Saturday, May 28, 2011

I'm talkin' about the FunGi here!

In the past weeks/months, you may have noticed severe warps in the cyberspace and time encapsulating my blog. It's lucky that cyberspace and time can so easily form wormholes to allow for time travel back and forth between posts of long ago and very recent. Sad that physical reality is not so accommodating. In any case, I'm not the kind of person who posts regularly, at least when I'm busy like I have been. I also like some time to digest and absorb my experiences, so that's why you might find new posts popping up, seemingly published on a date that previously had no post. I'm just trying to keep things chronological-ish, for my own personal convenience, really. It's probably confusing for everyone, but in the end of the day works for me. This will be way better to return to later on. Apologize but that's just the way it is.

Something that I've been neglecting to write about, despite it's being my major time commitment here, is Research. That's right, I'm about to talk about the R-word. I guess I've been so busy marveling at the places I've visited, people I've met, food I've eaten, landscapes and wildlife I've observed, and even mental journey I've had here, that I've not spent enough time marveling at the meaningful research I've had the pleasure to work on.

But recently I have really come to appreciate the incredible research opportunity I've had staring me in the face the whole time, and also just how many lucky breaks I've had on my journey to this point. So to recap, back at Yale in my sophomore year Spring, I took this course called 'Rainforest Expedition and Laboratory.' This was far from any mundane lab or science course most people are offered in pretty much any part of their education. Pretty much from the get-go, we were sold. This would be the coolest class any of us would ever take. Our Professor, Scott Strobel, wielded a combination of brilliance and charm, to go along with his wicked stories of seemingly magical microbes, living symbiotically in plant tissue. Woah woah woah. Sorry to whip out the big science term, symbiotically, but it just means that the microbes live in cahoots with the plant, and they help each other out, where possible. And these little cuties were known as "endophytes." So we were very much captivated after hearing about how new many of these microbes (mostly we were after fungal endophytes, because the bacterial ones were less likely to be new) were to science, and about how their range of chemical and biochemical diversity was in many ways unprecedented. Think fungi that spit out jet fuel (Myco-diesel), or others that emit antimicrobial gases (smelly fungus kills pathogens) or yet others that make the same anticancer compound as their host plant (Taxol, from the Pacific Yew, Taxus Brevifolia: cancer sucks). So we spent the first half of the term learning about these organisms, alongside lessons about pharmacology, patenting drugs, botany, ethnobotany, rainforest deforestation and the conservation efforts aimed to preserve these forests. Then, it was our turn to isolate these little rascals. Our group of 15 undergrads including myself, Professor Scott Strobel, Lori, a Post-doc, Carol, an Associate Professor, and Lauren, a graduate student Teaching Assistant, all got to visit Ecuador over spring break. How rad, right?
      In the Amazonion rainforest in Eastern Ecuador, we swam with piranhas, went birdwatching on the canopy, checked out an Ecuadorian family's farm and medicinal garden. Our trip also took us to the cloud forest to the North of Ecuador's capital, Quito, and the dry forests in the Southern city of Guayaquil. We even partied it up in Quito. But our primary goal was to collect plant stem samples to bring back to Yale in order to isolate [hopefully] new endophytes. We also enlisted the services of a most eccentric, brilliant, and loveable character, one by the name of Percy Nunez, a botanist from the university of Cuzco, Peru. This man has knowledge the likes of which I had never before seen or hear of. He can identify plants to the species level oftentimes, without flowering structures. This is unheard of for most of the world's experts. Not only that, but he does it with serious style, as he memorized our preconceived list of target plants and would sometimes put his hand out of our speeding bus' window, only the reveal he had in an instant, identified and grabbed one of our group's plants. Other times he would employ a slingshot method to collect a sample hanging far overhead. Not that we didn't have enough characters, Scott's father, Gary, also joined along. Actually Gary kind of got this endophyte field started, having discovered many of it's big superstar strains. And his research was a major motivating force for Scott, who applied to the Howard Hughes Medical Institute for a special grant to fund this course. Anyways, Gary was the one who first mentioned Borneo as a potential destination for research, as he had just returned from his visit at SBC...and he raved about how good their lab equipment was.

So, we came back, all jungle-ridden and jungle-juiced up, and isolated many microbes. Over the summer and subsequent semesters, we tested them for the ability kill human and plant pathogens, and tried to purify the single active compound, though no one quite got to the holy grail, a totally purified novel active compound, with a crystal structure to hit the nail on the coffin. We also did some genetic work to give these microbes some taxonomic context. That is to figure out where each one fits in on the 'tree of life', so that we could assess just how new our microbes were (which would indicate whether they were worth pursuing and devoting any more time to). Then some of us began working on testing our class' isolates in entirely different screens for new types of chemical/biochemical capabilities ('bioactivities'). I got super interested in the gas producing fungi, and as it so happened, my faculty adviser was John Carlson, who's trying to  understand the molecular biology and biochemistry of fruit fly (and mosquito) odor perception in order to develop better insect repellents. Somehow different ideas collided in my head and I decided that I wanted to test these microbes for the ability to repel fruit flies. And then wanted to characterize the gases that my fly repelling microbes made, by using a nifty and expensive new machine Scott acquired for his lab- Gas Chromatographer-Mass Spectrometer. In the end the assay I was doing with the flies worked and it didn't work...behavioral tests I learned were incredibly frustrating, in their variability and lack of real reliability. But the gas identity studies were still most fascinating, and left a mark on me.


         So when I got to SBC, my main goal was to isolate new endophytic fungi able to churn out interesting and useful gases. One theory has held that such fungi tend to grow in plants that similarly produce potent gases; plants like Cinnamomum (Cinnamon) and Mystica fragans (Nutmeg). During my time at SBC, I've tried to put this theory to the test, by collecting plants that do not necessarily have special essential oils of strong aromatic quality. And indeed, I've found some success in isolating some potentially new members of this genus, Muscodor, known for the antimicrobial power of their gases. So it's not always that the plant's properties determine its endophytes', though I also have not proven chemically that the host plants are without gases.
     Next, it turns out that SBC has a GC-MS (same machine from Scott's lab, though different model) and so since my return from Bhutan and Thailand in the end of March, I've been working with Ruth, a research officer in the Analytical Chemistry lab, to figure out how to use this machine. I've now done a 22 day time course (4 time points) of the gas production by several of the strains isolated by myself and Noreha, who's doing her PhD on these types of organisms. The data is many and takes some serious time commitment to analyze fully, but it's also very exciting. For one, we'll have identities of  molecules somewhich must be involved in our observed antimicrobial effects. Also then might make a case for some of these organisms as potential producers of biofuels, something of great promise in our current age of perpetual energy crises.
         Aside from this work, I'm involved in several side projects with other research officers, like one involving phylogenetics (using certain genetic markers roughly representative of evolutionary history of the organism or its biochemical make up) of actinomycetes, which are an order of bacteria known for profound chemical and biochemical diversity (one genus, Streptomyces, has by itself contributed HALF of the world's antibiotic drugs). Better phylogenetic data for these microbes would help up the ante when we try to collaborate with pharmaceutical companies, because it might hint at biochemical and chemical diversity (i.e. NEW ANTIBIOTICS?). Then in the last month, I also initiated a journal club (very long on the backburner, but we just recently got a subscription to Science, which I thought would be excellent for this club) modeled after those back in the U.S.- basically informal short discussion sessions in which groups of researchers try to take apart journal papers (sometimes but not always in their field), convey experimental logic, highlight new methodologies, and/or just eat some cookies and take a much needed break. Journal clubbing it has had its challenges, since English is obviously not the first language of people at SBC, and scientific literature, especially from impactful journals like Science, is packed full of complex jargon and plainly confusing writing (kind of like this post and the rest of my writing).

Suffice to say, I have my work cut out for me, but at least it's something. And the good thing is that people are interested to give this new activity a try, I just hope it can be continued after I'm gone.

And somehow things are all spiraling to an endpoint, which I am not entirely ready to approach. I'm going to miss this place so very dearly. Recent contemplations involve returning here sometime in the future (unclear when).

Update (June 16, 2011): I meant to include photos of my organisms, so I've now added them following this note. I've also included other data, like bioassay results and the Gas-chromatography/Mass spectrometry results (identities of gases made by these fungi). I had to put this all together for a lab presentation some days ago, and I pasted some of this stuff straight from that. Apologies for my laziness.

Bioassay results: Some of the suspected gas-producing fungal strains were grown for 7 days on Potato Dextrose Agar (PDA). Fungal and bacterial pathogens (The bacterial ones mostly affect humans, and fungal ones more relevant to plants) were inoculated onto media, and then exposed to each of the strains (most of which I isolated, with several positive controls) to see if gaseous compounds made by the strains could prevent growth of these pathogens. Basically, you should look at the plates labeled "control" and you'll notice very clear growth of the pathogens. Then compare the other plates and notice there is not always growth for the pathogen strains. This means that our isolate's gases are killing or inhibiting the growth of these pathogens, which is great news. This suggests some potential for these buggers to be used in the clinic, or in agricultural applications.

Gas Analyses (Phew, these are some Funky Fungi)

Just a small selection of the compounds the endophytic strains make (just pay attention to the structural diversity, I'll paste in a clearer version of this later on):

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