Scientific Sleuthing

I’ve always had a soft spot for detective novels. I even fancy myself a detective. Not like the ones you read about in pulp fiction made famous by Daly, Hamlet, and Spillane (to name a few), but a sleuth no less. I have spent the last couple of weeks playing detective in lab, you see. I am out on a hunt for molecules produced by these microbes I am studying. This type of investigative work is what attracted me to chemistry and biochemistry. At hand I have a set of known microbial metabolic pathways, a pretty good idea of what some of the intermediate and products are, a lot of chemistry knowledge, and a whole slew of really cool high tech tools.

The method is pretty straightforward. I feed my favorite microbe a known carbon source; it helps if I can radio-label a carbon or another atom on the substrate. I wait for a defined period of time, collect the media, and start the analysis. Step one: spin down cells, take off the supernatant, extract with dichloromethane, …

Yes, it is quite tedious, but many steps later, I have a small sample with metabolites extracted from the experimental microbial growth sample. Now the fun begins. First, what technique do I start with? The simplest is ultraviolet/visible (UV/vis) light spectra. What does the absorption spectra of your samples look like? Can you see something that resembles a conjugated backbone? How about an aromatic ring? Hints of structure here and there.  But then you ask, does the sample contain one or many kinds of molecules? Can I somehow separate these molecules?

In this next step I might choose liquid chromatography (LC) or gas chromatography (GC). Which technique I use depends on certain properties of the molecules I am looking for, are they water-soluble? Are they volatile? For water-soluble molecules I select LC and for volatile molecules I select GC. Both of these separation techniques have a mass spectrometry instrument (MS) attached, allowing me to get a sense of the molecular mass of each separated compound. I inject and watch the molecules fly. Every peak I see reveals a wealth of information about the compounds ‘vitals’. What is the mass of the molecule? Does it fractionate into smaller defined compounds with a known mass? Do the fractionation patters I see match a compound previously characterized?

Next, I isolate and purify the compound using LC, collecting the fractions that have my compound of interest and use Nuclear Magnetic Resonance (NMR) spectroscopy to try to resolve ambiguities due to configuration around enantiomeric carbon atoms. I use proton NMR and carbon NMR and study the chemical shifts I see in my purified sample. From these set of data, I can deduce whether I have a ‘R’ or ‘S’ configuration around my enantiomeric carbon and a host of other structural relationships.

In the end, I have a larges set of ‘clues’ from which I will build a chemical structure of the compounds isolated from the microbial growth media. But that is not enough. In order to stake a claim to a new molecule and possibly a whole new molecular pathway, I have to present my findings to other chemists and microbiologists, convincing them that I have done my sleuthing well. It is very tedious work and most of the time you do not find a new molecule, but every once in a while, you isolate something, a molecule never seen before. The thrill of finding what has not been observed before, drives me to explore the secret metabolic pathways hidden in these microbial species.

Stay tuned to see how this tale turns out. HHH

Padma Lakshmi opens the MIT Center for Gynepathology Research

The room was packed. We had received an invitation to sit in the special seating section and to attend the ‘after party’. Lily and I waited, in the third row, front and center.  Padma Lakshmi would be walking though those doors, stage left, momentarily. We’ve watched her on Bravo’s Top Chef for 6 seasons now.  Always so beautifully dressed, with her smile, knowing that she was ready to challenge the next set of top chef’s almost beyond their culinary limits. And for all but one, past that breaking point where they hit that wall and fell short of their gastronomic dreams.

Padma Lakshmi was at MIT for the official launch of the MIT Center for Gynepathology Research. This research center, the child of Professor Linda Griffith, is the first interdisciplinary academic research institute which brings together biologists, clinicians, and engineers with the goal of understanding the basic biology, physiology, and pathophysiology of the female reproductive tract.

Professor Linda Griffith began the afternoon by introducing Susan E. Whitehead, Lifetime Member of the MIT Corporation and Vice Chairman of the Whitehead Institute for Biomedical Research. Ms. Whitehead remarked on the bold research initiative embodied in the center. Dr. Tamer Seckin, President and Founder of the Endometriosis Foundation of America (EFA), followed her and introduced Padma Lakshmi.

There she was, tall, beautiful, poised, radiant, and very much pregnant. Ms. Lakshmi began to share the account of her personal struggle and eventual diagnosis with endometriosis. This disease slowly wrestled control of her life and body away from her.  She recalled of how she suffered alone with this recurring, debilitating disease, learning to tolerate excruciating pain.  Scheduling her life around the monthly assaults by this unbearable condition. Living every day with this incapacitating disease, one which until recently had no name or meaning to her. Her pain was apparent in her heart felt rendering of how she had suffered through misdiagnosis and unnecessary medical procedures before a friend referred her to Dr. Seckin. Padma told of the relief she felt hearing Dr. Seckin’s words.  He understood what she was going through.  His life’s work has been dedicated to understanding the molecular underpinnings of this debilitating disease.  Now, almost three years later, Padma tells of how she has regained control of her life and her body.

Through out her talk, Padma emphasized how the lack of awareness and education regarding endometriosis had shaped the medical diagnosis, treatment and response to her disease. She spoke of how social taboos deterred her from asking the right questions and demanding answers regarding her body and her physical state. She also described how the people who were closest in her life at times misunderstood or played down her symptoms, leaving her doubting her ability to properly describe her symptoms to others. It was not until Padma met Dr. Seckin that she finally understood the impact that endometriosis had had on her life.

What can you do as a woman, a partner, a loved one, for someone who is suffering with endometriosis? Education and information are the best tools to help you understand and identify the symptoms of this disease. Awareness of your body is key.  For those of us who care for our close ones, understanding and believing that when they complain, the pain and the discomfort they feel is real.  Do not brush it aside.  For all of us, do not hesitate to obtain a second opinion on a diagnosis.

In her closing remarks, Padma extended her heart felt thanks to Professor Griffith and the other members of the research center for making the MIT Center for Gynepathology Research a reality. For more information regarding endometriosis or other chronic non-cancerous diseases of the female reproductive tract please visit the MIT Center for Gynepathology Research or The Endometriosis Foundation of America.  Also, please read the article that appeared in the Boston Globe on Friday, December 4th.

Thankful for being able to do what it is that I do …

This is what four days in lab looks like.
This is what four days in lab looks like.

It is 7:24 on the day before Thanksgiving. AC/DC is cranked up so loud my ears are starting to bleed and I have at least four more hours to go before I go home tonight.  There are papers and three lab notebooks strewn all over my bench top.  This is my fourth 18-hour day in a row.  A crazy look in my eyes and a five-day growth on my face.  A marathon set of experiments trying to decipher the growth curve of the community of deep earth microbes that I am trying to identify and characterize.

I started this morning over thirteen hours ago by coming in, turning on the gas to exchange the atmosphere in the anaerobic tent loading chamber, and promptly blowing out the seals in the CO2 regulator.  You know that when this happens at 6:30 in the morning, it is not going to be a good day.  I couldn’t find another one in the building (nor in friends labs in a couple of other buildings) so I settled in and waited until AirGas opened later that morning.  Needless to say, it was closer to 1:00 in the afternoon before I got the tent back operational, putting me at least five hours behind schedule for an already jam packed day.  So this is how I find myself with one more hour to wait while my microbial cells sit in the first incubation of many in a long protocol with which I will fix them in paraformaldehyde for later DAPI stain and FISH analysis.

Why do I do this? Why do I put in the long hours crazy hours?  Because I love it.  Plain and simple, that is the only answer I can give.   I delve into the unseen, the unreal, the unknown world of extreme microbes, and try to make it visible, real, solving the mystery of who lives where and how the hell they do what they do in those most inhospitable places in which they thrive.  I have to design an experiment with the full understanding that the equipment does not exist for me to do these experiments.  I cannot go to a shelf and just pick up one of these and three of those and have some technician come over and set it all up for me.  This is truly science driven by your capacity to design, invent, and assemble the equipment that you will need, while doing the experiments at the same time.

Sometimes I forget how incredibly crazy and out of this world what I do is.  I get reminded of this when I try to explain what it is that I do to friends and family.  I get this look of fear and awe when I explain that the things I study grow at 100-200 atmospheres, at temperatures between 50-75 degreed centigrade, and under acidic conditions so extreme it would peel the skin off your bones.  Like I said, this is pretty cool stuff.

Well, I got to go now, the next cell wash and incubation is about to begin.  While all this is extremely exciting, getting to that final answer is laborious, painstaking, and tedious.  Still, I would not trade this life for any other one.  This is what I am thankful for on this week.  I get to do what I love (and sometimes get a little frustrated at) every day.  Take care and have a great Thanksgiving.

Center for Alternative Technology – Paul Allen

Last Friday, Paul Allen from the Centre for Alternative Technology (CAT) in Machynlleth Mid-Wales spoke about the centers work during seminar in the Parson’s Lab.  He is the CAT Development Director and co-author of the Zero Carbon Britain (ZCB) report. Mr. Allen has spent the last month traveling the US from the East Coast to the West Coast by train giving his presentation about the CAT and the ZCB initiative.

The CAT advocates for an environmental policy that integrates current and emerging environmental management practices, new technology, and education to promote sustainable living communities.  Recently they partnered with University of East London to offer graduate programs in Architecture specializing in Environmental and Energy Studies, Renewable energy in the Built Environment, and Ecological Building Practices.  Mr. Allen’s work with CAT and the ZCB report is providing a policy and lifestyle framework which if adopted, can lower their dependency on fossil fuels and imported energy taking Britain a long way on the road to zero carbon emissions and energy independence.  Along the way, I hope they can provide a successful, sustainable environmental management model that can be used by other developed nations.  If you are interested in finding out more about the CAT, make sure to visit them online.