The Search for High Quality Data

Kelly Townsend, Elmhurst College

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Photo Cred: Ashley Shaw

The Approach: In my previous post, I mentioned the importance of sea turtles to ecosystems and ecotourism. While very important, the populations of these endangered animals are declining largely due to human impacts on our oceans. Headlines of sea turtles washing up on shore from such things as being strangled by plastic or boat strikes is no new occurrence. Since sea turtles are declining for reasons largely caused by us, so it is up to us to save these beloved animals. This study aims to investigate the stability of two important health indices, RNA and plasma protein in sea turtle blood, at different temperature treatments over time.  These indices are frequently used by researchers to answer health related questions. Therefore, my study will hopefully aid other researchers in determining if their samples are of the right quality to measure these indices.

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Collecting blood from a Loggerhead Sea Turtle (Caretta caretta) Photograph authorized by NMFS Section 10(A)(1)(a) permit 19621

For this project, whole blood was obtained from loggerhead sea turtles off the coast of South Carolin. The blood was collected in either Vacutainer blood collection tubes containing sodium heparin or PAXgene tubes. Sodium heparin tubes contain an anticoagulant and are typically used for blood collection. PAXgene tubes contain an RNA preservative; therefore, are best suited for RNA analysis. The sodium heparin tubes used for plasma were centrifuged on the boat to separate the blood components (i.e, plasma, white blood, and remaining cells) while the PAXgene tubes for RNA were left unspun. Once in the lab, the tubes were divided out into approximately 1.5ml aliquots in order to subject them to different treatments. Plasma was used for the plasma protein treatments while whole blood was used for the RNA treatments. Treatments in this study included 4⁰C for seven days, 20⁰C for three days, delayed freeze time, and never frozen. There were also treatments that lasted twenty-eight days consisting of storage in cryogenic conditions (< -150⁰C), -80⁰C, and -20⁰C (frost-free and non-frost-free freezers). Once the treatments end, the plasma will be analyzed for protein concentrations via plasma electrophoresis, and the whole blood will be analyzed for RNA quality via RNA isolation followed by a bioanalyser to obtain RNA integrity numbers (RINs).

 

As a result of the study, I hope to determine the conditions at which plasma proteins and RNA are most stable and begin to lose stability in order to aid scientists with their research. By knowing these conditions, this will hopefully guide others in deciding how to store samples along with which ones are best suited for a variety of analyses. In order to help the sick and endangered sea turtles, the highest quality of research will be necessary which means high quality data. I hope this study will guide researchers to make that possible.

I would like to thank Dr. Jennifer Lynch, Jennifer Trevillian, and Jennifer Ness with the National Institute of Standards and Technology for being my supportive and awesome mentors. This project was made possible by the samples collected by Dr. Michael Arendt and the funding from the National Science Foundation (NSF DBI-1757899) supported by the Fort Johnson REU program.

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Small Steps to Save the Sea Turtles

Kelly Townsend, Elmhurst College

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Turtle trawl on the R/V Lady Lisa. Photograph authorized by NMFS Section 10(A)(1)(a) permit 19621.

The problem: Do you like sea turtles? As for me, I have fallen in love with these cute creatures who occupy parts of the ocean. Seeing them pop their heads up or glide through the water always amazes me, but many species are endangered. A lot of effort has gone into saving them since sea turtles play an important role in the marine ecosystem. The marine ecosystem makes up a part of our world that is deeply loved but also threatened. Sea turtles help marine ecosystems function by limiting the amount of seagrass beds and sponges through consumption (McClenachan et al., 2006). Therefore, sea turtles presence in the environmental community is key to ecosystem restoration where their numbers have dropped and seagrass disease has been able to spread and coral overgrowth has increased. In addition, sea turtles also play an important role in ecotourism. Places like Costa Rica, United States, and Australia use sea turtles as a source of income by promoting tourism in areas where they live or nest, offering turtle walks, and selling souvenirs (Campbell, 2003). Since sea turtles act as an important resource for humans, there has been much effort into rehabilitating injured sea turtles and researching them in order to determine better prognostic indicators and courses of treatment. Sea turtles are important to us environmentally and economically, so saving them from going extinct requires the most reliable research and data possible to make that happen

 

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Turtle nesting beach located in Tortuguero, Costa Rica.

RNA and plasma proteins are both potential indicators for overall organismal health, but they can degrade quickly if not properly stored. Plasma protein concentrations in sea turtles can help wildlife veterinarians diagnose a disease and create a proper treatment plan (Gicking et al., 2004). Therefore, measuring plasma proteins in archived samples can indicate when or if a . particular disease developed in sea turtles. In addition, RNA concentrations and quality are good indicators of general health. High ratios of RNA/DNA has shown indications of increased cellular protein synthesis along with increased growth potential which means the sea turtle is growing properly (Vieira et al., 2014). However, in order to use archived samples to accurately track health indicators such as plasma proteins and RNA, it is vital to know if storage conditions allowed degradation of these molecules.

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Whole blood tubes used for RNA analysis.

This study aims to investigate RNA and plasma protein stability at different temperature treatments over periods of time. Samples will be maintained in favorable conditions along with unfavorable conditions to analyze the difference between the qualities. By knowing what happens on a molecular level to blood when storage conditions go wrong, we hope to eliminate the use of low quality samples used in research. Freezers malfunction, people forget to put samples away, and blood may not be put in the proper place so the results of this study will become a reference to those researchers who experience these tragedies.

I would like to thank Dr. Jennifer Lynch, Jennifer Trevillian, and Jennifer Ness with the National Institute of Standards and Technology for being my supportive and awesome mentors. This project was made possible by the samples collected by Dr. Michael Arendt and the funding from the National Science Foundation (NSF DBI-1757899) supported by the Fort Johnson REU program.

References:

Campbell L. 2003. Contemporary culture, use, and conservation of sea turtles. In: Lutz PL, Musick JA, and Wyneken J (Eds). The biology of sea turtles, volume 2. Boca Raton, FL:   CRC Press.

Gicking JC, Foley AM, Harr KE, Raskin RE, Jacobson E. 2004. Plasma protein electrophoresis of the atlantic loggerhead sea turtle, Caretta caretta. Herpetological Medicine and Surgery 14:13-18.

McClenachan L, Jackson JBC, Newman MJH. 2006. Conservation implications of historic sea turtle nesting beach loss. Front Ecol Environ 4:290-296.

Vieira S, Martins S, Hawkes LA, Marco A, Teodosio MA. 2014. Biochemical indices and life traits of loggerhead turtles (Caretta caretta) from cape verde islands. PLoS ONE 9:e112181.

Manatees and PFCs- The Future of Contaminant Studies

Kady Palmer, Eckerd College

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In my previous post, “The Problem with PFCs- Seeking Answers in Plasma”, the abundance of perfluorinated chemicals, or more specifically perfluoroalkyl acids (PFAAs), was analyzed in manatee plasma and correlated to variables such as site, water temperature, and blood chemistry. The purpose of this study was to develop a greater understanding of these chemical contaminants in regards to their routes of exposure and subsequent health effects.

Accumulation of PFAAs within organisms is proposed to be predominantly attributed through diet. Therefore, apex predators, like alligators, dolphins, and humans are found to be at a higher risk for increased concentrations of these chemicals in their body (Bangma et al., 2017, Fair et al., 2012). This is a result of biomagnification, or increasing levels of a compound as one continues up the food chain or trophic hierarchy. Manatees, however, are not predators, and are considered lower on the trophic hierarchy due to their herbivorous diet. With that knowledge, the amount of PFAAs within them, if any, was hypothesized to be very small.

After obtaining data from chemical extractions and liquid chromatography tandem mass spectrometry (LC-MS/MS), concentrations of at least two perfluoroalkyl acids were detected in all 69 manatee plasma samples. What that means is that PFAAs are integrating into the biological systems of manatees and accumulating within their bloodstream, presenting different results than our initial hypothesis.

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One of the most common PFAAs found in manatee plasma, known as perfluorooctanesulfonic acid (PFOS). Photo from: http://pubs.sciepub.com/ces/2/1/3/

Data and statistical analyses determined location-based differences in PFAA concentrations. In addition, correlations were found between high PFAA burden, blood chemistry measurements, and water temperature at the time of sampling. With this information, a basis for further investigations is possible to begin determining potential health effects of PFAAs in not only manatees, but in humans as well.

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Because manatees cannot tolerate cold water, they congregate in warm waters during the winter seasons. Interestingly, correlations between water temperatures and PFAA values were found in this study. Photo from: http://proof.nationalgeographic.com/2014/07/21/floridas-manatees-the-search-for-warmer-water/

In summary, the purpose of this experiment was to answer two questions: 1) Are PFAAs present in manatee plasma? 2) If so, can heavy burdens of PFAAs be statistically correlated to health variables?

The first question was answered within the first week of analysis, simply by identifying detectable levels of these chemicals in manatee plasma. The second question, however, is more complicated to answer. The statistics say that there are associations between PFAAs and differing health measurements, however, the significance and meaning of that data is something future research must focus on. The reasons behind the correlations are still unknown, even though some explanations may be proposed.

I would like to extend an enormous thank you to everyone who made this project possible, including Dr. Jacqueline Bangma, Dr. Jessica Reiner, and my extremely motivating mentor, Dr. John Bowden. I would also like to thank the National Science Foundation for their funding, the College of Charleston’s Grice Marine Lab for hosting this REU, and the USGS Sirenia project for supplying the samples I utilized in this project.

References:

Bangma, Jacqueline T., John A. Bowden, Arnold M. Brunell, Ian Christie, Brendan Finnell, Matthew P. Guillette, Martin Jones, et al. “Perfluorinated Alkyl Acids in Plasma of American Alligators (Alligator Mississippiensis) from Florida and South Carolina.” Environmental Toxicology and Chemistry, no. 4 (2017a): 917. doi:10.1002/etc.3600.

Fair, Patricia A., Magali Houde, Thomas C. Hulsey, Gregory D. Bossart, Jeff Adams, Len Balthis, and Derek C.G. Muir. “Assessment of Perfluorinated Compounds (PFCs) in Plasma of Bottlenose Dolphins from Two Southeast US Estuarine Areas: Relationship with Age, Sex and Geographic Locations.” Marine Pollution Bulletin 64 (January 1, 2012): 66–74. doi:10.1016/j.marpolbul.2011.10.022.

 

The Problem with PFCs- Seeking Answers in Plasma

Kady Palmer, Eckerd College

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I previously outlined the problem of perfluorinated chemicals (PFCs) in the environment and their unknown health effects.  In order to gain this knowledge, it is essential to determine what types of PFCs are frequently used and the mechanisms by which an individual would be exposed to them. Here, we are measuring the presence or absence of 15 PFCs that are commonly associated with non-stick cookware, firefighting foam, and water-resistant materials.

This compiled list of PFCs is the basis of my research procedure. From here, I must learn how these compounds interact with biological components in organisms in order to understand their subsequent health effects. With that being said, the type of samples I am analyzing is a topic worth explaining. PFCs are known to be “proteinophilic” or, attracted to proteins in the bloodstream of organisms like humans and, in the case of my study, manatees. Therefore, I am using manatee plasma to test for the total individual burden of PFCs. 

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Fig 1. 69 collection tubes containing manatee plasma samples (left). Aliquots of 22 samples of manatee plasma for future studies (right). Photos taken by me!

With 69 different plasma samples, I am performing a series of procedures that allow me to extract the PFCs. After completing multiple chemical processes (methodology proposed by Reiner et al., 2012), I am left with a liquid (containing the PFCs), measuring no more than 1 mL to be placed into a small vial. From here the vials are inserted into a liquid chromatography tandem mass spectrometer (LC-MS/MS), a machine that reads each of the 15 unique chemical structures of the outlined PFCs of interest and determines their abundance in each vial. This system isolates the concentration of each perfluorinated chemical for every one of the 69 manatee samples.

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Fig 2. The basic process a mass spectrometer performs in order to provide the concentration of chemicals being studied. Photo from: http://www.emdmillipore.com/US/en/water-purification/learning-centers/Anwendungen/organic-analysis/lc-ms/lWib.qB.vb4AAAFA5fIBvVBh,nav?ReferrerURL=https%3A%2F%2Fwww.google.com%2F&bd=1

The concentrations of these chemicals is the ultimate goal of my research study. This data will be compared to manatee location, morphometrics, body condition, sex, and more, in order to gain a better understanding of the overall PFC burden on these animals. These factors, or variables, may also provide insight into what may be influencing the burden intensity an individual may face. Once this knowledge is gathered, potential links to the health effects of PFC accumulation can be investigated in both manatees and humans.

I’d like to thank the National Science Foundation for funding this research opportunity and the College of Charleston’s Grice Marine Laboratory REU program for making this experience possible. A special thanks to the NIST team who has been teaching and supporting me throughout this process, specifically, Dr. Jessica Reiner, Jacqueline Bangma, and my mentor, Dr. John Bowden.

Note: These samples were collected as part of a health assessment of manatees by the USGS Sirenia Project. No manatees were harmed in the process of obtaining them.

References

Reiner, Jessica, Karen Phinney, and Jennifer Keller. “Determination of Perfluorinated Compounds in Human Plasma and Serum Standard Reference Materials Using Independent Analytical Methods.” Analytical & Bioanalytical Chemistry 401, no. 9 (January 15, 2012): 2899–2907. doi:10.1007/s00216-011-5380-x.z

Gas Chromatography for Fatty Acid Analysis

Jack McAlhany, Wofford College

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In applying to College of Charleston REU at Grice Marine Laboratory, I assumed I would be researching the well-being of a local fishery or the biomechanics of an organism in response to different variables, but this was not the case. My research to this point has not focused on a living organism, but rather tiny portions of blood plasma that we vaporize into a gas to determine the basic constituents of the blood. A solid understanding of chemical structures, or a wild imagination, is necessary for this research because unlike working with live samples, our desired product is not visible. To give you a better understanding, the fatty acids we are detecting have between 8 and 24 carbon atoms while a 70 kg human has approximately 7X10^26 carbon atoms (Kross, B.).

In order to detect such tiny fat molecules I am using a gas chromatography with a flame ionization detector (GC-FID). This instrument converts a liquid sample to a gas that then travels through a 100 meter column with an inert gas carrier. The time at which certain samples exit the column is reproducible, so the retention time in the column is a way to determine which fatty acids are in a blood plasma sample (E.T.S. Laboratories).

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Figure 1: In a GC-FID, a sample (red) is vaporized in a vaporizing column (blue) and an inert gas carrier is the “liquid” phase that carries the vaporized fatty acid methyl ester (FAME) through a 100 meter column (gold). This column has a stationary phase that interacts differently with each FAME, causing the FAME’s to exit the column at different times. The detector responds to compounds that form ions when combusted in the hydrogen-air flame. This response is converted to electrical signals and produce a gas chromatogram we can analyze (E.T.S. Laboratories).

These retention times are represented on a gas chromatogram, which consists of peaks of varying sizes (Figure 2). Each fatty acid interacts in a unique, but constant, way with the stationary phase of the column, so fatty acid peaks are in the same order and have the relatively same retention time every trial. This constant retention time allows us to determine the fatty acid composition of a sample. Not only can we find the blood plasma fatty acid composition, but by analyzing the area under each peak, we can determine the concentration of each fatty acid in the blood. The concentration of each fatty acid or relationships in these concentrations hopefully will provide a biomarker that we can test in the field for the fatty acid disease, pansteatitis.

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Figure 2: Gas Chromatogram of 19 fatty acids. Each peak is labeled with its corresponding fatty acid (Christie W. 2011).

Acknowledgements

I would like to thank the College of Charleston for this internship, the National Science Foundation for funding, and Dr. John Bowden for his guidance as a mentor.

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Works Cited

Christie, W. 2011. Gas Chromatography and Lipids. The Oily Press” Chapter 10.http://lipidlibrary.aocs.org/GC_lipid/10_misc/index.htm

E.T.S. Laboratories https://www.etslabs.com/analysis.aspx?id=$GCP

Kross, B. Questions and Answers. Jefferson Lab. http://education.jlab.org/qa/mathatom_04.html.