WHat is the point of fences when there is nothing to keep out? why does this wall or fence give them comfort when they dont desire to intrude on each other? It seems he wants to say something about them not needing it but doesnt want to give the wrong impression and somehow mess up the bond they already have. But if that were to somehow get in between the relationship they do have, how strong was their neighborly friendship to begin with?

This setup reminds me most of Reddit, where there are different subreddits, or in this case discussion boards, where people can contribute their own ideas, either commenting under others' posts or beginning a new one. It is quite fascinating to think of the WELL as one of the first kinds of social media that people engaged in. Equally important, I find it important to address what groups of people (white, male, middle-to-upper class) initially engaged in online community and conversation, and how the beginnings of social media have influenced the way it is today. I also find it fascinating that there wasn't much advertising and actual shopping that stemmed from posting these products online, but rather more of a conversation about them and it is quite interesting to see how social media has flipped on its head to engage with consumer purchases and influencer marketing.

I love that Brandon Jacob Jenkins goes in and out of being in character and being himself. I also love this disclaimer because it's true. We know how movies and our culture depicted slaves but we don't have any proof to whether or not that was true. We can make assumptions but those are just assumptions and not facts. I like that BJJ is trying to get the reader out of their head and forget about any preconceived notions they might have about slaves and give in to his play and his ideas.

A preface that I find extremely rare to be honesty. The casual tone Jacobs-Jenkins is speaking about race already is setting us up for a play that takes Boucicault's Octoroon and turns it on it's head. From this opening scene, to this very line, we are opened up to a world of questioning every damn thing. This line particularly struck me because it immediately challenges the stereotypes of slaves I have gained through my years of public school education. I have been in acting classes where characters that were slaves were written and read as extremely illiterate. And more importantly, many stories have slaves lumped into the one category of speech with NO diversity. This very simple direction in parenthesis challenged YEARS of a racist bias I had been carrying. Theatre is supposed to challenge you and give you a new perspective, this absolutely already did and I am on page 17.

I thought this bit here was odd. Bjj has just struck the audience with the fact that he in fact does not have a therapist. The conversation that he just had, was probably between himself, in his head (although another actor could have said those lines). I don't get why he does this. He gives the audience eye contact, while giving himself an intense wedgie without showing emotion.

Throughout most part of the play, George is portrayed as a young man from Paris with artistic background, who is purely in love with Zoe regardless of their difference in class or race, and who tries to save the slaves that have been working for the family. However, there are some scenes here and there that hint that no matter how nice he acts on the surface he still holds this discriminative thought. This line was just stuck in my head as this is such a disrespectful, inaccurate, and racist thing to say.

THIS IS SO TRUE. I resonate with this so much as a woman of color. It reminded me of a time I wrote a short scene about a boy getting caught drinking alcohol by the decorative moose head on his wall on his prom night. I chose to make the character Indian simply because I am Indian. I remember writing it and thinking it was fun, silly, and light. The next day, one of my classmates said she admired how I was trying to combat stereotypes within Indian / South Asian families about drinking alcohol being taboo within the culture. I felt exactly how BJJ feels here. Sure, sometimes things do have other deeper meanings, but other times, it's just stream of consciousness, writing about whatever you want. People always try to put anything people of color do into a box to fit what they think we're trying to do, when really, sometimes, we're just doing whatever we want. Not everything has some super deep, profound, cultural meaning all the time.

Compassion from the native Americans, showing that kindness is a universal trait not exclusive to White Christians.

Contrary to what is stated right above she is able to find a wigwam after two tries and it was "comfortable lodging". The Native American who ran that wigwam showed her kindness and even stated if she came back he would help her again. This shows a very different version from "being angry and sent back".

Veiled threat? What is Rowlandson perceiving in this?

When she is comfortable she is happy .

Reality check of what's really happening to her.

Despite everything, they still treat her with at least some respect.

attributing the thoughtfulness of the Natives to God. does she ever actually thank the Natives?

This line, through a menacing tone, is intended to signify the beginning of Hektor’s decline. Before this major shifting point in the story, Hektor has been portrayed as among the most “reasonable” in the ranks of the Trojans. Before this point his authority had not been put into any question, and had it been, his character would’ve been expected to take others into account. Instead this confrontation between Hektor and Poulydamas is reminiscent of the encounter between Agamemnon and Thersites in the beginning of the epic. Recalling this incident, Thersites had acted as a voice of reason-- bringing into question Agamemnon’s pride as well as the end goal of the Trojan War. The similarities between these two encounters-- both involving authority figures, both offering warning, and both resulting in the anger of those called into question-- reveals a parallel between the opposing forces that rule the epic, reinforcing the blurred distinctions of good and bad in the fight for glory.

Some interesting finds Josh.

Related to some of the bullet journal (aka BuJo) and journaling space you will eventually come across the idea of "morning pages" which is a technique where you spend a block of time (usually in the morning, but ideally just before you want to do your creative thinking work) where you write for a set amount of time or number of pages. The goal of this method (and to some extent bullet journaling) is to clear the cruft and extraneous details out of your head to be able to better prioritize and focus on your creative work. There's a relatively large group of people doing this as a technique, so even knowing the phrase can help one to find the literature.

Tangentially related to this and memory (via our old friend rhetoric), I've been doing some significant research into the commonplace book tradition and general note taking with an eye towards knowledge acquisition, creation, and spaced repetition systems. This has led into research into the areas of the zettelkasten, digital note taking, digital gardens and the like. All fascinating areas which overlap memory via rhetoric. I suspect that many mnemonists in the Renaissance used commonplace books as physical written memory palaces, though I've yet to find anything in my research that directly links them other than the relationship they have in the long tradition of rhetoric in Western culture. Since you mention music and writing lyrics, I recently noted that Eminem has a commonplace technique which he calls "stacking ammo" by which he compiles ideas for his lyrics. His method is certainly less structured than a traditional commonplace book, but the overall form traces back to our friends Aristotle, Cicero, and Quintilian.

If you delve into some of the Bullet Journal and journaling literature you'll find a subculture of people (YouTube has hundreds of people with entire channels dedicated to the topic) who write into their daily/weekly planners and decorate them with stickers, washi tape, photos, calligraphy, drawings, etc. I've called some of this "productivity porn" before, but if you search commonplace book on Instagram or Pinterest you'll find examples of people whose journals and notes are becoming physical memory palaces where the visuals are likely helping them remember portions of their lives or what they're writing. The stickers and images to some extent are serving the purpose of drolleries seen in Medieval manuscripts as mnemonic devices.

And finally, tangentially related to all of this is another interesting sub-genre of memory and note taking called sketchnotes which combines active listening, writing, and drawing into a mnemonic related note taking activity. I'm actually a bit surprised to find so little on the technique here on the forum. Searching for sketchnotes on social media will provide lots of examples and there are many "What are sketchnotes" short videos on YouTube that will give you an idea of what's going on. Many of these talk about a memory component, but not being mired into the sub-topic of rhetoric, they're usually not using the same framings we would (here on the forum), though the effects one might expect are the same.

Some related richer resources for these areas, to help people from going down the rabbit hole within the performative social media spaces:

• How to Take Smart Notes: One Simple Technique to Boost Writing, Learning and Thinking–for Students, Academics and Nonfiction Book Writers by Sönke Ahrens
  • This touches on note taking within a zettelkasten framing, but is also applicable to the commonplace book tradition
• Sketchnote Handbook, The: the illustrated guide to visual note taking by Mike Rohde
  • This is one of the bibles in the space and gives a solid overview of what, why, how, etc.
• A Brief History & Ethos of the Digital Garden by Maggie Appleton

The risk highlighted within the sentence is contributed to her long captivity and the hope of being sold to her husband and watching it tear apart in front of her. She is losing her faith therein her will and hope to survive and thrive.

is this a knock in the head as in a singular K.O or beat to death because comparing it to starvation is a bit much considering she still gets food when she does jobs for it

Id probably say the same thing

Is she growing less afraid of them or is she losing her will to live? Both?

It is interesting to see why the bishops of Palestine decreased dramatically during the 11th century. Was this the result of an Islamic development between the Fatimids and the Seljuks, or the Crusaders justifying that only one bishop could be the head of the communal body?

This is exactly how I was taught to identify subjects, and it's kind of difficult to break the habit that's been drilled into my head.

This whole part sounds like a supervillain monologue explaining their overly-complicated plan,

It is an odd circumstance that tail bands are still used on modern books that don't need them. From a manufacturing standpoint, the decrease in cost would dictate they disappear, however they must add some level of bookiness that they're worth that cost.

I knew that Sol Golomb had been collaborating on a textbook going back almost fifteen years. It's great to see it not only finally come out, but to see it published with his name in the title!

I had the pleasure of taking Sol's combinatorics class at USC several years before he passed away, so I also got an early look at much of the material as he was using it in class. It was scheduled at my lunchtime, so I took the time to drive over to USC at lunch twice a week to sit in. My favorite part was seeing proofs for various things I'd seen in other branches of mathematics, but done in a combinatorial way.

Somewhere knocking around I think I've got audio recordings and notes of the class that I'll have to do something with one day.

Many talk about Sol's ability to do calculations in his head, but like most mathematicians he knew the standard tricks and shortcuts. To me this was underlined by the fact that he always did long division on the board when there wasn't a simple short cut.

Although amazing that Pegasus was created from the beheading of Medusa, why is not considered a demi-god as he is the son of Poseidon?

But before Zeus ate Metis, she put Athena into the mind of Zeus. Soon after that, Zeus’ headache became so overbearing that he asked Hephaestus to cut his head open to let out what was inside there. That’s how Athena came to being born.

Why create endless punishment for the titans instead of killing them?

Creation of more gods, creatures, and monsters. Mentions of famous heroes

Hesse must have felt alone without being able to trust anyone, so he had to keep to himself as Siddhartha does.

This is showing how serious this could be and how easily it could lead to catastrophic events.

Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

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Reply to the reviewers

Reviewer #1:

Summary

Copy number variations in the 1q21.1 loci, deletions and duplications, have been associated with neurodevelopmental disease. In particular, deletions of this locus result in a variety of neuronal phenotypes including microcephaly and schizophrenia in varying levels of severity. Duplications of the 1q21.1 locus are often associated with autism and/or macrocephaly.

In this study Nomura et al. generated 1q21.1 deletion and duplication hESC lines to study the impact of these CNVs on neuronal development. They generated brain organoids and observed a bidirectional effect of this CNV on organoid size, with 1q21.1 deletion showing smaller brain organoids whereas, the 1q21.1 dup lines grew large than controls. This in line with observed micro and macrocephaly observed in patients. They further analyzed these organoids at the gene expression level using single cell RNAseq and performed some electrophysiological assessment on neurons from of dissociated organoids.

This study is certainly of interest given the association of this loci with NDDs such as autism, epilepsy and schizophrenia. At this stage, the study is mainly a descriptive study, showing differences between the 1q21.1 del/dup versus controls but also between both the del/dup lines. There is no mechanistic insight provided. For example the 1q21.1 CNV encompasses several genes, of which some have already been linked to micro/macrocephaly (eg. NOTH2NL). More importantly, most of the conclusions drawn by the authors are based on a limited set of experiments/analysis which are not always carefully performed and/or presented. In general, the data presented are premature, therefore not supporting the claims/conclusion made by the author (eg title) This makes the overall impact of this study limited.

As the reviewer pointed out, NOTCH2NL (both A and B) have been regarded as micro/macrocephaly-related genes (Fiddes et al., Cell, 2018; Suzuki et al., Cell, 2018). In this study, however, we focused on the distal region of 1q21.1 between BP3 and BP4, which contains neither NOTCH2NLA nor NOTCH2NLB, because the target site is thought to be the core region of clinical 1q21.1 microdeletion/microduplication syndrome (Mefford et al., NEJM., 2008; Brunetti-Pierri et al., Nat. Genet., 2008; Van Dijck et al., EJMG, 2015). Although both NOTCH2NLA and B are located outside of our target, these genes are important for human neocortical development and neurogenesis, so we cite these papers (Fiddes et al. and Suzuki et al.) and discuss them in the discussion of the revised manuscript.

Main comments

In general, the interpretation of the data is too premature:

1. The title is not supported in any means by data

As requested by the reviewer, we have corrected the title as “Modeling reciprocal CNVs of chromosomal 1q21.1 in cortical organoids reveals alterations in neurodevelopment”.

1. Brain organoids size and development: In figure 2 the authors analyzed the development of the organoids. Based on the human phenotype the deletion would lead to smaller brain and the duplication to larger brain organoids. The presented data to support these claims are rather scarce. They indeed provide data on organoid size, however there is no information as to regard how this micro/macrocpehaly comes about. Only limited amount of cell types are being investigated with immunocytochemistry, which give little insight into the mechanism. Fig 3. The authors performed some very basic immunostaining and concluded that the neuronal maturity of 1q del seemed to be accelerated, whereas 1q dup decelerated from the NPC stage. However, there is no direct evidence provided for this. With simple additional immunostainings authors could already get a much better idea of what is going on. For example the authors could measure the amount of differentiating versus proliferating cells, cell cycle exit, etc (eg BrDU, KI67, pHH3 staining,...)

We thank the reviewer for the suggestion. In response to this, we plan to analyze additional markers such as phosphor-histone H3 (pHH3) to evaluate the late-G2/M status by immunostaining. In addition, to explain the smaller organoid size observed in 1q del organoids, we will check apoptosis markers such as cleaved-caspase3 by immunostaining and western blotting.

Further there are some technical aspect that would need to be resolved:

There is a general lack of brain organoid characterization of the controls. It is unclear on how many independent clones these experiments were performed.

We constructed one clone per genotype (1q21.1 deletion (1q del), 1q21.1 duplication (1q dup) and CTRL) from one human ES cell strain (khES-1) by next-generation chromosome engineering using the CRISPR/Cas9 system. According to the reviewer’s comment, we have added the information of each clone, including the actual number of each clone in the results section. Following the reviewer’s comment, we also recognized the importance of comparing targeted clones even in the same genotype to verify cellular phenotypes in a targeted clone. However, we consider that at least isogenic ES cell lines are less affected by genetic variances on other regions and epigenetic changes than patients-derived iPS cells.

• Fig 2C: it is unclear why brain organoid sizes reduce over time. Is this an indication of increased apoptosis? Did the authors measure this?

In order to respond to the reviewer’s comment, we plan to examine apoptotic markers such as cleaved caspase-3 by immunostaining or western blotting, as mentioned above.

• What is the reason for using t-test with Bonferroni correction as opposed to one -way (or even two-way) Anova is unclear in Fig 2C

Analysis of variance (ANOVA) has been regarded as optional when multiple comparisons without F-statistics are performed (Jason Hsu. 1996. Multiple Comparisons: Theory and Methods (Guilford School Practitioner)). We selected the Bonferroni test because we thought we could evaluate our data more strictly with the Bonferroni test than with the Tukey-Kramer test. In response to the reviewer’s request, we analyzed our data using one- way ANOVA with the Tukey-Kramer test. We confirmed that statistical significances were consistent (we can provide both data if requested). We have changed the description in the figure legend and methods section of the revised manuscript.

• 2E is unclear how they came to the conclusion that dosage dependent size difference in NPC organoids was caused by the number of cells within an organoid, not by the size of each cell or different cell types. Since they only measured the amount of Sox 2 positive cells and used Sox2 to measure cell diameter, whereas Sox2 is mainly expressed in the nucleus.

We thank the reviewer’s comment. We used images of SOX2 staining because contrasts of each cell in bright-field images were too obscure to be detected using the fluorescent microscopy, BZ-X analyzer, and because we found cell sizes seemed similar between bright-field images and SOX2 staining images. However, this method was not desirable. To respond to the reviewer’s comment, we have counted the number of cells in the images of each NPC organoid using the BZ-X analyzer and calculate the cell number per 1000 µm2. We found the cell density was not significantly different among the 3 genotypes. We understand that counting the cell number of a single organoid would be ideal, but it was impossible because each NPC organoid was too small. We have changed Figure 2E, descriptions in the methods and results section, and the corresponding figure legend in the revised manuscript.

• How do the authors explain that the Dup cells do not express Tubb neither CTIP2, do they only express NPCs and no neurons?

We consider this finding supports the immaturity in the cortical organoids with 1q21 duplication. However, we have checked only a few markers for intermediate progenitors and mature neurons so far. We plan to examine immature neuronal markers such as DCX and other mature neuronal markers such as NeuN by immunocytochemistry (ICC) to confirm this finding. Similarly, we will perform expression analysis by real-time qPCR to check mature and immature neuronal cell markers.

In short, the characterization of the brain organoids at the level of general development, cell types, proliferation, differentiation is underdeveloped.

We will examine the characterization of the brain organoids in more detail by different techniques as described above.

1. Electrophysiological assessment of brain organoids derived neurons:

In figure 4 the authors claim that both CNVs (Del/Dup) show hyperexcitability and altered expressions of glutamate system as common features between the Del/Dup lines. The data to support this are however scarce and far from being convincing:

The poor quality of the data is represented by images in 4B-E:

• First the authors choose to dissociate the organoids prior to measure the cells on MEA's. This takes away the advantage of 3D brain organoids, will add a lot of non-physiological stress, cause cell death and lead to unequal distribution of cells over the electrodes, see fig 4B.

We are afraid that the reviewer might misunderstand our experiment. In this experiment, we used not 3-D brain organoids but 2-D neurons. Based on established neural differentiation protocol (Fujimori et al., Stem Cell Reports, 2017, Toyoshima et al., Transl. Psychiatry, 2016, Matsumoto et al., Stem Cell Reports, 2016), we seeded single-cells dissociated from neurospheres on MEA dishes at the same density (8 x 105 cells per dish) on day 33 and continued culturing for 28 days on the MEA dish before analysis. Thus, we didn’t dissociate cells just before analysis. We could avoid adding non-physiological stress because we kept on culturing on the MEA dish for 28 days.

• MEA recording are meant to measure network activity and heavily (read: fully) dependent on the network being formed. Cherry picking electrodes for analysis is not justified, analysis should be performed per MEA chip not per electrode. Inclusion/exclusion parameters should be defined before analysis

We have performed statistical analysis with all chips (electrodes) per genotype in response to the reviewer's request. Even though the distributions of firing rate were not consistent among electrodes, we found the significant differences between CTRL and each mutant (Ctrl vs 1q del: p< 0.001, Ctrl vs 1q dup: p< 0.001, 1q del vs 1q del: p=1.0). We have changed Figure 4E, the descriptions in the methods section, and the corresponding figure legend in the revised manuscript this time. We also reanalyzed burst rates so that all electrodes were included in the statistical analysis. We have changed supplementary Figure 3 and edited the descriptions in the methods and the corresponding figure legend in this revised manuscript.

• MEA parameters such as Mean firing rate (spike/min) and burst rate are very sensitive to plating conditions, especially number of cells and clustering of cell around electrodes (see 4B). Given that the organoids already differ in size and according to the authors in cell number, but also in the amount of starting NPCs, one can expect very different cell densities/cell types per experiment/genotype. The authors should therefore show for every genotype the matching cell culture images. Also with regard to the claims made about GABAergic neurons the cell type composition at the time of the MEA recording should be characterized for every genotype.

As mentioned above, in MEA analysis, we used 2-D neuronal culture and seeded cells on each chip at the same density. The distribution patterns of cells were similar among the 3 genotypes. We will show the images of cultured neurons from 3 genotypes in the revised figure. As for the cell type composition, we plan to examine the expressions of GABAergic markers using extracted RNAs from neuronal cells on around 28 days post- dissociation (dpd). As reviewer #2 suggested, we also considered that drug treatment with bicuculine in this MEA system was meaningful. We plan to perform this experiment if the experimental conditions can be optimized.

• Fig 4B illustrates the points made above. The fact that no activity is observed in the control cells can be due to many different reasons: unequal plating, stress after dissociating cells, poor coverage of the electrodes, poor maturation, too early measuring time point, etc Because the authors have no control over the amount of cells covering the electrodes the data presented here carry very little carry little information. Fig 4B, best illustrates this with large cell clumps and areas without cell bodies. Measurements from these cell cultures are irrelevant and no conclusion can be drawn.

We suggest that the authors first benchmark this technique with their own differentiation protocol, show robust and reliable recordings on control cells, and only compare to the CRISPR lines at a time point at which the control cells show a decent amount of activity 1Hz. When doing so, also reduced activity can be monitored (For examples see, Trujillo et al, Cell Stem Cell2019 or Frega et al 2019 Nat comm).

As mentioned above, we seeded dissociated neurospheres in equal numbers on MEA dishes and kept culturing neurons gently for 28 days before analysis. Cell distribution was similar among the 3 genotypes and we could observe cell bodies in the area outside aggregates (we will provide additional bright-field images in the revised manuscript later). Low activities in CTRL neurons at 28 dpd could be observed even in the electrodes covered with dense cells, which were consistent among 3 independent experiments as described above. Nonetheless, we agreed with the reviewer that cellular conditions which could show stable activities even in CTRL neurons were more desirable. We have already tried longer cultures three times, but we could not perform sufficient analyses because neuronal cells became unhealthier after 35 dpd. We will try to improve the experimental conditions and perform analyses if the experimental conditions could be optimized.

• MEAs measure the output of the network (action potentials). In a network, this can be influenced by virtually every neuronal property (morphology, synaptic input, types ofsynapses, intrinsic excitability, etc). Therefore, the authors cannot conclude only based on fig 4E that the Del/Dup cells are intrinsically hyperactive. To make this conclusion they should measure this directly by assessing that passive and active intrinsic properties of individual neurons.

In control condition many electrodes do not give any signal. From these experiments it is impossible to know whether this is because of lack of cell on the particular electrode or real absence of activity. Certainly one could not conclude that the del en dup cell are intrinsically hyperexcitable.

As described above, we could observe the similarity of cell distributions among 3 genotypes. However, as the reviewer mentioned, the assessment of the individual neuronal activity would be better. Thus, we will perform patch-clamp recordings in addition to MEA analysis.

It seems that from the introduction the authors try to link 1q21 CNVs to epilepsy and ASd, thereby justifying the observed phenotypes.

• How do the authors reconcile the fact that more mature GABA system is observed in the Del lines with the so called increased activity compared to controls but not to the Dup lines.

We assumed that cell type compositions differed between 1q del and 1q dup, although network excitabilities were commonly observed in both mutants. We agree that this assumption lacks sufficient evidence even though we have shown the results in scRNAseq (Figure 6E). We consider that checking cell type compositions would be needed to ensure this. Although mature GABAergic neurons were increased in 1q del lines as mentioned by the reviewer, we think GABAergic signals and unknown factors such as epilepsy- associated genes (e.g., GRIN2A and SCN1A) may be involved in the abnormal neuronal firing. We will check the expression of these genes and examine the expressions of GABAergic markers in neuronal cells.

Single cell RNAseq

• I'm not a specialist on single cell RNAseq, however it seems that the analysis is underdeveloped and conclusion drawn for these experiments premature. It would be essential to validate some of the generated hypothesis, eg GABA maturity and not merely state as a conclusion (eg title).

We thank the reviewer for the suggestion. We have revised the title as we mentioned above, and we will revise the main text based on our results appropriately.

• How do the authors explain that a majority of the cells are Glial cells at day 27, and no presence of neurons.

On day 27 in our 3-D organoid protocol, cells were still in the developmental stage. That’s why we consistently described it as “NPC organoid” but not “brain organoid” in this paper. Indeed, our rationale for the scRNA-seq study was to determine gene(s) or gene regulatory network(s) when the difference of circumference was significant among genotypes (Fig. 2C). Although the underlying mechanism was not fully understood from our results, we interpreted this result. Radial glial cells (RGs) have the ability to self- renewal with symmetric divisions and play a role in both neurogenesis and gliogenesis (Lui et al. Cell 2011, A Kriegstein et al., Annu Rev Neurosci 2009). A recent study showed that the reduction of NF1, a tumor suppressor protein in the RAS/MAPK pathway, induced excessive production of glial cells, i.e., mainly oligodendrocyte precursor cells (OPCs) accompanied with astrocyte precursor cells, from RGs; furthermore, the reduction of NF1 also enhanced the cell divisions of generated OPCs (Z Shen, BioRxiv 2020). We have checked that the expression of NF1 in the glial cluster was also downregulated in our scRNA-seq data. Thus, we reasoned that the predominance of 1q dup cells in the glial cluster reflected the excessive production of glial cells from RGs, which were related to the alteration of the RAS/MAPK pathway. We will add this interpretation in the revised manuscript next time.

• How relevant is the changes in the extremely low amounts of GABAergic neurons in the Del cells, no excitatory neurons are present, only NSCs

In a previous paper, CA Trujillo et al. showed the cell type composition in 3-D human cortical organoids at different time points. GABAergic cells were restricted to later stages and the ratio was still very limited at 6 months (Figure 1J in CA Trujillo et al., Cell Stem Cell 2019). From this fact, we regarded the emergence of GABAergic neurons as meaningful even if the ratio was very low. As for excitatory neurons, we will further check the expressions of excitatory neuronal markers. (According to the screening chart we used, we did not explore excitatory neuronal markers as far as cells did not express SLC17A7 significantly).

Minor comments

• It is unclear how many clones were assessed per genotype

We constructed one clone per genotype. As we mentioned above, we have added the information in the results section of this preliminary revised manuscript.

• The authors should properly annotate the genotypes 1q21.1 instead of 1q del (line 134)

We have already annotated the abbreviations of 1q21.1 deletion and duplication in lines 87 and 93.

• Introduction seems to be somehow off topic since 1q21.1 locus is associated with several neurodevelopmental disorders, including SCZ, but is certainly not specific to ASD and epilepsy. So the premiss on line 86: to study 1q21.1 locus to understand ASD/epilepsy is somewhat misleading. I propose that the introduction would be focussed on the 1q21.1 and not on general on ASD/epilepsy.

As the reviewer pointed out, 1q21.1 CNVs are associated with other neurodevelopmental and neuropsychiatric disorders. Since our research aims to elucidate the underlying mechanism of ASD, we mainly focused on two representative comorbidities (abnormal brain size and epilepsy), which seemed relatively reproducible in vitro. However, we agree with the reviewer that the lack of information about clinical symptoms of 1q21.1 microdeletion and microduplication syndrome besides ASD was not appropriate. Thus, we will revise the introduction to mention the neurodevelopmental phenotypes of 1q21.1 CNVs in the revised manuscript next time.

• It is unclear whether they generated heterozygous or homozygous deletions.

We thank the reviewer for pointing it out. We have generated clones with heterozygous deletion and duplication. We have added the information in the results section of this revised manuscript.

• The authors should cite Fiddes, I. T. et al. Human-Specific NOTCH2NL Genes Affect Notch Signaling and Cortical Neurogenesis. Cell 173, 1356-1369.e22 (2018).

As the reviewer suggested, we will cite two papers regarding NOTCH2NL (NOTCH2NLA: Fiddes, I. T. et al., Cell 173, 2018; NOTCH2NLB: Ikuo K Suzuki et al., Cell 173, 2018) when we discuss the alteration of neuronal maturity and brain size. We will add the information in the revised manuscript next time.

• Many unclear statements eg line 138: Next, we analyzed each single-cell in an organoid

We thank the reviewer for noticing it. We have made an effort to remove inappropriate sentences in this revised manuscript.

• Discussion on E/I is very speculative, not supported by any evidence

In response to the reviewer’s suggestion, we will cut the descriptions which contain too speculative contents in the discussion section of the revised manuscript later.

Significance

The general topic of this study is high interest given the strong association of the 1q21.1 with disease. The authors developed interesting ESC line to study in parallel del and duplication. Unfortunately the level of of analysis performed on these organoids is not up the current stat of the art, are of low experimental quality, analyses are limited. Therefore no clear conclusion can be drawn except for the size of the organoids, very little mechanism is provided. This therefore remains a purely descriptive study for which the presented data are rather on low quality and limited impact in its current shape.

We thank the reviewer for the interest and criticism of our paper. As discussed above, we plan to perform additional analyses and experiments to justify our hypothesis more clearly and try to meet the reviewer’s requests.

Reviewer #2

This study was initiated to look at specific cellular and molecular mechanism of the duplication and deletion CNV frequently observed at the 1q21.1 gene locus in an isogeneic human embryonic stem (hES) cell model. The authors note that these CNVs are associated with higher than normal penetrance of ASD and epilepsy and aim to elucidate gene expression differences with single cell RNAseq and functional changes in this model system. The authors further sought to proliferation and differentiation states, in addition to neuronal activity, using both 2D cultures and 3D organoid models. The 1q21.1 gene locus model system made here is unique and the results broadly recapitulate the patient phenotype particularly with observations of macrocephaly in the "1q dup" and microcephaly in the "1q del".

Reviewers statement:

We have joint expertise in GABAergic neuronal development, iPSC 2D and 3D culture and ASD human molecular genetics.

Major comments:

• Not sure why ASD (if used it should also be spelled out) is mentioned in the title if ASD is only seen in a proportion of human 1q21.1. duplication (~36% will have autism) and 1q21.1 deletion (<10% will have autism) carriers. I would prefer to use 'neurodevelopmental phenotype'. A good update review that is accurate with respect to this CNV role in autism is PMID: 29398931. The authors should also put into the context of their results what is known with other neuropsychiatric phenotypes also seen in these CNV events;

We thank the reviewer for the suggestion and valuable information. We have corrected the title in the revised manuscript this time. We will also refer to the paper by Fernandez and Scherer (Dialogues Clin. Neurosci., 2017) to discuss the detail of roles and neuropsychiatric phenotypes of targeted CNVs.

• In Fig 1D the ddPCR validation for the genetic alterations in 1q del shows a normal return to 2 copies of GPR89B. However, in the 1q dup the CNV level is still elevated for GPR89B. Please determine how much further the duplication goes as there are five more potentially affected genes in this region (eg PDZK1P1). Modify the text appropriately to note the potential influence of any of these other genes on the experimental outcomes.

We thank the reviewer for pointing it out. Figure 1D showed the results of aCGH analysis to confirm the copy number alteration of the targeted region in each clone. This analysis expected that the target region contained GPR89B, as confirmed by PCR shown in Fig. 1B. However, as the reviewer’s comment, the cleavage sites shown in Figure 1D seem not consistent with the result of Fig. 1B. We think it reflects the limitation of the microarray-based CGH technique. Since the locus between GPR89B and LOC101927468 contains extensive repeat sequences, aCGH may not be an appropriate method. Thus, we will apply quantitative PCR (or ddPCR) to determine copy number alternation of each clone in addition to microarray-based CGH.

• The authors' claim that dosage dependent size differences in NPC organoids is caused by a change in the number of cells within the organoid rather than size - from Fig. 2D, cells in 1qdel organoid appears more compact; a quantification of cell number should be done to support this claim. IHC of D27/28 organoids with GABAergic markers would support authors' claim of alterations of GABAergic components in 1qdel cells. These suggested experiments would take 2-3 days if the organoids are available.

In response to the reviewer’s suggestion, we have counted the number of cells in the images of each NPC organoid using the fluorescent microscopy, BZ-X analyzer, and calculated the cell number per unit area (1000 µm2). We found the cell density was not significantly different among the 3 genotypes. We have changed Figure 2E, descriptions in the methods and results sections, and the corresponding figure legend in the revised manuscript this time. As for exploring GABAergic components in the NPC organoids, we plan to perform immunocytochemistry (ICC) and RT-qPCR analysis.

• Fig 4 E shows MEA data from "top 10". What is the top ten? Do you mean data points? There are batch differences in 1q dup with one batch having a lower expression than the other. Increasing the n value to accommodate the high variance observed in this group will greatly increase the validity of the data generated. Also, change the figure legend to indicate the age of these cultures. Given that the controls are not spiking, this data should be extended to probe the developmental profile further to week 9 when normal cells should be spiking so that the baseline activity of this isogenic line can be determined.

Top 10 meant the ten electrodes with the highest spike rates within one MEA dish. To respond to the reviewer’s suggestion, we have performed statistical analysis with all electrodes per genotype. Even though the distributions of firing rate were quite heterogeneous among different electrodes, we found significant differences between CTRL and each mutant per MEA dish. We have changed Figure 4E, descriptions in the methods section, and the corresponding figure legend in the revised manuscript this time.

The reviewer is correct that the spike rates in 1q dup were quite different between different batches. We noticed from our experiments that spike rates were easily affected by the health conditions of cells. Some mutant batches showed mild spike activities like circles in 1q dup, and some had very vigorous activities. We have even checked the reproducibility of significant differences between CTRL and each mutant per MEA dish with 3 independent experiments. As for the extended cultures to detect more frequent signals in CTRL neurons, we have already tried longer cultures three times. However, we could not perform sufficient analyses because neurons became unhealthier after 35 dpd. We will further try to improve the experimental setup and perform analyses if the experimental conditions could be optimized.

• Single cell RNAseq data suggests a cluster of GABAergic cell types that are appearing in the 1q del condition, but not in the 1q dup or control groups. The authors suggest that these GABAergic cells are excitatory because the chloride gradient has not yet been altered (no change to KCC2 expression). The authors should substantiate this idea in the MEA system with bicuculline treatment to block GABAergic transmission (drug washed in and out) to show that the spike activity observed in the 2D MEA experiments is due to GABAergic excitatory transmission. Ideally, this should be done for both the 1q dup, 1q del as well as controls.

We thank the reviewer for the suggestion. We agreed with the reviewer that drug treatment with bicuculine in this MEA system was meaningful to identify cellular properties. We will try to set up the experimental conditions and perform this experiment if the condition can be optimized.

• Fig 5A. The clustering method for single cell RNAseq seems shows a large proportion of "other" class cells begging the question as to what they are. Is there another cluster analysis, which might be used eg partially supervised/unsupervised clustering methods from the Allen Institute to help determine what these might be?

We initially made the screening chart for cell-type specifications according to cellular markers from Allen brain map (http://celltypes.brain-map.org/rnaseq/human_ctx_smart- seq) and a published paper (CA Trujillo et al., Cell Stem Cell 2019). We defined this cluster as “other” because this cluster did not have any significant genes in the 1st screening, although we understood that the specifications of all clusters were desirable. To investigate the cellular property in this cluster, we tried to put significant genes into Metascape to check gene ontology. We found some terms about immune cells (mainly lymphocytes and macrophages), cancer cells, roles for inflammation, and apoptotic process, although miscellaneous terms were also included. We have provided the screening chart as supplementary Table 4 in this revised manuscript. Next time, we will add a more detailed description of the ‘other’ cluster in the revised manuscript.

• Fig 5 B. The manuscript requires additional markers used in the cluster analysis. Particularly, expression of the GABAergic progenitor markers DLX5 and 6 as well as EMX1 for the progenitor cells. Details of all markers and cluster algorithms should be made available in supplementary tables and R scripts, so that others can repeat this analysis.

In response to the reviewer’s suggestion, we will check these GABAergic progenitor markers and add them to the revised figure and manuscript later. As we mentioned above, we performed the cell type specification of each cluster manually using our screening chart and did not use R scripts. We have provided the information on the screening process in supplementary Table 4 of this revised manuscript.

• Fig 6. Expanding the heat map of 1q del and 1q dup with CTRL expression would help with context for baseline levels in this isogenic cell line. Please also include additional GABAergic markers GABRA1, GABARB2and GABARG2, (subunits of the most common GABA-A receptor) SOM, VIP, NPY, (other GABAergic interneurons in addition to PVALB) DLX6, EXM1 and for excitatory markers GRIA2, GRIA3 and GRIA4 (all of which have developmentally regulated expression patterns) that will provide more context with the synaptic receptor literature. GRIN2D is expressed only in GABAergic cell types and so I would suggest including this NMDA receptor subunit as well.

We thank the reviewer for the valuable suggestions. To further explore the cellular properties in 1q del and 1q dup, we will check these cell markers additionally and show the results in the revised figure and manuscript next time.

Minor comments:

1. Additional references (eg. Schafer et al. 2019) should be discussed in relation to the authors' suggestions of altered neuronal maturity.

As the reviewer suggested, we will include the paper in our references and discuss the associations between neurodevelopmental disorders and altered neuronal maturity.

1. The authors show no change in PAX6 expression between genotypes, but significant differences in TBR2 expression between genotypes (Fig. 2C) - this alteration in normal cortical development should be included in results and discussed.

Radial glial cells (RGs) have abilities of both self-renewal and neurogenesis (Lui et al. Cell 2011, Fiddes, I. T. et al., Cell 2018). Fiddes et al. showed that if the balance leans toward neurogenesis, premature differentiation with higher TBR2 expressions was observed in week 4 human cortical organoids (Fiddes, I. T. et al., Cell 2018). However, the predisposition to neurogenesis is thought to cause the earlier shortage of RGs. Finally, these cells remain abundant in week 4 organoids. We considered this was why TBR2 expression was significantly different in 1q del, but PAX6 was not. We will add this interpretation in the revised manuscript next time.

1. In the introduction (Line 67): The author's state that "alterations in brain size is common in patients with ASD" using one meta-study to support this claim. Further primary studies should be consulted and the authors should give the proportion of the population with ASD and altered brain size to support this statement. In addition, the age range should be supported with primary papers.

As the reviewer suggested, we have cited some primary studies about the prevalence of altered brain size in ASD patients and its age range in this revised manuscript. Since it seems still controversial whether the enlargement of brain size persists or not until adolescence and adulthood (E H Aylward et al., Neurology 2002; J Piven et al., Am J Psychiatry 1995), we have also modified the description in this manuscript.

1. Line 73. The authors suggest that the brain growth deviations are "Postnatal stage restrictive". Citations are needed to support this statement.

As the reviewer suggested, we have cited some primary studies as described above and revised the manuscript.

1. In the scRNAseq data results please report total cell numbers counted for each cluster and for genotype group.

We apologize for the lack of information and thank the reviewer for noticing it. We have added the information in the results section of the revised manuscript this time.

1. In the results section (line 269-270) the authors suggest that 1q del cells are in a more mature state because the GABAergic cells are present and glutamatergic genes are similarly altered in 1q dup and 1q del. However, the results from the gene cluster data suggests that there is a very high proportion of progenitor cells (Progenitor 1 and 2 clusters), which seems to argue against faster maturation. This suggests to me that cell fate is being modified here.

We thank the reviewer for the valuable suggestion. Schafer et al. (the suggested paper in minor comment 1) reported that altered gene expressions in neuronal modules have already been observed in NSCs derived from ASD patient-derived iPSCs. As the reviewer suggested, we plan to consider our results in terms of the alteration of cell fate and neuronal maturity in the revised manuscript later.

1. Label figures on each page for ms.

As the reviewer suggested, we have labeled figures at the bottom right of each page.

1. Fix typos and heat map legends (currently no colors for log2 fold change in Fig 5 or 6)

We apologize to the reviewer for typos and grammatical errors. We made an effort to remove them. We also apologize for the lack of color information in the legends of Figure 5 and Figure 6 and thank the reviewer for noticing it. We have added the color information in the figure legends of the revised manuscript this time.

Significance

Overall the study is clearly described, and the outcomes have been substantiated to a certain degree, but requires a bit more work. This paper does represent a technical 'tour de force' and the authors should be applauded for sticking it out where other labs have so far failed. It might be useful to mention even in brief, of the number of 'failed' (failed or inaccurate) events. The availability of the lines should also be clearly stated.

We thank the reviewer for the positive comments. In addition to the plans described above, we have added more detailed information, e.g., how many screenings were carried out to get positive clones, in the revised version of the methods and results section. We have also added the descriptions about the availability of the 1q21.1 CNV cell lines in the data availability section of this revised manuscript.

Reviewer #3

In this research study by Nomura et al., the authors develop novel hESC-based models of reciprocal CNVs in distal 1q21.1 using CRISPR/Cas9 genome editing technology. Specifically, the authors genome edit KhES-1 cells to produce two isogenic hESC line that contain either a deletion or duplication of this chromosomal region. Patients with 1q21.1 deletion and 1q21.1 duplication syndromes show abnormal head size in conjunction with multiple neurodevelopmental co-morbidities such as epilepsy, developmental delay, and neuropsychiatric abnormalities. This is an important study since it provides robust research tools to understand molecular and cellular mechanisms that may underly these syndromes. Through generation of cortical organoid models, the authors demonstrate 1q21.1 deletion and duplication organoids show deficits in growth and over-growth, respectively. Additionally, the authors provide data that 1q21.1 deletion and duplication organoids show altered signaling cascades which may underly growth deficits and also abnormal neurodevelopment which may underly hyperexcitable neurons as demonstrated by multi-electrode array analysis. While my enthusiasm for this study remain high, I do have a significant number of major and minor reservations specific to the experimental design and analysis that if addressed would provide for an excellent contribution to the field.

Major concerns:

1. Though the authors provide extensive data in this study, major revisions are necessary to interpret all of their data in the context of the phenotypes they are observing in organoids and MEA analyses. In addition, the current study lacks cohesiveness throughout the various experiments and does not provide text that clearly unifies the results of the study. For example, no interpretation of higher TBR2 levels in 1q21.1 deletion is provided. Does this mean these organoids show accelerated neuronal differentiation? Also please see my comment regarding TBR2 staining the next section.

Other examples throughout the manuscript in which there is no clear interpretation of the data or inadequacies of unifying the results of the experiments.

We thank the reviewer for pointing out that our manuscript had inadequacies of the integrity and cohesiveness throughout our data. With additional data as follows, we plan to improve these issues in the revised manuscript later. As for TBR2 expression, we considered that higher TBR2 expressions in week 4 human cortical organoids showed the predisposition to neurogenesis in 1q del as demonstrated in a previous paper (Fiddes, I. T. et al., Cell 2018). We will add the description in the revised manuscript later.

• a. Additional interpretation why 1q21.1 duplication organoids show increased growth is lacking. The single cell RNA sequencing results show there are more glia, but no further interpretation is giving why these organoids show an overgrowth phenotype. Inversely, the 1q21.1 deletion organoids show more progenitor cells, but it is not apparent why this should result in decreased cell growth.

As we have mentioned above, we considered that the predominance of 1q dup cells in the glial cluster reflected the excessive gliogenesis from radial glial cells and enhanced cell divisions in relation to the alteration of the RAS/MAPK pathway (Z Shen, BioRxiv 2020). We plan to analyze additional markers related to cell proliferation and cell division by immunostaining to validate the above hypotheses. To investigate how 1q del organoids showed smaller size, we plan to examine apoptotic markers such as cytochrome C and caspase 3 by culturing NPC organoids again.

• b. The authors suggest that 1q21.1 duplication organoids are resistant to neuronal differentiation. What data supports this hypothesis other than the fact there are no mature neuronal cells are present in their single cell RNA sequencing data.

We considered that the results in Figure 3B and Figure 3D also supported this hypothesis that 1q dup organoids expressed the lower intensity of neuronal markers. Since we have only checked a few markers by immunocytochemistry (ICC), we plan to examine additional markers, i.e., immature neuronal markers such as DCX and other mature neuronal markers such as NeuN, as well as proliferation markers such as phospho histone H3 to ensure this hypothesis.

• c. The MEA analyses show hyperexcitability in both 1q21.1 deletion and duplication cultures. Since the authors suggest 1q21.1 duplication organoids are resistant to neuronal maturation, no interpretation is given why they show hyperexcitable phenotypes.

In the MEA analyses, we used not 3-D cortical organoids but 2-D neurons because the required culture period to emit electrical activities was thought to be much shorter in 2-D neurons according to some previous studies with human pluripotent cells (A Taga et al., Stem Cells Transl Med 2019; CA Trujillo et al., Cell Stem Cell 2019). We considered that 2-D neurons on 28 dpd (day 63) had much higher maturity than NPC organoids and even 1q dup neurons had already become mature enough to emit spike activities. We will also check neuronal marker expressions using 2-D neurons around 28 dpd by RT-qPCR to ensure this.

• d. The current study is lacking extensive immunohistochemical stains of representative markers that validate their findings from their single cell RNA sequencing experiments. For example, glial cell markers such as GFAP should be analyzed in 1q21.1 duplication organoids. Additionally, progenitor cell markers such as PAX6 and neuronal markers such as MAP2 and synaptic markers such as SYNAPSIN and others should be incorporated in the study.

We thank the reviewer for the suggestions. We plan to perform additional IHC staining for NPC organoids with the suggested markers and OPC markers.

1. Major details are lacking for the single cell RNA sequencing experiments.

• a. How many cells were analyzed from each group? How many organoids and what age of organoids were analyzed from each group, were they pooled together? Why was a log2FC 1.2 used as a threshold? It is unclear how the authors identify Progenitor 1 and 2 cell clusters? Are they distinct clusters or is this a continuum of differentiation. The progenitor 1 and 2 clusters were chosen based on expression of the ID transcription factors, but no text was provided why these genes specify progenitor cells.

We apologize for the lack of information and thank the reviewer for noticing it. We described the number of analyzed cells (32,171 cells: 1q del; 10,682, 1q dup; 11,987, CTRL; 9,502) in the results section (line 186) of the original manuscript. However, we could not count how many organoids were analyzed because they were too tiny (diameter; 400-700µm). Many organoids were needed to get the prescribed number of cells (25,000 cells per genotype). According to the analyzed data of size measurement for NPC organoids by fluorescent microscopy, at least 1,500 organoids were collected per genotype. We gathered all cultured organoids in the same batch, dissociated them, and then loaded the prescribed number of cells into the machine. We have added the description of the number of input cells in the methods section of this revised manuscript.

We used the threshold of log2FC > |1.2| so that the total number of DEGs became around 100-1000 in both bulk and the NSC cluster to avoid a very high or low number of DEGs. Some previous transcriptome studies used the same or even smaller thresholds (Xiaoming Ma et al., Front in Genet 2020; J Zhong et al., Brain Res 2016; Y Wang et al., BMC genomics 2016). We have added these descriptions in the methods section of this revised manuscript.

As for progenitor-1 and 2, we regarded them as a continuum based on the marker expressions. We chose ID transcription factors for progenitor cells, referring to a published paper (CA Trujillo et al., Cell Stem Cell 2019) as we have described in the methods section (line 633). Several articles have reported that ID transcription factors regulate proliferation and differentiation of neural precursor cells (K Yun et al., Development 2004; D Patel et al., Biochim Biophys Acta 2015).

Minor concerns:

1. I would suggest rephrasing the title of the study as it does not clearly convey the advancement to the field. I would suggest the following or something similar this is more concise: " Modeling Reciprocal CNVs of Chromosomal 1q21.1 in Cortical Organoids Reveals Alterations in Neurodevelopment."

We thank the reviewer for the concrete suggestion. We have revised the title as the reviewer suggested in this preliminary revised manuscript.

1. The length of the discussion is over extended and should be revised to become more concise.

We thank the reviewer for pointing it out. We will shorten the beginning part and delete unnecessary sentences in the discussion section of the revised manuscript later.

1. Additional experiments should be performed to characterize pluripotency of hESC clones generated after genome editing other than staining for alkaline phosphatase activity.

At minimum, karyotyping in addition to measuring pluripotency markers such as NANOG and OCT3/4 should be performed.

Karyotyping of wild-type ES cells has been checked by Institute for Frontier Medical Sciences, Kyoto University before being provided. After genome editing, we performed aCGH analysis for all 3 genotypes using the wildtype ES cells as reference genes and confirmed no chromosome aberrations were generated. We have added the information about karyotyping in the methods section of this preliminary revised manuscript.

As for pluripotency markers, we performed RT-qPCR analyses with ES cells after genome editing and confirmed that OCT3/4 was highly expressed than internal control genes. (We can provide the raw data if requested).

4) There are several dozen instances of spelling/grammatical and word choice errors throughout the manuscript. For example, line 24 reads "We generate isogenic..." should read "We generated isogenic... "

• a. Line 25: "opposite organoid size" as written is confusing to interpret.
• b. Line 46: "have been considered in the context of ASD" would read more clearly as "have been thought to underly ASD etiology."
• c. Line 53: "in the study of neurological development" should read "nervous system development".
• d. Line 118: ".. to detect the CRISPR target site for deletion" should read "to detect the CRISPR target site. For the deletion, we checked... "
• e. <![endif]>Line 119: "...flanking the CRISPR target site; for duplication, we amplified.. " should read "flanking the CRISPR target site, and for the duplication, we amplified..... ".
• f. Line 127: "we prepared control cells (CTRL) that transfected.... should read ""we prepared control cells (CTRL) that were transfected. ".
• g. Line 185: "organoid size and mature level" should read "organoid size and developmental maturity."
• h. In line 40, "We made cryosections of .... should read.... "We performed IHC for the three organoid genotypes on day 27... " i. <![endif]>In Supplementary Figure 8, line 554, "replictes" is misspelled.

We apologize to the reviewer for many typos and grammatical errors and thank the reviewer for pointing them out in detail. We have corrected these errors as the reviewer suggested.

5) Line 181: "with a little higher degree of.. " should be re-written more precisely and with more scientific accuracy.

As the reviewer requested, we have corrected the sentence in this revised manuscript.

6) Line 216, The use of the colloquial phrase: "On the other hand.. " should be replaced with more formal language. For example, "In contrast, the number of downregulated....

We thank the reviewer for pointing it out. We have corrected this colloquial phrase at 4 locations.

7) In line 201, Pprogenitor is misspelled.

We apologize and thank the reviewer for noticing it. We have corrected it in this preliminary revised manuscript.

8) In Figure 3, images showing TBR2 staining does not appear correct as this protein should be localized to the nucleus similar to SOX2 staining. I would suggest optimizing conditions such as utilizing antigen retrieval or other methods to reduce non-specific cytoplasmic staining.

We thank the reviewer for the valuable suggestion. We plan to optimize the condition and try other neuronal lineages markers such as DCX and NeuN.

9) I would suggest simplifying the text describing the primers utilized in this study and display them in a table format.

As the reviewer requested, we will make a supplementary table of primer sequences in the revised manuscript later.

10) Information regarding the number of technical replicates used in this study is lacking throughout the manuscript. For example, how many hESC clones were analyzed? How many organoids were analyzed for each specific assay such as single cell RNA sequencing and MEA analyses? How many independent experiments were used for these studies?

We apologize for the lack of information. We have constructed one clone per genotype one human ES cell strain (khES-1) and performed all further analyses. The precise number of NPC organoids in scRNA-seq could not be counted, as we mentioned above. As for MEA analysis, 8 x 10^5 cells were seeded on each dish as described in the original manuscript. However, it was unclear how many neurons were observed on each electrode because multiple cells and neurites covered each electrode. Thus, spike activities were detected as the network of many neurons. We have added the information in the methods section of this preliminary revised manuscript.

11) It is not clear why the authors choose two types of organoid methods in the study. The first protocol referred to as the "NPC organoid method" is synonymous to neurosphere culturing and should be referred to as neurospheres throughout the manuscript.

One protocol (Fujimori et al., Stem Cell Rep., 2017) was not for 3-D organoids but 2-D neurons (Figure 4A). Thus, we considered neurosphere and NPC organoid were different.

12) In Figure 4, panel C should be referred to as a local field potential trace and not a waveform.

We thank the reviewer for pointing it out. We have corrected the description as the reviewer suggested.

Reviewer #3

This is an important study since it provides robust research tools to understand molecular and cellular mechanisms that may underlie 1q21.1 deletion and duplication syndromes.

We thank the reviewer for the positive comments. We plan to perform additional analyses and experiments as described above and try to meet the reviewer’s requests.

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 #3

Evidence, reproducibility and clarity

In this research study by Nomura et al., the authors develop novel hESC-based models of reciprocal CNVs in distal 1q21.1 using CRISPR/Cas9 genome editing technology. Specifically, the authors genome edit KhES-1 cells to produce two isogenic hESC line that contain either a deletion or duplication of this chromosomal region. Patients with 1q21.1 deletion and 1q21.1 duplication syndromes show abnormal head size in conjunction with multiple neurodevelopmental co-morbidities such as epilepsy, developmental delay, and neuropsychiatric abnormalities. This is an important study since it provides robust research tools to understand molecular and cellular mechanisms that may underly these syndromes. Through generation of cortical organoid models, the authors demonstrate 1q21.1 deletion and duplication organoids show deficits in growth and over-growth, respectively. Additionally, the authors provide data that 1q21.1 deletion and duplication organoids show altered signaling cascades which may underly growth deficits and also abnormal neurodevelopment which may underly hyperexcitable neurons as demonstrated by multi-electrode array analysis. While my enthusiasm for this study remain high, I do have a significant number of major and minor reservations specific to the experimental design and analysis that if addressed would provide for an excellent contribution to the field.

Major concerns:

1. Though the authors provide extensive data in this study, major revisions are necessary to interpret all of their data in the context of the phenotypes they are observing in organoids and MEA analyses. In addition, the current study lacks cohesiveness throughout the various experiments and does not provide text that clearly unifies the results of the study. For example, no interpretation of higher TBR2 levels in 1q21.1 deletion is provided. Does this mean these organoids show accelerated neuronal differentiation? Also please see my comment regarding TBR2 staining the next section. Other examples throughout the manuscript in which there is no clear interpretation of the data or inadequacies of unifying the results of the experiments.
   • a. Additional interpretation why 1q21.1 duplication organoids show increased growth is lacking. The single cell RNA sequencing results show there are more glia, but no further interpretation is giving why these organoids show an overgrowth phenotype. Inversely, the 1q21.1 deletion organoids show more progenitor cells, but it is not apparent why this should result in decreased cell growth.
   • b. The authors suggest that 1q21.1 duplication organoids are resistant to neuronal differentiation. What data supports this hypothesis other than the fact there are no mature neuronal cells are present in their single cell RNA sequencing data.
   • c. The MEA analyses show hyperexcitability in both 1q21.1 deletion and duplication cultures. Since the authors suggest 1q21.1 duplication organoids are resistant to neuronal maturation, no interpretation is given why they show hyperexcitable phenotypes.
   • d. The current study is lacking extensive immunohistochemical stains of representative markers that validate their findings from their single cell RNA sequencing experiments. For example, glial cell markers such as GFAP should be analyzed in 1q21.1 duplication organoids. Additionally, progenitor cell markers such as PAX6 and neuronal markers such as MAP2 and synaptic markers such as SYNAPSIN and others should be incorporated in the study.
2. Major details are lacking for the single cell RNA sequencing experiments.
   • a. How many cells were analyzed from each group? How many organoids and what age of organoids were analyzed from each group, were they pooled together? Why was a log2FC >1.2 used as a threshold? It is unclear how the authors identify Progenitor 1 and 2 cell clusters? Are they distinct clusters or is this a continuum of differentiation. The progenitor 1 and 2 clusters were chosen based on expression of the ID transcription factors, but no text was provided why these genes specify progenitor cells.

Minor concerns:

1. I would suggest rephrasing the title of the study as it does not clearly convey the advancement to the field. I would suggest the following or something similar this is more concise: " Modeling Reciprocal CNVs of Chromosomal 1q21.1 in Cortical Organoids Reveals Alterations in Neurodevelopment."
2. The length of the discussion is over extended and should be revised to become more concise.
3. Additional experiments should be performed to characterize pluripotency of hESC clones generated after genome editing other than staining for alkaline phosphatase activity. At minimum, karyotyping in addition to measuring pluripotency markers such as NANOG and OCT3/4 should be performed.
4. There are several dozen instances of spelling/grammatical and word choice errors throughout the manuscript. For example, line 24 reads "We generate isogenic...." should read "We generated isogenic...."
   • a. Line 25: "opposite organoid size" as written is confusing to interpret.
   • b. Line 46: "have been considered in the context of ASD" would read more clearly as "have been thought to underly ASD etiology."
   • c. Line 53: "in the study of neurological development" should read "nervous system development".
   • d. Line 118: "...to detect the CRISPR target site for deletion" should read "to detect the CRISPR target site. For the deletion, we checked....".
   • e. Line 119: "...flanking the CRISPR target site; for duplication, we amplified..." should read "flanking the CRISPR target site, and for the duplication, we amplified......".
   • f. Line 127: "we prepared control cells (CTRL) that transfected.... should read ""we prepared control cells (CTRL) that were transfected...."
   • g. Line 185: "organoid size and mature level" should read "organoid size and developmental maturity."
   • h. In line 40, "We made cryosections of .... should read.... "We performed IHC for the three organoid genotypes on day 27...."
   • i. In Supplementary Figure 8, line 554, "replictes" is misspelled.
5. Line 181: "with a little higher degree of..." should be re-written more precisely and with more scientific accuracy.
6. Line 216, The use of the colloquial phrase: "On the other hand..." should be replaced with more formal language. For example, "In contrast, the number of downregulated....
7. In line 201, Pprogenitor is misspelled.
8. In Figure 3, images showing TBR2 staining does not appear correct as this protein should be localized to the nucleus similar to SOX2 staining. I would suggest optimizing conditions such as utilizing antigen retrieval or other methods to reduce non-specific cytoplasmic staining.
9. I would suggest simplifying the text describing the primers utilized in this study and display them in a table format.
10. Information regarding the number of technical replicates used in this study is lacking throughout the manuscript. For example, how many hESC clones were analyzed? How many organoids were analyzed for each specific assay such as single cell RNA sequencing and MEA analyses? How many independent experiments were used for these studies?
11. It is not clear why the authors choose two types of organoid methods in the study. The first protocol referred to as the "NPC organoid method" is synonymous to neurosphere culturing and should be referred to as neurospheres throughout the manuscript.
12. In Figure 4, panel C should be referred to as a local field potential trace and not a waveform.

Significance

This is an important study since it provides robust research tools to understand molecular and cellular mechanisms that may underlie 1q21.1 deletion and duplication syndromes.

Malpass, D. D. (n.d.). The impact of Covid-19 on students, staff and Departments of Psychology in UK Universities. This report was prepared by Dr Lisa Morrison Coulthard (Head of Research and Impact) and Laura Devlin (Research and Impact Lead). 124.

This is not even that surprising, it's amazing how during these insane moral panics how people will completely shut off their brains. From Mr. Potato Head to Dr. Seuss there is constantly some boogieman that we are supposed to believe will be the downfall of Western civilization or whatever.

"Critical Race Theory poisoned our water supply, burned our crops, and delivered a plague unto our houses!"

"It did?"

"No, but are we just gonna wait around until it does?"

SciScore for 10.1101/2021.09.13.21262971: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

No key resources detected.

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

None of the participants on follow up showed any abnormity in electrocardiogram and fundus examination This study had few limitations. Firstly, the design of the study was a cohort study, just observing the participants taking HCQs or not. The sample size in the intervention arm was very small and we cannot exclude the possibility of a modest prophylactic effect that remained undetected. Moreover, the frequency of exposure to COVID-19 was not quantified and the drug concentrations were not monitored. The cohort comprised mostly of young and healthy healthcare workers. Therefore, the results cannot be generalised for the more vulnerable sections of the population. The study was conducted at a single centre in the North India and therefore, may not be representative of disease prevalence and exposure in other regions. The design of the study was such that the participants got themselves tested as per the clinical need only and as a result, 66 % of the total participants were never tested during the study period. Hence, asymptomatic COVID-19 infections were not accounted for. Drugs like ivermectin and antiretroviral agents have also been tried and tested for the prevention of COVID-19, but head-to-head trials have been lacking. Of these, ivermectin received governmental approval for mass distribution in various states in India.(20) However, as per the available evidence, none of these agents should be used for chemo-prophylaxis outside the context of a clinical trial.(21,22) Eve...

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

SciScore for 10.1101/2021.09.10.459410: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

it seems that only in disaster does the family come together

mother and harri connect when harri came into car crash

electra complex

father has healed her

draws parallels to herself,

but she doesn't refuse

at least she doesn't want to refuse

similar to the pictures of the dad, you had to "imagine" what the dad looked like because they were cutout

I think that this line is perhaps the most important line in the entire piece. Bush had already seen how someone could theoretically delegate the automatic and logical operation of arithmetic to a machine by aligning the pattern of machine behavior with patterns of human thought. In my opinion, his chief insight in this piece is the alignment of machine behavior with human thought in the specific context of information retrieval. And where else could this insight have sprung into his head but from his own personal experiences coordinated the vastly complicated work of no less than 6000 premiere American scientists!

The trails also serves as a visualization of the way a person assocaites/thinks. Very interesting to think we can model this digitally now.

Repentance and waiting for the Lord. Here is Psalm 27 ESV : The Lord Is My Light and My Salvation Of David. 27 The Lord is my light and my salvation; whom shall I fear? The Lord is the stronghold[a] of my life; of whom shall I be afraid?

2 When evildoers assail me to eat up my flesh, my adversaries and foes, it is they who stumble and fall.

3 Though an army encamp against me, my heart shall not fear; though war arise against me, yet[b] I will be confident.

4 One thing have I asked of the Lord, that will I seek after: that I may dwell in the house of the Lord all the days of my life, to gaze upon the beauty of the Lord and to inquire[c] in his temple.

5 For he will hide me in his shelter in the day of trouble; he will conceal me under the cover of his tent; he will lift me high upon a rock.

6 And now my head shall be lifted up above my enemies all around me, and I will offer in his tent sacrifices with shouts of joy; I will sing and make melody to the Lord.

7 Hear, O Lord, when I cry aloud; be gracious to me and answer me! 8 You have said, “Seek[d] my face.” My heart says to you, “Your face, Lord, do I seek.”[e] 9 Hide not your face from me. Turn not your servant away in anger, O you who have been my help. Cast me not off; forsake me not, O God of my salvation! 10 For my father and my mother have forsaken me, but the Lord will take me in.

11 Teach me your way, O Lord, and lead me on a level path because of my enemies. 12 Give me not up to the will of my adversaries; for false witnesses have risen against me, and they breathe out violence.

13 I believe that I shall look[f] upon the goodness of the Lord in the land of the living! 14 Wait for the Lord; be strong, and let your heart take courage; wait for the Lord!

This entire section made me weep because I understand how she is feeling and it brought back memories for me. I've been in battle since I was a child, and I've seen injured infants and parents in anguish. It's really visual and emotive.

Personally, this stanza brought back memories of battle, and I could sense the anguish and terror that people feel when they are attacked. I went through everything and could picture the images in my mind.

I like that life, at least for me seems to portray itself as a comedian because this is a conversation we had in another class of mine. The conversation being how reading can make people create a civil society and we spoke about the counter to that how some people can get last in translation. Blurring the line between the written word and their reality and forgetting that there’s a separation there. You’ll never be your favorite writer because there’s only one of them and even yet that writer might of not even believed in themselves. So what I’d have to say to that is grab the hammer, make a mess of everything and jam all the pieces together no matter if they fit or not.

so it isn't dead yet but will be put out of its misery

they're going to kill the child so she won't suffer anymore

The text says that the author was able to complete this challenge of writing a poem while not being fluent in writing in English. The author wrote about the spring time and used words from T.V. shows and dictionaries to make the poem come to life. He had surrended to the box both his family and his school put him in and waved the white flag which was his words put on paper.

The function of the text is to reveal a challenge or obstacle the author was able to overcome. despite not being able to write fluently, the author wanted to challenge themselves .

I mean to be fair, at least they let her keep her child with her this time

She's calling them inhumane, but they did give her a horse to ride on for her and her wounded child (didn't make them walk).

Perhaps this is some of the value behind the ability to resort index cards within a zettelkasten over the prior staticness of the commonplace tradition? The ideas aren't anchored to the page, but can be moved around, rearranged.

42:07 - 46:47

"I think as painful as those times are, they are so rewarding, because it requires that you reassess and decide to really commit. It's like, I could be doing this, I could be doing that. Actually, I'm doing this because this is not just my passion. There is something else that is driving me to do it. But I can't even describe it, I don't know what it is. Once you get to that place, windows start to open, and you start to realize things. I don't know if I would have come to, had I not gone through a rough time. 2018 was the year that I said, ‘What we're doing today should exist a hundred years from now.’ People should be growing food in their communities and creating an economic engine that provides jobs to the residents in the community, food, to the residents. And up to that point, I wasn't thinking. And the moment I thought, ‘a hundred years,’ I had to question a whole bunch of shit because I have always been inspired by anger. Anger is an amazing motivator. I realized once I had this notion that Project Eats should exist a hundred years from now, I realized you can't grow something with anger. You can start it with anger, it will motivate you and get you to levels of creativity, resourcefulness and imagination, but you can't grow it with that.

And then the next level of, ‘Wow. I've got to grow this thing.’ I've got some shortcomings. I hate to ask people for anything. And so if you're going to grow it, you have to grow it with resources. And we, for the most part, have used the most valuable resources that we all have, which is the ones we have. Well, we can't really grow for a hundred years with that. How do you build that? And building that requires you to ask them that. And so I have these battles that I'm having in my head and out loud, I'm not asking that. And then having to resolve that, you know as uncomfortable as that may be, you know, if you want this thing bad enough, then embrace asking. Figure out how to love asking.

And now I am really kind of psyched about fundraising. I'm really into it, I'm going to raise a whole lot of money. And I'm going to do it on my terms. So how do I do it and what do I do? You can always do it the way that is right for you to do it. And I think we get, again, socialized out of thinking that we can, that we have to follow through it again.

The most profound thing for me has been how I view approaching art. And that evolved from the end of 2018. I believe art should be discovered. I believe we should engage the cause we discover. The notion is this, this stuff makes no sense to me, that we have to schedule time to see art. That's not how art feeds our soul. I actually want people to engage with whatever I make on their own. Get rid of those text labels for Christ's sake. Don't bombard me with how I'm supposed to see something. 'Cause when you do that, you disrupt the very reason that we are creating this conversation, and it’s a conversation, it’s not a mediated moment where I have to bow to your schedules and bow to the way to say it. And in that, my notions of what I create now have expanded."

OM SA

OM SA

OM SA

OM SA

Utter pathos. And it is implying that while he sang, although he was singing by himself, he was not alone. That's why the previous line is "Until the whole harmonious landscape rang-" It makes the regret filled raising of the jug, the shake of the head, and the being "again alone" so poignant. I feel like we've all been to Mr. Flood's Party, at one New Year's eve or another.

We can only guess why the author would provide the "jug" in this text not only once but several. Using context clues we can believe it is wine and or beer. If that is true the character is drunk and is deep into their feelings and that's why they feel so lonely. Being under the influence is effecting the character so much they don't see much "ahead of him" This makes the ending so sad for we are only left knowing the character is having negative thoughts of loneliness and death.

Ending and beginning the poem with the idea that he's very alone. Seeing it at the beginning of them poem, I didn't really feel bad for him but now I do. It's clear that he has no one and he even talks to himself.

You can really hear the emotion in his voice. You know something is good writing when you automatically hear the voice of the narrator in your head without having to put thought into it.

I agree with the statement that "the act of writing is an act of thinking". When we write we tend to let our thoughts take over. That's why people write the best when they have a clear head space, any distraction can alter the way you write. You can write something when you are distracted and revise it later when you are in a clear head space, and you'll think what you just wrote could have been better.

The author's process.

Im pretty sure that is rape

My parents did this to my cross country teammates all the time and I have been called so many different names while racing. I have also done this to my teammates on accident it has never been due to racism. Sometimes your mind just slips when you are cheering your head off like a psycho cause you want them to do their best.

Should the polling capability be featured here?

Also- the ones linked to Zoom are messing with my head. It seems to me now that Zoom is the mechanism by which students can meet virtually, but I see how students could also present and meet in groups and faculty can present. Maybe because Zoom is just the water in which we swim (swam?) for the past year, it seems like a more general tool than one that can be named pedagogically.

Reviewer #1 (Public Review):

Lenz et al have shown that IP injection of atRA does not affect sEPSC amplitude, sIPSC amplitude and frequency in the denta gyrus of both ventral and dorsal hippocampus. Interestingly, they observed a strong promoting effect of atRA on sEPSC frequency in the denta gyrus of dorsal, but not ventral, hippocampus. Lastly, they did not observe an difference in I/O in vivo, but did observe enhanced in vivo LTP in denta gyrus of mice injected with atRA which is abolished in the synaptopodin KO mice. The effect of atRA on LTP is very interesting as on sEPSC frequency in dorsal denta gyrus.

1) I do not agree with the authors' claim that atRA does not have a major effect on excitatory synaptic transmission. It seems that the sEPSC frequency increase by ~100%. Even if the 4 outlier points are excluded, the rest of the data points still clearly indicate an increase of sEPSC frequency.

What is the possible explanation of increased sEPSC frequency by atRA in dorsal region? Increased excitability of presynaptic neurons? (use TTX to decipher this?) Increased spine density? It seems that the authors did dye fill already... Count spine density? AND/OR increased glutamate release probability? (PPR measurement?) Did the authors perform I/O measurement in slice?

It is imperative that the authors tackle this issue head on.

2) The author need to specify which part of the denta gyrus for their in vivo study, as they discovered difference between ventral and dorsal in sEPSC frequency in slice preparation.

Reviewer #3 (Public Review):

Telacebec (Q203) is a novel first-in-class antituberculosis drug that targets Mycobacterium tuberculosis respiration and cellular energy production through inhibition of the mycobacterial cytochrome bcc-aa3 (CIII2CIV2) super complex and thus represents the third class of energetic inhibitors against M. tuberculosis. Q203, currently in phase II clinical trials, has proven anti-tuberculosis activity in humans. Q203 also has the potential to be used in the treatment of nontuberculosis mycobacterial infections e.g. Buruli ulcer, a neglected tropical skin disease.

The manuscript by Rubinstein and colleagues reports the atomic-resolution entire structure of the Mycobacterial super CIII2CIV2 complex from Mycobacterium smegmatis inhibited by the anti-TB drug Q203. Other groups have reported the structure of CIII2CIV2 complex at high resolution, but none of this previous work has captured the drug inhibited structure. Yanofsky's new CIII2CIV2 complex captures the binding site of Q203 providing the mechanism (protein-inhibitor contacts) of inhibition of the complex and importantly also sheds light on the mechanism of enzyme catalysis. The structure reveals that Q203 binds with its head group deep within the Qp-binding pocket in a pose similar to UQ thus blocking menaquinol oxidation by the complex in both menaquinol binding modes. Multiple interactions are apparent leading to stabilised inhibitor binding.

The paper provides further structural data on the role of the SOD subunit in the complex and suggests that SOD may indeed transfer electrons from periplasmic superoxide to CIV thus contributing to the PMF and energy generation. The paper is a comprehensive, well written in an easy to follow style and will have broad interest and readership.

The way the John describes death is very interesting he does it in a way where the kids do not fully understand it, and makes them less scared. He makes it seem like the way the man died was that he hit his neck and then couldn't see and because of that he starved and died. This is a concept that would normalize death for children and is a very good way of not making death so gore and violent.

Okay, I don't know if I'm the only one that is highly disturbed by this. I know it's a family business, but I'm disturbed. the fact that the kids are not fazed my death structs me highly weird. then on the next page the kids just goes into details about how a crack head hangs from the head and just death in general. they were just talking about death like it's a normal conversation between kids. well I mean they are in a funeral home

Some of the theories early on did not explain reactions of higher functioning animals such as Chimpanzees. For example taking an infant from its mother because the mother abandons it, and then that same mother having a second infant, and refusing to give it up. To reduce drives to simple hunger, pain, sex, and maternity and learning is difficult as it does not explain all drives or motivations. Especially when the subject has never been exposed to an object/person/thing that causes a reaction. Thus a new drive would need added, curiosity drive which could explain investigatory and manipulatory activities. Why an animal would take a more difficult (although interesting) path to get food.

The Greek tradition is indeed a fascinating one.

Can you see any ways to simplify the task of learning 30 words?

The task of learning 30 words by simply going over them in your head over and over again until you get them right. Also putting each word into practice as it will retain in your memory better.

The love between the two is there but there is an obstacle in the two that prohibit them just blatantly admitting it.

Author response

September 9, 2021

We would like to thank ASAPbio for selecting our preprint for review! We are excited to contribute to this new process and hope others will find it as helpful as we have. The comments generated by the “crowd” were detailed and thoughtful. Below we respond to the major discussion points and if there were specific reviewer comments relevant to the discussion point, we also included that statement. We also responded to each specific comment. We would love to continue this discussion, so we invite further feedback and responses! Thanks so much for your time.

-Chelsea Kidwell, Joey Casalini, and Minna Roh-Johnson

Major Discussion Point #1:One of the most important claims is that mitochondria are the organelles responsible for the activation of the signals of cell proliferation. However, a previous report by the last author reported that macrophages transfer cytoplasm to recipient cells. It cannot be excluded that other organelles or cellular fragments are transferred as well and contribute to the observed effects (ERK activity). Perhaps a good way to solve this would be the use of macrophages that are devoid of mitochondria. At least, this aspect should be discussed in the manuscript.

🡪 We had first considered two approaches to test the requirement and sufficiency of macrophage mitochondria in cancer cell proliferation. The first was to generate rho-zero macrophages (mtDNA-deficient), as you mention in your comment, such that the macrophages did not have functional mitochondria. However, we use primary human macrophages for all of our studies, and these cells would not survive long enough to generate rho-zero cells (which requires that the cells be treated with low levels of ethidium bromide for weeks). The second is to biochemically purify mitochondria from macrophages and directly inject these mitochondrial preps into breast cancer cells. We actually did this experiment, and cancer cells injected with purified mitochondrial preps exhibited higher proliferation (by live timelapse microscopy) compared to control cells. However, we also found that the mitochondrial purifications were not clean, and contained other membranous components in the cytoplasm. We tried centrifugation-centric approaches, as well as IP-ing against a mitochondrially-localized tag, but in all cases, the mitochondrial preparations contained other cytoplasmic components. Therefore, we did not feel that this approach was an adequate way to test effects of specifically the mitochondria. We certainly wanted to discuss this aspect in the manuscript, but unfortunately, we were limited due to space. If folks have suggestions on how to best purify mitochondria, we’d love to know, so please reach out.

However, in terms of the bigger question of whether the induced proliferation in cancer cells is specifically due to ROS accumulation in transferred macrophage mitochondria, we tried to address this question with the mito-KillerRed experiments, where we generate ROS using optogenetics, and ask whether this accumulation is sufficient to induce cancer cell proliferation (which we showed it was). We also showed that this same approach could induce Erk activity, and then in separate experiments, we show that macrophage mitochondrial transfer results in accumulation of ROS and increased Erk activity. We feel that these experiments support our conclusions, however, we’d love for a way to link it all together. Unfortunately, we are not convinced that such experiments are possible at this time.

Major Discussion Point #2: Most of the positive examples of transferred mitochondria discussed appeared in a small clump. However, there also appears to be another population that was more diffuse and co-localizes with host mitochondria (e.g., Fig2B, bottom right panels). It would be helpful to show results of these sibling mitochondria for assays performed on their clumpy siblings. If they behave differently, it would be helpful to provide some explanation.

Specific Comment: Figure 2 Majority (57%) of donated mitochondria do not colocalize with LysoTracker signal (N=24 cells, 4 donors) - Here the paper implies that some transferred mitochondria do co-localize with lysoTracker signal. More importantly, they co-localize with host mitochondria. It raises the question of whether they signal through ROS and ERK like their clumpy siblings who are in the limelight of most figures.

🡪Yes, you are correct. There does appear to be a diffuse population of macrophage mitochondria. The majority of these mitochondria co-localize with lysotracker, suggesting that they are being actively degraded. We can’t say that they tend to co-localize with endogenous cancer cell mitochondria, however, it’s possible that this diffuse population is comprised of both mitochondria that are being degraded and mitochondria that are fusing with the endogenous network. We do not know if this population has a different effect on cancer cell behavior because we did not follow this population (mostly because once the mitochondria are degraded or fuse with the network, we can no longer follow those mitochondria!). However, we did follow cancer cells that contained punctate macrophage mitochondria. Often times this was the only population we could observe in the cell at that time, and this is the population in which we observe accumulated ROS.

Major Discussion Point #3:The effects that are attributed to the transferred mitochondria are highly variable (figures 1F, 3A,E) and often due to a subpopulation of samples that show a few extreme values (e.g. figures 2D, 3E, S4B, S4D). This might be expected from effects that are caused by a single mitochondria (which has a small volume) that is transferred to a complete cell. This complicates the study of the transfer process and effects and should be discussed. Also, do the authors have ideas how to improve the system, to make it more robust and easier to study the effects?

🡪The variability in the assays likely reflects the heterogeneity within the biology - Each experiment contains macrophages derived from primary monocytes that are harvested from different human blood donors! Due to the primary nature of these cells, we do expect a range of phenotypes as each donor would have a different genetic background and the monocytes were likely exposed to different environmental stimuli. In fact, even though working on this study was a giant pain due to the variability, we felt more confident about our findings because despite the heterogeneity in the system, we still observed consistent phenotypes. Below we indicate where we took a sample set and removed “outliers”, and ran the statistical tests again. The differences were still statistically significantly different, further suggesting robustness of our findings.

However, we are always on the lookout for ways to make the system easier to study. One way that we will follow up on is using M2-like macrophages since they transfer mitochondria at a higher rate than unstimulated macrophages.

Major Discussion Point #4: The authors conclude that the transfer of dysfunctional mitochondria generated a signal mediated by ROS that activates cell proliferation signals. The statement that "transferred mitochondria act as a signaling source that promotes cancer cell proliferation" is too strong. There is increased ROS production from mitochondria, yes, but an experiment in which ROS are decreased would be needed to properly sustain that conclusion. The title and abstract could be changed to better reflect the data.

Specific Comment: ‘Furthermore, treatment with an ERK inhibitor (ERKi) was sufficient to inhibit ERK activity ‘- curious as to whether antioxidant treatment would reverse any proliferative phenotypes?

🡪We wish we could quench the ROS at macrophage mitochondria. We really tried. We used a combination of ROS quenchers (NAC, mitoTempo, Tempo) and ROS readouts (mitoSOX, CellRox, DCFDA, and the 2 biosensors used in our study: Grx and Orp1), and treated cells for various amounts of time, and no matter what we tried, we could not reliably detect reduction of ROS levels in the host network or the transferred mitochondria (without killing the cells, that is). Another issue that we faced was that any pharmacological treatment would have a global effect on the mitochondrial network in the recipient cells and therefore it would not be possible to distinguish effects from global inhibition of ROS versus specifically at the site of the transferred mitochondria, and we certainly observed cell death upon treatment of ROS quenchers because of this fact. We talked to a couple of ROS experts, and they indicated that this issue is not unique to us, although we unfortunately did not have viable solutions, so if people have ideas or suggestions, please let us know!!

However, despite our failed attempts at quenching ROS, the comment that "transferred mitochondria act as a signaling source that promotes cancer cell proliferation" is too strong of a statement… well, we don’t entirely agree given that we do perform sufficiency experiments in which weinduce ROS and observe both proliferation and ERK signaling, so we do feel reasonably justified to provide the title that we did. However, we will continue to mull over this comment. Thanks for sharing your thoughts.

Major Discussion Point #5:The study may benefit from more direct evidence to support its conclusion of increased proliferation after mitochondrial transfer. While the RNA-seq, flow cytometry, counting of completion of cytokinesis and dry mass measurements provided in the present study do lend some support to the proliferation hypothesis, they all seem indirect. With the biomarkers labeling the mitochondria of donor and potential recipient cells, high content imaging and tracking of cells could be used to monitor cell division. A comparison of cell division rates of transfer-positive cells and transfer-negative cells will provide a more pertinent test of whether mitochondrial transfer promotes recipient cell proliferation.

🡪We should probably do a better job at describing the dry mass measurements (QPI, quantitative phase imaging) because we view this quantification as one of the most direct measurement to monitor cell growth/division. The approach measures the changes in dry mass as the cells prepares for cell division. So not only do we get the final readout of division (complete cytokinesis), but we also get a measure of that growth rate (the cell getting ready to divide) before cytokinesis. This is why we are so tickled to collaborate with Tom Zangle’s lab because we could finally get a direct proliferation readout in real-time. We could also use this approach to follow thousands of cells at a time, a very critical aspect since mitochondrial transfer is rare event, and therefore, we need to follow many cells to have enough statistical power to quantify the growth rates. Check out some of the Zangle lab’s other papers (PMC5866559; PMC6917840; PMC4274116), and please let us know if you disagree with us!

Major Discussion Point #6: The authors have used such a tracking-based approach on a very small scale (n=5) to measure daughter cell growth rate. However, the data do not show a statistically significant difference between the growth rates of daughters that inherited transferred mitochondria and those who did not (Fig S3). Increasing the case number via high content imaging would help obtain sufficient data points for a reliable statistical test. In addition, as suggested above, an accounting of the daughter cells' division rate for transfer positive and negative cells would provide another line of evidence to either prove or disprove the increased proliferation rate hypothesis. The same suggestion goes to the optically induced ERK activation experiments shown in Fig3F. It is also helpful to include references that studied how ERK signaling promotes proliferation and compare the evidence here with evidence or assays used in those studies as a benchmark.

Specific Comment:Figure S3 - There is no statistical test to check for ‘increase in their rate of change of dry mass over time versus sister cells that did not inherit macrophage mitochondria’. What are the colours indicative of in S3B? Can this be reported in the figure legend.

🡪You are right – the tracking-based approach on daughter cells is based on a small ‘n’. However, the tracking itself is performed on 1000s of cells. It’s just that in order to capture daughter cell data, we have to find a cancer cell with macrophage mitochondria (which is only ~1% of the population), and then follow that cell until it divides, and then follow BOTH daughter cells. So, even with the 1000s of cells that we followed, we could only capture a small number of daughter cells. The colors in S3B represent each individual triads – parent and 2 daughters. We will make this info clearer in the legend.

In terms of the optically-induced ERK activation experiments, yes, it would be great to have a higher sampling. These experiments were performed at 63x so we could reliably draw small ROIs to mimic the size of a macrophage mitochondria. While we switched to lower magnification to follow cell division, we still were limited to only a few cells for the actual photoactivation. The technical aspects of this experiment were the reason for the low sampling. Despite these limitations though, we still observed increased cell division upon mito-killerred photoactivation, which we were honestly pretty surprised (and stoked) about.

Other specific comments:

-Figure S1A - The authors could perhaps use a more aggressive gating strategy here, clipping closer to the 231 population described in Fig S1A - picking only the center of the cluster in the upper left of the RFP vs CD11b plot would likely not affect results but make them more convincing by unequivocally excluding macrophages.

-Figure 1D - Not sure about the 0.2% baseline assigned for the monoculture of cancer cells (that does not have the macrophages with the Emerald mitochondria). It is determined with cytometry - I am no expert on that topic, so maybe I missed something - but it looks weird to see some cells with transfer when there is a monoculture.

🡪Due to the variable nature of the mito-mEm signal in the recipient cancer cells (i.e. transfer of one mitochondrion vs transfer of three), we found that an overlap of 0.2% set on a fully stained monoculture control was the most accurate way to gate for the recipient cancer cells. The final gating strategies used in our study were determined by FACS-isolating populations of interest based on several different gating strategies, and directly visualizing cancer cells with macrophage mitochondria without capturing macrophages or cancer cell/macrophage fusions (which is cool, but not what we wanted). To further clarify, there is no transfer occurring in the monoculture – the overlap of mEmerald signal into the transfer gate in that control sample is likely reflective of normally occurring autofluorescence. This is a very important point, so we will make this aspect clearer in the Methods section.

-Figure S1B - Could perhaps be an interesting follow-up question for future works re: differences between cell lines and propensities to transfer mitochondria. Did the authors attempt to use other cell lines (ie, MDCK, HeLa, iPSCs, etc)?

🡪Great question and something that we have also been thinking about. To date the only recipient cells we have used are 231, MCF10A, and PDxO cells. This would be a great avenue for future studies.

-Figure S1B - Did the authors see an increase in growth rate in MCF10A line despite the lower growth rate?

🡪We have not measured the growth rate in MCF10a recipient cells but something that would be great to follow up on in future studies.

-‘physically separated from macrophages by a 0.4μM trans-well insert’ - should this read 0.4 micrometer?

🡪Yes, great catch.

-Figure S1F - The authors wrote that they used a two-way ANOVA analysis, could you report the factors used for that analysis in the Figure legend.

🡪Noted!

-Figure 1B - It is difficult to see the arrowheads in 1B, suggest moving them so they are not covering the magenta fluorescence, have them point from a different angle, and make them more brightly colored. Insets here would help the reader. A negative control image from a monoculture would also be helpful, to ensure the GFP signal is not an artifact of culture conditions.

🡪Thank you for your feedback – we will take note of this.

-Figure 1F - For graphs that do not show zero (as in 1F), the bar should be omitted. In these cases the length of the bar does not reflect the average of the data (as it does in 1D).

-Figure 3C - Please omit bar, see comment on panel 1F.

🡪 In the case of Fig 1F, we modified the y-axis to eliminate empty space. The bar is representative of mean of the data displayed in both 1D as well as 1F, but we can add a broken y-axis to help make this point.

-Figure 1 - Given that these data are fractions of a population (ie. can be described via a contingency table), isn't something like a Fisher's exact test a better measure of significance here?

🡪We think you are referring to Figure 1D? If so, we thought that we could not use Fisher’s exact test because that test assumed parametric distributions (which we do not observe). We have been working with a biostatistician for our statistics, but please do let us know if we have it wrong.

-Single cell RNA- sequencing - In the methods section the authors mention doing a differential analysis between the cells that received the mitochondria and the cells that didn’t. It might be worth introducing a figure (a heatmap or a U-MAP) relating to this analysis. Single cell sequencing would not only affirm the heterogeneity between these two populations but also help in highlighting the novel cell surface markers associated with the two populations.

🡪Yeah, good point – we can add a UMAP.

-‘mito-mEm+ mitochondria remained distinct from the recipient host mitochondrial network, with no detectable loss of the fluorescent signal for over 15 hours’- It is surprising that the transferred mitochondria do (or cannot) fuse with the host 231 mitochondria.

🡪We were also initially surprised to find that the transferred mitochondria do not fuse with the host 231 network! We think that the lack of fusion is due to the fact that the transferred mitochondria do not exhibit membrane potential (which is required for mitochondrial fusion). We also think that these results open interesting lines of questioning: Why are these depolarized mitochondria not degraded? Is this an active avoidance of the mitophagy pathway? How dynamic are these punctae? Many fun and interesting questions regarding the long-lived nature of these transferred mitochondria.

-It is unclear in these images, but the 231 mitochondria appear fragmented too. Is it possible that the mitochondrial fusion machinery (Opa1 or Mfn1/2) are inactive?

🡪231 cells are capable of fission and fusion (PMC7275541, PMC3911914, and in our own timelapse recordings), so we think that the machinery is functional. However, we don’t know whether the 231 mitochondrial machinery changes after receipt of macrophage mitochondria. Interestingly, the references above both investigate how mitochondrial dynamics promote tumor metastasis. A fascinating future direction could include an investigation to how macrophage mitochondrial transfer influences tumor cell mitochondrial dynamics.

-Figure 2B - What does the MTDR staining of the macrophage mitochondria prior to transfer look like? Important to check this to confirm that only the transferred mitochondria had lower membrane potential.

-‘significantly higher ratios of oxidized:reduced protein were associated with the transferred mitochondria versus the host network’-Here too, it would be important to check the mito-Grx1-roGFP2 readout of macrophage mitochondria prior to transfer.

🡪The way that these comments are written is as if we already know that the mitochondria are dysfunctionalbefore transfer to cancer cells. But we actually do not know if that is the case. It’s also possible that macrophage mitochondria become dysfunctional once they are in the cancer cell, which would be equally cool. So, we are actively investigating this biology.

-Figure 2A, 2BB and S1D - How were the colocalizations assessed? Was it just a visual assessment? Given the importance of these experiments for the whole story, having a quantification of the level of colocalization with each dye would be important.

🡪This is a good point and it should be straightforward to include a Pearsons coefficient for these markers.

-Figure S1D - The paper makes an argument about mitochondria transferred from Macrophages (marked green) having positive DNA stain (gray), but appearing depolarized (negative TMRM stain). The image in FigS1D is peculiar, as the majority of the 231 cells' mitochondria appear to not have any DNA stain but maintain membrane potential (positive in TMRM), while some (just above the green macrophage mitochondria) do have both DNA stain and membrane potential. The authors might want to clarify whether this is a typical scenario, and if so perhaps offer an explanation as to why the 231 mitochondria exhibit such heterogeneity.

🡪The images in S1D are of a single z-plane image therefore the DNA signal in the endogenous network is more readily visible in planes that are not shown.

-‘we confirmed that 91% of transferred mitochondria were not encapsulated by a membranous structure, thus excluding sequestration as a mechanism for explaining the lack of degradation or interaction with the endogenous mitochondrial network’ - This is based on co-staining with MemBrite 640/660, which is a dye that "covalently labels the surface of live cells", thus there is a concern as to whether this approach allows to study whether the mitochondrium is encapsulated by an endomembrane.

🡪Thank you for your feedback. We actually do think that Membrite can label endomembrane in addition to the plasma membrane. This is from the published Membrite protocol: “MemBrite™ Fix dyes are designed to be fixed shortly after staining, when they primarily localize to the plasma membrane/cell surface. Cells also can be returned to growth medium and cultured after staining, however, dye localization in live cells changes over time. Labeled membranes become internalized, so staining gradually changes from cell surface to intracellular vesicles, usually becoming mostly intracellular after about 24 hours. Internalized MemBrite™ Fix dye is usually detectable for up to 48 hours after staining, though this may vary by cell type”.

In our hands, we found that the dye started to become internalized and labeled vesicles within the cell within a few hours of staining. The images in the panels that you refer to came from time-lapse imaging experiments of between 10-15 hours, therefore the cells have internalized the MemBrite signal allowing for the visualization of internal vesicles. Also, in other studies not in the preprint, we perfused purified mitochondrial preparations onto 231 cells. The 231 cells took up the mitochondria from the environment, and all of these engulfed mitochondria were surrounded by a MemBrite positive membrane! These results further suggest that if the transferred mitochondria were encapsulated by a membrane, we would be able to visualize it.

_-‘macrophage mitochondria are depolarized but remain in the recipient cancer cell’ -_Did the authors examine the extent of cancer cell death in their co-culture system (due to the activation of apoptosis by the depolarized mitochondria)?

🡪We do not find any evidence of abnormal levels of cell death by both flow cytometry assays as well through our QPI image analysis.

-Figure 2C–D - Like in Fig 2B, in the bottom left of panel of Fig 2C there are a lot of donor mitochondria not in highly oxidized state and the growth/proliferation phenotypes apply mostly to donor mitochondria that appear 'clumpy'.

-Perhaps it is worth commenting on whether there is a link between donor mitochondrial morphology and the suspected proliferation-enhancing phenotype.

🡪The images in Fig. 2C are of the same cell – a single recipient cancer cell which is expressing the Grx biosensor. The donor mitochondria are labeled with an arrowhead, the rest of the yellow/green signal (bottom right) is from the endogenous host network and therefore we do not expect it to be in a highly oxidized state (ie. more yellow than green).

Regarding the mito morphology and proliferation – great question, and one that we are actively working on!

-‘At 24 hours, we observed a similar trend, but no statistically significant difference (Fig. S4D). These results indicate ROS accumulates at the site of transferred mitochondria in recipient cancer cells’ - if a specific sensor fails to show a significant oxidation at 24 hours compared mito-Grx1-roGFP2 which reports on mitochondrial glutathione redox state, does that mean there are ROS independent ways to oxidize Glutathione? The authors did see cell growth phenotype both in 24 and 48 hours which suggests that something is happening in 24 hours despite no significant difference in ROS H2O2 sensor.

🡪The additional biosensor that we used – mito-Orp1-roGFP2 - has been engineered to be a readout of one type of ROS – H2O2. The Grx probe is a surrogate for ROS of any type, of which there are many! To us, it is not completely unexpected that they would behave differently over time since they are readout for two separate things, and it generates an interesting possibility that different types of ROS accumulate over time. Given that the Grx probe shows an increase at 24 hours, which is when we observe the proliferation phenotype, we think we are on the right track. If you have ideas on robust ways to directly observe specific types of ROS, we would love to know!

-The differences in ratio for the two sensors used are not very convincing. In Fig 2D and Fig S4B and D the “host” and “transfer” populations are very similar. The difference seems only due to the presence of a few outliers in the “transfer” populations. More importantly, sometimes it seems that these outliers come mostly from one donor rather than being present in all 3 donors. It could be good to show histograms of the two populations for each replicate/donor and maybe redo the stats excluding these outliers.

🡪We think that the heterogeneity that is observed is due to the biology in the system – we are using primary macrophages derived from blood donors. However, for the data represented in Fig 2D, just as a test case, we took out the top four “outliers” in that data set and re-ran the Wilcoxon matched-pairs signed rank test and the p-value was 0.0010 (***), further suggesting that the ROS biosensors are revealing consistent and robust results.

-Figure S5C - it seems like the percentage of cells that divided is the same for unstimulated cells and cells with stimulated mito-KillerRed. Isn't this contrary to the expectation? The figure shows that photobleaching cytoplasm decreased % cell division, which is puzzling.

🡪The mean percent of cells that divided in unstimulated and mito bleach are very similar and was not significantly different. One point to be made that may not be well illustrated in our graphical representation is that if you look at the matched data (points connected are averaged per FOV for each condition in the same experiment) the trend shows that the mito bleach does seem to have an increase in cell division which is washed out with the average bar overlay. We should note that this experiment is very “noisy” and therefore we needed a lot of N to be able to detect significant changes. We are currently thinking about other ways to demonstrate sufficiency as it relates to cell proliferation – any experimental suggestions would be very welcome! Thanks for the feedback.

-Figure 3A - In the 'cyto' condition 6 out of 13 fields have no cells that divide. Is that expected? What is the percentage of dividing cells for cells that were not illuminated at all (a control that is lacking)? There is large variation, ranging from 0% to 22%. The evidence that illumination of KillerRed leads to increased proliferation is rather weak. Also, since Cyto and Mito are different cells, is a "paired" statistical test the right kind of test to use here?

🡪Additional data pertaining to Fig. 3A can be found in Fig. S5C, which includes the control for cells not illuminated at all. Having no cells that divide in a field of view is not surprising to us – the doubling time for these cells is ~35 hours, and we imaged for 18 hours. Also, for each field of view, our ‘n’ for each field of view was often 6-8 cells because we performed these experiments at 63X to allow for accurately drawn regions of interest for photoactivation. We also internally controlled every experiment (each experiment consisted of fields of view that had either mito activation, cyto activation, or no-activation controls, all of which were imaged overnight with multiple x/y positions). Cells that left the field of view over the 18 hours of imaging could not be quantified. It’s this sampling that caused the large variation in the graph. But again, as with many of our experiments, despite this variability, we still observe a significant difference in our experimental conditions over control cyto bleach. As for the statistical test, our understanding is that given each experiment is internally controlled, and we compare within each experiment, a paired statistical test is appropriate here. We will consult with our biostatistician to confirm, though.

-‘ROS induces several downstream signaling pathways’ - We would not expect the authors to investigate every signaling pathway, but wonder if the PI3K pathway was explored? It seems to be the other major cancer/proliferative pathway induced by ROS.

🡪Yes, this is a very good point! We actually assessed three different pathways at first – ERK, PI3K-AKT, and NLRP3/inflammasome. While analyzing these 3 pathways simultaneously, we discovered that ERK inhibitors resulted in decreased proliferation in cancer cells with macrophage mitochondria. As a result, we then focused on the ERK pathway. We still do not know if PI3K-AKT or NLRP3/inflammasome pathways play a role in this biology because we have not gone back and revisited these experiments yet, however in figure 3F, ERKi treated recipient cells exhibit a partial ‘rescue’ of baseline proliferation. This suggests that other pathways may indeed be involved and we plan to investigate this possibility.

-‘Recipient 231 cells had significantly higher cytoplasmic to nuclear (C/N) ERK-KTR ratios compared to cells that did not receive transfer’-Since two different quantification styles with opposite fraction values were used, is it possible to please specify which one was used here.

🡪Will do!

-Figure 3B - Please show the outlines of the nuclei and that of the cell.

🡪That would be helpful, wouldn’t it? Will do!

-Figure 3D - it is peculiar that ERK-KTR in Fig 3D is so strongly cytosolic while in Fig 3B it is almost exclusively nuclear. If this sensor behaves differently in different situations, the authors may want to comment on how that would affect their conclusions.

🡪The panels in Fig. 3B were taken with the ImageStream flow cytometer which takes a lower resolution image of a single plane of a cell in suspension in the flow stream. In Fig. 3D, those images are from confocal spinning disk microscopy which allows for higher resolution, z-stack images of adherent cells on glass. Therefore, we think the differences that you point out are likely due to the fact that the two images come from very different imaging systems.

-Figure 3E - The effect of 'opto-induced' ERK activity is weak. The initial ERK-KTR is 1 at time point zero (as the data is normalized to this timepoint) and around 1 for both the cyto and mito condition. A statistical difference is observed, but the effect is minor and it is unclear whether it is biologically meaningful. The 'cyto' condition shows an average below 1 and the mito condition remains 1, suggesting that ERK activity remains constant when ROS are produced in the mitochondria.

-Also from S8C and 3E it appears cyto actually shows a decrease rather than mito showing an increase, could the authors comment on this?

🡪We have a few thoughts on this. The first is that we don’t expect a dramatic change in ERK signaling because the ROS accumulation is localized to a small region in the recipient cell. This is not a situation where we would expect a large-scale change because we are adding a growth factor. We can understand that the change in ERK activity may appear to be minor, but our data suggest that these subtle changes in kinase signaling translate into significant changes in downstream behavior – proliferation. The way that we interpret differences as “biological meaningful” is whether they exhibit a functional response, and in our study, we show that inhibiting the induction of ERK activity in cancer cells with macrophage mitos inhibits proliferation. What is most interesting to us is that cancer cells that do not have macrophage mitochondria have an unchanged fraction of cells in G2/M phase of the cell cycle in response to the concentration of ERK inhibitor we used, suggesting that the ERK inhibition specifically blocks macrophage mitochondria-induced proliferation.

In Fig. S8C, bleaching a region of cytoplasm does seem to cause a decrease in ERK activity over time. We really can’t explain this result. However, we do think that ERK activity is higher in mito-bleached cells because mt-ROS is generating an increase in ERK activity which compensates for the decrease in activity that occurs when the cytoplasmic region of interest is photobleached. It’s still a head scratcher, though, but we did perform internal controls for every experiment (as we describe above), and the mito-bleach, cyto-bleach, and no-bleach conditions were run side-by-side such that we can make apples-to-apples comparisons.

-‘patient-derived xenografts (PDxOs)’ - As a control it would be relevant to include a normal mammary organoid model perhaps from the same patient to demonstrate that the transfer of mitochondria specifically to the cancer cells is more beneficial.

🡪Using a normal mammary organoid cells as a control to compare efficiency of transfer and downstream phenotypes would be very interesting. Due to the fact that these are patient-derived organoids, we are unable to acquire non-malignant cells from the same patient. Expanding our studies in the MCF10A cell line that we utilized in this paper would be an alternative to what you propose and would also expand our understanding of general biology underlying mitochondrial transfer.

-‘macrophages to both HCI-037 and HCI-038 PDxO cells (Fig. 4G)’ - Why is M0 able to transfer efficiently to HCL-037 tumour when its mitochondrial network is less fragmented as M2?

🡪These results really stood out to us. It was quite surprising that in HCI-037, both M0 and M2 macrophages were able to transfer their mitochondria at similar efficiencies, but in HCI-038, M2 macrophages were more efficient at transfer. HCI-037 is a primary tumor, and HCI-038 is a metastases from the same patient, so there are some exciting avenues of study to examine how macrophage mitochondria transfer differs at the primary versus metastatic site. There is still very little known about how donor cell dynamics influence mitochondrial transfer!

-Are mito transfer from M0 depolarised and accumulate ROS or show increased ERK activity or increased cell proliferation?

🡪Yes – all studies, except studies pertinent to fig 4 (where we assessed macrophage differentiation states), were done with M0 macrophages.

-‘M2-like macrophages preferentially transferred mitochondria to the bone metastasis PDxO cells (HCI-038) when compared to primary breast tumor PDxO cells (HCI-037)’ -The authors may want to check this statement here as it is in consistent with their data plot. In Fig. 4G, M2/PDxO transfer percentages for HCI-037 and HCI-038 are about the same, unless the authors provide statistical tests to prove otherwise. Instead, M0 appears to transfer mitochondria to HCI-037 much more efficiently than it does HCI-038.

🡪Upon re-reading our sentence again, we now realize that it’s actually quite poorly written, so we can understand the confusion! What we meant to articulate is that M2-like macrophages are better at transferring mitochondria to HCI-038 than M0 macrophages. Whereas in HCI-037, we do not observe the same preferential transfer (ie. M0 and M2 can transfer at the same efficiency). We will certainly clarify this language in the manuscript.

-‘M2-like macrophages exhibit mitochondrial fragmentation’ - Is there a correlation between the status of the mitochondrial network in the donor and the % of transfer to the recipient? If so, this would be a correlation that would support the conclusions.

🡪Yes, please see Fig. 4C for transfer rates with different donor subtypes and Fig. 4H for a general working model on how we think these data fit into the larger picture.

-‘accumulate ROS, leading to increased ERK activity’ - Did the authors obtain similar results with the PDXOs? It would be an interesting observation if the primary samples also exhibit a mechanism similar to established cell lines wherein there are more accumulated genetic changes.

🡪Our main limitation with PDxOs is overcoming the technical hurdles related to our downstream assays. These include introducing relevant reporters and generating stable lines in the PDxOs, and imaging at high-resolution when the PDxOs are cultured in 3D. However, we are very interested in this question as well, and are actively thinking about ways to overcome these hurdles.

-It would also be interesting to examine whether there is any difference in the ROS-ERK mechanism for primary and metastatic tumour.

🡪We agree and this is an active avenue of investigation for us. We agree and are currently pursing models to understand how our findings fit into the larger picture of tumorigenesis and metastatic potential. We had spent months pursuing anin vivo approach using a murine Cre/LoxP system to genetically label mouse macrophage mitochondria with GFP. We crossed mice which express Cre under a monocyte-specific promoter (Jax, SN: 004781) and mice with germline expression of Lox-Stop-Lox-3xHA-EGFP-OMP25 (Jax, SN: 032290) with the expectation of seeing Cre-based excision of the stop cassette – thus resulting in offspring with macrophages expressing mitochondrial-localized GFP. However, the macrophages of the resulting offspring do not express GFP (by flow cytometry, imaging, and western blot analysis), despite the PCR-verified presence of both transgenes and the excision of the stop cassette. Needless to say, this was quite frustrating! We are currently in the process of developing a newly available MitoTag model which has been optimized for visualization purposes (Jax, SN: 032675). If you have any suggestions or advice on this matter we would much appreciate your thoughts!

-‘in cancer cells that receive exogenous mitochondria’ - Since these macrophages also transfer mitochondria to non-malignant cells, such as MCF10A cells shown in Fig S1B, perhaps the authors could comment on whether this is part of a physiological process that would also promote normal cell growth?

🡪 There are so many questions regarding when and why macrophages might transfer mitochondria. In general, mitochondrial transfer is observed in stressed cells. Our data suggest that transfer happens to MCF10A cells although at a much lower rate than their malignant counterparts, 231 cells, but we do not know whether similar downstream mechanisms and phenotypes are also occurring in the non-malignant cells. Thanks for your feedback – more to come here!

This is certainly true, a lot of things that we see and come across affect our thinking and is also highlights that some seemingly obvious answers are wrong when seen in a superficial manner, every question needs to be thought deeply.

This is an example of divine providence. According to Stanford Encyclopedia of Philosophy, Divine Providence is a traditional theism that holds that God is the creator of heaven and Earth. In which all that takes place in the Earth is called Divine Providence- which is under God's sovereign guidance and control. This is a notion that no matter how bad something's going if you keep faith in God, he will in turn bless you. We see this in the highlighted text because this an example of how they came over a hardship through the grace of God. This young man condemned and curse the poor people because of their sickness, and in return died from a painful disease in which he was thrown overboard.

McCann, Hugh J. and Daniel M. Johnson, "Divine Providence", The Stanford Encyclopedia of Philosophy (Spring 2017 Edition), Edward N. Zalta (ed.), URL = https://plato.stanford.edu/archives/spr2017/entries/providence-divine/

But, in case of the US or Egypt, how can we describe the state? I find the difference between the state and the government a little confusing.

I understand this point but it should be done carefully as it can easily go wrong and the students don't believe you know what you're doing - i.e. it can have the opposite effect

This Comment resonated with me because if I look back and think of what I may possibly have, economy wise. I would have more to show for today. However, I must admit, if I died tomorrow, I did have one heck of a good time!

I feel like these kind of mistakes tend to happen a lot when people are starting to save up money or when they suddenly have a large sum of it. In the moment you feel on top of the world and people end up blowing it all in the moment because of all that rush and excitement. But when people look back on it they sometimes really regret their decisions. I know I've made purchases I regret as well but it's good to learn from it and make better ones.

I noticed in this quote that alot of american citizens do this. They do end up making the money they need to do nice things like get a house, get a car, get some nice clothes but instead they waste their money and waste their lives working at a job they probably dont like to continue spending it. I feel like this is a big part of american society that goes under the carpet alot.

This is the lifestyle millions of people in America can relate to. A nine to five job that doesn't pay what it should and leads people to barely meet ends meats for biils, food, roof over their head, running clean water that cost an extreme amount. America is glorified and so far from the truth of how millions actually live and barely meet ends meat and may take a long time to get out of debts.

What a terrible thing, for this child to grow to know nothing of compassion in this world of slavery. How many people from this story have been rasied the way that Mr. D's son was.

These seem like odd associations. It feels like there is a double meaning here.

( p. 263 ) Malcolm X not only was fighting for Black peoples rights but for all BI&POC. In his head the enemy or “devil” was the white man.

Manto’s description of Sirajuddin’s daughter in this section of the story flips the tone of a seemingly happy ending on its head. As readers, we should be elated; the one problem Sirajuddin has been overcome with is finally resolved. Sakina has been found safe and well, at least, that’s how it appears. Upon first reading the ending, I was met with the same joy as Sirajuddin. However, after digesting the story I find that certain words and phrases Manto uses to describe the state of Sakina seem to bring a darker connotation to the ending of the story. “Painful slowness” is used to describe Sakina’s movements. It is not wrong to assume that something sinister happened to Sakina when she was “rescued” by the men who found her on the side of the road. Through the description of her encounter, it is clear that she was uncomfortable. Sakina is immediately described as “pretty” and, once she joins the men, she attempts to cover herself. “Nervous” and “ill-at-ease” are also used in this section, clear indicators of discomfort. Due to context clues and Sakina’s obvious physical collapse since her rescue, it is clear that she was abused or sexually assaulted in some way. It’s interesting to see how the elation Sirajuddin feels once he sees his daughter creates a mask of oblivion that shields his judgment. Sirajuddin is not able to see how Sakina is both emotionally and physically affected by her encounter with the men who found her. Even the doctor appears concerned. Manto states, “the doctor broke into a cold sweat,” perhaps revealing some sort of physical injury when Sakina parts her legs. Overall, it makes sense that Sirajuddin is happy to see Sakina alive, but the emotional baggage that Sakina must work through as a result of her assault forces a sense of despair upon the reader when the pieces are put together.

This was a tough read. Definitely mostly went over my head. The conclusion helped, but I feel like this paper was more an overview for people who already knew about classification stuffs rather than an introduction for people... Could just be me, though.

What does this mean by the "brain will set us free". Does it mean as in logic or is it referring to something else.

A common thread I have noticed between characters in this play is the lack of empathy they posses. First, with Marie handling her child. She does not come across as tender. She teases him and tells him that the sandman will cause him to go blind. She does not seem to be aware of his physical comfort. Woyzeck has to ask her to adjust him because he was pinched by a chair while she looked at herself in the mirror. The Doctor does not register the emotional turmoil of anyone around him, rather just noted their physical state. And here, the captain watches Woyzeck and the doctor run away views them as grotesque.

This moment was quite disturbing for me, as it foreshadows Marie's murder and displays Woyzeck's driven madness. It has become clear at this point that Woyzeck has highly suffered mentally and physically, as result of taking part in a medical trial. He not only experiences visual hallucinations, but has now gone on to hear voices in his head that encourage violence/ irrationality. Although he displays quite a few alarming behaviors, the audience can almost sympathize with him, as he is humiliated and victimized through the trials/ affair.

this sounds like a bit of a foreshadowing as to what's to come...he sounds as if he's contemplating something and he's torn between his emotions and his head. These malicious thoughts are eating him alive.

It seems that this person has had a really long life were there were some things that might have made the person as but in the end it seems that the person seemed to have a content kind of life that was well lived and that there also seemed like there was a lesson to keep your head held high.

This doesn't sound/make any sense in our head but English can be complicated when it comes to things like this.

!!!!!!!! Yes

This exactly describes my experience with Spanish in college. During class periods when I was required to speak or write, I would get lost in my head trying to remember how to do every little thing correctly. One of the assignments was to have a half hour conversation with someone who speaks Spanish as a first language, and during those I felt more confident because even though I wasn't getting things 100% grammatically right, I was still being understood.

ERD RED FLAMINGO ROAD

humm ... pointing out missing text.

RED ALERT

cease and desist your entire operation #IMH0

MORONIC DISAMBIGUATION

AZRAEL DENOUNCES "FRAMES" AND THE AZAZELIAN SET UP THIS BULLSHTI HAS TURNED INTO.

Respecto a este punto siempre me surgió la misma duda. Si iniciamos con una hipotesis sin tener total conocimiento de como vienen los datos, y luego al trabajarlos nos damos cuenta que en realidad vamos a llegar a otro punto, esto implica que no teníamos un buen conocimiento del negocio para plantear la hipotesis inicial o son situaciones que pueden darse por la mera naturaleza de desconocer puntos claves de la información ??

The fact "the paint and paper look as if a boys' school had used it" the trying to describe the broken appearance and state of the paper is very telling. When trying to find the perfect words to describe a wretched pice of paper, the author used boys to do that. This paragraph h reflects the careless and rough nature of masculinity. Just as they boys left patches in the paper, toxic masculinity has left patches in society.

At this point, John's wife is sick of the "Yellow wallpaper" which frustrates her to be in the room. I think it's interesting that the word 'because' is capitalized since it emphasizes she’s tired of following orders from John. The thought is on the back of her head 24/7. The narrator hinted the wife is trying to get a better understanding of the wall-paper. This similarity reminds me of Adam and Lansley having different thoughts to understand how the dynamo worked and how it was going to change their society.

what does the vine do?

I think it's interesting that these two species convergently evolved camouflage on only their head despite these continents being apart for over 500 million years

The Uncomfortable Truth is the Difficult and Unpopular Decisions are Now Unavoidable.

Topic is relevant across a span of global issues. Natural resources are Finite.....period! Timely decisions are critical to insure intelligent use of resources. DENIAL is the enemy and 800lb gorilla in the room. Neoliberisim and social dysfunction feed on any cognitive dissonance and poop it out as "crap". True believers of American Capitalism (yes there is a difference) have become "cult-like" and drink the fluid of the cult to the very end, human consequence is of no concern.

Point being: Reality is always elusive within a cult controlled (authoritative) mindset. Cult members are weak sheep, incapable of individual logic/reason. Authority can not be challenged. -- Denial, a human defense mechanism has been and is the common denominator in all personal and global conflict. Denial can be traced throughout modern history and rears its ugly head whenever the stakes are high.

He's also really smart. Admirable!!

Novak provides great emphasis on the subject's grin. As a reader I appreciate this detail because without being able to see this painting, I am able to create one in my head.

I see the privilege discussion arise on Facebook quite often among a particular group of my friends. A heated argument ensues when someone disagrees with white privilege existing. Specifically, a couple who has a strong Irish background. They always deny white privilege then immediately give us a history lesson on how Irish immigrants were treated in the past. They are good people but they just refuse to admit they have certain privileges other don't. I grew up poor, under developed, in an abusive home then parents divorced and I always envied those “rich kids” on the hill. The kids that would come to school with brand new clothes, brag about how many Christmas gifts they received, or they got exactly what they asked for on their birthday. As I grew older, I slowly noticed, my privileges that others didn’t have (we didn’t use that terminology though). A roof over our head, government provided groceries, free school lunch, and used clothing are all privileges not everyone has.<br> I believe an attribute that will help you inventory your privileges is gratitude. Just be thankful for what you do have, be appreciative and return the kindness with compassion and understanding to those less fortunate than you. Also, just except you have certain privileges others don’t and be an advocate for a better, all inclusive, world.

Schools prioritizing inclusion over success has lead to less sucess

In saying he is a "stranger in his own house", is powerful because this automatically pants a picture in the readers head that he feels as though he does not belong, or is negatively different in a space he and everyone else should coexist and be equal in. He also uses many words like prison or of/relating to enslavement because although African Americans are no longer slaves, he is expressing there is still systemic and social forms of oppression and discrimination.

These three areas seem to be lofty goals that really need to be expanded for the individual teacher. What does it mean to create space for teachers to reflect on and improve their own teaching practice? How do we intentionally foster teacher relationships with other teachers and with students? How do we improve the accessibility of classrooms for all students? I think each teacher needs to really reflect on how these are achievable in their own classroom and it seems the largest barrier is that it is difficult to reflect meaningfully when one is just trying to keep their head above water.

Points to the quality of new or potential recruits and shows how guards are also capable of violence.

If turning to analogies helps you as a writer and you are good at creating them in your head, you are one step ahead of the game. I know a lot of people who struggle creating analogies while writing.

The inhumane representation of thought

Reviewer #2 (Public Review): 

In this work, Katzen et al. aim to model the feeding behavior of C. elegans in terms of well-established economic theories of value assignment and rational decision-making. This rigorous, quantitative analysis takes advantage of a powerful experimental system in which food quality and "cost" can be precisely controlled while carefully monitoring consumption. By immobilizing worms in a chamber in which they are simultaneously exposed to two streams of food of varying quality and abundance/cost, the authors measure changes in the geometry of head swings, pump rates, and therefore relative food consumption. Interestingly, they present evidence that worms are able to make these decisions as though they are maximizing economic utility, based on the idea that the "price" of the food is a function of its density. Interestingly, these choices seem to be most robust after worms have learned something about the relative nutritional value of the two food sources, and the authors provide evidence that this process requires the AWC chemosensory neurons as well as dopamine signaling. The authors also identify the behavioral mechanism that optimizes rational choice: animals modulate the angle of their head swings but not their rate of pumping to bring about utility maximization. By examining AWC's physiological responses to stimulation by food, the authors attempt to determine whether AWC's responses might encode a quantity that the worm's nervous system uses in calculating economic utility, but the results of these analyses provide no obvious simple answer. One significant concern about the work is a technical one: the authors infer bacterial density from spectrophotometry (OD600), which can be problematic when trying to make accurate assessments of this quantity. It's possible that errors in concentration estimation could undermine the conclusions drawn from these analyses.

Literally wtf was the point of this I'm sorry

Does: Introduces the subject or flight attendant who is serving them. Sensory details like red-faced, blond hair, etc are used to paint an image of this subject.

Using detail to describe the differences between characters.

says: A couple hours went by and it is dinner time. does: He gives a detailed description of the fight attendant.

Does: Gives you a visual of who was serving the dinner and what they were wearing.

In society today, colored people get less chances at being hired for work and at succeeding in life because they aren't given the chance too. Before they can even go in for a job interview they are already analyzed just by the color of their skin, they aren't given the chance to live because of the racial stereotypes that take place in society today.

From the jump, Manoj Dora and Eleni Iacovidou mention the counterargument to the claim they plan to pursue. This is a good strategy as they are tackling their "competition" head on. This will leave more room to persuade the audience into taking efforts in their favor. In addition, starting off in this manner is an effective way to hook their intended audience.

Here, Dave Lewis hooks his audience by relating the issue he wants to discuss to a well-known subject. Based on the article commentary above, I can assume that Lewis is aware that his argument comes with a lot of counterarguments. Therefore, using this hook to exaggerate the issue head on is very persuasive.

ok, honestly at this point in the reading, I've come to agree with Jean on the fact that Miss Julie is a little crazed in the head. while reading throughout the previous few pages, I sense That she might be in attention getter, or maybe she lacks love or perhaps something like that. she sound like a spoiled Brat to be honest. she loves to order Jean around like he's a dog and she loves wanting to hear about people feeling towards her, but she never want to reveal her own feelings. At first I thought Jean was in love with Kristen, because he is always saying what a wonderful wife she would make, but turned out that he Loves Julie, or maybe He is just messing with her feelings. for her to literally say "have you no feelings", it's confusing, because on the other hand, it's her that does not have any feelings. Also I feel like maybe she's afraid of letting people in to her life, perhaps not quite sure, but I get the feeling.

The first unethcial decision toby made is lieing and know how great of a lie it was. He was consiously aware of his actions. If we take a look of the framework of deontology, it staes the one is able to determine if there actions are right or wrong bt examining their choices. Toby decided to lie therfore he knew that his choice of telling the bank he makes 350,000 to get a loan was ethically wrong.

This gives us a sense of familiarity because we all have been through something similar to this in our lives.

Even though this paints nice imagery, it shows a dating to the material as not many kids use public libraries besides at the university level. Library usage in USA

This is giving us a very specific example but also making it very relatable. We are likely to be in a similar situation.

Confused on the repetition of the term regarding neck breaking. Clearly he is not speaking about a physical neck breaking... so I wonder what he trying to insinuate with that word choice.

If anyone sees this annotation and know, lmk!

I think that it is very interesting to note that infants have a different response to hearing their mother's voice than hearing the voice of a stranger. This shows that even at such a young age, they have the capability to differentiate between people because of their voice being different than that of another persons. They also know who their mother is based on voice which is interesting.

Upon reading this description, I noticed that it reminded me of a channel I found on YouTube called English Heritage that has a series where they bring Avis Crocombe (a domestic servant who was the head cook at Audley End House in England during the 1880’s) back to life and use actors to reenact her day-to-day life. I say all of that to say that it helped me to have a more vivid view of the scene these stage directions are outlining, and I think getting to make that kind of connection, albeit unrelated and small, kind of piques my interest in the play a bit more, as odd as that might sound.

Old testament is judaic and priestly which offers two views, but how/why in the new testament?

Pathos/Logos; also provides insight not only into Jones troubled past, but his mental state and how it got out of hand and may have led to murder.

tabs or read it later sometimes really make me anxious.I think we just need to take it easy.

It's rare to come across a take so truly contrarian.

I opened browser windows before I had a browser with tabs; in the days before whatever fun TCP multiplexing they have now, it helped maximize the juice I got out of our creaking dialup. I loved that when traversing Wikipedia, if your windows opened just to the right of the open window, you could go all the way down one depth-first rabbit hole and pop back up to the next path. Even if that non-linearity was less efficient somehow, I love it fiercely.

I saw a TikTok recently that resonated with me. I can't find the actual video, but the video was talking about how you will never enjoy your life if you are trying to mimic someone else's. That really stuck with me. So often we look for something super specific and out of our realms of reach for contentment, but once we accept our lives as they are, we begin to enjoy the story that we are individually and uniquely crafting. We have to look past the standards or the ideals that we set in our head to really begin to see the story of ourselves. This quote reminded me of that, and the message is so important because in the age of social media and technology, it is so easy to want someone else's life, but it reaches a point where we can forget to love our own.

Reminds me of conversations had within the black community today regarding the need for black people to go above and beyond for the same recognition to be given that is easily afford by their white counterparts for doing far less.

What is "peculiar" about Du Bois's mode of perception? How does his "double consciousness" compare to Emerson's experience of the "bare earth" in "Nature"? Why doesn't Du Bois just head out for the "perfect sweetness of solitude"?

At this point he is starting to show a language development.

OM SA

This is sort of hard to wrap my head around this idea. I get what they are saying, just because somebody doesnt believe in it doesnt mean its not there, but how would you even begin to come to the same conclusion of what is right and wrong if there is no religion or culture behind it. To me ethics is all about religion and what god says is right and wrong. When you take religion out of the picture it is hard to understand because what makes it right and wrong at that point?

I love this idea that we are not the head or some sort of power figure, but we are learners along with our students.

Makes me think of Head Start, which helps children and families find doctors, dentists, and other family resources. However, only children who are at the poverty level or children with special needs qualify. I would like to learn more about Reggio Emilia's funding sources.