Author Archives: Yilin Yang

Reflections on the summer

8 weeks ago, I stood in the airport on the other side of the planet, alone, with a 50-pound suitcase. I was kind of depressed, not only because I would again be 8300 miles away (I actually looked this number up) from home for 6 straight months, but also because I was nervous about the summer. I have to admit that Duke has been quite a stressful and challenging place to be in the past year, and I was not sure what this summer had held for me. Two parts of me kept debating: “You will be fine! You’ve had experience working in a lab before.” “I know, but it’s a totally different lab this time. I’ll have to build new relationships with people, and learn from scratch about the research my new lab is doing.” “Yeah, it takes time, but eventually you will feel as comfortable as you are in your old lab.” “What if I don’t understand the science? What if I don’t know how to present my work to other people? What if I just somehow screw up?”… Anyway, me at the airport had no idea how these 8 weeks were going to be, which, me sitting at my bench now, can tell her, the past 8 weeks have been absolutely the most relaxing and rewarding experience she has ever had.

My project itself was very interesting – I’ve always wanted to work on human biology, and my project involved looking at human genetic diseases with human DNA samples. It’s been much of an excitement for me to simply hold the box of DNA samples labelled “Bipolar DNA samples – NIMH” in my hand, not to mention the awe that struck me when I was actually looking at the sequencing results. I feel like I am delving into the cryptic codes that constitute of our bodies and minds, although I know they are too exquisitely designed to be fully deciphered. But through the experience this summer, I’ve become more certain of my interest in genetics. The more complicated genetics is, the more rewarding it is to be part of the “deciphering squad”. So I will definitely be looking for more opportunities to learn about this field after the summer.

Other than my project itself, I’ve also found doing research a very amazing and relaxing lifestyle. Of course, it’s fun when experiments work and you get data. However, even though experiments fail 99% of the time, I still revel in the process of troubleshooting. Sometimes after a long day in lab, I was looking up literatures related to my project. I would suddenly somehow get inspired and come up with a new idea that might improve the outcome of my experiment. And then I really became that “crazy scientist”, wishing that I could go in lab right away and try it out, if only I had after-hour access to my lab. Other times, I got my sequencing results back at night. Occasionally, just browsing through them, I would have a big discovery (aka a heterozygous mutation), and I again became very excited. I would send a screenshot to my mentor immediately, even though I knew he would not check email until the next morning. Life as a researcher involves lots of “moments of truth”. For instance, when you are looking at a gel for the first time through the little glass window on top of the UV gel reader, or after doing a bacterial transformation, when you are taking the plates out of the incubator the next morning. It’s just those little moments of taking a deep breath, praying “please work”, and then exclaiming “oh my god it worked!”, that always make my day. Working in a lab everyday means that I always have something to be looking forward to, whether it be getting the results from yesterday’s PCR, or just giving myself a little pep talk like “okay, I’m going to redo this experiment, and it is going to work this time.” and this is what I find absolutely amazing and enjoyable about doing research.

Of course, I will still keep my options open and keep exploring other career paths. But looking back, I feel extremely fortunate and grateful to have participated in the BSURF program during this summer. It has really created a stress-free environment for me to figure out if I truly like doing research, and to occasionally have the feeling flash on me that, oh, I’m actually doing great things.

Reflecting on Dr. Gersbach’s Talk

The morning seminars have all been absolutely amazing. It’s great to hear about how all the speakers got to where they are, and most importantly, the science that they are doing. From fruit flies to lemurs, from evolution to medicine, it has never occurred to me that there are so many distinct yet interrelated branches under the huge umbrella of biological science, and every single one of them deserves being delved into.

As someone who is extremely fascinated by genes, I found Dr. Charles Gersbach’s talk on his life path and his research particularly fascinating. Dr. Gersbach focuses on cellular and molecular engineering. Specifically, one of the major aspects of his research is using genome editing technology to correct mutations that cause genetic diseases. For example, he and colleagues have successfully corrected dystrophin mutations in vivo in mouse models with CRISPR/Ca9 system, and this technology can potentially be translated to bedside, curing Duchenne Muscular Dystrophy in human patients. I found gene/cell therapy very promising because it can get deep down to the fundamental level of genetic diseases and correct the mistakes at that level. In contrast, most of the therapies currently are only able to ameliorate the symptoms at the surface level rather than actually cure the disease. There is a fundamental difference between drugs targeting bad proteins produced by bad genes, and drugs that directly target and fix those bad genes. I believe that once the gene editing technology becomes mature enough, it will have wide-ranging effects on healthcare.

Also from Dr. Gersbach’s talk, I learned that being a biomedical engineer does not mean dealing with brain signals / machines all the time, as how biomedical engineers are stereotypically pictured. Editing genomes and regenerating tissues are also part of problem-solving, therefore also part of engineering. The use of the CRIPR/Cas9 system for genome modification is an excellent example. The technology is adapted from bacterial adaptive immune system, and therefore part of basic science. On the other hand, it is utilized to solve problems on actual human patients, and therefore part of engineering. Dr. Gersbach’s research is really at the intersection between basic science and engineering, and for me personally, working at the interface between these two would be a very enjoyable and rewarding experience.

Of course, Dr. Gersbach’s life path is very inspiring too. Started off as a chemical engineer, he didn’t follow the majority and went into chemical industry. Instead, he tried to figure out what he wanted and followed it. This let me know that, as an aspiring scientist, there are really thousands of different roads ahead. No matter it’s the main street or it’s the road less taken, it’s important to always find my passion and follow my heart, alway do things I truly enjoy. As the program is almost coming to conclusion, I want to thank all the faculty members who shared about their life paths and research with us. All the seminars so far have been really fun and inspiring, and I can’t wait to hear about the life and research of the last two scientists next week!

Troubleshooting -> Working -> Failing

My project seemed very simple in the beginning: no difficult biochemistry, no complicated experimental procedures, and honestly, the fanciest machine ever involved in my experiments is probably the thermal cycler, aka the PCR machine. But in reality, as I’ve slowly found out, problems never stop, frustrations never stop. They come and go one after another, and I not only have to solve them scientifically, but also have to deal with them mentally.

The first real problem occurred as early as I was testing the primers I designed to amplify the gene of interest. I designed 4 different primer sets to amplify 4 different portions on the gene. Despite that theoretically, they should all work perfectly, oftentimes the case was, either the PCR reaction didn’t work at all and I didn’t see my target on the gel, or multiple nonspecific amplifications occurred and lots of bands of the wrong size showed up on the gel. I spent almost the entire first half of the program perfecting PCR — redesigning primers, playing with the annealing temperature of each primer, adjusting the amount of reagents in the reaction, etc. Everyday just involved an enormous amount of PCR and gel electrophoresis and attention to details: keeping detailed notes of what changed and what remained unchanged, while making sure I didn’t use up too much of a patient’s DNA for testing and troubleshooting.

When I got my PCRs working after almost 4 weeks of troubleshooting, I could finally send the products to sequencing. My hopes were high: after the sequences are back, I could start searching for mutations! But again, the sequencing reactions didn’t go smoothly. For mutation-searching, the quality of the signals is very important. Any contamination could bias the result, but the sequences were seldom free of contamination/noise. As a result, I had to start troubleshooting the sequencing reaction: designing new primers for sequencing, adjusting the concentration of the template, etc. As of today, there are still a few samples of which I haven’t got perfect sequences, and I’m still working hard on perfecting the results.

For the samples whose sequences were clean enough, I started to actually search for mutations. Mutations are, however, really, really, rare. So far, I’ve only spotted three, among which two are synonymous (they don’t cause a change in amino acid) and therefore clinically irrelevant. The most exciting one showed up just two days ago because it actually caused an amino acid change from tyrosine to cysteine. My mentor and I spent the entire afternoon almost frantically looking up this mutation, trying to figure out the details of it such as its functional relevance. Disappointingly however, it turned out that it was just a common variant across human population: it has an allele frequency of 1.8%, which is way too high to be considered rare or pathogenic.

In summary, I’ve not gotten any real “results” in my project. But in retrospect, I actually enjoyed this whole troubleshooting->working->failing process rather than got beaten by it. What’s exciting about scientific research is not entirely the groundbreaking discoveries, but all the different paths that lead to that destination as well as the views along them. I will continue working hard on my project and try my best to search for mutations, even if they are not there.

Chalk Talk Reflection

    I’ve always viewed the human body as a machine – one that’s so elegantly designed that somewhat ironically, we humans can never fully replicate or even understand. This is why I found Ricardo’s chalk talk on brain-machine interface particularly interesting.
    Ricardo’s project studies the lag time difference of unimanual versus bimanual tasks. Specifically, he is looking at velocity models and neuron firing rate models of a monkey performing these two kinds of tasks. His project definitely has a broader implication. It helps us better understand how the neurons in our brains work and how the signals interact with motor-control cells in other parts of our body. We can then decode, or translate, the action potentials generated in the brain cortex, and program them in a way that can control machine movement mimicking the voluntary muscles. These machines can be then designed in the form of a robot that almost becomes the continuation of an individual. Whatever the individual is thinking, the robot will perform the task for him/her. Moreover, these machines can also be designed in the form of an exoskeleton for paralyzed patients, so that these patients can regain self-controlled muscle movement. These two are the most apparent applications of brain-machine interface, but I believe that once the technology becomes more mature, it can be more far-reaching and even inspire other branches of biomedical engineering.
    I think brain-machine interface very well exemplifies how humans have finally started to decode themselves – not only having a better understanding of how our own bodies work, but also trying to “learn” from the delicate mechanisms that construct ourselves and design parts that can replace parts of our bodies when necessary. This is what I am most excited about biomedical engineering: learning from the biology of human bodies, and more importantly, coming up with solutions to tackle human body problems.
    Finally, thank everyone for their awesome chalk talk! Your talks have all been very fun and inspiring. Thank you for sharing your project and I can’t wait to hear more about the results/progresses of everyone’s research during the final poster presentation!

Week 4: Three Typical Days in Lab

    Instead of a daily schedule, I have more of a three-day schedule – well, if I am lucky enough and nothing goes wrong, which doesn’t normally happen.
    As I mentioned in my blog last week, I am searching for mutations on the gene of interest that can potentially lead to bipolar disorder, so my project is really a combination of bench work and computer work. On the first of the three days, I will start by aliquoting a new set of patient samples into strips PCR tubes just so that later on in my experiments, I will be able to take 8 samples at the same time with a multi-channel pipette. Then, I will be sitting (or rather, standing, because I feel more functioning when standing) by my bench and preparing for PCR reactions. This involves calculating how much of each reagent I need, actually mixing those reagents, and trying to do things as quickly and as accurate as possible because the tubes can’t stay out of the ice bucket for too long. Depending on the number of samples I have, how long this step takes varies. Last week, I did 96 PCRs at the same time (which I should probably never try again…) and it took me almost 3 hours merely setting things up. The actual PCR reaction takes place in a thermo cycler and takes about 2 hours. As I am waiting, I will be either reading papers and scribbling down notes, or helping out my mentor with his experiments. So far, I have helped him genotype mice and do Western blots. I really enjoy helping him because my personal project involves mostly repetitive work, and I always learn new skills when observing or doing his experiments. After my PCR finishes, I will take the tubes out and put them in the fridge, so that they stay fresh for the next day.
    The second day is mostly gel electrophoresis. I do gel electrophoresis on each of my PCR reaction so than I can 1) see if the PCR actually worked and 2) determine if the PCR products are of good enough quality to be sent directly for sequencing. Unfortunately, my gels always disappoint me, because a lot of the times, although I do see my target band on the gel, there are also some nonspecific bands which really interfere with sequencing. I have been trying to optimize the PCR reaction by redesigning primers and adjusting the primer annealing temperatures to reduce nonspecific binding. The results are getting better, but problems still come up every once in a while. At the end of the day, after I finish running all my samples on a gel, I will send the clean and working ones to sequencing.
    The third day usually starts early, because the sequencing company always sends me the results at 3am. So the first thing I wake up in the morning will be opening up my laptop and clicking on one of the chromatograms to check if the sequencing reactions worked. The rest of the day will involve mostly computational work. I will look carefully through each chromatogram, align the called sequence with the disease-free sequence from the online database and look for mutations in my patient samples. At the end of the day, I will document the mutations, if any, and the overall sequencing quality in my notebook. This will also be a day of discussion and Q&As with my mentor. We look through the chromatograms together and try to find out what we can improve on to make the sequencing results prettier. We seldom get perfect results, so there is always room for improvement. Figuring out what went wrong and how to fix it has been an enjoyable experience.
    Ideally, I only need to do around 400 PCRs to finish my project. In reality though, I have done way more than 400 PCRs in the first half of the program. Things go wrong. Sometimes the PCR just won’t work – you can see how the polymerase had slipped off the template from the chromatogram. More often, I see mixed wave peaks in the chromatogram, which means that I have contamination in my PCR. I spent most of the time reading and asking about, and trying out ways to optimize PCR and to get rid of contamination. So although my project sounds simple, what I am learning in lab is not just how to do a PCR, but how to perfect my techniques by trial and error, how to fail again and again but stay resilient. I am also on my way of becoming an “expert” in looking at sequences, and the best way of accomplishing this is: well, I’ll just have to look at enough of them.

Searching for Genetic Mutations Causing Bipolar Disorder

    The Jiang lab in the Department of Pediatrics, Division of Medical Genetics at Duke University focuses on finding the genetic or the epigenetic basis of various mental disorders such as autism, Angelman syndrome and Prader-Willi syndrome. Each of the mental disorders has its unique genetic or epigenetic link and pathophysiology. However, interestingly, there is evidence that three seemingly unrelated mental disorders: autism, bipolar disorder and schizophrenia have a genetic overlap [Carrol et al. “Genetic overlap between autism, schizophrenia and bipolar disorder”. Genome Medicine 2009], possibly due to similar pathophysiology underlying these disorders. It would be interesting to identify these specific mutations and determine how these mutations affect protein structures that further affect neuron function. Then, targeted drug treatment could be developed, and these diseases, which are now considered almost incurable, might finally have a potential cure.
    My project is a sub-project of this broader scheme. Specifically, I’m interested in looking at bipolar disorder. According to the National Institute of Mental Health, “bipolar disorder, also known as manic-depressive illness, is a brain disorder that causes unusual shifts in mood, energy, activity levels, and the ability to carry out day-to-day tasks.” As its name suggests, it is characterized by periodic mood swings between depression and mania. Surprisingly, bipolar disorder has seldom been as extensively studied as autism and schizophrenia, and almost no genetic mutations have been held responsible for causing the disease. So I’m interested in searching for candidate mutations that may have an effect on the structure of certain brain proteins and potentially cause the disease.
    To accomplish this goal, I have genomic DNA samples from 96 bipolar human patients to start with. I’m trying to use PCR to amplify the portion of interest from the entire genome. Gel electrophoresis is then utilized to determine the purity of the PCR product. As simple as this may sound, the whole process is actually quite an effort because it is very hard to get a PCR product clean and pure enough to be sent directly for sequencing. So my current task is still mostly optimizing PCR (changing the amount of primers added, playing with the annealing temperature etc.) in order to get rid of nonspecific bands and minimize primer dimers, both of which interfere with sequencing. Once PCR is perfected, my work will involve more computational biology such as sequence alignment and comparison in order to search for any mutation that are common to these patients. This is the primary goal of my short-term project.
    If there is any mutation that either causes a change in amino acid sequence (and therefore a change in protein structure), or introduces an early stop codon that blocks the expression of the protein, it is very likely that neuron functions, especially post-synaptic transmission will be impaired. So looking ahead, in the long run, if I indeed find any significant mutations, I will be looking at whether they are just common variants in the human species, and if the mutated sites are evolutionarily conserved. If they are not common variants and are indeed evolutionarily conserved (and therefore functionally relevant), that would be a huge deal. However, my short term project is a rather long shot, both because my sample size is very limited, and because the genetic cause of bipolar disorder has not been extensively studied. But this is exactly why I am very excited about this project: even if I end up finding nothing significant, I am still exploring something new, and this process itself is truly appealing.

The Life and Career of Dr. Yong-hui Jiang

    I saw only four people during my 20-minute walk to lab on Saturday morning, but Dr. Yong-hui Jiang was already waiting for me in his office when I got there. 9 a.m., in his office, this is how Dr. Jiang starts his weekend every week.
    Dr. Jiang went to one of the top medical schools in China straight from high school (which is still the educational system now in most Asian countries). However, unlike most of everyone else who “wanted to become a doctor since five”, he didn’t choose this path himself. At the time, China was under the Cultural Revolution, during which most of the intellectual population was harassed, attacked, and eventually put to death.  As a result, Dr. Jiang’s parents saw doctor as a safe and stable occupation and urged him to go to medical school. However, Dr. Jiang soon found passion in what started off as an involuntary decision. “A lot of technologies were lacking at that time,” Dr. Jiang explained, “There was no whole genome sequencing. Indeed, the entire field of genetics was still nascent. So a lot of diseases which are now known to have a genetic basis remained undiagnosable. It was just those seemingly mysterious diseases that ignited my passion. I like solving mysteries, so I wanted to tackle those diseases.”
    Opportunity came when he received a UNICEF fellowship for training in the United States. As a visiting pediatrician, Dr. Jiang worked closely with kids with Down Syndrome, which is one of the first identified genetically-related intellectual disabilities. “I became very interested in childhood intellectual disability disorders after my training. You know they are somehow genetically related, but you don’t know the exact cause or the pathophysiology. I was very determined to study those diseases, which meant I would need more training.” So he went on to the Baylor College of Medicine in Texas, where he both did his pediatric residency and got his PhD degree in Molecular and Human Genetics. That was when Dr. Jiang realized that he enjoyed not only interacting with patients clinically, but also working at the lab bench and coming up with ways to cure the diseases he saw at the bedside. To him, the two are mutually-reinforcing. “My young patients, especially their parents, they look into your eyes. They trust you and are counting on you to help them. This is what motivates me to go back in lab and try to figure out a cure for them. On the other hand, you also want to apply your research and see tangible results. I really enjoy both, and I think my job is very rewarding.”
    Indeed, the Jiang lab sticks to a three-step agenda: for any disease, first, identify the genetic cause (“what”). Second, understand the pathophysiology (“how”). And third, develop a cure and bring it to the bedside. Current areas of research in the Jiang lab focus on the genetics of autism spectrum disorder and epigenetic disorders such as Angelman and Prader-Willis syndrome. Dr. Jiang was very excited when he talked about his research: “Studying the genetic cause of these childhood disorders, I really feel my dream more than twenty years ago coming true. Now I am really tackling those previously undiagnosable and ‘mysterious’ cases, thanks to the development of whole genome sequencing and other resources and technologies. This was unimaginable decades ago, but even decades later, this still really fascinates me.”
    Now, as both a clinician and a researcher, Dr. Jiang himself spent approximately 25% of the time seeing patients in the hospital, and the rest in his office or in lab. “A life as both a clinician and a scientist can be tough and stressful sometimes,” Dr. Jiang admitted, “but as long as you really have the passion, you will deal with the pressure positively instead of letting failures stress you out. Passion for what you are doing also makes you very willing to work hard and devote yourself – otherwise, why do you think I’m here every Saturday morning?” This is also the piece of advice Dr. Jiang gives to all his students. Be a keen observer of the world around you and find your talent and passion inside it. Know what you really like and what you are really good at. How do you know if something is really your true passion then? “Well, you will know it when you find it.”

My “Selfish” Expectations

     Going in lab always gives me peace of mind: during my first year at Duke, whenever I felt stressed, I would just go to the plant genetics lab that I was volunteering in, get a PCR done, do a transformation, or run some assays. I stayed in lab for three hours before my interview for BSURF, and I spent an entire day in lab before my ECE final. It was always only when I had gloves on and pipets in hand that I could truly appreciate the elegance of biology, forget everything else, and feel the thrill of doing something I love.
     And then there comes BSURF. Surprisingly, the first day I walked into the Jiang lab in the Department of Medial Genetics, all I felt was excitement. No sweat in my hand. No stutter in my speech. Yes, plant genetics was cool – but this summer, I get to work on actual humans. A box of DNA samples from real, human patients already awaited me in the refrigerator. And looking around, there were all these fancy machines that I had never used or seen before. As my mentor introduced all the machines to me one by one – real-time PCR machine, ultracentrifuge, etc. – I felt more and more awestruck. I kept picturing how amazed I would feel if I could operate these machines on my own. I know I will get beautiful results and ugly ones, but either way, I marvel at how people had discovered all these ways to visualize and quantify the teeny tiny cell world. The machines are elegant. The whole lab is scenic. And I easily get addicted to scenic places with elegant designs.
     What’s even more exciting is that during the eight weeks, besides lab bench, I will also be doing a lot of computational work such as DNA sequence comparison and analysis. This is a new skill to me and I am very excited to learn. In the past week, I’ve already learned to how to find the cDNA of a gene, how to find different isoforms of a given protein-coding sequence, and how to design primers for PCR. I believe these are fundamental to any research related to genetics, and they will help me become a more independent researcher.
     Looking ahead, I don’t expect to understand the biochemistry of the operating mechanisms of all the fancy machines in lab, but I do want to get an idea of their functions, and how they help with research. I don’t expect to find or confirm the genetic causes of some neurological diseases, but I do want to get to learn as much as possible about the research of other people in lab, who are actually finding these causes. I don’t even expect my experiments to work all the time, but I just want to enjoy the view of lab everyday. I want to enjoy the machines humming, the “click” when a pipet tip is discarded and the attentive eyes of scientists. These expectations might sound a little selfish – I’m not even expecting to contribute to the field even the slightest amount – but I really can’t wait spend my eight weeks appreciating the elegance of biology and find my peace of mind within it.
Update: Here is a picture of me working (?) in the Jiang lab.