Fun in the Forest

Let’s play a word association game: Climate change

Rising sea level, acidifying oceans, species migration and extinction, extreme weather, and an ever-warming climate. Yes, all of these things and more. And while these are all of global concern, how about the impacts of climate change on a smaller, more regional level?

Here at Harvard Forest, we’re asking exactly that. Currently, Harvard Forest has three soil warming experiments running, each of which has cables installed under the soil to warm the plots 5° C above ambient temperature in an attempt to measure how a warmer climate may impact New England hardwood forests.

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Through routine measurements of gas fluxes, nutrient concentrations in the soil, and tree growth for over more than a decade, my mentor Jerry Melillo and his crew have been able to put together a really interesting story, which can be summed up in a few bullet points:

  • Warmer soils made for a more productive soil microbe community. This means that microbes were able to burn through soil organic matter – basically plant and animal debris in various stages of decomposing – and release a surplus of nutrients into the surrounding soil.
  • In particular, the breakdown of soil organic matter led to a huge spike in nitrogen availability. Because nitrogen is typically seen as a limiting nutrient in these ecosystems, having an abundance of free nitrogen meant that the trees in the heated plots were able to grow at a faster rate and store more woody biomass in comparison to the control plot.
  • Once nitrogen was freely available, trees stopped investing as much energy and carbon into forming fine root systems. Seeing as many species of fungi colonize fine root biomass by forming a sheath over the root tip, decreasing the number of fine roots also has the potential to impact root tip colonization

While these trends held up for the first 10 years of the experiment, in the past two years, one aspect has noticeably changed. The trees. Their growth spurt has come to an end.

And this is a really interesting observation, as it indicates that nitrogen might not be the only limiting factor in this ecosystem anymore. Perhaps a decade of soil heating has desynchronized nutrient cycles or depleted the system of another critical nutrient. And we’re hedging our bets on phosphorus.

To test out our hypothesis, my time at the Harvard Forest REU has been a whirlwind of different experiments and approaches that aim to tackle this question from as many angles as possible. And these can be summed up in a four-part game plan:

  1. We want to know what the availability of phosphate is in both the heated and control plots. To do this, we deployed charged membranes in the soil, which essentially act as roots to absorb free phosphate in the soil over the course of a two-week incubation.
  2. In addition to the availability of phosphate, we’re interested in the overall storage of phosphorus in the forest. This involved doing a series of sequential extractions to determine the size of labile and recalcitrant phosphorus pools in the soil.
  3. And what makes all this phosphate available? Phosphatase! So another part of my project involved measuring the potential enzymatic activity of phosphatase. On top of that, because the big time commitment in this experiment is preparing the soils, not running the assay, we also decided to measure the activity of beta-galactosidase, cellobiohydrolase, N-acetylglutamate, and a handful of other enzymes as a bonus.
  4. Finally, while those cover the parts of my project look at soil, I’m also trying to determine if decreasing fine root biomass and nitrogen availability has had any impact on the fungal community. Different species of fungi utilize different exploration strategies, which makes them more or less carbon expensive for the tree to maintain a relationship with. So our thinking is that if there is a surplus of nutrients available in the heated plots, then maybe some species of fungi will be more or less favorable for the tree to support. screen-shot-2015-06-16-at-1-06-14-pm

While I’ll likely be posting a few more updates as the summer goes on, if you’re interested in following the rest of my adventure this summer and all the nitty-gritty details of my projects, I also keep a science blog, which you can find here: environmentalbrigade.wordpress.com/ !

 

Soybeans, Blood, and Robots: The Epigenetics of Cancer

This summer I’m working in the lab of Dr. Laura Rozek, a molecular epidemiologist who specializes in environmental carcinogenesis. This lab is part of a team of researchers at the University of Michigan receiving a SPORE grant (Specialized Program of Research Excellence) from the NIH’s National Cancer Institute to investigate head and neck cancer. Most of the time when I tell people “head and neck cancer,” they hear “brain cancer,” which is not the case. More specifically, the cancers we are studying are called head and neck squamous cell carcinomas (HNSCC), which are tumors that form in the mouth and throat (oral cavity, oropharynx, and larynx). The most common contributing factors the the development of this disease is tobacco use and certain strains of HPV (human papilloma virus). While this kind of cancer is a small fraction of cancers in the US, it is much more prevalent in other regions of the globe, such as Southeast Asia.

The researchers here at Michigan are interested in understanding how epigenetic changes to certain genes arise and turn normal cells into cancerous cells, and how we might be able to reverse the process. For those of you who don’t know or have forgotten what epigenetics is, it’s the study of the chemical changes to DNA that activate or deactivate different parts of the genome at specific times. The specific kind of change studied in the Rozek lab is methylation, which involves methyl groups being attached to different nucleotides. Too much methylation, called hypermethylation, in the promoter region of a gene can turn it off. In cancers, genes for proteins called “tumor suppressors” are often turned off in this way. Research has also shown that cigarette smoking is directly related to the hypermethylation of the important tumor suppressor p16.

The project I’m working on is a phase II clinical trial to investigate the cancer preventative (“chemopreventative”) properties of compounds found in soybeans and other legumes called soy isoflavones. The study recruited both smokers and nonsmokers who had developed the cancer and gave them capsules containing soy isoflavones, mainly the one called genistein, which they ingested daily. The cancer patients who smoked were helped to quit. Tumor tissue and blood samples were taken from the patients before and after the soy treatment to analyze the changes in methylation levels.

Why soy? There are a couple reasons why soy is seen as a promising agent for nontoxic preventative therapy to undo damaging methylation levels. Past projects at Michigan have looked at diet in connection with HNSCC survival rates. They found a correlation to better rates of survival with higher consumption of fruits and vegetables, in particular soy and other leguminous foods high in soy isoflavones. Other research has found that genistein can inhibit the growth of other cancers (breast and prostrate) in vivo and in vitro through regulation of signaling pathways.

My role has been focused on the blood DNA component of the soy study. First, I extracted the DNA from the blood samples using a robot called the QIAsymphony from Qiagen. It basically uses a pipette-equipped arm controlled by a very smart computer to distribute reagents. It looks like this:

yes this is actually a robot

DNA extraction robot. I wanted to add a video of it running, but videos were too big to upload.

After obtaining the pure DNA, I bisulfite converted (BSC) the samples along with controls. This makes the methylation levels readable to the pyrosequencing machine. After this step, I have to copy the DNA using PCR (polymerase chain reaction) for each of seven genes that I am investigating. I have done two of these plates so far and run them through the Pyrosequencer, giving me data for two out of seven total genes. One of these genes is particularly tricky so I will not be sequencing it myself but sending it to a different department at UMich.

the computer for this thing is like from 1999 but the camera inside it is like $$$$$$$$$$$$$

The PyroMark MD pyrosequencer and the special plate that goes in it. The black tick marks indicate wells with controls.

So now I am equipped and trained on all the techniques needed to finish collecting data. I’ve been delayed by the PCR process because we were having difficulty making primer mixes that were not contaminated. My lab manager suspects that the primers are just old so we’ve ordered fresh new batches. In the meantime, I’ve been helping out in another phase of the HNSCC research by receiving and entering clinical information about tumor blocks and slides from patients who are enrolled in Michigan studies.

I don't even have to wear gloves

Tumor suspended in a block of wax to be stored at room temperature. This technique is called FFPE.

I thought they looked pretty

Stacked slides containing stained slices of tumor.

 

 

 

 

 

 

 

 

 

 

My next steps involve PCR of the next five genes and measuring their methylation levels. I’m on track to finish this with plenty of time left in July, so I may be helping out on other projects or shadowing the bio statisticians who will analyze my data.

-Claire Côté, ’17