Preliminary functional annotation of O104:H4 genes/proteins by Era7 Bioinformatics

Just a quick follow-up to my last post... here is a pdf of a paper (link) from the Oh No Sequences group at Era7 Bioinformatics that lists the full functional annotation of O104:H4.  It represents an amazing amount of work and is a great reference for anyone studying the O104:H4 strain. Major kudos to the ONS group!!

Using O104:H4 EHEC data... an example

I've had a few requests for an example of how to work with the new EHEC data.  I agree it can be very overwhelming to have  hundreds of Megabytes of genomic data, so here is a fairly simple example of what one might do and what you might encounter.  Suppose you had a drug (antibody, peptide, small molecule) and you knew it hit a protein called EprK.  EprK is an approximately 250 amino acid protein that is part of the Type III Secretion System (T3SS).  The T3SS is the cell-surface protein complex that attaches the pathogenic bacteria to the host cells.  Blocking proteins like EprK is one possible way to prevent EHEC pathogens from attacking normal cells and causing disease. Your drug works on other EHEC strains (such as O157:H7, the strain responsible for the 2006 outbreak in the US) but will it work on O104:H4?  Testing it directly is the best way to know, but obtaining the new strain is likely to be very difficult.  Another option is to go to the sequence data. 

I went to one of the sites that has the new sequence information (based on 'crowdsourcing' from various labs) on O104:H4 (I used the oh no sequences blog -- the blog for the R&D section of era7 bioinformatics) and found the identifier code for the EprK protein (here's the link).  Some of the data has been annotated based on sequence homology and EprK was one that has been identified.  Using this code, I found the DNA sequence and copied it to the clipboard.  Then I went to the NCBI website (link) and pasted the DNA sequence into the search box to do a BLAST search of all microbial genomes that have been sequenced.  There were dozens of hits, and nearly all of them were EprK proteins from various strains.  I found the O157:H7 strain and the alignment is impressive.  More than 95% of the DNA bases are identical between the two, suggesting that the two proteins are very similar. I've included the BLAST results of my search below using O104:H4 EprK (Query, top strand) and it's alignment with O157:H7 EprK (bottom strand). So, your drug probably works on the new strain too.  If you want the amino acid sequence of the O104:H4 strain, simply take the DNA sequence to ExPaSy (link) and translate it.  It actually took me a bit to get the protein sequence because there is a frameshift mutation in the O104:H4 sequence read.  If you scroll down to my alignment and find the part highlighted in red, you will see there is an extra adenosine (an 'A' base) in the O104 sequence.  This throws off the protein translation.  I assume it is a mis-read in the O104 sequence (a common mistake when the sequencing machine reads through a string of the same base) and deleted it when I translated from DNA to protein.  The resulting amino acid sequence (pasted below) is very similar to EprK from other EHEC strains.  I'll double check this and follow up with them.

     Anyhow, I don't think there is a structure for the EprK protein, but if there was, you could use the existing structure as a model and make the amino acid changes seen in the O104:H4 strain to give you a decent starting point for the structure-based design of new drugs.

 Find a pathogenic protein of interest and try this yourself... it's not too hard.  When the topic of EHEC comes up at the next party, you can impress your friends by saying you blasted several virulence factors and found them to be quite similar/different from strains of previous outbreaks.  I would do this myself but, oddly enough, I don't get invited to parties anymore.  Anyhow, as a final disclaimer... although I have tried to be careful please verify anything I have posted before use.

Query  1       GTTGAGGATGAATATAACTAATTGGATCATATATAATCTTTCTTAGGGCAAGATTCATAA 

               |||||||||||||||||||||||||| |||||||||||||||||||||||||||||||||

Sbjct  443403  GTTGAGGATGAATATAACTAATTGGAGCATATATAATCTTTCTTAGGGCAAGATTCATAA 

Query  61      CGCTCTCATATGTCTACTTAATTTTCAACCTGACTAAATTAGTTAGAATGGCCCTATACT 

               || |||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct  443343  CGTTCTCATATGTCTACTTAATTTTCAACCTGACTAAATTAGTTAGAATGGCCCTATACT  443284

Query  121     TCCATAACAGCCAGCAAGTCGCTACGGATATTAATGCAAGTAAGATAGAAACCGGCATAG 

               ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct  443283  TCCATAACAGCCAGCAAGTCGCTACGGATATTAATGCAAGTAAGATAGAAACCGGCATAG  443224

Query  181     CCTTATCATAAGCAAAAACAGGTTCGCTAATTTCATATGTTGGTGCTTGCTCAATAATGT 

               ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct  443223  CCTTATCATAAGCAAAAACAGGTTCGCTAATTTCATATGTTGGTGCTTGCTCAATAATGT  443164

Query  241     CTCTTCGTTTTGACAATACAACAGAAATATTTTCATATTGTACGCTTGCAGAGCTATTAA 

               ||||||||||||||||||||||||||||||| |||||||||||||||||| | |||||||

Sbjct  443163  CTCTTCGTTTTGACAATACAACAGAAATATTCTCATATTGTACGCTTGCAAAACTATTAA  443104

Query  301     CAATAAATCTCTTGATATCATTTATTTTTATTTCTGGGTTGATATCTTTTTCATATACTG 

               ||||||||||||| || |||||||||||||||||||| ||||||||||||||||||||||

Sbjct  443103  CAATAAATCTCTTTATGTCATTTATTTTTATTTCTGGATTGATATCTTTTTCATATACTG  443044

Query  361     CAAGTACAGAAATATGAATTGGTAAAGCAGTTTTACCACTATCGCCATTATCAACATCGT 

               ||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||

Sbjct  443043  CAAGTACAGAAATATGAATTGGTAAAGCAGTTTTACCACTATCGCCAGTATCAACATCGT  442984

Query  421     AACTAACATGTACTCTCGAAGAAATAATGCCATCCATAATTTTGAGAGATTGCTCTAACC 

               |||||||||||||||||||||||| ||| |||||||||||||||||||||||||||||||

Sbjct  442983  AACTAACATGTACTCTCGAAGAAACAATACCATCCATAATTTTGAGAGATTGCTCTAACC  442924

Query  481     GCTGCTCAATAGCAGAATATAGCCTTGCTTTTTCCGCTCGTGGAGATGAAAACGAGTGCA 

               ||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||

Sbjct  442923  GCTGCTCAATAGCAGAATATAGCCTTGCTTTTTCCGCTCGTGGAGATGAAA-CGAGTGCA  442865

Query  541     TCTGCAGGGAACATCTGCGATATTTGAATATCAGGCTTACCCGGTAGATTGTAGATTTTT 

               |||||||||||||||||||||||||||||||||||||||||||| |||||||||||||||

Sbjct  442864  TCTGCAGGGAACATCTGCGATATTTGAATATCAGGCTTACCCGGGAGATTGTAGATTTTT  442805

Query  601     AGCCAATCCACCGCAGAAGCAAAATCCGTTGGTTCGACAAATATTGAAAATCCTGTTTTG 

               ||||||||||||||||||||||||||||||||||| ||| | ||||| ||||| ||||||

Sbjct  442804  AGCCAATCCACCGCAGAAGCAAAATCCGTTGGTTCAACATAGATTGAGAATCCAGTTTTG  442745

Query  661     CCTTGATCCTTCTTTTCAGCATTAATATTATGTCTTTGTAAAACAGCAAGGACATCATTA 

               ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct  442744  CCTTGATCCTTCTTTTCAGCATTAATATTATGTCTTTGTAAAACAGCAAGGACATCATTA  442685

Query  721     GCTTGCTGTTGATCAAGATGGTTCAATAATTCCTGCTGCTTGCAGCCGCACAACAGCAGG 

               ||||||||||||||||||||||||| ||||||||||||||||||||||||||||||||||

Sbjct  442684  GCTTGCTGTTGATCAAGATGGTTCAGTAATTCCTGCTGCTTGCAGCCGCACAACAGCAGG  442625

Query  781     ATAAACAATAATA  793

               |||||||||||||

Sbjct  442624  ATAAACAATAATA  442612


Predicted amino acid sequence for O104:H4 EprK protein, (corrected for gap): 

L L F I L L L C G C K Q Q E L L N H L D Q Q Q A N D V L A V L Q R H N I N A E K K D Q G K T G F S I F V E P T D F A S A V D W L K I Y N L P G K P D I Q I S Q M F P A D A L V S S P R A E K A R L Y S A I E Q R L E Q S L K I M D G I I S S R V H V S Y D V D N G D S G K T A L P I H I S V L A V Y E K D I N P E I K I N D I K R F I V N S S A S V Q Y E N I S V V L S K R R D I I E Q A P T Y E I S E P V F A Y D K A M P V S I L L A L I S V A T C W L L W K Y R A I L T N L V R L K I K

Rapid characterization of the EHEC outbreak by “crowdsourcing”

Now we’ll be moving from papayas and fish to something a bit more sinister: EHEC O104:H4.  That is the name of the E. coli responsible for the recent German outbreak.  When a new outbreak begins sickening patients, researchers all around the world are mobilized to try and characterize the pathogen.  Since new strains often have similarity to well-understood strains, one of the critical first steps is to sequence parts the genome.  When SARS was first flaring up in Asia, I worked for a sizable biotech company focused on developing drugs for viral diseases.  Very early data suggested that the SARS virus may have a similar pathogenesis to related coronaviruses, particularly with regard to viral entry.  However, we couldn’t design drugs to combat SARS until we had the DNA sequence for that part of the genome.  Once that became available, I used our in-house analysis software to design the initial set of lead drugs and we were off and running.

                Sequencing the entire genome is extremely time consuming, but BGI (formally known as the Beijing Genomics Institute) is utilizing ‘crowdsourcing’ to help assemble the EHEC genome faster (here’s the press release).  Using open source software, Twitter feeds (@BGI_Events), and several sites for uploading data, they hope to pull together data from researchers around the world in an organized, efficient manner.   Here’s the bioproject link for this work at NCBI (link).  This exchange of data is great for biopunks because one can analyze the data almost in real time and there is a significant potential for finding interesting and important aspects of the EHEC strain, based on sequence similarities/differences with other strains.  Mike the Mad Biologist had a blog post a couple days ago that offers a glimpse of the type of analysis people are doing (link).  The more eyes there are on the data, the quicker the strain can be characterized and as I have mentioned before, the potential of using ‘citizen scientists’ or ‘crowdsourcing’ for efforts of this type are enormous.  With the advent of rapidly accessible data, and the power of on-line DNA analysis tools, the gap between the scientist and everybody else has never been smaller.

Using DNA 'barcodes' to combat fish fraud



Snapper fillets



Now that Memorial Day has passed, we tend to do a lot more grilling here in the Dark Lab.  The weather here in SoCal falls into a predictable perfection and any given evening is perfect for throwing something on the grill.  So, I head out to my local grocery store and look for a nice fish… snapper maybe?  Looking at the package, it’s definitely a fish but is it really snapper?  I can’t tell.  In fact, studies show that up to 70% of fish sold as snapper is actually something else.  The FDA tries to monitor fish but they are probably more focused on safety rather than accuracy.  However, there has been a lot of press lately about mislabeling of fish.  Last week, the New York Times ran an article (link) with some shocking statistics about how frequently fish are mislabeled.  According to a report by the non-profit group Oceana (2.3 Mb pdf here), for every three packs of fish you buy, one of them will be wrong. 

Oceana references a number of scientific studies, including a paper by Wong and Hanner (abstract), who use a PCR-based approach to analyze the DNA sequences of fish in the marketplace.  They found that some substitutions are obvious fraud.  For example, fish labeled as red snapper (sold at $3 per pound) was actually redfish (that would cost 72 cents a pound).  Fish labeled as white tuna sushi was actually tilapia.  These are flagrant mistakes, and it is not at all clear whether this is done on purpose or is the product of the complex network of processors and middle-men that are required to bring a fish out of the sea and to your dinner plate.  However, some mistakes are less apparent… for example, Atlantic halibut was labeled as Pacific halibut.  No big deal, right?  What if you knew that Atlantic halibut was endangered?  Would you still buy it? This type of mislabeling suggests some fishermen may be catching more than their quota of threatened or endangered fish and packaging them as something else.  Another recent article goes into more detail about the social and financial implications of fish fraud (abstract).

Want to know what fish you are buying?  It’s a great DIYbio project.  If your hackerspace has the ability to do DNA sequencing (or you can send sequencing samples via the hack shack) then checking your fish can be pretty easy.  You will probably want to sequence several spots in the genome and will need sequencing primers for each (which are cheap and easy to design).  Once you have the DNA sequence from your fish, you can use an online tool called Blast (link) to search the genome database for your sequence and it will tell you what species it is from.  If you already know the sequence (from the primer design, for example), then you can simply align the correct sequence with your fish’s DNA and see if you get a perfect match.  This method will give you a pretty good idea if you have the right fish as long as there are differences in the DNA sequence between the various species.  Sometimes, they can be very similar.

If you have access to a hackerspace with a PCR machine (and the reagents!) and a way to run an agarose (DNA) gel, there are several other options.  You can do an AFLP analysis, which is a very sensitive way to look for polymorphisms (changes) in DNA.  A recent paper by Maldini (abstract) outlines the approach and applies it to identifying fish.  They claim that even closely related fish can be identified.  Another PCR-based option is to amplify a gene using a species-specific primer.  In this case, you see good amplification (ie, a band on a gel) only when the DNA of that species is present.  Two advantages with the PCR approach are that you don’t need much DNA and it doesn’t need to be all that pure (both are big advantages for the biohacker). One thing you will need is a set of PCR primers for the species of fish you are buying.  I hope that someday these will also be readily available in any decent hackerspace, but until then, you will have to get them yourself.  The Wong and Hammer paper has some primers listed and primers for key genes from the most common types of market fish are freely available on the internet.  If they can’t be found directly, you can also design them from the fish’s genome.  Genbank (link) has some of this information but another good source is the website for the Fish Barcode of Life (link).  This great organization is trying to catalog all fish, including those we eat.  Eventually, they will have links to the genome of every fish so you can use that for primer design.  As an added DIYbio bonus, they are also looking for additional data from people like YOU!  Not with the DNA sequencing, but with the development of range maps that indicate where the different species of fish are found.  This is a great opportunity for all you fishermen out there (go here to see how you can report a sighting).  It’s also a way for biopunks to make important contributions to this effort while doing a little home-based food surveillance. 

So, did you notice?  The snapper picture is mislabeled... it's actually tilapia. At least you didn't pay 10 bucks for this blog post.

Biopunks in USA Today

There’s a pretty nice blurb on the DIYbio movement in today’s USA Today.  It covers the basics and highlights a few of the controversies but doesn’t describe many practical applications.  It touches on DNA sequencing and open access to lab equipment and basic molecular biology tools (expression vectors and strains, common reagents, etc) and hints at one cool application (the blue yogurt).  Nice plug for BioCurious (a hackerspace in the Bay area) and the OpenPCR machine.  They even talk about the risk of making “unstoppable Franken-microbes”.  Not that we would ever do such a thing in the Dark Lab, but I do know of three fictional teen biopunks who had a basement experiment go horribly wrong…

You can read the USA Today article here but you’ll have to wait awhile to read what happened to the teens.

More winemaking magic

A few posts ago, I described the outstanding lecture by Kerith Overstreet of Bruliam Wines on the biochemistry of wine (link). Although I tried to capture the essence of her talk, there is now a recording available including her slides. If you are interested in the topic but kept thinking ‘WTF is this guy talking about’, I recommend you hear it straight from the source. Here is the link to the audio.

Bruliam Wines also has a blog and today’s post (here’s the link) has a great discussion of how sulfur dioxide is used in wines. Added as potassium metabisulfite, the acidic wine converts it into sulfur dioxide (as well as bisulfite and the conjugate base, sulfite). Each of these reducing agents has a role in protecting or preserving the wine and just like anything else, too much of a good thing is bad. Finding the right balance so that the wine is free of bacteria, protected from oxidation over the long term, but does not lose its color or subtle flavors is yet another example of the magic of winemaking. Enjoy!

What's in YOUR papaya?



Papaya: The fruit with it's
own symposium



Some people out there are concerned over whether the food they are eating has been genetically modified (GM).  We don’t worry much about that in the Lab since there is nothing even remotely genetic about Twinkies or Mountain Dew.  Still, I mentioned before that a hackerspace was a great place to do some food surveillance.  During my research into traditional methods (PCR detection, etc), I ran across an article published a few years ago that described a really simple method to test for GM Papayas.  If you are a big papaya eater, this could be important, but the method could also be more general.

Papaya are susceptible to a nasty virus called Papaya Ringspot Virus (PRSV) which creates havoc in the fields.  Long ago, scientists found that by introducing a gene for the coat protein of this virus into the papaya genome, they could block the virus from attacking the plant.  However, another gene comes along for the ride – namely, a gene used for genetic selection in the lab.  When scientists introduce a gene into a plant cell, they often use a reporter to show them which clones successfully received the gene and which do not.  This marker is an enzyme called beta-glucuronidase (GUS).  What makes this such a good indicator of a GM papaya is that non-transgenic papaya do not have this enzyme, so if you can detect this in your fruit, it has to be transgenic.  One way to this, of course, is to use DNA primers specific for GUS and use PCR to try and amplify the DNA specific for the enzyme.  You could also try to sequence the DNA extracted from the papaya and look for the sequence for GUS (or any other inserted gene, such those for the PRSV coat proteins).  The complete genome of one type of GM papaya (the SunUp variety) was published in 2008 (Nature abstract) and is freely available at GenBank (link).  Others have also been subsequently published.



X-Gluc: A GUS substrate



Fortunately, for papaya lovers, there is an easier way.  5-bromo-4-chloro-3-indoyl glucuronide (X-Gluc) is a substrate for GUS and gets converted to glucuronic acid and a precipitate which happens to be blue.  Therefore, a very easy way to look for the presence of the enzyme is to screen directly with X-Gluc.  You don’t even have to purify DNA!  Simply take the seeds from the fruit of interest and smash them up really well.  Then incubate them in the presence of X-Gluc for about a day and if you see blue, you’ve got a GM papaya!  Here is a link to a recent article that describes the general idea (abstract).  In principle, if your hackerspace has access to X-Gluc, testing for the presence of GM papaya is extremely easy.  Scientists have used this same method to track cross-pollination between GM plants and non-transgenic neighbors.  One example was reported at the International Symposium on Papaya (abstract).  Yes, a papaya conference… and I thought I got a lot of grief for doing phage display.  Anyhow, I’ll have another post or two about this type of food monitoring and pretty soon you’ll be just like the USDA – only without the bureaucracy and bad jokes.

Be a Martian tourist!!

Wanna really get away?  Go to Mars!  You could use a commercial, deep-space rocket and travel for months in cramped quarters with a bunch of other tourists.  (Imagine getting on a plane for a 4000 hour, non-stop flight with all those screaming kids… better be lots of free booze on that flight!)  Or, you could take your touristy pictures from the comfort of home!  Check out the High Resolution Imaging Science Experiment (HiRISE).  It’s run out of the University of Arizona in conjunction with NASA and is focused on using high resolution images of Mars to study the weather, geology, topology, tectonics, and climate change.  Scientists have been using this data for years to develop models for these processes but Mars is a pretty big planet and the number of interesting sites to study is pretty vast.  I mean, we haven’t finished exploring Earth yet!  So, there is an opportunity for us Dark Lab types to dig into this treasure trove of data.  Much like the fuzzy blob site (blobs post), you can sift through pictures and highlight interesting or unusual features.  But they also take it one step further… with HiWish (link) you can actually suggest (using a Google-Mars like platform) Martian features to be photographed.  If selected, the Mars Reconnaissance Orbiter will be sent commands to zoom in on your target and take high resolution images.  Amazingly cool!  Many of the gallery pictures are really cool.  The image above is a shot of a very recent (like, within the last year) meteorite strike.  Other examples of interesting features would be odd canyons, rock formations, and that little green man with the Death Blaster.  Apparently, Martians don’t like being peeped at with telescopes.

Hack-shacks, biopunks, and the next revolution

Imagine this is 1976 (try to block out the bell-bottoms and disco music), and I wrote a blog about a couple of pimple-faced teens who had built this swell micro-computer in their garage.  They put the thing in a box and bring it to a local do-it-yourself computing club where a bunch of other computer junkies say things like “that’s totally rad, man”.  Yup, they’re computer hackers and two guys like this went on to start Apple Computer.  For all of us, life was never the same again.  Now fast-forward 35 years. 

The iPhone's great-great-grandad

The DIY biotechnology (ie, biohacking) movement has been growing for a few years but is still very much in its infancy. I remember going to a local community college with my dad and seeing him load the punched card machine to get a computer the size of a room do a routine calculation.  I could probably do that on my cell phone now.  The concept of a world wide web with a blogging program that could transmit my drivel around the world instantly was so far beyond what was possible at that time.  We are currently in the “computers as big as a room” phase with biohacking.  Doing projects is cumbersome.  Some ideas are still beyond the scope of current technology.  But it will not be long before the biological equivalent of the PC will be developed and it will rock the world.

Will biopunks cure cancer? Unlikely. But the discovery of new genetic targets for attacking cancer or infectious diseases is a good niche for garage drug hunters.  Most of the protein engineering work I do could be done at home with free software on the web, so the theoretical design of biologic drugs is also likely within reach.  Another likely application is the development of diagnostic kits.  With the big push into personalized medicine, I see huge opportunities there.  A biohacker can search the literature for biomarkers for a particular disease, or even try to find them on their own.  A few proof-of-concept experiments done in a well-equipped hack-shack (more on these in a future post) and you could have a prototype diagnostic kit.  Would you spend a couple hundred bucks for a toaster-sized cube that monitors the detailed health profile of your family?  I would.  Exposure to a cold virus? You know it on Day 1. Infection brewing?  You know about it before it even gets sore, plus you also know what type of infection you have and the resistance profile.  Want to make sure you’re your food is all-natural and pathogen-free?  No problem.  Monitor metabolic pathways for signs of imbalance (very early indicators of a number of diseases)?  We need to learn more about these pathways, but in principle this is also very possible.  A primitive version of this type of device could probably be made today.

The OpenPCR machine: The great-great grandad of ????

How about less practical gadgets like the iPod?  Designer plants?  Plants with leaves that glow in the dark?  Well within reach.  Carrots that taste like cotton candy? Probably doable.  How about genetic genealogy?  How fun would it be to try and find DNA samples from long-lost relatives (hair strands?) and map genetic contributions from them.  Kids might even be into collecting genes.  Pick up a leaf or a feather or a dead bug and process the DNA.  Compare genes, find rare and unusual enzymes, make trading cards.  It could be bigger than Pokemon!  Ok, maybe not, but still, somebody out there is going develop these things and the next Apple Computer is going to be born.  Check out the OpenPCR that is being developed by a couple of early biohackers (link)… could this be Apple 1?

Someday I’ll be drinking a glass of cab and telling my granddaughter that back in the old days, I had a whole group of scientists with a lab full of expensive equipment trying to identify and target genes. And it took years. Blah, blah, blah… and she’ll wander off and play with the genetically engineered mouse she made from a kit under the soft light of a bioluminescent tree.

Find the bubbles and fuzzy red blobs

Wow~ bubbles, green knots, red blobs

I mentioned in an earlier post that it had never been a better time to participate in exciting research projects.  Unfortunately, this no longer involves injections of semi-purified plant extracts in an effort ‘to see what happens’.  Today, you can participate in important research efforts in the comfort (and safety!) of your own home.  The common term for this is ‘citizen science’, which is a (dorky) term I hate.  The research doesn’t have to be dorky.  NASA Science News just highlighted one such effort called The Milky Way Project (link) where volunteers can contribute to the analysis of deep sky pictures obtained by the Spitzer space telescope.  After a free sign up, you view images taken by this instrument (many of them never before seen by a human) and identify interesting features in the picture.  One particular project is looking for ‘bubbles’ in interstellar gas.  A simple tutorial explains how to identify and mark the images, which are then submitted for further analysis.  What is so great about this is that anybody can do it!  I’ve ‘bubbled’ quite a few of these images and I can tell you, some of them are truly breath-taking.  The process reminds me of those gem mining attractions in the mountains of North Carolina (I’m sure they’re everywhere).  You get a bag of dirt and sift through it in a slough.  Most bags contain a number of small emeralds or rubies, but every once in awhile you find a honker.  That’s the scientific term for a big-ass emerald.  These images are much the same.  Some are pretty dull, some have a number of interesting features, and every so often you find a honker.  All of these ‘bubbled’ pictures are then fed back through the image analysis software with the ultimate goal of figuring out what the bubbles are, how they are formed, what purpose they serve in star formation. 

Two UFRBs (unidentified fuzzy red blobs)

Bubbles are boring?  You also learn to identify green knots of gas, which are unknown objects that scientists are very interested in learning more about.  These are a fairly new discovery as are the so-called ‘fuzzy red objects’,  which is the technical term for ‘WTF is that red blob’.  Somewhere there are happy graduate students who no longer have to stare at these images 24/7 until they start drawing bubbles in the freckle patterns of their significant others.  What’s in it for you?  Aside from the opportunity to contribute to research on the cutting edge of astronomy, there is also a chance to discover something totally new.  Something unknown to mankind.  Something which may contribute to a major breakthrough in the origin of the universe.  New data comes in from these instruments on a regular basis, so get off Facebook, pull up a chair next to your favorite astrophysicist and stare into the unknown.  I find that a good cabernet goes well with the fuzzy red blobs.

Winemaking: Science or Magic?

Red wine necklace --
 (from madewithmolecules.com)

Last night I attended a networking event advertised through the San Diego Biotech Network and sponsored by PGC200.org (link).  The topic of discussion?  The Biotechnology of Enology (Oenology for you Brits).   Some days, when experiments are particularly uncooperative, I wonder what it would be like to own a winery.  Seems like a good fit since I like wine, I like science, and I like growing things.  However, I have always been intrigued by how different wines can taste even though they are made from the same grape.  There is also huge variability in different vintages from the same winery.  And unlike beer, if you let wine sit in a bottle for a few years, it can become extraordinary.   How does this happen?  I mean, how hard can it be?  Pick some grapes, stomp them with your feet, pour the shit in a vat and wait a couple years.  Seriously.  The grapes come from the same plant each year so their genetic makeup is the same.  The winemaker knows how to prepare the grapes, add the yeast, control the temperature, uses the same bins, etc.  Budweiser can make the same beer year after year… why can’t the winemakers?

Reading the abstract for the talk (biochemical pathways, genetics, chemical reactions, blah, blah, blah) I figured science must have the answers to this.  Then I saw the bit about wine tastings and immediately signed myself up.  I’ve been to a ton of scientific conferences and let me tell you, almost any mind-numbing talk can be improved with a glass or two of wine.   The speaker was Kerith Overstreet, co-founder and CSO of Bruliam Wines (link)(Bruliam sounds like something off the periodic table, but is actually a portmanteau of her children’s names).   Her talk was nothing short of fantastic.  Life-altering, in fact.   She was an MD (pathology) before switching careers to winemaking and had a sense of humor as dry as a good cabernet.  We gathered in a beautifully modern conference room and during the reception, we admired the views of the surrounding mini-mountains.  She started us off with the 2009 Hayley Pinot Noir.  It was light and pleasant (she described it as feminine).  Lucy, my partner in crime on this trip, described it as kind of bitchy.  Not sure what that means but she knows a hell of lot more about wine than I, so I just nodded knowingly.  Maybe it had too much whine.  (Thank you, thank you.  I’m here all week.) Next up was the 2009 Doctor’s Vineyard Pinot Noir.  This was from the Monterey area (Santa Lucia Highlands) and we both agreed it was really nice.  Bruliam Wines has only been operational for three years and even I can tell that these wines kick ass.  After a couple glasses of wine and some great food, we were all ready for a nap – I mean, the talk.

(The scientifically squeamish may want to sit down now.  Or skip ahead a few paragraphs.)

Kerith started us off gently, talking about how the root stocks were different from the vines and that the two are actually selected independently, like picking out a top and a bottom.  Then she went into fermentation like a bat out of biochemical hell.  Early on, it’s all about the glucose pathway.  The added yeast converts sugars to ethanol and the genetic composition of the yeast determines the speed and efficiency of the process.    Alcohol is toxic to yeast, so there is a beautiful biochemical feedback loop at this stage.  As the yeast becomes stressed, pathways are activated (and are up-regulated by the dropping sugar concentrations) that release fatty acids, sterols, and other chemoprotectants (let’s call this ‘the good stuff’).  During the fermentation process, oxygen is bad – unless you are trying to make vinegar -- however, there is always a little bit in there and so some of ‘the good stuff’ gets oxidized to make “magical stuff” (ie, tannins).  These are my scientific terms, not Kerith’s.  You know when a girl gives you that “come hither” look?  That’s the female equivalent of a tannin.

Pretty simple so far, right?  Just control the reaction and you get your wine.  Not quite.  After the yeast is done, you then have to add bacteria for the second fermentation.   This step stabilizes the wine by converting nasty biochemical byproducts to more storage-friendly chemicals.  In the case of red wine, this is also where the magic occurs.  As the sugar level is crashing and the yeast are starting to die, you sprinkle bacteria ‘like fairy dust’ into the fermenting goo.  Choice of strain is again critically important (homofermenters, heterofermenters, malolactic strains, zzzzz) but whatever you add activates different biochemical pathways which, in turn, gives you different levels of “good stuff” and “magical stuff”.  Timing and balance is everything.   You can’t let the yeast or the bacteria over-process the biochemical byproducts or you wind up with wine that is “all alcohol and no sex”.   I’m calling these chemicals “magic” because honestly, people don’t know what most of these chemicals are or why they are important.  But they are mission-critical for body, flavor, mouthfeel, and aging, particularly in red wine.  These magic tannins are the key to turning a good bottle of cab into a mind-blowing experience.  How does that work?

Catechin - one of the magic molecules

One wine characteristic I know well is the dry, leathery feel of a cabernet.  It makes your mouth feel weird and some really young cabs are like drinking liquid desert.  The a-ha moment in Kerith’s talk came at the end when she was talking about aging.  When a cab is young, there are a lot of tannins in there and the taste can be very harsh.  That sensation in your mouth is because tannins bind very strongly with proteins in spit, so they cling to the sides of your mouth.  One of the magic tannins is from the flavin-3-ol subgroup  of flavinoids (not to be confused with the famous rapper) and is commonly known as catechin.  Catechin is a small chemical (see picture) but when it gets oxidized, it can form a dimer.  Dimers can be oxidized to form trimers, and so on.  As the chemical polymerizes, it has a harder time binding to the proteins in spit.  So as the wine ages, it seems “softer” or more mellow.  Other phenols and flavinoids can also be oxidized and combine to form very exotic chemicals that cause changes in the character of the flavor (more chocolate, less berry, hint of woodsmoke, etc).  I can’t even begin to comprehend how many different combinations of chemicals you could create inside that bottle.  You might be wondering how this happens -- I said that oxidation was bad (vinegar) and oxygen levels are low during fermentation.  Where does this tiny bit of oxygen come from?  The cork.  The OTR (oxygen transmission rate) of the cork results in micro—oxygenation of the wine.  These magic tannins are basically buffering the wine from a slow oxygen leak, protecting it from the bad types of oxidation that lead to enological disaster and creating tasty flavinoids in the process.  The more tannins in the wine, the longer it can be stored and potentially the better it gets.  Wine is truly an amazing witches’ brew! 

  So, is winemaking driven by science or magic?  If it’s science, is sure isn’t governed by classical mechanics.  There is little to no predictive power and the system is far too complex to control.  Even comparing it to quantum mechanics is a stretch.  Every batch that is set up will come out different because at the end, the distribution of tannins (type and concentration) cannot be predicted.  It will taste generally like a cabernet, but the specific taste will be different from batch to batch and sometimes even bottle to bottle.   Since there are multiple biochemical pathways involved, each with interdependent feedback loops, and the final output is so exquisitely dependent on the initial conditions, I wonder if the process is actually chaotic.  What if winemaking was described by something like a Lorenz attractor?  These non-linear dynamical systems are characterized by being globally deterministic (it tastes like a cab) but locally unpredictable (I taste bell peppers!).  The weather is a good example as is the stock market.  This means that good winemaking will likely come from empirical observations, intuition, and a little luck.  I do wonder if someday scientists will understand which tannins correspond to what taste or sensation.  One could then make synthetic wines using a simplistic fermentation process followed by the addition of supplemental tannins to create the right body.  But somehow, I don’t think that scientists will ever be able to, um, convincingly fake it.

The final bottle of the night was a 2009 Rockpile Rocky Ridge Zinfandel – “a zin for cab lovers”.  The description said it was “the perfect yin-yang of dark berries and savory spices” with “spectacular structure, layered complexity, and balance”.  Blah, blah… whatever.  This stuff was orgasmic. I really didn’t care what the hell was in there.   If that’s what a really good Zinfandel tastes like, I might be a believer.  So I poured myself another glass, let it open up a bit, and chalked one up for magic.