Category Archives: Molecular and Cell Biology

Monkeying around in the lab to find a good egg

In vitro fertilization (IVF) treatment is a procedure in which a woman’s mature eggs are removed via surgery, combined with sperm in a petri dish in a lab, and then the fertilized egg is placed in the uterus to continue growing into an embryo. Unfortunately, IVF is not covered by all insurance companies and is successful less than 50% of the time. Consequently, undergoing IVF can be a significant burden financially, physically, and emotionally for those who seek out this procedure.

What makes a “good” fertilizable egg? In this week’s special episode, we’re joined by Sweta Ravisankar, a 5^th year PhD candidate in the Cell and Developmental Biology program at OHSU (Oregon Health & Science University), who is trying to answer this question in hopes that being able to screen for the “more likely to succeed” eggs, will lower the economic, financial, and physical hurtles of IVF.

Sweta works at the at Oregon National Primate Research Center, OHSU within the division of Reproductive and Developmental Sciences OHSU. She is a graduate student mentored jointly by Dr Shawn Chavez and Dr. Jon D. Hennebold.

The Hennebold lab studies reproduction before the egg is fertilized. This stage involves studying the female reproductive system, the oocyte (egg) itself, and the development of the follicle (region that holds the immature eggs) before ovulation (dropping of immature egg into the ovary). In contrast, the Chavez lab looks at what happens after fertilization such as chromosome abnormalities and how these abnormalities effect embryo development. This joint mentorship allows Sweta to study a more complete story of development.

Screenshot from a video of development from 1C stage to a blastocyst stage. Complex human being development can be traced back to these 120-150 cells implanting in the uterus.

Sweta is always excited to share her science!

Looking at reproduction from these two perspectives allows Sweta to correlate the environment the egg exists in with how the embryo develops. For example, what is the impact of a western style diet (high in fat) on the biochemistry and development of follicles and embryos long term? How does polycystic ovarian morphology (POM) mimicked by prolonged exposure to high fat diet and high testosterone levels in females impact reproductive success at the biomolecular level?

Will work when needed: in the lab on a weekend with a cast on my foot (visible on the left leg).

Being at the Oregon National Primate Center, Sweta’s model organism is the “Rhesus macaque” monkey. These monkeys have a genome ~97.5% similar to humans, meaning that the work she does is very relevant and translatable to humans. Working with the monkeys also means that her research is variable depending on the day. The monkeys will sometimes undergo treatments similar to those done in human IVF (in vitro fertilization) clinics, including surgeries to collect eggs for further research. After harvesting these eggs, they can be fertilized and the cells’ growth, division, and development can be monitored in a plate. When these experiments are not taking place, Sweta conducts various molecular biology experiments.

Sweta has become a true Pacific northwestener: hiking in rain with her husband through the Washington Park, Portland, OR.

In India, Sweta completed her Bachelor’s degree at Dr. D. Y. Patil university in biotechnology and her first Master’s at SRM Institute of Science and Technology. During this time, Sweta happened to have several of family and friends undergoing IVF treatments and also worked in a fertility clinic for a time, bringing her attention to scientific needs within this field. Sweta then completed a second Master’s in Biological Sciences with a fellowship from the California Institute for Regenerative Medicine, and fell in love with fertility-related research during an internship at Stanford where she worked on embryo development. Her passion for this field of research led her to OHSU.

In addition to a being an accomplished researcher, Sweta is also an accomplished Indian Classical Dancer! She teaches bharatanatyam dance classes out of her home and travels around the US to perform. Long term, she hopes to continue research and also run a dance company.

Sweta will be presenting a piece on “depression” to work towards mental health awareness October 25th through 27th. The piece will be in Bharatanatyam and presented as a part of the 12th residency performance at N.E.W.

Sweta writes her own blog posts about her journey through grad school which can be found here:

To hear more about Sweta’s graduate work, personal struggles, and classical Indian dance moves, tune in on Sunday, October 20^th at 7 PM on KBVR 88.7 FM, live stream the show at http://www.orangemedianetwork.com/kbvr_fm/, or download our podcast on iTunes!

Genes & Body Metabolism: How our Muscles Control Outcomes

The basic human body plan is fairly similar (most have eyes, arms, and legs) but how efficiently our bodies’ function is unique and depend heavily on our genes. Although our brains use a lot of the simple energy compounds (like glucose), our skeletal muscles use 70% of our body’s total energy production such as fats, sugars, and amino acids. All of this energy demand from our skeletal muscles means our body’s metabolism is highly regulated by our muscles. If you want a higher metabolism then you should work out more to gain muscle; this process of muscle formation or repair is a complicated sequence of events requiring hundreds of genes all working together at the right time to promote muscle development. However, if one or many genes do not function properly this sequence of events have inefficiencies that diminish our muscle production capability; for some this means more time at the gym but for others it could lead to diseases like diabetes.

Vera working with her mouse models to better understand how a body’s metabolism is controlled by their genes

Our guest this evening is Vera Lattier (Chih-Ning Chang) who is a PhD candidate in the Molecular and Cellular Biology Program focusing on one gene in particular that orchestrates the muscle formation process at various stages of life development. This PITX2 gene is implicated in regulating the activity of other genes as well as formation of the eyes, heart, limbs, and abdominal muscles during embryonic stages. During later stages of life the amount of skeletal muscle you have dictates your bodies metabolism, and if you are unable to build muscles you tend to have a lower metabolism that encourages excess food to be stored as fat. This is the first step towards obesity and is also a precursor to developing diabetes that affects nearly 26 million people in the United States. Although eating right and exercising can have a substantial impact to your health, if your genes are not functioning correctly poor health may ensue at no fault of the patients.

Vera’s research uses mice as models to better understand this complex interaction between our genes and our body’s metabolism. As part of a decade’s long research through Dr. Chrissa Kioussi’s research lab at Oregon State University they examined the role of this PITX2 gene in three main stages of muscle formation. By mutating the gene to affect it’s expression (effectively ‘turning off’ the gene) during early embryonic formation the mice bodies were unable to effectively create the physical structures for basic bodily functions and they were not viable embryos. When mutating the gene near the time of birth the mice were fully functional at the early stage of life and seemed normal. However, when they grew older they quickly became obese, in fact three times as heavy as the average mice, that lead to fatty liver disease, enlargement of the heart, obesity, and of course diabetes. Vera’s work continues to try and elucidate the mechanisms behind the connection of our genes and our body’s metabolism through structural muscle formation that could help us to identify these limitations earlier and help save lives.

Vera giving presentations to scientific conferences to help people understand the importance of muscle in body metabolism.

There is so much more to discuss with Vera on tonight’s show. You’ll hear about her first experience with a microscope at a young age and how she dreamed of one day becoming an evil scientist (luckily her parents changed her mind). Be sure to tune in for what is sure to be an enlightening discussion on Sunday April 8^th at 7PM on KBVR Corvallis 88.7FM or by listening live.

Exploring a protein’s turf with TIRF

Investigating Otoferlin

Otoferlin is a protein required for hearing. Mutations in its gene sequence have been linked to hereditary deafness, affecting 360 million people globally, including 32 million children. Recently graduated PhD candidate Nicole Hams has spent the last few years working to characterize the activity of Otoferlin using TIRF microscopy. There are approximately 20,000 protein-coding genes in humans, and many of these proteins are integral to processes occurring in cells at all times. Proteins are encoded by genes, which are comprised of DNA; when mutations in the gene sequence occur, diseases can arise. Mutations in DNA that give rise to disease are the focus of critical biomedical research. “If DNA is the frame of the car, proteins are the engine,” explains Nicole. Studying proteins can provide insight into how diseases begin and progress, with the strategic design of therapies to treat disease founded on our understanding of protein structure and function.

Studying proteins

Proteins are difficult to study because they’re so small: at an average size of ~2 nanometers (0.000000002 meters!), specific tools are required for visualization. Enter TIRF. Total Internal Reflection Fluorescence is a form of microscopy enabling scientists like Nicole to observe proteins tagged with a fluorescent marker. One reason TIRF is so useful is that it permits visualization of samples at the single molecule level. Fluorescently-tagged proteins light up as bright dots against a dark background, indicating that you have your protein.

Another reason why proteins are hard to study is that in many cases, parts of the protein are not soluble in water (especially if part of the protein is embedded in the fatty cell membrane). Trying to purify protein out of a membrane is extremely challenging. Often, it’s more feasible for scientists to study smaller, soluble fragments of the larger protein. Targeted studies using truncated, soluble portions of protein offer valuable information about protein function, but they don’t tell the whole story. “Working with a portion of the protein gives great insight into binding or interaction partners, but some information about the function of the whole protein is lost when you study fragments.” By studying the whole protein, Nicole explains, “we can offer insight into mechanisms that lead to deafness as a result of mutations.”

Challenges and rewards of research

Nicole cites being the first person in her lab to pursue single molecule studies as a meaningful achievement in her graduate career. She became immersed in tinkering with the new TIRF instrument, learning from the ground up how to develop new experiments. Working with cells containing Otoferlin, in a process known as tissue culture, required Nicole to be in lab at unusual hours, often for long periods of time, to make sure that the cells wouldn’t die. “The cells do not wait on you,” she explains, adding, “even if they’re ready at 3am.” Sometimes Nicole worked nights in order to get time on the TIRF. “If you love it, it’s not a sacrifice.”

Why grad school?

As an undergraduate student studying Agricultural Biochemistry at the University of Missouri, Nicole worked in a soybean lab investigating nitrogen fixation, and knew she wanted to pursue research further. She had worked in a lab work since high school, but didn’t realize it was a path she could pursue, instead convinced that she wanted to go to medical school. Nicole’s mom encouraged her to pursue research, because she knew that it was something she enjoyed, and her undergraduate advisor (who completed his post-doc at OSU) suggested that she apply to OSU. She feels lucky to have found an advisor like Colin Johnson, and stresses the importance of finding a mentor who is personally vested in their graduate student’s success.

Besides lab work…

In addition to research, Nicole has been actively involved in outreach to the community, serving as Educational Chair of the local NAACP Chapter. Following completion of her PhD, Nicole intends to continue giving back to the community, by establishing a scholarship program for underrepresented students. Nicole remembers a time when she was told and believed that she wasn’t good enough, and while she was able to overcome this discouraging dialogue, she has observed that many students do not find the necessary support to pursue higher education. Her goal is to reach students who don’t realize they have potential, and provide them with resources for success.

Tune in on December 3rd at 7pm to 88.7 KBVR Corvallis or stream the show live right here to hear more about Nicole’s journey through graduate school!

Thanks for reading!

You can download Nicole’s iTunes Podcast Episode!

Earlier in the show we discussed current events, specifically how the tax bill moving through the House and Senate impact students. Please see our references and sources for more information.

Elucidating protein structure with crystals

Kelsey in the lab pipetting one of her many buffers!

Proteins are the workhorse molecules of the cell, contributing to diverse processes such as eyesight, food breakdown, and disabling of pathogens. Although cells cannot function without helper proteins, they’re so small that it’s impossible to view them without the aid of special tools. Proteins are encoded by RNA, and RNA is encoded by DNA; when DNA is mutated, the downstream structure of the protein can be impacted. When proteins become dysfunctional as part of disease, understanding how and why they behave differently can lead to the development of a therapy. In Andy Karplus’ lab in the Department of Biochemistry & Biophysics, PhD candidate Kelsey Kean uses a technique known as protein x-ray crystallography to study the relationship between protein structure and function.

Protein crystals. On the left, each blade making up this cluster is an individual crystal that needs to be separated before we can use them.

Protein diffraction. An individual crystal is placed in front of an x-ray beam and we collect the diffraction resulting from the x-ray hitting each atom in the protein crystal . Using the position and darkness of each spot (along with some other information), we can figure out where each atom in the crystal was originally positioned.

An electron density map. After collecting and processing our diffraction images, we get an electron density map (blue)- this shows us where all the electrons for each atom in the protein are- and this guides us in building in the atomic coordinates (yellow) for each part of the protein. It’s like a puzzle!

Crystallization of protein involves many steps, each of which presents its own unique challenges. A very pure protein sample is required to form an ordered crystal lattice, and hundreds of different buffer solutions are tested to find the ideal crystallization conditions. Sometimes crystals can take weeks, months, or a year to grow: it all depends on the protein. Once a crystal is obtained, Kelsey ships it to the synchrotron at Lawrence Berkeley National Laboratory, which provides a source of ultra powerful x-ray light beams. Exposure of the protein crystal to x-ray light results in a diffraction pattern, which is caused by the x-ray light diffracting off of all the atoms in the crystal. A map of electron density is generated from the diffraction pattern, and then the electron density map is used to determine where the atoms are located in the protein, like a complex puzzle. X-ray protein crystallography is really amazing because it allows you to visualize proteins at the atomic level!

In addition to her lab work, Kelsey is extensively involved in teaching and STEM outreach. For the past 3 summers, she has organized a week-long summer biochemistry camp through STEM Academy, with the help of a group of biochemistry graduate students. Kelsey has also been involved in Discovering the Scientist Within, a program providing 150 middle school girls with the opportunity to perform science experiments, including isolation of strawberry DNA and working with mutant zebrafish.

Kelsey completed her undergraduate degree in biochemistry with a minor in math at the University of Tulsa, where she was also a Division I athlete in rowing. She attributes her work ethic and time management skills to her involvement in Division I athletics, which required a significant commitment of time and focus outside of lab and coursework. During one summer when she wasn’t busy with competitive rowing, she performed experiments related to protein crystallography at the Hauptman-Woodward Medical Research Institute associated with the University at Buffalo.

Kelsey knew she wanted to pursue science from an early age. She grew up surrounded by scientists: her mom is a biochemist and her dad is a software engineer! She recalls playing with Nalgene squirt bottles as a kid, and participated in the Science Olympiad in middle school, where she engineered a Rube Goldberg machine. She cites early exposure to science from her family as one reason why she feels strongly about STEM outreach to students who might not otherwise receive encouragement or support. In the future, Kelsey would like to teach at a primarily undergraduate institution.

Please join us this Sunday, April 23rd on KBVR Corvallis 88.7FM at 7 pm PST to hear much more about x-ray protein crystallography, STEM outreach, and to hear an awesome song of Kelsey’s choosing! You can also stream this episode live at www.kbvr.com/listen.

The Sweetest Genes

Tonight we have the pleasure of speaking to Natalia Salinas who comes all the way from South America to work on producing more (and delicious) strawberries! Think about how often you see strawberries in the grocery store, but strawberries typically only produce one harvest per year. Some of Natalia’s work focus on identifying if the seeds’ DNA have the perpetual flowering characteristic so there are more potential harvests throughout the year. Just as important as quantity is quality; a second aspect of Natalia’s work is searching DNA markers to try and predict the sugar content in strawberries.

Ideally growers would like many harvests and sweeter strawberries, so tune in tonight at 7PM Pacific time to 88.7FM KBVR Corvallis or stream the show live at http://kbvr.com/listen to find out how Natalia can help your next milkshake be even more delicious!

Natalia is working to amplify the DNA sequences in strawberries to identify desirable traits.

The fruits of Natalia’s labor. Yum!

Genomics on the Farm: Breeding A More Resistant Rice

Photo courtesy the Jaiswal Lab

Tonight Noor Al-Bader of OSU’s Molecular and Cell Biology department joins us on the show to discuss her doctoral research concerning genomics and plant breeding. Working in Dr. Pankaj Jaiswal’s lab, Noor deals with large data sets of genetic information concerning varieties of Rice and Chia. The goal of her study is to determine which genes relate to the expression of traits implicated in stress resistance and nutritional content. Often the varieties of these crops grown for their value to farmers are susceptible to environmental stressors such as high salinity in water, drought, and high temperatures. These environmental concerns unfortunately promise to be increasing concerns in many areas such crops are grown due to the increasing impact of climate change. Wild types are often hardier, and genetic studies of both types hold promise for producing a “happy medium” capable of producing high yield, nutritious rice and chia that is also highly prosperous under less than desirable environmental circumstances. These new varieties are not produced via genetic modification in the lab, but bred on the farm, crossing strains generation after generation and recording the results with painstaking attention to detail- the old fashioned way. The contrast between the hands on work of horticulture and the hard science of genetics in the lab may seem a surprising pair, but in this case the genetics research is utilized to facilitate traditional methods of horticulture by simply speeding along a process that could normally take lifetimes. Just like in her research, Noor strives to have the best of both worlds.

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Inspiring Stories From Oregon State Graduate Students on 88.7 KBVR FM