Anatomy of a Research Paper
An Interview with Nikolas Chmiel
Nikolas Chmiel is a Research and Development Manager in the Life Science Group at Bio-Rad Laboratories. He earned his Ph.D. in DNA repair at the University of Utah and pursued his postdoc, researching RNA splicing in Jennifer Doudna's lab at the University of California, Berkeley. You can just tell by one sentence that Chmiel has done a lot of interesting things in his lifetime, but that doesn’t mean he won’t share it. In actuality, I was fortunate enough to speak with Chmiel, and he gave me the wonderful opportunity to take a sneak peak of his work as a scientist.
“[M]y graduate work centered around the enzyme MutY, part of a DNA repair pathway called Base Excision Repair. This pathway works to identify specific mutations in the nucleotide bases of DNA caused by oxidative damage,” he says. Oxidative damage occurs when there is an imbalance in the amount of reactive oxygen species (substances containing oxygen). These imbalances can be bad or good, depending on what they are doing, but they all affect cellular function. Reactive oxygen species commonly will damage nucleic acids or oxidize lipids and proteins. “For instance, the guanine in a G:C base pair may be chemically modified by oxidative stress brought on by environmental factors such as cigarette smoking (among many others),” Chmiel says.
Base excision repair, or BER, is a way a cell repairs damaged DNA. “Damaged DNA,” by the way, does not mean the DNA is battered and blue, but the DNA has a part that can lead to a mutation during DNA replication.
Abstract
The abstract of a research paper tells you basically a quick summary of what the team did in their research -- they let you know why they needed to research what they did and how they did it.
Chmiel and his team looked at how the cell can help correct these mutations caused by oxidative damage. He says, “During DNA replication, this chemical modification will cause the guanine residue to pair with an adenine, instead of the normal cytosine, causing a DNA mutation. MutY is one of the enzymes in the repair pathway that locates and removes these mutations.” As a result, Chmiel needed to look at how MutY was being able to do this on a biochemical level, and if this enzyme, which was present in E. coli bacteria, could be similarly observed in humans.
Introduction
The introduction of a research paper gives you an overview of the knowledge that we already know about on our research topic.
Oxidation damage to DNA can lead to complications in aging and cancer. But it wasn’t clear if mutations in BER enzymes could be a factor. This is because BER helps in repairing oxidation damage done on DNA, and if there was a problem with BER, it may help solve a genetic mystery behind “age-old” diseases.
Chmiel’s team was studying a family with a history of colorectal cancer, the second leading cause of cancer death in women and third in men. They noticed something interesting: the traditional genetic markers for the cancer were not present. Once, it was thought that defective mismatch repair (MMR) genes were the cause. MMR genes usually fix specific nucleotides that are out of place, and defective MMR genes can lead to a mutated adenomatous polyposis coli (APC) gene, which is one of the hallmarks of familial adenomatous polyposis. Otherwise known as “FAP,” the multiple polyps that form on the large intestine endothelium from this disease may become malignant tumors if not removed.
But this particular family did not have the typical inherited, called “germline,” mutations in their APC genes that would be usually found in FAP patients. Instead, the team found different mutations caused by non-inherited, or “somatic,” mutations. These types of mutations happen to DNA after a person is born, such as DNA damage due to prolonged radiation exposure. Sequencing suggested the clue: the number of transversions of the guanine-cytosine (G:C) base pairs were significantly higher in their colorectal tumors. Transversions are when a nucleotide base is incorrectly tied to another base. In this case, these patients had an abnormally large amount of mutations due to the G:C pairs getting switched to T:A base pairs. (We can think of the cell forgetting that instead of “Apple on the Tree” and “Car in the Garage,” someone told them “Ape and Grapes” or “Cat in the Tree.” The cell gets mixed up, and during DNA replication, a G:C pair can accidentally turn to a T:A pair. It’s your AP Biology nightmare.)
To take this one step further, these transversion mutations result in different codons being presented to the ribosome during translation, and these mutations cause different amino acids to be formed. Originally, a functioning APC gene would code for tyrosine at position 165 and a glycine residue (another word for amino acid) at position 382 in the resulting polypeptide. Instead, the transversions caused the tyrosine to be read as cysteine and glycine as aspartic acid. Moreover, the change from glycine proved to be even more disruptive. In a functioning APC gene, it would translate into a polypeptide with a sidechain of glycine with a simple hydrogen attached. When it gets read as aspartic acid due to the mutation, this sidechain becomes larger and more charged. This results in a major structure, and then function, change in the polypeptide.
Observing E. coli bacteria, there are certain enzymes, MutM, MutY, and MutT, that help fix mutations like these. In humans, there are homologous genes that do similar things. These enzymes have a special name: “DNA glycosylases.” They take out the “glyco-”, or sugar, from the damaged nucleotide base so it can be fixed later. Out of the main three DNA glycosylases, gene sequencing found missense mutations in the human homolog of MutY -- called the MYH gene -- that led to changes for two amino acids. This sounds familiar to how the family Chmiel’s team was studying had two incorrect amino acids. Moreover, transversion mutations are rare in people with fully functioning MYH enzymes, which made the team suspect it even more. As a result, they compared the MYH genes in cancer patients as well as 100 control patients.
Results
The findings section of a research paper is self-explanatory -- explain what you found from your research. The trick is, though, the scientists have to be truthful of what they see from the data, not what they think they see. This is often harder than it sounds. What they found should be clear and detailed, and the data shouldn’t be ambiguous. As Chmiel tells me, “But the interpretation often is, though that’s part of the fun of doing science!”
The team did not find conclusive evidence that the MYH gene was responsible for the studied siblings’ cancer in comparison to their control patients. However, the team did find evidence where those same two amino acids that result from a mutation in MYH, Tyr165Cys and Gly382Asp, would result in a significant decrease in base excision repair activity and a corresponding increase in G:C to T:A transversion mutations, such as those found in the APC genes of their studied family. Ultimately, this paper points to a proof of concept, and it could in theory mean that there could be a relationship in a mutation in MYH and colorectal cancer in humans.
Methods
The methods section of a research paper tells the audience how the team brought together the research. This is important because some research is re-done in order to test if it is true or not.
Chmiel’s team used a variety of methods in their research.
For example, histology, the study of cell structures, is extremely important when extracting tissues from the patients themselves. DNA has to be extracted in a precise manner, followed by a detailed protocol with chemicals that play different parts (e.g. buffers maintain the DNA’s structure or enzymes that digest any contaminants).
Chmiel’s team also had to do basic PCR in order to amplify particular exons from the APC gene. Moreover, Chmiel’s team had to do RT-PCR, which is significantly more rigorous and difficult than regular PCR that you may see in high school. RT-PCR or reverse transcriptase PCR, is when you amplify RNA by using reverse transcriptase to transcribe it into DNA and then amplify it that way. RNA is notoriously less stable than DNA, so Chmiel’s team had to handle amplifying genetic information from the E. coli bacteria with much more care.
Assays were also a big part in measuring enzymatic activity to check whether a mutation in the gene can cause a difference in the DNA glycosylases.Most of the assays used where polyacrylamide gel electrophoresis, or “PAGE.” This can be compared to the gel electrophoresis you may have used in your high school biology lab -- instead of using the agarose gel, though the team used polyacrylamide gel to separate small proteins that normal agarose gel would not be able to separate.
Discussion (Limitations and the Future)
And finally, the discussion of a research paper is almost like a conclusion -- except research never ends. In this section, we discuss what happened, and what could have been improved for further research. While most of the discussion is what happened and what it means, knowing what can be improved is essential to finding meaningful discoveries. Molecular biology is an expanding field, and a big part of it is collaborating with other experts to build on each other’s ideas.
Chmiel’s team found evidence for a possible relationship between colorectal cancer and DNA glycosylases. Instead of thinking of the conventional explanation for the disease, which were MMR gene mutations, Chmiel and his team questioned what regulated these mutations.
When I asked if he had any advice for high school students interested in separation techniques or just research in general, Chmiel explained, showing a genuine interest in the world around him:
Reading, asking questions and just keeping a general curiosity about the world around me. I was very fortunate that I had really good science teachers in high school, who I think were also a tad bit crazy and did a lot of neat experiments as part of their class. They inspired me to keep going and keep learning, which eventually led to a career in biotechnology. I was a teacher’s assistant for one of them during high school and was able to talk to them and try out a few experiments that I had researched on the internet. It was very exciting! I also volunteered at a science museum through most of high school. It was a great experience to learn the detailed science behind the exhibits and be able to explain it to others.
For Chmiel, he continues to search for new information even if it wasn’t right under his nose. For those who are interested in applying to jobs, for example, his advice is to learn everything about the company/organization that you are applying to.
Look at their website, download their annual reports and any technical documents regarding their products. The more you know going in, the better your chances of standing out from the rest of the applicants.
And science isn't limited to just specific people in the world. It’s important to do it yourself, because that’s what research is!
Talk to your teachers, and they can help you find opportunities to engage in scientific research. Also, you don’t need fancy equipment to do some really neat stuff. YouTube makes it easy to learn how to do just about anything, and they have some exceptional tutorials on experiments you can do at home with basic items found at a grocery store.
References
Al-Tassan N, Chmiel NH, Maynard J, et al. Inherited variants of MYH associated with somatic G:C→T:A mutations in colorectal tumors. Nat Genet. 2002;30:227–32.
Cover picture of dividing cancer cell from the National Cancer Institute at the University of Pittsburgh Cancer Institute.