Mike Gallagher

Nvm!

I’m confused, is this satire or real?

Very nice analysis. I may have missed it, but did this take into account the baseline 10% MYF that NIH has most years?

Where do you see 40%? I thought MYF was going to be limited to 2025 levels, which was about 25%

Looks like early neuro differentiation to me. Maybe SOX2 and TUJ1 or MAP2?

No it’s actually the overall acceptance rate. 65% of submissions get peer reviewed.

I’m surprised at the relatively high (35-45%) acceptance rate, given the quality of the journal! They must get very good submissions in general.

3/ also, in the context of PTA’s other films, I think it’s even less likely that this movie should be interpreted as anything other than artists trying to make interesting art.

2/ or go -100% and make up an entirely new universe with new laws of physics. My impression is the author thought this movie was something that it wasn’t, i.e. a vessel to promote (or disparage) specific political and cultural movements.

1/ I didn’t interpret the movie as making any attempt to do anything other than be a movie. I don’t think it was designed as political messaging, or to perfectly reflect any specifics about current or past American culture or politics. Movies can go documentary style with 100% adherence to real life

Help me, I don’t get it

Wow!

I think this is likely, at least in steady state. They may be more important in cell lineage or cell state changes, but overall they seem to do very little for gene expression. E/P interactions seem to form before, and independently from, TADs, after mitosis

2/ dynamic, doesn’t fuse or exhibit fission, and doesn’t exhibit concentration-dependent behavior, it can’t have formed through LLPS right?

1/ I think it’s falsifiable (in theory) to hypothesize that a specific body formed through LLPS, but not that LLPS exists *somewhere* in the cell. You can’t falsify the latter bc it’s not feasible, but you could falsify whether a specific body formed through LLPS, right? If it is not spherical,

Can LLPS exist in cells without scaffolds, as defined here? IOW is your critique about the proposed mechanism of LLPS, or LLPS in cells in general?

I’d appreciate a nomination

Is there any evidence that this is an academia problem, rather than a society problem? I find we academics tend to talk about academia like everything about it is "special" in some way, but 99% of the ppl I know well are non-academics, and all the bad stuff happens outside too, often more so

Systematic characterization of existing and novel inducible transgenic systems in human pluripotent stem cells after prolonged differentiation The ability to control transgene expression both temporally and quantitatively in human-relevant cells and tissues is a cornerstone of biomedical research. Additionally, precise transgene control is c...

Oops, here’s the right link www.biorxiv.org/content/10.1...

Great job market we scientists are in

20/ in summary, this "iPSC-Xon" system overcomes critical limitations in current transgene systems in hPSC-based work, and will benefit from additional improvements. I'm on the job market and would love to bring and improve this technology to other researchers!

19/ strangely, leaky SALL1 is not due to unwanted splicing in the absence of drug, but rather the unspliced pre-mRNA somehow escapes the nucleus and is translated in-frame to make the full-length protein. We still don't know how this works.

18/ we thought larger transgenes (Cas9 is ~4kb) might be more prone to disrupting the Xon splicing switch, so we tried the ~4kb SALL1 TF, a critical driver of microglia homeostatic function. In contrast with EGFP, Xon-SALL1 is fully leaky and shows aberrant splicing patterns.

17/ we also show that we can get inducible gene editing with Xon hPSCs, but only if sgRNAs are introduced transiently (panels C-E). Cas9 must have enough leakiness for maximal editing with constitutive lentiviral sgRNA introduced into hPSCs (A-B)

16/ what about other genes? Here we show Xon works for inducible control of GRN expression, and we can overexpress GRN in iPSC-derived microglia, which already express boatloads of it. P2A peptides improve both GRN and SMAD, and importantly reduce N-terminal tag to a single aa.

15/ a couple important points here: in addition to resisting silencing and being highly tunable, Xon adds only 24aa N-terminal tag (see below where we reduce it to 1aa) and needs only low nM drug doses, whereas degrons add ~500bp tags to BOTH termini to be effective, and need low uM drug doses.

14/ a note of caution is that while background EGFP is minor compared to even the lowest drug induced expression levels, this may need mitigation when using TFs or other strong cell state regulators

13/ Here's the commonly used H1 ESC background, differentiated into microglia with an embryoid body protocol (we used EB and monolayer)

12/ quantification shows how tunable this system really is! Below the 100nM dose, linear correlations between drug dose and EGFP is basically perfect both before and after differentiation

11/ again with AAVS1-targeted EGFP, what we saw was remarkable: first time we've ever seen robust inducibility after long differentiations, this time 4-7 weeks, across hPSC backgrounds and differentiation protocols!