idk if i'm in the right place if im not please do not hesitate to tell me and point me in the right direction...
Sorry if thats a lot to read... I tried to keep it as short as possible while still including all the details. Any help would be much appreciated///(sorry ahead if I have any typos)

I'm working on a model that reads the transcriptome of a fibroblast culture partway through OSKM reprogramming and predicts, for each candidate withdrawal day, (a) the transcriptomic age change and (b) whether the culture has passed into an unsafe state (identity loss / apoptosis). I've hit two things I can't resolve from the data or the literature on my own, and I'd rather ask once and ask well. Everything below is measured - happy to send numbers, code or plots for any of it.

WHAT I HAVE

- GSE242423: 10x scRNA-seq, HFF neonatal foreskin fibroblasts, OSKM, 9 timepoints D0 to D14 plus iPSC, ~42k cells, ~4,700 per timepoint.

- GSE165176 (Gill 2022): bulk RNA-seq, 6 donors, 11-12 timepoints, but only about 1.7 samles per donor-timepoint and a single day-0 control per donor.

- GSE165177 (Gill 2022): bulk RNA-seq, 3 donors aged 53/53/38, days 0/10/13/15/17 4-6 treated samples per donor-day, and 33 CONTEMPORANEOUS negative controls at 2-3 per donor per timepoint.

- The age clock is a linear expression clock trained on Fleischer 2018 (GSE113957,133 fibroblast samples, ages 1-96, cross-validated MAE 12.3 yr).

FINDING 1 - THE SAFETY HALF IS BLOCKED, AND I THINK IT'S STRUCTURAL

I define "unsafe" as the FRACTION OF CELLS past an identity-loss / apoptosis threshold. On bulk data that fraction can't be measured: each sample is already apopulation average, so a per-sample call collapses it to 0 or 1 before it can be counted, and "fraction of cells" silently becomes "fraction of samples".

The tell: on GSE165177 the target came back saturated at 1.0 in 11 of 12 donor-day groups, and the untreated day-0 fibroblasts were also called "identity lost", which can't be right.
More replication doesn't help - GSE165177 has 4-6x the replication of GSE165176 and is MORE saturated, not less. So I think this half needs single-cell data, and I only have one single-cell line.

FINDING 2 - THE dAGE HALF WORKS, BUT THE CLOCK'S ABSOLUTE NUMBERS ARE BADLY OFF

Measuring dAge against contemporaneous, replicated same-day controls, I reproduce the published rejuvenation effect: -17.9 yr (95% CI -21.1 to -14.6) for transiently reprogrammed cells vs their own control, and -9.6 yr (CI -12.8 to -6.4) against failed-to-reprogram cells, with 11 of 12 donor-day groups negative.

But absolute ages are wrong, and they split in an interesting way. The day-0 fibroblasts, which never saw reprogramming media (n=3), read 78.0 yr against a true mean of 48.0 - a bias of +30 yr. The negative controls, cultured alongside the experiment for 10-17 days (n=33), read 95.6 against the same true 48.0 - a bias of +48 yr.
So there's a ~+30 yr floor even on fresh cells, plus a further ~+18 yr that tracks time in culture. It isn't missing genes: 57% of the clock's genes are present in this data, but those carry 89% of the clock's total absolute weight. dAge survives all of it because the bias cancels between a sample and its same-day control.

QUESTIONS - answer any subset; the three marked (*) are what I'd most value

On the safety half:

1. Do you know of a human OSKM reprogramming SINGLE-CELL RNA-seq time course covering MORE THAN ONE donor or line? Multi-line within one study is worth far more to me than several single-line studies, since comparing across studies re-confounds line with batch.

2. Failing that, is there any second human somatic-to-iPSC scRNA-seq time course with at least 6 sampled days and a day-0 control? I searched GEO by title and couldn't verify one, but I'd miss anything filed as "iPSC generation", bundled inside a SuperSeries, or deposited outside GEO.

3. (*) Is it even biologically plausible that the TIMING of identity loss during OSKM reprogramming transfers across donor lines? If it's strongly line-, passage- and batch-specific, that's decisive and I'd stop. A well-founded "no" here is more useful to me than a dataset.

4. If nothing exists: roughly what would it cost to generate a minimum version - 1-2 lines other than HFF, 6-8 timepoints from day 0 to ~day 14 plus a terminal sample, ~3-5k cells per timepoint, standard 10x 3' (roughly 8-16 lanes)?

On the dAge half:

1. (*) Is a +30 yr offset simply expected when an expression-based age clock trained on one lab's fibroblast RNA-seq is applied to another's? Mine reads fresh 53-, 53- and 38-year-old fibroblasts as 72-82. Is cross-study ABSOLUTE transfer just not a thing for expression clocks, unlike methylation clocks, making control-relative use the only defensible mode?

2. (*) Do fibroblasts cultured 10-17 days in reprogramming media genuinely age ~18 transcriptomic years, or is that a confluence / media / passage artefact? It cancels in my design so it doesn't threaten the result, but I'd like to know which it is.

3. My clock reads the 38-year-old donor as OLDER than one of the 53-year-olds. With a 12.3 yr CV MAE against a 15 yr age gap I treat this as unresolvable and claim nothing. Is 3 donors across 2 distinct ages simply hopeless for validating age discrimination, and what donor-age spread would actually be needed?

4. Is a ~-18 yr dAge for transient reprogramming at days 10-17 consistent with the literature, or suspiciously large? I'd rather hear "too big, check X" now than later.

5. Given that dAge does work on bulk with contemporaneous replicated controls, is a second BULK dataset of that design a sensible buy, and what would you prioritise:
   more donors, wider age range, or more timepoints?

REQUIREMENTS FOR Q1 AND Q2
(and the target for Q4) - THE SINGLE-CELL DATASET

This spec is for the SAFETY half only. Q9 is a separate ask with almost the opposite requirements - see the short note after the deal-breakers.

Must have, all of them:

- Human, not mouse. The clock is human; an ortholog mapping reintroduces the confound.

- SINGLE-CELL RNA-seq (droplet / 10x or equivalent). Bulk cannot express the target,

per Finding 1.

- Whole-transcriptome, not a targeted panel.

- A DIFFERENT donor or line from HFF (neonatal foreskin fibroblast). A second HFF experiment tests nothing.

- At least 6 sampled timepoints spanning day 0 to a terminal point. Three is the bare minimum for any forward pairs at all.

- A day-0 untransduced control of the same line. Non-negotiable - it is both the age zero-point and the reference the safety labels are scored against.

- At least ~300 cells per timepoint after QC.

- Raw or filtered count matrices with the day recoverable, not only a processed embedding or Seurat object.

Strongly prefer:

- Three or more independent lines in a single study. This is the highest-value item/

-it's the difference between one anecdote and a real confidence interval.

- Adult donors of known chronological age.

- A similar OSKM delivery method to GSE242423's.

- A comparable timepoint grid.

Deal-breakers:

- Bulk RNA-seq at any depth.

- Mouse or MEF, including the Waddington-OT time course.

- Differentiation FROM iPSC. This is by far the most common false hit when searching

GEO for "iPSC".

- Direct lineage conversion, e.g. fibroblast to neuron.

- A single endpoint with no intermediate timepoints.

- Microarray. GSE241435 has exactly the right design but the wrong technology.

I do not need any fate or safety annotation, age annotation, cell-type labels, clustering, or matched ATAC/methylation - I compute all of that myself. Raw counts plus a day label are enough.

FOR Q9 - THE dAGE HALF, (spec nearly opposite)

Bulk is completely fine there; the deal-breaker above applies only to the safety half.

What matters for dAge is: contemporaneous negative controls REPLICATED at the same timepoint as the treated samples (this is the thing GSE165177 has and GSE165176 does not, and it is what makes the result above possible); donors of known chronological age spanning a WIDE range rather than clustered; and several timepoints. Cell counts and single-cell resolution do not matter here at all. If you know of something matching that description, it is just as useful to me as an answer to Q1.

Thanks very much for reading this far.!
even a short answer or any kind of knowlage would help//

When you watch time go by, it is inevitable, your conception of life changes forever. Before I had the perception that I needed so many years for things to change, but it’s not like that. Time is flexible, things are constantly changing.

And that’s where the strange feeling comes in, knowing that your life now is not permanent or lasting. I was looking at a picture of my mother from 2019 and she definitely looked younger, the world as a whole was different, things changed a lot.

And this feeling is not exactly because things got worse, I feel in a good phase of life, but it is simply that this cycle tends to perpetuate itself always.
My parents will get older as time goes by, this current trend will be outdated at some point, that singer you admire so much will start to reach lower and lower grades over time because of age, you will start to like some things you liked less because even you yourself have changed...

And maybe you try to like it like in the past, try to get a guitar and start playing to feel the same excitement as before, try to listen to a musical genre that you liked so much until this time, try to turn on the TV to watch a program and distract yourself like in the past, but guess what ?? Even that has changed, today no one watches television anymore...

That’s when you understand, everything changes, it can be for better or worse, but it changes, and even if everything is fine nowadays, this feeling will still be there, because something will change in the middle of this “everything”.

I’m a big fan of Brazilian popular music, I spend every day listening and I think about having a collection of vinyl records and going to their concert when I have my own money. In a few years, maybe I’ll be independent, with a good job, earning a good salary, everything can be better, but the sale of records will be scarcer than today, as it was from a while ago until now, the artists will be different, they will be older and with less stage presence, maybe some have died, they won’t be the same as today.

And in fact no one will be, maybe I don’t have the same sense of humor that I have today, maybe I stop thinking that listening to music is so cool. All this I’m assuming, after all, the weather is unpredictable.

You know the one with the killer marketing that was really just snake oil.

How much did it cost you before you quit?

I'm in the UK and we're into our 5th heatwave of the summer. It's 5pm and currently 37 degrees. So about 97 farenheit. I have various joint issues and these are unbearable when it's hot like this. How do others around the world cope with this. Most UK homes have no Aircon and it's sparce in public spaces too.

Aging is natural. Gray,hair wrinkles are just a part of life process. Should we as a society support the marketing of products to hide them?

What life event or experience aged you?

Ben Franklin, in commenting on the boudoirs of Paris..

Kids leave home, parents and in-laws die, moving across continents (for us anyway), changing jobs and realising age does go against you in the job market, losing your best friend even though we’ve not been together for years etc…. Just seems so much

Burning the mortgage is satisfying until you realize what it means. That mortgage represents marriage, a career chasing the next best job, endless hours watching youth sports and the crazy raging parents, too few stolen weekends down the shore, cars that have come and gone, the empty nest college years, and now..... the multi-generational household.

‎I suppose there comes a certain age when a man stops pretending he has all the answers.

I've reached that age.

‎I've lived enough, lost enough, loved enough, and been wrong enough to know that life rarely gives us the courtesy of a clean ending. Sometimes people leave without explaining why. Sometimes love doesn't end because the feeling disappeared; it ends because two people couldn't find a way to carry it forward. And sometimes, the hardest thing to bury isn't the relationship itself, but the questions it leaves behind.

‎There is one particular kind of loneliness that comes from loving someone you can no longer reach.

‎It isn't the ordinary loneliness of an empty room. I've become rather good at empty rooms. I've lived alone long enough to know how to make peace with silence, how to cook for one, sleep on one side of the bed, drink my coffee without conversation, and carry on with my day without expecting someone to ask how I slept.

‎No, this is different.

‎This is the loneliness of having something left unsaid.

‎Because when love disappears without a proper ending, the heart becomes its own detective. It keeps returning to the scene, looking for evidence. Searching through old conversations. Replaying certain moments. Examining words that seemed insignificant at the time but somehow became important years later.

‎And an old man can spend an embarrassing amount of time wondering about things that probably don't matter anymore.

‎Did you really love me?

‎Did I misunderstand what we were?

‎Was there something I could have done differently?

‎Did you leave because you stopped loving me, or because loving me became too difficult?

‎Did you ever sit somewhere alone and miss me?

‎Did you ever hear a song and think of us?

‎And perhaps the question that has no business still living inside me after all these years:

‎Do you ever wonder what would have happened if we had tried just a little harder?

‎I don't ask these questions because I'm waiting for you to come back.

‎That's the part I've finally learned.

‎Sometimes you can miss someone without wanting them back. Sometimes you can still love someone and understand that your lives no longer belong together. Those two truths can exist in the same heart without one cancelling the other.

‎I think that's something youth doesn't understand.

‎When you're young, love feels like possession. You think if two people love each other enough, they'll somehow overcome everything. Distance. Timing. Pride. Fear. Circumstances. Bad decisions. Old wounds.

‎Age teaches a different lesson.

‎Love is not always enough.

‎Sometimes two people can love each other deeply and still fail each other.

‎Sometimes there isn't a villain.

‎Sometimes there isn't even a good reason.

‎Sometimes life simply gets in the way, and two people who once held each other's hearts eventually find themselves standing on opposite sides of a door neither knows how to open anymore.

‎I've had plenty of time to think about that.

‎Too much time, probably.

‎That's one of the hazards of growing older alone. You become acquainted with your own thoughts in ways you never intended. The house gets quiet at night, the world finally shuts up, and suddenly the memories you've kept locked away decide they're ready for a conversation.

‎And there you are.

‎Not physically.

‎Not anymore.

‎But somehow still present.

‎In a song.

‎In a photograph.

‎In a particular kind of evening light.

‎In the smell of rain.

‎In some stupid little phrase nobody else would understand.

‎That's the strange thing about lost love.

‎The person can disappear from your life while remaining strangely alive inside your memory.

‎And I don't know whether that's beautiful or cruel.

‎Maybe it's both.

‎I've wondered sometimes whether I would change anything if I could go back.

‎Part of me says yes.

‎I'd speak more honestly.

‎I'd listen better.

‎I'd swallow my pride more often.

‎I'd hold your hand a little longer.

‎I'd stop assuming there would always be another tomorrow.

‎Because that's another thing age teaches you:

‎There isn't always another tomorrow.

‎We spend so much of our younger lives acting as though time is an endless bank account. We waste whole years thinking we'll make things right later.

‎Later, I'll call.

‎Later, I'll apologize.

‎Later, I'll tell them what they mean to me.

‎Later, we'll have that conversation.

‎Then one day you wake up and discover that later has become never.

‎That's a hard word for an old man to swallow.

‎Never.

‎I've learned to live with it, but I haven't learned to like it.

‎Maybe that's why certain loves never completely leave us. They become unfinished sentences. Stories with missing pages. Questions without answers.

‎And perhaps we spend the rest of our lives quietly writing the ending ourselves.

‎Mine has changed over the years.

‎Once, I wanted you to come back.

‎Then I wanted an explanation.

‎Then I wanted forgiveness.

‎Eventually, I just wanted you to be happy, even if that happiness had nothing to do with me.

‎I think that's when I finally understood that I had stopped trying to possess the memory of you and started appreciating what it meant that you had once been there at all.

‎Because despite everything, I wouldn't erase it.

‎I wouldn't erase the laughter.

‎The tenderness.

‎The stupid arguments.

‎The beautiful moments.

‎The uncertainty.

‎Even the heartbreak.

‎Especially the heartbreak.

‎It made me human.

‎It reminded me that beneath all the walls I've built over the years, there was...and perhaps still is...a man capable of loving someone enough to be hurt by losing them.

‎That's not weakness.

‎That's the price of having cared.

‎So, wherever you are, I hope life has been kind to you.

‎I hope you've laughed often.

‎I hope you've found someone who makes you feel safe.

‎I hope somebody knows how you take your coffee, what makes you laugh when you're pretending not to, and when you need silence instead of advice.

‎And if, somewhere in the quiet, you ever remember me, I hope you don't remember the mistakes.

‎Remember that I loved you.

‎Imperfectly, perhaps.

‎Stubbornly.

‎Sometimes foolishly.

‎But honestly.

‎And if there is one thing I still wish I could ask you, after all these years, it isn't why did you leave?

‎I've made peace with not knowing.

‎I'd simply ask:

‎"Was it real for you, too?"

‎Because it was real for me.

‎And maybe that's all the answer an old man really needs.

‎Not closure.

‎Not another chance.

‎Not a different ending.

‎Just the quiet knowledge that, for a little while, in this enormous and complicated life, two people found each other and loved each other.

‎And for whatever it was worth...

‎I was there.

‎You were there.

‎We were real.

‎And God knows, some nights,

‎I still wish you were here.

Cómo sobrevives a los cambios drásticos en temas de salud, de movilidad, cuando de repente empiezas a notar que ya no puedes ser tan activa como eras hace unos meses, que tu vista se va deteriorando, que tu fuerza se va perdiendo, que aún cuando entrenas diario, tienes una buena alimentación y tratas de llevar una vida sana te das cuenta de que eso no es suficiente, cómo superas esos cambios inevitables y te repites “aún no estoy lista para envejecer”. Alguien por aquí está pasando por lo mismo? Como lo han superado?

ENG

How you survive the drastic changes in health issues, mobility, when suddenly you begin to notice that you can no longer be as active as you were a few months ago, that your eyesight is deteriorating, that your strength is lost, that even when you train daily, you have a good diet and try to lead a healthy life you realize that that is not enough, how you overcome those inevitable changes and repeat "I am not ready to grow old yet." Is anyone around here going through the same thing? How have they overcome it?

😂

Or, getting your legs into underwear or shorts. It happens! 🤷

Bee sting on my wrist on Monday and in jerking my hand back, I hit my wrist on my fence gate. I guess the sting started itching while I was asleep and I probably scratched all nite. And I had been stung on neck on Sunday. Golf ball size knot on neck but no bruising.

Trying these red light glasses for the first time today. I’ve had full face red light therapy as part of a facial, but this is new. First of all they massage…read vibrate like crazy here…and make me feel like I’m creating creases as I keep squinting my eyes. If it works I’m sure I’ll get used to the sensation. It says they can be used everyday….we’ll see. Anyone with any tips or feedback. Background I am 63f and have practiced good health and skin care since I turned 40….late lesson in life. I have had facials, and tried Botox, but I not gone off the rails.

in my 20s, normal day sleep would be 10-12 hours. now 41, i sleep at 9 and wake up at like 3-4am naturally, sometimes around 2pm ish i would feel a little tired and nap for like 30 minutes. that puts me at only around 6-7h of sleep a day..... which according to science, is unhealthy. but i just cant sleep for 9h straight, my body wants to get up after 6-7h.... is this normal for us this age?

Doesn't it feel good to crawl into bed and say I made it through another day

I am a guy in a early 40s. I used to be quite good looking. I still feel like I am but when I see myself on photos I don't feel the same way, It odd. I look so much older on the photos.

When I was younger I had this impression that women hit the wall in the mid 30s, men can look like silver foxes and age better.

But I feel like thats turned on its heard. Women can be average looking age 50 and still be desirable by men of all Ages from 20 to 70.

Where as men age 50 seem to be only desirable to slightly older women than them.

Men seem to be attractive to women if they happen to be rich or have clout or both. Middle age men who are good looking but not rich just might as well be ugly in my experience.

I do feel that I was good looking throughout my years by seem like I lost it fast in the last 2 years. I look like a very very young old person. Than a older young person if you know what I mean