this seems inconsistent with her character

inconsistent voice here

this is funny but it takes away from the tension in the scene

how would she know this?

you could make this sentence sharper to mirror the feeling of the paper cut

this is a great way of showing us her internal monologue without taking us out of the usual style

this is funny but keep in mind who is narrating

where has this come from?

'she tried, unsuccessfully, to cut the image of her mother's new face out of her mind. taking a deep breath and adopting an expression of composure, she opened the door.'

too long

i see what you're trying to do but this doesn't give us a deeper image as much as just hammering home the same thing over and over

one or the other, you don't need both

too long

Jamaica became independent in 1962

Reggae came to be during a time of subjugation to Britain?

Emerged in Jamaica

Mainstream perception of Reggae Music

eLife Assessment

This manuscript describes an important study of a new lipid-mediated regulation mechanism of adenylyl cyclases. The biochemical evidence provided is convincing and will trigger more research in this mechanism. This manuscript will be of interest to all scientists working on lipid regulation and adenylyl cyclases.

Reviewer #1 (Public review):

Summary:

The authors show that the Gαs-stimulated activity of human membrane adenylyl cyclases (mAC) can be enhanced or inhibited by certain unsaturated fatty acids (FA) in an isoform-specific fashion. Thus, with IC50s in the 10-20 micromolar range, oleic acid affects 3-fold stimulation of membrane-preparations of mAC isoform 3 (mAC3) but it does not act on mAC5. Enhanced Gαs-stimulated activities of isoforms 2, 7, and 9, while mAC1 was slightly attenuated, but isoforms 4, 5, 6, and 8 were unaffected. Certain other unsaturated octadecanoic FAs act similarly. FA effects were not observed in AC catalytic domain constructs in which TM domains are not present. Oleic acid also enhances the AC activity of isoproterenol-stimulated HEK293 cells stably transfected with mAC3, although with lower efficacy but much higher potency. Gαs-stimulated mAC1 and 4 cyclase activity were significantly attenuated in the 20-40 micromolar by arachidonic acid, with similar effects in transfected HEK cells, again with higher potency but lower efficacy. While activity mAC5 was not affected by unsaturated FAs, neutral anandamide attenuated Gαs-stimulation of mAC5 and 6 by about 50%. In HEK cells, inhibition by anandamide is low in potency and efficacy. To demonstrate isoform specificity, the authors were able to show that membrane preparations of a domain-swapped AC bearing the catalytic domains of mAC3 and the TM regions of mAC5 are unaffected by oleic acid but inhibited by anandamide. To verify in vivo activity, in mouse brain cortical membranes 20 μM oleic acid enhanced Gαs-stimulated cAMP formation 1.5-fold with an EC50 in the low micromolar range.

Strengths:

(1) A convincing demonstration that certain unsaturated FAs are capable of regulating membrane adenylyl cyclases in an isoform-specific manner, and the demonstration that these act at the AC transmembrane domains.

(2) Confirmation of activity in HEK293 cell models and towards endogenous AC activity in mouse cortical membranes.

(3) Opens up a new direction of research to investigate the physiological significance of FA regulation of mACs and investigate their mechanisms as tonic or regulated enhancers or inhibitors of catalytic activity.

(4) Suggests a novel scheme for the classification of mAC isoforms.

Comments on revised version:

The issues I raised have largely been addressed. A minor concern relates to the legend for Figure 2C, where, according to the author's rebuttal, the vertical axis is "The ratio would be (Gsα + oleic acid stimulation) / (Gsα stimulation)" Otherwise, my general evaluation of the importance of the manuscript stands as stated in my initial review, namely, that the manuscript presents data and results that add a new dimension to existing paradigms for AC regulation, and will prompt future research into the role of physiological lipids in isoform-specific activation or inhibition of AC in tissues.

Reviewer #3 (Public review):

Summary:

Landau et al. have submitted a manuscript describing for the first time that mammalian adenylyl cyclases can serve as membrane receptors. They have also identified the respective endogenouse ligands which act via AC membrane linkers to modify and control Gs-stimulated AC activity either towards enhancement or inhibition of ACs which is family and ligand-specific. Overall, they have used classical assays such as adenylyl cyclase and cAMP accumulation assays combined with molecular cloning and mutagenesis to provide exceptionally strong biochemical evidence for the mechanism of the involved pathway regulation.

Strengths:

The authors have gone the whole long classical way from having a hypothesis that ACs could be receptors to a series of MS studies aimed at ligand indentification, to functional studies of how these candidate substances affect the activity of various AC families in intact cells. They have used a large array of techniques with a paper having clear conceptual story and several strong lines of evidence.

Comments on revised version:

In general, the authors have addressed my comments satisfactorily apart from the suggestion to use a lower ISO concentration in their assay or at least to discuss this issue, cite relevant literature etc. Pending this small amendment I would to fine to proceed.

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

Summary:

The authors show that the Gαs-stimulated activity of human membrane adenylyl cyclases (mAC) can be enhanced or inhibited by certain unsaturated fatty acids (FA) in an isoform-specific fashion. Thus, with IC50s in the 10-20 micromolar range, oleic acid affects 3-fold stimulation of membrane-preparations of mAC isoform 3 (mAC3) but it does not act on mAC5. Enhanced Gαs-stimulated activities of isoforms 2, 7, and 9, while mAC1 was slightly attenuated, but isoforms 4, 5, 6, and 8 were unaffected. Certain other unsaturated octadecanoic FAs act similarly. FA effects were not observed in AC catalytic domain constructs in which TM domains are not present. Oleic acid also enhances the AC activity of isoproterenol-stimulated HEK293 cells stably transfected with mAC3, although with lower efficacy but much higher potency. Gαs-stimulated mAC1 and 4 cyclase activity were significantly attenuated in the 20-40 micromolar by arachidonic acid, with similar effects in transfected HEK cells, again with higher potency but lower efficacy. While activity mAC5 was not affected by unsaturated FAs, neutral anandamide attenuated Gαs-stimulation of mAC5 and 6 by about 50%. In HEK cells, inhibition by anandamide is low in potency and efficacy. To demonstrate isoform specificity, the authors were able to show that membrane preparations of a domain-swapped AC bearing the catalytic domains of mAC3 and the TM regions of mAC5 are unaffected by oleic acid but inhibited by anandamide. To verify in vivo activity, in mouse brain cortical membranes 20 μM oleic acid enhanced Gαs-stimulated cAMP formation 1.5-fold with an EC50 in the low micromolar range.

Strengths:

(1) A convincing demonstration that certain unsaturated FAs are capable of regulating membrane adenylyl cyclases in an isoform-specific manner, and the demonstration that these act at the AC transmembrane domains.

(2) Confirmation of activity in HEK293 cell models and towards endogenous AC activity in mouse cortical membranes.

(3) Opens up a new direction of research to investigate the physiological significance of FA regulation of mACs and investigate their mechanisms as tonic or regulated enhancers or inhibitors of catalytic activity.

(4) Suggests a novel scheme for the classification of mAC isoforms.

Weaknesses:

(1) Important methodological details regarding the treatment of mAC membrane preps with fatty acids are missing.

We will address this issue in more detail.

(2) It is not evident that fatty acid regulators can be considered as "signaling molecules" since it is not clear (at least to this reviewer) how concentrations of free fatty acids in plasma or endocytic membranes are hormonally or otherwise regulated.

Although this question is not the subject of this ms., we will address this question in more detail in the discussion of the revision.

Reviewer #2 (Public review):

Summary:

The authors extend their earlier findings with bacterial adenylyl cyclases to mammalian enzymes. They show that certain aliphatic lipids activate adenylyl cyclases in the absence of stimulatory G proteins and that lipids can modulate activation by G proteins. Adding lipids to cells expressing specific isoforms of adenylyl cyclases could regulate cAMP production, suggesting that adenylyl cyclases could serve as 'receptors'.

Strengths:

This is the first report of lipids regulating mammalian adenylyl cyclases directly. The evidence is based on biochemical assays with purified proteins, or in cells expressing specific isoforms of adenylyl cyclases.

Weaknesses:

It is not clear if the concentrations of lipids used in assays are physiologically relevant. Nor is there evidence to show that the specific lipids that activate or inhibit adenylyl cyclases are present at the concentrations required in cell membranes. Nor is there any evidence to indicate that this method of regulation is seen in cells under relevant stimuli.

Although this question is not the subject of this ms., we will address this question in more detail in the discussion of the revision.

Reviewer #3 (Public review):

Summary:

Landau et al. have submitted a manuscript describing for the first time that mammalian adenylyl cyclases can serve as membrane receptors. They have also identified the respective endogenouse ligands which act via AC membrane linkers to modify and control Gs-stimulated AC activity either towards enhancement or inhibition of ACs which is family and ligand-specific. Overall, they have used classical assays such as adenylyl cyclase and cAMP accumulation assays combined with molecular cloning and mutagenesis to provide exceptionally strong biochemical evidence for the mechanism of the involved pathway regulation.

Strengths:

The authors have gone the whole long classical way from having a hypothesis that ACs could be receptors to a series of MS studies aimed at ligand indentification, to functional studies of how these candidate substances affect the activity of various AC families in intact cells. They have used a large array of techniques with a paper having clear conceptual story and several strong lines of evidence.

Weaknesses:

(1) At the beginning of the results section, the authors say "We have expected lipids as ligands". It is not quite clear why these could not have been other substances. It is because they were expected to bind in the lipophilic membrane anchors? Various lipophilic and hydrophilic ligands are known for GPCR which also have transmembrane domains. Maybe 1-2 additional sentences could be helpful here.

Will be done as suggested.

(2) In stably transfected HEK cells expressing mAC3 or mAC5, they have used only one dose of isoproterenol (2.5 uM) for submaximal AC activation. The reference 28 provided here (PMID: 33208818) did not specifically look at Iso and endogenous beta2 adrenergic receptors expressed in HEK cells. As far as I remember from the old pharmacological literature, this concentration is indeed submaximal in receptor binding assays but regarding AC activity and cAMP generation (which happen after signal amplification with a so-called receptor reserve), lower Iso amounts would be submaximal. When we measure cAMP, these are rather 10 to 100 nM but no more than 1 uM at which concentration response dependencies usually saturate. Have the authors tried lower Iso concentrations to prestimulate intracellular cAMP formation? I am asking this because, with lower Iso prestimulation, the subsequent stimulatory effects of AC ligands could be even greater.

The best way to address this issue is to establish a concentration-response curve for Iso-stimulated cAMP formation using the permanently transfected cells. We note that in the past isoproterenol concentrations used in biochemical or electrophysiological experiments differed substantially.

(3) The authors refer to HEK cell models as "in vivo". I agree that these are intact cells and an important model to start with. It would be very nice to see the effects of the new ligands in other physiologically relevant types of cells, and how they modulate cAMP production under even more physiological conditions. Probably, this is a topic for follow-up studies.

The last sentence is correct.

Appraisal of whether the authors achieved their aims, and whether the results support their conclusions:

The authors have achieved their aims to a very high degree, their results do nicely support their conclusions. There is only one point (various classical GPCR concentrations, please see above) that would be beneficial to address.

Without any doubt, this is a groundbreaking study that will have profound implications in the field for the next years/decades. Since it is now clear that mammalian adenylyl cyclases are receptors for aliphatic fatty acids and anandamide, this will change our view on the whole signaling pathway and initiate many new studies looking at the biological function and pathophysiological implications of this mechanism. The manuscript is outstanding.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

It is not clear from the methods section how free FAs were applied to membrane preparations or HEK293 cells. Were FAs solubilized in organic solvents, or introduced as micelles?

The requested info is inserted into the M&M section

Could the authors comment on what is known about the concentration of oleic acid and other non-saturated fatty acids in plasma membranes relative to those required to produce allosteric effects on cyclase activity?

This info is now included in the last paragraph of the discussion.

It would be worthwhile to test the effect of FAs on basal (not Gαs-stimulated) activity of mACs.

This has been carried with mAC isoforms 2, 3, 7, and 9 in which oleic acid enhances Gsα-stimulated activity. Due to the low levels of basal activities interpretable data were not obtained.

Do triglycerides esterified with oleic acid stimulate mAC3 and other sensitive isoforms?

Experiments were done with triolein and 2-oleoyl-glycerol (the answer is no). The data are presented in Fig. 3 and in the appendix Fig.’s 8, 9, 14; structural formulas in appendix 2 Fig. 4 were updated.

Does the quantity plotted on the vertical axis of Figure 1, right panel represent "Fractional Stimulation by Oleic acid" rather than simply "Fold Stimulation"? Clearly, as shown in the two left-most panels, Gαs stimulates both mAC and mAC5. Rather it seems that the ratio (oleic acid stimulation) / (Gαs stimulation) remains constant. This observation supports the statement in the discussion that "We suppose that in mAC3 the equilibrium of two differing ground states favors a Gαs-unresponsive state and the effector oleic acid concentration-dependently shifts this equilibrium to a Gαs-responsive state". It could also be said that the effect of oleic acid is additive, and in constant proportion to that of Gαs.

This comment certainly is related to Fig. 2:

The ratio would be (Gsα + oleic acid stimulation) / (Gsα-stimulation), i.e., fractional stimulation by addition of oleic acid is identical to fold stimulation.

We have amended the legend to fig. 2C for clarification.

The last sentence is wrong because oleic acid alone does not stimulate.

It is stated on page 3, 2nd to last line that "The action of oleic acid on mAC3 was instantaneous...". Since the earliest time point is taken at 5 minutes, the claim that the action of the lipid is instantaneous cannot be made. Information about kinetics would be useful to have, since it is possible that the lipid must be released from a micelle and be incorporated into the AC membrane fraction before it is active.

The first point is 3 min.

We deleted the word “instantaneous” and added the correlation coefficients for both conditions in the legend to appendix 2; fig. 1 for clarification.

The data spread in Figure 4 and other figures showing similar data is significant, to the extent that the computed value for EC50 may not be of high precision. Authors should cite the correlation coefficient for the overall fit and uncertainty for the EC50 value (in addition to significances by t-test of individual data points).

This will not add valuable information. Pearsons correlation coefficients are only for linear relationships.

(cf. N.N. Kachouie, W. Deebani (2020) Association Factor for Identifying Linear and Nonlinear Correlations in Noisy Conditions. Entropy 22:440)

The "switch" between relatively low potency and high efficacy in membrane preps to high potency and low efficacy in cells is remarkable. Could this have a methodological basis or is it reflective of the mechanism by which FAs access mACs in membrane preps vs. cell membranes, or perhaps some biochemical transformation of the lipid in cells?

Honestly, we do not know.

The authors should note that there is some precedence for this work:

J Nakamura , N Okamura, S Usuki, S Bannai, Inhibition of adenylyl cyclase activity in brain membrane fractions by arachidonic acid and related unsaturated fatty acids. Arch Biochem Biophys. 2001 May 1;389(1):68-76. doi: 10.1006/abbi.2001.2315.

The effects of FA deficiencies on AC and related activities have been noted:

Alam SQ, Mannino SJ, Alam BS, McDonough K Effect of essential fatty acid deficiency on forskolin binding sites, adenylate cyclase, and cyclic AMP-dependent protein kinase activity, the levels of G proteins and ventricular function in rat heart. J Mol Cell Cardiol. 1995 Aug;27(8):1593-604. doi: 10.1016/s0022-2828(95)90491-3. PMID: 8523422

The latter publications are supportive of, and provide context to, the author's findings.

Both references are mentioned and cited.

Minor points:

The significance of the coloring scheme in Figure 5C bar graph should be stated in the legend.

Done.

In the introduction, it is stated that "The protein displayed two similar catalytic domains (C1 and C2) and two dissimilar hexahelical membrane anchors (TM1 and TM2)". In both cases, the respective domains can be said to be similar in overall fold, but - certainly in the case of the catalytic domains - different in amino acid sequence in functionally important regions of the domain.

Done: Changed wording.

The statement in the introduction that "The domain architecture, TM1-C1-TM2-C2, clearly indicated a pseudoheterodimeric protein composed of two concatenated bacterial precursor proteins" The authors refer to the fact that mammalian enzymes are pseudo heterodimers whereas bacterial type III cyclases are dimers of identical subunits.

Done.

Reviewer #2 (Recommendations for the authors):

The title need not state that a 'new class of receptors' has been identified. There is no direct evidence that the lipids bind to the enzymes, and the affinities can only be surmised from the EC50 graphs. To call a protein a receptor requires evidence to show that the binding is specific by showing that binding can be inhibited by a large excess of 'unlabelled' ligand. This could have been done by procuring labelled lipids for experimental verification.

As is well known, lipids easily bind to proteins. In this study no purified proteins were used. Therefore, binding assays most likely would result in unreliable data.

The paper would have benefitted from showing sequence alignments in the TM domains of the ACs discussed in the paper. Further, a phylogenetic tree of mammalian ACs would also reveal which enzymes from other species may be regulated similarly to those described in the paper. This would be important for researchers who use other model organisms to study cAMP signalling.

Such data are in multiple papers accessible in the literature. Where deemed appropriate we inserted references.

Figures 1A and 1B show data from only two experiments. A third experiment would have been useful in order to show the statistical significance of the data.

At this stage more experiments would not have affected further experimental plans.

Statements made in the text (for example, the last paragraph on page 6) state only the mean value and not the SDs. This would have been important to include even if the data is shown in the appendix. The same is true in the Legend of Figure 2. Why have the authors decided to use SEM and not SDs?

The reason is specified in M&M.

Concentrations of lipids used in biochemical assays are in the micromolar range. This suggests that we have moderate affinity binding, more in the range of an enzyme for a substrate rather than a receptor-ligand interaction.

We happen to disagree. Clearly, the differential activities, enhancing or attenuating Gsα-stimulated mAC activities is most plausibly explained by mAC receptor properties. mACs have enzyme activities using fatty acids as substrates.

The authors add lipids to cells and show changes in cAMP levels in their presence and absence. They also discuss how these extracellular lipids could be produced. Do you think this is necessary in vivo, though? Could the lipids present in membranes naturally act as regulators? Do specific lipid concentrations differ in different cell types, suggesting tissue-specific regulation of these mammalian Acs?

These are things that could be discussed in the manuscript.

The last paragraph of the discussion deals with these questions.

This guy learns music creation efficiently, by learning the theory first and really analyzing worked examples (the masters). Positively surprises me. I rarely come across a non-learning expert who intuitively uses proper processes for skill acquisition.

Levels of understanding genres: - 0) No understanding Like the song, never heard anything like it before, but no idea about anything. - 1) Basic Understanding Knowing a bit about the name of the genre and subgenres, but you can be wrong. - 2) Immersion Really dive into subgenres and flavors of the main genre... Also a bit of history about the genre. Research. - 3) Structure Breaking down the structure of the tracks in the genre. For example through DAW. Basically first-principles thinking.

To level 1: Song Analyzer tools (for example musicstax or AI). The author recommends everynoise.com too to gain a basic understanding of genres.

To level 2: Find similar songs and artists for your playlists with that genre. Perhaps playlists. Important to understand the origin of the genre.

for - reply to - @Michael - annotation for annotation sake

reply to- @Michael - annotation for annotation sake - I think of annotation in the broadest possible sense - as social learning - Annotating is "making a note" and that is effectively noticing how I respond to the idea of another person. - If I am digesting ideas and suddenly a particular idea resonates with an idea in my salience landscape, my attention will be drawn to it. - That is, there is a salient reaction of my own consciousness with the ideas of another consciousness - If I react strongly to an idea, with my own ideas and feelings, then that moment of social learning is worth noting and recording, and hence annotating. - There's absolutely no point in annotating unless it is relevant to you - For me, it is the most powerful way to keep track of the evolution of my own intertwingled individual / collective learning journey - If that becomes a modus operandi for your annotation, then by definition they are all relevant, and not done simply for some external, dogmatic reason of conventionality

Yes, this would mean we are ERGODIC and not just a 501c3 and not just an FSC

YEs Kauffman will fund a company that is NOT a not for profit if its terms of assocaition say it has a charitable function. 501c3 for kauffman is all about TAX ANd yes, we can set up an LLC with an FSC bye laws just as easily

Paul will createa 501c3 as a RESEARCH instrument to apply for Kauffman;s research grant My hope is that IAN's 501c3 will apply for the PROJECT Grant that holds the eco-system of 501c3 fair shares commons

So nto everyone has a telegram account so that is even a splinter group even though it was asked for by some members of the team So we still have an email list and a telegram grop and they are not synchrinised

The meeting with Gyuri was a request from him to meet PAUL so that we could ground on the threads for the RESEARCH application I asked Donan to attend but I did not not ask others to attend as I do not want to place demands on people's time and space This is why I recorded it and sent it around If people then ANNOTATE the call the way I am doing here, Gyuri has data that he can use to develop technology based on what he is himself involved in and not just as a service provider

This is where SROI (Social Return on Investment) DATA will help us bridge the gap In a recent call in Ireland a football team scored a 17:1 SROI score for their impact on community

Author response:

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

We thank you for sending our manuscript for the second round of review.  We are encouraged by the comments from reviewer #2 that our supplementary work on naïve T cells and antibody blockade work satisfied their previous concerns and is important for our work.

The Editors raised concerns that we have shared preliminary data on Nrn1 and AMPAR double knockout mice.  We apologize for our enthusiasm for these studies.  Because of the publication model by eLife, we shared that data not because we needed to persuade the reviewer for publication purposes but rather to agree with the reviewer that the molecular target of Nrn1 is important, and we are progressing in understanding this subject.

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

To Reviewer #1:

Thank you for your thorough review and comments on our work, which you described as “the role of neuritin in T cell biology studied here is new and interesting.”.  We have summarized your comments into two categories: biology and investigation approach, experimental rigor, and data presentation.

Biology and Investigation approach comments:

(1) Questions regarding the T cell anergy model:

Major point “(4) Figure 1E-H. The authors assume that this immunization protocol induces anergic cells, but they provide no experimental evidence for this. It would be useful to show that T cells are indeed anergic in this model, especially those that are OVA-specific. The lack of IL-2 production by Cltr cells could be explained by the presence of fewer OVA-specific cells, rather than by an anergic status.”

T cell anergy is a well-established concept first described by Schwartz’s group. It refers to the hyporesponsive T cell functional state in antigen-experienced CD4 T cells (Chappert and Schwartz, 2010; Fathman and Lineberry, 2007; Jenkins and Schwartz, 1987; Quill and Schwartz, 1987).  Anergic T cells are characterized by their inability to expand and to produce IL2 upon subsequent antigen re-challenge. In this paper, we have borrowed the existing in vivo T cell anergy induction model used by Mueller’s group for T cell anergy induction (Vanasek et al., 2006).  Specifically, Thy1.1+ Ctrl or Nrn1-/- TCR transgenic OTII cells were co-transferred with the congenically marked Thy1.2+ WT polyclonal Treg cells into TCR-/- mice.  After anergy induction, the congenically marked TCR transgenic T cells were recovered by sorting based on Thy1.1+ congenic marker, and subsequently re-stimulation ex vivo with OVA323-339 peptide. We evaluated the T cell anergic state based on OTII cell expansion in vivo and IL2 production upon OVA323-339 restimulation ex vivo.  

“The authors assume that this immunization protocol induces anergic cells, but they provide no experimental evidence for this.”

Because the anergy model by Mueller's group is well established (Vanasek et al., 2006), we did not feel that additional effort was required to validate this model as the reviewer suggested. Moreover, the limited IL2 production among the control cells upon restimulation confirms the validity of this model.

“The lack of IL-2 production by Cltr cells could be explained by the presence of fewer OVAspecific cells, rather than by an anergic status”.

Cells from Ctrl and Nrn1-/- mice on a homogeneous TCR transgenic (OTII) background were used in these experiments. The possibility that substantial variability of TCR expression or different expression levels of the transgenic TCR could have impacted IL2 production rather than anergy induction is unlikely.

Overall, we used this in vivo anergy model to evaluate the Nrn1-/- T cell functional state in comparison to Ctrl cells under the anergy induction condition following the evaluation of Nrn1 expression, particularly in anergic T cells.  Through studies using this anergy model, we observed a significant change in Treg induction among OTII cells. We decided to pursue the role of Nrn1 in Treg cell development and function rather than the biology of T cell anergy as evidenced by subsequent experiments.

Minor points “(6) On which markers are anergic cells sorted for RNAseq analysis?”

Cells were sorted out based on their congenic marker marking Ctrl or Nrn1-/- OTII cells transferred into the host mice.  We did not specifically isolate anergic cells for sequencing.

(2) Question regarding the validity of iTreg differentiation model.

Major point: “(5) Figure 2A-C and Figure 3. The use of iTregs to try to understand what is happening in vivo is problematic. iTregs are cells that have probably no equivalent in vivo, and so may have no physiological relevance. In any case, they are different from pTreg cells generated in vivo. Working with pTreg may be challenging, that is why I would suggest generating data with purified nTreg. Moreover, it was shown in the article of Gonzalez-Figueroa 2021 that Nrn1-/- nTreg retained a normal suppressive function, which would not be what is concluded by the authors of this manuscript. Moreover, we do not even know what the % of Foxp3 cells is in the iTreg used (after differentiation and 20h of re-stimulation) and whether this % is the same between Ctlr and Nrn1 KO cells.”.

We thank Reviewer #1 for their feedback. While it is true that iTregs made in vitro and in vivo generated pTregs display several distinctions (e. g., differences in Foxp3 expression stability, for example), we strongly disagree with this statement by Revieweer#1 “The use of iTregs to try to understand what is happening in vivo is problematic. iTregs are cells that have probably no equivalent in vivo, and so may have no physiological relevance.”  The induced Treg cell (iTreg) model was established over 20 years ago (Chen et al., 2003; Zheng et al., 2002), and the model is widely adopted with over 2000 citations. Further, it has been instrumental in understanding different aspects of regulatory T cell biology (Hurrell et al., 2022; John et al., 2022; Schmitt and Williams, 2013; Sugiura et al., 2022).   

Because we have observed reduced pTreg generation in vivo, we choose to use the in vitro iTreg model system to understand the mechanistic changes involved in Treg cell differentiation and function, specifically, neuritin’s role in this process. We have made no claim that iTreg cell biology is identical to pTreg generated in vivo or nTreg cells. However, the iTreg culture system has proved to be a good in vitro system for deciphering molecular events involved in complex processes. As such, it remains a commonly used approach by many research groups in the Treg cell field (Hurrell et al., 2022; John et al., 2022; Sugiura et al., 2022). Moreover, applying the iTreg in vitro culture system has been instrumental in helping us identify the cell electrical state change in Nrn1-/- CD4 cells and revealed the biological link between Nrn1 and the ionotropic AMPA receptor (AMPAR), which we will discuss in the subsequent discussion. It is technically challenging to use nTreg cells for T cell electrical state studies due to their heterogeneous nature from development in an in vivo environment and the effect of manipulation during the nTreg cell isolation process, which can both affect the T cell electrical state.   

“Moreover, it was shown in the article of Gonzalez-Figueroa 2021 that Nrn1-/- nTreg retained a normal suppressive function, which would not be what is concluded by the authors of this manuscript.” 

We have also carried out nTreg studies in vitro in addition to iTreg cells. Similar to Gonzalez-Figueroa et al.'s findings, we did not observe differences in suppression function between Nrn1-/- and WT nTreg using the in vitro suppression assay. However, Nrn1-/- nTreg cells revealed reduced suppression function in vivo (Fig. 2D-L). In fact, Gonzalez-Figueroa et al. observed reduced plasma cell formation after OVA immunization in Treg-specific Nrn1-/- mice, implicating reduced suppression from Nrn1-/- follicular regulatory T (Tfr) cells. Thus, our observation of the reduced suppression function of Nrn1-/- nTreg toward effector T cell expansion, as presented in Fig. 2D-L, does not contradict the results from Gonzalez-Figueroa et al. Rather, the conclusions of these two studies agree that Nrn1 can play important roles in immune suppression observable in vivo that are not captured readily by the in vitro suppression assay.

“Moreover, we do not even know what the % of Foxp3 cells is in the iTreg used (after differentiation and 20h of re-stimulation) and whether this % is the same between Ctlr and Nrn1 KO cells.”

We have stated in the manuscript on page 7 line 208 that “Similar proportions of Foxp3+ cells were observed in Nrn1-/- and Ctrl cells under the iTreg culture condition, suggesting that Nrn1 deficiency does not significantly impact Foxp3+ cell differentiation”. In the revised manuscript, we will include the data on the proportion of Foxp3+ cells before iTreg restimulation.

(3) Confirmation of transcriptomic data regarding amino acids or electrolytes transport change

Minor point“(3) Would not it be possible to perform experiments showing the ability of cells to transport amino acids or electrolytes across the plasma membrane? This would be a more interesting demonstration than transcriptomic data.”

We appreciate Review# 1’s suggestion regarding “perform experiments showing the ability of cells to transport amino acids or electrolytes across the plasma membrane”.  We have indeed already performed such experiments corroborating the transcriptomics data on differential amino acid and nutrient transporter expression. Specifically, we loaded either iTreg or Th0 cells with membrane potential (MP) dye and measured MP level change after adding the complete set of amino acids (complete AA).  Upon entry, the charge carried by AAs may transiently affect cell membrane potential. Different AA transporter expression patterns may show different MP change patterns upon AA entry, as we showed in Author response image 1. We observed reduced MP change in Nrn1-/- iTreg compared to the Ctrl, whereas in the context of Th0 cells, Nrn1-/- showed enhanced MP change than the Ctrl. We can certainly include these data in the revised manuscript.

Author response image 1.

Membrane potential change induced by amino acids entry. a. Nrn1-/- or WT iTreg cells loaded with MP dye and MP change was measured upon the addition of a complete set of AAs. b. Nrn1-/- or WT Th0 cells loaded with MP dye and MP change was measured upon the addition of a complete set of AAs.

(4) EAE experiment data assessment

Minor point ”(5) Figure 5F. How are cells re-stimulated? If polyclonal stimulation is used, the experiment is not interesting because the analysis is done with lymph node cells. This analysis should either be performed with cells from the CNS or with MOG restimulation with lymph node cells.”

In the EAE study, the Nrn1-/- mice exhibit similar disease onset but a protracted non-resolving disease phenotype compared to the WT control mice.  Several reasons may contribute to this phenotype: 1. Enhanced T effector cell infiltration/persistence in the central nervous system (CNS); 2. Reduced Treg cell-mediated suppression to the T effector cells in the CNS; 3. Protracted non-resolving inflammation at the immunization site has the potential to continue sending T effector cells into CNS, contributing to persistent inflammation. Based on this reasoning, we examined the infiltrating T effector cell number and Treg cell proportion in the CNS.  We also restimulated cells from draining lymph nodes close to the inflammation site, looking for evidence of persistent inflammation.  When mice were harvested around day 16 after immunization, the inflammation at the local draining lymph node should be at the contraction stage.  We stimulated cells with PMA and ionomycin intended to observe all potential T effector cells involved in the draining lymph node rather than only MOG antigen-specific cells.  We disagree with Reviewer #1’s assumption that “This analysis should either be performed with cells from the CNS or with MOG restimulation with lymph node cells.”. We think the experimental approach we have taken has been appropriately tailored to the biological questions we intended to answer.

Experimental rigor and data presentation.

(1) data labeling and additional supporting data

Major points

(2) The authors use Nrn1+/+ and Nrn1+/- cells indiscriminately as control cells on the basis of similar biology between Nrn1+/+ and Nrn1+/- cells at homeostasis. However, it is quite possible that the Nrn1+/- cells have a phenotype in situations of in vitro activation or in vivo inflammation (cancer, EAE). It would be important to discriminate Nrn1+/- and Nrn1+/+ cells in the data or to show that both cell types have the same phenotype in these conditions too.

(3) Figure 1A-D. Since the authors are using the Nrp1 KO mice, it would be important to confirm the specificity of the anti-Nrn1 mAb by FACS. Once verified, it would be important to add FACS results with this mAb in Figures 1A-C to have single-cell and quantitative data as well.

Minor points  

(1) Line 119, 120 of the text. It is said that one of the most up-regulated genes in anergic cells is Nrn1 but the data is not shown.

(2) For all figures showing %, the titles of the Y axes are written in an odd way. For example, it is written "Foxp3% CD4". It would be more conventional and clearer to write "% Foxp3+ / CD4+" or "% Foxp3+ among CD4+".

(4) For certain staining (Figure 3E, H) it would be important to show the raw data, in addition to MFI or % values.

We can adapt the labeling and provide additional data, including Nrn1 staining on Treg cells and flow graphs for pmTOR and pS6 staining (Fig. 3H), as requested by Reviewer #1.

(2) Experimental rigor:

General comments:

“However, it is disappointing that reading this manuscript leaves an impression of incomplete work done too quickly.”

We were discouraged to receive the comment, “this manuscript leaves an impression of incomplete work done too quickly.” Our study of this novel molecule began without any existing biological tools such as antibodies, knockout mice, etc.  Over the past several years, we have established our own antibodies for Nrn1 detection, obtained and characterized Nrn1 knockout mice, and utilized multiple approaches to identify the molecular mechanism of Nrn1 function. Through the use of the in vitro iTreg system described in this manuscript, we identified the association of Nrn1 deficiency with cell electrical state change, potentially connected to AMPAR function. We have further corroborated our findings by generating Nrn1 and AMPAR T cell specific double knockout mice and confirmed that T cell specific AMPAR deletion could abrogate the phenotype caused by the Nrn1 deficiency (see Support Figure 2).  We did not include the double knockout data in the current manuscript because AMPAR function has not yet been studied thoroughly in T cell biology, and we feel this topic warrants examination in its own right.  However, the unpublished data support the finding that Nrn1 modulates the T cell electrical state and, consequently, metabolism, ultimately influencing tolerance and immunity.  In its current form, the manuscript represents the first characterization of the novel molecule Nrn1 in anergic cells, Tregs, and effector T cells. While this work has led to several exciting additional questions, we disagree that the novel characterization we have presented Is incomplete. We feel that our present data set, which squarely highlights Nrn1’s role as an important immune regulator while shedding unprecedented light on the molecular events involved, will be of considerable interest to a broad field of researchers.

“Multiple models have been used, but none has been studied thoroughly enough to provide really conclusive and unambiguous data. For example, 5 different models were used to study T cells in vivo. It would have been preferable to use fewer, but to go further in the study of mechanisms.”

We have indeed used multiple in vivo models to reveal Nrn1's function in Treg differentiation, Treg suppression function, T effector cell differentiation and function, and the overall impact on autoimmune disease. Because the impact of ion channel function is often context-dependent, we examined the biological outcome of Nrn1 deficiency in several in vivo contexts.  We would appreciate it if Reviewer#1 would provide a specific example, given the Nrn1 phenotype, of how to proceed deeper to investigate the electrical change in the in vivo models.

“Major points

(1) A real weakness of this work is the fact that in most of the results shown, there are few biological replicates with differences that are often small between Ctrl and Nrn1 -/-. The systematic use of student's t-test may lead to thinking that the differences are significant, which is often misleading given the small number of samples, which makes it impossible to know whether the distributions are Gaussian and whether a parametric test can be used. RNAseq bulk data are based on biological duplicates, which is open to criticism.”

We respectfully disagree with Reviewer #1 on the question of statistical power and significance to our work. We have used 5-8 mice/group for each in vivo model and 3-4 technical replicates for the in vitro studies, with a minimum of 2-3 replicate experiments. These group sizes and replication numbers are in line with those seen in high-impact publications. While some differences between Ctrl and Nrn1-/- appear small, they have significant biological consequences, as evidenced by the various Nrn1-/- in vivo phenotypes. Furthermore, we believe we have subjected our data to the appropriate statistical tests to ensure rigorous analysis and representation of our findings.

To Reviewer #2.

We thank Reviewer #2 for the careful review of the manuscript. We especially appreciate the comments that “The characterizations of T cell Nrn1 expression both in vitro and in vivo are comprehensive and convincing. The in vivo functional studies of anergy development, Treg suppression, and EAE development are also well done to strengthen the notion that Nrn1 is an important regulator of CD4 responsiveness.”

“The major weakness of this study stems from a lack of a clear molecular mechanism involving Nrn1. “  

We fully understand this comment from Reviewer #2. The main mechanism we identified contributing to the functional defect of Nrn1-/- T cells involves novel effects on the electric and metabolic state of the cells. Although we referenced neuronal studies that indicate Nrn1 is the auxiliary protein for the ionotropic AMPA-type glutamate receptor (AMPAR) and may affect AMPAR function, we did not provide any evidence in this manuscript as the topic requires further in-depth study.   

For the benefit of this discussion, we include our preliminary Nrn1 and AMPAR double knockout data (Author response image 2), which indicates that abrogating AMPAR expression can compensate for the defect caused by Nrn1 deficiency in vitro and in vivo. This preliminary data supports the notion that Nrn1 modulates AMPAR function, which causes changes in T cell electric and metabolic state, influencing T cell differentiation and function.  

Author response image 2.

Deletion of AMPAR expression in T cells compensates for the defect caused by

Nrn1 deficiency. Nrn1-/- mice were crossed with T cell-specific AMPAR knockout mice (AMPARfl/flCD4Cre+) mice. The following mice were generated and used in the experiment: T cell specific AMPAR-knockout and Nrn1 knockout mice (AKONKO), Nrn1 knockout mice (AWTNKO), Ctrl mice (AWTNWT). a. Deletion of AMPAR compensates for the iTreg cell defect observed in Nrn1-/- CD4 cells. iTreg live cell proportion, cell number, and Ki67 expression among Foxp3+ cells 3 days after aCD3 restimulation. b. Deletion of AMPAR in T cells abrogates the enhanced autoimmune response in Nrn1-/- Mouse in the EAE disease model. Mouse relative weight change and disease score progression after EAE disease induction.  

Ion channels can influence cell metabolism through multiple means (Vaeth and Feske, 2018; Wang et al., 2020). First, ion channels are involved in maintaining cell resting membrane potential. This electrical potential difference across the cell membrane is essential for various cellular processes, including metabolism (Abdul Kadir et al., 2018; Blackiston et al., 2009; Nagy et al., 2018; Yu et al., 2022). Second, ion channels facilitate the movement of ions across cell membranes. These ions are essential for various metabolic processes. For example, ions like calcium (Ca2+), potassium (K+), and sodium (Na+) play crucial roles in signaling pathways that regulate metabolism (Kahlfuss et al., 2020). Third, ion channel activity can influence cellular energy balance due to ATP consumption associated with ion transport to maintain ion balances (Erecińska and Dagani, 1990; Gerkau et al., 2019). This, in turn, can impact processes like ATP production, which is central to cellular metabolism. Thus, ion channel expression and function determine the cell’s bioelectric state and contribute to cell metabolism (Levin, 2021).

Because the AMPAR function has not been thoroughly studied using a genetic approach in T cells, we do not intend to include the double knockout data in this manuscript before fully characterizing the T cell-specific AMPAR knockout mice.  

“Although the biochemical and informatics studies are well-performed, it is my opinion that these results are inconclusive in part due to the absence of key "naive" control groups. This limits my ability to understand the significance of these data.

Specifically, studies of the electrical and metabolic state of Nrn1-/- inducible Treg cells (iTregs) would benefit from similar data collected from wild-type and Nrn1-/- naive CD4 T cells.”

We appreciate the reviewer’s comments. This comment reflects two concerns in data interpretation:

(1) Are Nrn1-/- naïve T cells fundamentally different from WT cells? Does this fundamental difference contribute to the observed electrical and metabolic phenotype in iTreg or Th0 cells? This is a very good question we will perform the experiments as the reviewer suggested. While Nrn1 is expressed at a basal (low) level in naïve T cells, deletion of Nrn1 may cause changes in naïve T cell phenotype.   

(2) Is the Nrn1-/- phenotype caused by Nrn1 functional deficiency or due to the secondary effect of Nrn1 deletion, such as non-physiological cell membrane structure changes?

We have done the following experiment to address this concern.  We have cultured WT T cells in the presence of Nrn1 antibody and compared the outcome with Nrn1-/- iTreg cells (Figure 3-figure supplement 2D,E,F). WT iTreg cells under antibody blockade exhibited similar changes as Nrn1-/- iTreg cells, confirming the physiological relevance of the Nrn1-/- phenotype.

Manuscript Revision based on the Reviewer’s suggestions:

Reviewer #1:

Major points (3) Figure 1A-D. Since the authors are using the Nrp1 KO mice, it would be important to confirm the specificity of the anti-Nrn1 mAb by FACS. 

Following the suggestion by Reviewer#1, We have included the Nrn1 Ab staining on activated Nrn1-/- CD4 cells in Figure 1D. We have also added the staining of cell surface Nrn1 on Treg cells in Figure 1-figure supplement 1D.

Major point: (5) “Moreover, we do not even know what the % of Foxp3 cells is in the iTreg used (after differentiation and 20h of re-stimulation) and whether this % is the same between Ctlr and Nrn1 KO cells.”

In the revised manuscript, we have included the proportion of Foxp3+ cells among Nrn1-/- and ctrl iTreg cells developed under the iTreg culture condition in Figure 2A.

Minor points  

(2) For all figures showing %, the titles of the Y axes are written in an odd way. For example, it is written "Foxp3% CD4". It would be more conventional and clearer to write "% Foxp3+ / CD4+" or "% Foxp3+ among CD4+".

Following reviewer#1’s suggestion, we have changed the Y-axis label in all the relevant figures.

(3) Would not it be possible to perform experiments showing the ability of cells to transport amino acids or electrolytes across the plasma membrane? This would be a more interesting demonstration than transcriptomic data.”

We appreciate Review# 1’s suggestion regarding “perform experiments showing the ability of cells to transport amino acids or electrolytes across the plasma membrane”.  We have used AAinduced cellular MP changes to confirm differential AA transporter expression patterns and their impact on cellular MP levels.  The data are included in the revised manuscript in Figure 3H and Figure 4K.

(4) For certain staining (Figure 3E, H) it would be important to show the raw data, in addition to MFI or % values.

We appreciated Reviewer #1’s suggestion and have included the histogram staining data for Figure 3E. We have moved the original Figure 3H to the supplemental figure and included the histogram staining data in Figure 3-figure supplement 1C.  Similarly, we have included the histogram staining data in Figure 4-figure supplement 1C.

Reviewer#2:

“Although the biochemical and informatics studies are well-performed, it is my opinion that these results are inconclusive in part due to the absence of key "naive" control groups. This limits my ability to understand the significance of these data.

Specifically, studies of the electrical and metabolic state of Nrn1-/- inducible Treg cells (iTregs) would benefit from similar data collected from wild-type and Nrn1-/- naive CD4 T cells.”

We greatly appreciate Reviewer#2’s suggestion and have carried out experiments on naïve CD4 cells derived from Nrn1-/- and WT mice. We have compared membrane potential, AA-induced MP change between Nrn1-/- and WT naïve T cells, and the metabolic state of Nrn1-/- and WT naïve T cells by carrying out glucose stress tests and mitochondria stress tests using a seahorse assay.  Moreover, to investigate whether the phenotype revealed in Nrn1-/- CD4 cells was caused by a secondary effect of cell membrane structure change due to Nrn1 deletion, we carried out Nrn1 antibody blockade in WT CD4 cells and investigated the phenotypic change. These new results are included in Figure 3-figure supplement 2.

Reference:

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Blackiston, D.J., K.A. McLaughlin, and M. Levin. 2009. Bioelectric controls of cell proliferation: ion channels, membrane voltage and the cell cycle. Cell Cycle 8:3527-3536.

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Fathman, C.G., and N.B. Lineberry. 2007. Molecular mechanisms of CD4+ T-cell anergy. Nat Rev Immunol 7:599-609.

Gerkau, N.J., R. Lerchundi, J.S.E. Nelson, M. Lantermann, J. Meyer, J. Hirrlinger, and C.R. Rose. 2019. Relation between activity-induced intracellular sodium transients and ATP dynamics in mouse hippocampal neurons. The Journal of physiology 597:5687-5705.

Hurrell, B.P., D.G. Helou, E. Howard, J.D. Painter, P. Shafiei-Jahani, A.H. Sharpe, and O. Akbari. 2022. PD-L2 controls peripherally induced regulatory T cells by maintaining metabolic activity and Foxp3 stability. Nature communications 13:5118.

Jenkins, M.K., and R.H. Schwartz. 1987. Antigen presentation by chemically modified splenocytes induces antigen-specific T cell unresponsiveness in vitro and in vivo. The Journal of experimental medicine 165:302-319.

John, P., M.C. Pulanco, P.M. Galbo, Jr., Y. Wei, K.C. Ohaegbulam, D. Zheng, and X. Zang. 2022. The immune checkpoint B7x expands tumor-infiltrating Tregs and promotes resistance to anti-CTLA-4 therapy. Nature communications 13:2506.

Kahlfuss, S., U. Kaufmann, A.R. Concepcion, L. Noyer, D. Raphael, M. Vaeth, J. Yang, P. Pancholi, M. Maus, J. Muller, L. Kozhaya, A. Khodadadi-Jamayran, Z. Sun, P. Shaw, D. Unutmaz, P.B. Stathopulos, C. Feist, S.B. Cameron, S.E. Turvey, and S. Feske. 2020. STIM1-mediated calcium influx controls antifungal immunity and the metabolic function of nonpathogenic Th17 cells. EMBO molecular medicine 12:e11592.

Levin, M. 2021. Bioelectric signaling: Reprogrammable circuits underlying embryogenesis, regeneration, and cancer. Cell 184:1971-1989.

Nagy, E., G. Mocsar, V. Sebestyen, J. Volko, F. Papp, K. Toth, S. Damjanovich, G. Panyi, T.A. Waldmann, A. Bodnar, and G. Vamosi. 2018. Membrane Potential Distinctly Modulates Mobility and Signaling of IL-2 and IL-15 Receptors in T Cells. Biophys J 114:2473-2482.

Quill, H., and R.H. Schwartz. 1987. Stimulation of normal inducer T cell clones with antigen presented by purified Ia molecules in planar lipid membranes: specific induction of a long-lived state of proliferative nonresponsiveness. Journal of immunology (Baltimore, Md. : 1950) 138:3704-3712.

Schmitt, E.G., and C.B. Williams. 2013. Generation and function of induced regulatory T cells. Frontiers in immunology 4:152.

Sugiura, A., G. Andrejeva, K. Voss, D.R. Heintzman, X. Xu, M.Z. Madden, X. Ye, K.L. Beier, N.U. Chowdhury, M.M. Wolf, A.C. Young, D.L. Greenwood, A.E. Sewell, S.K. Shahi, S.N. Freedman, A.M. Cameron, P. Foerch, T. Bourne, J.C. Garcia-Canaveras, J. Karijolich, D.C. Newcomb, A.K. Mangalam, J.D. Rabinowitz, and J.C. Rathmell. 2022. MTHFD2 is a metabolic checkpoint controlling effector and regulatory T cell fate and function. Immunity 55:65-81.e69.

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eLife Assessment

The neurotrophic factor Neuritin can moderate T-cell tolerance and immunity through both regulatory T (Treg) and effector T cells, promoting Treg cell expansion and suppression while dampening effector T cells to mediate the inflammatory response. Neuritin expression influences the membrane potential, ion channels, and nutrient transporter expression patterns of CD4+ T cells, contributing to differential metabolic states in Treg and effector T cells. These findings are solid and important for understanding immune regulation involving Treg cells and effector T cells.

Reviewer #1 (Public review):

The manuscript by Yu et al seeks to investigate the role of neuritin (Nrn1), identified as a marker of anergic cells, in the biology of regulatory (Tregs) and conventional (Tconv) T cells. Although the role of Nrn1 expressed by Tregs has already been explored (Gonzalez-Figueroa 2021 cited in the manuscript), this manuscript shows original new data suggesting that this molecule would be important in promoting Treg function and inhibiting Tconv effector function by acting at the level of membrane potential and molecule transport across the plasma membrane. However, multiple models have been used, but none has been studied thoroughly enough to provide really conclusive and unambiguous data. For example, 5 different models were used to study T cells in vivo. It would have been preferable to use fewer, but to go further in the study of mechanisms. In the absence of more in-depth study, the conclusions drawn by the authors are often open to questions. Major points concern the fact that there are not enough biological replicates for most experiments and some critical controls and data are lacking. Also, the authors have used iTregs rather than nTregs for many experiments (see below). This is unfortunate because the role of neuritin in T cell biology studied here is new and interesting.

Major points (in the order in which they appear in the text).

(1) A real weakness of this work is the fact that in most of the results shown, there are few biological replicates with differences that are often small between Ctrl and Nrn1 -/-. The systematic use of student's t test may lead to think that the differences are significant, which is often misleading given the small number of samples, which makes it impossible to know whether the distributions are Gaussian and whether a parametric test can be used. RNAseq bulk data are based on biological duplicates, which is open to criticism.<br /> (2) The authors use Nrn1+/+ and Nrn1+/- cells indiscriminately as control cells on the basis of similar biology between Nrn1+/+ and Nrn1+/- cells at homeostasis. However, it is quite possible that the Nrn1+/- cells have a phenotype in situations of in vitro activation or in vivo inflammation (cancer, EAE). It would be important to discriminate Nrn1+/- and Nrn1+/+ cells in the data or to show that both cell types have the same phenotype in these conditions too.<br /> (3) Fig 1A-D. Since the authors are using the Nrp1 KO mice, it would be important to confirm the specificity of the anti-Nrn1 mAb by FACS. Once verified, it would be important to add FACS results with this mAb in Figs 1A-C to have single-cell and quantitative data as well.<br /> (4) Fig 1E-H. The authors assume that this immunization protocol induces anergic cells, but they provide no experimental evidence for this. It would be useful to show that T cells are indeed anergic in this model, especially those that are OVA-specific. The lack of IL-2 production by Cltr cells could be explained by the presence of fewer OVA-specific cells, rather than by an anergic status.<br /> (5) Fig 2A-C and Fig 3. The use of iTregs to try to understand what is happening in vivo is problematic. iTregs are cells that have probably no equivalent in vivo, and so may have no physiological relevance. In any case, they are different from pTreg cells generated in vivo. Working with pTreg may be challenging, that is why I would suggest to generate data with purified nTreg.<br /> (6) Fig 2D-L. The model is designed to study the role of Nrn1 in nTreg. However, the % of Foxp3+ among CD45.2 nTreg cells fell to 5-15% of CD4+ cells (Fig 2F). Since we do not know what is the % of Foxp3 among the injected cells, we do not know whether this very low % is due to very high Treg instability or to preferential expansion of contaminating Tconvs. It is possible that the % of Tconv contaminant is high since Treg were sorted using beads and not FACS on some experiments. As it is very likely that there are Tconv contaminants that would be Nrn1-/- in the group transferred with Nrn1-/- "nTreg", the higher tumor rejection could be due to an overactivation of Nrn1-/- Tconvs (rather than a defect in Nrn1-/- Treg function).

Reviewer #2 (Public review):

Summary:

This manuscript explores the role of Nrn1 in T cell tolerance. A previous study has demonstrated that Nrn1 is up-regulated in the Tfr fraction of Foxp3+ T regulatory cells. These authors now confirm expression of Nrn1 in iTregs as well as report here that Nrn1 is also greatly over-expressed in anergic CD4 T cells, and this is the stepping off point for this investigation.

Most remarkably, experiments show that anergy induction is defective when T cells cannot express Nrn1. Furthermore, differentiation to a Foxp3+ iTreg phenotype is inhibited in the absence of Nrn1, and the iTregs that do develop appear functionally defective. On the other hand, the differentiation and expansion of Teff cells appears to be enhanced following deletion of Nrn1. With such defects in anergy induction as well as dysregulated Treg and Teff cell survival and function, auto reactive effector T cell activation becomes unrestrained and Nrn1-/- mice are more susceptible to severe EAE development.

Strengths:

The characterizations of T cell Nrn1 expression both in vitro and in vivo are comprehensive and convincing. The author's use of both Nrn1-/- T cells as well as anti-Nrn1 neutralizing Ab to achieve similar results is a strength. The in vivo functional studies of anergy development, Treg suppression, and EAE development are also well performed and strengthen the notion that Nrn1 is an important regulator of CD4 responsiveness.

Weaknesses:

The major weakness of this study stems from a lack of a clear molecular mechanism involving Nrn1. Previous studies of Nrn1 have suggested its role as a soluble molecule involved in intracellular communication, perhaps influencing cellular ion channel function and/or triggering downstream NFAT and mTOR activation. However, a unique receptor for Nrn1 has not been discovered and it remains unclear whether it acts in a cell-intrinsic or cell-extrinsic fashion for any particular cell type.

Data shown here provide evidence for alterations in the electrical and metabolic state of iTreg and Teff cells when the Nrn1 gene is deleted. Nrn1-/- Tregs and Teff cells each express a unique pattern of genes associated with Neurotransmitter receptor, Metal ion transmembrane transport, Amino acid transport, and mTORC1 signaling activities, different than that seen in wild-type mice. It remains unclear how Nrn1 reinforces the membrane potential and facilitates aerobic glycolysis during and after iTreg differentiation, and yet suppresses the membrane potential and restrains aerobic glycolysis during Teff cell differentiation. Importantly, naive cells lacking Nrn1 expression show normal electrical and metabolic behaviors.

eLife Assessment

In this valuable study, Li and others identified cell membrane receptors for juvenile hormone (JH), a terpenoid hormone in insects that regulates their development and reproduction. While intracellular receptors for JH have been well characterized, membrane receptors for JH remained elusive for many years. Although the authors provide solid evidence to indicate that the receptor tyrosine kinases they identified bind to JH in vitro and induce non-genomic responses in cultured cells, their loss-of-function phenotypes are not consistent with known JH functions, so additional work is required to define physiological roles of these receptors.

eLife Assessment

This important work presents data showing that all non-proneural phenotypes of the Inhibitor of DNA binding (Id) protein Emc are mediated through inappropriate nonapoptotic caspase activity. Using the developing Drosophila retina as a model the authors show that Emc acts by transcriptionally regulating the Death-Associated Inhibitor of Apoptosis 1 (diap1) gene, which impacts on Notch signaling by caspase-dependent increase of Delta protein. These are compelling findings, interesting for the caspase/apoptosis field as they add more non-apoptotic functions of caspases to the list, as well as for the Id field, which examines how Id proteins inhibit cell differentiation.

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Smith Corona Manual Vintage Typewriter Main Spring Removal Flush and Re-Lube Draw Band Attached by [[Phoenix Typewriter]]

Duane puts a few drops of red oil on the mainspring of typewriters as a lubricant before blowing it out to thin it down.

Here he demonstrates how to re-tension the mainspring of a Smith-Corona typewriter.

Smith Corona Typewriters 1935 - 1980 Type Alignment / Shift Motion Upper Lower Case Adjustment by [[Phoenix Typewriter]]

Duane starts out by showing the two adjustment screws for the upper and lower case motion adjustment on a 5 Series Smith-Corona portable. (This should be the same across several decades of machines and include the 4 and 6 series as well.)

This sets the stage by explaining that under typical conditions, nitrogen and oxygen are stable and do not form compounds. This explains that high temperatures are needed for the reaction to occur and are key to forming nitrogen oxides.

Reviewer #2 (Public review):

Summary:

In this study, the authors combine the study of clinical samples of antibiotic resistant bacteria with experimental evolution and evolutionary genomics to address important questions about the propensity for reversion in two different schema: de novo resistance arising within a patient, and transmitted resistance. The authors' use of a combination of methods help to answer the question outlined in their hypothesis, that de novo resistance mechanisms appear to revert to sensitive phenotypes more readily in a drug-free environment.

Strengths:

This study is exceptionally well-written and organized. The authors state their hypothesis clearly, and follow it up with an impressive effort that is truly translational-they make direct use of clinical samples of bacteria, and combine that with approaches in experimental evolution and evolutionary genomics. The conclusions follow naturally from the results, and there are no irresponsible leaps made.

Weaknesses:

I will divide my criticism into two areas, conceptual (most of my critique), with a very small methodological question.

(1) In the end, the authors offer findings that appear to be correct, and (again) are reported very clearly. However, this study is very surface-level in its theoretical underpinnings and construction, which is puzzling, because the field of antibiotic resistance and adaptation more broadly, is full of relevant studies and explanatory tools. Below I'll identify several areas where this manifests.

For one, the authors do not engage with a large recent literature on reversion, reversal, and compensation. It provides much more conceptual grounding for what the authors observe, much of it compatible with the findings from this study:

To offer two quick examples:<br /> - Avrani S, Katz S, Hershberg R. Adaptations accumulated under prolonged resource exhaustion are highly transient. MSphere. 2020 Aug 26;5(4):10-128.<br /> - Pennings, P.S., Ogbunugafor, C.B. and Hershberg, R., 2022. Reversion is most likely under high mutation supply when compensatory mutations do not fully restore fitness costs. G3, 12(9), p.jkac190.

Examinations of the studies on adaptation and reversion offer a richer mechanistic take on what was observed. But this literature alone is less of a problem than the general offering of different takes for the results. One can turn to a different literature - from ecology - to find a different explanation that is compatible with the findings.

De novo evolution involves the strong selection and rapid fixation of populations that are evolving largely to a relatively simple ecological milieu: their only primary function is to promote replication and survival of populations experiencing the negative fitness effects of drug pressure. Alternatively, transmitted resistant populations must deal with a multitude of selective pressures, working dynamically across time and space. In such a scenario, one would expect populations to locate places on the fitness landscape that are commensurate with survival in both drug-poor and drug-rich environments, as this is the ecological reality of the transmitted resistant bacteria. I could envision selection for "generalism" in this setting, corresponding to populations that have fixed mutations that promote resistance, but also those that ensure replication in drug-free environments. This regime might even reflect selection for "generalism" or "increased niche breadth." That is, transmitted resistance may have adopted a "jack of all trades, master of none" phenotype. The de novo resistance strains, alternatively, are selected for "generalism."

See the following for examples (there are many):

- Kassen R. The experimental evolution of specialists, generalists, and the maintenance of diversity. Journal of evolutionary biology. 2002 Mar 1;15(2):173-90.<br /> - Bell TH, Bell T. Many roads to bacterial generalism. FEMS microbiology ecology. 2021 Jan;97(1):fiaa240.

Note that this classically ecological explanation is only one of several other literatures that offer models for the findings in this study.

To the authors' credit, their study was about the very real-world problem of antibiotic resistance, using a system that is far less tractable than the model systems research that has generated a lot of data and theory. And sure: the study is valuable because it communicates an interesting finding using a combination of methods (impressively). But in some ways, the study almost reads as a descriptive exercise: it offers a good question (does de novo or transmitted resistance revert more readily), and tells you what they found (de novo does). However the explanatory mechanisms do not advance our understanding much. Reporting the presence of unstable and disruptive mutations in the de novo populations is hardly an explanation. That is, alternatively, data in support of a proper explanation. There is nothing magical about de novo evolution that should be selected for disruptive mutations.

The reasons for the different sorts of mutation could have to do with the population genetic particulars of the de novo regime: large populations, strong selective pressure, relatively static fitness landscape. In such a setting, selection marches a population greedily up a peak. Alternatively, a transmitted population arises from a lineage that has observed a multitude of ecologies, across different fitness landscapes and has fixed mutations that confer survival across all of them.

There's a literature that speaks to this:<br /> - Miller CM, Draghi JA. Range expansion can promote the evolution of plastic generalism in coarse-grained landscapes. Evolution Letters. 2024 Apr 1;8(2):322-30.<br /> - Bono LM, Draghi JA, Turner PE. Evolvability costs of niche expansion. Trends in genetics. 2020 Jan 1;36(1):14-23.

The findings are simple enough (a testament to the strong study design and execution) that supporting population genetic simulations, or analytical descriptions (maybe not relevant) could offer insight as to what really happened here.

(2) I recognize the challenge of working with clinical samples. It is very difficult to understand everything about them. But even having considered that, I might be missing something.

My main question here involves the origin of the putatively transmitted strains. The authors state that " Isolates were also obtained from six patients with a putatively transmitted resistant bacteria (hereafter PT), where a daptomycin-resistant, E. faecium bacteremia was identified on their first culture."

This seems like an awfully dubious way to identify transmitted resistance. I suppose I understand the logic (de novo evolution requires the observer to have seen the evolution happen in real-time). But this definition leaves the study wide open for an "apples to oranges" comparison that might render the other aspects questionable.

The de novo strains are being compared to transmitted strains that may have been part of lineages that had passed between many, many patients. If this were true, then we should expect the genomic architecture of the transmitted strains to be far different. The transmitted strains might have undergone more selection in different regimes and genetic drift. Drift might have fixed mutations in transmission bottlenecks, altering the genetic architecture. In such a scenario, one might expect these populations to have a more difficult time unwinding their resistance phenotype.

In the end, I applaud the authors on a well-done and well-written study.

eLife Assessment

This important study, which will be of interest to those studying the evolution and maintenance of antibiotic resistance, addresses the hypothesis that antibiotic resistance arising de novo during treatment will carry a higher fitness cost and will revert to susceptibility more readily than resistance that has been transmitted between hosts. There are, however, concerns that the 'putatively transmitted isolates' in this study do not necessarily represent resistant isolates that have been transmitted between hosts. The support for the central claim of different patterns of reversion between isolates with de novo resistance and putatively transmitted resistant isolates is currently incomplete.

Reviewer #1 (Public review):

Summary:

Tracy and colleagues study the loss of daptomycin resistance in Enterococcus faecium isolates from bloodstream infections using in vitro evolution experiments in the absence of antibiotics. They test the hypothesis that antibiotic resistance arising de novo during treatment will carry a higher fitness cost and will revert more readily than resistance isolates which have been transmitted and have therefore already survived in the absence of antibiotic selection pressure.

Strengths:

This is an important question as a fitness cost to resistance is typically found in lab evolution experiments and assumed in modelling studies, but often not identified in clinical isolates. Here the authors find examples of clinical isolates which do and don't revert to sensitivity in in vitro evolution in the absence of antibiotics. Sequencing of the lab evolved isolates revealed that reversal of resistance was often due to mutations in the same gene that evolved in vivo, which is nice evidence that these resistance mutations did confer a fitness cost.

Weaknesses:

Although this is an interesting study on an important topic, currently the results are overinterpreted do not justify the title of the paper 'Reversion to sensitivity explains limited transmission of resistance in a hospital pathogen' for several reasons. Firstly, the patient group, e.g. 'putatively transmitted' isolates vs 'de novo' isolates was not a significant predictor of change in MIC. Instead the change in MIC in the absence of antibiotics was significantly associated with the starting MIC of the isolate in the evolution experiments, but this would be expected since isolates with a higher MIC have more potential to decrease in MIC in the evolution experiments. The abstract and some conclusion do not match the results in some instances, for example the abstract states 'resistance that arose de novo within patients was higher level but exhibited greater declines in resistance in vitro'. In the discussion: they state "these findings support our hypothesis that transmitted resistance strains are less likely to revert". However, on page 14 the initial MICs between DNR and PTR were not significantly different and patient group was not a significant predictor of change in MIC. Sequencing of the lab evolved isolates revealed that reversal of resistance was often due to mutations in the same gene that evolved in vivo. However, there were also some example of mutations in the same genes within the PTR isolates, so it remains unclear if there is a significant difference in behaviour between the DNR and PTR isolates in terms of reversion mutations. Significance testing, controlling for the starting MIC, would help confirm this.

Secondly, the 'putatively transmitted isolates', i.e. isolates that were resistant in the first positive blood culture, do not necessarily represent resistant isolates that have been transmitted between hosts. E. faecium is primarily a commensal of the intestinal tract, but which can cause opportunistic extra-intestinal infections. These bacteremia cases were most likely caused by within-host translocation of a strain already colonizing the intestine to the bloodstream - indeed, it has been shown that antibiotics can lead to Enterococcus overgrowth in the intestine and subsequent bloodstream invasion (DOI: 10.1172/JCI43918). The 'putatively transmitted isolates' may have initially colonised the intestine via between host transmission in an already resistant state, as assumed by the authors, but they may also have evolved resistance de novo within the host's intestine prior to causing bloodstream infections. Since they do not have data on past daptomycin exposure in these individuals it cannot be assumed that these isolates were transmitted with high resistance between hosts. An alternative explanation for any differences between the 'de novo' and 'putatively transmitted' could be the environment where resistance evolved, e.g. the intestine with strong competition from other strains and species, or within the otherwise sterile bloodstream environment. The authors hypothesise that "newly resistant population must continue to transmit between hosts in antibiotic free conditions to ensure its survival" and that "transmission acts as a filter to select for resistance with a lower cost or lower chance of reversion". Rather than transmission per se, it is equally plausible that survival of the newly resistant population within the primary niche, the intestinal microbiota, is the crucial to filter for resistance with a lower cost.

Die von Waldbränden außerhalb der Tropen verursachten Emissionen haben sich seit 2001 fast verdreifacht. Weltweit haben die Emissionen durch Waldbrände in dieser Zeit um 60% zugenommen. Ursache dafür ist die Kombination von heißerem und trockenerem Wetter mit dem schnelleren Wachstum der Wälder durch die höheren Temperaturen. Die Wälder können durch die Brände jahrzehntelang zu Emittenten werden. Damit ist die Funktion der Wälder als Kohlenstoffsenken gefährdet. Das bedeutet auch, dass sie andere anthropogene Emissionen weniger kompensieren und die Fähigkeit verlieren, nach einem Überschreiten der 1,5°-Grenze C0₂ aus der Atmosphäre zu entfernen. Außerdem müssten diese von Menschen verursachten Emissonen den C0₂-Budgets der Nationalstaaten zugeordnet werden.

https://theconversation.com/forest-fires-are-shifting-north-and-intensifying-heres-what-that-means-for-the-planet-241337

So there is a distinction between certain lipid serums but overall there does seem to be a correlation between environmental factors and your epigenome / serum lipid + apolipoproteins

I find it interesting that there is a contradiction in the data they found and this warrants further study.

This is an important thing to clarify as this data is taken from Northern Europe. This also brings up a key point, who were the ethnic groups that were studied? I presume mostly Northern Europeans, I think this study should be replicated for other ethnic groups to see if theres a difference between how much your epigenome is affected by your environment taking into account ethnicity.

I find it interesting that there is chance your epigenome is also affected by your sex and that it actually can affect one sex more than another on average.

I like that they admitted that one model didn't really work for them and that another was better but they left in why they didn't use the first model and why the second was more fitting.

This even further helps prove our case that as time goes on these differences become even more apparent and even more noticeable due to environmental factors.

This section helps further prove that there is an environmental affect on your gene expression, that yes genetics plays a factor but so does your environment. Nurture and Nature are true.

I think the sex correlation was expected, at least from me, usually we have different levels of almost everything in our bodies due to sex.

I believe the prediction model is very important to emphasize the differences as if the prediction is accurate it proves the null, while if it is inaccurate it helps reject the null.

This explanation is very useful and makes it easy to understand how they went about comparing the data of two different groups.

Relying on subject self-reporting is not always the best, if they could've gained access to relevant medical and prescription records I think it would've been better but this is sufficient

Sample size is a large factor in determining the ability to reject the null and while this sample size isn't the biggest, for what they're studying I believe its sufficient.

I'm glad the twins weren't forcefully or purposefully separated, as far as we know at least. The ethics remain intact for now.

I wonder why this is? Is it because the older you get the more environmental factors compound on top of each other and it makes it hard to differentiate what has been caused by what?

While I agree that environmental factors can confound the study, I think that its actually beneficial for them to have similar environments (at least for the purposes of our paper) because it shows that even very minor differences such as outlook (which affects stress/dopamine) can change your epigenome. I also disagree with adoption studies ethically as oftentimes these twins are deliberately separated and siblings should have the chance to grow up together.

Many of these factors being determined by single-genes makes it a lot easier to study and experiment with.

The idea that not all lipids are affected is interesting and I want to learn more

Wise (2022) original comments: “350 years into this society’s existence, laws were finally passed to prohibit the discrimination that had been its hallmark for 15 generations — not because of some grand moral epiphany, mind you, but because the resistance of that society’s victims had finally forced a comeuppance.” “If you’re trying to control for non-racial explanations for racial disparities, you need to make sure your alternative variables are genuinely independent, Chad.”

Conceptionally.org (2024) original comments:<br /> “Public officials cannot enact any policy they please like they’re ordering dessert from a menu. They have to choose from among policies that are politically acceptable at the time. And we believe the Overton window defines that range of ideas.” “Instead of joining a side and pulling on the rope (of the Overton window), pull it sideways in a direction no one will resist.”

can you explain the derivation of this

What is the physical significance of \ddot{v}_y

I can see it looks like \ddot{x} = -\omega^2 x

In electrodynamics div E is a scalar product.

Where E is the electric field which is a vector. And E = nabla • f

But in the context of electrodynamics, what is our scalar field, f?

Erstmals wurde genau erfasst, welcher Teil der von Waldbränden betroffenen Gebiete sich auf die menschlich verursachte Erhitzung zurückführen lässt. Er wächst seit 20 Jahren deutlich an. Insgesamt kompensieren die auf die Erhitzung zurückgehenden Waldbrände den Rückgang an Bränden durch Entwaldung. Der von Menschen verursachte – und für die Berechnung von Schadensansprüchen relevante – Anteil der CO2-Emissione ist damit deutlich höher als bisher angenommen https://www.carbonbrief.org/climate-change-almost-wipes-out-decline-in-global-area-burned-by-wildfires/

why in that case, the second term of the equation is not ignored?

wild that there's shit this fucking weird. you couldn't make it up

we dont need money

Ada berapakah nomina dalam paragraf tersebut? Ada berapa kalimatkah dalam paragraf tersebut?

This is common even outside of the care sector. I think we expect that technology can fix many of our problems in the future.

I wonder if some of the hesitation to use these robots is concern for job replacement by these machines.

Do these care homes really need a robot or just a machine like a automated lifting program.

I think its very shortsighted to not interact with the future users of these robots. The engineers need to see what these users need in addition to the basic care if they want to develop advanced care robots.

This is very forward thinking of the government. I think safety standards for a lot of robots need to be created even before the technology is truly finalized.

So how would they be able to truly come up with an effective solution to the problem that they have not seen themselves?

Which one is the most beneficial for the elderly?

From this perspective, the television workplace operates as what Daphne Spain calls a “spatial institution,” in which “the properties of a social system express themselves through daily activities at the same time

a counteroffer: the freedom to “hire an all-woman staff, producer, director . . . everybody” and to program what she wanted. To her surprise, WBZ management immediately agreed to these terms,

Hamlin recalls coming into a room with eighteen men seated around a big table who assured her that she could retain her position as a cultural reporter for the station while she hosted the morning show because of the formulaic and simplistic nature of programming for women. “It’s easy,” they told Hamlin, since she would have “dodo birds for viewers” and the labor required by the program would involve only “a little make-up, a little hairdo, a chef you can cook with, and from time to time there will be a star coming through.” (intro 3)

With 16 bits, you can represent 2^16 different values, because each bit can have 2 possible states (0 or 1), and there are 16 bits total.

Lets Suppose the roundtrip takes 5ms and retransmission happens every 4ms Then roundtrip exceeds retransmission, means new sync request will be triggered before recieving ack from prev req, which will lead to high number of packet req until the saturation point is reached

They again are referencing their sources. The majority of this article is using information from the prosecutor's office to explain the timeline and event of Payne's passing.

They are using other sources to explain how he died and the author clearly states how they know what they know.

Because Liam Payne is a prominent person, this event is going to attract a lot of attention and the news will effect a lot of fans.

This story focuses on timeliness, magnitude and human interest. It begins with really focusing on timeliness with this mention that there was already information being put out the day after.

Included testimony from the U.S. president of McDonalds. Quoted so that it is not confused with opinion.

BBC includes this statistic to show the reader the effect that this spread is having on the consumers, reiterating why this is a big deal.

BBC is using a credible, factual source for their information about the E. Coli spread.

Of course CD40 signaling inhibits differentiation. The data shows that longer treatment with CD40 antibody decreased differentiation compared to 24h of treatment.

.

UMD's robotics department has student projects in collaboration with gemstone that do very similar things.

I think that's a good idea, especially for elderly who might get confused by a humanoid robot talking to them if they have a cognitive disability.

Why do we think older people will especially resist care from robots?

Japan has a very large elderly population and a smaller population of young people, which is what makes it so difficult to find enough people to take care of the elderly.

This would be very helpful for older people who cannot control their bladder. When people have memory loss, they can forget that they have to use the bathroom, so maybe by having a robot remind them, it can help them slightly with their memory.

oh, um, that escalated quickly.

Is Magic about to happen? I hope so!

I'm confused with the punctuation.

What exactly is this trying to say? Did they steal it, was it out of kindness? I'm confused.

Who's the "her" referring to?

Self view

Love the word choice.

the grayness mentioned again

i think this is the first piece of action Ive read in this piece omggggg

im wondering the same

interesting statement

im still wondering if the underscores have significance. does it represent them maybe saying it in a different language and it's translated?

I like this phrase idk why

wow

I hate the terms owner and master yuck

if they want to be like the emigrant ship then why aren't they trying harder than they are to be like it?

weaponizing humans

I would be the same ngl. why does kindness matter when you're being physically tortured?

is this worded weird or can I just not read?

wow hello generalization and racism

reminding of before: the calm before the storm. are we now getting to the storm?

ooo gossip

never seen this term before. is it the plural feminine form?

do the underscores have significance?

hmmm

so is the stranger the boat? maybe I missed some context

traditional garment consisting of a skirt and veil to disguise the wearer's identity

the ship? gendered?

this captain can't steer apparently

In general this document is an educational description of the process of enzymes being used as biological catalysts and the different mechanisms that are utlized in the process as a whole

Does a process like this occur when enzymes are used by bacteria to counter antibiotics?

How substrates bond and what happens once they do

Another key term, includes link with extra information(read later)

Key terminology

The first statement appears to be a general statement of the material to be discuessed further in the document based off the title

General introduction to the purpose of the following subsection of this article

Spending hours on reading the documentation and guide lines of the host Canadian unitiverity

is the only options

This sentence is interesting because it is true that media is a skill to be learned. We must learn how to learn the different types of media in order to gain the knowledge that we need to progress in this world as well as use it in our everyday lives. Without this skill, how can we progress or advance in the world since everything is digitalized?

The term "mediatized environment" reflects the context in which we operate, implying that media is not just a tool but a significant context that influences thoughts, behaviors, and societal structures.

The benefits outlined (e.g., GPS navigation, and finding restaurants) showcase how these technologies enhance convenience and accessibility, making daily tasks more manageable and efficient. The ease of maintaining relationships through social media illustrates how ICTs facilitate social connectivity, enabling users to engage with a broad network of friends without significant effort. The robust interconnection highlights the role of ICTs in shaping and enriching our social and cultural environments, suggesting that technology serves practical purposes and influences our social lives and identities.

This framework underscores the complexity of interactions between various dimensions of human experience and media technologies. Each grouping (technological, sociocultural, etc.) represents a critical lens for understanding how individuals engage with media. Highlighting technological relations indicates a prioritization of how digital tools shape human behavior and identity, suggesting that these tools are central to modern existence.

preload = volume of blood returning to heart to the right atrium

afterload = resistance

contractility = how hard the heart gotta work

Beta 1 adrenergic receptors = gas pedal for the heart, increases HR and contractility

Beta 2, alpha 1 = vasoconstriction

M2 = acetylcholine binds to M2 leads to slowing of heart rate

heart cant pump properly, less blood is reaching the rest of the body

don't take phenylephrine which is worsening the workload of her heart

Dilated cardiomyopathy can lead to atrial fluttering, because heart muscles are stretched and damaged, lead to abnromal condcution

Amlodipine = non-DHP, only works to dilate the blood vessels and has no effects on heart like verapamil (which lowers AV conduction / decreases contractility of the heart)

Phenylephrine = selective alpha 1 agonist, causes vasoconstriction which worsens HTN

more calcium = more contractility

when calcium levels rise it bind to troponin and increase contraction, which increases stroke volume -- more blood pumped out with each beat

Orthopnea is the sensation of shortness of breath (dyspnea) that occurs when a person is lying flat and improves when sitting or standing up. Associated with HF (When lying down, the redistribution of fluid from the lower extremities into the chest increases the pressure on the lungs, exacerbating the difficulty in breathing.)

pulse oximetry = difficulty delivering blood to body

central cyanosis = related too the low oxygen delivery to the body

Jugular Venous Distension = related to right sided HF, dilated and restrictive cardiomyopathy (right atrium pressure builds up because blood keeps regurgitating back to RA, then it backds up into the veins))

Peripheral edema/crackles = from pulmonary congestion since blood be backing up

How will climate change affect this weather? Will it make this area less conducive to farming and growing crops and sustaining life?

Is the conflict being referred to mainly between ethnic groups or tribal conflict? Or is it referring to conflict between the state and the citizens?

• E1 activates ubiquitin and transfers it to E2
• E2 is positioned close to the target by E3
• E3 transfers ubiquitin from E2 to the target

"Eukaryotic cells contain multiple versions of the E3 ubiquitin ligase known as the SCF (Skp1/cullin/F box), each of which is distinguished by a different F box protein that uses a domain at the carboxyl terminus to recognize substrates for ubiquitination. The F box protein Dia2 is an important determinant of genome stability in budding yeast"

Because Mcm7 cannot be ubiquitinated, CMG is constitutively bound to DNA, and will not be removed at any point in the cell cycle. This means that when the cell divides, it will divide it's DNA with CMG still loaded, and the next round of DNA replication will be impaired.

The authors mutated a region of the Mcm complex, specifically Mcm7, seemingly through a mutation at residue 10 from lysine to arginine, making it impossible to be ubiquitinated by SCF-Dia2.

Ubiquitnation leads to CMG unloading, via ubiquitylation, via disassembly by Cdc48/p97, so this mutation makes the CMG constitutively bound to DNA.

Cells with the Mcm7-10R mutation could not be ubiquitated and therefore CMG could not be disassembled.

However, this did not lead to cell death, because a secondary disassembly pathway using Rrm3 and Pif1 helicases was enacted.

Without CMG disassembly, cells were not viable and the ability to ubiquitinate new helicases was lost? It seems.

Cells that have the MCM7-10R mutation will carry the CMG complex on their DNA into the next cell cycle. This helicase must be removed to make way for new helicases and further DNA replication.

The authors found that Rrm3 acts during S-phase to disassemble the CMG complexes from previous cell generations.

Pif1 is a helicase responsible for the maintenance of nuclear and mitochondrial DNA in S. cerevisiae.

Pif1 tightly couples ATP hydrolysis, single-stranded DNA translocation, and duplex DNA unwinding.

This is the recommended reading from our textbook for =Week 9=

Please, try to annotate it as much as possible!

female body is the states means of reproduction!!!!!!!!!!!!

Korean gov institures 3rd plan, effort to improve state of declining marriage and threat of lower birth rate. emphasis on female fertility, extra support for newly married couples blamed womens ferility

blame childless generatoin on neoliberlism

policies and laws did not help

The ship has sailed for Harris when it comes to Middle Eastern voters. Between her administration's involvement in the middle east in the last 4 years and her now attempt to sway voters with Cheney, I think it is best for her to move on from this sector.

I don't like when politicians, on any part of the political spectrum, use people as spearheads for their political campaign. If Michigan voters like Kamala enough, they'll vote for Kamala, I don't believe Liz Cheney(Ex-Rep of Wyoming) is going to sway anyone, especially not Michigan republicans.

Internet access for queer men gave freedom to learn and understand themselves and where they belonged, ultimately helping them feel less alone. It did not matter where they lived, the internet was a safe place they could go to not worry about the confines imposed on them in the real world. Without such opportunities and freedom gay men would have otherwise succumbed to the loneliness and rampant homophobia of the 90s

This is crucial to note because although the competition seems cut and dry there is a loophole for legal action.

This is important because if the DOJ decided to speak it could of backfired and caused chaos.

The most well-known example in the hacking of metadata is the 2014 celebrity photo leak, or as most know it, "The Fappening". A number of celebrities were hacked through poor security on their iCloud accounts to reveal private photos. Although the content itself was private, they knew more through metadata in those photos, location data encoded in these photos, for example.

This is a good thong for republicans. If I recall, last election there were countless more early voters for the democratic party.

When people see a couple who fit this description in the community I often overhear conversations and comments which reinforce this stereotype. It is a stigma based around an assumption that both are trying to get one thing off the other. Moreover, wealth from the older white man, or youth and beauty from the Asian woman. It is less likely people will believe they are truly in a loving interracial relationship.

Asian women are more sexualized as they are often depicted as a male fantasy in the West, however the same does not go from Asian men. This may explain the contrast in interest between the participants as although Hannah needed to adapt she was not outright avoided like her male Chinese counterparts.

Metadata can sometimes be surprising in the way that it can violate privacy. For example, many photos and messages have embedded location data in them-meaning that others can know where you have been at any given time, and you might never know that such a trail exists. Moreover, the websites collect browsing metadata with the intention of tracking your online activity. Even combining metadata from different sources, companies can create an extremely detailed picture of you without needing to access the actual data.

This is so important and we should welcome the assets of all individuals.

These days, my friends talk about the inferences that could be made based on the types of accounts that they follow. Sexual orientation is one that frequently pops up along with what kind of personality they have or what hobbies they enjoy. An easy giveaway is if they are following certain brands or locations.

Thank you for sharing this work! Negative data is published much less frequently, it's great that you put this out here.

Regarding your results, you mention several times that the Trim7 antiviral phenotype comes from an in vitro overexpression system. It seems like you may be attributing the lack of activity in vivo to due to lower expression levels in the mouse. I would be curious to know if in vitro you also see a strong dose dependence, where Trim7 can only restrict viral replication at high expression levels.

If the differences are driven by expression levels, one interesting explanation might be that MNV has a natural inhibitor of Trim7, and only when you overexpress Trim7 do you see antiviral effects. I used to study anti-CRISPRs (phage encoded inhibitors of bacterial immunity), and we would see a very similar dynamic. In our experiments, if a phage had an anti-CRISPR, it would appear to be unimpacted by the CRISPR-Cas immune system, but if we overexpressed CRISPR, we could "overwhelm" the anti-CRISPR and the phage would fail to replicate.

If you have any genomic libraries of MNV it could be interesting to see if any MNV ORFs are able to rescue MNV growth in the the Trim7 cell line.

this is now the second time in the past couple of weeks this drug has come up in recent news stories

according to this article his cause of death was due to the fall but the drugs taken lead to his fall.

the people who were with Liam when he died foresaw what ended up happening. This source is directly from the hotel receptionist

this drug is very unstable and how people react to the drug can vary drastically.

pink cocaine is a drug that is on the rise and is a mix of multiple different illegal substances.

I like taking written notes for a lot of things, mainly because I not only feel more engaged with the information being presented to me, but it also ensures that I'm only writing down what's important as I typically can't write as fast as the presenter is talking. That way I'm not left with a bunch of extra stuff in my notes that makes them more confusing and "stuffy" if you will.

Does this not amount to a suggestion that we outright abandon the principle that the law should apply to everyone equally?

This is a stronger critique of the argument - here the point made is that you can hardly argue there's a moral responsibility to make the victim whole where proper consideration as to why the defendant acted in that way has not been given - however here the author appears to be assuming that the only remedy a court will award is that the plaintiff be made whole

The weakness in this argument is that the author fails to explain how this sort of compensation is more than ordinary morality would demand. Upon reading this point it seems entirely reasonable for the wrongdoer to be responsible of ensuring the victim of their careless action retains the same standard of living they had, had the defendant not acted in this manor - the argument would be more effective if the author actually delve deeper in explaining how this contravenes 'ordinary morality'.

In all of these examples you could apply Perry's theory of responsibility, the law in nature is all centred around holding wrong doers responsible for their actions, however the main difference here in the civil recourse theory is that its simply analysing this dynamic through the provision of aid by the state in assisting in the endeavour of holding wrongdoers accountable.

Rights of Action and remedy

.

Case#: Patient 2, Turkish, 4 years old (report)

DiseaseAssertion: Accumulation of guanidinoacetate in brain and a deficiency of creatine in blood. GAMT deficiency in the liver.

FamilyInfo:

CasePresentingHPOs: HP:0011344, HP:0001252, HP:0001251 (Severe developmental delay, muscular hypotonia, ataxia)

CaseHPOFreeText: Intractable seizures, Bio- chemical and spectroscopy findings were similar to those of patient 1

CaseNotHPOs:

CaseNotHPOFreeText:

Biochemical analyte testing:

Brain Magnetic Resonance Spectroscopy (MRS):

GAMT activity assay: Total RNA from liver, fibroblasts, or leukocytes. The residual GAMT activity varied between levels below the limit of detection and 1.9 units/g liver. When 1:1 mixtures of liver extracts from the two patients and control livers were incubated, the GAMT activity exceeded the calculated activity by 40%-60%. This excludes the possibility that the deficiency of GAMT in the patients' livers is due to an inhibitor. It, rather, points to the presence of an activator of GAMT in the patients' livers. The activities of two unrelated enzymes (cytosolic lactate dehydrogenase and lysosomal ,B-hexosaminidase) and the protein content, which served as reference parameters, were within the same range of controls (table 1). Taken together, these results confirm the suspected deficiency of GAMT activity in the liver of the two patients.

Zygosity: Homozygous

Variant 1:

ClinVarID:

CAID:

gnomAD:

Variant 2:

ClinVarID:

CAID:

gnomAD:

ParentalGenotypes:

PreviouslyPublished:

Case#: Patient 1, German, male, 22 months old (report)

DiseaseAssertion: Accumulation of guanidinoacetate in brain and a deficiency of creatine in blood. GAMT deficiency in the liver.

FamilyInfo:

CasePresentingHPOs: HP:0008947, HP:0007153, (Infantile muscular hypotonia, Progressive extrapyramidal movement disorder,

CaseHPOFreeText: emiballistic voluntary movements, at the age of 22 mo his developmental age was that of a 8-wk-old infant,

CaseNotHPOs:

CaseNotHPOFreeText:

Biochemical analyte testing:

Brain Magnetic Resonance Spectroscopy (MRS): Bilateral abnormal signal intensities in the globus pallidus. A severe deficiency of creatine and creatine phosphate and simultaneous accumulation of guanidinoactetate were detected by in vivo proton and phosphorous magnetic resonance spectroscopy of the brain.

GAMT activity assay: Total RNA from liver, fibroblasts, or leukocytes. The residual GAMT activity varied between levels below the limit of detection and 1.9 units/g liver. When 1:1 mixtures of liver extracts from the two patients and control livers were incubated, the GAMT activity exceeded the calculated activity by 40%-60%. This excludes the possibility that the deficiency of GAMT in the patients' livers is due to an inhibitor. It, rather, points to the presence of an activator of GAMT in the patients' livers. The activities of two unrelated enzymes (cytosolic lactate dehydrogenase and lysosomal ,B-hexosaminidase) and the protein content, which served as reference parameters, were within the same range of controls (table 1). Taken together, these results confirm the suspected deficiency of GAMT activity in the liver of the two patients.

Zygosity: Homozygous

Variant 1:

ClinVarID:

CAID:

gnomAD:

Variant 2:

ClinVarID:

CAID:

gnomAD:

ParentalGenotypes:

PreviouslyPublished:

This signifies control. People simply looking for job prospects and opportunities were being played like chess pieces for the pleasure of these high-power men.

I think it is so important for victims to feel comfortable to come forward and to not let their abusers to continue having power over them. I think the public needs to be more accepting to victims of these crimes and not just turn a cold shoulder because the person being accused of the crimes is influential.

Similar to Jeffrey Epstein. That case was so important for all the cases following after that, it encouraged victims to speak up and let their voices be heard.

These NDAs signifies the use of intimidation and sparking fear within people.

I feel like these sorts of cases continue to come out more and more. The Hollywood industry, which I would assume these men were apart of, does a terrible job at protecting vulnerable people who are looking to make social connections. Rather, people in high power continually take advantage of innocent people.

Were these victims also people who worked under him? The party setting also reminds me of the huge case revolving around P. Diddy at the moment.

call to action for players

players should be able to disconnect after a game the same way employees disconnect from work. These messages don't allow them to do so

direct evidence of how these messages impacts a player's thinking

same idea that was expressed in the Blame Game article I read

proof that this is a relevant issue and should not be overlooked

the city centers of large urban centers