This Is How We Do It ♫

Julianna Duran, Virginia Tech

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First and foremost, if you didn’t get the reference in the title please click here!

Now that I have educated you on the topic of music, let’s switch to science.

 The Approach: In my previous post I mentioned that I am studying the lipids of Nile Crocodile and Mozambique Tilapia. So the first thing I did is wrestle the reptile like Steve Irwin and hand catch my fish – just kidding, but imagine how cool that would be! My samples were collected from Lake Loskop, South Africa in 2014. Once they were in my possession, here is what I did.

  1. Sample Preparation
    • The muscle tissue samples I received looked like chicken breasts you buy from the grocery store – except the size of a fat bean. These solid chunks need to be turned into a fine powder for me to analyze them. This was done by freezing the sample in the cryomill machine – where the samples were shaken extremely fast and broken up

      Cryomill

      Cryomill

  2. Extraction
    • Think of what happens when you pour oil in water. They go to different ends and don’t mix, right? (Yes) That is exactly what I’m doing with my samples. We are adding lots of chemicals to break down fats into their building blocks: Fatty Acids! The muscle layer (organic layer) hates touching the chemicals, so I take that out and can use it for my next step!
    • Check out a video I made of one of my extractions
  3. Gas Chromatography
    • This instrument is how I will measure the amount of each fatty acid in my samples.
    • How does it work?
      • The sample is injected into the system and enters a narrow glass column. The sample separates in this column based on its weight and boiling point. The particle encounters a flame at the end of the glass, which detects what specific fatty acid it is. The computer then gets this signal and generates a graph showing a fatty acid profile. Each peak on the graph is a different fatty acid, and the height of the peak indicates how much of it there is in the sample.
      • For help envisioning this process, take a look at this video (I used it when I learned about this instrument!)

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        Chromatogram

Summary:

I will be physically and chemically breaking down my samples, then getting fatty acid profiles for each of my individual species. This is all to see if there is a difference between healthy and diseased species and what lipids are most affected by Pansteatitis!


Supported by the Fort Johnson REU Program (NSF DBI-1757899), Dr. Mike Napolitano, Dr. John Bowden, The College of Charleston, NOAA, and NIST. 


References:

CryoMill. https://www.retsch.com/products/milling/ball-mills/mixer-mill-cryomill/function-features/ (accessed Jun 18, 2019).

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Life in Plastic, It’s not Fantastic

Samuel Daughenbaugh, DePauw University

2DA71FE7-975A-4AA8-8A78-DF3D1E545F05The Problem: We live in a plastic world. Plastics have saturated all aspects of our daily lives and, as a consequence, have also entered the natural world.  About 8.3 billion metric tons have been produced in the past 60 years, playing a pivotal role in the advancement of modern society (Parker, 2018). Although they are used to create many things we enjoy and benefit from, there are serious consequences for the health of humans and the environment that are associated with their use.

We have found plastics in unexpected places, everywhere from human guts to the most remote locations on earth (Schwabl, 2018; Woodall, 2014). Plastics have a long list of negative effects on living organisms, but their impact in the ocean is of special concern. Pictures of turtles with straws up their noses, bottle caps spilling out of dead bird stomachs, and penguins strangled in plastic beverage rings are often posted on social media sites. Less widely known are the chemical additives that leach from plastics. Phthalates are one such group of additives that pose threats to the health of humans and marine life.

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Current Fort Johnson REU Interns (Julianna Duran not pictured) collecting plastic and sand dollars on Otter Island. (Photo credit: R. Podolsky)

Phthalates have been valuable to the plastic industry because they promote flexibility and durability in many plastics (EPA, 2017). An astounding 470 million pounds of phthalates are used in the United States every year (EPA, 2017). This presents a significant problem because phthalates interfere with the production of important hormones that regulate growth and metabolism in humans and other animals (Boas et al., 2012).

This summer I am exploring the effects of three different phthalates– dimethyl phthalate (DMP), di-n-butyl phthalate (DBP), and di-2-ethylhexyl phthalate (DEHP)–on the larval development of marine invertebrates, using the purple-spined sea urchin (Arbacia punctulata) as a model. Sea urchin larvae float freely in the water column for an extended period of time and, therefore, are vulnerable to many marine pollutants.

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Purple-spined sea urchin (Arbacia punctulata)

Sea urchins are an important model because they are closely related to humans. Both humans and sea urchins use a signaling hormone called thyroxine, which is especially important for growth in early developmental stages (Heyland et al., 2006). Exposure to phthalates can disrupt the production of thyroxine. Additionally, larvae are very important to study because they form the base of food webs. Being at the bottom of the food chain means they feed animals at higher levels, many of which humans rely on for protein. Therefore, understanding how phthalates affect sea urchin growth and metabolism can lead to new insights into how these pollutants directly and indirectly impact human health.

 Acknowledgements

I would like to thank my mentor, Dr. Robert Podolsky, for his continued support, guidance, and encouragement. This project is supported by the Fort Johnson REU Program, NSF DBI-1757899.

References

Boas, M., Feldt-Rasmussen, U., & Main, K. M. (2012). Thyroid effects of endocrine disrupting chemicals. Molecular and Cellular Endocrinology, 355(2), 240-248. 

Environmental Protection Agency (Ed.). (2017). Phthalates. America’s Children and the Environment, 3, 1-19.

Heyland, A., Price, D. A., Bodnarova-Buganova, M., & Moroz, L. L. (2006). Thyroid hormone metabolism and peroxidase function in two non-chordate animals. Journal of Experimental Zoology Part B: Molecular and Developmental Evolution, 306B(6), 551-566.

Parker, L. (2018, December 18). A whopping 91% of plastic isn’t recycled. Retrieved from  http://www.nationalgeographic.com

Schwabl, P. (2018, October). Assessment of Microplastic Concentrations in Human Stool. Conference on Nano and microplastics in technical and freshwater systems, Monte    Verità, Ascona, Switzerland.

Woodall, L. C., Sanchez-Vidal, A., Canals, M., Paterson, G. L., Coppock, R., Sleight, V., . . . Thompson, R. C. (2014). The deep sea is a major sink for microplastic debris. Royal      Society Open Science, 1(4), 140317-140317. doi:10.1098/rsos.140317

Crikey! What’s in the Water?

Julianna Duran, Virginia Tech

1B7047D7-DD01-4D65-B081-9D809AC07271The Problem: South Africa is home to some of the most extraordinary wildlife and culture. This diverse ecotourism plays a major role in their economy and conservation efforts.

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Nile Crocodile (Photo credit: Darren Poke)

The Olifants River System in the Mpumalanga Province is a large source of water that provides a habitat for several species. Over the last 30 years in this region, there have been dramatic declines of Nile Crocodile (Crocodylus niloticus), fish, and waterfowl.

The cause of this is a disease called Pansteatitis. It is hypothesized that contaminants from coal mining and agriculture contributed to the emergence of the disease. Invasive species and the stagnant water may also be enhancing the intensity of its effects.

Pansteatitis is an inflammatory disease that affects the lipids, or fats, of an animal. The fats become tough which cause pain and a reduction in mobility that can make the species easier prey or unable to hunt for food.

Mozambique Tilapia (Oreochromis mossambicus) have been frequently diagnosed with pansteatitis and maintain a large population size. These characteristics make them a perfect model organism to use for researching pansteatitis – which is why they were selected for my project. I will be analyzing muscle tissue samples of these fish to compare the fatty acid profiles between healthy and diseased specimen; infected Nile Crocodile muscle will also be key in understanding how pansteatitis affects different organisms.

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Mozambique Tilapia – Photo taken from John Snow

It is important that we study Mozambique Tilapia to influence management efforts for top predators like Nile Crocodile, whose presence and actions impact the food web. In addition, tilapia and other fish are harvested and I want to ensure that any diseased fish caught are safe to eat. Although there have been no studies that have found whether or not this disease can directly affect humans, I hope that my study can give us an indication of the indirect human health risks.

Research Questions

  1. What is the difference in Fatty Acid Profiles between healthy and diseased Mozambique Tilapia?
  2. What is the difference between diseased Mozambique Tilapia and Nile Crocodile?
  3. What lipids are most affected by Pansteatitis?

This Summer, I will be investigating these questions and reporting back my findings. To find more information on the topics check out these links:

Blood Chemistry of Pansteatitis-Affected Tilapia

Life History of Mozambique Tilapia

Life History of Nile Crocodile


Supported by the Fort Johnson REU Program (NSF DBI-1757899), Dr. Mike Napolitano, Dr. John Bowden, The College of Charleston, NOAA, and NIST. 


References:

Bowden, J., Cantu, T., Chapman, R., Somerville, S., Guillette, M., Botha, H., Hoffman, A., Luus-Powell, W., Smit, W., Lebepe, J., Myburgh, J., Govender, D., Tucker, J., Boggs, A. and Guillette, L. (2016). Predictive Blood Chemistry Parameters for Pansteatitis-Affected Mozambique Tilapia (Oreochromis mossambicus). PLOS ONE, 11(4), p.e0153874.

Poke, D. 5 Interesting Facts About Nile Crocodiles. https://haydensanimalfacts.com/2015/03/04/5-interesting-facts-about-nile-crocodiles/ (accessed Jun 27, 2019).

Snow, J. Mozambique Tilapia. https://www.mexican-fish.com/mozambique-tilapia/ (accessed Jun 17, 2019).

Getting warmer…

Kaylie Anne Costa, University of Miami

IMG_6879Findings: In my previous post, I outlined how lipidomics and metabolomics would be used with mass spectrometry to study changes in the lipids and metabolites in manatee plasma in response to cold stress syndrome. The purpose of this study to provide deeper understanding how cold stress syndrome impacts Florida manatees

Our original research question was: Can changes in the lipidome and metabolome of plasma samples of Florida manatees be seen in response to CSS? Although the metabolomics data is still being processed, lipidomics has already shown promising results. Through our research we have found an interesting correlation between an

individual having a plasma Serum Amyloid A (SAA) value outside the healthy range and changes seen in their plasma lipidome. SAA is an acute phase protein produced in response to inflammation. When comparing the healthy manatee plasma samples to the CSS plasma samples with a Serum Amyloid A value greater than 50 µg/mL, we have found 81 lipids that differ significantly between plasma samples from healthy manatees and manatees with cold stress syndrome (Figure 1).

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Figure 1: Percentages of each lipid category out of the 81 total significant lipids that differed between CSS and healthy manatees

Our results indicate that the plasma lipidome of Florida manatees can differ as a result of cold stress syndrome. Now the next question is: what does this difference mean in context of manatees’ physiological response to cold stress syndrome?

This question is harder to answer, but we hope to be able to trace these lipids back to specific biological pathways that are altered by CSS. When the analysis of the metabolomic data is complete, we will have more pieces to the puzzle that may allow us to hone in on specific biological pathways affected by CSS that produce a change in both the lipidome and metabolome.

This pilot study will hopefully pave the way for future studies that will help protect this threatened species and conserve them as a sentinel species for studying how environmental changes will impact human health for the future.

This summer I have gained crucial research experience by using advanced techniques of analytical chemistry to address a threat to health in the marine environment. Through this REU program, I have learned about the diverse ecosystems in the Charleston area as well as the history that makes Charleston such a unique place. I would recommend the Fort Johnson REU program to any student looking for an opportunity to further their marine science education through research.

I cannot say thank you enough to my mentors Dr. John Bowden and Dr. Mike Napolitano. Their knowledge and eagerness to guide me through this process made this project possible. I would also like to thank the College of Charleston’s Grice Marine Lab for hosting the Fort Johnson REU program, National Science Foundation (NSF DBI-1757899)for funding, and our collaborators with the USGS Sirenia project for supplying the samples used in this study.

References:

Harr, K., Harvey, J., Bonde, R., Murphy, D., Lowe, M., Menchaca, M., … & Francis-Floyd, R. (2006). Comparison of methods used to diagnose generalized inflammatory disease in manatees (Trichechus manatus latirostris). Journal of Zoo and Wildlife Medicine37(2), 151-159.

 

Methods for the Manatees

Kaylie Anne Costa, University of Miami

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The Approach: In my previous post, I described cold stress syndrome (CSS) in Florida manatees and the major threat it poses to the survival of this integral species. To expand the current scientific knowledge of CSS, I will be analyzing the lipids (aka fats) and metabolites, which are the products remaining after biological processes such as digestion, respiration, and maintenance of homeostasis, in 12 healthy and 21 CSS-affected manatee plasma samples in hopes of learning more about the metabolism of this condition and potential avenues for therapeutic applications.

In order to study the lipids and metabolites in manatee blood, I will be using liquid chromatography and mass spectrometry (LC/MS) with an electrospray ionization source. Metabolomics and lipidomics will be separately analyzed. After a chemical extraction is performed to selectively separate either the lipids or metabolites in the plasma, each extract will be individually injected into the chromatographic column to separate the chemical compounds present so that only similar compounds are analyzed in any moment of time (methodology proposed by Bligh & Dyer, 1959 and Cambridge Isotope Laboratories, Inc.). Once the separated compounds reach the end of the column, they are

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Rescued manatee showing signs of cold stress syndrome. (Photo from: https://savethemanateenewtech.weebly.com/endangerment.html)

transferred to the electrospray ion source where a high temperature and voltage will be applied to evaporate the solvent and give the compounds a charge to form ions that are then directed into the mass spectrometer. Within the mass spectrometer, the ions will first be filtered by electric fields to remove anything other than either lipids or metabolites and then detected by mass to charge ratio. The most abundant ions will be fragmented and the mass to charge ration of the fragments will also be detected using an MS/MS scan. To see an animation of the flow of ions through the mass spectrometer, please click the following hyperlink: https://www.youtube.com/watch?v=_A6NBBBcdts

As a result of the above processes, retention times for each ion are displayed in a graphical form called a chromatogram and the mass spectrum is recorded. Since the masses and retention times will not change between scans, these parameters for each ion can be matched to known databases of known lipids and metabolites. By applying multivariate statistics, we can determine if there is a difference in the lipids and/or metabolites in the plasma of manatees with CSS compared to healthy manatees.

flow chart

The top left graph shows a chromatogram. The highlighted peak is then shown on the mass spectrum below with a mass to charge ratio (m/z) of 760.58607. By locating this m/z and the m/z of its fragments in the mass spectrum of a MS/MS scan and matching the values with a database, we know the original peak represents Phosphatidylcholine (16:0_18:1).                                        (Graphic by Dr. Mike Napolitano)

The goal of my project is to see if CSS alters the lipid and metabolite contents of manatee plasma. If differences exist, I will study them to learn more about the progression of cold stress syndrome in manatees and the particular systems and metabolic pathways that are affected. It is our hope that this information leads to developing both diagnostic and treatment options for these animals thereby reducing the impacts of this syndrome.


A huge thank you goes to my mentor Dr. John Bowden and co-mentor Dr. Mike Napolitano as well as everyone at NIST, HML, and Fort Johnson for all of their help and guidance. I would also like to thank the National Science Foundation for funding and the Fort Johnson REU program for making this research possible (NSF DBI-1757899).


References:

Bligh, E. G., & Dyer, W. J. (1959). A rapid method of total lipid extraction and

purification.Canadian journal of biochemistry and physiology, 37(8), 911-917.

Cambridge Isotope Laboratories, Inc. Metabolomics QC Kit For Untargeted/Targeted Mass

Spectrometry: User’s Manual. Tewksbury, MA: Author.

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.

Bacteria in the Ocean? That Eat Iron??

Lauren Rodgers, Rutgers University

Version 2The problem: Have you ever asked yourself, what is iron? It is an element? A rock? Some weird orange-ish substance? Is it the tool that you use to get the wrinkles out of clothes? And what does iron even do? Does it just sit there? Does anything eat it? Can we make things out of it? Iron is one of the most abundant elements on earth, yet not many people know much about the important role it plays in our lives.

Iron is more than just an element, or something found within a rock. It’s a nutrient, something necessary for the growth and metabolism of almost every living organism on Earth (Hedrich & Johnson, 2011). In the ocean, iron is found in two different forms, ferrous iron or Fe(II), which is soluble in water, and ferric iron or Fe(III), which is insoluble in water (Hedrich & Johnson, 2011). Because ferrous iron is soluble it is the form of iron that can be used by most organisms in the water (Hedrich & Johnson, 2011). This ferrous iron, however, is limited in the ocean despite its abundance in the Earth’s crust. In fact, Fe(II) is present only in incredibly small concentrations, making it a major limiting factor of growth for all of the plants and algae in the ocean. This is important because these plants and algae serve as the base of many food chains, so if there is a limitation on the growth of these organisms, it affects every other organism throughout the food chain. Though iron is an extremely important nutrient for many living organisms, it is still not well understood. One of the least understood aspects is how iron specifically cycles through different marine environments. Does it ever change form? Does anything add iron to the ocean? Does anything take iron out of the ocean? These questions bring us to Zetaproteobacteria.

Zetaproteobacteria is a recently discovered class of iron-oxidizing microbes. This just means that the bacteria eat iron in the form of Fe(II) and produce Fe(III) as a waste product (Emerson et al., 2007; Chiu et al., 2017). In fact, these waste products can take on the form of hollow tubes, also called tubular sheaths, or twisted stalks that you can see under the microscope!

 

Zetaproteobacteria were initially described in 2007 near hydrothermal vents, utilizing the large concentrations of Fe(II) that were present in the fluid that spewed from the vents (Emerson et al., 2007).

Iron Mat

Iron mat composed of Zetaproteobacteria on a lava rock near the submarine Loihi volcano. (A. Malahoff, Hawaii, Loihi Volcano, July 1988)

How do Zetaproteobacteria relate to the cycling of iron? 

Zetaproteobacteria, with their role in eating iron and transforming it from its soluble Fe(II) state into its insoluble Fe(III) form may have an important role in the cycling of iron through the environment, functioning as an important source of iron removal.

Since their discovery, Zetaproteobacteria have also been observed in many other habitats, including coastal estuarine habitats with lower levels of iron, similar to that of Charleston, SC. (Laufer et al., 2017; Chiu et al., 2017). Our study will try to identify if these Zetaproteobacteria are present in the muddy soils around Charleston, as well as measure the levels of Fe(II) and Fe(III) in the rivers where these bacteria may be found.

 

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Hopefully, through the study of the distribution of Zetaproteobacteria across the globe, including the chemical characteristics of the different environments that they inhabit, we may get a clearer picture of how iron cycles in aquatic environments and the role that these Zetaproteobacteria play.


I would like to thank my mentor, Dr. Heather Fullerton, for guiding me through this research. I would also like to thank the National Science Foundation for funding this research as well as the College of Charleston and Grice Marine Lab for their support.


References 

Chiu, B. K., Kato, S., McAllister, S. M., Field, E. K., & Chan, C. S. (2017). Novel pelagic iron-oxidizing Zetaproteobacteria from the Chesapeake Bay oxic-anoxic transition zone. Frontiers in Microbiology, 8(JUL), 1–16. https://doi.org/10.3389/fmicb.2017.01280

Emerson, D., Rentz, J. A., Lilburn, T. G., Davis, R. E., Aldrich, H., Chan, C. S., & Moyer, C. L. (2007). A novel lineage of proteobacteria involved in formation of marine Fe-oxidizing microbial mat communities. PLoS ONE, 2(8), e667. https://doi.org/10.1371/journal.pone.0000667

Hedrich, S., Schlömann, M., & Johnson, D. B. (2011). The iron-oxidizing proteobacteria. Microbiology,157(6), 1551–1564.

Laufer, K., Nordhoff, M., Halama, M., Martinez, R. ., Obst, M., Nowak, M., … Kappler, A. (2017). Microaerophilic Fe(II)-oxidizing Zetaproteobacteriaisolated from low-Fe marine coastal sediments – physiology and characterization of their twisted stalks. Applied and Environmental Microbiology, 83(February), AEM.03118-16. https://doi.org/10.1128/AEM.03118-16

Mori, J. F., Scott, J. J., Hager, K. W., Moyer, C. L., Küsel, K., & Emerson, D. (2017). Physiological and ecological implications of an iron- or hydrogen-oxidizing member of the Zetaproteobacteria, Ghiorsea bivora, gen. nov., sp. Nov. ISME Journal, 11(11), 2624–2636. https://doi.org/10.1038/ismej.2017.132