Monthly Archives: March 2015

Penn Sit University Issue Brief

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The following is a draft of my issue brief about why Penn State should encourage students take breaks from prolonged sitting and stand more often:

Sitting is positively associated with markers for cardiovascular risk. Glucose and insulin peak higher for those who remain continuously seated after eating, relative to those who punctuate sitting with low-intensity movement, such as walking, every twenty minutes, among 19 obese but non-diabetic people, according to a 2012 study by Dunstan et al. According to the same study, lower levels of blood sugar and insulin following meals are associated with a lower risk for type 2 diabetes mellitus. Additionally, people burn fewer calories while sitting, whether they are of healthy weight or obese (Levine, Schleusner, & Jensen, 2000). Together, these findings explain the report of Hamilton et al. (2007) linking sitting time to increased risk for type 2 diabetes mellitus and obesity.

Lipoprotein lipase (LPL) decreases while sitting. LPL is an enzyme that appears to directly interact with cholesterol in the blood and promote its uptake, reducing LDL (bad) cholesterol levels and indirectly increasing HDL (good) cholesterol levels. LPL is produced by skeletal muscles, which produce higher amounts of LPL during exercise. However, continuous low levels of activity seem to be needed for continuous LPL production. Studies in rats show that after four hours of activity analogous to sitting, LPL levels decline for a period of about fourteen hours by 90 – 95%, at which level they remain; thus a single day spent sitting abrogates most LPL activity. Since LPL improves cholesterol levels, sitting indirectly raises triglycerides and LDL cholesterol and lowers HDL cholesterol (Hamilton, Hamilton, & Zderic, 2007; Hamilton, Healy, Dunstan, Zderic, & Owen, 2008). LDL cholesterol levels positively correlate with risk for fatal atherosclerosis and heart attacks, as do high triglyceride to HDL ratios (Assmann & Schulte, 1992). Together, these findings provide a mechanism to support the research by Katzmarzyk and Lee (2012) showing that sitting increases the risk of death by any cause and that, on average, people who sit for more than six hours per day die between 1.8 to 2.0 years sooner than those who sit for fewer than three hours per day.

How much Penn State students are sitting, & when.

College students are not invincible against the maladies of prolonged sitting—not even at Penn State University. Penn State professors David Conroy, Steriani Elavsky, and Shawna Doerksen and graduate students Jaclyn Maher and Amanda Hyde (2013) monitored the activity of 128 undergraduates and found that on average, they sat for 67% of their waking hours—over eleven hours per day. Conroy et al. also found, paradoxically, that students both sat and purposefully exercised the most on Tuesdays, Wednesdays, Thursdays, and Fridays, since, they reasoned, students have classes and sports practices on weekdays.

 

Curious about what factors besides the day of the week lead to intrapersonal differences in sedentary behavior, Conroy et al. found, unsurprisingly, that habitually sedentary students tended to sit more during the study, but that the stronger their daily wills to avoid sedentary behavior, the less time they spent sitting. This suggests that Penn State students, though predisposed towards sitting in class and while studying, can sit less if they choose. Therefore, if Penn State can convince students to avoid prolonged sitting, it is likely that students would begin to habitually sit less.

Indeed, some colleges are already piloting standing workstations. At the College of Saint Benedict and Saint John’s University, a pair of liberal arts colleges in Minnesota, exercise science professor Mary Stenson prompted her department to install standing desks in half of the classrooms in Murray Hall, a gym facility on campus. Although a controlled study to determine whether these standing workstations benefit students’ performance and well-being is still underway, Stenson reported that she feels more alert and productive at her standing desk (Dittberner, 2014). These desks were provided by JustStand, a national organization that markets Ergotron brand sit-stand workstations and other products, which it has donated to over 4,000 organizations (The Mission for JustStand.org, 2015).

Students at Penn State will likewise benefit from a well-designed program to encourage them to reduce the time they spend sitting. Admittedly, implementing a standing desk program at the College of Saint Benedict and Saint John’s University would be easier than implementing one at Penn State. The former pair of institutions enrolls only 3,744 undergraduates and charges over $40,000 in annual tuition (The College of Saint Benedict and Saint John’s University, 2015), while Penn State’s University Park campus has 40,541 undergraduates, who pay $17,502 (in state) or $30,452 (out of state) per year (The Pennsylvania State University, 2015). Penn State is therefore less flexible and receives fewer private funds; consequently, implementing a standing desk program would likely take more time and effort, although it could implement a small pilot program as an initial step towards determining how to design a larger program.

Recommendations for Penn State to improve

There are a number of mutually inclusive components of this program to reduce the amount of time that students spend sitting. However, Conroy et al. (2013) found that both students’ habits and intentions to avoid being sedentary strongly influence their sedentary behavior. Simply providing students with standing workstations without educating them as to why they should avoid sitting would likely change the engrained habits of few students, so the investment in standing desks would be wasted. In order to change students’ sedentary habits, Penn State must first make students intend to sit less, which could be accomplished by educating students first about the health impacts of sitting. After students want to sit less, they would be more likely to use equipment that Penn State would provide later on to change their habits.

The endeavor to encourage Penn State students to interrupt long sitting breaks would comprise three main goals: first, educate students about why they should sit less and for shorter periods of time; second, provide students with the equipment that they would need to accomplish this; third, develop ways to continuously motivate students to avoid protracted sitting. To develop these goals, it must be noted that Professor Conroy himself said, “It took a little while to get used to standing while working…. It’s best to ease your way into it” (LaJeunesse, 2013). That being said, students should not immediately try to stand up continuously as they work. Rather, they should first focus on interrupting habitual protracted sitting. As Professor Conroy again said, “Frequent interruptions to the amount of time spent sitting are likely to accumulate over time into valuable health benefits” (LaJeunesse, 2013). As students become accustomed to standing, they can stand for longer periods of time.

This overall goal begins with the first goal of educating students, which would be the responsibility of those who develop the classes and design the curricula of different majors, as well as of students to spread the message. Students already take interest in their fitness by frequenting the gym and taking some of the 65 fitness-based kinesiology classes that Penn State offers for credit, presumably because they have learned throughout their lives that exercise is important for health (The Pennsylvania State University, 2015). The aim of this goal is to create a culture in which students view prolonged sitting as being even more detrimental to their health than lack of vigorous exercise. Initially, this could be accomplished by incorporating lectures on the scientific risks of sitting into every physical fitness class. Additionally, a new 1.5-credit kinesiology class consisting of lectures on the risks of prolonged sitting could be created. This class would meet for less class time than most fitness classes, but most of the grade would be based on one assignment: wearing a device to measure the amount of time spent sitting and standing and being graded on the frequency of breaks while sitting.

Not every student takes physical education classes, however. Students are required to take at least 3 general health credits at Penn State, but these can come from sedentary biobehavioral health and nutrition classes as well (The Pennsylvania State University, 2015). Therefore, the general education requirements could be changed to mandate at least 1.5 to 3 physically active classes to ensure that all students would learn about the risks of prolonged sitting. To reach students who are not currently taking a physical education class, infographics about the adverse effects of prolonged sitting could be placed in popular locations around campus to encourage health-minded students to take notice. All together, these measures could educate Penn State students that sedentary behavior is as influential as physical activity in determining health. “The health message has been delivered really successfully that physical activity is good for you…. However, very few people think about the dangers of sitting all day,” (LaJeunesse, 2013), said Professor Conroy.

After Penn State students begin to realize the importance of avoiding prolonged sitting, Penn State can begin the second goal: provide students with the resources they need to avoid prolonged sitting. This goal would fall to the administrators who purchase classroom and commons building furniture. As purchasing new equipment may be expensive, these administrators have several options to mitigate the expenses. First, they can replace furniture as it wears out, as they would need to purchase new furniture anyway. This option has the upside of being relatively inexpensive but the downside of being slow, as it may be many years before all of the furniture wears out and is replaced. Second, they could sell the existing furniture to other schools that need to replace theirs; this option would recuperate some of the costs of buying standing-compatible desks but would involve effort to sell the furniture, and Penn State would not be able to sell some of it, such as large, built-in desks and lecture hall seats. Third, Penn State could raise tuition by the amount (small, compared to the overall tuition) needed to defray the cost of the standing equipment. Additionally, there are already products that use a harness to support a desk in front of one’s chest, on which one can place a laptop or notebook. Penn State could design such a product and sell it at the bookstore to help defray the costs of new furniture. Combined, these options would provide students with materials that they would need to stand up and minimize the cost to Penn State.

To continuously encourage students to interrupt sitting, with or without using these materials, is the third goal of this program. Professor Conroy is himself developing interventions to encourage this behavior, which I will interview him about. Penn State University has developed a wellness initiative, Take Care of Your Health, in which faculty who complete a WebMD wellness profile and get a preventative physical exam and biometric screening are rewarded $100 each year they complete it (Take Care of Your Health Initiative, 2015). Through an initiative, Walking Works, faculty of the Hershey Medical Center and the Penn State College of Medicine can compete to take the most steps (Walking Works returns to medical center campus next month, 2006). Combining Walking Works with Take Care of Your Health, Penn State could develop a program for all campuses in which teams of faculty, and even students, could compete to take the most steps and spend as little time seated as possible. Top teams could be given modest prizes. Deliberate programs such as these could encourage students and faculty to sit for shorter periods of time and be more active.

Meanwhile, Penn State could implement a number of other, informal strategies to reduce sedentary time. The campus is under constant renovation, and new buildings could be designed to better accommodate standing or sit-standing behavior. Food courts, especially, could be changed to eliminate buffets, since students can take too much food and spend a long uninterrupted time sitting to eat their meal. Even more simply, the buffets could do away with trays (just as there are no trays in the a la carte area of Redifer), so that students could carry only one or two dishes at once and would need to get up periodically for more food; this strategy could discourage both uninterrupted sitting and overeating. To encourage students to take breaks to move around while studying, Penn State could design a smartphone app that would remind them every half hour to get up for a few minutes and record, using the phone’s pedometer, whether the students did. This information could be used in the aforementioned wellness competition and physical education classes contingent on reducing sitting. During class, professors could have students stand up for two minutes every 25 minutes (once in a 50 minute class, twice in a 75 minute class). Research suggests that elementary school students lose focus when sitting for long periods of time and that in children, adolescents, and even the elderly, aerobic fitness positively correlates with working memory. Moreover, obesity, to which prolonged sitting can lead, is correlated to brain damage (Ratey & Sattelmair, 2012). These informal interventions to break up prolonged sitting may therefore improve students’ health and academic performance.

Concession of the drawbacks of these approaches

Before such initiatives go into effect, Penn State should consider the potential drawbacks. Cost and effort is the foremost tangible obstacle to providing students with the resources to stand in class and while studying. However, the strategies proposed, including selling current furniture, replacing furniture as it wears out, redesigning buildings to be renovated to accommodate standing, designing and selling wearable notebook or laptop harnesses, and raising tuition to cover the unmet costs would enable Penn State to provide standing or sit-stand workstations for students. Another drawback of implementing standing desks is the potential for students to begin standing too much, too soon. Indeed, Krause et al. (2000) show that prolonged standing, while already a risk factor for the development of varicose veins, may lead to atherosclerosis more so than prolonged sitting, at least in those with ischemic heart disease. Clearly, standing all day is not the solution; rather, the consensus among all studies seems to be that prolonged periods of being stationary, rather than specifically sitting or standing, impair health.

Conclusion, and why this matters

A large amount of research shows that prolonged inactivity, especially sitting, leads to a broad range of chronic diseases, including obesity and heart disease, and to a greater risk of death from any cause over time. Moreover, sedentary behavior impairs learning and focus. Schools are now taking notice and piloting programs to encourage students to move more. While elementary school is the ideal time to merge school and physical activity, college is not too late, and students who in college form healthy work habits may carry these habits into the workplace. While research has revealed risks of prolonged sitting, a smaller amount of research suggests that prolonged standing in one position may also be risky; therefore, the goal is not to encourage students to stand for long periods of time, but rather to avoid sitting for long periods of time. Penn State can encourage students by providing furniture—such as the high tables in Atherton and Simmons—at which students can either stand or sit, developing wellness initiatives, educating students on the risks of prolonged inactivity, and a myriad of other strategies. This endeavor will require time, effort, and money, but the short-term cost of investing in students’ health is outweighed by the long-term benefits for students: improved academic performance, health, and lower medical expenses later in life. By habitualizing students to avoid sedentary behavior now, Penn State can give its students longer, more productive, and healthier lives.

 

References

Assmann, G., & Schulte, H. (1992). Relation of high-density lipoprotein cholesterol and triglycerides to incidence of atherosclerotic coronary artery disease (the PROCAM experience). The American Journal of Cardiology, 70(7), 733 – 37. Retrieved from http://www.sciencedirect.com/science/article/pii/000291499290550I#

Benden, M. E., Blake, J. J., Wendel, M. L., & Huber, J. C., Jr. (2011). The Impact of Stand-Biased Desks in Classrooms on Calorie Expenditure in Children. American Journal of Public Health, 101(8), 1433 – 36. doi:10.2105/AJPH.2010.300072

The College of Saint Benedict and Saint John’s University. (2015). Retrieved from http://www.csbsju.edu/

Conroy, D. E., Maher, J. P., Elavsky, S., Hyde, A. L., & Doerksen, S. E. (2013). Sedentary behavior as a daily process regulated by habits and intentions. Health Psycology, 32(11), 1149 – 57. doi:10.1037/a0031629

Dunstan, D. W. et al. (2012). Breaking Up Prolonged Sitting Reduces Postprandial Glucose and Insulin Responses. Diabetes Care, 35(5), 976 – 83. doi:10.2337/dc11-1931

Dittberner, A. (2014). Standing workstations appear on campus. Retrieved from http://www.csbsju.edu/news/standingdesks

Hamilton, M. T., Hamilton, D. G., & Zderic, T. W. (2007). Role of Low Energy Expenditure and Sitting in Obesity, Metabolic Syndrome, Type 2 Diabetes, and Cardiovascular Disease. Diabetes, 56(11), 2655 – 67. doi:10.2337/db07-0882

Hamilton, M. T., Healy, G. N., Dunstan, D. W., Zderic, T. W., & Owen, N. (2008). Too Little Exercise and Too Much Sitting: Inactivity Physiology and the Need for New Recommendations on Sedentary Behavior. Current Cardiovascular Risk Reports, 2(4), 292 – 98. doi:10.1007/s12170-008-0054-8

Katz, A., Mulder, B., & Pronk, N. (2014). Sit, Stand, Learn: Using Workplace Wellness Sit-Stand Results to Improve Student Behavior and Learning. American College of Sports Medicine’s Health & Fitness Journal, 19(1), 42 – 44. doi:10.1249/FIT.0000000000000089.

Katzmarzyk, P. T. & Lee, I-M. (2012). Sedentary behaviour and life expectancy in the USA: a cause-deleted life table analysis. BMJ Open, 2(4). doi:10.1136/bmjopen-2012-000828

LaJeunesse, S. (2013). Probing Question: is sitting bad for your health? Retrieved from http://news.psu.edu/story/295260/2013/11/14/research/probing-question-sitting-bad-your-health

Levine, J. A., Schleusner, S. J., & Jensen, M. D. (2000). Energy expenditure of nonexercise activity. American Journal of Clinical Nutrition, 72(6), 1451 – 54. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/11101470.

The Mission for JustStand.org. (2015). Retrieved from http://www.juststand.org/tabid/960/language/en-US/default.aspx

The Pennsylvania State University. (2015). Retrieved from www.psu.edu

Krause, N. et al. (2000). Standing at work and progression of carotid atherosclerosis. Scandinavian Journal of Work, Environment & Health, 26(3), 227 – 36. doi:10.5271/sjweh.536

Take Care of Your Health Initiative. (2015). Retrieved from http://ohr.psu.edu/benefits/take-care-of-your-health/

Ratey, J. J., & Sattelmair, J. (2012). The Mandate for Movement: Schools as Agents of Change. In Physical Activity Across the Lifespan (12). Retrieved from http://link.springer.com/chapter/10.1007%2F978-1-4614-3606-5_12.

Roy, B. A. (2012). Sit Less and Stand and Move More. American College of Sports Medicine’s Health & Fitness Journal, 16(2), 4. doi:10.1249/01.FIT.0000413046.15742.a0

Van der Ploeg, H. P., Chey, T., Korda, R. J., Banks, E. B., & Bauman, A. (2012). Sitting Time and All-Cause Mortality Risk in 222 497 Australian Adults. Archives of Internal Medicine, 172(6), 494 – 500. doi:10.1001/archinternmed.2011.2174

Walking Works returns to medical center campus next month. (2006). Retrieved from http://news.psu.edu/story/206019/2006/01/19/walking-works-returns-medical-center-campus-next-month

Small interference; significant interpretation

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To discover what something does, often we remove it and observe what changes. This is especially true in biology. Jinquan observed that under certain conditions, cells start producing a lot of a protein called ATF4. Proteins in cells are frequently linked in pathways, wherein one protein turns on another protein, which turns on another, and so on, until the final protein actually carries out the cells’ responses to a stimulus. Jinquan wants to figure out if ATF4 turns on several other proteins in a pathway. If she can make some cells stop producing ATF4 while allowing other cells to produce it, and then show (using western blotting, discussed previously) that the cells without ATF4 are producing less of those other proteins than the cells that are still producing ATF4, then she has evidence that ATF4 is part of that pathway. But how to turn off ATF4 protein?

RNA interference is one method we use to stop making a protein. Normally, to make a protein, a cell first uses the DNA segment encoding the protein (a gene) to create a piece of RNA, a molecule similar to DNA, in a process called transcription. This type of RNA, which codes for a protein, is messenger RNA (mRNA). The cell then uses a large group of molecules known as a ribosome to read the mRNA and make a protein from its code of bases, a process called translation.

DNA → mRNA → protein

Eukaryotic cells (cells with nuclei, such as those of all macroscopic organisms) have evolved a way to prevent the translation of a specific protein by destroying the mRNA encoding it: RNA interference. RNA interference is useful in the lab, where we can use it to dramatically reduce the amount in which a cell produces a specific protein, and, by observing the differences between cells with and without the protein, infer the protein’s function. We can induce RNA interference by adding, or transfecting, another type of RNA called small interfering RNA (siRNA) into cells. siRNA, like DNA, is a double-stranded helix, but, unlike DNA, is automatically chopped up into small fragments by a protein called Dicer. Then, a collection of proteins known as an RNA-induced silencing complex binds to each fragment, discards one strand of siRNA, and displays the strand it retains. Just like two complementary DNA strands in a double helix bind together, mRNAs that have sequences that match the siRNA sequences will bind to the siRNA. A protein called Slicer (or Argonaute), part of the RNA-induced silencing complex, then cuts up the mRNA, preventing it from being translated into a protein. RNA-induced silencing complexes can work over and over, degrading many mRNAs, and shutting down the production of the protein that the mRNA encodes. For an in-depth video on RNA interference, follow this link.

Mechanism_of_RNA_interference

 

Simone Mocellin and Maurizio Provenzano: Mechanism of RNA interference. Wikimedia Commons. Here, the double-stranded siRNA (abbreviated dsRNA) is chopped by Dicer to form siRNA fragments, which associate with the RNA-induced silencing complex (RISC) and cleave mRNA (blue) into fragments, preventing translation.

Earlier this week, I watched Jinquan transfect cells with siRNA that specifically targets the mRNA that produces a protein called ATF4. A day before the transfection, Jinquan had cultured the cells she wanted to transfect in 4 plastic wells.

Two wells would be transfected with siRNA for ATF4. To prepare this siRNA, Jinquan added in one tube 8 microliters (µL) of siRNA for ATF4 to 200 µL of a special siRNA transfection medium containing solutes that facilitate the transfection. Next, to 200 µL of siRNA transfection medium in a second tube, she added 10 µL of siRNA transfection reagent, which binds to the siRNA and helps to carry it into the cells. She pipetted the dissolved siRNA into the transfection reagent tube and waited 30 minutes while the two bound together.

(8 µL siRNA + 200 µL medium) + (10 µL transfection reagent + 200 µL medium) =  ~ 400 µL

To understand what the lack of ATF4 does, cells without ATF4 must be contrasted with cells with ATF4—the negative control cells. To make sure that any differences between the negative controls and the transfected cells arose from differences in ATF4 levels, and not from the process of transfection itself or the addition of the transfection reagent, Jinquan also mixed 10 µL of transfection reagent with 400 µL of transfection medium.

10 µL transfection reagent + 400 µL medium = ~ 400 µL

Using a small vacuum pipette, Jinquan sucked out the liquid medium in which the cells were growing (the cells remained attached to the bottom of the plastic wells), and she washed them with transfection medium, which she removed. After adding 800 µL more of transfection medium, she pipetted the approximately 200 µL of siRNA with transfection reagent into 2 wells and 200 µL of transfection reagent alone into the other 2 wells, drop by drop, each drop in a different place. She gently rocked the wells to distribute the siRNA, then incubated them for 6 hours to let the wonders of transfection and RNA interference occur.

Now, a few days after the transfection, Jinquan is making western blots of proteins extracted from the cells. If she finds that ATF4 is much lower in transfected cells, then the transfection worked. What she is curious about, though, is whether the other proteins she suspects are part of the ATF4 pathway are much lower in transfected cells. If they are, her results will be evidence that ATF4 is upstream of the other proteins in the same pathway. Slowly, using RNA interference, the pathways of cellular proteins can be elucidated, which can lead to novel drugs to inhibit proteins in cancer pathways or to turn on more salubrious proteins.

Colleges in diStress!

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The problem was evident before my class was in kindergarten, but it affects us today as college freshmen—and today, it is worse. College students were stressed fifteen years ago, according to this 2000 article on the website of the American Institute of Stress, and though a 2008 addendum stated that the problem had worsened in the intervening years, the article’s opening sounds eerily relevant today: “No time for sleep, no time for playing games, no time for going to parties. You must get that six-figure job, you have to get an “A” in this class, and you must succeed.”

I imagine that most of you have thought this, as I have for a little over three years. While I had participated in marching band and quiz bowl in ninth grade, I added on pit orchestra, traveling jazz band, a local music competition, County Band, and my first AP class—World History—in tenth grade. By the middle of March, I had burned out: I couldn’t devote enough time to any one thing to do it very well. But did I learn? While conceding pit orchestra, I took five AP classes in eleventh grade and four in twelfth, and became more involved in quiz bowl and PMEA bands. After all, there was so much to do, and colleges would be scrutinizing my accomplishments.

Somehow, I imagined without reading into the issue that once the college admissions process was over, college would be much less stressful than twelfth grade. But application essays have not ceased. Now there are scholarships, internships, and research opportunities, ways to earn money, gain skills, and prepare my resume for me to secure a career that will yield even more money later on, if I pass the interviews. Combine this with my hazy and labile conception of my career, and you can see some of the roots of stress and insecurity for me and many other college students.

Indeed, this is a national trend. This NY Times article from last month cited a recent survey, “The American Freshman: National Norms Fall 2014,” that reported that significantly more freshmen feel depressed or overwhelmed than they did in 2009. Likely, many changes are driving down students’ mental health. That article posited that increased college student pressure to “compete in a global economy, and think they have to be on top of their game all the time” is one cause, as is the anxiety they may have developed by being overworked in high school. It also implicated students spending less time socializing and more time studying as a probable cause of stress and argued there is an optimal balance between work and play, and that universities should encourage students to find that balance rather than incessantly work harder. While the article said that drinking and smoking rates are declining nationally, signs that students are not resorting to unhealthy ways to cope with stress, students are increasingly socializing more with media and less in person, which can contribute to feelings of isolation.

Seventeen more causes for declines in students’ mental health appeared in this Psychology Today article. These causes range from students not developing personal life philosophies anymore, to students becoming narcissistic, to poor mental health care. Putting all of the causes together, I think that a large part, though certainly not the only part, of declines in mental health is increasing materialism, which dove-tails with an “I want it NOW because I DESERVE it” attitude. The aforementioned article mentions high school grade inflation fostering a sense of superiority, praising of assertiveness fostering a sense of entitlement, development of pills fostering underestimates of how difficult it can be to recover from diseases, and increased financial pressure fostering a sense that students must succeed, or else! To this, I would add that faster communication between students and ubiquity of Internet access has made us think that we deserve instant rewards for minor accomplishments, instant replies to our messages, and, even worse, that we must always be on our guard to make sure we don’t do anything stupid, or else our stupidity will be spread across social media in five seconds. Also, that our peers at Penn State are earning internships and scholarships pressures those of us who are not now to seek out these opportunities. Since many of us students come into college with (perhaps unrealistically) high expectations, we may feel like failures if we don’t get accepted into the programs that admitted our peers. Others’ success stories motivate us to succeed but don’t prepare us to fail.

All of this centralizes on materialism. Wanting lucrative careers, prestigious resumes, and flawless facades, we overlook why we really want them, why we need them as soon as possible, and what they really mean to us. Answering those subjective questions is certainly much harder than answering the questions of how much money your scholarship was worth or how many weeks your internship was. How to find fulfillment is a question many people ask, and beyond the scope of this blog—even if I did have the answers, which, as a 19-year-old freshman, I do not. I encourage all of you, though, to ask yourselves why you really want the things that you do and to do things that mean something to you, not to everyone else looking upon you. It will take some courage and discomfort, but is it worth the trouble if the alternative is more and more stressed and depressed college students across the country?

Making a Western

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A project of my own, finally. The reason I joined a research lab. Though learning lab techniques is invaluable preparation to do one’s own experiments, I had been getting bored earlier this semester when I would merely come in, ask what work there was to do, and make gels or run a PCR. Since Jinquan was planning the experiments and working eight hours or more per day, I couldn’t keep up with the progress she was making and felt excluded.

In my BMB 488 research class, which is a great way for any BMB major undergraduate to get involved in research, my classmates and I had the opportunity to present original research at the Undergraduate Poster Exhibition. My Principal Investigator, Dr. Wang, gave me five papers to get ideas; one especially intrigued me, but first, some background. Just as DNA’s ATGC base sequence encodes genes, histone proteins, which DNA is always wrapped around, carry a sequence of modifications that encodes how genes are turned on and off. An important modification is the addition of three methyl groups to the 36th amino acid—a lysine—on the third histone; this is called histone 3 lysine 36 trimethylation. Without lysine 36 trimethylation, cells cannot effectively repair DNA, accruing lethal or carcinogenic mutations.

Nucleosome

DNA wrapped around a particle made of 8 histones, forming a so-called nucleosome

The intriguing paper written by Peter Lewis et al. showed that when cells are given DNA coding for a mutated histone 3, in which lysine 36 is replaced by another amino acid—methionine—virtually all lysine 36 trimethylation disappears, even on normal histone 3s; such a mutation could precipitate cancer. I want to figure out why the methionine histones abrogate lysine 36 trimethylation. Given that I have two weeks before the poster session in which to do experiments, I don’t expect to solve the mystery, but I do plan to test several hypotheses—namely, that the methionine histones affect how much the cells produce certain proteins. To measure the levels of proteins, I will need to use the lab skills I have been learning but will also need to learn a new skill: western blotting.

Western blotting (named after Southern blotting, which is named after Edwin Southern), is a way to see if a cell culture is producing a protein and, if so, to estimate how much. I have watched, but not performed one myself, so I am referencing this and this other protocol from Abcam.

Step 1: Extract the protein. All of the first steps should be done on ice with ice-cold liquid media. Start by washing the cell culture dish with phosphate-buffered saline solution, aspirating, and then adding a lysis buffer to split the cells open and release the proteins within. Scrape the cells out of the dish, transfer them to chilled microcentrifuge tubes, and then agitate them for 30 minutes, to ensure all cells lyse. Centrifuge the tubes at maximum speed for 20 minutes to precipitate large fragments of the cells in each tube into a pellet at the tube’s bottom, while leaving the proteins, too light to precipitate, dissolved in the supernatant—the fluid above the pellet. Pipette the supernatants into new tubes.

Step 2: Denature the protein. Aliquot 50 µL of the contents of each tube and measure the concentration of protein in each 50 µL sample. Then, unless otherwise specified, add sodium lauryl sulfate (to give the proteins a negative charge) and beta-mercaptoethanol (to break disulfide bonds that hold proteins together), and heat the tubes to 100°C for 5 minutes to denature the proteins. This changes the proteins from their highly specific shapes to linear chains of amino acids, necessary for the next step.

Step 3: SDS-PAGE Electrophoresis. As the cell extracts have many proteins, different proteins must be separated in order to determine how much of each is present in the cells. To do this, take a previously prepared polyacrylamide gel with a number of slots on top. Load an equal mass—20 to 30 µg—of each protein sample into a slot atop the gel, and then place the gel vertically in a machine that gives puts the top of the gel at –50 volts and the bottom at +50 volts for 1 to 2 hours; this is called electrophoresis. Since the proteins have negative charges, they will move down towards the positive voltage, and the larger the protein, the more it will bump up against other molecules in the gel, so the slower it will run. After the gel is finished running, the proteins will be separated based on size, and smaller proteins will have moved further from the starting wells. Additionally, a so-called protein ladder, comprising proteins of known size, is loaded into one slot; the distances that each protein in the cell samples run are compared with the markings on the ladder to estimate the size of each protein. The proteins, however, are invisible, so they must be stained before visualization.

Load_a_sample_into_a_polyacrylamide_gel_electrophoresis_chamber

Photo by rox. Loading a protein sample into a gel for electrophoresis.

Step 4: Blotting. Blotting transfers the proteins from the gel to a nitrocellulose membrane on which the proteins can be stained. Cut the slots off of the gel, and then, on the flat blotting machine, place, in turn, moist filter paper, the gel, the membrane, and more moist filter paper, close the lid, and place a heavy weight on top to press the gel and membrane together. The machine applies a voltage, but this time, the positive voltage is on top, so that the proteins move upward, out of the horizontal gel and into the membrane. After the transfer, briefly stain the membrane with Ponceau Red, which binds to proteins, to determine if the blotting worked.

Step 5: Antibodies. Ponceau Red binds to all proteins; to determine if there is a specific protein that the cells produced, antibodies, which bind to only one type of protein, are used. First, “block” the membrane to prevent random antibody binding by rocking the membrane back and forth for 1 hour at 4°C in a protein-rich fluid, such as skim milk or BSA. Wash off the blocker with TBST and add a solution containing a primary antibody, which binds only to the protein that you want to detect, and rock it back and forth it with the membrane for 3 to 18 hours, depending on the protein, and probably at 4°C. Finally, wash off the primary antibody with TBST and add a secondary antibody, which not only binds to the first antibody but also carries a molecule that fluoresces when exposed to UV light. Rock it back and forth it for 1 to 2 hours at room temperature.

Antibodies

Diagram by Imoen. Antibodies in western blotting: B is the blocker, A is the protein of interest, “Rabbit anti-A” is the primary antibody that binds to A, and “Goat anti-rabbit” is the secondary antibody that binds to Rabbit and also carries the fluorescent tag.

 

Step 6: Imaging. Finally, with the fluorescent secondary antibody bound, the membrane is ready to be imaged. There are several ways to do this, but in our lab, we take the membrane to a darkroom, and place it in a UV emitting machine along with film. The UV excites the secondary antibody, causing it to fluoresce and release light onto the film, developing it. Wherever the film turns black, secondary antibody had bound, and, if everything went well, the protein of interest had also bound.

Using western blotting, I aim to determine if the mutated histone with methionine in place of lysine 36 changes the amount in which several proteins are produced. If one or more of these proteins does change, then it will support my hypothesis that changes in these proteins’ levels abrogate lysine 36 trimethylation. If not, well, then, I have other hypotheses to test, probably after the poster session, but I will have ruled out ones that don’t work.

qPCR – SYBR green to measure a gene

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If the genome is a library filled with both useful and useless information, then gene regulation is the librarian who directs you to only the books you find useful. All of the labs on the fourth floor of North Frear are trying to determine how cells find and express—that is, turn on—useful genes while repressing genes that they don’t need at the moment, or ever. Lately, Jinquan has been using a technique called either real-time PCR or quantitative PCR (qPCR) to measure how much of certain genes her cell samples are expressing. When a cell expresses a gene, it first transcribes it, which is the process of making a messenger RNA (mRNA) molecule from the DNA. Thus, if a cell has a lot of mRNA for a certain gene, it is highly expressing that gene.

To measure how much mRNA cells are making for a given gene, we first extract the mRNA from the cell and then synthesize DNA from it, which I described in an earlier post. In order for PCR to work, we need to add primers, which are short sequences of DNA that are complimentary to regions of the DNA strand we want to replicate. This is fortunate, because we only want to measure the DNA of one gene; if every strand was replicated, we would have a lot of DNA, but we wouldn’t know how much of it was for our gene of interest. Since every gene has a different DNA sequence and we know the sequence of the gene that we want to replicate, we can choose a “forward” primer that binds to the beginning of the gene and a “reverse” primer that binds to the end. Only DNA in between the primers (inclusive of the primers) will be replicated.

We add all of the reagents in qPCR to a plastic plate that has 96 wells in it. This is very useful if we want to measure the expression of several different genes, because we can add the primer for one gene to the wells in one column, the primer for a different gene to the wells in another column, and so on. Additionally, we can add DNA from different types of cells to different rows, so that way, we can simultaneously measure the expression of several genes in several types of cells. Here, we measured twelve genes, including the genes for PUMA, MCL1, and actin, in DNA samples from four different kinds of cells.

96-well PCR plate

 

 

 

 

 

 

 

Life technologies: “MicroAmp Optical 96-Well Reaction Plate.” A plate used to perform qPCR.

With 96 wells to fill, it makes sense to make a master mix of reagents common to every tube and then to add the specific reagent to each tube—this saves you 96 tedious pipette actions. Whether the specific reagent is the set of primers or the DNA sample from the type of cell depends on the number of genes and types of cell being measured. If you are measuring more genes than cell types, make master mixes that include the DNA from a specific cell type; otherwise, make master mixes that include the primers for a specific gene. In our case, we had four types of cells and twelve genes. If we made a master mix with the primers for each gene, we would have needed to make twelve different master mixes; instead, we made only four different master mixes by adding the DNA from each cell type to the common reagents.

The common reagents—the DNA polymerase, the free nucleotides, and the all-important SYBR green dye (more on that later)—were pre-mixed for us, so for each DNA sample, we simply added 100 µL of DNA sample to 150 µL of common reagents, making 250 µL total for each DNA sample. Since we had four cell types and 96 wells, each one occupied 24 wells; for each 250 µL mixture, Jinquan pipetted 10 µL into each well, leaving 10 µL as an error margin (no pipette measures perfectly). Then, she added 2 µL of each mixture of forward and reverse primers to each well—twelve different primer sets, eight wells per set.

After adding the reagents, Jinquan sealed the wells closed with a sheet of sticky plastic and placed them in the qPCR thermocycler machine. The thermocycler cycles through several temperatures to allow the DNA polymerase to replicate the DNA. In my earlier post on PCR, I was incorrect about the number of different temperatures used in PCR. The correct cycle is as follows, although specific temperatures and times vary slightly from PCR to PCR:

Temp.             Time                Purpose

94°C                3 min               initially denature: heat to separate the DNA strands from each other

94°C                30 sec              dentature: heat to separate the DNA strands from each other

55°C                30 sec              anneal: cool to let the primers bind to the DNA

72°C                40 sec              elongate: warm to allow DNA polymerase to replicate the DNA

72°C                10 min             finally elongate: allow the DNA polymerase to finish replicating DNA

4°C                  indefinitely      stop: chill for storage of the DNA to prevent DNA breakdown

PCR steps

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Madprime: “PCR,” from Wikimedia Commons. The original DNA template is in blue, primers in red, and newly synthesized DNA in green; the DNA polymerase enzyme that synthesizes new DNA is the green circle.

While the first, penultimate, and last steps are performed once, the bolded steps are repeated for the number of times we want to double the amount of DNA. qPCR usually takes between 30 and 40 cycles. Each cycle, the thermocycler measures the amount of DNA in each well. I mentioned earlier that the master mix contains SYBR green, which is a molecule that binds to DNA. When and only when it is bound, SYBR green will emit green fluorescent light when it is illuminated by blue light. To measure the amount of DNA, then, the thermocycler emits a flash of blue light at the end of the 72°C step, and it measures the amount of green light emitted; more light means more SYBR green is bound to DNA means more DNA.

Below a certain number of DNA molecules, there will be so few SYBR green molecules bound to DNA that the thermocycler cannot detect the green light produced. Therefore, there is a certain threshold number of molecules required for DNA detection. The thermocycler measures the number of cycles that it takes to first detect DNA and calls that number the threshold cycle, abbreviated Ct. Since the amount of DNA doubles every cycle, if one gene reaches threshold one cycle later than another, there were approximately half of the number of DNA molecules to begin with, and thus half of the expression for that gene in the cell that made the DNA. If a gene reaches threshold two cycles later, it had about one fourth of the expression; n cycles later means 1 / 2n times the expression.

qPCR thus provides a way to measure the relative expression of genes in cells, although it does not measure the absolute number of DNA molecules in the original samples. Usually, the expression of each gene is compared to the expression of a reference gene whose expression varies little between different types of cells; actin, a popular choice, is the one which we used in the aforementioned experiment. Comparing the expression of a certain gene to the expression of actin, we can determine how much the cells express it and how the gene is regulated in different types of cells or cells exposed to different chemicals.