Scientist in the garden: tomato crop builds, faces giant enemy

As regular readers of this blog know, one of my hobbies is gardening and I like to bring a scientific perspective to the garden. This year in my garden I’ve planted a whole bunch of tomatoes including unusual and fun varieties. Little did I know that a giant tomato enemy would arrive. More on that in a bit.

Below is a plate of the ones I picked today including the blue one Dark Galaxy and the funny pointy-tipped yellow cherry ones called Barry’s Crazy Clusters, which in both cases are from Wild Boar Farms, a local place known for creating amazing new tomatoes (@WildBoarFarms). Note that big Celebrity tomato on the right that weighed nearly a pound.

Paul's tomatoes

Returning from the big ISSCR stem cell meeting in SFO I found my vegetable garden doing pretty well despite the constant near-100 degree temps we’ve had in the Sacramento-Davis area for weeks. The heat has been nearly unrelenting and remarkably both on the way to and from San Francisco there were two brush fires going both directions that mucked up traffic. This doesn’t bode well for the fire season.

Giant tomato hornworm

My tomato plants looked a bit thirsty despite sprinklers and some watering by the family, but pretty good overall. However, while watering I did find one tall plant that had nearly all of its leaves missing right from the top.

My first thought was, “Oh, no, must be a giant tomato hornworm!” It didn’t take long to find the massive beast that had been gorging itself on this plant (see image above).

One of the most striking things about these caterpillars is their spine on their rear end and this guy’s was bright red. This particular monster was longer and wider than my pointer finger. No wonder that plant was half-stripped of its leaves.

Rather than kill it, we decided to capture and study this thing for a while. I don’t know if it will turn into a giant five-spotted hawk moth before we lose patience with having it in the house in a huge jar with tomato leaves for food.

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Fiona Watt excellent talk on epidermal stem cell plasticity #ISSCR2016

Yesterday at ISSCR 2016, Fiona Watt gave a great talk about plasticity of epidermal stem cells. As with my other #ISSCR2016 posts, this one is a stream of impressions, quotes, and key points. For my other blog posts from the meeting see here.

First she provided a nice overview of human skin architecture and regulation. The position of cells within this tissue directly impacts their fate and this is true of the stem cells as well. How do stem cells in the basal layer adopt specific fates as they commit and then differentiate?

Fiona Watt

Her team looked for surface markers that link to clonogenic potential of stem cells. The ability to form clonal colonies is an assay for the actual stem cells in the epidermis. They found specific markers that are elevated in human epidermal stem cells.

Different microenvironmental cues trigger differentiation via different signal transduction pathways, which includes cues such as micropatterned island versus soft, porous gel.

What regulates commitment?

They looked at gene expression in suspension (t-SNE statistical analysis of different time points). There was a poor correlation between mRNA and protein at 4h in suspension (adding my two cents–so studies like Affymetrix and RNA-Seq clearly do not always tell the whole story). Are there post-transcriptional/post-translational changes taking place?

A major finding is that protein dephosphorylation is a characteristic of commitment. Spike in phosphatase activity at the same time.

Phosphatase KD gave mixed results of more or fewer colonies depending on the phosphatase targeted. Thus, the balance of difference phosphatase activities will be central. Changes were apparent in differentiation and proliferation in a neat in vitro epidermis model (building epidermis on de-epidermalized skin). There are pro-commitment phosphatases that impact AP1 factors.

Some focus on DUSP10 (opposite effect of pro-commitment factors and they may antagonize). Why is commitment state transient?

Exit the niche, downregulation of MAPK, increase in pro-commitment phosphatases. DUSP10 makes commitment transient.

Open questions.

  • Do different external stimuli trigger a common commitment state?
  • Does cell position influence commitment?
  • Are different commitment states linked to different terminal differentiation outcomes?

I thought this was an outstanding talk, one of the best at the meeting.

Poll for #ISSCR2016 attendees: what’s your view of REGROW Act?

Shinya Yamanaka at #ISSCR2016 on reprogramming of cells & scientists

The second day of ISSCR 2016 started off with a great session on pluripotency and plasticity, and the first talk was by Shinya Yamanaka. He changed the title of his talk to “Reprogramming of Cells and Scientist”. As with my other posts on this meeting, this one is a stream of quotes and impressions from the talk. The beginning was more autobiographical on his part and then the second half was on basic science. I really enjoyed this talk overall.

It’s now been a decade since Shinya Yamanaka’s seminal paper on mouse IPS cells. Shinya started his talk going back in time to the early 1990s of his postdoc at the Gladstone. He cloned NAT1 as a postdoc (Yamanaka et al. Genes & Dev, 1997).

Shinya Yamanaka

He found that NAT1 is required for early mouse development. He made NAT1 null mESCs and found that the NAT1 KO mESCs could not differentiate.

He got his own lab in 2000 and he and his group tried to induce pluripotency in somatic cells. It was 6 years later that they published the first IPS cell paper.

IPS cell technology has “reprogrammed me too” he said. One of the things I enjoy most about Shinya’s talks over the years (besides the wonderful science) is that he is very free with discussing what it means to be a scientist and how science effects scientists including on a personal level.

He noted that after human IPS cells, “I have been spending a lot of time in talking with people in government and industry and banks, and also spending a lot of time in fund raising.” I think this is what he meant by reprogramming of him by IPS cells.

“Some portion of myself is refractory to reprogramming. That part tells me I should enjoy basic research” and then he said that’s what my talk will be on: basic science.

He focused on NAT1 and its knockout in mESCs. Could NAT1 KO mESCs be in the ground state even without 2i treatment? NAT1-nulls even without 2i have the same morphology as WT cells in 2i. They did single cell RNA analysis. 2i makes WT mESCs more uniform in gene expression with higher Oct4 levels, etc. NAT1 nulls even without 2i are very similar to 2i WT cells. It seems NAT1 is an inhibitor of the ground state.

What about NAT1 in human ES cells?

Conventional gene targeting in human cells didn’t work. They could only get hets but no homozygous KOs (unpublished work of Kazu Takahashi). So it seems NAT1 is essential to human ES cells. Importantly, Kazu could get homozygous in the context of Dox NAT1 transgene. When you then remove Dox you get basically a complete NAT1 knockout. 2i LIF supports self-renewal of NAT1 null IPS cells. The NAT1 null IPS cell show higher than WT levels of OCT4 and NANOG as well as other pluripotency factors.

What does NAT1 do as a protein?

NAT1 is similar to eiF4G and it is known itself also as eiF4G2. They function in translational control. eiF4G is an essential linker in translational initiation. They searched for NAT1 binding proteins by doing flag tag IP. It binds to many translational proteins and many similar factors as eiF4G. There are a few things that eiF4G binds that NAT1 doesn’t.

Does NAT1 have general or specific translational regulatory functions? There might be some specific ones.

When NAT1 is turned off some specific proteins are elevated including KLF4 and PRDM14, two key TFs that are required for transition from primed to naive state. RNAs of these two are not changed so the change is at the translational level.

I can’t wait to hear more in the future about NAT1’s role in pluripotency.

Thought provoking talk from John Dick at #ISSCR2016 on cancer stem cells

Professor_John_Dick_FRS

Wikipedia photo

Dr. John Dick gave a great talk yesterday on cancer stem cells here at ISSCR 2016. Below I summarize his talk and as always with these meeting blogs, the post is not polished and is more of a stream of the speaker’s main points. He started out broadly with a nice introduction to this area of research.

There’s a lot of controversy around cancer stem cells (CSC). How many tumors have CSCs? How different are cells within the same tumor?

The normal hierarchical organization of hematopoiesis is disrupted in AML. Are CSC properties clinically relevant in leukemia?

Here the focus is on leukemic stem cells (LSC).  If a patient’s cells can engraft a mouse then that patient has much worse survival. This engraftment predicts relapse. They have developed a LSC prognostic score. NMP1mut FLT3-ITD neg cells are mentioned. miRNA signatures and epigenetics matter for survival. Big picture conclusion: stem cell properties are very important to the disease.

More genetic studies. Branching tumor evolution during leukemia development: what is the role of stem cells? They did deep targeted sequencing of the genes known to be important for AML. They discovered a common ancestor gene commonly mutated in AML. (Shlush, Nature 2014). DNMT3a mutation was present in the common ancestor cell. Leukemia blasts can have the DNMT3a and NPM1C alleles, but many only have one marker (suggesting clonal evolution).

This raises many interesting questions.

Where does relapse come from? What is cellular origin? Did the chemo induce changes? Or are there residual cells that then spur a tumor comeback?

There’s no definitive marker for LSC.

Evidence of a long evolution in the preleukemic phase. One model is that relapse originates from rare LSC that evolve before diagnosis and survive therapy.

A fascinating point–cells that preferentially grow in the mouse xenograft are not the predominant one in the patient at presentation but rather the ones that will later kill the patient through relapse. Another model is that relapse stems from a rare CD33+ subset.