almost the "self definer" connecting my connection of the jinn, the Jotunn and the "idea" the Yotunnheim is now very much and very obviously connected by "joven" to Yovenheim ... viiiia Odinheim, via the Cherubim of the "chair you beam" people, who of course are one in the same with the "Frost Gaints" or whomever are the women of renl<nown, known only to those who know ... "ren own" in this day in age;

because of Ren, Wren, and of course "Stumpy" aka the Scotts of Prescott and Scottsdale people who are searrching high and low for reasons to shun Flagstaff and consider it outside of the union of NVUS, FLUS, and of course the few, the proud, the CAUS. :)

(ROT-moond-TOM-sun SIN-drome) A rare, inherited disorder that affects many parts of the body, especially the skin, eyes, bones, hair, and teeth. The main sign or symptom is a red blistering rash on the face that begins in early infancy.

Trichothiodystrophy (TTD) is a rare autosomal recessive multisystem disorder characterized by sulfur-deficient brittle hair, mental and physical retardation, ichthyosis, and, in many patients, cutaneous photosensitivity but no cancer incidence.

Can we substitute this defintion with the one from Bloome and B?

Five Fairly Fun Fixes For Free by [[Joe Van Cleave]]

Advice for typewriter ribbon printing, especially as silk ribbons age and more quickly gunk up the loops or letters like "e" or "a". Nylon ribbon and a thin plastic backing sheet can be helpfu.

Use of bookbinder's glue on fabric of typewriter cases, then layers of shoe polish.

General advice for replacing feet on typewriter cases.

Small incremental improvements to your typewriter can be easier and more sustainable than trying to do everything at once.

El eritema infeccioso, también conocido como megaloeritema o 5.º enfermedad, es una enfermedad infecciosa producida por el virus de DNA Parvovirus B19,2​ de la familia Parvoviridae.

Bloom syndrome (BSyn) is a rare genetic disorder characterized by short stature; a sun-sensitive, red rash that occurs primarily over the nose and cheeks; mild immune deficiency with increased susceptibility to infections; insulin resistance that resembles type 2 diabetes; and most importantly, a markedly increased susceptibility to many types of cancer, especially leukemia, lymphoma and colorectal tumors.

por calor o radiación uv

Smith-Corona 5-Series Touch Adjustment by [[Joe Van Cleave]]

This is quite a statement. There is more "true terror" in the line of the boat than in the whales themselves! Suggesting or implying that the most frightening dimensions of the voyage are internal to the ship, the men and their tools--the mundane conditions of their labor--rather than external, spectacular nature. Maybe we shouldn't generalize too far, but it is a crucial, if subtle revision of, say, Ahab's view.

Philosophical/metaphysical leap at the end (as he often provides). He suggests that as mortals we are all constantly subject to danger and death; our domestic safety is an illusion. Just when I was thanking my stars that I never have to encounter whale line in the midst of a hunt, he reminds us that if we open ourselves to the true precariousness of life and limb, we would recognize the ubiquitous risk of life itself. (This reminds me in some ways of Montaigne's "To Philosophize Is to Learn How to Die": through practice they get to a point where they can shrug at such immediate terror as the line.)

fear, anticipation, apprehension

Reminds me of what Meursault remembers Maman saying as he sits in his prison cell, that you can get used to anything.

When has that ever stopped you before? Haha.

Eek.

Wow. Problematic, to say the least. Clear racial hierarchy, particularly with regard to beauty.

This shows Fleece is acting under orders, against his will. It reminds me of Jim humoring Tom and Huck because he has to. This might tend to seem like the portrayal here of Fleece reinforces racial stereotypes, but as Wyn said with the novel James it seems to me that Fleece's performance here is underscored by a whole different internal perspective on Stubb's abuse. He is putting on the show Stubb demanded.

Shouts out for Fleece rudely, repeatedly, again disturbing his sleep. (Meanwhile he widens his legs "as if to form a more secure base for his supper"; he has no intention of moving from his comfortable repast.

Stubb commands a (the) Black harpooneer to go overboard and fetch him a whale steak. This might not stand out as much without Stubb's subsequent abuse of the Black cook Fleece. I'm guessing this "tapering extremity" is retrieved at significant inconvenience, and probably risk, to Daggoo.

Would be funny if he were not dogging Fleece with this nonsense. This feels like jolly, happy-go-lucky Stubb on the surface, but this whole scene shows that Stubb is happy to amuse himself at the expense of his perceived lesser. (This whole scene also resonates again when he tells Pip, "We can't afford to lose whales by the likes of you; a whale would sell for thirty times what you would, Pip, in Alabama" ("The Castaway" 307). Sure, there Stubb is trying to use any means available to convince Pip not to jump again, but he clearly sees him as Black and therefore fair game for bullying and devaluation (or, monetary valuation). I feel like Stubb shows his true colors (so to speak) as far as race in his obnoxious treatment of Fleece. He has no qualms about using race to put Pip "in his place.")

He has the last word, but has to mutter it to himself. If he spoke back directly, Stubb would not let him go yet. There would be some sort of consequences. His words are "sage," and he entirely perceives that Stubb is acting the shark in this scene.

More, more, more...

C'mon. Just let him go to bed.

More orders.

So... burn yourself so that my steak can be more perfectly rare. Your injury/discomfort/etc. is not as important as my whims.

Right. Jerk.

Again, this bullying makes me physically ill. He is physically manipulating him here with his orders. Fleece just wants to do whatever he needs to in order to get away and go back to bed. This is clearly harrassment.

He's deliberately trying to make Fleece look foolish and have to articulate things that most people would not be forced to articulate.

He may know what Stubb is getting at but instead chooses to emphasize how he ought to be in bed and intends to end this meeting and go there.

Is this your business? (What does that have to do with the steak, as Fleece asked before about his age.)

Forces Fleece to do as he says. No bodily autonomy. He has to stand where Stubb says to, eat what Stubb says to, etc.

Again, this is Fleece registering his objection as strongly as he dares.

What a jerk. Complaining while he eats and makes Fleece stand there.

He demands justification for this interrogation, but Stubb silences him and demands an answer.

Poor Fleece. He is old, disabled, and tired, and Stubb is subjecting him to humiliation.

He again registers his objection.

Swearing again against Stubb's orders.

Note the diction here.

Here, Fleece stands up for himself and refuses to play along further-- until Stubb orders him again, repeating "go on, go on."

Ironic. This swearing while admonishing against Fleece's swearing shows how Stubb is deliberately messing with Fleece for his own amusement.

This is bullying. I feel in my childhood bones here the times when older kids or kids with more social power ordered me to do awkward things just because they could and wanted to show that they had to power to command. This is clear abuse of power as Stubb is both a mate and white.

Hassles Fleece. Complains just to complain. Ingratitude. Bully.

Horrifying fact. Ironic tone: "decently buried," casual language equates a parcel to a dead human being (but self-aware?).

I wish this independent clause was not a parenthetical. It is Melville's approach to writing; find the aesthetic in this topic, whatever the chapter may be about. Maybe I will make a T-shirt with the line emblazoned on the front: "There is an aesthetic in all things." And on the back, If you haven't seen it today, you're not looking well enough.

The pigmented spindle cell tumor is a distinct benign melanocytic lesion with characteristic clinical and histopathologic features. Until recently, it has been poorly documented in the literature and is frequently misdiagnosed as malignant melanoma

The junctional melanocytic nevus is a well-circumscribed brown to black macule, which may clinically resemble a lentigo. It may develop anywhere on the body surface. Usually it appears during childhood or early adolescence, and matures with time into a compound nevus and later into an intradermal nevus.

Lentigines, or liver spots, are benign lesions that occur on the sun-exposed areas of the body. The backs of hands and face are common areas.

Interface dermatitis includes diseases in which the primary pathology involves the dermo-epidermal junction. Lupus erythematosus, dermatomyositis, lichen planus, graft versus host disease, erythema multiforme, fixed drug eruptions, lichen striatus, and pityriasis lichenoides are considered major interface diseases

This section highlights the role of internet-based interactions in the professional development of medical students. It emphasizes that during their education, students acquire essential professional communication skills, which they apply on social media to build their digital professional identities. The Internet facilitates the formation of virtual communities, where frequent interactions, enabled by the convenience of handheld devices, support the continuous development and reinforcement of their professional identity.

La dermatitis artefacta (DA) es un diagnóstico excepcional, que genera perplejidad y ansiedad al dermatólogo al encontrase ante una patología autoprovocada y de la que el paciente sabe más que el médico en cuanto a su etiología. Al contrario que otras dermatosis en las que existen pruebas histológicas o bioquímicas, en la DA el diagnóstico es de exclusión, lo que exige un gran consumo de recursos y de tiemp

Royal Model "O" Typewriter Adjust Ribbon Lift for each Type Bar Lever, Works on Quite Deluxe too. by [[Phoenix Typewriter]]

Forming individual type bars, particularly for capital letters to get the tops to print evenly when they're more faint than other similar typefaces.

There are different speeches and speech time requirements. .

There seems to be no written responses mentioned here.

Since there are no online zoom session I just have to complete work before its due date/

Royal QDL Quiet Deluxe Typewriter, Left Margin Stop, Alignment Adjustment, Repaired uneven edge by [[Phoenix Typewriter]]

How to adjust the margin stops on a Royal Quiet De Luxe and related portable models.

this is a pge

study

good note here

Y (casos o no), a (recibido tratamiento o no)

What is a sharp causal null hypothesis?

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Reply to the reviewers

We thank all three reviewers for their insightful comments. Based on this feedback, we have performed additional experiments, and revised our manuscript. Below, we address each comment and describe the revisions.

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

Summary: Ponomarova et al. showed that neomorphic idh-1 mutation results in increased levels of cellular D-2HG. The authors compared the high D-2HG phenotypes by D-2HG dehydrogenase mutant and identified vitamin B12 dependent vulnerability differences. The downregulated gene function of glycine cleavage system involved in one-carbon donor units exacerbates the phenotypes while adding one-carbone donors suppresses the phenotype. They concluded that the idh-1neo mutation imposes a dependency on the one-carbon pool. The manuscript is very interesting but I think the manuscript should be modified to be more clear for broad audiences.

Concerns: The authors mention a number of examples for metabolic changes of D-2HG in the first paragraph of introduction. I think that a metabolic map explaining the changes helps readers to understand the questions proposed by the authors.

Thank you for this suggestion. A figure illustrating the contributing factors in D-2HG metabolism has been added to the manuscript (Figure 1A).

The authors say that D-2HG affects carcinogenesis in many ways, citing previous works. They should say a higher concentration of D-2HG does affect carcinogenesis or not in dhgd loss of function, if they assume the concentration is most important for carcinogenesis.

Thank you for pointing this out. We have added this information in lines 70-72 of the revised manuscript: "Increased levels of D-2HG caused by the inhibition of D-2-hydroxyglutarate dehydrogenase activity have also been associated with different cancers (PMID: 29339485, PMID: 34296423, PMID: 35007759)."

Line 110, mode should be read as model, I guess.

Thank you - we have corrected this error.

In Figure 4C, concentrations of formate are shown; 0. 20, 40, 80, 160 mM. Is this correct? the high concentration of substrates changes the osmotic pressure of the medium. Also, high concentration of formic acid is toxic to animals. Considering the concentration of vitamin B12 was 64 nM, I wonder concentration unit of formate is also nM.

We confirm that we supplemented the media with formate in the millimolar range. The highest doses of supplemented formate somewhat slowed the development of P0 animals, but they consistently produced viable progeny. To clarify this we have added the following line to the text on lines 184-187: "The highest doses of supplemented formate somewhat slowed the development of P0 animals, but restored the survival of idh-1neo embryos to wild-type levels on a regular diet of E. coli OP50 as well as the diet of RNAi-competent E. coli HT115."

Additionally, the use of sodium formate ensured that the pH of the media remained unchanged.

I could not understand how embryonic and larval lethality confer the same mechanisms on animal carcinogenesis. Could you explain the logic link between lethal mutation and carcinogenesis. Or do the two phenotypes share only a part of metabolic changes?

Thank you for this suggestion. We have added this in lines 242-246 of the Discussion:

"While our results have focused on how the neomorphic idh-1 mutation affects the developing embryo, proliferating cancer cells also have been shown to have increased demand for 1C units, for instance, to synthesize nucleosides (33)(PMID: 24657017). Thus, we can speculate that cancers with mutated IDH1 may be increasingly sensitive to depletion of the 1C pool, also."

Vitamin B12 is an essential substance and deficiency in humans results in sever diseases. Is the lethal phenotype by treatment of idh-1neo mutants comparable to humans? Is the concentration of vitamin B12 similar in humans?

The daily dose of human vitamin B12 (cobalamin) in supplements can reach 12.5 µg per kg (PMID: 18606874), while we supplement the media fed to worms with approximately 55 µg cobalamin per kg (64 nM adenosylcobalamin). No known adverse effects are associated with excessive intake of vitamin B12 by healthy individuals; therefore, no tolerable upper intake level has been set (PMID: 23193625). However, the impact of vitamin B12 on patients with IDH1neo-positive cancers has not been studied.

Reviewer #1 (Significance (Required)):

I think that the manuscript is interesting and may lead an important progress of this field. However, in general, metabolic disorders are difficult to understand for the people outside the speciality. The authors should explain carefully the structure/property, pathways, enzyme functions, and concentration effects of substances of interest.

See above, we hope these edits are sufficient.

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

Increased levels of the metabolite D-2HG (derived from alpha-KG) are associated with multiple disorders. In a previous study, the authors showed that in C. elegans dhgd-1 deletion mutants, embryonic lethality resulting from the accumulation of D-2HG in is caused by a lack of ketone bodies. In this study, the authors generated a new model of D-2HG accumulation in C. elegans, idh-1neo, in order to further understand how D-2HG exerts its toxic effects in different contexts. This allele mimics mutations found in neomorphic mutations of human IDH1 that lead to abnormal D-2HG production from alpha-KG. Interestingly, the authors find that idh-1neo mutants are distinct from animals lacking the D-2HG dehydrogenase dhgd-1 previously reported. Specifically, while vitamin B12 rescues the embryonic lethality in dhgd-1 deletion animals, it enhances the lethality of idh-1neo animals. Through an elegant genetic screen, and complementation studies with specific metabolites, they provide compelling evidence that this vitamin B12-dependent enhancement is due to depletion of the 1C pool. Specifically, a reverse genetic screen revealed that inactivation of components of the 1 C-producing glycine cleavage system (GCS) results in embryonic lethality in idh-1neo, but not wildtype animals. Complementation studies with specific metabolites show that replenishing C groups is sufficient to reverse embryonic lethality.

This is a very clear, well written paper. Experiments are well controlled and executed, figures are of the highest quality and conclusions are convincing. Prior studies are appropriately referenced. No additional experiments are required by this reviewer.

Minor points 1) In Figure 2A could authors explain how beta-alanine (increased) is different from alanine (decreased). As a non-specialist this is not clear to me.

Thank you for pointing this out. We added this explanation to the figure legend (lines 510-512).

2) Did the authors test inactivation of the lipoamide dehydrogenase (dld-1) has the same effect as the other identified components of the GCS?

The dld-1 RNAi clone was present in the metabolic library that we screened but was not identified as a "hit." We have added the following in lines 164-168 of the revised manuscript: "Two other GCS genes, gcsh-2 and dld-1 were not identified as 'hits'. gcsh-2 is associated with the same reaction as gcsh-1, indicating that the latter encodes an active enzyme (30). dld-1 functions in other metabolic processes, particularly in lactate/pyruvate metabolism, and confers embryonic lethality when knocked down in wild type animals (31)".

**Referees cross-commenting**

Comments to Reviewer #3: 1/ The authors treat the idh-1neo worms with vitamin B12 to reduce 3HP concentrations. The authors should consider conducting experiments to reduce 3HP by other means also. This would help establish a causal relationship between the D-2HG accumulation and observed phenotypes.

The authors show that adding vitamin B12 to the diet of the idh-1neo significantly increased their D-2HG levels. Furthermore, dhgd-1 RNAi drives a further increase in D-2HG in idh-1neo animals and led to 100% penetrant embryonic lethality among the F1 generation of idh-1neo animals. Together I think this provided strong evidence for a causal relationship between the D-2HG accumulation and observed phenotypes. Further characterizing these phenotypes would be interesting but is beyond the scope of this paper.

4/ The authors should clarify whether it is really vitamin B12 or any other metabolite from the bacteria (like methionine) that is bringing about the phenotypes. Have they tested metabolically inactive bacteria?

the authors show that supplementing B12-treated idh-1neo animals with formate (another 1C donor) restored the survival of idh-1neo embryos, supporting a role for B12 in depletion of the 1C pool. They also show that suppressing Met/SAM cycle genes in idh-1neo prevent 1C depletion and restore availability of 1C units. So the evidence that 1C unit depletion is at the core of the observed phenotypes is pretty convincing

7/ The authors should conduct metabolomic profiling to examine changes in metabolic pathways, including 1C, glycine metabolism, glucose metabolism etc, in idh-1neo animals subjected to GCS gene knockdown, and vitamin B12 supplementation.

Not clear how these experiments would add to this story. Open up another line of research

8/ The audience will be limited to the field although the study pertains to an oncometabolite. The study value would have improved if the authors had included cancer cell data. Also, the phenotype studied has not been mechanistically linked to the oncometabolite function, making the study academic in nature.

The intetest of this study is that it is being carried out in an organismal context.

Reviewer #2 (Significance (Required)):

As a geneticist with a general interest in metabolomics I find this an elegans study that offers new insight into how IDH-1 and -2 neomorphic mutations affect metabolic rewiring in the context of a whole animal. Although similarities are observed between idh-1neo mutants and animals lacking the D-2HG dehydrogenase dhgd-1, both of which have increased levels of the metabolite D-2HG, specific metabolic differences are observed. The identification of 1C unit deficiency as a driver of lethality in idh-1neo mutants is highly significant given the central importance of 1C metabolism. This study should therefore be of interest to a wide audience.

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

Ponomarova et al presents a short follow up of their previous study to elucidate the role of a oncogenic variant of idh-1 that increases the 3HP levels, similar to the Ddhgd-1 mutant. Using a combination of metabolomics and genetics, they show that the defect in idh-1neo worms on high vitamin B12 diet is the draining of the 1C pool, distinct from the mechanisms of lethality observed in the Ddhgd-1 mutant. While the findings are interesting, there is a lack of mechanistic understanding of the basis of the phenotype observed. Moreover, the authors do not establish the link between the oncometabolite, that should support uncontrolled cell division, with the observed phenotype. Some control experiments are missing and should be included in the revised manuscript. there could be many other The comments on the manuscript are as follows, in no particular order:

1. The authors treat the idh-1neo worms with vitamin B12 to reduce 3HP concentrations. The authors should consider conducting experiments to reduce 3HP by other means also. This would help establish a causal relationship between the D-2HG accumulation and observed phenotypes.

To further examine the link between 3HP and idh-1neo embryonic lethality, we targeted hphd-1 by RNAi, which increases 3HP levels (Ponomarova et al., 2023). Hphd-1 knockdown did not induce lethality in the wild-type or exacerbate lethality in idh-1neo animals (Figure S3), further demonstrating that lack of 3HP degradation is not linked to this phenotype (lines 143-145).

Also, see cross-comments from Reviewer #2 above.

The authors should investigate the functional impact of HPHD-1 inhibition on 3-hydroxypropionate levels and D-2HG accumulation by RNAi knockdown of HPHD-1 in idh-1neo animals.

We have now performed the suggested experiment please see response to comment 1 above.

The authors do not clearly mention clearly which diet in some of their experiments. This is imporant since the two diets used (OP50 and HT115) differ in their vitamin B12 content, and thus could have different consequences.

We added this information in figures, figure legends, and lines 259-260 of the revised manuscript.

The authors should clarify whether it is really vitamin B12 or any other metabolite from the bacteria (like methionine) that is bringing about the phenotypes. Have they tested metabolically inactive bacteria?

The reviewer correctly points out that bacterial metabolism may play a role in the effects exerted by vitamin B12. We have not tested metabolically inactivated bacteria, however, our RNAi experiments (Figure 4E) demonstrate that supplemented vitamin B12 acts through the Met/SAM cycle in idh-1neo animals. Please also see cross-comments from Reviewer #2.

The authors consistently use 64 nM of Vitamin B12. Will the hphd-1 mutant and the idh-1neo mutant have different vitamin B12 thresholds for the observed phenotypes?

Thank you for raising this interesting point. While 64 nM vitamin B12 virtually eliminates 3HP accumulation in idh-1 animals (Figure 2D), we have not tested if this dose is sufficient to eliminate 3HP accumulation in hphd-1 mutant. However, potential differences in 3HP levels in idh-1neo and hphd-1 animals treated with vitamin B12 would not contradict our conclusion that 3HP is not the cause of embryonic lethality in idh-1neo mutant animals.

Figure 3b: HT115 has inherently high levels of vitamin B12 so the RNAi effect of genes should be seen on the OP50 diet supplemented with B12.

Despite reports of elevated B12 levels in E. coli HT115, vitamin B12-induced embryonic lethality of idh-1neo on a diet of OP50 is more severe than on a diet of HT115 bacteria (Figure 4C). Therefore, it may be harder to quantify synthetic lethal interaction of idh1-neo with GCS RNAi knockdown using OP50 strains (which would need to be created).

The authors should conduct metabolomic profiling to examine changes in metabolic pathways, including 1C, glycine metabolism, glucose metabolism etc, in idh-1neo animals subjected to GCS gene knockdown, and vitamin B12 supplementation.

While these results would be interesting and further our understanding of metabolic changes that occur in idh-1neo mutant animals we think they are beyond the scope of the manuscript. Also, please see cross-comments from Reviewer #2.

Perform rescue experiments using different one-carbon donors (e.g., formate, serine) to restore embryonic viability in idh-1neo mutants under conditions of vitamin B12-induced stress. Quantify the efficacy of these interventions using developmental assays.

In addition to formate rescue experiments (Figure 4C), we supplemented idh-1neo animals with serine (Figure 4D and S7). Similar to formate, serine supplementation resulted in the rescue of idh-1neo embryonic lethality on an E. coli OP50 diet (lines 187-189). The lack of rescue on an HT115 diet could be due to HT115 bacteria containing more glycine (Gao et al., 2017), which might limit the efficiency of serine conversion to glycine needed for 1C unit production.

Provide experimental evidence to show that idh-1neo animals possess an alternative source of energy.

We have previously found that diminished production of ketone bodies in ∆dhgd-1 mutants causes embryonic lethality that can be rescued by exogenous supplementation of ketone body 3-hydroxybutyrate (Ponomarova et al., 2023). In contrast to dhgd-1 mutants, idh-1neo embryonic lethality fails to respond to supplemented 3-hydroxybutyrate (Figure S4), indicating the lethality associated with the idh-1neo mutation is caused by a different mechanism, i.e., a depletion in 1C-units.

The authors use vitamin B12 to inhibit the shunt pathway (line 127). They should explore alternate strategies to do the same, like gene knockdown.

Please see our response to comment 1 above where we discuss RNAi knock-down of the shunt pathway gene, hphd-1.

It is not clear why the authors did not follow up with the other phenotypes of the idh-1neo that were visible without the Vitamin B12 supplementation. They should follow up with this and also other phenotypes to explore the broader physiological consequences of D-2HG accumulation.

We agree that the other physiological consequences of D-2HG accumulation are interesting, and we plan to investigate them in our future studies.

The authors should include control experiments without supplementation of vitamin B12, ketone bodies etc. in each of their figures.

We thank the reviewer for this suggestion. We have added these data (Figures S5, 6, 7, and 8).

The authors posit that the idh-1neo depletes the 1C pool leading to the observed lethality. So, when they supply formate to replenish it, they rescue the lethality of the B12-treated worms. Similar results are obtained by knocking down the enzymes. So where are the 1C units going? Understanding this will provide the much-needed mechanistic understanding to this study.

We appreciate this insightful comment and expand our discussion to elaborate on this issue (lines 224-227). "We propose that a lack of 1C units in idh-1neo can impede pyrimidine biosynthesis via thymidylate synthase tyms-1, which uses 1C units to generate dTMP. Supporting this hypothesis, RNAi of tyms-1 causes embryonic lethality (36-38)."

It may be important to measure the D-2HG levels in the mitochondria vs the cytosol.

While this is an interesting point, we think that this line of inquiry is beyond the scope of this work (and is technically challenging).

The idh-1neo is an oncometabolite. The authors do not show any data to indicate whether this mutant has any defect in cell division/cell cycle in the somatic tissue or germline.

In this study we primarily focused on the molecular changes in the metabolic network that occur in idh-1neo mutant animals, which we think is an important advance in understanding the basis for how this mutation affects IDH function. Additional phenotypic outcomes of these perturbed metabolic processes will be the basis of future studies.

Reviewer #3 (Significance (Required)):

The audience will be limited to the field although the study pertains to an oncometabolite. The study value would have improved if the authors had included cancer cell data. Also, the phenotype studied has not been mechanistically linked to the oncometabolite function, making the study academic in nature.

While we agree that the link between idh-1neo, 2HG production and oncometabolite function has not been directly shown we think that our study adds important molecular understanding of metabolic changes that occur in relation to idh-1neo function which are important for future studies of how this mutation affects carcinogenesis. Also, please see cross-comments from Reviewer #2.

In addition, we specified statistical significance in Figure 2, described statistical tests used (lines 361-363) and corrected a few grammatical errors throughout the text.

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Referee #3

Evidence, reproducibility and clarity

Ponomarova et al presents a short follow up of their previous study to elucidate the role of a oncogenic variant of idh-1 that increases the 3HP levels, similar to the dhgd-1 mutant. Using a combination of metabolomics and genetics, they show that the defect in idh-1neo worms on high vitamin B12 diet is the draining of the 1C pool, distinct from the mechanisms of lethality observed in the dhgd-1 mutant. While the findings are interesting, there is a lack of mechanistic understanding of the basis of the phenotype observed. Moreover, the authors do not establish the link between the oncometabolite, that should support uncontrolled cell division, with the observed phenotype. Some control experiments are missing and should be included in the revised manuscript. there could be many other The comments on the manuscript are as follows, in no particular order:

1. The authors treat the idh-1neo worms with vitamin B12 to reduce 3HP concentrations. The authors should consider conducting experiments to reduce 3HP by other means also. This would help establish a causal relationship between the D-2HG accumulation and observed phenotypes.
2. The authors should investigate the functional impact of HPHD-1 inhibition on 3-hydroxypropionate levels and D-2HG accumulation by RNAi knockdown of HPHD-1 in idh-1neo animals.
3. The authors do not clearly mention clearly which diet in some of their experiments. This is imporant since the two diets used (OP50 and HT115) differ in their vitamin B12 content, and thus could have different consequences.
4. The authors should clarify whether it is really vitamin B12 or any other metabolite from the bacteria (like methionine) that is bringing about the phenotypes. Have they tested metabolically inactive bacteria?
5. The authors consistently use 64 nM of Vitamin B12. Will the hphd-1 mutant and the idh-1neo mutant have different vitamin B12 thresholds for the observed phenotypes?
6. Figure 3b: HT115 has inherently high levels of vitamin B12 so the RNAi effect of genes should be seen on the OP50 diet supplemented with B12.
7. The authors should conduct metabolomic profiling to examine changes in metabolic pathways, including 1C, glycine metabolism, glucose metabolism etc, in idh-1neo animals subjected to GCS gene knockdown, and vitamin B12 supplementation.
8. Perform rescue experiments using different one-carbon donors (e.g., formate, serine) to restore embryonic viability in idh-1neo mutants under conditions of vitamin B12-induced stress. Quantify the efficacy of these interventions using developmental assays.
9. Provide experimental evidence to show that idh-1neo animals possess an alternative source of energy.
10. The authors use vitamin B12 to inhibit the shunt pathway (line 127). They should explore alternate strategies to do the same, like gene knockdown.
11. It is not clear why the authors did not follow up with the other phenotypes of the idh-1neo that were visible without the Vitamin B12 supplementation. They should follow up with this and also other phenotypes to explore the broader physiological consequences of D-2HG accumulation.
12. The authors should include control experiments without supplementation of vitamin B12, ketone bodies etc. in each of their figures.
13. The authors posit that the idh-1neo depletes the 1C pool leading to the observed lethality. So, when they supply formate to replenish it, they rescue the lethality of the B12-treated worms. Similar results are obtained by knocking down the enzymes. So where are the 1C units going? Understanding this will provide the much-needed mechanistic understanding to this study.
14. It may be important to measure the D-2HG levels in the mitochondria vs the cytosol.
15. The idh-1neo is an oncometabolite. The authors do not show any data to indicate whether this mutant has any defect in cell division/cell cycle in the somatic tissue or germline.

Significance

The audience will be limited to the field although the study pertains to an oncometabolite. The study value would have improved if the authors had included cancer cell data. Also, the phenotype studied has not been mechanistically linked to the oncometabolite function, making the study academic in nature.

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

Learn more at Review Commons

Referee #2

Evidence, reproducibility and clarity

Increased levels of the metabolite D-2HG (derived from alpha-KG) are associated with multiple disorders. In a previous study, the authors showed that in C. elegans dhgd-1 deletion mutants, embryonic lethality resulting from the accumulation of D-2HG in is caused by a lack of ketone bodies. In this study, the authors generated a new model of D-2HG accumulation in C. elegans, idh-1neo, in order to further understand how D-2HG exerts its toxic effects in different contexts. This allele mimics mutations found in neomorphic mutations of human IDH1 that lead to abnormal D-2HG production from alpha-KG. Interestingly, the authors find that idh-1neo mutants are distinct from animals lacking the D-2HG dehydrogenase dhgd-1 previously reported. Specifically, while vitamin B12 rescues the embryonic lethality in dhgd-1 deletion animals, it enhances the lethality of idh-1neo animals. Through an elegant genetic screen, and complementation studies with specific metabolites, they provide compelling evidence that this vitamin B12-dependent enhancement is due to depletion of the 1C pool. Specifically, a reverse genetic screen revealed that inactivation of components of the 1 C-producing glycine cleavage system (GCS) results in embryonic lethality in idh-1neo, but not wildtype animals. Complementation studies with specific metabolites show that replenishing C groups is sufficient to reverse embryonic lethality.

This is a very clear, well written paper. Experiments are well controlled and executed, figures are of the highest quality and conclusions are convincing. Prior studies are appropriately referenced. No additional experiments are required by this reviewer.

Minor points

1. In Figure 2A could authors explain how beta-alanine (increased) is different from alanine (decreased). As a non-specialist this is not clear to me.
2. Did the authors test inactivation of the lipoamide dehydrogenase (dld-1) has the same effect as the other identified components of the GCS?

Referees cross-commenting

Comments to Reviewer #3:

1/ The authors treat the idh-1neo worms with vitamin B12 to reduce 3HP concentrations. The authors should consider conducting experiments to reduce 3HP by other means also. This would help establish a causal relationship between the D-2HG accumulation and observed phenotypes.

The authors show that adding vitamin B12 to the diet of the idh-1neo significantly increased their D-2HG levels. Furthermore, dhgd-1 RNAi drives a further increase in D-2HG in idh-1neo animals and led to 100% penetrant embryonic lethality among the F1 generation of idh-1neo animals. Together I think this provided strong evidence for a causal relationship between the D-2HG accumulation and observed phenotypes. Further characterizing these phenotypes would be interesting but is beyond the scope of this paper.

4/ The authors should clarify whether it is really vitamin B12 or any other metabolite from the bacteria (like methionine) that is bringing about the phenotypes. Have they tested metabolically inactive bacteria?

the authors show that supplementing B12-treated idh-1neo animals with formate (another 1C donor) restored the survival of idh-1neo embryos, supporting a role for B12 in depletion of the 1C pool. They also show that suppressing Met/SAM cycle genes in idh-1neo prevent 1C depletion and restore availability of 1C units. So the evidence that 1C unit depletion is at the core of the observed phenotypes is pretty convincing

7/ The authors should conduct metabolomic profiling to examine changes in metabolic pathways, including 1C, glycine metabolism, glucose metabolism etc, in idh-1neo animals subjected to GCS gene knockdown, and vitamin B12 supplementation.

Not clear how these experiments would add to this story. Open up another line of research

8/ The audience will be limited to the field although the study pertains to an oncometabolite. The study value would have improved if the authors had included cancer cell data. Also, the phenotype studied has not been mechanistically linked to the oncometabolite function, making the study academic in nature.

The intetest of this study is that it is being carried out in an organismal context.

Significance

As a geneticist with a general interest in metabolomics I find this an elegans study that offers new insight into how IDH-1 and -2 neomorphic mutations affect metabolic rewiring in the context of a whole animal. Although similarities are observed between idh-1neo mutants and animals lacking the D-2HG dehydrogenase dhgd-1, both of which have increased levels of the metabolite D-2HG, specific metabolic differences are observed. The identification of 1C unit deficiency as a driver of lethality in idh-1neo mutants is highly significant given the central importance of 1C metabolism. This study should therefore be of interest to a wide audience.

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

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Referee #1

Evidence, reproducibility and clarity

Summary:

Ponomarova et al. showed that neomorphic idh-1 mutation results in increased levels of cellular D-2HG. The authors compared the high D-2HG phenotypes by D-2HG dehydrogenase mutant and identified vitamin B12 dependent vulnerability differences. The downregulated gene function of glycine cleavage system involved in one-carbon donor units exacerbates the phenotypes while adding one-carbone donors suppresses the phenotype. They concluded that the idh-1neo mutation imposes a dependency on the one-carbon pool. The manuscript is very interesting but I think the manuscript should be modified to be more clear for broad audiences.

Concerns:

The authors mention a number of examples for metabolic changes of D-2HG in the first paragraph of introduction. I think that a metabolic map explaining the changes helps readers to understand the questions proposed by the authors.

The authors say that D-2HG affects carcinogenesis in many ways, citing previous works. They should say a higher concentration of D-2HG does affect carcinogenesis or not in dhgd loss of function, if they assume the concentration is most important for carcinogenesis.

Line 110, mode should be read as model, I guess.

In Figure 4C, concentrations of formate are shown; 0. 20, 40, 80, 160 mM. Is this correct? the high concentration of substrates changes the osmotic pressure of the medium. Also, high concentration of formic acid is toxic to animals. Considering the concentration of vitamin B12 was 64 nM, I wonder concentration unit of formate is also nM.

I could not understand how embryonic and larval lethality confer the same mechanisms on animal carcinogenesis. Could you explain the logic link between lethal mutation and carcinogenesis. Or do the two phenotypes share only a part of metabolic changes?

Vitamin B12 is an essential substance and deficiency in humans results in sever diseases. Is the lethal phenotype by treatment of idh-1neo mutants comparable to humans? Is the concentration of vitamin B12 similar in humans?

Significance

I think that the manuscript is interesting and may lead an important progress of this field. However, in general, metabolic disorders are difficult to understand for the people outside the speciality. The authors should explain carefully the structure/property, pathways, enzyme functions, and concentration effects of substances of interest.

.https://www.youtube.com/watch?v=iARAHKBdbUg

Wiki-How Video Project

https://www.nytimes.com/2024/06/24/climate/extreme-wildfires-have-doubled-in-2-decades-study-finds.html

https://www.nytimes.com/2024/06/25/world/australia/record-heat-deaths-events.html

https://taz.de/Klimakonferenz-in-Aserbaidschan/!6016105/

https://taz.de/Rueckschlag-bei-Waermepumpen/!6016273/

https://www.reuters.com/business/energy/exxon-led-guyana-oil-groups-net-jumps-2023-margins-top-nvidias-2024-06-25/

https://www.repubblica.it/green-and-blue/2024/06/25/news/incendi_estremi_aumento_crisi_clima-423284040/

https://www.repubblica.it/economia/2024/06/25/news/trino_fotovoltaico_centrale_nucleare_gas_transizione_energetica-423289236/?ref=RHLF-BG-P11-S2-F

https://www.liberation.fr/environnement/biodiversite/proteger-12-des-terres-du-monde-pourrait-eviter-les-extinctions-despeces-les-plus-imminentes-20240625_T53I57MOFNBMBGUZDA6BALPBSY/

Music Library

Music Library

Music Library

Qual imagem ilustrará o texto? Podemos talvez produzir uma...

Vejam meu comentário abaixo para a última imagem

Não podemos por Fonte: fornecido pelos autores. Isso não diz nada. Ou a imagem da plantação de dendê foi feita por eles ou eles fornecem o nome de que a fotografou ou da insituição responsável

Se for possível, dar maior espaço do final do texto para a foto. Me pergunto se, a nível de diagramação, não pomos a imagem do Edson alinhada à esquerda e a do Márcio à direita para distribuir o peso da página

vírgula após salientar

;

por uma vírgula após ambiental

entre vírgulas

aqui seria ou óleo de palma, não? Seria interessante dar o significado/etimologia da palavra 'dendê'

nosso conhecimento científico me parece mais apropriado

Isso é uma afirmação? uma realidade? ou ainda uma possibilidade? Se for o caso, por o verbo 'poderia'. Aí é preciso dar um fecho ao resumo introdutório, fazendo uma 'chamada' para o texto propriamente dito.

Por então entre vírgulas

por uma vírgula após CO2

I wonder when comparing the evolutionary tree of a Cnidaria species to the evolutionary tree of a snake, or spider, how similar would they be?

These are all valid reasons that support the claim that the Starlet Sea Anemone is a prime model organism for studying the venom of an animal with a complex life cycle.

Music Library?

This is an interesting correlation that I haven't thought about before, but makes sense. I frequently tell my students that the world is too hard, we need to be kind to one another.

sobre la "madre".

Srive para prevenir el cáncer

history of impact measurement

no wonder he's arya's friend

a 14 yr old with that much muscle?

theyre fucking sorry

pls be important later

eww

whattt

younger??

lol

don't call shireen that

aww

LMAO

THATS KINDA COOL THO

NOOOOO

oh i know them

nah thats heinous

HIS MOM

those cloesest to him

yay hes not scared of wolves

this is gonna sound so mean but pleasw that sunds so funny

yeah hes from highgarden

jon and his outliers

lol

I DO ACTUALLY

oh i didnt know he was a noble

YES JON CHAPTERR

YUP

NO YOU WERE RIGHT

if henry branwell could then so can bran

if only bran knew how much they love him

the queen...

old nan mention!

pinning it on varys lol but also lys mention!

URHURNFRSNZ.,XKLFGAEUWI3

I NEED A FANFIC OF THIS ASAP

i want his pov so BADLY

pls i'm so hyped to see them

hotheaded like jon

lannisters

he's trying his best

i like how he wasn't the first to call him

he's pretty good at this comforting thing

interesting

interestingg

LMAO THE LINE

i feel so bad for rickon like every single family member is leaving him when he's only THREE

:(

i wish he knew how much his mother loves him and her long vigallance over him

nu uh

robb and rickon bonding, he's basically his parent now

the wolves match them all so perfectly

A BABY

ah hell nah

an eclipse?

good idea but also come on man

should check if anything has changed from the time this journal was published

Just spend all GC in one go

Blubell is a plain-text language, similar to Markdown, which simplifies the authoring of Akoma Ntoso documents.

Deeper

Hermeneutics

I like this definition of Stewardship. We have character traits at my school, and Stewardship is one of them. Often times, students think of stewardship as just cleaning up after themselves. I like that this definition puts more emphasis of people.

I like how she worded "judgments in disguise", it can be frustrating when someone is asking a question that they already have an answer to in their head.

Interesting. This was just surprising to me!

Culmination of Ideation

Author Response

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

Reviewer #1 (Public Review):

The manuscript by Sejour et al. is testing "translational ramp" model described previously by Tuller et al. in S. cerevisiae. Authors are using bioinformatics and reporter based experimental approaches to test whether "rare codons" in the first 40 codons of the gene coding sequences increase translation efficiency and regulate abundance of translation products in yeast cells. Authors conclude that "translation ramp" model does not have support using a new set of reporters and bioinformatics analyses. The strength of bioinformatic evidence and experimental analyses (even very limited) of the rare codons insertion in the reporter make a compelling case for the authors claims. However the major weakness of the manuscript is that authors do not take into account other models that previously disputed "rare or slow codon" model of Tuller et al. and overstate their own results that are rather limited. This maintains to be the weak part of the manuscript even in the revised form.

We are glad the reviewer thinks our evidence makes “a compelling case for the authors claims”. This was our main aim, and we are satisfied with this.

The reviewer believes the major weakness of the manuscript is that we do not take into account other models and do not (see below) cite numerous other relevant papers. The reviewer made essentially the same criticism at the first review, at which time we looked quite hard for papers generally meeting the reviewer’s description. We found a few, which we incorporated here. Still, we did not find the body of evidence whose existence the reviewer implies. We are citing every study we know to be relevant, though of course we will have inadvertently missed some, given the huge body of literature. After the first round of review, we wrote “the reviewer did not give specific references, and, though we looked, we weren’t always sure which papers the reviewer had in mind.” We hoped the reviewer would provide citations. But only two citations are provided here, both to A. Kochetov, and these don’t seem central to the reviewer’s points.

The studies that authors do not mention argue with "translation ramp" model and show more thorough analyses of translation initiation to elongation transition as well as early elongation "slow down" in ribosome profiling data. Moreover several studies have used bioinformatical analyses to point out the evolution of N-terminal sequences in multiple model organisms including yeast, focusing on either upstream ORFs (uORFs) or already annotated ORFs. The authors did not mention multiple of these studies in their revised manuscript and did not comment on their own results in the context of these previous studies.

Mostly, we do not know to what papers the reviewer is referring. This may be our failing, but it would have helped if the reviewer had cited one of them. There are papers discussing the evolution of N-terminal sequences, but as far as we know, these do not discuss translation speed or codon usage. Of course, we may have missed some papers.

As such the authors approach to data presentation, writing and data discussion makes the manuscript rather biased, focused on criticizing Tuller et al. study and short on discussing multiple other possible reasons for slow translation elongation at the beginning of the protein synthesis. This all together makes the manuscript at the end very limited.

We think the reviewer may be considering our paper as being generally about translation speeds, whereas in our minds, it is not. This difference in views as to what the paper is “about” is perhaps causing friction. To us, it is indeed a limited paper. We are narrowly focused on the finding of Tuller that there is an enrichment of rare, slow codons at the 5’ end of genes, and we have sought an explanation of this particular fact. This is not a paper about rates of translation generally—it is a limited paper about the reason for the 5’ enrichment of rare, slow codons.

To expand on this, the encoded slow 5’ translation due to rare, slow codons (of Tuller et al.) is a small effect (1% to 3%). The possible unencoded slow 5’ translation of unknown mechanism discussed by some other papers (e.g., Weinberg et al. 2016, Shah et al. 2013) is a much larger effect (50% or more). Just from the different magnitudes, it seems likely these are different phenomena. And yet, despite the small size of the encoded effect, it is for some reason this paper by Tuller et al. that has captured the attention of the literature: as we point out below, Tuller et al. has been cited over 900 times. Partly because of the wide and continuing influence of this paper, it is worth specifically and narrowly addressing its findings.

Reviewer #2 (Public Review):

Tuller et al. first made the curious observation, that the first ∼30-50 codons in most organisms are encoded by scarce tRNAs and appear to be translated slower than the rest of the coding sequences (CDS). They speculated that this has evolved to pace ribosomes on CDS and prevent ribosome collisions during elongation - the "Ramp" hypothesis. Various aspects of this hypothesis, both factual and in terms of interpreting the results, have been challenged ever since. Sejour et al. present compelling results confirming the slower translation of the first ~40 codons in S. cerevisiae but providing an alternative explanation for this phenomenon. Specifically, they show that the higher amino acid sequence divergence of N-terminal ends of proteins and accompanying lower purifying selection (perhaps the result of de novo evolution) is sufficient to explain the prevalence of rare slow codons in these regions. These results are an important contribution in understanding how aspects of the evolution of protein coding regions can affect translation efficiency on these sequences and directly challenge the "Ramp" hypothesis proposed by Tuller et al.

I believe the data is presented clearly and the results generally justify the conclusions.

We thank the reviewer for his/her attention to the manuscript, and for his/her comments.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

As mentioned in the public review major weakness of the manuscript is the lack of analyses for confounding effects, overstatements of the results (using single amino acid sequence reporter) and the lack of discussion of previous work that argues against Tuller et al model. In my previous review I mentioned multiple other studies that addressed "slow codons" model in more detail.

No, the reviewer did not cite any specific studies.

While some of these studies are mentioned in the revised manuscript, authors are still rather biased and selective in their discussions. I should also point out that previous studies, that authors fail again to mention, were focused on either translation initiation, initiation to elongation transition or early elongation effects in relation to mRNA sequence, structure, codons as well as amino acid sequence. Also additional studies with bioinformatic analyses of N-terminal conservation and existence of start sites at the beginning of the protein sequences in multiple model organisms were also omitted.

Again, we do not know to what papers the reviewer is referring. But this sounds like a lot. Our paper is aimed at a specific, narrow topic: Why is there an excess of rare, slow codons in the 5’ region of genes? We are not trying to make general statements about all things affecting and affected by translation speed, we are just trying to explain the excess of rare, slow codons.

In general manuscript seems to be too much focused-on discussion of Tuller's paper . . .

Yes, we are focused on the Tuller findings, the excess of rare slow codons in 5’ regions.

. . . and arguing with the model that was already shown by multiple other studies to be limited and not correct.

We find it unsatisfactory that the reviewer states in a public review that there are multiple other studies showing that the Tuller model is not correct, and yet does not cite any of them. Furthermore, for the reviewer to say that Tuller et al. is “not correct” is too sweeping. The core finding of Tuller et al. was the excess of rare, slow codons in the 5’ regions of genes. We confirm this; we believe it is correct; we are not aware of any literature disputing this. Then, Tuller interpreted this as an adaptation to promote translational efficiency. On the interpretation, we disagree with Tuller. But if one is to disagree with this interpretation, one needs an alternative explanation of the fact of the excess rare, slow codons. Providing such an alternative explanation, and doing an experiment to distinguish the explanations, is our contribution. We are not aware of any other paper making our interpretation.

There are of course many papers that discuss various aspects of translation at the 5’ ends of genes, and we do cite quite a few such papers in our manuscript, though certainly not all. But papers of this general kind do not, and cannot, show that Tuller et al. is “not correct”. As far as we know, no paper provides an alternative explanation for the rare slow codons, and no paper does an experiment to modulate translation speed and look at the effect on gene expression. Notably, the slow translation phenomenon associated with the rare codons found by Tuller et al. is a very small effect—a change of about 1% to 3% of translation speed. Some other papers on translation speed are dealing with possible changes in the range of 50% or more. These are presumably some other phenomenon (if indeed they are even real changes in translation speed), and, whether they are true or not, the results and interpretations of Tuller et al. could still be true or not. Of course, if we knew of some previous paper showing the Tuller paper is not correct, we should and would cite it.

To expand on the current view of Tuller in the literature, Tuller et al. has been cited 956 times according to Google Scholar. This makes it an extremely influential paper. After finding Tuller et al. in Entrez Pubmed, one can look under “Cited by” and see the five most recent papers that cite Tuller et al. The five papers given on May 23 2024 were Bharti . . . Ignatova 2024; Uddin 2024; Khandia . . . Choudhary 2024; Love and Nair 2024; and Oelschlaeger 2024. We went through these five most recent papers that cite Tuller et al., and asked, did these authors cite the Tuller results as fully correct, or did they mention any doubts about the results? All five of the papers cited the Tuller results as fully correct, with no mention of any kind of doubt. For instance, Kandia et al. 2024 state “The slow “ramp” present at 5’ end of mRNA forms an optimal and robust means to reduce ribosomal traffic jams, thus minimizing the cost of protein expression40.”, while Oelschlaeger (2024) states “Slow translation ramps have also been described elsewhere and proposed to prevent traffic jams along the mRNA [51,52,53].” Although Uddin (2024) cited Tuller as fully correct, Uddin seemed to think (it is a little unclear) that Tuller found an enrichment of highly-used codons, opposite to the actual finding. The multiple contrary studies mentioned by the reviewer do not seem to have been very influential.

There are papers containing skepticism about the Tuller interpretation, and also papers with results that are difficult to reconcile in a common-sense way with the Tuller interpretation. But skepticism, and a difficulty to reconcile with common sense, are far from a demonstration that a paper is incorrect. Indeed, Tuller et al. may have been published in Cell, and may be so highly cited, exactly because the findings are counter-intuitive, colliding with common sense. Our contribution is to find a common-sense interpretation of the surprising but correct underlying fact of the 5’ enrichment of rare, slow codons.

Having wrote that in the previous review, I have to admit that Sejour et al manuscript in the main text has a minimal amount of novelty with experimental evidence, the conclusions are based on three reporters with and without stalling/collision sequence with the same amino acid sequence and varying codons. Some more novelty is seen in bioinformatic analyses of multiple yeast sequences and sequence conservation at the N-termini of proteins. However, even this part of the manuscript is not discussed fully and with correct comparison to previous studies. Authors, based on my previous comments discuss further experimental shortcomings in their new and "expanded" discussion but the use of a single reporter in this case cannot relate to all differences that may be coming from ORFs seen in complete yeast transcriptome. There are multiple studies that used more reporters with more than one amino-acid and mRNA sequence as well as with similar variation of the rare or common codons. The handwaving argument about the influence of all other mechanisms that can arise from different start sites, RNA structure, peptide interaction with exit channel, peptidyl-tRNA drop-off, eIF3 complex initiation-elongation association, and etc, is just pointing up to a manuscript that is more about bashing up Tuller's model and old paper than trying to make a concise story about their own results and discuss their study in plethora of studies that indicated multiple other models for slow early elongation.

We don’t understand why the reviewer is so grudging.

Discussion of the ribosome's collisions and potential impact of such scenario in the author's manuscript is left completely without citation, even though such work has relevant results to the author's conclusions and Tuller's model.

This is not true. We cite Dao Duc and Song (2018) “The impact of ribosomal interference, codon usage, and exit tunnel interactions on translation elongation rate variation.” PLoS Genet 14, and Tesina, . . . and Green (2020) “Molecular mechanism of translational stalling by inhibitory codon combinations and Poly(A) tracts. EMBO J., which are two excellent papers on this subject. We also cite Gamble et al. (2016), who found the underlying result, but at that time did not attribute it to ribosome collisions.

Previous studies (not cited) for example clearly indicate how the length from stalling sequence to start codon is related to ribosome collisions. Moreover such studies are pointing out differences in initiation vs elongation rates that may impact ribosome collisions and protein expression. Both of these topics would be very valuable in discussions of evolutionary changes in the current yeast ORFs. Not to mention that authors do not really discuss also possibilities for differences in 5'UTRs and uORFs in relation to downstream ORFs sequence and codon composition.

It is not clear to us that such papers are highly relevant to the issue on which we are working.

The argument about whether cycloheximide or not is doing 5' ribosome slowdown (lines 425-443) is just rambling about Weinberg's paper from 2016 without any real conclusion. In this section authors are just throwing down hypothesis that were more clearly explained in Weinberg's manuscript or shown experimentally in studies done after the Weinberg et al. paper was published.

Earlier, the reviewer had the criticism that “The studies that authors do not mention argue with "translation ramp" model and show more thorough analyses of translation initiation to elongation transition as well as early elongation "slow down" in ribosome profiling data.” The main study we know of dealing with these issues like these is that of Weinberg et al. 2016. In our opinion, this is a thoughtful paper on these issues. But now, at this point, the reviewer seems to criticize the fact that we do extensively cite results from Weinberg et al. It is true that there is no ultimate conclusion, but why there is no conclusion is a little bit interesting. Weinberg et al show that even in studies that do not use cycloheximide as the first step in ribosome profiling, there is some left-over high density of ribosomes near 5’ ends. But, all these ribosome profiling experiments do use cycloheximide at a later step in the procedure. Until someone does a ribosome profiling experiment without the use of any cycloheximide at any step, there will be no firm conclusion. This is not our fault—and also not the issue we are writing about. And, the reason this paragraph is in the manuscript at all is that the reviewer (we thought) had asked for something like this in the first review.

At the end, even in the limited novelty of evolutionary arguments about non-existing N-terminal conservation of codons or amino acids they fail to cite and discuss previous work by Kochetov (BioEssays, 2008 and NAR, 2011) which have additional explanation on evolution of N-terminal sequences in yeast, human or Drosophila.

These two papers of Dr. Kochetov’s have some relevance and we now cite them. These are the only papers cited by the reviewer in his/her two reviews.

Probably the reviewer would have preferred a paper on a different subject.

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

Response to Reviewers:

We thank the reviewers for their comments, and their evident close reading of the manuscript. Generally, we agree with the reviewers on the strengths and weaknesses of our manuscript. Our revised manuscript has a more extensive discussion of alternative explanations for initial high ribosome density as seen by ribosome profiling, and which more specifically points out the limitations of our work.

As a preface to specific responses to the reviewers, we will say that we could divide observations of slow initial translation into two categories, which we will call “encoded slow codons”, and “increased ribosome density”. With respect to the first category, Tuller et al. documented initial “encoded slow codons”, that is, there is a statistical excess of rare, slowly-translated codons at the 5’ ends of genes. Although the size of this effect is small, statistical significance is extremely high, and the existence of this enrichment is not in any doubt. At first sight, this appears to be a strong indication of a preference for slow initial translation. In our opinion, our main contribution is to show that there is an alternative explanation for this initial enrichment of rare, slow codons—that they are a spandrel, a consequence of sequence plasticity at the 5’ (and 3’) ends of genes. The reviewers seem to generally agree with this, and we are not aware that any other work has provided an explanation for the 5’ enrichment of rare codons.

The second category of observations pertaining to slow initial translation is “increased ribosome density”. Early ribosome profiling studies used cycloheximide to arrest cell growth, and these studies showed a higher density of ribosomes near the 5’ end of genes than elsewhere. This high initial ribosome density helped motivate the paper of Tuller et al., though their finding of “encoded slow codons” could explain only a very small part of the increased ribosome density. More modern ribosome profiling studies do not use cycloheximide as the first step in arresting translation, and in these studies, the density of ribosomes near the 5’ end of genes is greatly reduced. And yet, there remains, even in the absence of cycloheximide at the first step, a significantly increased density of ribosomes near the 5’ end (e.g., Weinberg et al., 2016). (However, most or all of these studies do use cycloheximide at a later step in the protocol, and the possibility of a cycloheximide artefact is difficult to exclude.) Some of the reviewer’s concerns are that we do not explain the increased 5’ ribosome density seen by ribosome profiling. We agree; but we feel it is not the main point of our manuscript. In revision, we more extensively discuss other work on increased ribosome density, and more explicitly point out the limitations of our manuscript in this regard. We also note, though, that increased ribosome density is not a direct measure of translation speed—it can have other causes.

Specific Responses.

Reviewer 1 was concerned that we did not more fully discuss other work on possible reasons for slow initial translation. We discuss such work more extensively in our revision. However, as far as we know, none of this work proposes a reason for the 5’ enrichment of rare, slow codons, and this is the main point of our paper. Furthermore, it is not completely clear that there is any slow initial translation. The increase in ribosome density seen in flash-freeze ribosome profiling could be an artefact of the use of cycloheximide at the thaw step of the protocols; or it could be a real measure of high ribosome density that occurs for some other reason than slow translation (e.g., ribosomes might have low processivity at the 5’ end).

Reviewer 1 was also concerned about confounding effects in our reporter gene analysis of the effects of different codons on efficiency of translation. We have two comments. First, it is important to remember that although we changed codons in our reporters, we did not change any amino acids. We changed codons only to synonymous codons. Thus at least one of the reviewer’s possible confounding effects—interactions of the nascent peptide chain with the exit channel of the ribosome—does not apply. However, of course, the mRNA nucleotide sequence is altered, and this would cause a change in mRNA structure or abundance, which could matter. We agree this is a limitation to our approach. However, to fully address it, we feel it would be necessary to examine a really large number of quite different sequences, which is beyond the scope of this work. Furthermore, mRNAs with low secondary structure at the 5’ end probably have relatively high rates of initiation, and also relatively high rates of elongation, and it might be quite difficult to disentangle these. But in neither case is there an argument that slow initial translation is efficient. Accurate measurement of mRNA levels would be helpful, but would not disentangle rates of initiation from rates of elongation as causes of changes in expression.

Reviewer 2 was concerned that the conservation scores for the 5’ 40 amino acids, and the 3’ 40 amino acids were similar, but slow translation was only statistically significant for the 5’ 40 amino acids. As we say in the manuscript, we are also puzzled by this. We note that 3’ translation is statistically slow, if one looks over the last 100 amino acids. Our best effort at an explanation is a sort of reverse-Tuller explanation: that in the last 40 amino acids, the new slow codons created by genome plasticity are fairly quickly removed by purifying selection, but that in the first 40 amino acids, for genes that need to be expressed at low levels, purifying selection against slow codons is reduced, because poor translation is actually advantageous for these genes. To expand on this a bit, we feel that the 5000 or so proteins of the proteome have to be expressed in the correct stoichiometric ratios, and that poor translation can be a useful tool to help achieve this. In this explanation, slow translation at the 5’ end is bad for translation (in agreement with our reporter experiments), but can be good for the organism, when it occurs in front of a gene that needs to be expressed poorly. Whereas, in Tuller, slow translation at the 5’ end is good for translation.

Reviewer 2 wondered whether the N-terminal fusion peptide affects GFP fluorescence in our reporter. This specific reporter, with this N-terminus, has been characterized by Dean and Grayhack (2012), and by Gamble et al. (2016), and the idea that a super-folder GFP reporter is not greatly affected by N-terminal fusions is based on the work of Pedelacq (2006). None of these papers show whether this N-terminal fusion might have some effect, but together, they provide good reason to think that any effect would be small. These citations have been added.

Rematch of the Parts

Author response:

Reviewer #1 (Public Review):

Abbasi et al. assess in this MEG study the directed connectivity of both cortical and subcortical regions during continuous speech production and perception. The authors observed bidirectional connectivity patterns between speech-related cortical areas as well as subcortical areas in production and perception. Interestingly, they found in speaking low-frequency connectivity from subcortical (the right cerebellum) to cortical (left superior temporal) areas, while connectivity from the cortical to subcortical areas was in the high frequencies. In listening a similar cortico-subcortical connectivity pattern was observed for the low frequencies, but the reversed connectivity in the higher frequencies was absent.

The work by Abbasi and colleagues addresses a relevant, novel topic, namely understanding the brain dynamics between speaking and listening. This is important because traditionally production and perception of speech and language are investigated in a modality-specific manner. To have a more complete understanding of the neurobiology underlying these different speech behaviors, it is key to also understand their similarities and differences. Furthermore, to do so, the authors utilize state-of-the-art directed connectivity analyses on MEG measurements, providing a quite detailed profile of cortical and subcortical interactions for the production and perception of speech. Importantly, and perhaps most interesting in my opinion, is that the authors find evidence for frequency-specific directed connectivity, which is (partially) different between speaking and listening. This could suggest that both speech behaviors rely (to some extent) on similar cortico-cortical and cortico-subcortical networks, but different frequency-specific dynamics.

These elements mentioned above (investigation of both production and perception, both cortico-cortical and cortico-subcortical connectivity is considered, and observing frequency-specific connectivity profiles within and between speech behaviors), make for important novel contributions to the field. Notwithstanding these strengths, I find that they are especially centered on methodology and functional anatomical description, but that precise theoretical contributions for neurobiological and cognitive models of speech are less transparent. This is in part because the study compares speech production and perception in general, but no psychophysical or psycholinguistic manipulations are considered. I also have some critical questions about the design which may pose some confounds in interpreting the data, especially with regard to comparing production and perception.

(1) While the cortico-cortical and cortico-subcortical connectivity profiles highlighted in this study and the depth of the analyses are impressive, what these data mean for models of speech processing remains on the surface. This is in part due, I believe, to the fact that the authors have decided to explore speaking and listening in general, without targeting specific manipulations that help elucidate which aspects of speech processing are relevant for the particular connectivity profiles they have uncovered. For example, the frequency-specific directed connectivity is it driven by low-level psychophysical attributes of the speech or by more cognitive linguistic properties? Does it relate to the monitoring of speech, timing information, and updating of sensory predictions? Without manipulations trying to target one or several of these components, as some of the referenced work has done (e.g., Floegel et al., 2020; Stockert et al., 2021; Todorović et al., 2023), it is difficult to draw concrete conclusions as to which representations and/or processes of speech are reflected by the connectivity profiles. An additional disadvantage of not having manipulations within each speech behavior is that it makes the comparison between listening and speaking harder. That is, speaking and listening have marked input-output differences which likely will dominate any comparison between them. These physically driven differences (or similarities for that matter; see below) can be strongly reduced by instead exploring the same manipulations/variables between speaking and listening. If possible (if not to consider for future work), it may be interesting to score psychophysical (e.g., acoustic properties) or psycholinguistic (e.g., lexical frequency) information of the speech and see whether and how the frequency-specific connectivity profiles are affected by it.

We thank the reviewer for pointing this out. The current study is indeed part of a larger project investigating the role of the internal forward model in speech perception and production. In the original, more comprehensive study, we also included a masked condition where participants produced speech as usual, but their auditory perception was masked. This allowed us to examine how the internal forward model behaves when it doesn't receive the expected sensory consequences of generated speech. However, for the current study, we focused solely on data from the speaking and listening conditions due to its specific research question. We agree that further manipulations would be interesting. However, for this study our focus was on natural speech and we avoided other manipulations (beyond masked speech) so that we can have sufficiently long recording time for the main speaking and listening conditions.

(2) Recent studies comparing the production and perception of language may be relevant to the current study and add some theoretical weight since their data and interpretations for the comparisons between production and perception fit quite well with the observations in the current work. These studies highlight that language processes between production and perception, specifically lexical and phonetic processing (Fairs et al., 2021), and syntactic processing (Giglio et al., 2024), may rely on the same neural representations, but are differentiated in their (temporal) dynamics upon those shared representations. This is relevant because it dispenses with the classical notion in neurobiological models of language where production and perception rely on (partially) dissociable networks (e.g., Price, 2010). Rather those data suggest shared networks where different language behaviors are dissociated in their dynamics. The speech results in this study nicely fit and extend those studies and their theoretical implications.

We thank the reviewer for the suggestion and we will include these references and the points made by the reviewer in our revised manuscript.

(3) The authors align the frequency-selective connectivity between the right cerebellum and left temporal speech areas with recent studies demonstrating a role for the right cerebellum for the internal modelling in speech production and monitoring (e.g., Stockert et al., 2021; Todorović et al., 2023). This link is indeed interesting, but it does seem relevant to point out that at a more specific scale, it does not concern the exact same regions between those studies and the current study. That is, in the current study the frequency-specific connectivity with temporal regions concerns lobule VI in the right cerebellum, while in the referenced work it concerns Crus I/II. The distinction seems relevant since Crus I/II has been linked to the internal modelling of more cognitive behavior, while lobule VI seems more motor-related and/or contextual-related (e.g., D'Mello et al., 2020; Runnqvist et al., 2021; Runnqvist, 2023).

We thank the reviewer for their insightful comment. The reference was intended to provide evidence for the role of the cerebellum in internal modelling in speech. We do not claim that we have the spatial resolution with MEG to reliably spatially resolve specific parts of the cerebellum.

(4) On the methodological side, my main concern is that for the listening condition, the authors have chosen to play back the speech produced by the participants in the production condition. Both the fixed order as well as hearing one's own speech as listening condition may produce confounds in data interpretation, especially with regard to the comparison between speech production and perception. Could order effects impact the observed connectivity profiles, and how would this impact the comparison between speaking and listening? In particular, I am thinking of repetition effects present in the listening condition as well as prediction, which will be much more elevated for the listening condition than the speaking condition. The fact that it also concerns their own voice furthermore adds to the possible predictability confound (e.g., Heinks-Maldonado et al., 2005). In addition, listening to one's speech which just before has been articulated may, potentially strategically even, enhance inner speech and "mouthing" in the participants, hereby thus engaging the production mechanism. Similarly, during production, the participants already hear their own voice (which serves as input in the subsequent listening condition). Taken together, both similarities or differences between speaking and listening connectivity may have been due to or influenced by these order effects, and the fact that the different speech behaviors are to some extent present in both conditions.

This is a valid point raised by the reviewer. By listening to their own previously produced speech, our participants might have anticipated and predicted the sentences easier. However, during designing our experiment, we tried to lower the chance of this anticipation by several steps. First, participants were measured in separate sessions for speech production and perception tasks. There were always several days' intervals between performing these two conditions. Secondly, our questions were mainly about a common/general topic. Consequently, participants may not remember their answers completely.

Importantly, using the same stimulus material for speaking and listening guaranteed that there was no difference in the low-level features of the material for both conditions that could have affected the results of our statistical comparison.

Due to bone conduction, hearing one’s unaltered own speech from a recording may seem foreign and could lead to unwanted emotional reactions e.g. embarrassment, so participants were asked whether they heard their own voice in a recording already (e.g. from a self-recorded voice-message in WhatsApp) which most of them confirmed. Participants were also informed that they were going to hear themselves during the measurement to further reduce unwanted psychophysiological responses.

(5) The ability of the authors to analyze the spatiotemporal dynamics during continuous speech is a potentially important feat of this study, given that one of the reasons that speech production is much less investigated compared to perception concerns motor and movement artifacts due to articulation (e.g., Strijkers et al., 2010). Two questions did spring to mind when reading the authors' articulation artifact correction procedure: If I understood correctly, the approach comes from Abbasi et al. (2021) and is based on signal space projection (SSP) as used for eye movement corrections, which the authors successfully applied to speech production. However, in that study, it concerned the repeated production of three syllables, while here it concerns continuous speech of full words embedded in discourse. The articulation and muscular variance will be much higher in the current study compared to three syllables (or compared to eye movements which produce much more stable movement potentials compared to an entire discourse). Given this, I can imagine that corrections of the signal in the speaking condition were likely substantial and one may wonder (1) how much signal relevant to speech production behavior is lost?; (2) similar corrections are not necessary for perception, so how would this marked difference in signal processing affect the comparability between the modalities?

One of the results of our previous study (Abbasi et al., 2021) was that the artefact correction was not specific to individual syllables but generalised across syllables. Also, the repeated production of syllables was associated with substantial movements of the articulators mimicking those observed during naturalistic speaking. We therefore believe that the artefact rejection is effective during speaking. We also checked this by investigating speech related coherence in brain parcels in spatial proximity to the articulators. In our previous study we also show that the correction method retains neural activity to a very large degree. We are therefore confident that speaking and listening conditions can be compared and that the loss of true signals from correcting the speaking data will be minor.

References:

• Abbasi, O., Steingräber, N., & Gross, J. (2021). Correcting MEG artifacts caused by overt speech. Frontiers in Neuroscience, 15, 682419.

• D'Mello, A. M., Gabrieli, J. D., & Nee, D. E. (2020). Evidence for hierarchical cognitive control in the human cerebellum. Current Biology, 30(10), 1881-1892.

• Fairs, A., Michelas, A., Dufour, S., & Strijkers, K. (2021). The same ultra-rapid parallel brain dynamics underpin the production and perception of speech. Cerebral Cortex Communications, 2(3), tgab040.

• Floegel, M., Fuchs, S., & Kell, C. A. (2020). Differential contributions of the two cerebral hemispheres to temporal and spectral speech feedback control. Nature Communications, 11(1), 2839.

• Giglio, L., Ostarek, M., Sharoh, D., & Hagoort, P. (2024). Diverging neural dynamics for syntactic structure building in naturalistic speaking and listening. Proceedings of the National Academy of Sciences, 121(11), e2310766121.

• Heinks‐Maldonado, T. H., Mathalon, D. H., Gray, M., & Ford, J. M. (2005). Fine‐tuning of auditory cortex during speech production. Psychophysiology, 42(2), 180-190.

• Price, C. J. (2010). The anatomy of language: a review of 100 fMRI studies published in 2009. Annals of the new York Academy of Sciences, 1191(1), 62-88.

• Runnqvist, E., Chanoine, V., Strijkers, K., Pattamadilok, C., Bonnard, M., Nazarian, B., ... & Alario, F. X. (2021). Cerebellar and cortical correlates of internal and external speech error monitoring. Cerebral Cortex Communications, 2(2), tgab038.

• Runnqvist, E. (2023). Self-monitoring: The neurocognitive basis of error monitoring in language production. In Language production (pp. 168-190). Routledge.

• Stockert, A., Schwartze, M., Poeppel, D., Anwander, A., & Kotz, S. A. (2021). Temporo-cerebellar connectivity underlies timing constraints in audition. Elife, 10, e67303.

• Strijkers, K., Costa, A., & Thierry, G. (2010). Tracking lexical access in speech production: electrophysiological correlates of word frequency and cognate effects. Cerebral cortex, 20(4), 912-928.

• Todorović, S., Anton, J. L., Sein, J., Nazarian, B., Chanoine, V., Rauchbauer, B., ... & Runnqvist, E. (2023). Cortico-cerebellar monitoring of speech sequence production. Neurobiology of Language, 1-21.

Reviewer #2 (Public Review):

Summary:

The authors re-analyse MEG data from a speech production and perception study and extend their previous Granger causality analysis to a larger number of cortical-cortical and in particular cortical-subcortical connections. Regions of interest were defined by means of a meta-analysis using Neurosynth.org and connectivity patterns were determined by calculating directed influence asymmetry indices from the Granger causality analysis results for each pair of brain regions. Abbasi et al. report feedforward signals communicated via fast rhythms and feedback signals via slow rhythms below 40 Hz, particularly during speaking. The authors highlight one of these connections between the right cerebellum lobule VI and auditory association area A5, where in addition the connection strength correlates negatively with the strength of speech tracking in the theta band during speaking (significant before multiple comparison correction). Results are interpreted within a framework of active inference by minimising prediction errors.

While I find investigating the role of cortical-subcortical connections in speech production and perception interesting and relevant to the field, I am not yet convinced that the methods employed are fully suitable to this endeavour or that the results provide sufficient evidence to make the strong claim of dissociation of bottom-up and top-down information flow during speaking in distinct frequency bands.

Strengths:

The investigation of electrophysiological cortical-subcortical connections in speech production and perception is interesting and relevant to the field. The authors analyse a valuable dataset, where they spent a considerable amount of effort to correct for speech production-related artefacts. Overall, the manuscript is well-written and clearly structured.

Weaknesses:

The description of the multivariate Granger causality analysis did not allow me to fully grasp how the analysis was performed and I hence struggled to evaluate its appropriateness. Knowing that (1) filtered Granger causality is prone to false positives and (2) recent work demonstrates that significant Granger causality can simply arise from frequency-specific activity being present in the source but not the target area without functional relevance for communication (Schneider et al. 2021) raises doubts about the validity of the results, in particular with respect to their frequency specificity. These doubts are reinforced by what I perceive as an overemphasis on results that support the assumption of specific frequencies for feedforward and top-down connections, while findings not aligning with this hypothesis appear to be underreported. Furthermore, the authors report some main findings that I found difficult to reconcile with the data presented in the figures. Overall, I feel the conclusions with respect to frequency-specific bottom-up and top-down information flow need to be moderated and that some of the reported findings need to be checked and if necessary corrected.

Major points

(1) I think more details on the multivariate GC approach are needed. I found the reference to Schaum et al., 2021 not sufficient to understand what has been done in this paper. Some questions that remained for me are:

(i) Does multivariate here refer to the use of the authors' three components per parcel or to the conditioning on the remaining twelve sources? I think the latter is implied when citing Schaum et al., but I'm not sure this is what was done here?

If it was not: how can we account for spurious results based on indirect effects?

Yes, multivariate refers to the three components.

(ii) Did the authors check whether the GC of the course-target pairs was reliably above the bias level (as Schaum et. al. did for each condition separately)? If not, can they argue why they think that their results would still be valid? Does it make sense to compute DAIs on connections that were below the bias level? Should the data be re-analysed to take this concern into account?

We performed statistics on DAI and believe that this is a valid approach. We argue that random GC effects would not survive our cluster-corrected statistics.

(iii) You may consider citing the paper that introduced the non-parametric GC analysis (which Schaum et al. then went on to apply): Dhamala M, Rangarajan G, Ding M. Analyzing Information Flow in Brain Networks with Nonparametric Granger Causality. Neuroimage. 2008; 41(2):354-362. https://doi.org/10.1016/j.neuroimage.2008.02. 020

Thanks, we will add this reference in the revised version.

(2) GC has been discouraged for filtered data as it gives rise to false positives due to phase distortions and the ineffectiveness of filtering in the information-theoretic setting as reducing the power of a signal does not reduce the information contained in it (Florin et al., 2010; Barnett and Seth, 2011; Weber et al. 2017; Pinzuti et al., 2020 - who also suggest an approach that would circumvent those filter-related issues). With this in mind, I am wondering whether the strong frequency-specific claims in this work still hold.

This must be a misunderstanding. We are aware of the problem with GC on filtered data. But GC was here computed on broadband data and not in individual frequency bands.

(3) I found it difficult to reconcile some statements in the manuscript with the data presented in the figures:

(i) Most notably, the considerable number of feedforward connections from A5 and STS that project to areas further up the hierarchy at slower rhythms (e.g. L-A5 to R-PEF, R-Crus2, L CB6 L-Tha, L-FOP and L-STS to R-PEF, L-FOP, L-TOPJ or R-A5 as well as R-STS both to R-Crus2, L-CB6, L-Th) contradict the authors' main message that 'feedback signals were communicated via slow rhythms below 40 Hz, whereas feedforward signals were communicated via faster rhythms'. I struggled to recognise a principled approach that determined which connections were highlighted and reported and which ones were not.

(ii) "Our analysis also revealed robust connectivity between the right cerebellum and the left parietal cortex, evident in both speaking and listening conditions, with stronger connectivity observed during speaking. Notably, Figure 4 depicts a prominent frequency peak in the alpha band, illustrating the specific frequency range through which information flows from the cerebellum to the parietal areas." There are two peaks discernible in Figure 4, one notably lower than the alpha band (rather theta or even delta), the other at around 30 Hz. Nevertheless, the authors report and discuss a peak in the alpha band.

(iii) In the abstract: "Notably, high-frequency connectivity was absent during the listening condition." and p.9 "In contrast with what we reported for the speaking condition, during listening, there is only a significant connectivity in low frequency to the left temporal area but not a reverse connection in the high frequencies."

While Fig. 4 shows significant connectivity from R-CB6 to A5 in the gamma frequency range for the speaking, but not for the listening condition, interpreting comparisons between two effects without directly comparing them is a common statistical mistake (Makin and Orban de Xivry). The spectrally-resolved connectivity in the two conditions actually look remarkably similar and I would thus refrain from highlighting this statement and indicate clearly that there were no significant differences between the two conditions.

(iv) "This result indicates that in low frequencies, the sensory-motor area and cerebellum predominantly transmit information, while in higher frequencies, they are more involved in receiving it."

I don't think that this statement holds in its generality: L-CB6 and R-3b both show strong output at high frequencies, particularly in the speaking condition. While they seem to transmit information mainly to areas outside A5 and STS these effects are strong and should be discussed.

We appreciate the reviewer's thoughtful comments. We acknowledge that not all connectivity patterns strictly adhere to the initial observation regarding feedback and feedforward communication. It's true that our primary focus was on interactions between brain regions known to be crucial for speech prediction, including auditory, somatosensory, and cerebellar areas. However, we also presented connectivity patterns across other regions to provide a more comprehensive picture of the speech network. We believe this broader perspective can be valuable for future research directions.

Regarding the reviewer's observation about the alpha band peak in Figure 4, we agree that a closer examination reveals the connectivity from right cerebellum to the left parietal is in a wider low frequency range. We will refrain from solely emphasizing the alpha band and acknowledge the potential contribution of lower frequencies to cerebellar-parietal communication.

We also appreciate the reviewer highlighting the need for a more nuanced interpretation of the listening condition connectivity compared to the speaking condition. The reviewer is correct in pointing out that while Figure 4 suggests a high-frequency connectivity from L-A5 to R-CB only in the speaking condition, a direct statistical comparison between conditions might not reveal a significant difference. We will revise the manuscript to clarify this point.

Finally, a closer examination of Figure 3 revealed that the light purple and dark green edges in the speaking condition for R-CB6 and L-3b suggest outgoing connections at low frequencies, while other colored edges indicate information reception at high frequencies. We acknowledge that exceptions to this directional pattern might exist and warrant further investigation in future studies.

(4) "However, definitive conclusions should be drawn with caution given recent studies raising concerns about the notion that top-down and bottom-up signals can only be transmitted via separate frequency channels (Ferro et al., 2021; Schneider et al., 2021; Vinck et al., 2023)."

I appreciate this note of caution and think it would be useful if it were spelled out to the reader why this is the case so that they would be better able to grasp the main concerns here. For example, Schneider et al. make a strong point that we expect to find Granger-causality with a peak in a specific frequency band for areas that are anatomically connected when the sending area shows stronger activity in that band than the receiving one, simply because of the coherence of a signal with its own linear projection onto the other area. The direction of a Granger causal connection would in that case only indicate that one area shows stronger activity than the other in the given frequency band. I am wondering to what degree the reported connectivity pattern can be traced back to regional differences in frequency-specific source strength or to differences in source strength across the two conditions.

This is indeed an important point. That is why we are discussing our results with great caution and specifically point the reader to the relevant literature. We are indeed thinking about a future study where we investigate this connectivity using other connectivity metrics and a detailed consideration of power.

Reviewer #3 (Public Review):

In the current paper, Abbasi et al. aimed to characterize and compare the patterns of functional connectivity across frequency bands (1 Hz - 90 Hz) between regions of a speech network derived from an online meta-analysis tool (Neurosynth.org) during speech production and perception. The authors present evidence for complex neural dynamics from which they highlight directional connectivity from the right cerebellum to left superior temporal areas in lower frequency bands (up to beta) and between the same regions in the opposite direction in the (lower) high gamma range (60-90 Hz). Abbasi et al. interpret their findings within the predictive coding framework, with the cerebellum and other "higher-order" (motor) regions transmitting top-down sensory predictions to "lower-order" (sensory) regions in the lower frequencies and prediction errors flowing in the opposite direction (i.e., bottom-up) from those sensory regions in the gamma band. They also report a negative correlation between the strength of this top-down functional connectivity and the alignment of superior temporal regions to the syllable rate of one's speech.

Strengths:

(1) The comprehensive characterization of functional connectivity during speaking and listening to speech may be valuable as a first step toward understanding the neural dynamics involved.

(2) The inclusion of subcortical regions and connectivity profiles up to 90Hz using MEG is interesting and relatively novel.

(3) The analysis pipeline is generally adequate for the exploratory nature of the work.

Weaknesses:

(1) The work is framed as a test of the predictive coding theory as it applies to speech production and perception, but the methodological approach is not suited to this endeavor.

We agree that we cannot provide definite evidence for predictive coding in speech production and perception and we believe that we do not make that claim in the manuscript. However, our results are largely consistent with what can be expected based on predictive coding theory.

(2) Because of their theoretical framework, the authors readily attribute roles or hierarchy to brain regions (e.g., higher- vs lower-order) and cognitive functions to observed connectivity patterns (e.g., feedforward vs feedback, predictions vs prediction errors) that cannot be determined from the data. Thus, many of the authors' claims are unsupported.

We will revise the manuscript to more clearly differentiate our results (e.g. directed Granger-Causality from A to B) from their interpretation (potentially indicating feedforward or feedback signals).

(3) The authors' theoretical stance seems to influence the presentation of the results, which may inadvertently misrepresent the (otherwise perfectly valid; cf. Abbasi et al., 2023) exploratory nature of the study. Thus, results about specific regions are often highlighted in figures (e.g., Figure 2 top row) and text without clear reasons.

Our connectograms reveal a multitude of results that we hope is interesting to the community. At the same time the wealth of findings poses a problem for describing them. We did not see a better way then to highlight specific connections of interest.

(4) Some of the key findings (e.g., connectivity in opposite directions in distinct frequency bands) feature in a previous publication and are, therefore, interesting but not novel.

We actually see this as a strength of the current manuscript. The computation of connectivity is here extended to a much larger sample of brain areas. It is reassuring to see that the previously reported results generalise to other brain areas.

(5) The quantitative comparison between speech production and perception is interesting but insufficiently motivated.

We thank the reviewer for this comment. We have addressed that in detail in response to the point (1&4) of reviewer 1.

(6) Details about the Neurosynth meta-analysis and subsequent selection of brain regions for the functional connectivity analyses are incomplete. Moreover, the use of the term 'Speech' in Neurosynth seems inappropriate (i.e., includes irrelevant works, yielding questionable results). The approach of using separate meta-analyses for 'Speech production' and 'Speech perception' taken by Abbasi et al. (2023) seems more principled. This approach would result, for example, in the inclusion of brain areas such as M1 and the BG that are relevant for speech production.

We agree that there are inherent limitations in automated meta-analysis tools such as Neurosynth. Papers are used in the meta-analysis that might not be directly relevant. However, Neurosynth has proven its usefulness over many years and has been used in many studies. We also agree that our selection of brain areas is not complete. But Granger Causality analysis of every pair of ROIs leads to complex results and we had to limit our selection of areas.

(7) The results involving subcortical regions are central to the paper, but no steps are taken to address the challenges involved in the analysis of subcortical activity using MEG. Additional methodological detail and analyses would be required to make these results more compelling. For example, it would be important to know what the coverage of the MEG system is, what head model was used for the source localization of cerebellar activity, and if specific preprocessing or additional analyses were performed to ensure that the localized subcortical activity (in particular) is valid.

There is a large body of evidence demonstrating that MEG can record signals from deep brain areas such as thalamus and cerebellum including Attal & Schwarz 2013, Andersen et al, Neuroimage 2020; Piastra et al., 2020; Schnitzler et al., 2009. These and other studies provide evidence that state-of-the-art recording (with multichannel SQUID systems) and analysis is sufficient to allow reconstruction of subcortical areas. However, spatial resolution is clearly reduced for these deep areas. We will add a statement in the revised manuscript to acknowledge this limitation.

(8) The results and methods are often detailed with important omissions (a speech-brain coupling analysis section is missing) and imprecisions (e.g., re: Figure 5; the Connectivity Analysis section is copy-pasted from their previous work), which makes it difficult to understand what is being examined and how. (It is also not good practice to refer the reader to previous publications for basic methodological details, for example, about the experimental paradigm and key analyses.) Conversely, some methodological details are given, e.g., the acquisition of EMG data, without further explanation of how those data were used in the current paper.

We will revise the relevant sections of the manuscript.

(9) The examination of gamma functional connectivity in the 60 - 90 Hz range could be better motivated. Although some citations involving short-range connectivity in these frequencies are given (e.g., within the visual system), a more compelling argument for looking at this frequency range for longer-range connectivity may be required.

Given previous evidence of connectivity in the gamma band we think that it would be a weakness to exclude this frequency band from analysis.

(10) The choice of source localization method (linearly constrained minimum variance) could be explained, particularly given that other methods (e.g. dynamic imaging of coherent sources) were specifically designed and might potentially be a better alternative for the types of analyses performed in the study.

Both LCMV and DICS are beamforming methods. We used LCMV because we wanted used Granger Causality which requires broadband signals. DICS would only provide frequency-specific band-limited signals.

(11) The mGC analysis needs to be more comprehensively detailed for the reader to be able to assess what is being reported and the strength of the evidence. Relatedly, first-level statistics (e.g., via estimation of the noise level) would make the mGC and DAI results more compelling.

We perform group-level cluster-based statistics on mGC while correcting for multiple comparisons across frequency bands and brain parcels and report only significant results. This is an established approach that is routinely used in this type of studies.

(12) Considering the exploratory nature of the study, it is essential for other researchers to continue investigating and validating the results presented in the current manuscript. Thus, it is concerning that data and scripts are not fully and openly available. Data need not be in its raw state to be shared and useful, which circumvents the stated data privacy concerns.

We acknowledge the reviewer's concern regarding the full availability of the dataset. Due to privacy limitations on the collected data, we are unable to share it publicly at this time. However, to promote transparency and enable further exploration, we have provided the script used for data analysis and an example dataset. This example dataset should provide a clear understanding of the data structure and variables used in the analysis. Additionally, we are happy to share the complete dataset upon request from research teams interested in performing in-depth secondary analyses.

how does a low-d manifold enable the warping of representations and thereby the mapping of sensory_inputs-> motor_outputs?

progress is being made.

https://en.wikipedia.org/wiki/Aldwych

Aldwych cognate with Aldrich?

?!

Reasonable attribution is the standard.

赵元任好厉害。《汉语口语语法》、《中国现代语言学的开拓和发展》和《语言问题》三本书，都找来读下。特别是《语言问题》。

没看懂。

typo キーに

typo　します

意味はよいのですが読みにくいと感じました

代案

Trueである限り

Don't see how they arrived at this "insight"