaling.

Reviewer #2 (Public review):

The study by Liu et al provides a functional analysis of lnc-FANCI-2 in cervical carcinogenesis, building on their previous discovery of FANCI-2 being upregulated in cervical cancer by HPV E7.

The authors conducted a comprehensive investigation by knocking out (KO) FANCI-2 in CaSki cells and assessing viral gene expression, cellular morphology, altered protein expression and secretion, altered RNA expression through RNA sequencing (verification of which by RT-PCR is well appreciated), protein binding, etc. Verification experiments by RT-PCR, Western blot, etc are notable strengths of the study.

The KO and KD were related to increased Ras signaling and EMT and reduced IFN-y/a responses.

Thanks for your positive comments. It did take us a few years to reach this scientific point for understanding of lnc-FANCI-2 function.

Although the large amount of data is well acknowledged, it is a limitation that most data come from CaSki cells, in which FANCI-2 localization is different from SiHa cells and cancer tissues (Figure 1). The cytoplasmic versus nuclear localization is somewhat puzzling.

Regarding lnc-FANCI-2 localization, it could be both cytoplasmic and nuclear in cervical cancer tissues, HPV16 or HPV18 infected keratinocytes, and HPV16+ cervical cancer cell line CaSki cells which contain multiple integrated HPV16 DNA copies. But surprisingly, it is most detectable in the nucleus in HPV16+ SiHa cells which contain only one copy of integrated HPV16 DNA (Yu, L., et al. mBio 15: e00729-24, 2024). No matter what, knockdown of lnc-FANCI-2 expression from SiHa cells induces RAS signaling leading to an increase in the expression of p-AKT and p-Erk1/2 (suppl. Fig. S6B).

Reviewer #3 (Public review):

Summary:

A long noncoding RNA, lnc-FANCI-2, was reported to be regulated by HPV E7 oncoprotein and a cell transcription factor, YY1 by this group. The current study focuses on the function of lnc-FANCI-2 in HPV-16 positive cervical cancer is to intrinsically regulate RAS signaling, thereby facilitating our further understanding of additional cellular alterations during HPV oncogenesis. The authors used advanced technical approaches such as KO, transcriptome and (IRPCRP) and LC- MS/MS analyses in the current study and concluded that KO Inc-FANCI-2 significantly increases RAS signaling, especially phosphorylation of Akt and Erk1/2.

Strengths:

(1) HPV E6E7 are required for full immortalization and maintenance of the malignant phenotype of cervical cancer, but they are NOT sufficient for full transformation and tumorigenesis. This study helps further understanding of other cellular alterations in HPV oncogenesis.

(2) lnc-FANCI-2 is upregulated in cervical lesion progression from CIN1, CIN2-3 to cervical cancer, cancer cell lines, and HPV transduced cell lines.

(3) Viral E7 of high-risk HPVs and host transcription factor YY1 are two major factors promoting lnc-FANCI-2 expression.

(4) Proteomic profiling of cytosolic and secreted proteins showed inhibition of MCAM, PODXL2, and ECM1 and increased levels of ADAM8 and TIMP2 in KO cells.

(5) RNA-seq analyses revealed that KO cells exhibited significantly increased RAS signaling but decreased IFN pathways.

(6) Increased phosphorylated Akt and Erk1/2, IGFBP3, MCAM, VIM, and CCND2 (cyclin D2) and decreased RAC3 were observed in KO cells.

Thanks for your positive comments. It has taken us almost nine years to reach this point to gradually understand lnc-FANCI-2 functions, which are more complex than our initial thoughts.  

Weaknesses:

(1) The authors observed the increased Inc-FANCI-2 in HPV 16 and 18 transduced cells, and other cervical cancer tissues as well, HPV-18 positive HeLa cells exhibited different expressions of Inc-FANCI-2.

Both HPV16 and HPV18 infections induce lnc-FANCI-2 expression in keratinocytes (Liu H., et al. PNAS, 2021). However, HPV18+ cervical cancer cell lines HeLa and C4II cells (Figure S1A and S1B) do not express lnc-FANCI-2 as we see in HPV-negative cell lines such as HCT116, HEK293, HaCaT, and BCBL1 cells. Although we don’t know why, our preliminary data show that the lnc-FANCI-2 promoter functions well and is sensitive to YY1 binding in lnc-FANCI-2 expressing CaSki and C33A cells in our dual luciferase assays but is much less sensitive to YY1 binding in HeLa and HCT116 cells, indicating some unknown cellular factors negatively regulating lnc-FANCI-2 promoter activity.

Author response image 1.

A firefly luciferase (FLuc) reporter containing either the wild-type (−600 wt) or YY1-binding-site-mutated lnc-FANCI-2 promoter was evaluated in CaSki, HeLa, C33A, and HCT116 cells for its promoter activity, with Renilla luciferase (RLuc) activity driven by a TK promoter serving as an internal control. The two YY1-binding motifs (A and B) with a X for mutation are illustrated in the right diagram.

(2) Previous studies and data in the current showed a steadily increased Inc-FANCI-2 during cancer progression, however, the authors did not observe significant changes in cell behaviors (both morphology and proliferation) in KO Inc-FANCI-2.

Thanks. We do see decreases in cell proliferation, colony formation, and cell migration, accompanied by increased cell senescence, from the lnc-FANCI-2 KO cells to the parent WT cells.  These data are now added to the revised Fig. 1 and the revised supplemental Fig. S3.

(3) The authors observed the significant changes of RAS signaling (downstream) in KO cells, but they provided limited interpretations of how these results contributed to full transformation or tumorigenesis in HPV-positive cancer.

As we stated in the title of this function of lnc-FANCI-2, the lnc-FANCI-2 intrinsically restricts RAS signaling and phosphorylation of Akt and Erk in HPV16-infected cervical cancer. Presumably, high RAS-AKT-ERK signaling inhibits tumor cell survival due to senescence induction as we show in our new Figure 1 and supplemental Fig. S3. A similar report was found in a lung cancer study (Patricia Nieto, et al. Nature 548: 239-243, 2017).

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

Major comments:

(1) A major issue is that parts of the manuscript read like a collection of experimental results. However, some of the results do not contribute directly to the central story. Besides confusing the reader, the large amount of apparently disparate results can raise more questions. For example:

a) Why is lnc-FANCI-2 highly expressed in HPV16-infected cervical cancer cell lines (but not in HPV18-infected cells)?

b) How do p53 and RB repress the expression of lnc-FANCI-2?

c) What regulates the sub-cellular localization of lnc-FANCI-2?

d) How does lnc-FANCI-2 negatively regulate RAS signalling?

e) How does MAP4K4 bind to lnc-FANCI-2?

f) Do lnc-FANCI-2 and MAP4K4 require each other to regulate RAS signalling?

g) How does RAS signalling regulate the transcription of MCAM and IGFBP3?

h) How does MCAM feedback on RAS? Do the different MCAM isoforms impact on RAS signalling differently?

i) How does IGFBP3 feedback on ERK but not AKT?

j) How do the other mentioned proteins like ADAM8 fit into the regulatory network?

k) Each question will require a lot more work to address. I think it would be good if the authors could think through carefully what the key message(s) in the current manuscript should be and then present a more focused write-up.

Thanks for the critical comments. Because this study is the first time to explore lnc-FANCI-2 functions, we would like to be collective. We believe these data are important to guide any future studies. We really appreciate our reviewer listing many questions related to HPV infection, cell biology, RAS signaling, cancer biology from questions a to k. To address each question in a satisfactory way will be a separate study, but fortunately, our report has pointed out such a direction with some preliminary data for future studies. Here below are our responses to each question from a to k:

a) Both HPV16 and HPV18 infection induce lnc-FANCI-2 expression in keratinocytes (Liu H., et al. PNAS, 2021). However, HPV18+ cervical cancer cell lines HeLa and C4II cells (Figure S1A and S1B) do not express lnc-FANCI-2 as we see in HPV-negative cell lines such as HCT116, HEK293, HaCaT, and BCBL1 cells. Although we don’t know why, our preliminary data show that lnc-FANCI-2 promoter functions well and is sensitive to YY1 binding in lnc-FANCI-2 expressing CaSki and C33A cells but is much less sensitive to YY1 in HeLa and HCT116 cells, indicating some unknown cellular factors negatively regulating lnc-FANCI-2 promoter activity.

b) We don’t know whether p53 and pRB could repress the expression of lnc-FANCI-2 although C33A cells bearing a mutant p53 and mutant pRB express high amount of lnc-FANCI-2. However, KD of E2F1 had no effect on lnc-FANCI-2 promoter activity in CaSki cells (Liu, H., et al. PNAS, 2021).

c) RNA cellular localization can be affected by many factors, including splicing, export, and polyadenylation. As lnc-FANCI-2 is a long non-coding RNA, its regulation of cellular location could be more complicated than mRNAs and thus could be a future research direction.  

d) The conclusion that lnc-FANCI-2 negatively regulates RAS signaling is based on both lnc-FANCI-2 KO and KD studies.  Please see the proposed hypothetic model in Figure 8E.

e) The MAP4K4 binding to lnc-FANCI-2 was demonstrated by our IRPCRP-Mass spectrometry (Fig. 8A and 8C), although the exact binding site on lnc-FANCI-2 was not explored. As you probably know, many enzymes today turn out an RNA-binding enzyme (Castello A., et al. Trends Endocrinol. Metab. 26: 746-757, 2015; Hentze MW., et al. Nat. Rev. Mol. Cell Biol. 19: 327-341, 2018)    

f) Yes, they are slightly relied on each other in regulating RAS signaling. We found that KD of MAP4K4 in parent CaSki cells (Figure 8D) led to more effect on RAS signaling (MCAM, IGFBP3, p-Akt) than that in lnc-FANCI-2 KO ΔPr-A9 cells. In contrast, the latter displayed more p-Erk1/2 than that induced by KD of lnc-FANCI-2 in the parental CaSki cells (Figure S7C).

g) We believe RAS signaling regulates most likely the transcription of MCAM and IGFBP3 through phosphorylated transcription factors (Figure 8E diagram).

h) As a signal molecule with at least 13 ligands/coreceptors (Joshkon A., et al. Biomedicines 8: 633, 2020), the increased MCAM appears to sustain RAS signaling (Fig. 7J and Fig. 8E). We are assuming the full-length cytoplasmic MCAM plays a predominant role in RAS signaling due to its abundance than the cleaved nuclear MCAM missing both transmembrane and cytoplasmic regions. Plus, RAS signaling mainly occurs in the cytosol.  

i) Exact mechanism remains unknown. Lnc-FANCI-2 KO cells exhibit high expression levels of IGFBP3 RNA and protein and p-Erk1/2, but not so much for p-Akt, possibly due to IGFBP3 regulation of MAPK for Erk phosphorylation, but not much so on PI3K for Akt phosphorylation.

j) The dysregulation of RAS signaling and ADAM protein activity is implicated in various cancers. ADAM proteins can modulate RAS signaling by cleaving and releasing ligands that activate or inactivate RAS-related pathways (Schafer B., et al. JBC 279: 47929-38, 2004; Ohtsu H., et al. Am J Physiol Cell Physiol 291: C1-C10, 2006; Dang M, et al. JBC 286: 17704-17713, 2011; Kleino I, et al. PLoS One 10: e0121301, 2015). Some ADAM proteins are Involved in the migration and invasion of cancer cells, and its loss can promote the degradation of KRAS (Huang Y-K., et al. Nat Cancer 5: 400-419, 2024). In this revision, we have a brief discussion on ADAMs and RAS signaling.

k) We agree with our reviewer that each question will require a lot more work to address. As this study is to explore the lnc-FANCI-2 function for the first time, however, we prefer to include all of these data that have been selectively included in this write-up. We hope reviewer 1 will be satisfied with our response to each question from a to j. 

(2) Figures S1A & S1C - Replicates are needed.

Yes, we have repeated all of the experiments. The quantification shown in Figure S1A and S1C was performed in triplicate, and error bars have been added to the updated figure.

3) Figure S1D - There seems to be some lnc-FANCI-2 RNA in the nucleus of CaSki cells as well. Please quantify the relative amount of lnc-FANCI-2 in the nucleus vs cytoplasm.

Yes, a small fraction of lnc-FANCI-2 is in the nucleus of CaSki cells as we reported (Liu H., PNAS, 2021, Movies S1 and S2). We did quantify by fractionation and RT-qPCR the relative amount of lnc-FANCI-2 in the nucleus vs cytoplasm in Figure S1C. 

(4) Figure S2B - (a) For ΔPr-A9 cells, it looks like there is an increase in E6 and a decrease in E7, instead of "little change" as the authors claimed. (b) I suggest checking the protein levels for all the control and KO clones.

Thanks for the questions. We had some variation in E6 and E7 detection and the submitted one was one representative.  We grew again the lnc-FANCI-2 KO clones A9 and B3 and reexamined the expression of HPV16 E6/E7 proteins and their downstream targets, p53 and E2F1. As shown in new Figure S3A expt II, we saw again some variations in the detections (~20-30%) and these variations do not reflect a noticeable change for their downstream targets. Thus, we do not consider these changes significantly enough to draw a conclusion in our study, but rather most likely from sampling in the assays.

(5) In the Proteome Profiler Human sReceptor Array analysis, multiple proteins were highlighted as having at least 30% change. But it is unclear how they relate to RAS signaling.

Thanks for this comment.  Cellular soluble receptors are essential for RAS signaling, EMT pathway and IFN responses. For example, the dysregulation of RAS signaling and ADAM protein activity is implicated in various cancers. ADAM proteins can modulate RAS signaling by cleaving and releasing ligands that activate or inactivate RAS-related pathways (Schafer B., et al. JBC 279: 47929-38, 2004; Ohtsu H., et al. Am J Physiol Cell Physiol 291: C1-C10, 2006; Dang M, et al. JBC 286: 17704-17713, 2011; Kleino I, et al. PLoS One 10: e0121301, 2015). Some ADAM proteins are Involved in the migration and invasion of cancer cells, and its loss can promote the degradation of KRAS (Huang Y-K., et al. Nat Cancer 5: 400-419, 2024). In this revision, we have a brief discussion on ADAMs and RAS signaling.

(6) Does knockdown of MAP4K4 lead to an increase in MCAM and IGFBP3?

Yes, the MAP4K4 KD from parental WT CaSki cells does lead an increase in MCAM (~70%) and IGFBP3 (~30%) which is like the knockdown of lnc-FANCI-2 shown in the revised Figure 8D.

Minor comments:

(7) In the opinion of this reviewer the title is somewhat unwieldy.

Thanks. We have shortened the title as “The lnc-FANCI-2 intrinsically restricts RAS signaling in HPV16-infected cervical cancer”

(8) The abstract can be more focused and doesn't have to mention so many gene names. In fact, the significance paragraph works better as an abstract. For the significance, the authors can provide another write-up on the implications of their research instead.

Thanks. We have revised the abstract and added the implications of this research.

(9) The last sentence of the introduction feels a little abrupt. It would be good to elaborate a little more on the key findings.

Thanks for this critical comment. We have revised as in the following: In this report, we demonstrate that lnc-FANCI-2 in HPV16-infected cells controls RAS signaling by interaction with MAP4K4 and other RNA-binding proteins. Ablation of lnc-FANCI-2 in the cells promotes RAS signaling and phosphorylation of Akt and Erk. High levels of lnc-FANCI-2 and low level of MCAM expression in cervical cancer patients correlate with improved survival, indicating that lnc-FANCI-2 plays a critical role in regulating RAS signaling to affect cervical cancer progression and patient outcomes.

(10) Typo on line 191: Should be ADAM8 and not ADMA8.

Corrected.

Reviewer #2 (Recommendations for the authors):

The paper contains a vast amount of data and would greatly benefit from an expanded version of the schematic of Figure 8E summarizing the main results. Including additional details on FANCI-2 regulation by HPV (primarily from previous studies) and its implications for HPV16-driven carcinogenesis would provide a more comprehensive overview.

Thanks for the suggestion. We have modified our Figure 8E to include HR-HPV E7 and YY1 in regulation of lnc-FANCI-2 transcription.

Further specific comments:

(1) The introduction may be shortened to increase readability (e.g. lines 77-90; 94-105).

We have shortened the introduction by deletion of the lines 94-105 from our initial submission.

(2) Lines 55-57 the number of cervical cancer diagnoses and mortality need to be updated to the latest literature. The reference is from 2012.

Thanks. We have revised and updated accordingly with a new citation (Bray F., et al: Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 74, 229-263 (2024))

(3) Line 61: Progression rate of CIN3 is incorrect (31% in 30 years according to reference 5).

Thanks. Corrected.

(4) Lines 108-112 are difficult to understand and should be rewritten.

Thanks. Revised accordingly.

(5) Line 116 Is this correct or should 'but' be 'and'?

Thanks. Corrected accordingly.

(6) Figure 1A top: The difference between cervical cancer and normal areas is hard to see in the top figure. The region labeled as "normal" does not resemble typical differentiating epithelium or normal glandular epithelium, though this is difficult to assess accurately from the image provided. I suggest adding HE staining and also the histotypes.

We have added an H&E staining panel in the corresponding region to Figure 1A, which clearly shows the normal and cancer regions. Both cervical cancer tissues were cervical squamous cell carcinoma.

(7) HFK-HPV16 & 18 cells (Figure 1B) are not described in the Materials & Methods.

Thanks. We revised our Materials and Methods by citing our two previous publications.

(8) Figure 2E (RNA scope on FANCI-2 KO) only shows 2 to 3 cells, which makes it somewhat difficult to assess downregulated expression in the KO. I suggest replacing these with pictures showing more cells (i.e. >10) to strengthen the results.

We have replaced the image in Figure 2E to include more cells.

(9) The spindle-like morphology in deltaPr-A9 cells shown in FigS2A is not very distinct. Including images at higher magnification could help clarify this feature.

Good comment. We have enlarged the images for better view and revised the context.

(10) Both protein and RNA expression analysis have been performed on WT CaSki cells and FANCI-2 KO cells. If I am correct there is little overlap between the significantly changed gene products. What does this mean? Have you looked into the comparison?

The DEGs identified from RNA-seq indicated a genome wide transcriptome change, while the protein array we used only covered 105 soluble protein receptors. However, we did find 9/15 (60%) membrane proteins in cell lysates (PODXL2, ECM1, NECTIN2, MCAM, ADAM9, CDH5, ADAM10, ITGA5, NOTCH1, SCARF2, ADAM8, TIMP2, LGALS3BP, CDH13, and ITGB6) exhibited consistent changes in expression (underlined) by both RNA-seq and protein array assays. We have revised the text with this information (page 11). Other six proteins (40%) had inconsistent expression correlation in two assays could be due to post-translational mechanisms, such as protein stability, modifications and secretion, etc.  

(11) Figure S7, which represents TCGA data and survival is quite complex. It would be more effective to display a similar figure for FANCI-2, as was done for MCAM in Figure 7I, to simplify the comparison and enhance clarity.

Thanks. However, the suggested figure for lnc-FANCI-2 was published in PNAS paper already (Liu H., et al. PNAS, 2021).  The Figure S8 in this revision is the result from our in-house GradientScanSurv pipeline, a new way to correlate the expression and survival more accurately.

What do the Figures look like if you analyse only HPV16+ patients versus HPV18+ patients, considering that FANCI-2 upregulation in cell lines is related to HPV16 and not 18? Is there an effect of histotype? Or tumor stage?

HPV18 infected keratinocytes express high level of lnc-FANCI-2. Two HPV18⁺ HeLa and C4II cell lines and HPV-negative cell lines, such as HCT116 cells, which do not express lnc-FANCI-2 could be due to the presence of some unknow repressive factors. We found that lnc-FANCI-2 promoter functions well in responding to YY1 binding in CaSki and C33A cells expressing lnc-FANCI-2 but does not so in HeLa and HCT116 cells in our dual luciferase assays. 

(12) It remains puzzling that FANCI-2 upregulation was previously shown to already occur in CIN lesions and increase further in cervical cancer, while the current data indicate that FANCI-2 suppresses AKT activation. If I am correct Akt activation has been linked to cervical carcinogenesis. Similarly, line 434 states that increased MCAM might promote cervical tumorigenesis, implying that low FANCI-2 would stimulate tumorigenesis. If I understand correctly, the increase in FANCI-2 observed in CIN lesions would reflect a "brake" on the carcinogenic pathway and its sustained increase in cancer might indicate that growth is still (partly) controlled. As mentioned earlier, a Figure illustrating the relation between FANCI-2, HPV, and the carcinogenic process would be beneficial for clarity.

Yes. Increased MCAM, but low level of lnc-FANCI-2, correlates with poor cervical cancer survival. We have revised Figure 8E to illustrate this relation better.  

(13) May part of the potentially conflicting findings be explained by CaSki cells being of metastatic origin? Related to this, does the expression of FANCI-2 or MALM depend on the tumor stage?

Thanks for this important suggestion. Unfortunately, we found that the expression of lnc-FANCI-2 and MCAM is not associated with cervical cancer stage based on the TCGA data (http://gepia.cancer-pku.cn/index.html). See the data below:

Author response image 2.

Despite some lingering uncertainty, the extensive experiments conducted using KO and KD cells do provide compelling evidence that lnc-FANCI-2 function is linked to RAS signaling and EMT.

Thanks for your positive review and instructive comments.

Reviewer #3 (Recommendations for the authors):

(1) The authors observed the increased Inc-FANCI-2 in HPV 16 and 18 transduced cells, and other cervical cancer tissues as well, HPV-18 positive HeLa cells exhibited different expressions of Inc-FANCI-2. I suggest authors provide more discussions on this difference, for example, HPV genotypes. HPV genome status in host cells? Cell types?

Thanks. We found the keratinocyte infections with HPV16, HPV18, and other HR-HPVs could induce lnc-FANCI-2 expression (Liu H., et al. PNAS, 2021). In this report, we found HPV18⁺ HeLa and C4II cells and other HPV-negative cell lines do not. Our preliminary data on lnc-FANCI-2 promoter activity assays showed the presence of a negative regulatory factor (s) in non-lnc-FANCI-2 expressing cells. See the data in Author response image 1.

We have revised our discussion by inclusion these sets of the luciferase data as data not shown.

(2) I suggest the authors discuss more details on how the changes of RAS signaling in KO cells help our further understanding of the molecular mechanisms for HPV-associated full-cell transformation and malignancy in addition to the well-known functions of HPV E6 and E7.

Thanks. We have modified the Figure 8E as suggested by reviewer 2 and revised the discussion further.

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Vorschlag: Wie können SuS für ethische Aspekte von KI-Systemen sensibilisiert werden?

Algorithms determine how data are computed.

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eLife Assessment

The paper addresses the question of gene epistasis and asks what is the correct null model for which we should declare no epistasis. By reanalyzing synthetic gene array datasets regarding single and double-knockout yeast mutants, and considering two theoretical models of cell growth, the authors reach the valuable conclusion that the product function is a good null model. While the justification of some assumptions is incomplete, the results have the potential to be of value to the field of gene epistasis.

Reviewer #1 (Public review):

Summary

Detecting unexpected epistatic interactions between multiple mutations requires a robust null expectation-or neutral function-that predicts the combined effects of multiple mutations on phenotype based on the individual effects of single mutations. This study evaluated the relevance of the product neutrality function, where double-mutant fitness is represented as a multiplicative combination of single-mutant fitness in the absence of epistatic interactions. The authors used a recent large dataset on fitness, specifically yeast colony size, to analyze epistatic interactions.

The study confirmed that the product function outperformed other neutral functions in predicting double-mutant fitness, showing no bias between negative and positive epistatic interactions. Additionally, in the theoretical portion of the study, the authors employed a previously established theoretical model of bacterial cell growth to simulate growth rates of both single- and double-mutants under multiple parameters. The simulations similarly demonstrated that the product function was superior to other functions in predicting the fitness of hypothetical double-mutants. Based on these findings, the authors concluded that the product function is a robust tool for analyzing epistatic interactions in growth fitness and effectively reflects how growth rates depend on the combination of multiple biochemical pathways.

Strength

By leveraging a previously published large dataset of yeast colony sizes for single- and double-knockout mutants, this study validated the relevance of the product function, which has frequently been used in genetics to analyze epistatic interactions. The confirmation that the product function provides a more reliable prediction of double-mutant fitness compared to other neutral functions is valuable for researchers analyzing epistatic interactions, particularly those working with the same dataset.<br /> Notably, this dataset has been previously used in studies exploring epistatic interactions with the product neutrality function. This study's findings affirm the validity of using the product function, which could enhance confidence in the conclusions drawn by those earlier studies. Consequently, both researchers utilizing this dataset and readers of prior research will benefit from the confirmation provided by this study.

Weakness

This study contains several serious problems, primarily stemming from the following issues: ignoring the substantial differences in the mechanisms regulating cell growth between prokaryotes and eukaryotes and adopting an overly specific and unrealistic set of assumptions in the mutation model. Below, the details are discussed.

(1) Misapplication of prokaryotic growth models

The mechanistic origin of the multiplicative model observed in yeast colony fitness is explained using a bacterial cell growth model. However, there is no valid justification for linking these two systems. The bacterial growth model, the Scott-Hwa model, heavily rely on specific molecular mechanisms, such as ppGpp-mediated regulation, which adjusts ribosome expression and activity during translation. In particular, this mechanism is critical to ensure growth-dependency of the fraction of ribosome in proteome in the Scott-Hwa model [https://doi.org/10.1111/j.1462-2920.2010.02357.x; https://doi.org/10.1073/pnas.2201585119]. Yeast cells lack this regulatory mechanism, making it inappropriate to directly apply bacterial growth models to yeast.<br /> The Weiße model is based on a larger set of underlying equations and involves more parameters than the Scott-Hwa model. In the original paper by Weiße et al. (PNAS, 2015), however, the model parameters were fitted solely to experimental data from E. coli, and the model's applicability to yeast was never assessed. In summary, for neither the Scott-Hwa model nor the Weiße model has it been demonstrated that the entire model quantitatively fits experimental data from yeast. A positive correlation between growth rate and RNA/protein ratio, often observed in yeast, supports only a limited portion of either model, and does not constitute validation of the models as a whole.

(2) Overly specific assumptions in the theoretical model

The theoretical model assumes that two mutations affect only independent parameters of specific biochemical processes. However, this overly restrictive assumption weakens the model's validity in explaining the general occurrence of the multiplicative model in mutations. Furthermore, experimental evidence suggests limitations of this approach. For example, in most viable yeast deletion mutants with reduced growth rates, the expression of ribosomal proteins remained largely unchanged, contrary to the predictions of the Scott-Hwa model [https://doi.org/10.7554/eLife.28034]. This discrepancy highlights that the Scott-Hwa model and its derivatives cannot reliably explain mutants' growth rates based on current experimental evidence.

(3) Limited reliability of the mechanistic origin of the multiplicative model

The authors seem to regard growth-optimizing feedback as the mechanistic origin of the multiplicative model. However, the importance of growth-optimizing feedback in explaining product neutrality heavily depends on the very specific framework of the Scott-Hwa model. As I pointed out above, the Scott-Hwa model is a bacterial growth model that considers only a narrowly defined set of biochemical reactions. Using such a narrow model to explore the mechanistic origin of product neutrality observed on a genome-wide scale appears to be inappropriate. Arguments based on either the Scott-Hwa model or the Weiße model fail to account for the generality of product neutrality across diverse genetic perturbations. These models, in their current form, do not explain the broader patterns of product neutrality observed experimentally.

Reviewer #2 (Public review):

The paper deals with the important question of gene epistasis, focusing on asking what is the correct null model for which we should declare no epistasis.

In the first part, they use the Synthetic Genetic Array dataset to claim that the effects of a double mutation on growth rate is well predicted by the product of the individual effects (much more than e.g. the additive model). The second (main) part shows this is also the prediction of two simple, coarse-grained models for cell growth.

I find the topic interesting, the paper well written, and the approach innovative.

Comments on revisions:

The authors have adequately addressed the comments raised in the review below, and I find that the paper has improved.

Author response:

The following is the authors’ response to the original reviews

Reviewer #1 (Public review):

Summary:

Detecting unexpected epistatic interactions among multiple mutations requires a robust null expectation - or neutral function - that predicts the combined effects of multiple mutations on phenotype, based on the effects of individual mutations. This study assessed the validity of the product neutrality function, where the fitness of double mutants is represented as the multiplicative combination of the fitness of single mutants, in the absence of epistatic interactions. The authors utilized a comprehensive dataset on fitness, specifically measuring yeast colony size, to analyze epistatic interactions.

The study confirmed that the product function outperformed other neutral functions in predicting the fitness of double mutants, showing no bias between negative and positive epistatic interactions. Additionally, in the theoretical portion of the study, the authors applied a wellestablished theoretical model of bacterial cell growth to simulate the growth rates of both single and double mutants under various parameters. The simulations further demonstrated that the product function was superior to other functions in predicting the fitness of hypothetical double mutants. Based on these findings, the authors concluded that the product function is a robust tool for analyzing epistatic interactions in growth fitness and effectively reflects how growth rates depend on the combination of multiple biochemical pathways.

Strengths:

By leveraging a previously published extensive dataset of yeast colony sizes for single- and double-knockout mutants, this study validated the relevance of the product function, commonly used in genetics to analyze epistatic interactions. The finding that the product function provides a more reliable prediction of double-mutant fitness compared to other neutral functions offers significant value for researchers studying epistatic interactions, particularly those using the same dataset.

Notably, this dataset has previously been employed in studies investigating epistatic interactions using the product neutrality function. The current study's findings affirm the validity of the product function, potentially enhancing confidence in the conclusions drawn from those earlier studies. Consequently, both researchers utilizing this dataset and readers of previous research will benefit from the confirmation provided by this study's results.

Weaknesses:

This study exhibits several significant logical flaws, primarily arising from the following issues: a failure to differentiate between distinct phenotypes, instead treating them as identical; an oversight of the substantial differences in the mechanisms regulating cell growth between prokaryotes and eukaryotes; and the adoption of an overly specific and unrealistic set of assumptions in the mutation model. Additionally, the study fails to clearly address its stated objective-investigating the mechanistic origin of the multiplicative model. Although it discusses conditions under which deviations occur, it falls short of achieving its primary goal. Moreover, the paper includes misleading descriptions and unsubstantiated reasoning, presented without proper citations, as if they were widely accepted facts. Readers should consider these issues when evaluating this paper. Further details are discussed below.

(1) Misrepresentation of the dataset and phenotypes

The authors analyze a dataset on the fitness of yeast mutants, describing it as representative of the Malthusian parameter of an exponential growth model. However, they provide no evidence to support this claim. They assert that the growth of colony size in the dataset adheres to exponential growth kinetics; in contrast, it is known to exhibit linear growth over time, as indicated in [Supplementary Note 1 of https://doi.org/10.1038/nmeth.1534]. Consequently, fitness derived from colony size should be recognized as a different metric and phenotype from the Malthusian parameter. Equating these distinct phenotypes and fitness measures constitutes a fundamental error, which significantly compromises the theoretical discussions based on the Malthusian parameter in the study.

The reviewer is correct in pointing out that colony-size measurements are distinct from exponential growth kinetics. We acknowledge that our original text implied that the dataset directly measured the exponential growth rate (Malthusian parameter), when in fact it was measuring yeast colony expansion rates on solid media. Colony growth under these conditions often follows a biphasic pattern in that there is typically an initial microscopic phase where cells can grow exponentially, but as the colony expands further then the growth dynamics become more linear (Meunier and Choder 1999). We have revised our text to state clearly what the experiment measured.

However, while colony size does not exhibit exponential growth kinetics, several studies have argued that the rate of colony expansion is related to the exponential growth rate of cells growing in non-limiting nutrient conditions in liquid culture. This is because colony growth is dominated by cells at the colony boundaries that have access to nutrients and are in exponential growth. Cells in the colony interior lack nutrients and therefore contribute little to colony growth. This has been shown both in theoretical and experimental studies, finding that the linear growth rate of the colony is directly linked to the single-cell exponential growth rate (Pirt 1967; Gray and Kirwan 1974; Korolev et al. 2012; Gandhi et al. 2016; Meunier and Choder 1999). In particular, the above studies suggest that the linear colony growth rate is directly proportional to the square root of the exponential growth rate. Therefore, one would expect that the validity of the product model for one fitness measure implies its validity for the other measure. In addition, colony size was found to be highly correlated with the exponential growth rate of cells in non-limiting nutrients in liquid culture (Baryshnikova et al. 2010; Zackrisson et al. 2016; Miller et al. 2022). For these reasons, we treated the colony size and exponential growth rate as interchangeable in our original manuscript. 

To address the important point raised by the reviewer, we now explain more clearly in the text what the analyzed data on colony size show and why we believe it is reflective of the exponential growth rate. Finally, we note that our results supporting the product neutrality function are consistent with the work of (Mani et al. 2008), which used smaller datasets based on liquid culture growth rates (Jasnos and Korona 2007; Onge et al. 2007).

The text in Section 2.3 now reads:

“Having verified empirically that the Product neutrality function is supported by the latest data for cell proliferation, we now turn our attention to its origins. Addressing this question requires some mechanistic model of biosynthesis. However, most mechanistic models of growth apply directly to single cells in rich nutrient conditions, which may not directly apply to the SGA measurements of colony expansion rates. In particular, colony growth has been shown to follow a biphasic pattern (Meunier et al. 1999). A first exponential phase is followed by a slower linear phase as the colony expands. Previous modeling and empirical work indicates that this second linear expansion rate reflects the underlying exponential growth of cells in the periphery of the colony (Pirt 1967; Gray et al. 1974; Gandhi et al. 2016; Baryshnikova, Costanzo, S. Dixon, et al. 2010; Zackrisson et al. 2016; Miller et al. 2022). More precisely, mathematical models show the linear colony-size expansion rate is directly proportional to the square root of the exponential growth rate under non-limiting conditions. Intuitively, this relationship arises because colony growth is dominated by the expansion of the population of cells in an annulus at the colony border that are exposed to rich nutrient conditions. These cells expand at a rate similar to the exponential rate of cells growing in a rich nutrient liquid culture. In contrast, the cells in the interior of the colony experience poor nutrient conditions, grow very slowly, and do not contribute to colony growth.

This intimate relationship between both proliferation rates allows us to explore the origin of the Product neutrality function in mechanistic models of cell growth. Indeed, if colony-based fitnesses follow a Product model, then

where the superscript c indicates colony-based values for the fitness W and the growth rate λ. Taking into account the relationship between single-cell exponential growth rates and colony growth rates, we can write

where the superscript l denotes liquid cultures. Combining these expressions, we obtain

In other words, from the perspective of the Product neutrality function, fitnesses based on colony expansion rates are equivalent to fitnesses based on single-cell exponential growth rates. The prevalence of the Product neutrality model—both in the SGA data and in previous studies on datasets from liquid cultures (Jasnos et al. 2007; Onge et al. 2007; Mani et al. 2008)—encourages the exploration of its origin in mechanistic models of cell growth.”

(2) Misapplication of prokaryotic growth models

The study attempts to explain the mechanistic origin of the multiplicative model observed in yeast colony fitness using a bacterial cell growth model, particularly the Scott-Hwa model. However, the application of this bacterial model to yeast systems lacks valid justification. The Scott-Hwa model is heavily dependent on specific molecular mechanisms such as ppGppmediated regulation, which plays a crucial role in adjusting ribosome expression and activity during translation. This mechanism is pivotal for ensuring the growth-dependency of the ribosome fraction in the proteome, as described in [https://doi.org/10.1073/pnas.2201585119]. Unlike bacteria, yeast cells do not possess this regulatory mechanism, rendering the direct application of bacterial growth models to yeast inappropriate and potentially misleading. This fundamental difference in regulatory mechanisms undermines the relevance and accuracy of using bacterial models to infer yeast colony growth dynamics.

If the authors intend to apply a growth model with macroscopic variables to yeast double-mutant experimental data, they should avoid simply repurposing a bacterial growth model. Instead, they should develop and rigorously validate a yeast-specific growth model before incorporating it into their study.

There is nothing that is prokaryote specific in the Scott-Hwa model. It does not include the specific ppGpp mechanism to regulate ribosome fraction that does not exist in eukaryotes.  The general features of the model, like how the ribosome fraction is proportional to the growth rate have indeed been validated in yeast (Metzl-Raz et al. 2017; Elsemman et al. 2022; Xia et al. 2022). Performing a detailed physiological analysis of budding yeast across varying growth conditions in order to build a more extensive model is beyond the scope of this work. Finally, we note that the Weiße model, which we also analyzed, is also generic and has replicated empirical measurements both from bacteria and yeast (Weiße et al. 2015).

To clarify this point in the text, we have added the following to Section 2.3: 

“Experimental measurements in other organisms suggest that the observations leading to this model, including that the cellular ribosome fraction increases with growth rate, are in fact generic and also seen in the yeast S. cerevisiae (Metzl-Raz et al. 2017; Elsemman et al. 2022; Xia et al. 2022).”

(3) Overly specific assumptions in the theoretical model

he theoretical model in question assumes that two mutations affect only independent parameters of specific biochemical processes, an overly restrictive premise that undermines its ability to broadly explain the occurrence of the multiplicative model in mutations. Additionally, experimental evidence highlights significant limitations to this approach. For example, in most viable yeast deletion mutants with reduced growth rates, the expression of ribosomal proteins remains largely unchanged, in direct contradiction to the predictions of the Scott-Hwa model, as indicated in [https://doi.org/10.7554/eLife.28034]. This discrepancy emphasizes that the ScottHwa model and its derivatives do not reliably explain the growth rates of mutants based on current experimental data, suggesting that these models may need to be reevaluated or alternative theories developed to more accurately reflect the complex dynamics of mutant growth.

In the data from the Barkai lab referenced by the reviewer (reproduced below), we see that the ribosomal transcript fraction is in fact proportional to growth rate in response to gene deletions in contradiction to the reviewer’s interpretation. However, it is notable that the ribosomal transcript fraction is a bit higher for a given growth rate if that growth rate is generated by a mutation rather than generated by a suboptimal nutrient condition. We know that the very simple Scott-Hwa model is not a perfect representation of the cell. Nevertheless, it does recapitulate important aspects of growth physiology and therefore we thought it is useful to analyze its response to mutations and compare those responses to the different neutrality functions.  We never claimed the Scott-Hwa model was a perfect model and fully agree with the referee’s statement above that “... these models may need to be reevaluated, or alternative theories developed to more accurately reflect the complex dynamics of mutant growth.” Indeed, we say as much in our discussion where we wrote: 

“While we focused on coarse-grained models for their simplicity and mechanistic interpretability, they might be too simple to effectively model large double-mutant datasets and the resulting double-mutant fitness distributions. We therefore expect the combination of high throughput genetic data with the analysis of larger-scale models, for instance based on Flux Balance Analysis, Metabolic Control Analysis, or whole-cell modeling, to lead to important complementary insights regarding the regulation of cell growth and proliferation.”

To further clarify this point, we discuss and cite the Barkai lab data for gene deletions see Figure 2 from Metzl-Raz et al. 2017.

(4) Lack of clarity on the mechanistic origin of the multiplicative model

The study falls short of providing a definitive explanation for its primary objective: elucidating the "mechanistic origin" of the multiplicative model. Notably, even in the simplest case involving the Scott-Hwa model, the underlying mechanistic basis remains unexplained, leaving the central research question unresolved. Furthermore, the study does not clearly specify what types of data or models would be required to advance the understanding of the mechanistic origin of the multiplicative model. This omission limits the study's contribution to uncovering the biological principles underlying the observed fitness patterns.”

We appreciate the reviewer’s interest in a more complete mechanistic explanation for the product model of fitness. The primary goal of this study was to explore the validity of the Product model from the perspective of coarse-grained models of cell growth, and to extract mechanistic insights where possible. We view our work as a first step toward a deeper understanding of how double-mutant fitnesses combine, rather than a final, all-encompassing theory. As the referee notes, we are limited by the current state of the field, which has an incomplete understanding of cell growth. 

Nonetheless, our analysis does propose concrete, mechanistically informed explanations. For example, we highlight how growth-optimizing feedback—such as cells’ ability to reallocate ribosomes or adjust proteome composition—naturally leads to multiplicative rather than additive or minimal fitness effects. We also link the empirical deviations from pure multiplicative behavior to differences in how specific pathways re-balance under perturbation, and we suggest that a product-like rule emerges when multiple interconnected processes each partially limit cell growth.

In the discussion, we clarify what additional data and models we think will be required to advance this question. Namely, we propose extending our approach through larger-scale, more detailed modeling frameworks – that may include explicit modeling of ppGpp or TOR activities in bacteria or eukaryotic cells, respectively. We also emphasize the importance of refining the measurement of cell growth rates to uncover subtle deviations from the product rule that could yield greater mechanistic insight. By integrating high-throughput genetic data with nextgeneration computational models, it should be possible to hone in on the specific biological principles (e.g., metabolic bottlenecks, resource reallocation) that underlie the multiplicative neutrality function.

Reviewer #2 (Public review):

The paper deals with the important question of gene epistasis, focusing on asking what is the correct null model for which we should declare no epistasis.

In the first part, they use the Synthetic Genetic Array dataset to claim that the effects of a double mutation on growth rate are well predicted by the product of the individual effects (much more than e.g. the additive model). The second (main) part shows this is also the prediction of two simple, coarse-grained models for cell growth.

I find the topic interesting, the paper well-written, and the approach innovative.

One concern I have with the first part is that they claim that:

"In these experiments, the colony area on the plate, a proxy for colony size, followed exponential growth kinetics. The fitness of a mutant strain was determined as the rate of exponential growth normalized to the rate in wild type cells."

There are many works on "range expansions" showing that colonies expand at a constant velocity, the speed of which scales as the square root of the growth rate (these are called "Fisher waves", predicted in the 1940', and there are many experimental works on them, e.g. https://www.pnas.org/doi/epdf/10.1073/pnas.0710150104) If that's the case, the area of the colony should be proportional to growth_rate X time^2 , rather than exp(growth_rate*time), so the fitness they might be using here could be the log(growth_rate) rather than growth_rate itself? That could potentially have a big effect on the results.

We thank the reviewer for their thoughtful remarks. As they rightly pointed out, a large body of literature supports that colonies expand at constant velocity both from a theoretical and experimental standpoint. 

As discussed in the answer to the first question of Reviewer 1, this body of work also suggests that the linear expansion rate of the colony front is directly related to the single-cell exponential growth rate of the cells at the periphery. Hence, although the macroscopic colony growth may not be exponential in time, measuring colony size (or radial expansion) across different genotypes still provides a consistent and meaningful proxy for comparing their underlying growth capabilities. 

In particular, these studies suggest (consistently with Fisher-wave theory) that the linear growth rate of the colony 𝐾 is proportional to the square root of the exponential growth rate 𝜆. Under the assumption that the product model is valid for a given double mutant and for the exponential growth rate, we would have that

The associated wave-front velocities would then be predicted to be

In other words, if the product model is valid for fitness measures based on exponential growth rates, it should also be valid for fitness measures based on linear colony growth rates. 

We now include this discussion in the revised version of Section 2.3.

Additional comments/questions:

(1) What is the motivation for the model where the effect of two genes is the minimum of the two?

The motivation for the minimal model is the notion that there might be a particular process that is rate-limiting for growth due to a mutation. In this case, a mutation in process X makes it really slow and process Y proceeds in parallel and has plenty of time to finish its job before cell division takes place. In this case, even a mutation to process Y might not slow down growth because there is an excess amount of time for it to be completed. Thus, the double mutant might then be anticipated to have the growth rate associated with the single mutation to process X. We now add a similar description when we introduce the different neutrality functions in Section 2.1.

(2) How seriously should we take the Scott-Hwa model? Should we view it as a toy model to explain the phenomenon or more than that? If the latter, then since the number of categories in the GO analysis is much more than two (47?) in many cases the analysis of the experimental data would take pairs of genes that both affect one process in the Scott-Hwa model - and then the product prediction should presumably fail? The same comment applies to the other coarse-grained model.

From our perspective, models like the Scott-Hwa model constitute the simplest representation of growth based on data that is not trivial. Moreover, the Scott-Hwa model is able to incorporate interactions between two different biological processes. We believe models, like the Scott-Hwa and Weiße models, should be viewed as more than mere toy models because they have been backed up by some empirical data, such as that showing the ribosome fraction increases with growth rate. However, the Scott-Hwa model is inherently limited by its low dimensionality and relative simplicity. We do not claim that such models can provide a full picture of the cell. As argued in the main text, we have chosen to focus on such models because of their tractability and in the hope of extracting general principles. We nonetheless agree with the reviewer that they do not have the capacity to represent interactions between genes in the same biological process. We now note this limitation in the text. 

(3) There are many works in the literature discussing additive fitness contributions, including Kaufmann's famous NK model as well as spin-glass-type models (e.g. Guo and Amir, Science Advances 2019, Reddy and Desai, eLife 2021, Boffi et al., eLife 2023) These should be addressed in this context.

We thank the reviewer for pointing out this part of the literature. We do believe these works constitute a relevant body of work tackling the emergence of epistasis patterns from a theoretical grounding, and now reference and discuss them in the text. 

(4) The experimental data is for deletions, but it would be interesting to know the theoretical model's prediction for the expected effects of beneficial mutations and how they interact since that's relevant (as mentioned in the paper) for evolutionary experiments. Perhaps in this case the question of additive vs. multiplicative matters less since the fitness effects are much smaller.

This is an interesting question. Since mutations increasing the growth rate generated by gene deletions or other systematic perturbations are rare, we did not focus on them. Of course, as the reviewer notes, in the case of evolution experiments, these fitness enhancing mutations are selected for. To address the reviewer's question, we can first consider the Scott-Hwa model. In this case, the analytical solution remains valid in the case of fitness enhancing mutations so that the fitness of the double mutant will be the product neutrality function multiplied by an additional interaction term (see Figure 3). The mathematical derivation predicts that the double mutant fitness can potentially grow indefinitely. Indeed, the denominator can be equal to zero in some cases. In simulations, we see that the observation for deleterious mutations does not seem to hold for beneficial mutations (new supplementary Figure S5 shown below). Indeed, no model seems to replicate double mutant fitnesses much better than any other. This suggests that the growth-optimizing feedback we discuss in section 2.3 may have compound effects that ultimately make double-mutant fitnesses much larger than any model predicts.

We recognize this may be an important point, and discuss it in detail in the revised section 2.3 as well as in the discussion.

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Osiąganiu

eLife Assessment

This paper highlights an important physiological function of PGAM in the differentiation and suppressive activity of Treg cells by regulating serine synthesis. This role is proposed to intersect with glycolysis and one-carbon metabolism. The study's conclusion is supported by solid evidence from in-vitro cellular and in-vivo mouse models.

Reviewer #1 (Public review):

Summary:

This work provides a new potential tool to manipulate Tregs function for therapeutic use. It focuses on the role of PGAM in Tregs differentiation and function. The authors, interrogating publicly available transcriptomic and proteomic data of human regulatory T cells and CD4 T cells, state that Tregs express higher levels of PGAM (at both message and protein levels) compared to CD4 T cells. They then inhibit PGAM by using a known inhibitor ECGC and show that this inhibition affects Tregs differentiation. This result was also observed when they used antisense oligonucleotides (ASOs) to knockdown PGAM1.

PGAM1 catalyzes the conversion of 3PG to 2PG in the glycolysis cascade. However, the authors focused their attention on the additional role of 3PG: acting as starting material for the de novo synthesis of serine.

They hypothesized that PGAM1 regulates Tregs differentiation by regulating the levels of 3PG that are available for de novo synthesis of serine, which has a negative impact on Tregs differentiation. Indeed, they tested whether the effect on Tregs differentiation observed by reducing PGAM1 levels was reverted by inhibiting the enzyme that catalyzes the synthesis of serine from 3PG.

The authors continued by testing whether both synthesized and exogenous serine affect Tregs differentiation and continued with in vivo experiments to examine the effects of dietary serine restriction on Tregs function.

In order to understand the mechanism by which serine impacts Tregs function, the authors assessed whether this depends on the contribution of serine to one-carbon metabolism and to DNA methylation.

The authors therefore propose that extracellular serine and serine whose synthesis is regulated by PGAM1 induce methylation of genes Tregs associated, downregulating their expression and overall impacting Tregs differentiation and suppressive functions.

Strengths:

The strength of this paper is the number of approaches taken by the authors to verify their hypothesis. Indeed, by using both pharmacological and genetic tools in in vitro and in vivo systems they identified a potential new metabolic regulation of Tregs differentiation and function.

Reviewer #2 (Public review):

Summary:

The authors have tried to determine the regulatory role of Phosphoglycerate mutate (PGAM), an enzyme involved in converting 3-phosphoglycerate to 2-phosphoglycerate in glycolysis, in differentiation and suppressive function of regulatory CD4 T cells through de novo serine synthesis. This is done by contributing one carbon metabolism and eventually epigenetic regulation of Treg differentiation.

Strengths:

The authors have rigorously used inhibitors and antisense RNA to verify the contribution of these pathways in Treg differentiation in-vitro. This has also been verified in an in-vivo murine model of autoimmune colitis. This has further clinical implications in autoimmune disorders and cancer.

[Editors' note: The authors addressed important comments by the reviewers.]

Author response:

The following is the authors’ response to the original reviews

Public Reviews:

Reviewer #1 (Public review):

This work provides a new potential tool to manipulate Tregs function for therapeutic use. It focuses on the role of PGAM in Tregs differentiation and function. The authors, interrogating publicly available transcriptomic and proteomic data of human regulatory T cells and CD4 T cells, state that Tregs express higher levels of PGAM (at both message and protein levels) compared to CD4 T cells. They then inhibit PGAM by using a known inhibitor ECGC and show that this inhibition affects Tregs differentiation. This result was also observed when they used antisense oligonucleotides (ASOs) to knockdown PGAM1.

PGAM1 catalyzes the conversion of 3PG to 2PG in the glycolysis cascade. However, the authors focused their attention on the additional role of 3PG: acting as starting material for the de novo synthesis of serine.

They hypothesized that PGAM1 regulates Tregs differentiation by regulating the levels of 3PG that are available for de novo synthesis of serine, which has a negative impact on Tregs differentiation. Indeed, they tested whether the effect on Tregs differentiation observed by reducing PGAM1 levels was reverted by inhibiting the enzyme that catalyzes the synthesis of serine from 3PG.

The authors continued by testing whether both synthesized and exogenous serine affect Tregs differentiation and continued with in vivo experiments to examine the effects of dietary serine restriction on Tregs function.

In order to understand the mechanism by which serine impacts Tregs function, the authors assessed whether this depends on the contribution of serine to one-carbon metabolism and to DNA methylation.

The authors therefore propose that extracellular serine and serine whose synthesis is regulated by PGAM1 induce methylation of genes Tregs associated, downregulating their expression and overall impacting Tregs differentiation and suppressive functions.

Strengths:

The strength of this paper is the number of approaches taken by the authors to verify their hypothesis. Indeed, by using both pharmacological and genetic tools in in vitro and in vivo systems they identified a potential new metabolic regulation of Tregs differentiation and function.

We are grateful to the reviewer for their thoughtful and constructive consideration of our work. We appreciate their comment that the number of approaches taken to test our hypothesis represents a strength that increases confidence in the conclusions.

Weaknesses:

Using publicly available transcriptomic and proteomic data of human T cells, the authors claim that both ex vivo and in vitro polarized Tregs express higher levels of PGAM1 protein compared to CD4 T cells (naïve or cultured under Th0 polarizing conditions). The experiments shown in this paper have all been carried out in murine Tregs. Publicly available resources for murine data (ImmGen -RNAseq and ImmPRes - Proteomics) however show that Tregs do not express higher PGAM1 (mRNA and protein) compared to CD4 T cells. It would be good to verify this in the system/condition used in the paper.

This is a fair comment. Although our pharmacologic and genetic studies demonstrated the importance of PGAM in Treg differentiation and suppressive function in murine cells, thereby corroborating the hypothesis formed based on human CD4 cell expression data, we agree that investigating PGAM expression in murine Tregs is important in the context of our work. In reviewing the ImmPres proteomics database, the reviewer is correct that PGAM1 expression was not higher in iTregs compared to other subsets, including Th17 cells. However, when compared to other glycolytic enzymes, expression of PGAM1 increases out of proportion in iTregs. In particular, the ratio of PGAM1 to GAPDH expression is much greater in iTregs compared to Th17 cells. This data is now shown in the revised Figure S5. The disproportionate increase in PGAM1 expression is consistent with the regulatory role of PGAM in the Treg-Th17 axis via modulation of 3PG concentrations, a metabolite that lies between GAPDH and PGAM in the glycolytic pathway. The divergent expression changes between GAPDH and PGAM furthermore support the conclusion that GAPDH and PGAM play opposite roles in Treg differentiation.

It would also be good to assess the levels of both PGAM1 mRNA and protein in Tregs PGAM1 knockdown compared to scramble using different methods e.g. qPCR and western blot. However, due to the high levels of cell death and differentiation variability, that would require cells to be sorted.

We appreciate this comment. As noted by the reviewer, assessing PGAM1 expression via qPCR and Western blot would require cell sorting, which we do not currently have the resources to pursue. However, we measured the effect of ASOs on PGAM1 protein expression using anti-PGAM1 antibody via flow cytometry, which allowed gating on viable cells. As shown in Figure S3A, PGAM-targeted ASOs led to an approximately 40% decrease in PGAM1 expression, as measured by mean fluorescence intensity (MFI). Furthermore, we now show in revised Figure S2 that ASO uptake was near-complete in our cultured CD4 cells.

It is not specified anywhere in the paper whether cells were sorted for bulk experiments. Based on the variability of cell differentiation, it would be good if this was mentioned in the paper as it could help to interpret the data with a different perspective.

Cells were not sorted for bulk experiments. In the revised manuscript, this point is made clear in the text, figure legends, and Methods. It is worth noting that all bulk experiments were conducted on samples with greater than 70% cell viability (greater than 90% for stable isotope tracing studies).

Reviewer #2 (Public review):

Summary:

The authors have tried to determine the regulatory role of Phosphoglycerate mutate (PGAM), an enzyme involved in converting 3-phosphoglycerate to 2-phosphoglycerate in glycolysis, in differentiation and suppressive function of regulatory CD4 T cells through de novo serine synthesis. This is done by contributing one carbon metabolism and eventually epigenetic regulation of Treg differentiation.

Strengths:

The authors have rigorously used inhibitors and antisense RNA to verify the contribution of these pathways in Treg differentiation in-vitro. This has also been verified in an in-vivo murine model of autoimmune colitis. This has further clinical implications in autoimmune disorders and cancer.

We very much appreciate these comments about the rigor of the work and its implications.

Weaknesses:

The authors have used inhibitors to study pathways involved in Treg differentiation. However, they have not studied the context of overexpression of PGAM, which was the actual reason to pursue this study.

We appreciate this comment and agree that overexpression of PGAM would be an excellent way to complement and further corroborate our findings. Unfortunately, despite attempting several methods, we were unable to consistently induce overexpression of PGAM1 in our primary T cell cultures.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

I would suggest increasing the font size for flow cytometry gates. Percentages are the focus of the analysis, and it is very hard to read any.

We have increased the font size on all flow cytometry gates, as suggested.

Moreover, most of the flow data show Tregs polarization based on CD25 and FOXP3 expression. However, Figure 3 A, Figure 4D and Figure S3 show Tregs polarization based on FSC and Foxp3. Is there any reason for this?

Antibody staining against CD25 was poor in the experiments noted, which is why Foxp3 alone was used to identify Treg cells in these experiments.

Especially for Figure 3A, other cells could also express Foxp3 making interpretation difficult.

This is a fair comment. With respect to Figures 4D and S3 (now revised Figure S4), these experiments were conducted in isolated CD4 cells, in which the population of CD25-Foxp3+ cells is minimal following Treg polarization (as evident in our other figures). Regarding Figure 3A, previous work has found minimal expression of Foxp3 in circulating non-T cells (Devaud et al., 2014, PMID 25063364), such that we have confidence the identified Foxp3 expressing cells are, in fact, Treg cells. Notably, Figure 3A was already gated on CD4+ T cells, and in the periphery of wild-type mice, these would be reasonably referred to as Tregs, although this does not apply to diseased states or specific cases such as the tumor microenvironment.

The level of murine Tregs differentiation varies a lot among experiments. The % of CD4+CD25+FOXP3+ is ranging from 14% to 77% (controls). It would be good to understand and verify why such differentiation variability.

For most of our Treg polarization experiments, % differentiation in the control group falls within the 35 – 55% range. We found that treatment with ASOs (even scrambled control ASOs) tended to decrease Treg polarization overall, leading to lower numbers of Foxp3 expression in these experiments. Differentiation was similarly low in a few experiments that did not involve the use of ASOs, which we believe was caused by batch variability in the recombinant TGF-b that was used for polarization. Despite this variability, experiments were conducted with sufficient independent experiments and biological replicates to observe consistent trends and to have confidence in the results, as corroborated by statistical testing and the wide variety of experimental approaches used to verify our conclusions. Notably controls were run in every experiment, allowing accurate comparisons to be made in each individual experiment.

Similar comments apply to the level of cell death observed in the cultures of polarizing Tregs.

Although there was some variability in cell viability between experiments, flow cytometry experiments were always gated on live cells, and we believe concerns about reproducibility are substantially mitigated by the number of independent experiments, biological replicates, and distinct experimental approaches used for verification of the experimental findings. For all bulk experiments, cell viability was greater than 70% and equal across samples. For the flux studies, viability was greater than 90% and equal across samples.

Figure 2 B and D: EGCG has been used at two different concentrations. Is it lower in Figure 2D because of one condition being a combination of inhibitors or is it a typo?

The doses stated in the original legend are correct. Yes, drug doses were optimized for combination-treatment experiments. This point is now clarified in the figure legend.

Figure 2G: The description in the results does not match figure legend - Text - serine/glycine-free media or control (serine/glycine-containing) media; figure legend - serine/glycine-free media or media containing 4 mM serine.

We thank the reviewer for pointing out this discrepancy, which was an error in the text. The two conditions used were 1) serine/glycine-free media, and 2) serine/glycine-free media supplemented with 4 mM serine. The text and figure legend have both been updated to clarify this point.

Figure 3 F and G: the graphs do not show the individual points.

Individual points were not shown in these graphs because they are derived from scRNA-seq data, with SCFEA calculated from individual cells. As such, there are far too many data points to display all individual values.

CD4+ T-cell isolation and culture: cells were cultured in 50%RPMI and 50% AIM-V.

I thought that AIM-V medium was intended to be for human cultures. Could some of the conditions explain the low level of differentiation observed in some experiments? If there is such variability it might be because the conditions used are not optimal and therefore not reproducible.

We appreciate this critique. Although AIM-V media is often used for ex vivo human T cell cultures, it can similarly be used for mouse T cell culture with the addition of b-mercaptoethanol, as suggested by ThermoFisher and as used in prior publications, such as PMID 36947105. As outlined in the responses above, the differentiation we observed was consistent in most experiments, with some variability based on experimental conditions (such as lower differentiation in the setting of ASO treatment). Furthermore, we believe the number of independent experiments, biological replicates, and independent experimental approaches used in the study supports the reproducibility of our findings.

Figures S1 A, S2 B, and S4: the flow data are shown using both heights (FSC) and area (zombie NIR dye). It would be better to use areas for both parameters.

In the revised manuscript, areas are now used on both the x- and y-axes for these figures.

Figure S1 B and S2 C: The bar graphs are both showing proliferation index, however, the graphs are labelled differently in the two figures and in the legend (proliferation index -Fig S1 B; division index -Fig S2 C and replication index in the legend of Fig S2 C). The explanation of how the index has been calculated should probably go in the legend of the first figure that shows it.

We thank the reviewer for this comment. In the revised manuscript, we have ensured consistency in the terminology (“proliferation index” is now used consistently), and the explanation of the proliferation index calculation is now included in the legend to Figure S1, where the proliferation index first appears.

Were Tregs PGAM1 KD used for RNAseq sorted or not? Based on the plots shown in Figure S2 B there is ~ 50% death which needs to be taken into consideration for the analysis if not depleted.

Similar question for all bulk experiments. It is not specified in the methods or figure legends.

The cells used for RNAseq and other bulk experiments were not sorted. This point is now made clear in the text, figure legends, and Methods. However, cultures were only used for bulk analyses if the viability in those particular experiments was greater than 70%. Given the sensitivity of stable isotope tracing analyses, cultures were only analyzed for those studies if viability was greater than 90%. In these experiments, viability was similar across samples.

It was mentioned in Figure 1 that the PGAM KD led to transcriptional changes that impacted MYC targets and mTORC1 signalling. It would be good to validate these findings maybe with more targeted experiments.

We appreciate this suggestion and agree that validation and further investigation of these critical targets would be worthwhile. However, because of limitations to resources and the fact that these findings are not critical to the main conclusions of the study, we consider these experiments as future directions beyond the scope of the current work.

Reviewer #2 (Recommendations for the authors):

Here are a few suggestions and recommendations to improve the research study.

(1) The authors have used the word 'vehicle' in most of the figures, however, this word is not explained well in the figure legend. The authors may want to clarify to readers whether vehicle is a plasmid or a solvent for control purposes. For example, in Figure 1D, if vehicle is a plasmid, then another sample for vehicle +/-EGCG should be considered for the rigor in results.

Thank you for identifying this point of confusion. For all drug treatment experiments, vehicle controls consisted of solvent alone without drug. For ASO experiments, the control condition consisted of scrambled ASO. This point is now made clear in the Methods (“Drug and ASO Treatments” section) as well as in the main text. Furthermore, the figure legends and axes have been edited such that “vehicle” is only used to refer to drug experiments (in which solvent vehicle alone was used as control), and “control” is used to refer to ASO experiments (in which scrambled ASO served as control).

(2) Figure 1H represents the RNAseq data for knockdown of PGAM1. It might be interesting to see similar data for the overexpression of PGAM1.

We appreciate this comment and agree that overexpression of PGAM1 would be an excellent way to complement and further corroborate our findings using PGAM1 knockdown and pharmacologic inhibition. Unfortunately, despite attempting several methods, we were unable to consistently induce overexpression of PGAM1 in our primary T cell cultures.

(3) The font in most of the data from flow cytometry experiments (for example 1I) is not legible. Please increase the font size to make it legible.

Font sizes have been increased.

(4) Figure S2, PGAM expression was measured by Flow cytometry experiments. A similar experiment using western Blot, the direct measurement of protein expression, will strengthen the evidence.

We appreciate this comment. As noted in the public reviews, Western blot would require sorting of viable cells, and unfortunately we do not currently have the resources to conduct additional experiments with FACS. However, we respectfully note that assessing protein expression via flow cytometry quantifies protein levels based on antibody binding, similar to Western blot (or in-cell Western blot), while also allowing gating on viable cells. We also note that nearly 100% of cultured CD4 cells took up ASO, as shown in revised Figure S2.

(5) Figure 1J, it is mentioned in the text that 10 datasets were studied. a normalized parameter such as overexpression or suppression could be studied with the variance. It will be good to understand the variability in response among different datasets.

We thank the reviewer for the opportunity to clarify this data. This data was taken from a single published dataset (Dykema et al., 2023, PMID 37713507) in which 10 distinct subsets of tumor-infiltrating Tregs (TIL-Tregs) were identified, rather than from 10 distinct datasets. After identifying the Activated (1)/OX40hiGITRhi cluster of TIL-Tregs as a highly suppressive subset that correlates with resistance to immune checkpoint blockade, Dykema et al. compared gene expression in this subset to the bulked collection of the other 9 subsets, and the data shown in Figure 1J is derived from this analysis. As such, the data in Figure 1J is, indeed, a normalized parameter of overexpression, showing overexpression of PGAM1 in this highly suppressive subset versus other subsets, out of proportion to proximal rate-limiting glycolytic enzymes. The main text and figure/figure legend have been edited to clarify this point.

(6) It will be good to rephrase that the roles of PGAM and GAPDH are opposite, this paragraph is confusing since words such as "supporting Treg differentiation" and "augments Treg differentiation" have been used, although the data in S3 and 1D are opposite. Any possible explanation for the opposing roles of PGAM and GAPDH, despite their involvement in the same pathway of glycolysis, can be added to build up the interest of readers. What is the comparison of the expression of GAPDH and PGAM in Figure 1J?

We thank the reviewer for this comment, as we appreciate that the language used in our initial manuscript was confusing. We have edited the main text, in both the Results and Discussion section, in order to clarify this point and provide explanation as suggested. Indeed, our experimental data indicate that GAPDH and PGAM play opposing roles in Treg differentiation; whereas inhibiting GAPDH activity leads to greater Treg differentiation (shown in revised Figure S4 and our previously published work), similarly inhibiting PGAM leads to diminished Treg differentiation. We view this point (that enzymes within the same glycolytic pathway can have divergent roles in T cells) as a primary implication of these findings, with the explanation that individual enzymes within the same pathway can differentially regulate the concentrations of key immunoactive metabolites. In our study, we identified 3PG as a key immunoactive metabolite whose concentration would be differentially impacted by GAPDH activity versus PGAM activity, since it lies downstream of GAPDH but upstream of PGAM.

To provide further evidence for the opposing roles of GAPDH and PGAM, we analyzed existing datasets. In the revised Figure S5, we show that the PGAM1/GAPDH expression ratio increases in both human and mouse Tregs compared to other CD4 subsets.

(7) Figure 2C, what is M+1, M+2 etc. Does it represent the number of hrs? If so, why are the results for 6 hrs are not shown since the study was for 6 hrs? And what is happening with M+2?

We appreciate the opportunity to clarify this point and apologize for prior confusion. The terminology “M+n” refers to mass-shift produced by incorporation of 13-carbon. When a metabolite incorporates a single 13-carbon atom, it has a mass-shift of one (M+1), whereas incorporation of three 13-carbon atoms produces a mass-shift of three (M+3). Because we used uniformly 13-carbon labeled glucose, 3PG derived from the labeled glucose will have all three carbons labeled (M+3), as will serine that is newly synthesized from 3PG. Because serine can enter the downstream one-carbon cycle and be recycled, we also see the appearance of recycled serine with a single 13-carbon (M+1). The critical point in Figure 2C is that labeled serine is higher in Th17 versus Treg cells, demonstrating that de novo serine synthesis from glycolysis is greater. The main text has been edited to clarify this important point.

(8) Including the quantification of inhibition and rescuing effect of EDCG and NCT will be helpful to readers.

The inhibition and rescuing effects of these drugs are quantified in Figures 2D and 2E as they relate to Treg differentiation. The reviewer may be referring to quantification of relative effects on 3PG levels and serine synthesis. If so, we unfortunately do not have the resources to complete these studies, which would require large-scale quantitative mass spectrometry studies or enzyme activity assays.

(9) Figure 2D and 2E: The authors could also experiment with a dose dependence curve on EGCG and NCT on this phenotype for Treg differentiation. That can help understand the balance between serine pathways and glycolysis pathways. Similarly, the dose dependence of 3PG for Figure 2E and comparing it to the kinetic constants of these enzymes involved and cellular concentrations, these details will be helpful to understand the metabolic dynamics, because this phenotype could be an interplay of both 3PG and serine concentrations.

We appreciate this suggestion and agree that establishing detailed dose-dependence curves and relating these findings to enzyme kinetics would yield additional insights into the biochemical regulation provided by PGAM and PHGDH. Unfortunately we do not have the resources to pursue these additional studies, which therefore lie beyond the scope of our current work.

(10) Figure 4: Explanation for no effect of methionine supplementation?

Thank you for raising this point. We speculate that methionine supplementation had minimal effect because physiologic levels of serine were sufficient to provide basal substrates for the one-carbon cycle. On the other hand, eliminating methionine produced enough of a decrease in one-carbon metabolism to potentiate the effects of excess serine. This point is now briefly addressed in the text.

(11) For direct connection between PGAM and methylation, methylation experiments could be worked out with NCT1 and SHIN1 (as in Figure 4H).

We very much appreciate this suggestion, which we agree would provide a strong complementary approach. Unfortunately we do not have the resources to pursue these studies currently. However, we believe the increased methylation observed following PGAM knockdown (Figure 4G) as strong evidence that PGAM activity directly modulates methylation.

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