Author Archives: Michael Williams

BSURF Series Finale

I came into this summer initially wanting to focus on being able to ask tough questions in order to further myself as a scientist. When I originally came up with this goal, I thought that I would be coming up with some grand paradigm changing question, but I found that the questions I learned to ask this summer were even more valuable. I learned how to question myself at every stage of the protocol in order to understand not just how to do something, but why I was doing it, providing me with better insight into this project. So while I was not coming up with earthshattering questions, learning to critically evaluate myself and not just the world around me has increased my ability as a scientist.

Over this summer I have also learned that failure is not the end all be all. In the lab, I learned that while experiments may not always go the way that I think they should, I can still learn from them by seeing how this experiment failed, and in turn improving myself my learning how to fix it. From the talks, I learned that while some of these scientists experienced setbacks in their early years, they were still able to lead amazing careers because they did not let those setbacks define them. That has been a hard lesson for me to learn as whenever something does not go right in my life I obsess over it and think that it reflects my shortcomings as a person. But I have learned that science is built more on failure than it is by success, and a failure can be significantly more valuable than a success if you are able to learn from it.

Overall, this has been a fantastic summer and while my project did not go as expected, I still learned quite a bit in the last 8 weeks. I would like to thank Dr. G and Jason for allowing me to even be part of this program, and I would like to thank everyone at the Murphy lab for putting up with me for these last 2 months.

 

 

Episode 7: d-cas9

Of all the Nobel Laureates and Basically-Nobel-Laureates that came to talk to us this summer, none stood out to me as much as Dr. Anne West. The main reasons that I found Dr. West’s talk so valuable was because it provided me with clarity on where to go in the study of epigenetics. She outline how it was one thing to show that there is a correlation between environmental stimuli and epigenetic changes, but it is an entirely different thing to show a causative relationship between epigenetic changes and phenotypic changes. Her talk also helped me to think about the practicality of the way that we approach an ever changing field with technologies that are over 20 years old.  When Dr. West started talking about how d-cas9 could be used to provide specific modulations to various epigenetic changes, I was particularly interested because it provided isnight for me into how current technological advancements can provide new insights into what is actually happening epigenetically. For instance, if I noticed signifigant methylation changes at a CpG site in a gene, I could use d-cas9 DNA Methyl Transferase to replicate that change(and ONLY that change) in a controlled cohort of mice to see what the true effects of that change are on an organism. It could also be used to rescue epigenetic damage that could be caused by environmental stimuli, without causing the errant mutations that regular cas9 causes. In short, Dr. West showed me a practical next step that could be taken in order to expand on my lab’s epigenetic research while also pointing out the inherent flaws in our approach.

 

An honorable mention for favorite talk would of course be Dr. Noor’s, as she showed me how awesome biological research could be even if a lot of people do not feel that way because it does not have any “practical applications” (see: Nobel Prize).

Episode 6: The abstract

Cannabis is one of the most popular drugs consumed in the United States. While work has been done to quantify whole genome transgenerational effects of cannabis exposure, there has been little done to quantify transgenerational effects on a gene-by-gene basis. Previously, Reduced Representation Bisulfite Sequencing identified Shank1 and Dlg4, genes associated with the post-synaptic density and the post synaptic membrane, as being differentially methylated in the sperm profile of cannabis(THC) exposed rats and controls. It is however unknown that this difference in methylation is significant  under specific gene methylation analysis. It is also unknown if any differences in methylation cause significant changes in the expression of Shank1 and Dlg4. That is why in this study, potential differential methylation of Shank1 and Dlg4* were measured through pyrosequencing of the somatic tissues of F1 rats. In addition, RT-PCR** was conducted in order to quantify changes in gene expression of somatic tissues in these F1 rats. It was found that in Shank1 there was hypermethylation in both CpG sites of interests, while in Dlg4 there was hypomethylation in one CpG site and hypermethylation in the other.*** The next steps for this study would be to quantify the gene expression and methylation changes for the F2 generation, while also quantifying the phenotype in the F1 generation.

*Biotin labeled primer for this gene(necessary for pyrosequencing) may not arrive due to inconsistencies with Dlg4’s primers in PCR

** RT-PCR may not be done at the time of this poster presentation

*** This data is from the RRBS whole genome methylation, not from specific gene methylation from pyrosequencing

A day in the life

When I first walk into lab, I check my email to see if there are any updates from my mentor on my project. Then I prepare PCR to test the primers for Shank1 and Dlg4, as I am finding that I have to adjust the master mix to minimize dimers that appear during this PCR, Once I stick that day’s PCR in the thermocycler, I then run a gel for PCR that I did overnight. After doing that, I consolt with my mentor about what the gel means, and what further alterations to the mastermix I should make. After this, I take my lunch break where I read protocols and the literature. After lunch, I spend a few more hours experimenting with optimal conditions for Shank1 and Dlg4 primers in preparation for pyrosequencing.

Once I am done optimizing these primers, it will be time to pyrosequence to test for methylation, and then use RT-PCR to test for gene expression.

Episode 4-The Phenotype approach

For this past week, I have been listening to my peers present chalk talks on theirs works, providing me with insight into other areas of biological research. Of these projects, Sweta Kafle’s  phenotypic analysis of transgenerational seed stability in Arabidopsis thaliana could act as an indicator for the stability of transgenerational affects in these plants. These transgeneraiontal affects would be induced by  placing Arabidopsis thaliana  F0’s in hot or cold conditions, then either keeping these conditions constant throuighout the F2 generation, or alternting this patern of hot and cold until the F2 generation, Or altering the hot and cold conditions then keeping those conditions constant ( Hot cold cold, cold hot hot) until the F2 generation..The reason that her project was so interesting to me is while we are analyzing a similar phenomenon, we are taking 2 very different approaches. While S’weta is testing for phenotypic changes and then going on to test for epigenetic ones, I am doing the exact opposite. This talk helped provide me with insight into how the next steps for my own project should be to analyze the phenotypic affects( behavioral, physiological) that an epigentic insult has on my F1 rats. But  coming back to S’weta’s project, I really liked the way that Sweta provided a comphrehensive look into both the flaws and strengths of her study, and it in turn provided a wonderful experience.  I am looking forward to the results of Sweta’s study, and any further insight that future related projects have.

 

Until Next Time

Episode 3: The Life of Dr. Susan K. Murphy

Susan K Murphy, a phenomenal mentor. Taken from obgyn.duke.edu

Susan K Murphy  received her undergraduate degree in Biology with a minor in Chemistry from UNC-Charlotte.  She then went on to graduate school at Wake Forest University and studied Virology(study of viruses) , where she received her PhD in Microbiology and Immunology. She initially wanted to focus on vaccine development, but during her time in graduate school her son suffered from a rare form of liver cancer, causing Dr. Murphy to shift gears in her research and pursue a path more relevant to cancer research. Dr. Murphy’s postdoctoral research was done here at Duke, in Dr. Randy Jirtle’s laboratory where she studied genomic imprinting and epigenetics. Because she was studying the imprinting of a gene that was highly expressed in ovarian tissue, she ended up with an offer for a faculty position in Gynecologic Oncology here at Duke, where she studies how epigenetic factors influence early stages of ovarian cancer.

This is not the first time that Dr. Murphy has taught someone. While Dr. Murphy used to “panic at the thought of speaking in front of a group of people”, she found out that she enjoys teaching students and appreciates the curiosity that they display. She has given guest lectures at a number of courses  here at Duke.

Outside of her love for the lab, Dr. Murphy loves animals. Her family has always had pets of one sort or another.  When she was about 12, her brother had  2 boa constrictors, a reticulated python and an Indian python which she took care of.   Her mother raised “teddy bear” hamsters and her family would go to many of the local pet shops every weekend to buy supplies. After high school, she spent 10 years working before she went back to college, where she ended up working as a pet store manager and developed a love for fish and birds, and she still has fish and birds today (a 48-year old parrot, 3 cockatiels, 2 of which she hand raised, and 2 parakeets), She volunteered at the Monterey Bay Aquarium before and after it opened, initially helping to raise sea otters, then as a docent in the aviary and the microscope lab; She also worked as a shelter manager for the Monterey County SPCA; when I moved to North Carolina I worked at a veterinary office for 5 years, assisting with all forms of animal care – including surgeries and cleaning dog’s and cat’s teeth.

 While Dr. Murphy loves how her career in science allows her to come up with questions about the nature of life and things we cannot see, and then figure out how best to go about finding answers to those questions – revealing truths about life, there are some things which she would like to change about the scientific process. Chief among these would be the peer review and the lack of funding for grants. There are many scientists like Dr. Christopher Kontos who can have years of work derailed due to the peer review process, or be unable to pursue projects like through a lack of funding, which ultimately contributes to the next generation of scientists having a very pessimistic outlook on academia. 
In lab, Dr. Murphy constantly stresses the importance of safety. The reason for this was when she was an undergrad, she worked in a lab on a project studying Vibrio vulnificus, a bacterium with an LD50 of 1. According to Dr. Murphy,  it is a flesh eating type organism, lives in the ocean and becomes concentrated in shellfish, particularly oysters, such that people with certain underlying risk factor (alcoholism, liver disease, older age) are more at risk of becoming infected with this bug, from eating raw oysters, or going in the ocean when they have an open wound.  Part of the research she was doing involved shucking the oysters that had dined on Vibrio vulnificus that she grew up and fed them.  She would have to shuck a bushel of oysters for each experiment (that’s about 350 oysters).  After a few hundred, I decided that the gloves she was wearing on top of the rubber gloves were too hot and restrictive so I took them off.  Shortly thereafter the oyster knife slipped and went straight into my hand, puncturing the skin.  She had to go admit to my PI that She neglected the safety requirements and now might die!  He took me immediately to the emergency room, with his published papers in his hand showing that tetracycline was the drug of choice for V. vulnificus infections and told the ER doc that he must give me tetracycline immediately!  They did, and obviously all was fine.  However, that very embarrassing incident has made her a much more aware PI and that is why she stresses lab safety to everyone who works in lab.
If you want to learn more about Dr. Murphy, feel free to shoot her an email at susan.murphy@duke.edu
Until Next Time,

Episode 2: Weed the People

The Murphy Lab focuses on studying heritable epigenetic effects that are induced by various environmental stimuli. One of the latest projects is  Cannabis-Induced Potential Heritability of Epigenetic Revisions in Sperm, or CIPHERS, which focuses on the heritable epigenetic changes associated with cannabis use in males.

For my project this summer, I will be analyzing genes that are critical to certain aspects of development in order to see if there is differential methylation in the progenitor (F0) and offspring(F1) generations. While I cannot go into specifics at this project, I can provide a general overview of expectations and  potential significance of this project. This work is essential to the overall focus of CIPHERS as it could provide evidence for cannabis use inducing transgenerational epigenetic changes that affect the viability of both progenitor and offspring. The data generated by CIPHERS could in turn be used to guide policymakers and the general public in the epigenetic effects of cannabis use. In the future, I will likely be working on the F2 generation for these genes in order to solidify that cannabis exposure is in fact a transgenerational stimulus.

BSURF Episode 1: Goals and Dreams

This summer I will be conducting research in Dr. Susan Murphy’s lab in the Department of Obstetrics and Gynecology through the B-SURF program. Here, under the mentorship of Dr. Kelly Acharya,  I will get insight into how scientists conduct epigenetic research, helping me in turn to understand the many complexities of the epigenome.

One of my major goals this summer is to learn how to ask insightful scientific questions that could potentially lead to independent research directions. To do this, I am making it my goal in my lab to constantly ask questions about various processes in the lab so that I could understand the intricacies of experiment and assay design. By doing this, I will not only better understand how to ask scientific questions but I will also understand how to answer them.

Building on my previous goal, I also want to learn how to communicate more effectively in science. I have always struggled with conveying shortcomings or failures when it comes to my work, so I want to learn how to comfortably convey these shortcoming to my peers. While this may seem minor, the ability to convey mistakes to my colleagues will be an essential first step in learning how to effectively convey ideas to them.

Another component to this goal would be learning to talk to my colleagues as more than just researchers. To do this, I attend social events in the lab and share details about myself whenever appropriate.. An essential element to learning how to communicate scientifically is learning to communicate.

My final goal this summer would be to make a contribution to Dr. Murphy’s lab through my work. While I learned skills in my laboratory experiences in school, I found that I did not contribute to science in any meaningful way for having learned these skills. This summer, I want to use the skills that I learned in this lab to further their epigenetic research projects. There is so bigger motive for this goal other than to make a contribution to science through my work in the lab.

To be continued on the next episode of B-SURF: The Williams Chronicles

Vsauce, Michael Here

https://warosu.org/sci/?search_op=op&search_int=dontcare&search_ord=new&search_del=dontcare&offset=24&ghost=yes&search_res=post&task=search2&search_capcode=all&search_text=vsauce