I wasn’t certain whether to post this, because it felt to me that many other posts by people in their late 20s or early 30s say the same things. But I also, in a sense, treat my blog as a series of messages to my future self to remember how I used to think and this is something I wanted to send. So, I’m posting it.

I’m 28. That’s a fabulous number - it’s the sum of the first consecutive integers, primes, and also non-primes. It’s a triangle number. But it’s also ~a decade since I moved to the valley, and 16 years since I started working in labs. I feel distinctly different than I did at those ages, and have recently been wondering what changed the most. 

I decided to write down what I could think of - these are just notes, not prescriptions. Some things are good, some are bad, and I’m not sure which fall into which category yet!

• I often doubt my perception of own motivations, and sometimes assume I don’t ‘really’ know I’m doing something until days, months, or years after the action.

• I’m much more aware of focusing explicitly on building a strong social ‘base’ - friends and family feel more like important pillars of care and affection to build and maintain and less like transitory connections.

• I spend almost no time with people I intuitively don’t like or am bored by, where I used to assume I should ignore those impulses.

• I know what I want - both immediately, and out of life in general. I used to have goals, but I didn’t know what I wanted.

• I’m much more tolerant, sometimes, of illegible, incoherent work. I’ve learned to highly value sometimes chasing beauty and intuitions, and to assume there are even some very practical things that are impossible to figure out without those instincts.

• I used to be default wild (live life as variably as possible), I now feel much more inclined to respect and value tradition (I feel that I don’t understand it, and better respect how much useful information can be stored up in it). Tradition is different from what the majority societal beliefs currently are.

• I understand my mind a lot better, in conventional ‘become an adult ways’ - I’ve explored most of the therapy and woo paths to some degree, and have practical useful tools from them to manage stress and reptilian brain responses to things.

• I’m much more open than I used to be, and default to transparency. If something feels off, I’ll usually mention it. I don’t view it as my job to solve other people’s problems, at least to the same degree that I used to.

• I don’t feel looked at all the time, and I feel confident that I’m valued for my skills rather than how I look. I used to often feel uncertain about this.

• I have much better heuristics for when a situation is ‘dangerous’. I’ve seen friends in the ER, and different mental problems, and have a better sense for how long it takes to recover from certain things and when you should go into ‘adult mode’ and take care of someone in trouble. I also know that you can’t fix people and that sometimes you have to accept how a friend wants to live their life.

• I’m more wary of love, but it also feels stronger when it does happen. I default to being myself in relationships, instead of trying to be someone else.

I'm naturally good at two things - not giving up on projects that take more than a decade, and feeling intense, transcendent joy in response to a scientific understanding of the world.

I'm confused by whether the latter 'skill' is a good thing to cultivate, and wrote this draft post to understand it better.

I was obsessed with scientific phenomena well before I could plausibly understand them. When I was a kid and saw a picture of crowded molecules in a cell, I wanted to die with pleasure. There’s something I feel when watching a Nima Arkani-Hamed talk, sitting in the undergraduate physics lecture hall at MIT, or visiting Los Alamos that gets me every time. Like I’ve been punched in the gut and need to double over to catch my breath, like some intense and transcendent beauty is directly adjacent to me. When I do many hours of work to understand the concepts involved, I’ve been able to stay in the state longer, gasping at the beauty of it.

When I went to the Western Wall in Jerusalem, I could see emotion on the faces of people around me, and that mirrored to me what I felt when I visited Isaac Newton’s house as a child. 

I feel conflicted about this state because you can be enamored with something without understanding it, so I'm not sure that I trust it. Was my childhood engineered to induce it in the presence of anything that looks scientific? Is it a misplaced religious impulse? Am I well-calibrated enough for it to mean something, or is pursuing it just a descent into meaningless hedonism? 

Many of smartest scientists I know don't seem to experience this emotion regularly. They don’t feel the need to curl into a ball, spasming in rapturous delight at simple equations, and probably get a ton more done because of that. Sometimes I get intensely afraid that my desire for this state is pure hedonism - and then wonder why I should care, if it is.

Conversely, it feels like one of the most wonderful things you could experience - a wholesome version of hedonism. I've wanted to cry when people think they understand this state but don’t. How many people who pursue art seeking this depth of emotion would find a more powerful version of it in science instead? I feel like you can tell by looking at someone’s life - if you really had been there, would you truly live as you do? It’s not happiness, not gentle comfort, not delighted understanding, not a state of inquisitive play. It’s not obviously useful, and can be voraciously distracting. But to me it feels like something to live for. 

This is a half-draft of a blogpost that I’ll probably never finish, but I’m posting it because I’m curious - how prevalent is this emotion? How many people experience it in the presence of scientific truths, despite not having the genius to immediately comprehend them? How is it good, and how is it bad? 

I've decided not to work with Aubrey de Grey or SENS in any capacity moving forward.

I had one bad experience with him when I was 17 - he told me in writing that he had an ‘adventurous love life’ and that it had ‘always felt quite jarring’ not to let conversations with me stray in that direction given that ‘[he] could treat [me] as an equal on every other level’.

He sent this from his work email, and I’d known him since I was 14. Stuff like that happened sometimes, and I wrote it off as a mistake - something that might be my fault for trying to work in an industry when I was younger than average or because I had mentioned concerns about mentors doing stuff like that in a previous email. In the past few months, in part through conversations with Celine Halioua (who interned at SENS), I’ve learned it’s a serial pattern he’s enacted with women over whom he’s in a position of power. You can read about Celine's experience here.

I almost left the field several times as a teenager because of stuff like this happening. I knew that sometimes there would be misunderstandings, but I didn’t expect a trusted mentor I’d known since childhood to hit on me so blatantly, and insinuate that it had been on his mind for a while. It felt wrong to voluntarily go into an industry where - I basically inferred - sexual harassment was the norm.

Sexual harassment isn't acceptable behavior in the longevity field, but Aubrey is a really bad counterexample who does a lot of outreach. So newcomers to the field - mostly students and minors - can get the wrong impression.

It feels very weird to write about this, because I have a separate deeply held belief that we shouldn’t penalize people for not fitting into social norms, or for being different in ways that we can’t understand today. I just personally have no interest in working in a culture that is okay with certain norms (for example, propositioning minors or employees), and I’m angry to realize that Aubrey inappropriately propositioned more than one woman over whom he was in a position of power, many in the community knew about it, and no one did anything.

Earlier this year, Aubrey told me he was trying to 'cleanup his board' in response to 'derogatory rumors', which was actually how I found out this wasn't something only I had experienced, and that he was trying to stop his board from doing anything about it. The SENS board is aware of claims of sexual harassment and hired a firm to investigate these concerns, but also recently took a ~$25M donation which was helped by Aubrey's fundraising capabilities and reputation despite the ongoing investigation. Aubrey's position as their major fundraiser has impacted their decision to work with him despite these concerns.

Lots of people are aware of these concerns, but no one has said anything for a decade, and given the recent donation and incentives involved neither Celine nor I have confidence that SENS will take the appropriate measures to stop Aubrey from harassing more young women. It might be an open secret in the longevity community that this is a problem, but kids on the internet don’t have access to that information, and Aubrey is still mentoring minors. So, we’re making our experiences public. We wish we had known what many in the community did about him when we were entering the field. 

I’d like to ask better questions about biology, quickly. This is hard, particularly in fields I have no experience with. There’s no general solution to this problem, but there are things that seem to help. 

I’d submit that reading papers is actually, in isolation, a potentially dangerous activity. Sure, it’s better than doing nothing. But papers are generated in a rich social context that you don’t have, with important subtext you might completely miss. A paper is to a reproducible result as a mouse is to a human - many things might work in one but not the other.

So, what on earth can you do?

The closest I’ve come to a satisfying answer is to form mental models of the underlying concepts that are robust, manipulatable, and with which you can run thought experiments. This isn’t a necessary or sufficient condition for deep biological understanding, but it’s one of the few things you can do outside of a lab and check. If your model results in an exploded cell or a single bit genome, you can make it better.

_______

What does a thought experiment look like in biology?

People whose thoughts I admire often say that they ‘don’t understand something’, despite a perfect ability to recite the textbook examples of it. So, I’ve always been interested in what it would mean to understand something, particularly in biology.

My current hypotheses: 

1. Taking a lot of time (at least 20-30% of the hours you expect to spend in a field) to create mental ‘toys’ that you can fluidly manipulate, will lead to net faster understanding and storage, and potentially more and better novel ideas. This would mean at least 20-30 hours for a field you expect to spend 100 hours interacting with. 
2. Letting yourself do a lot of weird things that don’t look like learning, if they ‘feel’ right in the first phase, is important. 
1. I dance and move to create spaces in which my brain ‘sees’ the answer to things. I’m sure everyone has a similarly unique way in which they assimilate new information. Our default way of intaking and processing new information (sitting at a desk craned over a computer) seems really, really terrible at enabling and discovering these modes of thought.

Specifically, to create a mental model, you might try the following:

1. Generate a list of numbers, as long as possible, relevant to the object under study.
1. Attempt to ‘play’ with each number at least once. Generate a problem in which you manipulate that number, and solve it mentally. Then, attempt to visualize an object that would represent the problem, and ‘see’ the solution.
2. Try to ‘see’ the thing you want to understand. Generate as many visualizations as possible, of objects in the cell you are already comfortable with. Watch each of them to see what happens - is the phenomenon you want to understand clear from the visual? If not, can you see where the photons or electrons came from in the machine that generated the result? What did they interact with, in the biology? Can you see any places where this feels uncomfortable to you, or as though it would miss something?
3. If you feel really uncomfortable with the areas of the cell involved, checking whether there are basic equations or simulations that might give you a clearer intuition for the objects involved. Would better mental models for relevant equations regarding the length, timescale, information content, force, or energy involved help you generate more visualizations?

'Visualizing' could mean anything from a several seconds-long attempt to tag a word with a picture, to a mental action requiring 30+ minutes of continuous concentration, analogous to what Tesla describes here. I mean the latter, not the former.

_______

There are two ways to solve a problem. Get a bunch of other people to work on it, or do it yourself. I do the former, professionally. I don’t expect a deeper understanding of biology to be the critical thing limiting returns in my venture fund - that would be stupid, because it’s only a peripheral aid in talent identification, and entrepreneurs don’t (typically) care if you’re intellectually quick.

But the longevity problem is hard, because it’s currently (still) decoupled from what companies are doing, ultimately. We don’t yet have the concepts to solve it completely, we just know enough to do useful things that will be very profitable. And conceptual progress is hard, particularly in companies motivated to get a product to market quickly. So, outside of the financial constraints of my day job, I think it’s worth really worrying a lot about personal conceptual understanding of the field, to make sure what I’m doing makes sense toward the ultimate goal I care about. And even if an academic has figured out an idea, it can take a surprisingly long time to be able to fluidly understand and use something that’s fast to parse. Thus, the above. 

_______

Notes:

[1] Perhaps these sound similar to Polya’s heuristics - I think it would be wonderful if we could just directly use those in biology, but generating the right question seems to be as big of a deal as solving it, so there’s some extra creative work required. 

[2] Great books to seed your mind here include “Cell Bio by the Numbers”, William Bialek’s “Searching for Principles’, “Principles of Physical Biology”, and a thorough review of David Goodsell illustrations. 

Thanks to Sebastien Zany for suggesting many of the personal experiments that lead to the ideas above.

A week ago, I was curious - can we make an immortal yeast?

I wasn't sure. In 5 minutes of thinking, it seemed potentially doable (on the order of a 5+ year scale project for a hardworking technical team, but testable along the way).

It would also be the first time humanity made an organism immortal. Not counting human cells or pre-existing immortal species.

But I wasn't really sure. So, I decided to do a bit of looking into the question.

tl;dr - It's not obviously infeasible. Good researchers in the field don't see obvious reasons it wouldn't work. There are some potential issues (for example, would bud scars = dumb upper cap on replicative lifespan). There are also a few potential hacks that might work surprisingly well (for example, sporulating yeast to regenerate them - thanks to those who sent this paper). It might be worth doing chronological vs replicative lifespan to get around the bud scar issue + using some marker of metabolic activity. The best way to use 'the power of yeast genetics' would be to do a genetic screen - a knockout screen was done, but you could still do a mutant or over expression screen.

I feel kind of unsatisfied, though. What I want is a clear idea of what causes this aging and how to reverse it. Instead, there are ~49 things that the literature notes are linked to yeast, and our best response is still 'screen every gene in the genome'. What I'd love is a list of physically plausible hypotheses to chase down, and some feeling that there wasn't an enormous amount of uncharacterized dark matter that could actually be the core problem.

I'd also love tiny nanobots that can move atoms wherever I want them, so I guess the universe doesn't really care about my desires. Ah well.

Stuff I did: 

#1 - Try to build a mental model of what it 'feels' like to be a yeast cell

This sounds kind of weird, but I have a hard time thinking about biology if I can't see the atoms. book.bionumbers.org is my personal 'Young Lady's Primer', so I went on their database and got ~806 bionumbers that correspond to yeast. I've included them in Appendix 3 below - I haven't Anki'd all these yet, but they were a useful reference when trying to get an intuition for yeast. I also just sat in a big room for ~2 hours thinking* about the consequences of each of the things we see change with aging. For example, aged cells seem to increase in size - how would this affect the concentration of everything? Does protein expression and metabolite transport correspondingly increase? Do mitochondria increase proportionately in volume, if not, would every protein see a decrease in average ATP concentration available? Are there more membrane proteins, or does their density increase? Were the transcriptional studies that looked at this normalized in a way that we could even tell re net vs just relative protein expression? 

And on and on. I've included a list of questions for all the hallmarks at the bottom of this page, complete with a bunch of #notsatisfied tags. At some point I'll probably just make my aging Roam graph public, would be easier to see the full context that way.

#2 - Go through the literature to understand what people currently think

My takeaway:

- It's labor-intensive  to manually do replicative yeast lifespans, and (aside from maybe Calico) the field doesn't yet have a very robust system for doing this at high-scale. There are lots of labs working on the problem with candidate approaches (microfluidics, genetics).

- Sporulation (a way to 'regenerate' yeast) looks promising! I have some unanswered questions (sporulation efficiency declines with age, so is this a selection artifact?), but Angelika Amon and Elçin Ünal might have addressed them. Maybe it's worth carefully optimizing the amount and timing of Ndt80 expression, given that the young cells only lived a bit longer?

- There are lots of candidate damage types relevant to aging (see Appendix 2), but no consensus on which are the most important.

- Yeast cells age in variable ways, and most measurements are population-wide.

#3 - Ask a bunch of questions about what the consequences might be of yeast aging in different ways.

Normally, I'd put more work into using the mental model from #1 to come up with new plausible things that could be going wrong with yeast, in addition to those from the literature. But this week was pretty busy, so as a first pass I just took the list of 49 hallmarks that had been published in a review and went through those. It feels bad and wrong to not have done more thinking about the physical modeling though. 

I've included the list of hallmarks (Appendix 1), and the questions generated by them below (Appendix 2). 

To sum things up - I'm still curious about this!

If I had infinite time, my next step would be to think more about being a yeast cell* and the kinds of things that could go wrong with age. Then, I'd try to do more concrete thought experiments inspired by a subset of the questions in Appendix 2. I'd carefully investigate the sporulation regeneration biology, and think a bit about mother/daughter asymmetric damage segregation (despite that potentially being not as actively regulated as people thought).

If I were a practicing yeast geneticist working towards a PhD, I might also start just trying to design some kind of experiment. But I'm not sure thinking about the problem more first would actually be a bad idea, despite having a huge personal tendency to overanalyze things.

Ultimately, if we had single atom pushers and microscopes that could see where all the stuff was, exactly, this would obviously be a cool problem to work on. Another constraint I didn't talk about is what we can do about all of this. Our 'stuff we can do' space seems to be 1) change the environment (temperature/pH/pressure/metabolites/etc), 2) give a small molecule or 3) express a protein. Yeast being a unicellular organism doesn't (that I know) open up amazing new ways to manipulate it, although I'm curious if I'm missing something interesting here. Particularly things that are physically possible but not yet implemented. So, proteins are still our most expressive and complex nanobots for doing what we want, in this scenario. I don't know enough about expected progress in protein engineering over the coming decade to know whether that should be an optimistic or pessimistic update on the ability to fix stuff that we directly know is going wrong and should be fixed.

Anyway. That's all from me. If you're interested in working on/thinking about this problem, I'd love to hear from you! (info@longevity.vc), even if just to shoot around all the confusing bits of it. 

Deep thanks to Mark McCormick, Martin Borsch Jensen, Adam Marblestone and Reuben Saunders for technical discussions that helped clarify some of the above, even though the opinions represented above are obviously just my wrong-headed and fuzzy attempt to understand the problem and not any kind of direct representation of their opinions on the topic! 

*my way of thinking about this is to pretend I am a yeast cell, and think about what I would feel like and what I would worry about as I got bigger. That sounds hard to explain in a non-crazy way on paper, so maybe I'll make a video about it at some point. It also may just be a totally ineffectual way of thinking about problems, who knows.

Appendix 1 (extracted manually from Janssens and Veenhoff 2016, Figure 1)

Appendix 2 (written using Hallmarks from Appendix 1)

• Standard questions (default apply to all of the below, in addition to specific questions)
  
  • [[standard quantitative questions]]
    • By how much does this occur?
    • How do we know it occurs?
    • What genes control this phenotype when expressed?
      • How do they control this phenotype, physically?
    • Does inducing this specific phenotype in a non-aging organism lead to faster death?
    • Has anyone reversed this phenotype? 
    • 4 most relevant publications showing this
    • What correlated things would you expect to see if this were true?
    • What physically causes this?
    • At what age was it first noticed?
    • Does it appear in all members of the population? Has a single-cell analysis been done?
    • How does it change over the lifetime of the organism?
    • Does this have predictive power for death?
    • Does this impair an obvious necessary function of the organism? To what degree?
    • What is the variability in this phenotype?
  • [[organelle quantitative questions]]
    • Diameter
    • Shape
    • How many in a cell
    • Lipid composition
    • Membrane protein composition
    • Internal composition
      • Density
      • pH
      • Macromolecular breakdown
    • What is it continuous with
      • How leaky is it to this thing
    • What is the flux of what across the cell membrane?
    • How was it first discovered?
    • What do we know that it does, today?
    • How do we look at it? How has that changed?
    • What is it tethered to?
    • What does it exchange membrane with?
    • What bounces off its cell surface by temporarily binding but stays on the outside?
    • How variable is it between cells?
    • How often it fuses with itself
      • How often it fissions with itself
      • How often it fuses/fissions with other organelles
    • Can we isolate it from the cell?
    • Under what conditions do the above parameters really change?
    • What are the major labs that study this? 
    • What are recently published techniques about this? What techniques would all labs want with regard to this?
  • [[protein quantitative questions]]
    • What is the protein concentration in the cell?
    • How is it degraded? At what rate is it degraded?
    • Does the protein lose function over time?
      • What causes this if so?
    • How much is it expressed?
    • What environmental signals do we know to change this protein concentration?
    • What directly turns on and off expression of the mRNA for this protein?
    • What known post-translational modifications does this protein have?
    • How does expression change with time?
    • How many genes encode for this protein?
• Cell size increases
  • By how much does this occur?
  • How do we know it occurs?
  • What genes control this phenotype when expressed?
    • How do they control this phenotype, physically?
  • Does inducing this specific phenotype in a non-aging organism lead to faster death?
  • Has anyone reversed this phenotype? 
  • 4 most relevant publications showing this
    • https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0167394
  • What correlated things would you expect to see if this were true?
    • Do things increase or decrease in concentration?
      • https://science.sciencemag.org/content/338/6108/822.abstract
    • Do old yeast take in nutrients in the same way? Is the flux higher?
    • When they get bigger does their composition change? Are they more dilute?
    • What does their cell surface look like?
      • Is it stretched?
      • Is it the same thickness?
      • Did they make more of it?
      • Is the protein density different? 
      • Is the organelle density different?
    • Does the nucleus also get bigger?
    • Do the cells get a bit bigger after each division? Is there a gene that can modulate that size difference?
    • Does the bud scar have anything to do with it?
    • Is it the cell wall vs inside of the cell
    • Do you ever see a bud come from the middle of a scar?
    • Does the cell wall become more porous at all?
      • What does it mean for the cell wall to become porous, what has holes in it?
      • How many proteins thick is the cell wall?
      • When something buds off, how does the cell wall deal with that?
        • If you got rid of all the chitin would the cell wall be okay?
        • How stiff is the cell wall in an older cell?
        • What is the pressure like of an older cell, does it get more or less?
        • pH?
        • How is an old cell transcriptionally and protein-wise different from a young cell?
        • Why can’t an old cell make another daughter?
        • Do daughters know how old the mother is?
        • Do cells ever try to half-make a daughter and fail? 
        • Can you regenerate an old cell right when it really thinks it can’t make daughters anymore?
        • How long can a cell just still be metabolically active after it stops making daughters? 
        • Do cells have some specific thing that happens at death like increased porosity or no more transcription?
    • How often do spores die?
      • Quiescent cells?
  • What physically causes this?
  • At what age was it first noticed?
  • Does it appear in all members of the population? Has a single-cell analysis been done?
  • How does it change over the lifetime of the organism?
  • Does this have predictive power for death?
  • Does this impair an obvious necessary function of the organism? To what degree?
  • What is the variability in this phenotype?
• Vacuole size increases
  • When does this start happening? Do the number of lipids change? Does the pressure change? Does everything inside of it get more dilute? How continuous is the vacuole with the cytosol? What causes the increase of size? Does the vacuole divide to create a new daughter vacuole? How is the new daughter vacuole created if not? What proteins are in the vacuole membrane, and what lipids? Can we isolate vacuoles? How big is a vacuole (~2um?). How much does vacuole size vary with cell size? How does it vary with conditions cells have experienced? How does it vary with age? Do any things obviously accumulate in the vacuole with age? What genes regulate vacuole size? Does the vacuole ever break open? How is the inside of the vacuole different from the outside? How was the vacuole first discovered? Do cells ever have multiple vacuoles? Do mutant cells ever have multiple vacuoles? What structure is the vacuole derived from? How is the vacuole made? Are there vacuole-specific proteins? What is the flux of things across the vacuole membrane? How does the vacuole relate to the outside of the cell? What is the vacuole analogous to in humans? Can cells exchange vacuoles? Do vacuoles play any role in cell-cell signaling? What other organelles do vacuoles signal to? What are vacuoles tethered to in the cell? 
• Decreased resistance to mutagen (EMS)
  • [[standard quantitative questions]]
  • What does it look like to have decreased resistance to a mutagen?
    • Does it mean death in response is increased? 
    • Is DNA more damaged in response?
    • What other things are more damaged in response?
  • What is EMS physically binding to?
  • What does the DNA repair pathway look like in cells that have been treated with EMS?
    • What does the [[yeast DNA repair pathway questions]] look like in general? How well have we characterized it?
    • How does resistance to mutagenesis change with age?
  • How do other DNA repair pathways change with aging? How does mutation load change with aging?
  • By how much does EMS increase the average amount of mutagenesis?
  • How was EMS first discovered?
    • What else does it do?
• Increased stress resistance (and trehalose production gene)
  • What kind of stress is the cell more resistant to?
  • Does this continue through the end of life, monotonically? What kinds of stress is the cell less resistant to?
  • How is stress resistance measured?
    • Instant death?
    • Lifespan?
    • Ability to turn on genes in annotated stress-response pathways? 
  • What is the therapeutic window for stress resistance?
  • How much additional resource does the cell put towards increased stress resistance?
    • Is this physiologically relevant? Could the cell maintain this normally?
  • What kinds of stresses does the cell normally encounter?
  • Why is the cell physically more stress resistant?
• Vacuolar pH decreases
  • See vacuolar questions above
  • Does the vacuole have a proton pump?
    • If so, do it's levels change with age?
  • Does the vacuole fuse with things?
  • Can protons diffuse across the vacuole membrane?
  • What can reverse the phenotype of vacuolar pH decreasing?
  • If you put alkaline in the vacuole, what does it do?
  • What would decreasing pH be expected to do to protein function of things in vacuole?
  • What other things in vacuole are regulated by pH?
  • How was it discovered that enzymes more active in a low pH-environment might be less liable to harm a cell if released?
• Oxidative protein damage (increased carbonyl levels)
  • How is oxidative protein damage defined?
  • How is oxidative protein damage measured?
  • In which compartments does it increase?
  • Where would oxidative protein damage be likely to occur?
  • What agents oxidatively damage proteins?
    • Do these agents increase in frequency? 
    • Does their location correlate to protein oxidation?
  • What does oxidative protein damage mean?
  • Are there non-oxidative forms of protein damage?
    • If so, do those also increase?
  • What is the baseline rate of protein oxidation?
  • What is the baseline rate of fixing protein oxidation?
    • How is protein oxidation fixed?
    • Is it directly recognized?
  • Does oxidatively damaged protein net increase over time with age? When does this start to happen if so?
  • How much can one protein be oxidatively damaged?
    • Beside the protein doing its normal thing less efficiently, what is bad about protein oxidative damage?
  • What other things get oxidatively damaged in a cell?
    • What other ways are there to structurally impair things?
  • Does oxidatively damaged protein aggregate?
  • Does the cell have specific pathways for recognizing and eliminating oxidative damage?
  • Is oxidative damage used by the cell for anything?
  • Do any cellular reactions directly cause oxidative damage?
  • Do any things not get oxidatively damaged that we would expect to see oxidatively damaged?
    • On a population scale, are all things oxidatively damaged roughly equally?
  • Are parts of the cell particularly protected from oxidative damage?
  • What are the most common oxidative damage-causing agents in a cell? How are they generated or how do they get in?
  • Does the cell ever make more oxidative stress for a certain purpose?
  • What is the half-life of agents that cause oxidative stress?
• Decreased retention of oxidatively damaged proteins
  • What are all the things that are asymmetrically segregated?
• Increased histone H4K16 acetylation
  • How do levels of histone acetylases/deacetylases/methylases change over time?
  • Could you predict histone modification changes from net changes in enzymes which modify histones? If not, what might cause the gap?
  • How do all histone marks change with age? How would you expect this to change DNA structure?
  • Can you predict gene expression from histone marks? If not, how do histone marks give you more information than gene expression? Maybe a better overall heuristic for average gene expression over a certain time period - but potentially you could just measure this? 
  • What are all known yeast histone marks?
  • How do they vary?
    • How often do they vary appreciably?
    • How do they vary during cell replication?
    • Do they vary on the order of minutes, hours, months?
  • Do yeast have a DNA methylation age clock? If not, how useful are histone marks to predicting age?
  • How do we look at histone marks? What would this method not catch? Can we correlate this to DNA structure and gene expression?
  • How do histone marks physically affect gene expression?
    • Do they recruit other proteins? What are those proteins if so?
  • How prevalent are the substrates of histone marks? Does their concentration ever limit the reaction? 
  • At a given time, how many histone-modifying enzymes are bound to DNA? How does this change with time? What DNA sequence controls where they bind?
  • In what other ways is yeast DNA modified?
  • Does yeast DNA have a 3D structure? How is this regulated?
  • Have we done Hi-C in yeast? How else would you look at 3D DNA structure.
  • Are histone modifications relevant for anything besides modulating gene expression?
    • Should it be possible for there to be a methylation clock but not a gene expression clock for aging?
  • #notsatisfied
• Decreased histone H3K56 acetylation
• Mitochondrial redox potential declines
  • What does the redox potential of the mitochondria mean?
  • How is this related to mitochondrial function?
  • #notsatisfied
• Decreased mitochondrial membrane potential
  • What causes the observed decrease in mitochondrial membrane potential in yeast?
  • Does the membrane get more leaky?
  • Does cytosol pH concentration change in a way that increases pH in the intermembrane space?
  • Is there a decrease in net activity of the proteins in the membrane that should maintain this potential? 
  • Is there a decrease in density of proteins that should maintain this gradient? 
  • Does the mitochondria get bigger with cell age?
    • If so, does protein expression for complexes to maintain mitochondrial pH incease?
      • If not, does the ratio of protein complex activity to maintain pH with the size of the intermembrane space go down?
      • Would this decrease correspond to the difference in intermembrane potential?
    • Does mtDNA copy # increase?
  • #notsatisfied
• Altered nuclear pore complexes
  • How many NPCs are there in a nucleus?
  • Are they homogenously distributed?
  • How often are NPCs turned over? How are they degraded?
    • How are NPCs damaged?
  • How are nuclear pore complexes altered?
  • Does their number decrease?
  • Does the nucleus increase in size with age?
    • Does NPC expression increase proportionately if so?
    • What would regulate this?
  • What activity do NPCs modulate? What goes through them? Are there different kinds of NPCs?
  • Do NPCs have subunits? Can they be arranged in different ways? Does this change with age if so?
  • Are nuclear pore complexes found anywhere except for the nucleus?
  • How are nuclear pore complexes made? Where do they go to get to the nucleus?
    • If they need to go to non-nuclear places, are those places affected with age?
  • Do nuclear pore complexes have post-translational modifications? What are they if so?
  • How many different nuclear pore complex types are there?
  • Do nuclear pore complexes do any active things? Do they bind to anything? What is associated to them?
  • How do we track nuclear pore complexes?
  • Do NPCs play any structural role in the cell?
  • How were they first discovered? How were they first characterized?
  • What does NPC complex expression respond to?
  • Does the relative ratio of NPC complex parts change with age (and is it possible for the complex to have different stoichiometry?)
    • Does the expression of the different NPC components change in proportion to each other with age?
    • Does it always change in the same way if so?
  • #notsatisfied
• Increased resistance to UV at age 8
  • What does it mean to have increased resistance to UV?
  • Are certain stress response pathways turned on more?
  • Is there less mutation?
  • #notsatisfied
• Decreased Multidrug REsistance Transporter (Tpo1) activity
  • How do all genes change with age?
  • What class of drugs does Tpo1 modulate resistance to?
  • What are all the drug transporters in yeast? Do they all decrease in activity with age?
  • Does Tpo1 have decreased expression, or decreased activity?
  • [[protein quantitative questions]]
  • #notsatisfied
• Increased gluconeogenesis
  • How much glycogen do yeast store?
  • What % of glucose in yeast is in glycogen?
    • How much does this vary with environmental conditions?
    • What environmental conditions regulate this?
  • Do yeast continuously do gluconeogenesis?
    • When do they do it if not?
  • #notsatisfied
• Decreased glycolysis
  • By how much does glycolysis decrease with age?
  • What is the flux through the glycolysis pathway normally, in glucose / second?
  • What other pathways take metabolites from the glycolysis pathway? How do they very?
  • What regulates glycolysis?
    • Is there one or a set of trasncription factors that proportionally regulate the expresion of glycolysis proteins?
    • Does the relevant amount of proteins in glycolysis change with age?
  • What causes the decrease in glycolysis? Are all protein components expressed at the same amount? 
    • Are all protein components expressed at the same density? 
  • Do all protein components function as efficiently as they did?
    • How do proteins in the glycolysis pathway get damaged?
    • What is the average damage level of a protein in the glycolysis pathway with aging?
    • Does the cytosol change with age? 
      • If so, would that change affect the function of these proteins?
  • Do the balances of metabolites for each of the reactions change over time?
    • How so?
  • How do we measure the decrease in glycolysis with age?
  • Does glucose density in the cell change with age?
    • What transports glucose into/out of the cell? Does that change with age?
  • How does the end product of glycolysis change in concentration with age?
  • #project is it possible to work backwards from the change in concentration of the metabolites involved in each reaction, to see if the rate of any of the proteins involved would be expected to change with time?
  • #notsatisfied #otherreasons #mapfullpathway
• Occurrence of HSP104 foci
  • What is an HSP104 foci?
  • How big are these foci?
  • How many are there?
  • Do all cells have them?
    • If not, what % of cells have them?
  • How does the occurrence of HSP104 foci change with age?
  • How many proteins are in them?
  • What characterizes a protein in this state?
  • Do any proteins or processes degrade HSP104 foci?
  • Do all cells get HSP104 foci?
  • Are these foci ever excreted?
  • How are HSP104 foci segregated between mother and daughter cell?
  • Do they have any proposed functional role? 
  • How were they first identified?
  • Where are the foci located in the cell? 
    • Are they tethered to anything?
    • Does anything bind to them?
  • How are HSP104 foci characterized?
  • Are foci inherited? 
  • How do foci grow? 
  • What is the baseline # of HSP104 proteins in a cell?
    • Does this number or concentration remain the same with age?
      • If not, how does it change?
  • Does the concentration or number of HSP104 proteins in a cell that are not in foci remain the same independent of foci?
  • Do any other proteins form foci?
  • What is the rate of foci growth?
    • What cellular factors does this rely upon?
  • What proteins form prions in the cell? What controls this?
  • #notsatisfied #prionquestions
• Increased ROS levels (in 22% of population)
  • What is ROS? How many chemical structures are covered by this word?
  • By how much does ROS increase? What does this look like over age?
  • How would you expect this to affect protein oxidation levels?
    • Do you see this effect?
  • What causes the increase in ROS levels?
    • Do we know what things create ROS?
    • If we know some of them, does their level of activity increase with age?
  • Are there some cells in which ROS does not increase?
  • Should mitochondrial stress in some way be proportional to ROS production?
  • What quenches ROS once it is produced?
    • How do these processes work?
  • #notsatisfied #moremechanismquestions
• Mitochondrial fragmentation
  • How is this measured?
  • By how much does this increase?
    • How many fragments do you see, with age?
  • Does this happen across cells?
  • Does mitochondrial fragmentation affect mitochondrial efficiency, or is the internal membrane surface area essentially the same?
  • Does mitochondrial fragmentation increase the flux of metabolites from the cytosol?
    • Is there ever a delay here?
  • Are there any cells in which more fusion is instead detected?
  • Does mitochondrial composition change with age?
  • When does the mitochondria fragment normally?
  • Is mitochondrial fragmentation thought to provide some kind of functional benefit?
  • What proteins might regulate mitochondrial fragmentation?
  • #notsatisfied
• Aggregation of carbonyl-damaged proteins (HSP104 associated)
  • How is aggregation of carbonyl-damaged proteins detected?
  • What does it mean for a protein to be carbonyl-damaged?
  • What is the approximate level of carbonyl-damaged proteins in the cell?
    • How much carbonyl damage does the average protein have?
      • What is the variability in this?
    • Is it normally in the same place?
    • What other kinds of damage occur to proteins?
  • Can yeast remove carbonyl damage from proteins?
    • How, if so?
    • How active is this pathway normally?
  • #notsatisfied
• Stress reporter MSN2/4 levels increase
  • What does this mean
  • #notsatisfied
• Reduced sporulation efficiency 16.7% instead of 69.8%
  • What is sporulation efficiency?
  • If sporulation efficiency decreases, does this mean that cells that do sporulate are selected to maybe be more healthy?
    • #sporulation
  • #notsatisfied
• Loss of silencing at chromosome ends
  • How much is silencing lost?
  • Does this result in new expression?
    • What does the expressed RNA do, if so?
  • Does this render the chromosome ends more liable to some form of DNA reorganization?
    • What, if so?
  • #notsatisfied #physicalquestions
• Detetable levels of ERCs
  • What is an ERC?
  • How big is it?
  • Is the sequence always the same?
  • How many ERCs are there in a young cell?
    • How many in an old cell?
  • Do ERCs increase in all cells?
  • Is there any correlation between ERC # and probability of death?
  • Do any mutants not show ERC increase?
  • Do ERCs replicate independently of the genome?
  • Could any enzymes extract ORIs from ERCs if so?
  • At what rate do ERCs divide?
    • Is this the same between budding and non-budding cells?
  • In cells with a lot of ERCs, what % of the cell volume is taken up by ERCs?
  • #notsatisfied #physicalquestions
• Enlarged nucleolus
  • What is the nucleolus?
  • What characterizes the nucleolus?
  • By how much does it increase in size?
  • Is there just one?
  • What adds in mass to the nucleolus to make it increase in size?
    • Does the average amount of each normal component of a nucleolus increase by the same proportionate amount?
  • #notsatisfied #moremechanismquestions
• Daughters can be born as petites (lacking mtDNA)
  • How often are daughters born as petites?
    • How does this increase with age?
  • Do petites have a different replicative or chronological lifespan to normal?
  • What does mitochondrial flux in a petit look like vs normal?
  • #nosatisfied
• Detection of glucose/energy-metabolism protein changes
  • What does this mean?
• Detection of oxidative stress response proteins
  • Which proteins?
  • By how much do the increase?
  • [[protein quantitative questions]]
• Invagination of vacuolar membranes
  • What physically causes this?
  • By how much do they invaginate?
  • Is there a structure holding them in place?
  • How many invaginations are there in the membrane?
  • Are they equally spread across the membrane?
  • Does this cause an increased flux over the vacuolar membrane?
  • #notsatisfied #physicalquestions
• Over 50% of population has a random (nonaxial) budding pattern
  • What is the normal budding pattern?
    • What enforces it?
    • Are these proteins and metabolites expressed at the same concentration in aged cells?
    • If you look at the cells that do and don't show this non-axial pattern, is this related to the expression of proteins and metabolites in the relevant pathway?
  • #notsatisfied
• Increase sterility mating frequency drops to 25% from 78%
  • Can repeated attempts at mating with the same yeast eventually lead to mating?
  • Does the expression of any gene fix this?
  • Does increased concentration of any mating factors fix this?
  • Are there any obvious phenotypes in the morphology of cells that cannot mate?
  • #notsatisfied #moremechanismquestions
• Accumulated ERCs
  • See ERC questions above
• ROS detected to be localized at mitochondria
  • See ROS questions above
  • Is ROS normally not in mitochondria?
  • Does the ratio of ROS in the mitochondria vs not change?
  • Is the ROS in the inner compartment, or the intermembrane compartment?
  • Can ROS diffuse between the cytosol and the intermembrane compartment?
• Age-associated sterility
  • see mating questions above
  • #notsatisfied
• 10-15% reduced lifespan of daughters
  • Does this always happen?
  • Does this happen for the spores of certain aged cells?
  • Does this happen to all old cells?
  • What correlates to this?
  • Do the daughters that show this phenotype have some phenotype in common?
  • #notsatisfied #morephenotypequestions #physicalquestions
• Increase of cells with a G2/M DNA content (population level)
  • What is a G2/M DNA content?
  • #notsatisfied #physicalquestions
• Histone mRNA levels increase
  • By how much do mRNA concentrations increase?
  • Do all histone mRNA levels increase?
  • Are there associated transcription factors whose levels have increased?
  • Has the concentration increased?
  • What have they increased relative to?
  • #notsatisfied
• Histone protein levels decrease
  • By how much do histone protein levels decrease?
  • Do all histone protein levels decrease?
  • Are there fewer nucleosomes in the cell?
  • Is there generally more expression of genes in the cell?
  • Is there more degradation of histone proteins? Is there less translation of histone mRNA?
  • #notsatisfied #moremechanismquestions #physicalquestions
• Increased ROS levels (in 22% of population)
  • see ROS questions above
• Chance of symmetric divisions between mother and daughter
  • What does a symmetric division mean?
  • Is there a pathway that causes this? Could this be induced in a mother from youth?
  • #notsatisfied #moremechanismquestions
• Amplification of the right segment of chromosome XII in 15% of cells
  • By how much is it amplified?
  • Are other chromosome segments amplified?
  • Do these cells have higher mortality?
  • #notsatisfied #moremechanismquestions
• Significant decrease of DNA breaks
  • How many DNA breaks are there normally?
  • By how much is this decreased?
  • How are DNA breaks detected?
  • Is it clear whether the baseline level of DNA breaks occurring is decreased, or the rate at which they are fixed increases?
  • Do DNA breaks seem to correlate with some measure of transcriptional activity in DNA?
  • Could DNA breaks actually be increasing in some way?
  • #notsatisfied #moremechanismquestions
• Two fold increase of retrotransposon DNA content
  • What is the baseline level of retrotransposon activity?
  • How many retrotransposons are there normally?
  • is it the same in all cells?
  • Does retrotransposon level predict death in some way?
  • Are retrotransposons particularly present in some part of the cell?
  • Would the increase in retrotransposon activity be likely to result in some amount of gene disruption?
    • How much, if so?
  • #notsatisfied #moremechanismquestions
• Genomic translocations in rDNA
  • What is this?
  • #notsatisfied #moremechanismquestions
• Genomic translocations in mtDNA
  • What is this?
  • #notsatisfied
  • #moremechanismquestions
• Four fold increase of mtDNA content
  • Is this related to an increase in mitochondrial size?
  • Is this true in all cells?
  • Does this change the density of mtDNA in mitochondria?
  • Is there any corresponding increase in mtDNA protein expression?
  • What proteins are on mtDNA but not the nucleus?
  • How is mtDNA divided between mother and daughter?
  • How does the mutation load of mtDNA change with time?
  • Is mtDNA tethered to anything?
  • Is there any mtDNA outside of the mitochondria?
  • In young mutants with increased mtDNA, what phenotype do we see?
  • #reallynotsatisfied #moremechanismquestions #physicalquestions
• Fuzzier nucleosome positioning
  • What does this mean?
    • Does this mean more movement in nucleosome positioning?
  • By how much has this increased?
  • Is it true of all nucleosomes?
  • Is it related to transcriptional activity at the site?
  • #notsatisfied
• Histone occupancy reduced 50%
  • What does this mean?
  • #notsatisfied #moremechanismquestions
• Decreased pheromone response 35% of cells respond to pheromone
  • What does this mean?
  • By how much is it decreased?
  • Does this correspond to anything interesting in those cells with the decreased response?
  • Are the cells with decreased mating response more likely to show sterility than those without?
  • What protein normally detects pheromone?
  • Does the concentration of this protein change with age?
  • What does the cell normally do in response to pheromone?
  • How much pheromone do older cells secrete?
  • #notsatisfied #moremechanismquestions
• Concepts
  • [[yeast DNA repair pathway questions]]
    • How many proteins are known to be part of the yeast DNA repair pathway?
    • How many different ways is DNA repaired?
      • What do each of these different ways do? 
      • What is the efficiency of each of these different ways?
      • What is the rate at which errors are repaired incorrectly in each of these different ways?
    • What is the baseline rate of DNA mutation in non-budding cells?
      • In budding cells?
        • What is the error rate of the normal yeast DNA polymerase?
        • How does DNA repair machinery affect this?
        • Are there less error-prone DNA polymerases?
        • Do we know of any essentially perfect DNA polymerases?
          • If not, why not? 
          • Is there a source of thermal noise that prevents this?
        • Is there a tradeoff between speed and fidelity in DNA replication? If not, is there another tradeoff?
      • What causes DNA mutation in non-replicating cells?
    • How do we measure the baseline rate of mutagenesis?
      • What mutagensis would this obviously not catch, if any? 
    • What causes mutagenesis?
      • Are UV rays relevant here? If so, if we shield yeast from UV rays, what happens?
      • Is thermal noise related to bond strength relevant? If we used nucleotides with a higher differnetial in bond strength, what would happen? Would there be negative effect related to the change in energy required to unzip DNA across the cell?
    • Are parts of DNA more targeted for mutagenesis than others?
    • Is there asymmetric seggregation of chromatids between mother and daughter?
    • Is DNA somehow repaired more during sporulation or budding? 
    • If you could specify any machine in order to make DNA less liable to mutagenesis, what would you specify? 
      • Is it possible for us to make this?
    • What DNA repair enzymes are people trying to make?
    • #project how does the division of yeast relate to how mutagenic it is?
      • How do populations of yeast diverge genetically over time?
      • How many mutations on average does it take for a yeast to become unable to divide?
      • How many mutations on average does a non-dividing yeast have per second or hour?
      • How many mutations does a dividing yeast have per division?
      • What is the ratio between the two?
      • Are important regions of the genome more protected?
      • How often does a yeast in a population become unable to divide further because of a mutation?
        • Is this ever fixed?
      • Are mutations to DNA repair or lethal mutations the only mutations that seem irreverslble?
        • Is there still a way to reverse them?
      • How often does a cell get better because it mutates?
      • Is mutation used for any non-obvious purpose? How important is it to allow a yeast population to adapt? How often does the genome of a population shift? How often in a way that seems selected for?
      • Given the above, when would a non-dividing cell be expected to die, on average? Is that what we see?
      • Does mutation rate change depending on rate of metabolism? What other things affect mutation rate?
    • Do yeast pass plasmids to each other?
• Other
  • What other things are there in the cell that could increase, but aren't covered by the above? 

Appendix 3 (pulled from https://bionumbers.hms.harvard.edu/search.aspx, extracted with pandas read_html)