Meet the Crew
Mike “Couniham” Counihan and the Skin Project
The stratum corneum (SC) is the outermost layer of the epidermis, the outer part of the skin in mammals. The SC consists of corneocytes (dead cells) suspended in an extracellular lipid matrix. This lipid matrix is composed of ceramides (Cers), cholesterol (Chol), and free fatty acids (FFAs). The vast majority of the hydrocarbon chains in the Cers and FFAs are fully saturated (no double bonds). Together with their relatively small headgroups, the lipids pack together more closely than a typical cell membrane (which is made up of phospholipids with a mix of saturated and unsaturated tails). Most commonly, the matrix is made up of three bilayers in between each corneocyte, giving it both lateral (horizontal, within a layer) and lamellar (vertical, between layers) organization.
Organization of the human stratum corneum (van Smeden et al., 2013)
The Skin Project looks at the lateral phase behavior of the lipid matrix and specifically how each individual component (Cers, Chol, and FFAs) affects the fluidity of the lipid layer. An example, topical medications need to penetrate the SC to reach living cells in deeper layers of the skin, so knowing what makes the SC lipid matrix more fluid, and thus more permeable to these types of medicines, will aid in the development of more efficient drugs.
SC matrix lipids: ceramide (top), cholesterol (middle), and free fatty acid (bottom)
To investigate lateral lipid organization, we create lipid monolayers on a water surface using a Langmuir trough. The lipids’ polar headgroups lie on the air-water interface, and the nonpolar hydrocarbon tails point in the air, which represents one layer in the SC lipid matrix. We then compress the lipids using barriers on the water surface and measure the change in surface tension at the interface as the lipids get closer and interact with one another. This gives us information about the fluidity of the lipid monolayer. By varying the lipid mixture composition (e.g., more Chol, less FFA), we can tease out which lipid produces greater lateral fluidity in the lipid matrix. Additionally, we can use fluorescence microscopy to visualize the monolayer and image the fluid region.
Our Langmuir trough with fluorescence microscope setup
Cartoon depiction of a lipid monolayer on a Langmuir trough
David “Comic Sans” Van Doren and the Nanoparticle Project
Nanoparticles have a large, and growing, array of applications in industrial and medical settings. Their increasing use and vast potential make the characterization of nanoparticle interactions an important task for not only developing technologies, but also for understanding their potential toxicity to biological systems. Nanoparticles can interact with cells by adhering or inserting into the lipid bilayer of the plasma membrane. This interaction can have various disruptive effects, such as changing the fluidity of the membrane, affecting one or more phases of the lipid domain system, or even creating pores in the membrane. This project aims to examine nanoparticle-lipid relationships by measuring various nanoparticle interactions on model membrane systems such as lipid monolayers or giant unilamellar vesicles (GUVs), with the intent of relating these simplistic, representative systems to cells that would be affected by similar encounters with nanoparticles.
Giant unilamellar vesicle made of DPPC/DOPC/cholesterol
GUVs, vesicles made up of a single bilayer of lipids, were prepared using DPPC, DOPC, and cholesterol to model the cell membrane. Polystyrene nanoparticles, functionalized with either amine or carboxyl groups, were applied to populations of GUVs to characterize the effects of positively and negatively charged nanoparticles on the membrane. Morphological changes in the membrane were monitored by fluorescence microscopy. By understanding basic factors that influence the nanoparticle-lipid interaction, such as charge and size of nanoparticles, researchers can begin to predict and anticipate adverse health impacts of similar nanoparticles used in products and treatments.
David in the microscope room.
The Mom-brane Lady
Dr. Shelli Frey, known as Dr. Almighty Supreme Pastry Chef Frey in this corner of the science center, puts up with a great deal to ensure that we have an enjoyable and meaningful research experience (including being referred to by ridiculous, but fitting, titles). She fields all of the struggles, complaints, and headaches that we bring to her and sends us on our way with plans of attack for fixing what ever trouble we’ve gotten ourselves into. Currently, Dr. Frey has a lovely toddler named Ellie and a second daughter on the way (yay!). Both of her children have made considerable contributions to lab work this summer. Ellie helped describe lipid domains with a fantastic drawing, while Dr. Frey and her child-on-the-way have been tag-teaming lab work together. One might argue that Dr. Frey is even more efficient in lab in her current state of pregnancy, as her baby bump acts as a convenient and portable lab bench. Dr. Frey has asked us to clarify that she should not be confused with the psychic medium Shelly Frey, who visited our area recently. While Dr. Frey is all knowing and all seeing, she thankfully does not charge $250 for advice.
Ellie’s depiction of lipid domains
Extreme Makeover: Lipid Lab Edition
Summer research this year kicked off just as any other summer must: with cleaning up the messes that we let accumulate over the two semesters of research during the school year. Cleaning vials in the Lipid Lab is a big deal. It is a process that requires patience, determination, and a heck of a lot of concentrated sulfuric acid. While the Lipid Lab crew knew that they would need to deal with the endless whine of the sonicator and the burning from the lactic acid built up after hours of manually jostling vials, they persevered through their first few days knowing that cleaning vials was a small price to pay for the countless hours of uninterrupted research they would enjoy further down the road.
Water usually isn’t hard to come by in the Lipid Lab; we get a few gallons cascading through the laboratory’s wall and ceiling every few rainstorms. However, cleaning out the tubing of the water heater requires ceiling particulate-free water. The lack of a sink in the trough room required the famous engineering prowess of the Lipid Lab’s senior chemistry research assistants to provide flowing water to the otherwise arid space.
Aquaduct construction in the P-Chem lab
New Toys and Gadgets
Look how cute this guy is! Last summer, the Lipid Lab purchased a small trough from KSV NIMA that was used for experiments in the interdisciplinary Chem 358 course titled Salty and Fatty. He is used for creating lipid monolayers and obtaining data about the material properties of cell membranes. He holds an adorable 180 mL. Compared to Papa Trough situated in the trough room, he is a tad smaller, but comes with updated software and a sleek control box that makes him pretty fantastic to run experiments on.
Vials weren’t the only components that needed attention. After finding homes for the piles of articles and clutter that had built up in the prep lab (courtesy of Warren Alexander “Your Data Disturbs Me” Campbell IV), we next turned to the trough room. If you are avid Lipid Lab blog readers or have mistakenly found your way into the trough room this summer, you may notice that the Papa Trough is no longer connected to the fluorescence microscope that is usually on the lab’s vibration-canceling table. The motorized microscope stage required maintenance to deal with a bit of corrosion, so Mike decided to take on the rust himself. Armed with chemistry know-how and razor-sharp reflexes, he managed to dismantle a good portion of the platform. However, a wrench was thrown in his plans when he did not have the proper tools to completely take it apart (#badpun). The Lipid Lab crew wrapped up the stage in a hand-crafted cocoon of plush foam and sent it to the only person guaranteed to be able to satiate our eternal need for lateral motion: Ernie (the instrument tech at Siskiyou).
Mike tirelessly tinkering with the microscope stage.
Mike contemplating screwdrivers.
Lipid Lab Strives to be X-SIG-tastic
Working in the far corner of the chemistry department all day can get lonely. One of the goals of the X-SIG summer experience is to have undergraduate research students engage with students and concepts outside of their immediate field of interest. Aching for more human interaction and cross-disciplinary hangouts, the Lipid Lab hosted an event for chemistry, biology, and physics students to connect over a movie, Exploring the Living Cell, which deals with the basics of cellular life in the hopes that all students, especially those outside of biology, could further their understanding of the life sciences. Those that attended know that the Lipid Lab now endorses plankton for therapeutic purposes.
Exploring the Living Cell. Kleiner, V; Sardet, C. 2006. CNRS. Film.
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This work is made possible by the efforts of the Gettysburg Chemistry Department, HHMI, Avanti Polar Lipids, and viewers like you. Thank you.
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