I have experienced this kind expectation to reality relationship in some of my personal relationships. These people had an idea of what they could be if they could just stop being inadequate that only served to generate shame and guilt. Often, there was never any real grounding for the things they expected of themselves, but they felt the weight of those expectations as if they were an undeniable reflection of their potential. I am sure many of this is related to external social expectations that are later internalized. These expectations seem to rarely serve as drivers for someone to be more productive and more often seem to break people down and make them overall less likely to engage with life.

One of our recent lectures identified the importance of an improvement mindset. Tools like these could help avoid developing unrealistic expectations that ultimately dissuade attempts at self improvement. They could provide an interpretive lens to contextualize feedback in ways that are more constructive.

Social media has achieved this goal long ago as this generation is on their phones all day. Such as every day when I check my screen time it's over 8 hours or more, and 70% time is spent on TikTok. By using data mining the app has fairly figured out what phase of life I am in and every TikTok that i see is relatable so I feel a connection with it. For example, if someone goes through a break-up, their whole FYP will be filled with tiktoks that would be about a break-up on how someone went through something same or something comforting, keeping them hooked to it. As for me whatever I am going through in my life it's like my TikTok knows all of it and shows exact same posts. In this way, I can think how data mining may be used to extract my conversations with my friends or what I like and repost depending on my mood is also being tracked.

Thực nghiệm: - Mô hình LLM: LLaMA-3-3B và Qwen-1.5.7B. - Bộ dữ liệu: Đối với tiếng Anh, bộ dữ liệu Pile-NER được sử dụng. Đối với tiếng Trung, bộ dữ liệu Sky-NER do chính nhóm tác giả xây dựng được sử dụng. LoRA được sử dụng trong quá trình huấn luyện LLM - Mô hình truy xuất: Sử dụng GTE-large để tạo ra các embedding câu và số lượng mẫu tương đồng được truy xuất là 2. - Phương pháp đánh giá: Tập trung vào đánh giá Zero-shot.

in termius, add "shift" to c/v

makes difficult for students to attend classes on campus

would allow lots of parents to seek help on their assignments and other questions if their kids were welcomed in the space. as mom, this would be extremely helpful.

sad that many students, especially student parents have to go through this.

staff should be more aware of the needs of student parents seeking help to achieve their goals

thus, student parents don't receive all the help they need.

Student parents seeking a higher education should recieve more help as they are trying to better themselves and the lives of their children.

making it harder for those parents seeking a higher education

There should be more resources for student parents so they can achieve their goals. They are just as important as the average student.

Beginning this stanza of “What the Thunder Said”, Eliot describes a woman who manipulated her hair, and “fiddled whisper music on those strings”. Interpreting “those strings” as the woman’s own hair we can interpret a curious instance of a woman using her body as an instrument to play music. Of course, we must acknowledge that realistically, one can’t make any substantial sounds with their hair, and thus we can interpret her “whisper music” as imagined, or only perceived by her. In terms of the human body, especially in relation to hair, we can further understand this passage by looking at page 298 of the Visuddhi-Magga. This page discusses the superficiality of beauty and the ego, as it declares that the human body is repulsive. The repulsiveness of the human body is argued, as the Visuddhi Magga reads, “When any part of the body becomes detached, as, for instance, the hair of the head … people are unwilling so much as to touch it”. According to the Visuddhi Magga, humans assign significance and beauty to discardable parts of their body, and when those parts are discarded, humans view them with disgust. When comparing the teachings of the Visuddhi-Magga with the long-haired woman, there seems to be a contrast in appreciation for the human body. While the Visuddhi-Magga argues that the body, especially the hair, is repulsive, the woman is using her own hair as an instrument, something of significance and beauty in and of itself. I believe another important aspect of this analysis lies in the consideration of Eliot’s notion of “conceptual death”. In “The Waste Land” Eliot has challenged the reader’s literal understanding of death, and instead seems to propose the idea that death is a complex and cultural state that cannot be so easily defined. Literally, our hair is dead, but when attached to our body, it becomes a part of a living thing, and thus seems to gain significance through what I argue is “conceptual vitality”. Interpeting the lesson of the Visuddhi-Magga, hair loses its “vitality” when it is cut off, and becomes recognizably repulsive. Though it was always dead, it has lost its significance to the body. I would argue that the woman using her hair as an instrument is an affirmation of the hair’s significance to herself, and thus, a part of her own conceptual vitality.

Psalm 63 describes longing for God in a place with no water, while this stanza describes longing for water whilst pointing out the abundance of rock. In Psalm 63, it even says of God, "My soul thirsteth for thee," which equates God to water in a sense. When looking at this section of "The Waste Land" together with Psalm 63, it makes this part seem notably unreligious.

This line, which seemingly emphasizes how water can kill/take apart human beings, draws a contrast to Corinthians, which states "All were baptized into Moses in the cloud and in the sea," which appears to show how water is used to "baptize" someone into a religion. I think the difference between the water usages in these respective works stems from Eliot looking at some of the more literal actions of water while Corinthians looks at more figurative, religious uses of it.

Eliot references the "Isle of Dogs". Matthew 7:6 states "Give not that which is holy unto the dogs, neither cast ye your pearls before swine, lest they trample them under their feet, and turn again and rend you." I find this interesting because I essentially understand to say "be careful who you associate with because not all people are good," which draws a contrast to this stanza which generally seems to lack intention. For example, it says "The barges drift" and references "Drifting logs," which implies a lack of control over the circumstances. This is all very interesting because in Matthew, "the dogs" seemingly refer to people you find yourself associating with if you become too careless with your actions, and going "Past the Isle of Dogs" feels similarly unintentional.

Interestingly, throughout this entire long stanza, the night seems to become darker as the actions become darker. First, we're just in the "violet hour", then time passes throughout the stanza, and it ends with "And gropes his way, finding the stairs unlit" (Eliot, 248), after Tiresias has raped a woman. The way light and darkness is used here draws a contrast to how it's used in Fragment 149 of a Sappho poem, where she refers to "Bringing everything that shining Dawn scattered, you bring the sheep, you bring the goat, you bring the child back to its mother" (Sappho). Here, darkness and nighttime are seen as things that bring people/animals together in a pleasurable way by reuniting them, whereas in this stanza Tiresias and a woman are brought together at night, but he rapes her, thereby correlating darkness and nighttime with darker actions in "The Waste Land".

This is a clear reference to Edmund Spenser's poem "Prothalamion". I think it might represent Eliot trying to get closer to that aesthetic of the 1500s, when the world was generally more untouched. In the next lines he says "The river bears no empty bottles, sandwich papers, / Silk handkerchiefs, cardboard boxes, cigarette ends / Or other testimony of summer nights" (Eliot, 176-178), which I think touches on the theme of industrialization. Therefore, Eliot may be referencing Spenser to feel closer to that pre-industrial world.

.h2

.h2

the majority of student parents attend community college, so why is most of the funding going to 4 year universities?

finding balance as a mother is so difficult and at times you feel burnt out.

the chart shows that on campus childcare has decreased, yet the demand for higher education has increased.

women single parents are trying to better themselves and earn a higher education

childcare is an important factor for student parents, without childcare they wouldn't be able to attend school.

large amount of college students are parents

This bit of information reminds me of a few studies and lawsuits that have occurred in the last decade or two regarding names on job applications. The inquiries focused on the concept that someone's name being less culturally familiar to a recruiter would negatively bias an applicant's chances of getting to the interview stage. This effect was studied using identical resumes with different names associated to measure employer responses. This seems like a great example of the primacy effect making biases that are sometimes difficult to identify more obvious.

I can see this effect happen where you may not think expectation would matter very much. There are many times where someone has stopped to say something to me, but the content of the statement is outside of my current mode of thinking. A perfectly understandable statement can become completely unintelligible purely because the context of the message did not prepare the receiver to comprehend it. If something like this can happen in the case of straight forward comprehension, the effect must me exacerbated by the complexity or obscurity of the intended communication.

Can I annotate an entire chapter?

30D: 726 trades, 40.53%pl

90% dd in 2022

why would it be that? bc there's too much work that requires management and headcount?

In the context of social media, replication refers to the process where content, or a modified version of it, is shared and distributed across platforms, reaching more viewers. When users share or remix content, the new version may include changes or additions, which are then carried forward as the content continues to spread. This creates a cycle where the modified version becomes the basis for future iterations, allowing both the original and the altered content to reach even larger audiences over time.

Knowing how recommendation algorithms work, users - especially content creators - will often adjust their strategies to expand their content, such as by increasing engagement and using trending topics. This is critical for creators who rely on social media for income, as higher visibility can lead to more opportunities for monetization. However, this also raises ethical issues, as it can sometimes encourage sensationalism or low-quality content exploitation systems.

Test annotation!

It's essential that Design Justice emphasizes not only the outcome of design but who is involved in the process. If only dominant groups are part of the decision-making, we risk creating systems that unintentionally harm or exclude marginalized groups. Ensuring that all voices are represented can lead to more inclusive, equitable design solutions that truly serve diverse communities.

I hear this phrase again. I have only heard it in this paper and in MLA Guide to Digital Literacy.

I understand these examples, but for some reason I don't think it is the same... Some songwriters don't "rely on a time-honored verse-chorus-verse", whilst some do many don't. That is why I think that writers can use their own language, and their own creativity, and their own writing styles to in turn make it a great paper.

This reminds me of how we have been talking about writing to meet the status quo of the perfect paper.

I am currently watching an episode of Gilmore Girls where one of the main characters bright up the question of how commonly used catch phrases can be used a plagiarism. Sometimes I live in fear of committing plagiarism. Sometimes a thought comes into my mind and I get nervous I read/heard it somewhere then I will get flagged for plagiarism. At times I worry more about the sources being peer reviewed or is it in MLA/APA format, more than the actual paper. It is hard to know what is/isn't plagiarism.

Return sentence is a great way of writing and I will make sure to use that in my next essay. Also, other template are very useful and give me a better understanding to what the writer try to teach us.

I like to use a statistic at the beginning of my essay because I think it grab people attention and make them curious about the rest of the essay.

Good advice for us is to start with what people say in other research papers and then add your points and discussion.

A writers should know their audience and work to make them engage and not lost during the speech. Also, it is better to give each information on the right time to make the audience comfortable about what we are saying.

It is important to show why are you bringing other words and how it really helps you introducing your points.

Please note that I corrected the typos here. Alvin

eLife Assessment

This important paper demonstrates that different PKA subtypes exhibit distinct subcellular localization at rest in CA1 neurons. The authors provide compelling evidence that when all tested PKA subtypes are activated by norepinephrine, catalytic subunits translocate to dendritic spines but regulatory subunits remain unmoved. Furthermore, PKA-dependent regulation of synaptic plasticity and transmission can be supported only by wildtype, dissociable PKA, but not by inseparable PKA.

Reviewer #1 (Public review):

Summary:

This is a short self-contained study with a straightforward and interesting message. The paper focuses on settling whether PKA activation requires dissociation of the catalytic and regulatory subunits. This debate has been ongoing for ~ 30 years, with renewed interest in the question following a publication in Science, 2017 (Smith et al.). Here, Xiong et al demonstrate that fusing the R and C subunits together (in the same way as Smith et al) prevents the proper function of PKA in neurons. This provides further support for the dissociative activation model - it is imperative that researchers have clarity on this topic since it is so fundamental to building accurate models of localised cAMP signalling in all cell types. Furthermore, their experiments highlight that C subunit dissociation into spines is essential for structural LTP, which is an interesting finding in itself. They also show that preventing C subunit dissociation reduces basal AMPA receptor currents to the same extent as knocking down the C subunit. Overall, the paper will interest both cAMP researchers and scientists interested in fundamental mechanisms of synaptic regulation.

Strengths:

The experiments are technically challenging and well executed. Good use of control conditions e.g untransfected controls in Figure 4.

Weaknesses:

The novelty is lessened given the same team has shown dissociation of the C subunit into dendritic spines from RIIbeta subunits localised to dendritic shafts before (Tillo et al., 2017). Nevertheless, the experiments with RII-C fusion proteins are novel and an important addition.

Reviewer #2 (Public review):

Summary:

PKA is a major signaling protein which has been long studied and is vital for synaptic plasticity. Here, the authors examine the mechanism of PKA activity and specifically focus on addressing the question of PKA dissociation as a major mode of its activation in dendritic spines. This would potentially allow to determine the precise mechanisms of PKA activation and address how it maintains spatial and temporal signaling specificity.

Strengths:

The results convincingly show that PKA activity is governed by the subcellular localization in dendrites and spines and is mediated via subunit dissociation. The authors make use of organotypic hippocampal slice cultures, where they use pharmacology, glutamate uncaging, and electrophysiological recordings.

Overall, the experiments and data presented are well executed. The experiments all show that at least in the case of synaptic activity, distribution of PKA-C to dendritic spines is necessary and sufficient for PKA mediated functional and structural plasticity.<br /> The authors were able to persuasively support their claim that PKA subunit dissociation is necessary for its function and localization in dendritic spines. This conclusion is important to better understand the mechanisms of PKA activity and its role in synaptic plasticity.

Weaknesses:

While the experiments are indeed convincing and well executed, the data presented is similar to previously published work from the Zhong lab (Tillo et al., 2017, Zhong et al 2009). This reduces the novelty of the findings in terms of re-distribution of PKA subunits, which was already established, at least to some degree.

Reviewer #3 (Public review):

Summary:

Xiong et al. investigated the debated mechanism of PKA activation using hippocampal CA1 neurons under pharmacological and synaptic stimulations. Examining all major PKA-R isoforms in these neurons, they found that a portion of PKA-C dissociates from PKA-R and translocate into dendritic spines following norepinephrine bath application. Additionally, their use of a non-dissociable form of PKA demonstrates its essential role in structural long-term potentiation (LTP) induced by two-photon glutamate uncaging, as well as in maintaining normal synaptic transmission, as verified by electrophysiology. This study presents a valuable finding on the activation-dependent re-distribution of PKA catalytic subunits in CA1 neurons, a process vital for synaptic functionality. The robust evidence provided by the authors makes this work particularly relevant for biologists seeking to understand PKA activation mechanisms, its downstream effects, and synaptic plasticity.

Strengths:

The study is methodologically robust, particularly in the application of two-photon imaging and electrophysiology. The experiments are well-designed with effective controls and a comprehensive analysis. The credibility of the data is further enhanced by the research team's previous works in related experiments. The study provides sufficient evidence to support the classical model of PKA activation via dissociation in neurons.

Weaknesses:

No specific weaknesses are noted in the current study; future research could provide additional insights by exploring PKA dissociation under varied physiological conditions, particularly in vivo, to further validate and expand upon these findings.

Author response:

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

New Experiments

(1) Activation-dependent dynamics of PKA with the RIα regulatory subunit, adding to the answer to Reviewers 1 and 2. To determine the dynamics of all PKA isoforms, we have added experiments that used PKA-RIα as the regulatory subunit. We found differential translocation between PKA-C (co-expressed with PKA-RIα) and PKA-RIα (Figure 1–figure supplement 3), similar to the results when PKA-RIIα or PKA-RIβ was used.

(2) PKA-C dynamics elicited by a low concentration of norepinephrine, addressing Reviewer 3’s comment. We have found that PKA-C (co-expressed with RIIα) exhibited similar translocation into dendritic spines in the presence of a 5x lowered concentration (2 μM) of norepinephrine, suggesting that the translocation occurs over a wide range of stimulus strengths (Figure 1-figure supplement 2).

Reviewer #1 (Public Review):

Summary:

This is a short self-contained study with a straightforward and interesting message. The paper focuses on settling whether PKA activation requires dissociation of the catalytic and regulatory subunits. This debate has been ongoing for ~ 30 years, with renewed interest in the question following a publication in Science, 2017 (Smith et al.). Here, Xiong et al demonstrate that fusing the R and C subunits together (in the same way as Smith et al) prevents the proper function of PKA in neurons. This provides further support for the dissociative activation model - it is imperative that researchers have clarity on this topic since it is so fundamental to building accurate models of localised cAMP signalling in all cell types. Furthermore, their experiments highlight that C subunit dissociation into spines is essential for structural LTP, which is an interesting finding in itself. They also show that preventing C subunit dissociation reduces basal AMPA receptor currents to the same extent as knocking down the C subunit. Overall, the paper will interest both cAMP researchers and scientists interested in fundamental mechanisms of synaptic regulation.

Strengths:

The experiments are technically challenging and well executed. Good use of control conditions e.g untransfected controls in Figure 4.

We thank the reviewer for their accurate summarization of the position of the study in the field and for the positive evaluation of our study.

Weaknesses:

The novelty is lessened given the same team has shown dissociation of the C subunit into dendritic spines from RIIbeta subunits localised to dendritic shafts before (Tillo et al., 2017). Nevertheless, the experiments with RII-C fusion proteins are novel and an important addition.

We thank the reviewer for noticing our earlier work. The first part of the current work is indeed an extension of previous work, as we have articulated in the manuscript. However, this extension is important because recent studies suggested that the majority of PKA-RIIβ are axonal localized. The primary PKA subtypes in the soma and dendrite are likely PKA-RIβ or PKA-RIIα. Although it is conceivable that the results from PKA-RIIβ can be extended to the other subunits, given the current debate in the field regarding PKA dissociation (or not), it remains important to conclusively demonstrate that these other regulatory subunit types also support PKA dissociation within intact cells in response to a physiological stimulant. To complete the survey for all PKA-R isoforms, we have now added data for PKA-RIα (New Experiment #1), as they are also expressed in the brain (e.g., https://www.ncbi.nlm.nih.gov/gene/5573). Additionally, as the reviewer points out, our second part is a novel addition to the literature.

Reviewer #2 (Public Review):

Summary:

PKA is a major signaling protein that has been long studied and is vital for synaptic plasticity. Here, the authors examine the mechanism of PKA activity and specifically focus on addressing the question of PKA dissociation as a major mode of its activation in dendritic spines. This would potentially allow us to determine the precise mechanisms of PKA activation and address how it maintains spatial and temporal signaling specificity.

Strengths:

The results convincingly show that PKA activity is governed by the subcellular localization in dendrites and spines and is mediated via subunit dissociation. The authors make use of organotypic hippocampal slice cultures, where they use pharmacology, glutamate uncaging, and electrophysiological recordings.

Overall, the experiments and data presented are well executed. The experiments all show that at least in the case of synaptic activity, the distribution of PKA-C to dendritic spines is necessary and sufficient for PKA-mediated functional and structural plasticity.

The authors were able to persuasively support their claim that PKA subunit dissociation is necessary for its function and localization in dendritic spines. This conclusion is important to better understand the mechanisms of PKA activity and its role in synaptic plasticity.

We thank the reviewer for their positive evaluation of our study.

Weaknesses:

While the experiments are indeed convincing and well executed, the data presented is similar to previously published work from the Zhong lab (Tillo et al., 2017, Zhong et al 2009). This reduces the novelty of the findings in terms of re-distribution of PKA subunits, which was already established. A few alternative approaches for addressing this question: targeting localization of endogenous PKA, addressing its synaptic distribution, or even impairing within intact neuronal circuits, would highly strengthen their findings. This would allow us to further substantiate the synaptic localization and re-distribution mechanism of PKA as a critical regulator of synaptic structure, function, and plasticity.

We thank the reviewer for noticing our earlier work. The first part of the current work is indeed an extension of previous work, as we have articulated in the manuscript. However, this extension is important because recent studies suggested that the majority of PKA-RIIβ are axonal localized. The primary PKA subtypes in the soma and dendrite are likely PKA-RIβ or PKA-RIIα. Although it is conceivable that the results from PKA-RIIβ can be extended to the other subunits, given the current debate in the field regarding PKA dissociation (or not), it remains important to conclusively demonstrate that these other regulatory subunit types also support PKA dissociation within intact cells in response to a physiological stimulant. To complete the survey for all PKA-R isoforms, we have now added data for PKA-RIα (New Experiment #1), as they are also expressed in the brain (e.g., https://www.ncbi.nlm.nih.gov/gene/5573). Additionally, as Reviewer 1 points out, our second part is a novel addition to the literature.

We also thank the reviewer for suggesting the experiments to examine PKA’s synaptic localization and dynamics as a key mechanism underlying synaptic structure and function. We agree that this is a very interesting topic. At the same time, we feel that this mechanistic direction is open ended at this time and beyond what we try to conclude within this manuscript: prevention of PKA dissociation in neurons affects synaptic function. Therefore, we will save the suggested direction for future studies. We hope the reviewer understand.

Reviewer #3 (Public Review):

Summary:

Xiong et al. investigated the debated mechanism of PKA activation using hippocampal CA1 neurons under pharmacological and synaptic stimulations. Examining the two PKA major isoforms in these neurons, they found that a portion of PKA-C dissociates from PKA-R and translocates into dendritic spines following norepinephrine bath application. Additionally, their use of a non-dissociable form of PKC demonstrates its essential role in structural long-term potentiation (LTP) induced by two-photon glutamate uncaging, as well as in maintaining normal synaptic transmission, as verified by electrophysiology. This study presents a valuable finding on the activation-dependent re-distribution of PKA catalytic subunits in CA1 neurons, a process vital for synaptic functionality. The robust evidence provided by the authors makes this work particularly relevant for biologists seeking to understand PKA activation and its downstream effects essential for synaptic plasticity.

Strengths:

The study is methodologically robust, particularly in the application of two-photon imaging and electrophysiology. The experiments are well-designed with effective controls and a comprehensive analysis. The credibility of the data is further enhanced by the research team's previous works in related experiments. The conclusions of this paper are mostly well supported by data. The research fills a significant gap in our understanding of PKA activation mechanisms in synaptic functioning, presenting valuable insights backed by empirical evidence.

We thank the reviewer for their positive evaluation of our study.

Weaknesses:

The physiological relevance of the findings regarding PKA dissociation is somewhat weakened by the use of norepinephrine (10 µM) in bath applications, which might not accurately reflect physiological conditions. Furthermore, the study does not address the impact of glutamate uncaging, a well-characterized physiologically relevant stimulation, on the redistribution of PKA catalytic subunits, leaving some questions unanswered.

We agreed with the Reviewer that testing under physiological conditions is critical especially given the current debate in the literature. That is why we tested PKA dynamics induced by the physiological stimulant, norepinephrine. It has been suggested that, near the release site, local norepinephrine concentrations can be as high as tens of micromolar (Courtney and Ford, 2014). Based on this study, we have chosen a mid-range concentration (10 μM). At the same time, in light of the Reviewer’s suggestion, we have now also tested PKA-RIIα dissociation at a 5x lower concentration of norepinephrine (2 μM; New Experiment #2). The activation and translocation of PKA-C is also readily detectible under this condition to a degree comparable to when 10 μM norepinephrine was used.

Regarding the suggested glutamate uncaging experiment, it is extremely challenging because of finite signal-to-noise ratios in our experiments. From our past studies, we know that activated PKA-C can diffuse three dimensionally, with a fraction as membrane-associated proteins and the other as cytosolic proteins. Although we have evidence that its membrane affinity allows it to become enriched in dendritic spines, it is not known (and is unlikely) that activated PKA-C is selectively targeted to a particular spine. Glutamate uncaging of a single spine presumably would locally activate a small number of PKA-C. It will be very difficult to trace the 3D diffusion of these small number of molecules in the presence of surrounding resting-state PKA-C molecules. Finally, we hope the reviewer agrees that, regardless of the result of the glutamate uncaging experiment, the above new experiment (New Experiment #2) already indicate that certain physiologically relevant stimuli can drive PKA-C dissociation from PKA-R and translocation to spines, supporting our conclusion.

Reviewer #2 (Recommendations For The Authors):

It was a pleasure reading your paper, and the results are well-executed and well-presented.

My main and only recommendations are two ways to further expand the scope of the findings.

First, I believe addressing the endogenous localization of PKA-C subunit before and after PKA activation would be highly important to validate these claims. Overexpression of tagged proteins often shows vastly different subcellular distribution than their endogenous counterparts. Recent technological advances with CRISPR/Cas9 gene editing (Suzuki et al Nature 2016 and Gao et al Neuron 2019 for example) which the Zhong lab recently contributed to (Zhong et al 2021 eLife) allow us to tag endogenous proteins and image them in fixed or live neurons. Any experiments targeting endogenous PKA subunits that support dissociation and synaptic localization following activation would be very informative and greatly increase the novelty and impact of their findings.

We agreed that addressing the endogenous PKA dynamics is important. However, despite recent progress, endogenous labeling using CRISPR-based methods remains challenging and requires extensive optimization. This is especially true for signaling proteins whose endogenous abundance is often low. We have tried to label PKA catalytic subunits and regulatory subunits using both the homologous recombination-based method SLENDR and our own non-homologous end joining-based method CRISPIE. We did not succeed, in part because it is very difficult to see any signal under wide-field fluorescence conditions, which makes it difficult to screen different constructs for optimizing parameters. It is also possible that, at the endogenous abundance, the label is just not bright enough to be seen. Nevertheless, for both PKA type Iβ and type IIα that we studied in this manuscript, we have correlated the measured parameters (specifically, Spine Enrichment Index or SEI) with the overexpression level (Figure 1-figure supplement 1). We found that they are not strongly correlated with the expression level under our conditions. By extrapolating to non-overexpression conditions, our conclusion remains valid.

To overcome the inability to label endogenous PKA subunits using CRISPR-based methods, we have also attempted a conditional knock-in method call ENABLED that we previously developed to label PKA-Cα. In preliminary results, we found that endogenously label PKA were very dim. However, in a subset of cells that are bright enough to be quantified, the PKA catalytic subunit indeed translocated to dendritic spines upon stimulation (see Additional Fig. 1 in the next page), corroborating our results using overexpression. These results, however, are not ready to be published because characterization of the mouse line takes time and, at this moment, the signal-to-noise ratio remains low. We hope that the reviewer can understand.

Author response image 1.

Endogeneous PKA-Cα translocate to dendritic spines upon activation.

Second, experiments which would advance and validate these findings in vivo would be highly valuable. This could be achieved in a number of ways - one would be overexpression of tagged PKA versions and examining sub-cellular distribution before and after physiological activation in vivo. Another possibility is in vivo perturbation - one would speculate that disruption or tethering of PKA subunits to the dendrite would lead to cell-specific functional and structural impairments. This could be achieved in a similar manner to the in vitro experiments, with a PKA KO and replacement strategy of the tethered C-R plasmid, followed by structural or functional examination of neurons.

I would like to state that these experiments are not essential in my opinion, but any improvements in one of these directions would greatly improve and extend the impact and findings of this paper.

We thank the reviewer for the suggestion and the understanding. The suggested in vivo experiments are fascinating. However, in vivo imaging of dendritic spine morphology is already in itself challenging. The difficulty greatly increases when trying to detect partial, likely transient translocation of a signaling protein. It is also very difficult to knock down endogenous PKA while simultaneously expressing the R-C construct in a large number of cells to achieve detectable circuit or behavioral effect (and hope that compensation does not happen over weeks). We hope the reviewer agrees that these experiments would be their own project and go beyond the time and scope of the current study.

Reviewer #3 (Recommendations For The Authors):

Please elaborate on the methods used to visualize PKA-RIIα and PKA-RIβ subunits.

As suggested, we have now included additional details for visualizing PKA-Rs in the text. Specifically, we write (pg. 5): “…, as visualized using expressed PKA-R-mEGFP in separate experiments (Figs. 1A-1C).”.

Double major so you know it's important.

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Author response:

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

Reviewer #1 (Public Review): 

Summary: 

The authors examined the salt-dependent phase separation of the low-complexity domain of hnRN-PA1 (A1-LCD). Using all-atom molecular dynamics simulations, they identified four distinct classes of salt dependence in the phase separation of intrinsically disordered proteins (IDPs), which can be predicted based on their amino acid composition. However, the simulations and analysis, in their current form, are inadequate and incomplete. 

Strengths: 

The authors attempt to unravel the mechanistic insights into the interplay between salt and protein phase separation, which is important given the complex behavior of salt effects on this process. Their effort to correlate the influence of salt on the low-complexity domain of hnRNPA1 (A1-LCD) with a range of other proteins known to undergo salt-dependent phase separation is an interesting and valuable topic. 

Weaknesses: 

(1) The simulations performed are not sufficiently long (Figure 2A) to accurately comment on phase separation behavior. The simulations do not appear to have converged well, indicating that the system has not reached a steady state, rendering the analysis of the trajectories unreliable.

We have extended the simulations for an additional 500 ns, to 1500 ns. The last 500 ns show reasonably good convergence (see Figure 2A).

(2) The majority of the data presented shows no significant alteration with changes in salt concentration. However, the authors have based conclusions and made significant comments regarding salt activities. The absence of error bars in the data representation raises questions about its reliability. Additionally, the manuscript lacks sufficient scientific details of the calculations.  

We have now included error bars. With the error bars, the salt dependences of all the calculated properties (exception for Rg) show a clear trend. Additionally, we have expanded the descriptions of our calculations (p. 15-16).

(3) In Figures 2B and 2C, the changes in the radius of gyration and the number of contacts do not display significant variations with changes in salt concentration. The change in the radius of gyration with salt concentration is less than 1 Å, and the number of contacts does not change by at least 1. The authors' conclusions based on these minor changes seem unfounded. 

The variation of ~ 1 Å for the calculated Rg is similar to the counterpart for the experimental Rg. As for the number of contacts, note that this property is presented on a per-residue basis, so a value of 1 means that each residue picks up one additional contact, or each protein chain gains a total of 131 contacts, when the salt concentration is increased from 50 to 1000 mM.

Reviewer #2 (Public Review): 

This is an interesting computational study addressing how salt affects the assembly of biomolecular condensates. The simulation data are valuable as they provide a degree of atomistic details regarding how small salt ions modulate interactions among intrinsically disordered proteins with charged residues, namely via Debye-like screening that weakens the effective electrostatic interactions among the polymers, or through bridging interactions that allow interactions between like charges from different polymer chains to become effectively attractive (as illustrated, e.g., by the radial distribution functions in Supplementary Information). However, this manuscript has several shortcomings: 

(i) Connotations of the manuscript notwithstanding, many of the authors' concepts about salt effects on biomolecular condensates have been put forth by theoretical models, at least back in 2020 and even earlier. Those earlier works afford extensive information such as considerations of salt concentrations inside and outside the condensate (tie-lines). But the authors do not appear to be aware of this body of prior works and therefore missed the opportunity to build on these previous advances and put the present work with its complementary advantages in structural details in the proper context.

(ii) There are significant experimental findings regarding salt effects on condensate formation [which have been modeled more recently] that predate the A1-LCD system (ref.19) addressed by the present manuscript. This information should be included, e.g., in Table 1, for sound scholarship and completeness. 

(iii) The strengths and limitations of the authors' approach vis-à-vis other theoretical approaches should be discussed with some degree of thoroughness (e.g., how the smallness of the authors' simulation system may affect the nature of the "phase transition" and the information that can be gathered regarding salt concentration inside vs. outside the "condensate" etc.). Accordingly, this manuscript should be revised to address the following. In particular, the discussion in the manuscript should be significantly expanded by including references mentioned below as well as other references pertinent to the issues raised. 

(1) The ability to use atomistic models to address the questions at hand is a strength of the present work. However, presumably because of the computational cost of such models, the "phase-separated" "condensates" in this manuscript are extremely small (only 8 chains). An inspection of Fig.1 indicates that while the high-salt configuration (snapshot, bottom right) is more compact and droplet-like than the low-salt configuration (top right), it is not clear that the 50 mM NaCl configuration can reasonably correspond to a dilute or homogeneous phase (without phase separation) or just a condensate with a lower protein concentration because the chains are still highly associated. One may argue that they become two droplets touching each other (the chains are not fully dispersed throughout the simulation box, unlike in typical coarse-grained simulations of biomolecular phase separation). While it may not be unfair to argue from this observation that the condensed phase is less stable at low salt, this raises critical questions about the adequacy of the approach as a stand-alone source of theoretical information. Accordingly, an informative discussion of the limitation of the authors' approach and comparisons with results from complementary approaches such as analytical theories and coarsegrained molecular dynamics will be instructive-even imperative, especially since such results exist in the literature (please see below). 

We now discuss the limitations of our all-atom simulations and also other approaches (p. 13; see below).

(2) The aforementioned limitation is reflected by the authors' choice of using Dmax as a sort of phase separation order parameter. However, no evidence was shown to indicate that Dmax exhibits a twostate-like distribution expected of phase separation. It is also not clear whether a Dmax value corresponding to the linear dimension of the simulation box was ever encountered in the authors' simulated trajectories such that the chains can be reliably considered to be essentially fully dispersed as would be expected for the dilute phase. Moreover, as the authors have noted in the second paragraph of the Results, the variation of Dmax with simulation time does not show a monotonic rank order with salt concentration. The authors' explanation is equivalent to stipulating that the simulation system has not fully equilibrated, inevitably casting doubt on at least some of the conclusions drawn from the simulation data. 

First off, with the extended simulations, the Dmax values converge to a tiered order rank, with successively decreasing values from low salt (50 mM) to intermediate salt (150 and 300 mM) to high salt (500 and 1000 mM). Secondly, as we now state (p. 13), our low-salt simulations mimic a homogenous solution whereas our high-salt simulations mimic the dense phase of a phase-separated system. The intermediate-salt simulations also mimic the dense phase but at a somewhat lower concentration (hence the intermediate Dmax value).

(3) With these limitations, is it realistic to estimate possible differences in salt concentration between the dilute and condensed phases in the present work? These features, including tie-lines, were shown to be amenable to analytical theory and coarse-grained molecular dynamics simulation (please see below).  

The differences in salt effects that we report do not represent those between two phases. Rather, as explained in the preceding reply, they represent differences between a homogenous solution at low salt and the dense phase at higher salt. We also acknowledge salt effects calculated by analytical theory and coarse-grained simulations (p. 13).

(4) In the comparison in Fig.2B between experimental and simulated radius of gyration as a function of [NaCl], there is an outlier among the simulated radii of gyration at [NaCl] ~ 250 mM. An explanation should be offered.  

After extending the simulations and analyzing the last 500 ns, the Rg data no longer show an outlier though still have some fluctuations from one salt concentration to another.

(5) The phenomenon of no phase separation at zero and low salt and phase separation at higher salt has been observed for the IDP Caprin1 and several of its mutants [Wong et al., J Am Chem Soc 142, 24712489 (2020) [https://pubs.acs.org/doi/full/10.1021/jacs.9b12208], see especially Fig.9 of this reference]. This work should be included in the discussion and added to Table 1. 

We now have added Caprin1 to Table 1 (new ref 26) and discuss this paper (p. 13).

(6) The authors stated in the Introduction that "A unifying understanding of how salt affects the phase separation of IDPs is still lacking". While it is definitely true that much remains to be learned about salt effects on IDP phase separation, the advances that have already been made regarding salt effects on IDP phase separation is more abundant than that conveyed by this narrative. For instance, an analytical theory termed rG-RPA was put forth in 2020 to provide a uniform (unified) treatment of salt, pH, and sequence-charge-pattern effects on polyampholytes and polyelectrolytes (corresponding to the authors' low net charge and high net charge cases). This theory offers a means to predict salt-IDP tie-lines and a comprehensive account of salt effect on polyelectrolytes resulting in a lack of phase separation at extremely low salt and subsequent salt-enhanced phase separation (similar to the case the authors studied here) and in some cases re-entrant phase separation or dissolution [Lin et al., J Chem Phys 152. 045102 (2020) [https://doi.org/10.1063/1.5139661]]. This work is highly relevant and it already provided a conceptual framework for the authors' atomistic results and subsequent discussion. As such, it should definitely be a part of the authors' discussion. 

We now cite this paper (new ref 34) in Introduction (p. 4). We also discuss its results for Caprin1 (new ref 18; p. 13).

(7) Bridging interactions by small ions resulting in effective attractive interactions among polyelectrolytes leading to their phase separation have been demonstrated computationally by Orkoulas et al., Phys Rev Lett 90, 048303 (2003) [https://journals.aps.org/prl/abstract/10.1103/PhysRevLett.90.048303]. This result should also be included in the discussion. 

We now cite this paper (new ref 41; p. 11).

(8) More recently, the salt-dependent phase separations of Caprin1, its RtoK variants and phosphorylated variant (see item #5 above) were modeled (and rationalized) quite comprehensively using rG-RPA, field-theoretic simulation, and coarse-grained molecular dynamics [Lin et al., arXiv:2401.04873 [https://arxiv.org/abs/2401.04873]], providing additional data supporting a conceptual perspective put forth in Lin et al. J Chem Phys 2020 (e.g., salt-IDP tie-lines, bridging interactions, reentrance behaviors etc.) as well as in the authors' current manuscript. It will be very helpful to the readers of eLife to include this preprint in the authors' discussion, perhaps as per the authors' discretion along the manner in which other preprints are referenced and discussed in the current version of the manuscript. 

We now cite this paper (new ref 18) and discuss it along with new ref 26 in Discussion (p. 13).

Reviewer #3 (Public Review): 

Summary: 

This study investigates the salt-dependent phase separation of A1-LCD, an intrinsically disordered region of hnRNPA1 implicated in neurodegenerative diseases. The authors employ all-atom molecular dynamics (MD) simulations to elucidate the molecular mechanisms by which salt influences A1-LCD phase separation. Contrary to typical intrinsically disordered protein (IDP) behavior, A1-LCD phase separation is enhanced by NaCl concentrations above 100 mM. The authors identify two direct effects of salt: neutralization of the protein's net charge and bridging between protein chains, both promoting condensation. They also uncover an indirect effect, where high salt concentrations strengthen pi-type interactions by reducing water availability. These findings provide a detailed molecular picture of the complex interplay between electrostatic interactions, ion binding, and hydration in IDP phase separation. 

Strengths: 

Novel Insight: The study challenges the prevailing view that salt generally suppresses IDP phase separation, highlighting A1-LCD's unique behavior. 

Rigorous Methodology: The authors utilize all-atom MD simulations, a powerful computational tool, to investigate the molecular details of salt-protein interactions. 

Comprehensive Analysis: The study systematically explores a wide range of salt concentrations, revealing a nuanced picture of salt effects on phase separation. 

Clear Presentation: The manuscript is well-written and logically structured, making the findings accessible to a broad audience. 

Weaknesses: 

Limited Scope: The study focuses solely on the truncated A1-LCD, omitting simulations of the full-length protein. This limitation reduces the study's comparative value, as the authors note that the full-length protein exhibits typical salt-dependent behavior. A comparative analysis would strengthen the manuscript's conclusions and broaden its impact.

Perhaps we did not impress on the reviewer how expensive the all-atom MD simulations on A1-LCD were: the systems each contained half a million atoms and the simulations took many months to complete. That said, we agree with the reviewer that, ideally, a comparative study on a protein showing the typical screening class of salt dependence would have made our work more complete. However, we are confident of the conclusions for several reasons. First, the three salt effects – charge neutralization, bridging, and strengthening of pi-types of interactions – revealed by the all-atom simulations are physically sound and well-supported by other studies. Second, these effects led us to develop a unified picture for the salt dependence of homotypic phase separation, in the form of a predictor for the classes of salt dependence based on amino-acid composition. This predictor works well for nearly 30 proteins. Third, recent studies using analytical theory and coarse-grained simulations (new ref 18) also strongly support our conclusions.

Reviewer #1 (Recommendations For The Authors): 

(1) In Figure 1, the color scheme should be updated and the figure remade, as the current set of color choices makes it very difficult to distinguish the magenta spheres.  

We have increased the sizes of ions in Figure 1 to make them distinguishable.

(2) Within the framework of atomistic simulations, the influence of salt concentration alteration on protein conformational plasticity is worth investigating. This could be correlated (with proper details) with the effect of salt-concentration-modulated protein aggregation behavior. 

We now use RMSF to measure conformational plasticity, which shows a clear salt-dependent trend with a 27% reduction in fluctuations from 50 mM to 1000 mM NaCl (new Fig. S1).

(3) The authors should mention the protein concentrations employed in the simulations and whether these are consistent with experimentally used concentrations.  

We have mentioned the initial concentration (3.5 mM). We now further state that this concentration is maintained in the low-salt simulations, indicating absence of phase separation, but is increased to 23 mM in the high-salt simulations, indicating phase separation. The latter value is consistent with the measured concentrations in the dense phase (last two paragraphs of p. 5).

(4) It would be useful to test the salt effect for at least two extreme salt concentrations at various protein concentrations, consistent with experimental protein concentration ranges.  

In simulation studies of short peptides (ref 37), we have shown that the initial concentration does not affect the final concentration in the dense phase, as expected for phase-separation systems. We expect that the same will be true for the A1-LCD system at intermediate and high salt where phase separation occurs. Though this expectation could be tested by simulations at a different initial protein concentration, such simulations would be expensive but unlikely to yield new physical insight.

(5) Importantly, the simulations do not appear to have converged well enough (Figure 2A). The authors should extend the simulation trajectories to ensure the system has reached a steady state.  

We extended the simulations for an additional 500 ns, which now appear to show convergence. In Figure 2A we now see Dmax values converge to a tiered order rank, with successively decreasing values from low salt (50 mM) to intermediate salt (150 and 300 mM) to high salt (500 and 1000 mM). 

(6) The authors mention "phase separation" in the title, but with only a 1 μs simulation trajectory, it is not possible to simulate a phenomenon like phase separation accurately. Since atomistic simulations cannot realistically capture phase separation on this timescale, a coarse-grained approach is more suitable. To properly explore salt effects in the context of phase separation, long timescale simulation trajectories should be considered. Otherwise, the data remain unreliable. 

Our all-atom simulations revealed rich salt effects that might have been missed in coarse-grained simulations. It is true that coarse-grained models allow the simulations of the phase separation process, but as we have recently demonstrated (refs 36 and 37), all-atom simulations on the μs timescale are also able to capture the spontaneous phase separation of peptides and small IDPs. A1-LCD is much larger than those systems, so we had to use a relatively small chain number (8 chains here vs 64 used in ref 37 and 16 used in ref 37). S2ll, we observe the condensation into a dense phase at high salt. We discuss the pros and cons of all-atom vs. coarse-grained simulations in p. 13.

(7) In Figure 5E, the plot does not show that g(r) has reached 1. If it does, the authors should show the full curve. The same issue remains with supplementary figures 1, 2, 3, etc.  

We now show the approach to 1 in the insets of Figs. S2, S3, S4, and 5E.

(8) None of the data is represented with error bars. The authors should include error bars in their data representations. 

We have now included error bars in all graphs that report average values.

(9) The authors state that "the net charge of the system reduces to only +8 at 1000 mM NaCl (Figure 3C)" but do not explain how this was calculated. 

We now add this explanation in methods (p. 16).

(10). The authors mention "similar to the role played by ATP molecules in driving phase separation of positively charged IDPs." However, ATP can inhibit aggregation, and its induction of phase separation is concentration-dependent. Given ATP's large aromatic moiety, its comparison to ions is not straightforward and is more complex. This comparison can be at best avoided. 

In this context we are comparing the bridging capability of ATP molecules in driving phase separation of positively charged IDPs in ref 36 to the bridging capability of the ions here. In ref 36 the authors show ATP bridging interactions between protein chains similar to what we show here with ions.

(11) Many calculations are vaguely represented. The process for calculating the number of bridging ions, for example, is not well documented. The authors should provide sufficient details to allow for the reproducibility of the data. 

We have now expanded the methods section to include more detailed information on calculations done.

Reviewer #3 (Recommendations For The Authors): 

Include error bars or standard deviations for all results averaged over four replicates, particularly for the number of ions and contacts per residue. This would provide a clearer picture of the data's reliability and variability. 

We have now included error bars in all graphs that report averaged values.

Strengthen the support for the conclusion that "each Arg sidechain often coordinates two Cl- ions, multiple backbone carbonyls often coordinate a single Na+ ion." While Fig. 3A clearly demonstrates ArgCl- coordination, the Na+ coordination claim for a 131-residue protein requires further clarification. Consider including the integration profile of radial distribution functions for Na+ ions to bolster this assertion. 

We now report the number of Na+ ions that coordinate with multiple backbone carbonyls (p. 7) as well as the number of Na+ ions that bridge between A1-LCD chains via coordination with multiple backbone carbonyls (p. 9). Please note that Figure 4A right panel displays an example of Na+ coordinating with multiple backbone carbonyls.

Address the following typographical errors in the main text: o Page 11, line 25: "distinct classes of sat dependence" should be "distinct classes of salt dependence" o Page 14, line 9: "for Cl- and 3.0 and 5.4 A" should be "for Cl- and 3.0 and 5.4 √Ö" o Page 14, line 18: "As a control, PRDFs for water were also calculated" should be "As a control, RDFs for water were also calculated" (assuming PRDF was meant to be RDF) 

We have now corrected these typos.

Consider expanding the study to include simulations of the full-length protein to provide a more comprehensive comparison between the truncated A1-LCD and the complete protein's behavior in various salt concentrations. 

As we explained above, even with eight chains of A1-LCD, which has 131 residues, the systems already contain half a million atoms each and the all-atom simulations took many months to complete. Full-length A1 has 314 residues so a multi-chain system would be too large to be feasible for all-atom simulations.

eLife Assessment

In this potentially important study, the authors conducted atomistic simulations to probe the salt-dependent phase separation of the low-complexity domain of hnRN-PA1 (A1-LCD). The authors have identified both direct and indirect mechanisms of salt modulation, provided explanations for four distinct classes of salt dependence, and proposed a model for predicting protein properties from amino acid composition. There is a range of opinions regarding the strength of evidence, with some considering the evidence as incomplete due to the limitations in the length and statistical errors of the computationally intense atomistic MD simulations.

Reviewer #1 (Public review):

Summary:

The authors examined the salt-dependent phase separation of the low-complexity domain of hnRN-PA1 (A1-LCD). Using all-atom molecular dynamics simulations, they identified four distinct classes of salt dependence in the phase separation of intrinsically disordered proteins (IDPs), which can be predicted based on their amino acid composition. However, the simulations and analysis, in their current form, are inadequate and incomplete.

Strengths:

The authors attempt to unravel the mechanistic insights into the interplay between salt and protein phase separation, which is important given the complex behavior of salt effects on this process. Their effort to correlate the influence of salt on the low-complexity domain of hnRNPA1 (A1-LCD) with a range of other proteins known to undergo salt-dependent phase separation is an interesting and valuable topic.

Weaknesses:

Based on the reviewer's assessment of the manuscript, the following points were raised:

(1) The simulation duration is too short to draw comprehensive conclusions about phase separation.<br /> (2) There are concerns regarding the convergence of the simulations, particularly as highlighted in Figure 2A.<br /> (3) The simulation begins with a protein concentration of 3.5 mM ("we built an 8-copy model for the dense phase (with an initial concentration of 3.5 mM)"), which is high for phase separation studies. The reviewer questions the use of the term "dense phase" and suggests that the authors conduct a clearer analysis depicting the coexistence of both the dilute and dense phases to represent a steady state. Without this, the realism of the described phenomena is doubtful. Commenting on phase separation under conditions that don't align with typical phase separation parameters is not acceptable.<br /> (4) The inference that "Each Arg sidechain often coordinates two Cl- ions simultaneously, but each Lys sidechain coordinates only one Cl- ion" is questioned. According to Supplementary Figure 2A, Lys seems to coordinate with Cl- ions more frequently than Arg.<br /> (5) The authors are requested to update the figure captions for Supplementary Figures 2 and 3, specifying which system the analyses were performed on.<br /> (6) It is difficult to observe a clear trend due to irregularities in the data. Although the authors have included a red dotted line in the figures, the trend is not monotonic. The reviewer expresses concerns about significant conclusions drawn from these figures (e.g., Figure 2C, Figure 5A, Supplementary Figure 1).<br /> (7) Given the error in the radius of gyration (Rg) calculations, the reviewer questions the validity of drawing conclusions from this data.<br /> (8) The pair correlation function values in Figure 5E and supplementary figure 4 show only minor differences, and the reviewer questions whether these differences are significant.<br /> (9) Previous reports suggest that, upon self-assembly, protein chains extend within the condensate, leading to a decrease in intramolecular contacts. However, the authors show an increase in intramolecular contacts with increasing salt concentration (Figure 2C), which contradicts prior studies. The reviewer advises the authors to carefully review this and provide justification.<br /> (10) A systematic comparison of estimated parameters with varying salt concentrations is required. Additionally, the authors should provide potential differences in salt concentrations between the dilute and condensed phases.<br /> (11) The reviewer finds that the majority of the data presented shows no significant alteration with changes in salt concentration, yet the authors have made strong conclusions regarding salt activity.

The manuscript lacks sufficient scientific details of the calculations.

Reviewer #2 (Public review):

This is an interesting computational study addressing how salt affects the assembly of biomolecular condensates. The simulation data are valuable as they provide a degree of atomistic details regarding how small salt ions modulate interactions among intrinsically disordered proteins with charged residues, namely via Debye-like screening that weakens the effective electrostatic interactions among the polymers, or through bridging interactions that allow interactions between like charges from different polymer chains to become effectively attractive (as illustrated, e.g., by the radial distribution functions in Supplementary Information). However, this manuscript has several shortcomings: (i) Connotations of the manuscript notwithstanding, many of the authors' concepts about salt effects on biomolecular condensates have been put forth by theoretical models, at least back in 2020 and even earlier. Those earlier works afford extensive information such as considerations of salt concentrations inside and outside the condensate (tie-lines). But the authors do not appear to be aware of this body of prior works and therefore missed the opportunity to build on these previous advances and put the present work with its complementary advantages in structural details in the proper context. (ii) There are significant experimental findings regarding salt effects on condensate formation [which have been modeled more recently] that predate the A1-LCD system (ref.19) addressed by the present manuscript. This information should be included, e.g., in Table 1, for sound scholarship and completeness. (iii) The strengths and limitations of the authors' approach vis-à-vis other theoretical approaches should be discussed with some degree of thoroughness (e.g., how the smallness of the authors' simulation system may affect the nature of the "phase transition" and the information that can be gathered regarding salt concentration inside vs. outside the "condensate" etc.).

Comments on revised version:

The authors have adequately addressed my previous concerns and suggestions. The manuscript is now significantly improved. The new results and analyses provided by the authors represent a substantial advance in our understanding of the role of electrostatics in the assembly of biomolecular condensates.

Reviewer #3 (Public review):

Summary:

This study investigates the salt-dependent phase separation of A1-LCD, an intrinsically disordered region of hnRNPA1 implicated in neurodegenerative diseases. The authors employ all-atom molecular dynamics (MD) simulations to elucidate the molecular mechanisms by which salt influences A1-LCD phase separation. Contrary to typical intrinsically disordered protein (IDP) behavior, A1-LCD phase separation is enhanced by NaCl concentrations above 100 mM. The authors identify two direct effects of salt: neutralization of the protein's net charge and bridging between protein chains, both promoting condensation. They also uncover an indirect effect, where high salt concentrations strengthen pi-type interactions by reducing water availability. These findings provide a detailed molecular picture of the complex interplay between electrostatic interactions, ion binding, and hydration in IDP phase separation.

Strengths:

• Novel Insight: The study challenges the prevailing view that salt generally suppresses IDP phase separation, highlighting A1-LCD's unique behavior.<br /> • Rigorous Methodology: The authors utilize all-atom MD simulations, a powerful computational tool, to investigate the molecular details of salt-protein interactions.<br /> • Comprehensive Analysis: The study systematically explores a wide range of salt concentrations, revealing a nuanced picture of salt effects on phase separation.<br /> • Clear Presentation: The manuscript is well-written and logically structured, making the findings accessible to a broad audience.

Weaknesses:

• Limited Scope: The study focuses solely on the truncated A1-LCD, omitting simulations of the full-length protein. This limitation reduces the study's comparative value, as the authors note that the full-length protein exhibits typical salt-dependent behavior. However, given the much larger size of the full-length protein, it is acceptable to omit it given the current computing resources available.

Overall, this manuscript represents a significant contribution to the field of IDP phase separation. The authors' findings provide valuable insights into the molecular mechanisms by which salt modulates this process, with potential implications for understanding and treating neurodegenerative diseases.

This is a good point how the struggle for power often comes with inhumane treatment.

Denk aan het vergiftigen van iemand met poedersuiker, dit moet gezien worden als ondeugdelijke poging

daarom nu ook kijken naar poging tot bevrijding!

What do we mean by preferred? What do we knwo about the the colelctive shadow that was not harvested?

Capture is a word from war Can we use a word like Gather?

Why reward modelling? Can this create a pavlovian affect / effect?

A very limited intention - just to maximise apporoval What abotu to access the quantum potential?

We like the term Undividual rather than Individual Individual means undivided whole but we have come to understand it as separate.

Why only opinions and critiques? What about potentials? What about shadows? What about Qualia?

This is the same process as Quaker Clerks of Meetings have been doing for nearly 400 years

This shows the importance of language Yet, the language can come FROM the group rather than be PUT TO the group

What if consensus at the group meeting does not last after the meeting is over?

In Dialogue, DISCUSS comes from Percuss or Concuss - beating an idea to death. In Dialogue, the idea is held open for us all to witness and explore beenath its symptom, its explicate, by delving into its implicate as an undivided wholeness

Yes. And the collective must also be able to reach disagreement and still stay in Dialogic relations

metaphor

jfc

what the fuck?? THIS IS LITERALLY PART OF WHY WE GO TO GRADUATE SCHOOL

for - separation - reference - The three great separations

separation - reference - The three great separations - https://hyp.is/go?url=https%3A%2F%2Finthesetimes.com%2Farticle%2Findustrial-agricultural-revolution-planet-earth-david-korten&group=world

for - questions - collaboration literacy - Donna Nelham - to - book - The Birth and Death of Meaning - Ernest Becker -

questions - collaboration - Donna Nelham - These three questions are all related - To get to the root of collaboration, it is helpful to examine the roots of human psychology to understand the fundamental relationship between - the individual and - the group - In his work "The Birth ad Death of Meaning, Ernest Becker argues, citing other peers, that - the self concept needs to emerge for effective group collaboration to develop and - the self concept requires others in order to construct it - Hence, other is already implicated in the construction of our own self - In Deep Humanity terminology, we call this intertwingledness of the self and other the "individual / collective gestalt"

to - book - The Birth and Death of Meaning - Ernest Becker - https://hyp.is/40fZHv9CEe6bTovrYzF92A/www.themortalatheist.com/blog/the-birth-and-death-of-meaning-ernest-becker

for - adder - for deep collaboration - article - Co-creative Collaboration - Donna Nelham

adder - for deep collaboration - article - Co-creative Collaboration - Donna Nelham - symmathesy - mutual learning - Nora Bateson - https://hyp.is/_V3NAk4UEe6Z6btu_1LIkA/norabateson.wordpress.com/2015/11/03/symmathesy-a-word-in-progress/

for - portmanteau - collaboration washing - Donna Nelham

portmanteau - collaboration washing - Donna Nelham - like greenwashing - nice!

I appreciate how they discussed being open to getting an answer wrong and working with those students, with patient to help them gain a better understanding of the topic.

Allowing access to certain resources that enhance a student's learning is the prime example of a well-structured classroom

By embracing "storientation," educators can learn about students' interests, families, and experiences outside of school. This approach counters the tendency to rely on a single narrative, fostering a deeper understanding t

This approach combines discipline and structure with a belief in each student's potential, fostering an environment where all students are challenged to reach their best. An equity-focused mindset ensures that these high expectations are paired with a genuine commitment to each child's success.

The main idea is that "lean-in assessment" is crucial for understanding each student's unique learning journey. By engaging with students and observing their approaches to tasks, strengths, and challenges, educators can gather valuable insights that standardized tests cannot provide.

The key point is that flexibility in teaching routines is essential for effective instruction. While structured mini-lessons can be useful, they may not meet the diverse needs of all learners.

The central message is that creating a safe space for failure in the classroom is essential for learning. By framing failure as valuable data rather than a source of shame, students can openly acknowledge their struggles.

The main idea is that culture should be viewed as a valuable resource in education. Ignoring students' identities diminishes their experiences and potential for learning. Recognizing and engaging with students' cultural backgrounds allows them to better understand and connect with challenging content. Encouraging students to share their backgrounds fosters a supportive environment that values diversity and enhances learning for everyone.

The key message is that achieving success for every child requires recognizing and addressing the inequalities faced by specific groups, including English language learners, students with special needs, and those impacted by trauma or poverty

Relaying to students that their point in their educational journey does not define them as an individual and instilling a joy for understanding and learning that differs among students is where the true importance lies

Although the implementation of mini lessons can be taught with the hope it will reach all students, understanding that not all students learn in the same way nor are they all at the same point in their educational journey, it is important to offer additional support when noticed to be prompted

Ensuring the success of every student individually is of greatest value is crucial to achieving an equitable classroom that values the learning point of all students

Yes its important to know about all of the students identities read books about different cultures to show everyone and boarder theyre horizons

Its important to teach them to learn from theyre mistakes so they won't be ashamed of the mistakes everyone makes because no one is perfect

Sometimes a student needs a break because theyre over simulated and its nothing to be uncomfortable about and you should explain we all have our moments for a little minting breather and come back in ready to effectively finish the lesson

The more you know about how a student works the more you can develop different lesson plans to build off those skills and expand off that

Its important to let know that theyre capable of being able to accomplish anything they wanna do give the the confidence to do what they wanna do and they'll succeed

Its important to know who your students are and understand where they come from to properly be able to communicate with them in way that would make it easy for them to understand.

eLife Assessment

This manuscript presents a valuable new quantitative crosslinking mass spectrometry approach using novel isobaric crosslinkers. The data are solid and the method has potential for a broad application in structural biology if more isobaric crosslinking channels are available and the quantitative information of the approach is exploited in more depth.

Reviewer #1 (Public review):

Summary:

Crosslinking mass spectrometry has become an important tool in structural biology, providing information about protein complex architecture, binding sites and interfaces, and conformational changes. One key challenge of this approach represents the quantitation of crosslinking data to interrogate differential binding states and distributions of conformational states.

Here, Luo and Ranish present a novel class of isobaric crosslinkers ("Qlinkers"), conduct proof-of-concept benchmarking experiments on known protein complexes, and show example applications on selected target proteins. The data are solid and this could well be an exciting, convincing new approach in the field if the quantitation strategy is made more comprehensive and the quantitative power of isobaric labeling is fully leveraged as outlined below. It's a promising proof-of-concept, and potentially of broad interest for structural biologists.

Strengths:

The authors demonstrate the synthesis, application, and quantitation of their "Q2linkers", enabling relative quantitation of two conditions against each other. In benchmarking experiments, the Q2linkers provide accurate quantitation in mixing experiments. Then the authors show applications of Q2linkers on MBP, Calmodulin, selected transcription factors, and polymerase II, investigating protein binding, complex assembly, and conformational dynamics of the respective target proteins. For known interactions, their findings are in line with previous studies, and they show some interesting data for TFIIA/TBP/TFIIB complex formation and conformational changes in pol II upon Rbp4/7 binding.

Weaknesses:

This is an elegant approach but the power of isobaric mass tags is not fully leveraged in the current manuscript.

First, "only" Q2linkers are used. This means only two conditions can be compared. Theoretically, higher-plexed Qlinkers should be accessible and would also be needed to make this a competitive method against other crosslinking quantitation strategies. As it is, two conditions can still be compared relatively easily using LFQ - or stable-isotope-labeling based approaches. A "Q5linker" would be a really useful crosslinker, which would open up comprehensive quantitative XLMS studies.

Second, the true power of isobaric labeling, accurate quantitation across multiple samples in a single run, is not fully exploited here. The authors only show differential trends for their interaction partners or different conformational states and do not make full quantitative use of their data or conduct statistical analyses. This should be investigated in more detail, e.g. examine Qlinker quantitation of MBP incubated with different concentrations of maltose or Calmodulin incubated with different concentrations of CBPs. Does Qlinker quantitation match ratios predicted using known binding constants or conformational state populations? Is it possible to extract ratios of protein populations in different conformations, assembly, or ligand-bound states?

With these two points addressed this approach could be an important and convincing tool for structural biologists.

Comments on latest version:

I raised only two points which they have not addressed: Higher multiplexing of Qlinkers (1) and experiments to assess the statistical power of their quantitation strategy (2).

I can see that point (1) requires substantial experimental efforts and synthesis of novel Qlinkers would be months of work. This is an editorial decision if the limited quantitative power of the "2-plex" approach they have right now is sufficient to support publication in eLife. While I like the approach, I feel it falls short of its potential in its current form.

For point (2), the authors did not do any supporting experiments. They claim "higher plex Qlinkers" would need to be available, but I suggested experiments that can be done even with Q2linkers: Using one of the two channels as a reference channel (similar the Super-SILAC strategy published in 2010 by Geiger et al; using an isotope-labeled channel as a stable reference channel between different experiments and LC-MS runs), they could do time-courses or ligand-concentration-series with the other channel and then show that Qlinkers allow quantitative monitoring of the different populations (e.g. conformations or ligand-bound proteins).

As an additional point, I was a bit surprised to read that the quantitation evaluation in Figure 1 is based on a single experiment (reviewer response document page 6, line 2 in the authors' reply). I strongly suggest this to be repeated a few times so a proper statistical test on experimental reproducibiltiy of Qlinkers can be conducted.

In summary, the authors declined to do any experimental work to address my concerns.

Reviewer #2 (Public review):

The regulation of protein function heavily relies on the dynamic changes in the shape and structure of proteins and their complexes. These changes are widespread and crucial. However, examining such alterations presents significant challenges, particularly when dealing with large protein complexes in conditions that mimic the natural cellular environment. Therefore, much emphasis has been put on developing novel methods to study protein structure, interactions, and dynamics. Crosslinking mass spectrometry (CSMS) has established itself as such a prominent tool in recent years. However, doing this in a quantitative manner to compare structural changes between conditions has proven to be challenging due to several technical difficulties during sample preparation. Luo and Ranish introduce a novel set of isobaric labeling reagents, called Qlinkers, to allow for a more straightforward and reliable way to detect structural changes between conditions by quantitative CSMS (qCSMS).

The authors do an excellent job describing the design choices of the isobaric crosslinkers and how they have been optimized to allow for efficient intra- and inter-protein crosslinking to provide relevant structural information. Next, they do a series of experiments to provide compelling evidence that the Qlinker strategy is well suited to detect structural changes between conditions by qCSMS. First, they confirm the quantitative power of the novel-developed isobaric crosslinkers by a controlled mixing experiment. Then they show that they can indeed recover known structural changes in a set of purified proteins (complexes) - starting with single subunit proteins up to a very large 0.5 MDa multi-subunit protein complex - the polII complex.

The authors give a very measured and fair assessment of this novel isobaric crosslinker and its potential power to contribute to the study of protein structure changes. They show that indeed their novel strategy picks up expected structural changes, changes in surface exposure of certain protein domains, changes within a single protein subunit but also changes in protein-protein interactions. However, they also point out that not all expected dynamic changes are captured and that there is still considerable room for improvement (many not limited to this crosslinker specifically but many crosslinkers used for CSMS).

Taken together the study presents a novel set of isobaric crosslinkers that indeed open up the opportunity to provide better qCSMS data, which will enable researchers to study dynamic changes in the shape and structure of proteins and their complexes.

Comments on latest version:

The authors have not really addressed most of the concerns. They have added minimal discussion points to the text. This is okay from my perspective as eLife's policy is to leave it up to the authors of how strongly to consider the reviewers' comments. I should add that I do fully agree with the other reviewer that the quantitative assessment from Figure 1 should have been done in triplicates at least and that this would actually be essential.

Author response:

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

Reviewer #1 (Public review):

Summary:

Crosslinking mass spectrometry has become an important tool in structural biology, providing information about protein complex architecture, binding sites and interfaces, and conformational changes. One key challenge of this approach represents the quantitation of crosslinking data to interrogate differential binding states and distributions of conformational states.

Here, Luo and Ranish present a novel class of isobaric crosslinkers ("Qlinkers"), conduct proof-of-concept benchmarking experiments on known protein complexes, and show example applications on selected target proteins. The data are solid and this could well be an exciting, convincing new approach in the field if the quantitation strategy is made more comprehensive and the quantitative power of isobaric labeling is fully leveraged as outlined below. It's a promising proof-of-concept, and potentially of broad interest for structural biologists.

Strengths:

The authors demonstrate the synthesis, application, and quantitation of their "Q2linkers", enabling relative quantitation of two conditions against each other. In benchmarking experiments, the Q2linkers provide accurate quantitation in mixing experiments. Then the authors show applications of Q2linkers on MBP, Calmodulin, selected transcription factors, and polymerase II, investigating protein binding, complex assembly, and conformational dynamics of the respective target proteins. For known interactions, their findings are in line with previous studies, and they show some interesting data for TFIIA/TBP/TFIIB complex formation and conformational changes in pol II upon Rbp4/7 binding.

Weaknesses:

This is an elegant approach but the power of isobaric mass tags is not fully leveraged in the current manuscript.

First, "only" Q2linkers are used. This means only two conditions can be compared. Theoretically, higher-plexed Qlinkers should be accessible and would also be needed to make this a competitive method against other crosslinking quantitation strategies. As it is, two conditions can still be compared relatively easily using LFQ - or stable-isotope-labeling based approaches. A "Q5linker" would be a really useful crosslinker, which would open up comprehensive quantitative XLMS studies.

We agree that a multiplexed Qlinker approach would be very useful. The multiplexed Qlinkers are more difficult and more expensive to synthesize. We are currently working on different schemes for synthesizing multiplexed Qlinkers.

Second, the true power of isobaric labeling, accurate quantitation across multiple samples in a single run, is not fully exploited here. The authors only show differential trends for their interaction partners or different conformational states and do not make full quantitative use of their data or conduct statistical analyses. This should be investigated in more detail, e.g. examine Qlinker quantitation of MBP incubated with different concentrations of maltose or Calmodulin incubated with different concentrations of CBPs. Does Qlinker quantitation match ratios predicted using known binding constants or conformational state populations? Is it possible to extract ratios of protein populations in different conformations, assembly, or ligand-bound states?

With these two points addressed this approach could be an important and convincing tool for structural biologists.

We agree that multiplexed Qlinkers would open the door to exciting avenues of investigation such as studying conformational state populations.  We plan to conduct the suggested experiments when multiplexed Qlinkers are available.

Reviewer #2 (Public review):

The regulation of protein function heavily relies on the dynamic changes in the shape and structure of proteins and their complexes. These changes are widespread and crucial. However, examining such alterations presents significant challenges, particularly when dealing with large protein complexes in conditions that mimic the natural cellular environment. Therefore, much emphasis has been put on developing novel methods to study protein structure, interactions, and dynamics. Crosslinking mass spectrometry (CSMS) has established itself as such a prominent tool in recent years. However, doing this in a quantitative manner to compare structural changes between conditions has proven to be challenging due to several technical difficulties during sample preparation. Luo and Ranish introduce a novel set of isobaric labeling reagents, called Qlinkers, to allow for a more straightforward and reliable way to detect structural changes between conditions by quantitative CSMS (qCSMS).

The authors do an excellent job describing the design choices of the isobaric crosslinkers and how they have been optimized to allow for efficient intra- and inter-protein crosslinking to provide relevant structural information. Next, they do a series of experiments to provide compelling evidence that the Qlinker strategy is well suited to detect structural changes between conditions by qCSMS. First, they confirm the quantitative power of the novel-developed isobaric crosslinkers by a controlled mixing experiment. Then they show that they can indeed recover known structural changes in a set of purified proteins (complexes) - starting with single subunit proteins up to a very large 0.5 MDa multi-subunit protein complex - the polII complex.

The authors give a very measured and fair assessment of this novel isobaric crosslinker and its potential power to contribute to the study of protein structure changes. They show that indeed their novel strategy picks up expected structural changes, changes in surface exposure of certain protein domains, changes within a single protein subunit but also changes in protein-protein interactions. However, they also point out that not all expected dynamic changes are captured and that there is still considerable room for improvement (many not limited to this crosslinker specifically but many crosslinkers used for CSMS).

Taken together the study presents a novel set of isobaric crosslinkers that indeed open up the opportunity to provide better qCSMS data, which will enable researchers to study dynamic changes in the shape and structure of proteins and their complexes. However, in its current form, the study some aspects of the study should be expanded upon in order for the research community to assess the true power of these isobaric crosslinkers. Specifically:

Although the authors do mention some of the current weaknesses of their isobaric crosslinkers and qCSMS in general, more detail would be extremely helpful. Throughout the article a few key numbers (or even discussions) that would allow one to better evaluate the sensitivity (and the applicability) of the method are missing. This includes:

(1) Throughout all the performed experiments it would be helpful to provide information on how many peptides are identified per experiment and how many have actually a crosslinker attached to it.

As the goal of the experiments is to maximize identification of crosslinked peptides which tend to have higher charge states, we targeted ions with charge states of 3+ or higher in our MS acquisition settings for CLMS, and ignored ions with 2+ charge states, which correspond to many of the normal (i.e., not crosslinked) peptides that are identified by MS. As a result, normal peptides are less likely to be identified by the MS procedure used in our CLMS experiments compared to MS settings typically used to identify normal peptides. Our settings may also fail to identify some mono-modified peptides. Like most other CLMS methods, the total number of identified crosslinked peptide spectra is usually less than 1% of the total acquired spectra and we normally expect the crosslinked species to be approximately 1% of the total peptides. 

We added information about the number of crosslinked and monolinked peptides identified in the pol I benchmarking experiments (line 173).  The number of crosslinks and monolinks identified in the pol II +/- a-amanitin experiment, the TBP/TFIIA/TFIIB experiment and the pol II experiment +/- Rpb4/7 are also provided.

(2) Of all the potential lysines that can be modified - how many are actually modified? Do the authors have an estimate for that? It would be interesting to evaluate in a denatured sample the modification efficiency of the isobaric crosslinker (as an upper limit as here all lysines should be accessible) and then also in a native sample. For example, in the MBP experiment, the authors report the change of one mono-linked peptide in samples containing maltose relative to the one not containing maltose. The authors then give a great description of why this fits to known structural changes. What is missing here is a bit of what changes were expected overall and which ones the authors would have expected to pick up with their method and why have they not been picked up. For example, were they picked up as modified by the crosslinker but not differential? I think this is important to discuss appropriately throughout the manuscript to help the reader evaluate/estimate the potential sensitivity of the method. There are passages where the authors do an excellent job doing that - for example when they mention the missed site that they expected to see in the initial the pol II experiments (lines 191 to 207). This kind of "power analysis" should be heavily discussed throughout the manuscript so that the reader is better informed of what sensitivity can be expected from applying this method.

Regarding the Pol II complex experiment described in Figures 4 and 5, out of the 277 lysine residues in the complex, 207 were identified as monolinked residues (74.7%), and 817 crosslinked pairs out of 38,226 potential pairs (2.1%) were observed. The ability of CLMS to detect proximity/reactivity changes may be impacted by several factors including 1) the (low) abundance of crosslinked peptides in complex mixtures, 2) the presence of crosslinkable residues in close proximity with appropriate orientation, and 3) the ability to generate crosslinked peptides by enzymatic digestion that are amenable to MS analysis (i.e., the peptides have appropriate m/z’s and charge states, the peptides ionize well, the peptides produce sufficient fragment ions during MS2 analysis to allow confident identification). Future efforts to enrich crosslinked peptides prior to MS analysis may improve sensitivity.

It is very difficult to estimate the modification efficiency of Qlinker (or many other crosslinkers) based on peptide identification results. One major reason for this is that trypsin is not able to cleave after a crosslinker-modified lysine residue.  As a result, the peptides generated after the modification reaction have different lengths, compositions, charge states, and ionization efficiencies compared to unmodified peptides. These differences make it very difficult to estimate the modification efficiencies based on the presence/absence of certain peptide ions, and/or the intensities of the modified and unmodified versions of a peptide. Also, 2+ ions which correspond to many normal (i.e., unmodified) peptides were excluded by our MS acquisition settings.

It is also very difficult to predict which structural changes are expected and which crosslinked peptides and/or modified peptides can be observed by MS.  This is especially true when the experiment involves proteins containing unstructured regions such as the experiments involving Pol II, and TBP, TFIIA and TFIIB. Since we are at the early stages of using qCLMS to study structural changes, we are not sure which changes we can expect to observe by qCLMS. Additional applications of Qlinker-CLMS are needed to better understand the types of structural changes that can be studied using the approach.

We hope that our discussions of some the limitations of CLMS for detecting conformational/reactivity changes provide the reader with an understanding of the sensitivity that can be expected with the approach.  At the end of the paragraph about the pol II a-amanitin experiment we say, “Unfortunately, no Q2linker-modified peptides were identified near the site where α-amanitin binds. This experiment also highlights one of the limitations of residue-specific, quantitative CLMS methods in general. Reactive residues must be available near the region of interest, and the modified peptides must be identifiable by mass spectrometry.” In the section about Rbp4/7-induced structural changes in pol II we describe the under-sampling issue. And in the last paragraph we reiterate these limitations and say, “This implies that this strategy, like all MS-based strategies, can only be used for interpretation of positively identified crosslinks or monolinks. Sensitivity and under sampling are common problems for MS analysis of complex samples.”

(3) It would be very helpful to provide information on how much better (or not) the Qlinker approach works relative to label-free qCLMS. One is missing the reference to a potential qCLMS gold standard (data set) or if such a dataset is not readily available, maybe one of the experiments could be performed by label-free qCLMS. For example, one of the differential biosensor experiments would have been well suited.

We agree with the reviewer that it will be very helpful to establish gold standard datasets for CLMS. As we further develop and promote this technology, we will try to establish a standardized qCLMS.

Reviewer #1 (Recommendations for the authors):

Only a very minor point:

I may have missed it but it's not really clear how many independent experiments were used for the benchmarking quantitation and mixing experiments for Figure 1. What is the reproducibility across experiments on average and on a per-peptide basis?

Otherwise, I think the approach would really benefit from at least "Q5linkers" or even "Q10linkers", if possible. And then conduct detailed quantitative studies, either using dilution series or maybe investigating the kinetics of complex formation.

We used a sample of BSA crosslinked peptides to optimize the MS settings, establish the MS acquisition strategies and test the quantification schemes.  The data in Figure 1 is based on one experiment, in which used ~150 ug of purified pol I complexes from a 6 L culture. We added this information to the Figure 1 legend. We also provide information about the reproducibility of peptide quantification by plotting the observed and expected ratios for each monolinked and crosslinked peptide identified in all of the runs in Figure S3.

We agree with the reviewer that the Qlinker approach would be even more attractive if multiplex Qlinker reagents were designed. The multiplexed Qlinkers are more difficult and more expensive to synthesize. We are currently working on different schemes for synthesizing multiplexed Qlinkers.

Reviewer #2 (Recommendations for the authors):

In addition to the public review I have the following recommendations/questions:

(1) The first part of the results section where the synthesis of the crosslinker is explained is excellent for mass spec specialists, but problematic for general readers - either more info should be provided (e.g. b1+ ions - most readers will have no idea why that is) - or potentially it could be simplified here and the details shifted to Materials and Methods for the expert reader. The same is true below for the length of spacer arms.

However - in general this level of detail is great - but can impact the ease of understanding for the more mass spec affine but not expert reader.

We have added the following sentence to assist the general reader: A b1+ ion is an ion with a charge state of +1 corresponding to the first N-terminal amino acid residue after breakage of the first peptide bond (lines 126-128).

(2) The Calmodulin experiment (lines 239 to 257) - it is a very nice result that they see the change in the crosslinked peptide between residues K78-K95, but the monolinks are not just detected as described in the text but actually go 2 fold up. This would have been actually a bit expected if the residues are now too far away to be still crosslinked that the monolinks increase. In this case, this counteraction of monolinks to crosslinked sites can also be potentially used as a "selection criteria" for interesting sites that change. Is that a possible interpretation or do the authors think that upregulation of the monolinks is a coincidence and should not be interpreted?

We agree with the reviewer that both monolinks and crosslinks can be used as potential indicators for some changes. However, it is much more difficult to interpret the abundance information from monolinks because, unlike crosslinks, there is little associated structural/proximity information with monolinks. Because it is difficult to understand the reason(s) for changes in monolink abundance, we concentrate on changes in crosslink abundances, which provide proximity/structural information about the crosslinked residues.

(3) Lines 267 to 274: a small thing but the structural information provided is quite dense I have to say. Maybe simplify or accompany with some supplemental figures?

We agree that the structural information is a bit dense especially for readers who are not familiar with the pol II system.  We added a reference to Figure 3c (line 177) to help the reader follow the structural information. 

As qCLMS is still a relatively new approach for studying conformational changes, the utility of the approach for studying different types of conformational changes is still unclear. Thus, one of the goals of the experiments is to demonstrate the types of conformational changes that can be detected by Q2linkers.  We hope that the detailed descriptions will help structural biologists understand the types of conformational changes that can be detected using Qlinkers.

(4) Line 280: explain maybe why the sample was fractionated by SCX (I guess to separate the different complexes?).

SCX was used to reduce the complexity of the peptide mixtures. As the samples are complex and crosslinked peptides are of low abundance compared to normal peptides, SCX can separate the peptides based on their positive charges.  Larger peptides and peptides with higher charge states, such as crosslinked peptides, tend to elute at higher salt concentration during SCX chromatography.  The use of SCX to fractionate complex peptide mixtures is described in the “General crosslinking protocol and workflow optimization” section of the Methods, and we added a sentence to explain why the sample was fractionated by SCX (lines 278-279).

(5) Lines 354 to 357: "This suggests that the inability to identity most of these crosslinked peptides in both experiments is mainly due to under-sampling during mass spectrometry analysis of the complex samples, rather than the absence of the crosslinked peptides in one of the experiments."

This is an extremely important point for the interpretation of missing values - have the authors tried to also collect the mass spec data with DIA which is better in recovery of the same peptide signals between different samples? I realize that these are isobaric samples so DIA measurements per se are not useful as the quantification is done on the reporter channels in the MS2, but it would at least give a better idea if the missing signals were simply not picked up for MS2 as claimed by the authors or the modified peptides are just not present. Another possibility is for the authors to at least try to use a "match between the run" function as can be done in Maxquant. One of the strengths of the method is that it is quantitative and two states are analyzed together, but as can be seen in this experiment, more than two states might want to be compared. In such cases, the under-sampling issue (if that is indeed the cause) makes interpretation of many sites hard (due to missing values) and it would be interesting if for example, an analysis approach with a "match between the runs" function could recover some of the missing values.

We agree that undersampling/missing values is an important issue that needs to be addressed more thoroughly. This also highlights the importance of qCLMS, as conclusions about structural changes based on the presence/absence of certain crosslinked species in database search results may be misleading if the absence of a species is due to under-sampling. We have not tried to collect the data with DIA since we would lose the quantitative information. It would be interesting to see if match between runs can recover some of the missing values. While this could provide evidence to support the under-sampling hypothesis, it would not recover the quantitative information.

We recommend performing label swap experiments and focusing downstream analysis on the crosslinks/monolinks that are identified on both experiments. Future development of multiplexed Qlinker reagents should help to alleviate under-sampling issues. See response to Reviewer #1.

(6) Lines 375 to 393 (the whole paragraph): extremely detailed and not easy to follow. Is that level of detail necessary to drive home that point or could it be visualized in enough detail to help follow the text?

We agree that the paragraph is quite detailed, but we feel that the level of detailed is necessary to describe the types of conformational changes that can be detected by the quantitative crosslinking data, and also illustrate the challenges of interpreting the structural basis for some crosslink abundance changes even when high resolution structural data exists.

To make it easier to follow, we added a sentence to the legend of Figure 5b. “In the holo-pol II structure (right), Switch 5 bending pulls Rpb1:D1442 away from K15, breaking the salt bridge that is formed in the core pol II structure (left). The increase in the abundances of the Rpb1:15-Rpb6:76 and Rpb1:15-Rpb6:72 crosslinks in holo-pol II is likely attributed to the salt bridge between K15 and D1442 in core pol II which impedes the NHS ester-based reaction between the epsilon amino group of K15 and the crosslinker.”

(7) Final paragraph in the results section - lines 397 and 398: "All of the intralinks involving Rpb4 are more abundant in holo-pol II as expected." If I understand that experiment correctly the intralinks with Rpb4 should not be present at all as Rpb4 has been deleted. Is that due to interference between the 126 and 127 channels in MS2? If so, then this also sets a bit of the upper limit of quantitative differences that can be seen. The authors should at least comment on that "limitation".

Yes, we shouldn’t detect any Rpb4 peptides in the sample derived from the Rpb4 knockout strain. The signal from Rpb4 peptides in the DRpb4 sample is likely due to co-eluting ions. To clarify, we changed the text to:

All of the intralinks involving Rpb4 are more abundant in the holo-pol II sample (even though we don’t expect any reporter ion signal from Rpb4 peptides derived from the ∆Rpb4 pol II sample, we still observed reporter ion signals from the channel corresponding to the DRpb4 sample, potentially due to the presence of low abundance, co-eluting ions)(lines 395-399).

(8) Materials and Methods - line 690: I am probably missing something but why were two different mass additions to lysine added to the search (I would have expected only one for the crosslinker)?

The 297 Da modification is for monolinked peptides with one end of the crosslinker hydrolyzed and 18 Da water molecule is added. The 279 Da modification is for crosslinks and sometimes for looplinks (crosslinks involving two lysine residues on the same tryptic peptide).

for - quote / critique - it is upon us, beyond our power to alter, and therefore to be accepted and made the best of. It is a waste of time to criticize the inevitable. - Andrew Carnegie - The Gospel of Wealth - alternatives - to - mainstream companies - cooperatives - Peer to Peer - Decentralized Autonomous Organization (DAO) - Fair Share Commons - B Corporations - Worker owned companies

quote / critique - it is upon us, beyond our power to alter, and therefore to be accepted and made the best of. It is a waste of time to criticize the inevitable. - Andrew Carnegie - The Gospel of Wealth - This is a defeatist attitude that does not look for a condition where both enormous inequality AND universal squalor can both eliminated - Today, there are a growing number of alternative ideas which can challenge this claim such as: - Cooperatives - example - Mondragon corporation with 70,000 employees - B Corporations - Fair Share Commons - Peer to Peer - Worker owned companies - Cosmolocal organizations - Decentralized Autonomous Organization (DAO)

for - quote / critique / question - Thus is the problem of Rich and Poor to be solved. The laws of accumulation will be left free; the laws of distribution free. Individualism will continue, but the millionaire will be but a trustee for the poor; intrusted for a season with a great part of the increased wealth of the community, but administering it for the community far better than it could or would have done for itself. - The Gospel of Wealth - Andrew Carnegie

quote / critique / question - Thus is the problem of Rich and Poor to be solved. The laws of accumulation will be left free; the laws of distribution free. Individualism will continue, but the millionaire will be but a trustee for the poor; intrusted for a season with a great part of the increased wealth of the community, but administering it for the community far better than it could or would have done for itself. - The Gospel of Wealth - Andrew Carnegie - The problem with this reasoning is that it is circular - By rewarding oneself an extreme and unfettered amount of wealth for one's entrepreneurship skills creates inequality in the first place - Competition that destroys other corporations ends up reducing jobs - At the end of life, the rich entrepreneur desires to give back to society the wealth that (s)he originally stole - If one had reasonable amounts of rewarding innovation instead of unreasonable amounts, the problem of inequality can be largely mitigated in the first place whilst still recognizing and rewarding individual effort and ingenuity

for - quote / critique - The price we pay for this salutary change is, no doubt, great - Andrew Carnegie

quote / critique - The price we pay for this salutary change is, no doubt, great - Andrew Carnegie - Carnegie goes on to write that the great freedoms offered by industrial mass production has an unavoidable price to be paid - Successful manufacturing and production cooperatives, B-Corporations, worker-owned companies, etc have disproved that it is an either-or situation. - Consider the case of the Spanish manufacturing giant, Mondragon, a federation of worker cooperatives employing 70,000 people located in Spain - where this price is NOT paid - Carnegie's essay reflects a perspective based on the time when he was alive - Were Carnegie alive today to witness the natural conclusion of his trend of progress in the Anthropocene, he would witness - extreme pollution levels of industrial mass production threatening to destabilize human civilization itself - astronomical wealth inequality - And these two are linked: - wealth inequality - a handful of elites have the same wealth as the bottom half of humanity - carbon inequality - that same handful pollutes as much as the bottom half of humanity

to - Mondragon cooperative - explore - https://hyp.is/GeIKao1rEe-9jA_97_KRBg/exploremondragon.com/en/ - Oxfam wealth and carbon inequality reports - https://jonudell.info/h/facet/?max=100&expanded=true&user=stopresetgo&exactTagSearch=true&any=oxfam

for - critique - destruction of Individualism - The Gospel of Wealth - Andrew Carnegie - individual / collective Gestalt - Deep Humanity

critique - destruction of Individualism - The Gospel of Wealth - Andrew Carnegie - From a Deep Humanity perspective, the individual and the collective are intertwingled - This is the individual / collective gestalt - Communism and Capitalism are both extreme poles - the truth lies somewhere in the middle - which acknowledges both are individual AND collective nature simultaneously - and works to balance them

for - critique - extreme wealth inequality cannot be avoided for the greater improvement of society - The Gospel of Wealth - Andrew Carnegie - stats - Mondragon corporation - comparison of pay difference between highest paid and lowest paid - adjacency - Gandhi quote - Andrew Carnegie beliefs in The Gospel of Wealth

critique - extreme wealth inequality cannot be avoided for the greater improvement of society - The Gospel of Wealth - Andrew Carnegie - It's a matter of degree - Wealth differences within US corporations of 344 to 1 are obscene and not necessary, as proven by - Wealth difference of 6 to 1 in Mondragon federation of cooperatives - To quote - Gandhi, there is enough to meet everyone's needs but not enough to meet everyone's greed - The great problem with such large wealth disparity is that those who know how to game the system can earn obscene amounts of money - and since the concept of luxury goods is made desirable and proportional to monetary wealth, it creates a positive feedback loop of insatiability - The combination of engaging in ever greater luxury lifestyle and power is intoxicating and addictive

to - stats - Mondragon corporation - comparison of pay difference between highest paid and lowest paid - https://hyp.is/QAxx-o14Ee-_HvN5y8aMiQ/www.csmonitor.com/Business/2024/0513/income-inequality-capitalism-mondragon-corporation

for - critique - extreme wealth a reward for rare management skills - Andrew Carnegie - The Gospel of Wealth - Mondragon counterexample - to - stats - Mondragon pay difference between highest and lowest paid - article - In this Spanish town, capitalism actually works for the workers - Christian Science Monitor - Erika Page - 2024, June 7

critique - extreme wealth a reward for rare management skills - Andrew Carnegie - The Gospel of Wealth - Mondragon counterexample - This is invalidated today by large successful cooperatives such as Mondragon

to - stats - Mondragon corporation - comparison of pay difference between highest paid and lowest paid - https://hyp.is/QAxx-o14Ee-_HvN5y8aMiQ/www.csmonitor.com/Business/2024/0513/income-inequality-capitalism-mondragon-corporation

for - quote / critique- Much better this great irregularity than universal squalor - Andrew Carnegie

quote / critique - Much better this great irregularity than universal squalor - Andrew Carnegie - Carnegie is writing from his perspective of the contrast between - life when he grew up, lived in an age of perceived universal squalor and - the world he helped shape through industrial mass production that produced high quality goods in such numbers that they became available to all - Yet, even before Carnegie, inequality had existed, for the world prior to Carnegie had its share of kings, queens, emperors and authoritarians - Even today, the best we might say of modern democracies is a decoupling of wealth and official governance - although even that is inaccurate as the thriving lobbying industry allows industrial magnates to decide upon rules of governance that are friendly towards their businesses - In contrast, from the commons perspective, and especially from the Cosmolocal movement of production, there is proposed a road that leads to - much less and much more tolerable levels of inequality and no universal squalor - a civilization existing within safe and just earth system boundaries

for - quote / critique - The Indians are today where civilized man then was

quote / critique - The Indians are today where civilized man then was - Andrew Carnegie - The Gospel of Wealth - Carnegie starts off his essay with this statement, that is meant to contrast how far industrial mass production has progressed society compared to the the rate of progress before it - It is an unfortunate choice of comparison as it is tainted with the mass genocide brought about by Carnegie's colonialist ancestors - Human civilization progressed in nonuniform spurts, with some parts of the world advancing greater than other parts at different times of human history

first Norman King

Now, that is a great line.

25%

A mouse the size of a New Your city rat jumped out at the cat, and the cat got scared for its life because the mouse was bigger than him

eLife Assessment

This useful study by Nandy and colleagues examined relationships between behavioral state, neural activity in cortical area V4, and trial-by-trial variability in the ability to detect weak visual stimuli. They present solid evidence indicating that certain changes in arousal and eye-position stability, along with patterns of synchrony in the activity of neurons in different layers of V4, can show modest correspondences to changes in the ability to correctly detect a stimulus. These findings are likely to be of interest to those who seek a deeper understanding of circuit mechanisms that underlie perception.

Reviewer #1 (Public review):

Summary:

In this study, Nandy and colleagues examine neural, physiological and behavioral correlates of perceptual variability in monkeys performing a visual change detection task. They used a laminar probe to record from area V4 while two macaque monkeys detected a small change in stimulus orientation that occurred at a random time in one of two locations, focusing their analysis on stimulus conditions where the animal was equally likely to detect (hit) or not-detect (miss) a briefly presented orientation change (target). They discovered two behavioral and physiological measures that are significantly different between hit and miss trials - pupil size tends to be slightly larger on hits vs. misses, and monkeys are more likely to miss the target on trials in which they made a microsaccade shortly before target onset. They also examined multiple measures of neural activity across the cortical layers and found some measures that are significantly different between hits and misses.

Strengths:

Overall the study is well executed and the analyses are appropriate (with some possible caveats discussed below).

Weaknesses:

I have two remaining concerns. First, with the exception of the pre-target microsaccades, the correlates of perceptual variability (differences between hits and misses) appear to be weak and disconnected. The GLM analysis of the predictive power of trial outcome based on the behavioral and neural measures is only discussed at the end of the paper. This analysis shows that some of the measures have no significant predictive power, while others cannot be examined using the GLM analysis because these measures cannot be estimated in single trials. Given these weak and disconnected effects, my overall sense is that the current results provide a limited advance to our understanding of the neural basis of perceptual variability.

In addition, because the authors combine data across stimulus contrasts, I am somewhat uneasy about the possible confounding effect of contrast. As expected, stimulus contrast affected the probability of hits vs. misses. Independently, contrast may have affected some of the physiological measurements. Therefore, showing that contrast is not the source of the covariations between the physiological/behavioral measurements and perception can be challenging, and I am not convinced that the authors have ruled this out as a possible confound. It is unclear why the authors had to vary contrast in the first place, and why the analyses had to be done by combining the data across contrasts or by ignoring contrast as a variable (e.g., in the GLM analysis).

Hi! Just showing you what an annotation looks like in context and also explaining what this is a bit more.

The FYW Committee is a committee comprised of full-timers at QC who teach in the first-year writing program and / or are experts in Writing Studies. We make decisions about stuff that happens in the FYW program. Formerly, we've also done things like review three-year contract materials.

Ah, I wish I could access their SIS and see the filter.

"Were" should be "was" as it is referring to the word "average," a singular quantity. "Were" would be used if the author was referring to multiple averages.

No values regarding sugar percent concentration are specified with the three repeated experiments. It is clear that the author repeated the experiment with different concentrations of sugar water when the reader refers to the graph. These values need to be included in the description.

It is unclear if the author is trying to make a general statement about calculations or if the author is trying to relay what actually happened during the experiment. The verb "is" must be changed to "was" to be in the past passive tense. And, the "liquid" needs to be specified. For ease of understanding, the previous sentence can be combined with this one: "Then, the mass of the liquid within the beaker was calculated by subtracting the initial mass from the final mass."

Abigail is doing what she can to provide for her family, she is currently very busy, this is probably why she hasn't attempted to make salt peter (white powder used to make gunpowder and preserve food)

In the second sentence of this paragraph Abigail mentions she is attending the sick, one of her neighbors and in this highlighted section she mentions how it has affected many other people and towns. Most likely just like Abigail was doing other women were also helping nurse the sick.

Thoughts: I find Abigail's actions important here because during a war it is vital to be in good health. Illnesses can bring death and also weaken, decreasing helping hands that could help them win this war.

Here Abigial is being resourceful. She has mention that her husband asked if she had made salt peter (white powder used to make gunpowder and preserve food)

But she has not attempted it yet, but she has information on who can provide that material to her husband and should he need it. She offers to make the purchase and send it to him.

Thoughts: Abigail is helping her husband, providing materials that would be vital to aide them in war and her husband is not taking her seriously about giving women equal rights.

Animal experimentation has been going on for hundreds of years and helped with pharmaceutical and illness research. Doing the same experiments on humans would not work because of the extent of the experiments and the types of tests preformed.

The comma is unnecessary. Also, using the preposition "by" when referring to subtraction creates confusion. The described values should be flipped: "The volume was calculated by subtracting the initial volume of the water in the graduated cylinder from the volume of the graduated cylinder after the metal rod had been added. To further clarify, numeric values should be used.

for - future annotation - Twitter post - AI - collective democratic - Habermas Machine - Michiel Bakker

I don't have a problem with this kind of poisoning. When you stand up to a company like Kellog's that has money, expensive lawyers and cares only about their bottom line it needs to be done. The David and Goliath of it all begs for action to fighting against unfair work conditions for the ordinary worker.

for - stats - Mondragon corporation - pay difference comparison between highest paid and lowest paid - from - essay - The Gospel of Wealth - Andrew Carnegie - Carnegie organization

from - essay - The Gospel of Wealth - Andrew Carnegie - Carnegie organization - https://hyp.is/dIoiDo16Ee-0n2OpOK3lwg/www.carnegie.org/about/our-history/gospelofwealth/

stats - Mondragon corporation - comparison of pay difference between highest paid and lowest paid - Modragon - 6 to 1 - typical US - 344 to 1 - typical Spain - 77 to 1

This makes a lot of sense but is also incredible scary to know that someone is being watched and most likely being manipulated. The susceptible to scams and prone to addiction is probably the most frightening thing. I feel that is just evil to track people's vulnerabilities and possible use it against them.

Author response:

Review #1:

Also, they observed no difference in the binding free energy of phosphatidylserine 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 directly note this contrast with experimental findings 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 in the R47H variant extends beyond decreased binding affinities and also impacts binding patterns. As stated in our manuscript: ‘Our observations for both sTREM2 and TREM2 indicate that R47H-induced dysfunction may result not only from diminished ligand binding but also an impaired ability to discriminate between different ligands in the brain, proposing a novel mechanism for loss-of-function.’

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.

The reviewer raises an interesting point regarding the repetition of individual simulations, a consideration we carefully evaluated during the design of this study. However, we believe our approach—running multiple independent models 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.

In our study, we demonstrate that within the 150 ns timescale of our protein/ligand (PL) simulations, the relatively small ligands are able to move from their initial docking positions to a specific binding site. While ideally, replicates of these independent models would further strengthen the findings, this was not computationally feasible given the unprecedented total duration of our simulations. Importantly, our conclusions are seldom based on the results of a single protein/PL simulation.

Moreover, the ergodic hypothesis suggests that over sufficiently long timescales, simulations will explore all accessible states. Additionally, we have performed several replicate simulations of our WT and R47H Ig-like domain models in solution, specifically to investigate CDR2 loop dynamics.

In this case, since the system involves only the protein and lacks the independent replicates seen in the protein/PL simulations, these runs were chosen to effectively capture the stochastic nature of CDR2 loop movement.

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. While R47H is the most common and prolific mutation in literature, an extensive catalog of other mutations is important to explore. We are currently preparing two separate publications that will delve into these gaps in more detail, as addressing them was beyond the scope of the present study.

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.

We are currently working to address this comment to strengthen the validity of our results and statistical conclusions in the revised manuscript.  

Review #2:

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 will adjust and refocus how we reference this evidence from Kober et al. in our revised manuscript. 

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. Hence, we are currently working toward a manuscript that will be the first biologically relevant model of TREM2 in a membrane and will challenge the current paradigm of using the Ig-like domain as an experimental surrogate for TREM2.

eLife Assessment

This useful manuscript addresses some key molecular mechanisms on the neuroprotective roles of soluble TREM2 in neurodegenerative diseases. Thw study will advance our understanding of TREM2 mutations, particularly on the damaging effect of known TREM2 mutations, and also explain why soluble TREM2 can antagonize Aβ aggregation. However, the primary experimental method, MD simulations, suffers from limited sampling, rendering the results incomplete for definite conclusions.

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).

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.

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.

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".

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.

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.

Weaknesses:

Unfortunately, the work suffers from two fundamental flaws.

(1) 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.

(2) 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.

Résumé de la vidéo [00:00:06][^1^][1] - [00:24:50][^2^][2]:

Cette vidéo traite de la prévention de la violence armée et des meilleures pratiques basées sur des données probantes. Elle aborde les facteurs de risque, les mesures de prévention et l'importance des preuves scientifiques pour documenter et évaluer l'efficacité des interventions.

Points forts : + [00:00:06][^3^][3] Objectifs de la présentation * Familiariser avec la notion de données probantes * Identifier les facteurs de risque associés à la violence armée * Discuter des mesures de prévention prometteuses + [00:01:00][^4^][4] Statistiques sur la violence armée * Augmentation des homicides en 2022 * Influence des crimes violents par arme à feu * Implication des gangs dans les homicides + [00:07:00][^5^][5] Importance des données probantes * Utilisation des meilleures preuves disponibles * Études longitudinales pour identifier les facteurs de risque * Outils actuariels pour évaluer le risque de récidive + [00:12:00][^6^][6] Synthèse systématique * Méthodologie pour évaluer l'efficacité des programmes * Inclusion et exclusion des études basées sur la qualité * Importance de la transparence et de la rigueur + [00:16:00][^7^][7] Facteurs de risque de la violence armée * Facteurs individuels, interpersonnels, communautaires et institutionnels * Influence de la pauvreté et de la désorganisation sociale * Accès aux armes à feu et densité des commerces d'alcool

Résumé de la vidéo [00:24:52][^1^][1] - [00:48:49][^2^][2]:

Cette vidéo traite des initiatives et des mesures pour prévenir la violence chez les jeunes, en mettant l'accent sur les actions non coercitives et les revues parapluies pour évaluer l'efficacité des programmes.

Moments forts: + [00:24:52][^3^][3] Initiatives non coercitives * Offrir des alternatives à la judiciarisation * Travailler sur les compétences des jeunes * Prévenir la probabilité ou la gravité des délits + [00:26:22][^4^][4] Revue parapluie * Synthétiser les connaissances sur l'efficacité des programmes * Comparer différentes mesures pour prévenir la violence * Identifier les meilleures options + [00:27:02][^5^][5] Exemple de subvention * Subvention du ministère de la Sécurité publique * Revue parapluie sur les violences par armes à feu * Consultation de bases de données et d'experts + [00:35:00][^6^][6] Mesures législatives et policières * Vérification des antécédents et système de permis * Dissuasion ciblée et confiscation des armes * Programmes policiers et initiatives communautaires + [00:42:00][^7^][7] Programmes de prévention développementale * Travailler sur les habilités sociales et comportementales * Aider les parents et les jeunes à risque * Réduire les comportements antisociaux de 25%

Résumé de la vidéo [00:48:50][^1^][1] - [01:00:02][^2^][2]:

Cette partie de la vidéo discute des méthodes de prévention de la violence chez les jeunes, en se concentrant sur les revues parapluies et les synthèses systématiques pour évaluer l'efficacité des programmes de prévention.

Moments forts: + [00:48:50][^3^][3] Revue parapluie * Comparaison des mesures de prévention * Consultation de 12 bases de données * Inclusion de 75 études + [00:52:00][^4^][4] Synthèse systématique * Prévention de la violence conjugale * Identification de cinq catégories de mesures * Réduction de 18 % des agressions + [00:56:00][^5^][5] Prévention de la violence dans les relations amoureuses * Programmes de résolution des conflits * Communication affirmée * Efficacité des mesures + [00:57:00][^6^][6] Prévention de la radicalisation * Contre-narratif * Efficacité limitée * Besoin de stratégies alternatives + [00:58:00][^7^][7] Programmes sportifs * Prévention de la violence * Effets modestes * Nécessité de revues systématiques supplémentaires

wichtig

eLife Assessment

The study provides a valuable showcase of a workflow to perform large-scale characterization of drug mechanisms of action using proteomics in which on-target and off-targets of 166 compounds using proteome solubility analysis in living cells and cell lysates were determined. The evidence supporting the claims of the authors is solid, however, the inclusion of more replicate experiments and more statistical rigor would have strengthened the study. This will be of broad interest to medicinal chemists, toxicologists, computational biologists and biochemists.

"I'm always trying to get back to the 20s a little bit." <br /> —John Dickerson, in Field Notes interview (2016) https://vimeo.com/169725470

Dickerson says he's got two screens on the computer in his office as well as an ipad and a phone. But he's also got a "notebook does only one thing". He's also got an old black lacquer Underwood (No. 4, 5, or 6?) on his office desk still.

Wonder if he uses it?

quote?

preconceived notion

look into?

sustainable agriculture backed up by financial incentives

economic incentive

soil is base for solving the problem

carbon sequestration

is this fast enough?

sustainable agriculture benefits for farmer, economy, and general public

harmful practices

rapidly declining

not specific but shows governmental policy needs to change

soil importance

potential source

getting worse

Mars CEO says he cares, but wheres the follow through

fertilizer problems

needs to be everyone

does he really care?

may have increased

2 year old data, but significant

needs to happen asap to mitigate permanent damage and destruction

incentives and productivity

needs to work within capitalism (boo)

midwest/great plains region

practices and data production

quantified economic incentive

yes exactly that thank you

can help clean pollutted area

numbers

important for soil health and plant growth

QUOTE

holy grail of quotes

quote?

microbes role in climate change

why it sequesters carbon

main ideas in my paper

hopefully we will adapt

style varies on location

continued investment

literally my paper

more barriers

barriers to entry

management

highlighted

highlight

synthetic and polluting fertilizer cheaper than sustainable ones

financial and time requirement needed for sustainable practices

lack of understanding leads to degredation

more nitrogen = more plant

fertilizer

Example of how this gets operationalized: There will be platforms (think Naviance on big data and personalized data steroids) that will help Learners discover right fit opportunities based in part on the credentials they already have. There will be savvy institutions connecting to others' credentials so as to increase the likelihood that Learners discover those institutions' program offerings. This will be akin to a sort of skill-based SEO approach as a recruitment/admissions strategy.

Presicion

Probably the funniest portrayal of academia.

for - Mondragon cooperative - explore

Insight: The energy efficiency of RISC processors makes them ideal for devices where power consumption is a critical factor, such as smartphones and other portable electronics.

Applications While RISC didn’t take off in desktop and server applications, it became dominant in embedded systems and mobile devices. RISC processors like ARM and MIPS are widely used in smartphones, tablets, gaming devices, and embedded systems.

Popular RISC Processors:

ARM: Dominates mobile and embedded markets (used in iPhones, Android devices). MIPS, PowerPC, SPARC: Found in various computing and embedded applications.

Advantages of RISC:

Speed: Simplified instructions mean faster execution per cycle. Cost: Fewer transistors reduce chip complexity and manufacturing costs. Energy Efficiency: Especially important in embedded systems and mobile devices.

Disadvantages of RISC:

Software Complexity: The need for more instructions places a heavier burden on software development. Blurring Lines: Modern CISC processors incorporate RISC-like features, reducing the distinction between the two architectures.

Insight: As the gap between RISC and CISC narrows, both architectures borrow elements from each other, creating hybrid designs in modern processors.

Performance RISC processors emphasize reducing the number of cycles per instruction (CPI), even at the cost of increasing the total number of instructions. This is in contrast to CISC, which focuses on reducing the number of instructions by using more complex ones.

Key Formula for Performance:

Time per program = Time per cycle × Cycles per instruction × Instructions per program

CISC vs. RISC:

CISC: Fewer instructions, but each instruction may take multiple cycles. RISC: More instructions but with fewer cycles per instruction, leading to faster performance in optimized scenarios.

Example: The multiplication process (MULT) in RISC involves multiple steps (LOAD, PROD, STORE), while CISC can perform the same task with a single command.

Insight: The programming model for RISC often increases the number of instructions required, but it optimizes the execution time for each instruction. This reduces the cycles per instruction (CPI), balancing overall performance.

Architecture and Programming The primary difference between RISC and CISC lies in how instructions are handled. RISC systems require that tasks be broken down into several smaller, simpler instructions, whereas CISC processors can perform complex tasks with fewer instructions.

RISC Characteristics:

Instructions executed in one clock cycle. Emphasizes pipelining to allow the processor to work on several instructions simultaneously. Large number of registers to reduce memory access time.

RISC Processors RISC (Reduced Instruction Set Computer) processors are designed to execute a smaller set of simple instructions. Historically, until the 1980s, CPUs were developed to handle complex instruction sets, known as CISC (Complex Instruction Set Computer) processors. RISC reversed this trend by simplifying instructions, which allows for faster execution and reduced hardware complexity.

Key Highlights:

Simplified Instructions: RISC processors handle fewer, simpler instructions than CISC, making execution faster. Lower Transistor Count: Fewer transistors in RISC chips make them cheaper and easier to design.

Insight: While simpler instructions often mean more lines of code for complex tasks, RISC systems are optimized for speed through streamlined execution.