PCR AND A DANCE IN THE MIND FIELD

PCR stands for “Polymerase Chain Reaction”, but it could just as well be “Polymerase Chain Revolution”.  I know that even those who do not work on DNA or molecular biology are aware that PCR is a tremendously powerful tool that influences many areas of our life.  Forensics is a classic example, when PCR is used not only to help a prosecutor’s case, but what I find even more fascinating, to prevent innocent people from paying for a crime they did not commit. Many people on death row have been released from prison thanks to one of the most elegant and surprisingly simple techniques in molecular biology. Through PCR, a specific segment of DNA is replicated over and over and over inside a tiny plastic tube. The ability to make a lot of DNA starting with a few molecules opened the doors to countless types of studies, from evolution to detection of genetic and infectious diseases. For biochemists, it is actually impossible to do research without PCR.  Taking our lab as an example, we use it almost on a daily basis, either to make precise alterations in bacterial genes, or to delete bacterial genes from the chromosome.  Without this technology, many of our experiments could not be performed, whereas others would take months instead of days, or even hours.

The genius behind the invention of PCR is Dr. Kary Mullis, who won the Chemistry Nobel Prize exactly twenty years ago, in 1993.  His own recollection of his scientific journey can be found in the fascinating (and at times controversial) book “Dancing Naked in the Mind Field”.

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“We were at mile marker 46.58 on Highway 128, and we were at the very edge of the dawn of the age of PCR. I could feel it”. (Dancing Naked in the Mind Field, page 7)

Phil and I happened to travel right through that highway several times last week, and we made sure to take the book with us so we could read it under the spell of that beautiful setting.

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“When you  get the hang of it, science, like everything else people do for a living, is pretty straightforward. You are in the business of solving puzzles. The way to approach a puzzle is to think about it for a while, look at all the facts you can find out about it, and then take a guess.  Propose a solution. The next step is to try your best to disprove your solution. Show that the pieces don’t fit together in the way  that you have proposed. If you can do that, then propose another solution.  And then do the same thing. Reality is a tricky little puzzle”.  (K. Mullis, Dancing Naked in the Mind Field, page 50).

Through my work, I had the chance to meet incredibly impressive people.  One such person was Joe Neilands, Phil’s PhD advisor from Berkeley.  The impact of Neilands on Phil’s scientific career and political views was huge. Even though Joe passed away many years ago, Phil always includes a picture of him in his talks, a well-deserved tribute to the man who discovered siderophores.  Siderophores (as I mentioned in the blog before)  are molecules that allow bacteria and other microorganisms to survive in a world where iron is virtually unavailable.   I knew that Kary Mullis was a PhD student in Neilands’ lab, in fact he was still around for a while when Phil joined the lab. I was thrilled to find out several references to his great mentor in the book.

“The lab in which I learned the most about life was presided over by Joe Neilands. (…) Joe Neilands made me aware of the present-day planet.  I already knew about the universe but had spent little time thinking about today and the people around me”. (Dancing Naked in the Mind Field, page 35).

Tomorrow will be a special day for us.  Kary Mullis will be in town to present a conference at our department, invited by Phil.  He will have lunch with graduate students, talk to faculty, and certainly fascinate us with his recollections of the discovery of PCR.   The talk is open to the public, so if you find yourself “in the neighborhood”, consider dropping by…    😉

NOTE ADDED AFTER PUBLICATION OF THIS POST:  Conference will be streamed live and open to the public, so if you want to listen to him, join us by clicking here (you can also watch it later, it will be saved on the site).

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I am so excited to finally meet him! I know it will be a great event for our department and a unique opportunity for graduate students to interact with someone who is not only brilliant, but is also not afraid to speak his own mind and to swim against the current, no matter how strong a current it is.

“The laws of science are demonstrable. They are not beliefs. When experiments in our century showed that Newton’s gravitational laws were not quite accurate, we changed the laws – despite Newton’s good name and holy grave in Cambridge. Relativity fits the facts better. This  is the way science has been done now for almost four centuries, and because of science – not religion or politics – even people like you and me can have possessions that only a hundred years ago kings would have gone to war to own. Scientific method should not be taken lightly”.  (Dancing Naked in the Mind Field, page 112).

ONE YEAR AGO: October 16: World Bread Day

TWO YEARS AGO: The US Listeria Outbreak 2011

THREE YEARS AGO: 36 Hour Sourdough Baguettes

FOUR YEARS AGO: October 16 is World Bread Day

THE US LISTERIA OUTBREAK, 2011

As some of the regular readers of this blog may know, our lab has been working on Listeria monocytogenes since 2003, studying how it transports iron from the environment (or from the human body) into the cell. After the recent outbreak involving cantaloupes from Colorado, I thought it would be worth writing about it.

First, how do bacteria contaminate a cantaloupe? Listeria normally exists  in the environment living on dead organic material in the soil (fallen leaves, rotting wood, dead plants … it’s called a saprophyte), and also in many different species of animals from birds to fish.  Indeed, some proportion (around 10%) of healthy humans have listeria in the intestinal tract, without causing any problems. Contamination of foodstuff happens in many ways. For instance, a worker involved in packaging fruits or handling equipment that harvests and packages fruits (or other foodstuff) may contaminate the produce if he or she has poor basic hygiene procedures.  But this source is quite limited in scope, and more likely, contamination results from fields that are treated with infected manure.

Where are the bacteria in the contaminated food? In the case of cantaloupes, bacteria can only infect the outside of the fruit.  Unless the husk or skin of the product is broken, the inside will remain sterile.   However, keep in mind that when  you cut the fruit open with a knife, you may transfer bacteria from the outside to the interior, edible part of the fruit.  One of the interesting characteristics of listeria, which is crucial to its contamination of food, is that unlike most bacteria it grows very well at low temperatures, like in the refrigerator (4 C). Therefore, if food is contaminated by this organism and refrigerated for later consumption, listeria will simply go on happily multiplying.  Another unfortunate characteristic of this nasty pathogen is that it has no odor, and so it doesn’t seem to affect the taste of the food. Some outbreaks in France were linked to chocolate milk, and people were drinking milk heavily contaminated by listeria without any noticeably bad flavor.

Can you get rid of Listeria by washing the fruit?
  Yes.  You can use dish-washing detergent and a soft brush, followed by a good rinse and blotting with a paper towel.  However, if  the fruit was bruised during transport, breaking the natural barrier provided by its skin, then the bacteria gain access to the fruit’s flesh.  Since refrigeration doesn’t slow its multiplication, in this case you are better off discarding any fruit that originated in the region suspected of contamination.

Why is it so hard to pinpoint the source of contamination? Most enteric bacterial pathogens, E.coli O157, Shigella dysenteria , and Salmonella typhimurium for examples, rapidly induce symptoms (within 12 to 24 hours) after ingestion of the contaminated food.   Listeria begs to differ.  It may take a couple of weeks, sometimes more, for symptoms to appear. You can imagine how tricky it becomes for epidemiologists to trace the origin of the outbreak.  Do you remember exactly what you ate 2 weeks ago, and where it was?  If you’re a food blogger  you might have a higher chance of answering yes to this question, but even then, it’s not easy.  😉  Also, the listerial incubation time differs from person to person, complicating the issue even further.

How dangerous is listeria anyway? It depends on who you are. Most healthy individuals will not even develop symptoms or become infected.  It takes a huge dose (about a billion) of bacteria to infect a healthy person.  However, the very young, the very old, pregnant women, and immunocompromised individuals (undergoing steroid treatments, HIV-infected) are at much higher high risk, because their immune systems are not up to the challenge. In France, where unpasteurized cheese is considered (as it should be) a delicacy, pregnant women are advised to avoid them because they are a source of listerial contamination.

What makes Listeria so deadly?  Once you eat contaminated food, the bacteria passes to the intestine, where it invades the epithelial cells,  white blood cells, and then reaches the bloodstream. It releases toxins, which make you sick with similar symptoms to those of other enteric pathogens like E.coli and Salmonella: fever, intestinal cramps, diarrhea, general discomfort. But that’s just the beginning. If your immune system can’t contain the bacterial growth, the strain has one more deadly trick up its sleeve: it can cross the delicate (and normally powerful) barrier between the blood and the brain. Once that happens, meningitis occurs, as well as other serious neurological problems like brain abscesses and paralysis.  Again, for the most part these problems don’t happen with healthy individuals, only those at high risk – young kids, aging people, pregnant women, and immunocompromised patients.

Can listerial infections be treated by antibiotics?  That’s the good side of this pathogen.  Most strains are sensitive to antibiotic treatment, so many  weapons are available to deal with it.  However, once the strain crosses the blood-brain barrier, antibiotics have difficulty clearing the infection, resulting in a high mortality & morbidity rate.

Some cool facts about listeria  Like many other species of bacteria, listeria can swim because they have little organelles called “flagella”  that propel them in the direction of food and other attractants.   However, it is not able to make flagella at the body temperature of mammals and humans – 37 C – so in our bodies, they cannot swim.   How do they move from cell to cell?  In a fantastic mechanism, that almost seems like the product of a science fiction movie director:  once inside our cells, the bacteria induces some of our own proteins to gather together  (in biochemical terms they  induce these proteins to “polymerize”) forming structures that act like jet propellers, and literally push each bacterium across the cell, making it go through the membrane and reach the neighbor cell.    In a classic cartoon depiction, here is what it looks like:

In this figure from Wikipedia (which originated from the laboratory of Dan Portnoy at UC Berkeley), we see in the outer edge images from electron microscope of the “real thing.”  In the center,  a cartoon depiction of what is taking place.  The bacterium is represented as a black, rod-shaped structure. InlA and InlB are two genes necessary for the initial invasion of epithelial cells.   Once inside the cell, the bacterium is briefly contained inside a little vesicle.  But listeria escapes this small “prison cell” by digesting the vesicle’s membrane with an enzyme called  listeriolysin (LLO).   It is then free inside the cell, and immediately starts the process of polymerizing actin (through the action of proteins like ActA), that act to propel the bacterium across the cell, allowing it to reach the cell adjacent to it.  And the process goes on, and on, and on…

What are we specifically working on?   For listeria to go on multiplying in our body, it needs iron.  It steals iron from us by several different mechanisms.  We are trying to understand what are the most important sources of iron, and how could we prevent listeria from using it.  By interfering with its iron uptake mechanism, we hope to prevent it from multiplying to a level that will cause disease.  Some of our published work can be found jumping here and here.

I hope you found this small overview helpful…

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“LIFE IS A MATTER OF TASTE…”

That was David Rosengarten’s closing line in his wonderful show “Taste,” years ago on FoodTV. Each episode centered around one dish, prepared by David while he talked about its origins, variations, and ultimately, his method to flawlessly reproduce it in your own kitchen. It was a simple format, but it delivered everything I expected from a cooking show. He almost always remained true to the classics. For instance, his shows on quiche Lorraine, cheese souffle and croissants were all based on recipes from Julia Child’s Mastering the Art of French Cooking. But sometimes he would wander off in new directions: his interesting show on “Compromise Clam Chowder” was an example of his gastronomic rebellion. After explaining the differences between the New England and Manhattan methods for chowder, he offered his own version, borrowing a little bit from each. Similarly, his macaroni and cheese included a few “extras” to make the dish less monotonous, and it was absolutely delicious.

Through his show I learned about many exotic dishes such as laarb, before I even had a chance to try it in a restaurant. And, I stopped twisting my nose at offal, because David Rosengarten’s recipes were perfect for any palate. From him I learned how to cook pork ribs in a stove-top smoker, mimicking as closely as possible the best ribs of Memphis, one of the homes of barbecue. I loved watching him prepare Brazilian moqueca, even if his pronunciation of the state of its origin (Bahia) was a little off ;-). His show was classy, informative and fun, and I was sad to see it end.

So, I guess you can imagine how I felt when I received an email from Daniel Boneville, who just produced a video with David, and sent me the link. If you’ve also been suffering from Rosengarten-withdrawal syndrome, allow me to ease your pain, and I cross my fingers that this is just the first of a very long series!

Click here for the one and only David Rosengarten, on a quest to re-create his favorite tomato sauce…

With many thanks to Daniel Boneville….

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PARIS, JE t’AIME…

When we finally took off for Paris our Boeing 777, the biggest and most comfortable of the jumbo jets, didn’t even notice the heavy rain and overcast enveloping Washington DC. And the happy couple it was transporting to the place they love so much didn’t’ mind the foul weather either. Clouds and rain, after all, are the norm in Parisian winter months, so we were ready for it. But, the initial day of such trips is the most difficult: an early morning arrival after a night on the plane with only a few hours of sleep, followed by the huge but requisite struggle to defeat jet lag. To reset our circadian clocks to the new schedule we walked outside as much as possible, and only saw the inside of our hotel room after night fell.

Having lived in Paris for several years we don’t visit many museums or tourist hot spots. “… Been there, done that,” from the Tour Eiffel to La Defense, from Montmartre to the Louvre, from Musee d’Orsay to Musee Salvador Dali, from cemetiere Pere Lachaise to the quartier Latin … Instead, what we love about Paris are the neighborhoods we used to call home. As we turn each corner we pass by each of our favorite cafes, brasseries, and boulangeries. We enjoy repeating our weekly Saturday afternoon walks, and that’s exactly what we did again yesterday… we departed our hotel close to Ecole Militaire and walked and walked, and walked some more….

We stopped at La Grande Epicerie de Paris, where I bought some “pimente d’espelette,” we strolled up rue de Rennes , then turned down to boulevard Montparnasse. We walked and walked some more, stood at the corner of Boulevards St Michel and St Germain, the gateway to Notre Dame, now so beautiful after its recent cleaning, and continued down to the cafe Les Deux Magots, which brings memories of dining outside at countless cafes throughout Paris, savoring the views of people passing by. Yesterday it was 40 F (3 C), but all the seats outside were filled by people from all over the world, each one living their own private love affair with Paris.

What’s for dinner? It was a simple meal at Le Bosquet, a classic brasserie two blocks from our hotel. The same waiter from several years ago handed us a menu that was almost unchanged. Why change oysters, confit de canard, and tarte Tatin? 😉

On a side note – If you haven’t seen the film Paris, Je t’Aime, I urge you to do so – it is a series of short stories by different directors, filmed in different Parisian neighborhoods. One of our favorites is the final vignette, the narrative (in broken, yet adorable French) of a lonely, middle-aged American woman who, after studying French for a few years, finally visits Paris for the first time in her life. Beautiful, touching, and a declaration of love for one of the most amazing cities in the world.

I feel the same way. It’s so nice to see it all again!

ANTIBIOTICS AND FOOD

If I were to define myself professionally, I’d say I am equal parts microbiologist, molecular biologist, and biochemist.  I obtained my doctorate in biochemistry in 1986, working on the genetic instability and antibiotic resistance of bacteria. Since then, I’ve studied bacterial pathogenesis, vaccine biotechnology, and more recently, the biochemistry of iron uptake by Escherichia coli and Listeria monocytogenes, two potentially lethal bacterial contaminants of food. As a result of these experiences, I have strong opinions about the use of antibiotics in food production.

Watching the news this week, I saw a pig farmer from my home state (Oklahoma) defending the practice of feeding pigs antibiotics to improve their health and weight.   I’d love nothing better than to invite that man over for a cup of coffee and a conversation, so I could ask him what exactly he knows about the use of antibiotics.  For example, has he ever heard of the term “plasmid?”  What about you?  If you care to read about it, then follow me to the next page….

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GOOD ARTICLE ALERT

From today’s food section of the Los Angeles Times, a very interesting article on the best recipes of the past 25 years.

To read the article, click here

I wonder how many of those will be on my endless list “to try soon”  😉

HAVE A WONDERFUL NEW YEAR’s EVE!