Dorothy Hodgkin: From Nobel Laureate to Google Doodle

http://www.google.com/doodles/dorothy-hodgkins-104th-birthday

The first time I heard of Dorothy Hodgkin was last May when Google featured her model of Penicillin as its doodle. Side note: I actually didn't see it myself, but was lucky enough to have a mother-in-law who was interested in my science and pointed it out to me. Having completed my dissertation in X-ray crystallography, I was surprised to not have heard of her before: the third woman to win the Nobel Prize in Chemistry for “her determinations by X-ray techniques of the structures of important biochemical substances (1).” The two other women were of course Marie Curie, and unbeknownst to me, her daughter, Irène Joliot-Curie. Dorothy Hodgkin determined the X-ray structures for important biomolecules, such as penicillin and Vitamin B12, as well as making her mark on protein crystallography. Her structure of Vitamin B12 allowed for the synthesis of the vitamin to treat megaloblastic anemia. I’ve written a separate post describing the field of X-ray crystallography in more detail here.

 Structure of Vitamin B12, one of the most complex structures that  had been determined by 

X-ray Crystallography at the time.  Image courtesy of chemicalheritage.org

It was apparent from my readings into her life that Dorothy Hodgkin is an example of the type of scientist I always hoped to be: committed, passionate, and steadfast about her work and its implications, well rounded and diverse in her interests, humble, yet confident in what she wanted in her life from an early age. Of course, sexism is not hard to find in her story. The headlines when she won the Nobel Prize in 1964 was "British woman wins Nobel Prize – £18,750 prize to mother of three." in the Telegraph and "Oxford housewife wins Nobel" in The Daily Mail. But she seemed to rise above it all.

Her father taught and worked in archaeology in Egypt, the Sudan, and Jerusalem and Dorothy would visit at different times in her life (2). She carried a love of Africa throughout her life, later making many trips to Ghana while her husband worked at the University of Ghana, and even for a time as advisor to the Ghanaian president (2,4). Eventually, her daughter would teach in Zambia (2).

Dorothy’s life is rife with the stuff of good dramas. According to a 1998 biography, her very influentional advisor, J.D. Bernal and she were lovers on and off for years, even after she married (3). The politics of this advisor, as well as her husband’s were of a communist persuasion and she was banned from travelling to the U.S. without a CIA waiver (4). It is unclear if she held the same political views but after being refused entry to the U.S. to attend a conference organized by Linus Pauling, she travelled to the Soviet Union, attempting to set up scientific collaborations. She also visited China many times throughout her career and had many collaborations with Chinese and Indian scientists. She campaigned for inclusion of countries into the International Union of Crystallography such as China and the Soviet Union, who were banned for political reasons (4).

Dorothy believed it was the responsibility of scientists to help make the world a better place. She was president of the Pugwash council from the mid 1970s to late 1980s (4). Never heard of the Pugwash conferences? Neither had I. Pugwash is an international organization established in the years following WWII, bringing together scholars with the goal towards international conflict resolution and nuclear and chemical weapon disarmament.

Dorothy Hodgkin, image courtesy of chemicalheritage.org

What drew me to her, more than her politics though was her dedication to solving a particular problem. She studied one protein, insulin for more than 35 years, eventually solving the structure. In her Nobel Lecture she recounts a sentence from the W.H. Braggs book “Concerning the Nature of Things” which peaked her interest in the field as a teenager, “”Broadly speaking, the discovery of X-rays has increased the keenness of our vision over ten thousand times and we can now ‘see’ the individual atoms and molecules.”” A few sentences later she writes, “The process of 'seeing' with X-rays was clearly more difficult to apply to such systems than my early reading of Bragg had suggested; it was with some hesitation that I began my first piece of research work with H. M. Powell…(5).” Four years after giving this lecture she finally determined the structure. When she originally set out in the 1930s, the technology and methodology was not advanced enough to study proteins. She continued to.. Her structure contributed to the knowledge of receptor/hormone binding and allowed for reliable synthesis of the hormone as a treatment for diabetes. 

This part of her story resonated with me as I remember seeing the beautiful pictures of protein structures in late bachelor studies and trying to wrap my mind around how one came to these pictures. Indeed, I would discover it was more complex than even I imagined. And I was lucky to benefit from all the amazing advances that semi-automate the steps along the way from setting up hundreds of crystal conditions (or in my case, thousands) to shooting the crystals with X-rays in California while sitting in my house in Baltimore to solving the structure using information gained by other crystallographers on similar proteins. Even with these advances, it took three and a half years to determine the structure of my one and only protein, Plasmodium falciparum Atg8.

It was with some sadness that I left the crystallography world behind, with its helpful and always willing to commiserate, yet highly critical, compatriots. My experiences make me appreciate all the more the contributions that Dorothy made in the then male-dominated and inchoate field of biomolecular and protein X-ray crystallography.

References:

1. (2014). Dorothy Crowfoot Hodgkin - Facts. Nobelprize.org. Nobel Media.

2. (2014). Dorothy Crowfoot Hodgkin – Biography. Nobelprize.org. Nobel Media AB 2014. Retrieved August 24, 2014. Retrieved from http://www.nobelprize.org/nobel_prizes/chemistry/laureates/1964/hodgkin-bio.html.

3. Ferry, Georgina (1998). Dorothy Hodgkin: A Life. London: Granta Books.

4. Howard, J.A.K. (2003). Dorothy Hodgkin and her contributions to biochemistry. Nat Rev MCB. 4, 891-896.

5. Hodgkin D.C. (1964). The X-ray Analysis of complicated molecules. Nobel Lectures. Chemistry 1963-1970, Elsevier Publishing Company, Amsterdam, 1972.

A quick introduction into protein X-ray crystallography:

What information do we get from X-ray crystallography?

Proteins, which are the machines inside our cells that use energy to do countless tasks, such as copy our DNA for the next cell, cause our muscles to contract, bind hormones, allow our neurons to release hormones and neurotransmitters. It is vastly helpful to understand what proteins look like, their 3-dimensional structure, to understand how they work. For example, take the protein, DNA helicase. In order for our genome to be passed on to newly-divided cells or our progeny (in the form of sperm and egg cells), it must be copied. In order to be copied, it must be temporarily pulled apart from its more stable double-stranded form, to a single stranded-form where the information is accessible. DNA helicase is a motor that uses energy from ATP to unwind the DNA. In the image below, you can see that the structure of one molecule of the protein, which alone doesn’t really explain its function. But the protein binds five other identical helicase molecules to form a donut-shape, which encircles one strand of the DNA. The motor then drives the helicase forward, pulling the two strands apart by shear force.

 

 (PDB code: 2R5U) (http://www.pdb.org/pdb/explore/explore.do?structureId=2R5U)

This crystal structure is specifically of the helicase protein from the tuberculosis bacteria. Knowing the structure allows scientists to design drugs that bind the protein in the bacteria, but not in humans, and to prevent the bacteria from replicating.

How do we get this information?

Proteins are extremely small, too small to see with the human eye and the structure is too small to see with a microscope. But if we can get the protein to crystallize, forming a regularly repeating structure that is much bigger than the single protein alone, we amplify the signal-to-noise ratio. X-rays are then shot at the crystal. When the X-rays hit the atoms that make up the protein, they are diffracted, that is, the direction and intensity change. The pattern of diffraction provides information about the position of the atoms in the protein and the diffraction pattern is captured by a special camera device behind the protein. Of course, back when Dorothy Hodgkin was solving the structures of penicillin and Vitamin B12, there was no camera, the diffraction pattern was recorded on X-ray film. Using mathematical formulas, we can then backtrack to the structure, but there is a big caveat; some information, the phases, are missing from the diffraction pattern. There are several ways, which I won’t go into here, for crystallographers to get the missing information and solve the structure.

Dorothy Hodgkin contributed to the methods available to get this missing information and solve the phase problem. This included using selenium derivatives and using microorganisms to incorporate the selenium into the protein.

The what and why of the Ebola virus disease

 Electron micrograph of Ebola virus (Cynthia Goldsmith, CDC (wikimedia commons)

With the death toll surpassing one thousand, this is the largest Ebola outbreak since its emergence in 1976. The outbreak is unusual in its occurrence on the west coast of Africa rather than central and also in its spread to urban areas. The outbreak has received much news coverage in recent weeks as the death toll rises and two Americans infected were flown home to receive “experimental therapy.” Why does the outbreak capture the international attention so much more than diseases such as malaria, which kills three times that number…in one day? Perhaps one reason is that malaria is perceived as a chronic condition in developing countries, with eradication a far-fetched goal. The horrific effects of Ebola, hemorrhagic fever, internal bleeding and death resulting from multiple organ failure are probably even more of a contributor. This, combined with a mortality rate of up to 90% (though the numbers are closer to 60% in the current outbreak), makes for a terrifying disease. With the announcement by the WHO this week that experimental drugs can be used in Africa, it seemed like a good time to write a post about the biology behind the Ebola disease and how these much-discussed experimental drugs work. I apologize in advance if this information seems basic to readers in the scientific field, especially virologists, but this post is aimed at those less familiar with science.

The pathogen that causes Ebola is a virus. Viruses are different from bonafide living organisms in that they lack cellular structure and cannot make their own proteins. Proteins are the workhorses of the cell, molecular machines that replicate the genome, allow cells to divide, secrete, move, etc. But viruses are just genetic material, either RNA or DNA, enclosed in a protein coat and possibly a lipid envelope (depending on the type of virus). In order to make more of themselves, the virus must invade cells, be it cells of bacteria, plants, or mammals, and hijack the host machinery. The Ebola virus is a single-stranded negative sense RNA virus. This refers to what has to happen inside the host cell in order to replicate. Its genome is encoded in RNA, which, although very similar to DNA (used by most organisms to encode their genome), is chemically less stable than DNA. The virus has a helical protein coat that protects its genome, giving it a striking long filamentous appearance.

Journey of the Ebola virus:
The virus can spread to a new person when blood or body secretions containing the virus come in contact with mucus membranes or broken skin. A glycoprotein (made of a chain of amino acids and sugar) forms spikes on the virus surface and attach to the host cells. The viral proteins bind receptors on the host cell and trick the host cell into engulfing the virus, bringing it inside the cell (Saeed et al., 2010). The virus then needs to make a version of its genome that the cell can read like a blueprint to make more RNA genome, as well as the structural elements of the virus. Luckily for the virus, it has brought a protein, made during its previous infection to convert the genome into a proper blueprint. The newly copied and packaged viruses then must get out of the cell and invade new ones. The Ebola virus “buds” from the host cell, enclosing itself in lipids from the plasma membrane of the host cell, which is the barrier that separates cells from their environment. One protein in particular on the virus envelope is central to this step and is therefore being investigated as a potential drug target (Jasenosky et al., 2001, Timmons et al., 2001). However this is not what is in the ZMapp drug given to three Ebola patients.

Why is Ebola so deadly?
While some viruses infect only certain types of cells, the Ebola virus spreads through the blood system to infect cells in many organs. Patients often suffer liver, kidney, and other organ failure. A major contributor of the mortality of the disease is “immune paralysis” where patients are unable to mount a defense. This is because many of the proteins the virus makes are specialized to interfere with our defense systems allowing the pathogen to wreak havoc unchecked (Lubaki et al., 2013). For example, while our immune system is designed to sense viral RNA (double stranded RNA), a protein made by the Ebola virus covers and hides the RNA from the host. And any amount of interferon signaling activation that is achieved is inhibited later on by the same and other viral proteins (Leung et al., 2010). Additionally one of the first cells infected by the virus are macrophages, immune cells required early on to recruit other immune cells.  This both prevents them from doing their job and causes them to secrete substances that leads to the cell death of other immune cells and makes capillaries leaky, causing internal bleeding (Geisbert et al, 2003).

What’s in this experimental drug?
When infected by bacteria or viruses, our immune systems produce antibodies, “Y”-shaped proteins that bind a protein on the foreign invader similarly to a lock and key mechanism, using the two tips of the “Y”. This neutralizes the pathogen and recruits other parts of the immune system. Therefore antibodies against Ebola virus injected into a patient could help fight the infection.

But where can we get these antibodies?
Mice similarly produce antibodies so scientists at Mapp Biopharmaceutical injected mice with the virus and harvested the antibodies the mice produced. After identifying the antibody reactive against Ebola, they modified the genetic blueprint encoding the antibody so it looked more human-like. This is to prevent an immune reaction in humans, in which the immune system would see the injected antibody as a foreign invader rather than a helping hand.

How to make large amounts of this antibody? 
Since all organisms use the same genetic code, scientists can make all kinds of cells produce the protein of their choice. In graduate school, I made E. coli bacteria produce proteins from the malaria parasite so that I could study the protein more easily (hence my comment about the deadliness of malaria in the introduction). However since bacteria aren’t very good at making complex antibodies, mammalian cell lines are often used instead. Mapp Biopharmaceutical uses tobacco plants because production is faster and cheaper. The DNA is inserted into the plant cells and the plant’s machinery makes the recombinant protein, which is then harvested. ZMapp, the serum that was used on the two Americans, is a combination of antibodies made in this way. It is important to keep in mind that although the two American patients appear to be responding positively to ZMapp, it is impossible to determine from this that the drug works. The only way to determine the effectiveness of a drug is through clinical trials in which a control group receives a placebo. However with both the ethical questions of testing drugs in developing countries and the extremely short supply of serum, clinical trials are unlikely to occur in the near future.

References:
Geisbert, T., et al. (2003). Pathogenesis of Ebola Hemorrhagic Fever in Cynomolgus Macaques. Am J Pathol. 163, 2347-2370.

Feldmann H., et al. (2001). Biosynthesis and role of filoviral glycoproteins. J Gen Virol. 82, 2839-48.

Jasenosky, L. D., Neumann, G., Lukashevich, I., and Kawaoka, Y. (2001). Ebola virus VP40-induced particle formation and association with the lipid bilayer. J Virol. 75, 5205–5214.

Lubaki, N.M., et al. (2013). The Lack of Maturation of Ebola Virus-Infected Dendritic Cells Results from the Cooperative Effect of at Least Two Viral Domains. J Virol. 87, 12506.

Leung, D.W., et al. (2010). Structural basis for dsRNA recognition and interferon antagonism by Ebola VP35. Nat Struct and Mol Biol. 17, 165–172.

Timmins, J., Scianimanico, S., Schoehn, G., and Weissenhorn, W. (2001). Vesicular release of ebola virus matrix protein VP40. Virol. 283, 1–6.

Saeed, M. F., et al. (2010). Cellular Entry of Ebola Virus Involves Uptake by a Macropinocytosis-Like Mechanism and Subsequent Trafficking through Early and Late Endosomes. Plos Path. 6, 1-15.

Wahl-Jensen, V., et al. (2005). Effects of Ebola Virus Glycoproteins on Endothelial Cell Activation and Barrier Function. J Virol. 79, 10442-10450.

What's in a miniprep? A "short" history of a taken for granted technique

If you’re a scientist chances are the miniprep was one of the first techniques you learned. I still remember opening my first blue box as an undergraduate and have done countless preps since. For non-scientists, a miniprep is a way to separate plasmid DNA from chromosomal DNA. Plasmids are DNA encoded on an enclosed circle that replicate inside bacteria independently from the much bigger, linear chromosomal DNA. Bacteria transfer these plasmids to each other, which give those bacteria special properties such as antibacterial resistance. Scientists have hijacked this system to put whatever DNA we want on this plasmid and put it inside bacteria which will quickly make more of that DNA, After purifying this foreign DNA away from bacterial junk, we can then do whatever it is we evil scientists do with foreign DNA…perhaps genetically modify some tomatoes just to make them less flavorful?  

The miniprep’s ease lies in its step-by-step instructions and pre-made buffers. The name doesn’t hurt either, sounding rather diminutive and much less threatening than a phenol/chloroform extraction. Many an older faculty has complained that science has become so automated young scientists don't even understand the basis for the miniprep technique. So for a description of how a miniprep kit works, see the end of the article, as "it's proprietary" probably won't work as a response on your GBO. 

But where did the miniprep technique come from? Probably not many people have wondered this and I hadn’t either until this week for some reason. Perhaps I’m starting to crack under the pressure of the new postdoc, but I decided to put some time into investigating, which, with the Internet, is not that difficult.

The basis of the miniprep kit, the alkaline lysis, was published by Birnboim and Doly in 1979 (Birboim and Doly 1979). In 1988, Dr. Birnboim recounted how he developed the technique in Citation Classics (Birnboim 1988), a feature in Current Contents that ran from 1977 to 1993 and sought to show the human side involved in putting out some of the most widely-cited papers. During a sabbatical in Paris, H. Chaim Birnboim decided he needed a fast and reproducible way to purify plasmid DNA. He was not sufficiently fluent in French and was therefore a little isolated in the lab, for which he attributes his ability to focus on solving this problem rather than working on another lab project. What Dr. Birnboim was actually interested in was identifying and studying a new class repetitive sequences in higher eukaryotes. The sequence (a polypyrimidine tract) was resistant to acid treatment so the idea was to examine clones from a mouse DNA library. He would screen for those regions with long pyrimidine tracts using acid treatment that would break other DNA sequences into short fragments. The DNA was labeled with ³²P, a radioactive isotope of phosphate, before acid treatment and the slow-moving piece of DNA containing the repetitive sequence would be identified with autoradiography. This screen required analysis of many clones, and hence, a quick way to purify the DNA. At the time of the article he had not received any awards, but said that it was “personally gratifying to have developed a procedure that has survived for nearly a decade.” 

Of course this is only the first part of the miniprep technique, the second being the spin column, which relies on the ability of nucleic acids to bind a silica membrane under certain conditions, such as high salt. Dr. Birnboim published the use of silica-glass powder to bind and purify nucleic acids in 1982 (Marko et al. 1982) and the technique was improved upon by other scientists using different chaotropic agents (Boom et al. 1990). By the mid 1980s, Qiagen began selling kits for purification of plasmids and in the early 1990s companies began patenting the technology with Promega filing a patent in 1995 for “Nucleic acid purification on silica gel and glass mixtures” (US Patent number: 5658548) and Qiagen filing a patent in 1994 for “A method for the purification and separation of a nucleic acid mixture by chromatography” (Patent number 6383393). And so the miniprep battle began. But that’s for the company reps to worry about.

I’ve always found minipreps to be pretty relaxing because of their foolproofness (aside from a mistaken miniprep attempt after Happy Hour). Despite their ease, there is enough room to make you feel as though you have somehow improved on the design. For example, “psst, if you heat the water before elution, it will increase your yield” or “hey, pass the elution over the column again, my friend.”

I am terrible with numbers. I can’t remember my anniversary and regularly invert the birth year and day for my husband. I also recently wrote 7/14/20 on an eppendorf tube. BUT if I get hit in the head and someone wants to assess my mental faculties you can ask me the protocol for a miniprep: 250 microliters P1, 250 microliters P2, 350 N2….and don’t forget to heat the water.

How a miniprep works: The first buffer (P1) is simply to resuspend the cells and digest RNA. The P2 lysis buffer contains a detergent to lyse the cell membrane and a high pH that denatures the DNA. This is because the hydroxide ions pull hydrogen ions off the DNA molecule, disrupting the hydrogen bond network that holds the DNA strands together. Addition of the neutralization buffer (N3) lowers the pH and allows the circular DNA to go back to being double-stranded while the large, bulky chromosomal DNA cannot and forms a precipitate. High-speed centrifugation pellets the chromosomal DNA away from the soluble plasmid DNA. The supernatant containing plasmid DNA is then added to a silica membrane under high salt conditions, which allow double-stranded, but not single-stranded RNA and DNA to bind. A chaotropic agent, such as guanidium HCl disrupts the hydration shell around DNA and allows positively charged ions to form a salt bridge between the phosphate backbone and negatively-charged silica membrane. The membrane is washed and then DNA is eluted with a low ionic strength buffer, such as water. And there you have it, more than you wanted to know about minipreps.

References:

Birnboim, H.C. and Doly, J. (1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7(6), 1513-23.

Birnboim, H.C. (Nov 7 1988). Citation Classic – A rapid alkaline extraction procedure for screening recombinant plasmid DNA. CC/Life Sci. 45, 12-1

Boom, R., Sol, C.J., Salimans, M.M., Jansen, C.L., Wertheim-van Dillen, P.M., van der Noordaa, J. (1990). Rapid and simple method for purification of nucleic acids. J Clin Microbiol. 3, 495-503.

Marko, M.A., Chipperfield, R., Birnboim, H.C. (1982). A procedure for the large-scale isolation of highly purified plasmid DNA using alkaline extraction and binding to glass powder. Anal Biochem. 2, 382-7.

Very good read and insight into a scientist's life and passion

Saw this article from one of my favorite news organizations: BBC News Magazine. It is about the scientist who sought to identify the causative agent of Ebola:

The virus detective who discovered Ebola in 1976

The summer of scientific missteps

What is going on with scientists this summer? Misplaced smallpox strains, contamination at the CDC? It took me over a week to decide what I really thought about all this before I could write about it. Is the media blowing things out of proportion, only increasing the public’s mistrust in science? How did this happen and is it part of a bigger problem?

First came news from the director of the Centers for Disease Control and Prevention (CDC) in Atlanta, GA, that two laboratories, working with highly pathogenic organisms would be shut down and all shipment of materials from BSL-3 and -4 level labs (high biosafety levels) stopped pending review. In one case, a low-pathogenic influenza virus, H9N2, was contaminated with the highly pathogenic H5N1 (of “bird flu” fame) and sent to the Department of Agriculture who discovered the mistake. In a separate incident the bacteria responsible for anthrax disease had been improperly inactivated before transferring it to a lower biosafety level lab. Apparently, approved sterilization techniques were not followed, nor was their a standard protocol in the lab for inactivating and transferring the anthrax bacteria to other labs.

Then in early July, 6 vials of the variola virus (responsible for smallpox) were found in a storage freezer (a freezer probably very much resembling a deep freezer) at the National Institute of Health (NIH) in Bethesda, MD. The samples were prepared and stored in 1954. Previously the only remaining stocks of virus (which was eradicated in 1981) were thought to be held at the CDC and at the State Research Center of Virology and Biotechnology in Siberia.

So how could this happen?
Upon hearing the news, I shook my head, but was not surprised. Scientists are quite fallible, especially when it comes to lab organization and thinking they are immune to lab safety practices. The fast pace required to publish and not perish can often lead to sloppiness: sloppiness in record keeping and in properly training and overseeing safety measures. I have experienced both of these first hand. It is not hard to imagine at all how 6 vials, even vials of deadly and potential agents to be used for bioterrorism, went unnoticed amidst the ~40,000 vials that fit inside a standard laboratory -80˚C freezer. Also, there is a lot of turnover in science: graduate students change every 4.5 years while postdoctoral fellows come and go as often as every year (though three is more likely). This combined with the high variability in record keeping practices often leads to loss of years of work, and in this case, a potentially deadly mistake. Because of time constraints for advisors, there is usually little oversight into the record keeping practices of the researchers actually making the samples. It is usually not until that researcher leaves that there is a mad scramble to make sure everything is in “order.” The best-case scenario is that samples will have been entered into a database. But in too many cases, there is no database and samples are poorly labeled (and anecdotally, written in another language), labels are worn off, and of course, encrusted in ice. Unless someone is immediately taking over that researcher’s work, these “orphaned” boxes of material are often shoved to the back of the freezer, apparently in some cases, waiting to cause a scandal half a century later.

Though there have been multiple calls from the World Health Organization for scientists to go through their inventories and find remaining vials of smallpox throughout the decades since it was eradicated, there was little impetus and no oversight for scientists to actually go through the arduous (and finger-numbing) procedure. Really, the virus poses little public health threat left alone at the bottom of a deep freezer. Also, on a positive note, the researcher who identified the virus notified his superior and the viral stocks were transfer to the CDC for analysis and destruction. One can imagine a worse case scenario where an unknowing researcher somehow disposed the vials in a receptacle that was not subsequently autoclaved (exposing the virus to pressurized steam that kills viruses and bacteria). However low, there was a chance for these viral stocks to make it into the environment alive.

Many scientists I talked to were frustrated by these glaring oversights because they worried it would put smallpox virus (and other “extinct” disease) research at risk. There has been an ongoing debate by countries around the world about not so much as whether to destroy the virus, but when. Why keep the virus around? Is there really a chance for smallpox to return or be used by terrorists? Well, in 2002, scientists were able to synthesize poliovirus de novo using mail-order DNA segments that were assembled using the genome sequence of the virus as a blueprint and molecular biology techniques known to any graduate student (Cello et al, 2002). There was immediate uproar from both the science community and general public and the potential creation of the smallpox virus specifically cited as a major concern. And so scientists argue, we need to keep a few smallpox stocks around just in case.

It seems like a silly response to the NIH incident to destroy the remaining known smallpox stocks when the real danger, it seems, is from the stocks we don’t know about, not from those in Russia or at the CDC…oh wait. I can understand why the general public mistrusts scientists. In 2003, the NIH accidentally sent anthrax to a Children’s Hospital in California for Pete’s sake! And the most recent investigations into the CDC discovered anthrax specimens in unlocked refrigerators in a hallway where many workers pass through. And again, record keeping was found to be inadequate. If we want to argue to keep around deadly pathogens, “just in case”, we need to get it together people.

Of course, this is easy for me to say from my budding yeast field. The worst my biological agent will do is give you a hang over (not true at all, but I couldn’t resist). And of course, I always follow proper safety precautions, never eating or drinking in lab and always wear long pants and closed shoes. But although I started off thinking the media was potentially blowing things out of proportion, I found myself in the end, angry at scientists but not really having any answers. Clearly more oversight into protocols is necessary and I believe Dr. Thomas Friedman, the director at the CDC, will do it. But as long as time is of such limited supply and demands are so high on supervisors, sloppiness will continue to contribute to incidents like these. But that's just my two cents, what do you all think?

References:
Cello J, Paul AV, Wimmer E (2002) Chemical synthesis of poliovirus cDNA: generation of infectious virus in the absence of natural template. Science 297: 1016–1018.

Transitioning from a life of woe to a life of wow: from malaria to yeast

When I started the blog not very long ago, the intention was to post an article once a week. But last week came and went without posting anything. I can’t even really blame this on my Birthday and having my family in town as this all happened after the self-imposed deadline of Thursday. I have some articles in the works but find all the legwork of diving into the primary literature unappealing at this moment. So instead I will discuss my personal experience with transitioning from biochemistry to yeast genetics. After years struggling with the malaria parasite Plasmodium falciparum in my PhD; cloning from a 70% AT-rich genome, trying to express, purify, and crystallize proteins that are generally larger and more disordered than their human homologues in E. coli, and trying to do western blots with not very good antibodies, I thought I would do myself a favor and focus on the model eukaryotic organism, Saccharomyces cerevisiae. I would still study the biological processes I was interested in, autophagy and protein trafficking, but in a system with some proper tools. Plus yeast smells a heck of a lot better than bacteria!

So how are things going nearly 3 months in?

One thing that has taken getting used to is the amount of planning that is required when working with yeast. I was very used to deciding to purify a protein from bacteria and two days later, having the materials to do so. Once the proteins were purified and stably frozen away, experiments could be conducted as soon as I planed them. But with yeast, more time is required to first grow on a plate from a glycerol stock, then transform a plasmid and wait three more days for colonies, then start a small overnight culture, and the next day: microscopy. A week can easily go by where I don’t really have anything tangible to show. This is thankfully changing, but requires a lot of multi-tasking and planning ahead so that while strains are growing for one project, I am doing experiments for another. Right now, my method of juggling this has evolved into each day reviewing the previous days “to-do” list and writing a new one for that day. I can only imagine there are or will soon be, lab apps that will send text reminders for different experiments.

I am also learning that yeast are tricky little guys. They readily undergo homologous recombination, which is great for manipulating the genome, but they will do anything to stay alive and seem to shuffle things around as need be so that I am left wondering where the heck in the genome did my GFP tag go? I cannot complain too much about this, as I have deleted a gene in less than a month, compared to the oft-predicted one-year time frame for P. falciparum. I was hoping the microscopy would be easier with yeast, but really they are not much bigger than the asexual forms of Plasmodium (5-10 µm vs 1-2 µM). I still find myself looking longingly at the images of human cells or tissues appearing on the microscope computer screen next to me. But at least tagging yeast genes is easy, allowing for live microscopy. However, tagging a gene can be deleterious for that protein’s function and I have struggled for weeks trying to C-terminally tag several proteins in a complex without success. So I am about to begin the laborious task (this is relative, of course) of N-terminal tagging several genes to hopefully get around this problem. Which is to say that science is never easy.

So how are things going? Generally pretty good. I am learning a lot about genetics and working with yeast. Maybe in 3 more months I will actually have some worthwhile results…or be working for a brewery.