Cells and Instruments, but no Folsom Prison Blues

Brian Wuertz, Warren Wilson College

Headshot

In my previous post, “Hiding in plain sight”, I introduced DOSS, a compound that has been recently identified as a probable obesogen. We are especially concerned about the potential of this compound to cause obesity symptoms in developing children through exposure from their mothers. While DOSS is in many products we use daily, such as homogenized milk and makeup products, it is commonly prescribed to pregnant women in the form of Colace stool softener. I am investigating both how much DOSS is in certain places in the body and how it may promote obesity.

One of the main concerns about obesity is that it elevates the risk of developing other diseases such as diabetes or cancer by causing a state of chronic inflammation (Bianchini 2002).  Chronic inflammation in  adipose tissue is regulated by immune cells, including macrophages. Macrophages are immune cells found throughout the body that help to fight against infection by recognizing invading bacteria and engulfing them in a process called phagocytosis, literally meaning to eat the other cells. In addition to phagocytosis macrophages are important regulators of the larger inflammatory response by secreting proteins that tell other cells to initiate or maintain a state of inflammation (Fujiwara 2005). This inflammatory reaction may be induced by DOSS. We have seen evidence of increased inflammation and obesity in mice treated with DOSS, so in order to figure out what causes that I am focusing on macrophages because of the way they regulate inflammation.

File_000 (1)

I am isolating macrophages from breast milk samples under this hood in a sterile environment to make sure they are not contaminated with bacteria.

One way to study the inflammatory response of macrophages is to expose them to DOSS and then see if they produce the inflammatory proteins. Instead of trying to measure the secreted proteins, we can measure how much RNA is made in the cell. The RNA is the translator molecule that takes the plan for the protein from the DNA and makes it available for the cell to read and make the right protein. I identified genes for four different inflammatory proteins to measure the RNA so we can test if DOSS causes the macrophages to make more of any of them. I am testing macrophages that I am isolating from human placenta and breast milk tissue because the developing child is influenced by inflammation in the placenta and breast milk. Macrophages in these tissues could be the source of inflammation that influences how the child develops.

Okay so we have talked about cells, but what about the instruments? In my last post I introduced my instrument of choice, but did not call it that. It is not a guitar or a saxophone, but the HPLC, or high performance liquid chromatograph. This is simply a fancy instrument used to separate chemical compounds by forcing them through a tiny filter column filled with tiny beads. Some compounds stick more to the beads than others, so when you flow a liquid through the column the compounds come out of the column at different times. It is essential to separate the compounds in a sample because then you can measure the amounts of individual compounds.

We want to know where DOSS goes in the body, so we need to be able to measure how much of it is in a sample. I am working to get a system up and running to measure the amounts of DOSS in samples from different cells and tissues. We want to be able to measure DOSS in humans and in marine mammals such as dolphins. Dolphins are exposed to DOSS in the COREXIT oil spill dispersal agent that is applied to large and small scale oil spill issues along coastlines and in harbors. Dolphins are an important sentinel species, meaning that they can provide insight into human health issues.

I have to prepare a column and get the right mixture of solvents to make DOSS come off of the column in a timely fashion and in a way that we can measure it. The measurement is actually done with a mass spectrometer, which measures allows us to identify the compound based on how much it weighs. The number of atoms and types of atoms in the compound determine the mass of the compound. This mass is how the instrument measures the compound. The technique I am using is therefore called liquid chromatography mass spectrometry or LC-MS and the instrument is also referred to by LC-MS. Hopefully by the end of the summer I will be able to find beautiful data with this instrument that will make a coherent tune rather than a jumble of notes.

IMG_3755

This is the MS part. It measures the mass of the compound and then breaks it apart and measures the mass of the pieces of the compounds and the amount of the compounds.

IMG_3754

This is the LC or liquid chromatography part of the LC-MS instrument. Most of the work is figuring out the best solvent system to the sample through the small column with the red tag on it.

Funding for this REU program is generously provided by the National Science Foundation and hosted by the College of Charleston. Dr Demetri Spyropoulos at the Medical University of South Carolina is graciously hosting my research project and providing mentorship.

CofClogo-300x139nsf1

References:

Bianchini, F., Kaaks, R., and Vainio, H. (2002). Overweight, obesity, and cancer risk. The Lancet Oncology 3, 565–574.
Fujiwara, N., and Kobayashi, K. (2005). Macrophages in Inflammation. Current Drug Target -Inflammation & Allergy 4, 281–286.

Living Life as a Sea Urchin Momma

Hailey Conrad, Rutgers University

20120643_1501407253250960_452947481_n (1)

Me working hard to make my sea urchin babies

For my project I am using the same technique that the father of genetics, Gregor Mendel, used to establish his Laws of Heredity: cross breeding. So, I have to breed and raise a whole lot of sea urchins. For a refresher, I’m trying to determine if there is heritable genetic variation in how sea urchin (specifically an Arbacia punctulata population from Woods Hole, Massachusetts) larvae respond to ocean acidification. To do this, I’m rearing sea urchin larvae in low and high carbon dioxide conditions and measuring their skeletal growth. I’m breeding 3 sea urchin males with 3 sea urchin females at a time, for a total of 9 crosses. To tease apart the impact of genetic variation on just the larvae themselves, I will be fertilizing the sea urchin eggs in water aerated with either current atmospheric levels of carbon dioxide, about 410 parts per million, or 2.5 times current atmospheric carbon dioxide levels, about 1,023 parts per million. Then, I will be rearing the larvae in water aerated with either 409 ppm CO2 or 1,023 ppm CO2. This will give me four different treatments for each cross, giving me 36 samples in total. By fertilizing and rearing them in the same and different levels of carbon dioxide I will be able to see how much of an impact being fertilized in water with a higher carbon dioxide concentration has on larval growth versus just the larval growth itself. It’s important for me to make that distinction because I just want to identify genetic variation in larval skeletal growth, and separate out any extraneous “noise” clouding out the data. I’m rearing the larvae in a larval rearing apparatus. Each of the 36 samples will be placed in jar with water aerated with the correct CO2 treatment. Each jar will constantly have atmosphere with the correct CO2 concentration bubbled in. Each has a paddle in it that is hooked to a suspended frame that is swayed by a motor. This keeps the larvae suspended in the water column. The jars are chilled to 14 C by a water bath.

IMG_9756_Moment

My larval rearing apparatus

After a 6-day period the larvae are removed from the jars and their skeletal growth is measured. They are preserved with 23% methanol and seawater and frozen.

DSCN7310

An Arbacia punctulata pluteus

You’re probably curious how the heck I am able to measure the larva’s skeletons. They’re microscopic! Well, I use a microscope coupled to a rotary encoder with a digitizing pad and a camera lucida. Which, looks like this:

IMG_0042 (1)

A microscope coupled to a rotary encoder with a digitizing pad and a camera lucid hooked up to a computer

This complicated-sounding hodge-podge of different devices enables me to do something incredible. I can look through the microscope at the larva, and also see the digitizing pad next to the microscope, where I hold a stylus in my hand. When I tap the pad with the stylus and the coordinates of various points on the anatomy of the plutei that I am tapping at get instantly recorded on my computer! The rotary encoder is the piece attached to the left side of the microscope and it enables me to record coordinates in three dimensions. Then, I can use those coordinates to calculate the overall size of the skeleton. My favorite part of doing science is learning how scientists are able to do the seemingly impossible- like measuring something microscopic.

After I gather all of my data, I will do some statistical analysis to see the affect that the male parents have on the skeletal growth of their offspring. I will not be focusing on the impact that females have on the skeletal growth of their offspring. The quality of the egg itself could be an influencing factor on the size of the offspring, whereas sperm is purely genetic material. Like how I’m trying to isolate the influence of ocean acidification during larval rearing from during the act of fertilization, I am trying to isolate just genetic influences on larval skeletal growth from egg quality. Check back to see how it goes!

Special thanks to the National Science Foundation for funding this REU program, the College of Charleston and Grice Marine Laboratory for hosting me, and Dr. Bob Podolsky for mentoring me!

 

 

 

The Problem with PFCs- Seeking Answers in Plasma

Kady Palmer, Eckerd College

Manatee_CR

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. 

PFAAs1       PFAAS2

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.

Mass Spec

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

Some Dramatic Microorganisms and Targeted Genetic Analysis

Emily Spiegel, Bryn Mawr College

IMG_4143

Genetic analysis has become the name of the game in many fields of biological research. Genes encode proteins, and in biology, proteins are king. Proteins guide biological pathways throughout the entire organism, so if you can track the genes, you can understand how the animal functions. Advances in technology like CRISPR, RNA sequencing, and PCR have improved the accessibility and accuracy of high-level genetic analysis in laboratories across the world. Some scientists utilize this technology to study the entire genome of an organism, while others attempt to understand the response of specific genes to various environmental factors or other external influences. This summer, I’m conducting an experiment focused on the latter. I’ll be studying how the polar algae species, Fragilariopsis cylindrus (affectionately known as Frag) copes with environmental stress by reproducing sexually. To do so, I’ll use targeted RNA sequencing to track genes related to sexual reproduction.

In order to understand how a Frag, responds to environmental stresses, you need a lot of algae. I reared nearly 100 liters of this algae in different artificial conditions. These conditions varied by two factors: photoperiod (the length of day and night), and nutrient levels. If you missed my previous post, “Stressing Out My Algae,” you should check it out for more details on the background for this experiment. We suspect that in conditions of stressful light energy (24 hours of continuous light), Frag will respond by reproducing sexually as opposed to its normal asexual mode of reproduction. This could possibly be a mechanism to rid itself of excess energy in times of stress, since sexual reproduction is more energetically expensive than asexual reproduction. By reproducing sexually, Frag may improve its chances of survival against this stress. Compounded with this is our hypothesis on nutrient deprivation. Previous experiments have shown that when a major nutrient, nitrogen, is limited, the algae cannot grow at full capacity and sexual reproduction is inhibited. We predict that when the stress of nitrogen limitation is combined with the stress of high light energy, we’ll see a reduction in the algae’s ability to survive in the stressful conditions due to the inhibition of sexual reproduction. So if we stress out the Frag enough and take away their ability to have sex, they’ll probably die. They’re some very dramatic microorganisms.

IMG_4131

24 bottles of algae were grown in six different experimental conditions varied by length of light exposure and nutrient levels. Algae was reared in 4-liter bottles filled with seawater.

So we grew our Frag, four bottles per six experimental conditions. Every day for eight days we extracted biomass from the bottle. From this sample we could test chlorophyll levels and cell counts, both of which give us a good idea of how well the algae in that bottle are growing in their conditions. We also took samples to be used for RNA extraction. Remember how genes encode proteins and proteins are king? Well before you can have your protein product, you need RNA. You’ve probably heard of DNA, which is the double stranded genetic cookbook. RNA is its single stranded offspring, which is then used as a the direct template to make proteins. A lot of genetic analysis therefore looks at RNA instead of DNA in order to understand how genes are being transcribed for protein production. We’re currently working on extracting the RNA from the original biomass sample and then running that pure RNA through a specialized machine called Nanostring. This is extremely targeted analysis, as Nanostring focuses in on the specific RNA we’re most interested in. In this case, we’re interested in RNA which is encoded from genes related to sexual reproduction. Using Nanostring will tell us how active the genes for sexual reproduction are in each bottle, which we can analyze to derive any correlation between our environmental stress factors and sexual reproduction.

If our hypothesis is correct, then we’ll see the greatest expression of sexual reproduction genes in the conditions of high light energy (24 hours of continuous light). We’d expect to also see low growth performance in nitrogen limited populations, indicated by low cell counts and chlorophyll levels. In these populations we predict we’ll see little if any expression of genes related to sexual reproduction. By the end, we’ll hopefully have a clearer picture of how phytoplankton like Frag deal with environmental stress.

Funding for this project is provided by the National Science Foundation in collaboration with the College of Charleston Grice Marine Laboratory and the National Oceanic and Atmospheric Administration. Acknowledgements to the entire lab of Dr. Ditullio and Dr. Lee in the Hollings Marine Laboratory facility.

Playing with Plutei

Hailey Conrad, Rutgers University

IMG_9461

Me! Photo Credit: Kady Palmer

Ocean acidification is known as climate change’s evil twin. When the pH of ocean water drops, carbonate ions in the water form carbonic acid instead of calcium carbonate. Calcium carbonate is the form of calcium that marine animals that have calcium-based skeletons (like us!) and shells use to build their bones and shells. Having smaller and weaker skeletons or shells impacts their ability to survive. However, some individuals within certain species or populations of species have genes that make them more resistant to ocean acidification. If these individuals are able to pass on these genes to their offspring, then the species has the ability to evolve in response to ocean acidification instead of going extinct. This summer I’m working with Dr. Bob Podolsky in College of Charleston’s Grice Marine Field Station to study the extent to which ocean acidification affects Atlantic purple sea urchins, Arbacia punctulata. We are specifically trying to see if any individuals within a population from Woods Hole, Massachusetts, have any heritable genetic resistance to the negative impacts of ocean acidification. We hypothesize that there will be genetic resistance given that the northern Atlantic coast naturally has lower levels of saturated calcium carbonate, so a population that has evolved to live in that type of environment should have some resistance to lower calcium carbonate levels already (Wang et al 2013). We’re using a basic cross breeding technique to rear Arbacia punctulata larvae to their plutei stage, when they have four main body rods. At this stage they look less like sea urchins than they do like Sputnik!

DSCN7245

A sea urchin pluteus larvae with four body rods

Then, we will look to see if any of the male parents consistently produce male offspring that are more resistant to ocean acidification.  If males like these exist within this population, then the species has the capacity to evolve in response to ocean acidification, instead of going extinct! This is a very big deal, and could potentially be very hopeful. Even if we don’t get the results that we are hoping for, the results of this research could inform policy and management decisions.

Literature Cited:

Wang, Z. A., Wanninkhof, R., Cai, W., Byrne, R. H., Hu, X., Peng, T., & Huang, W. (2013). The marine inorganic carbon system along the Gulf of Mexico and Atlantic coasts of the United States: Insights from a transregional coastal carbon study. Limnology and Oceanography, 58(1), 325-342. doi:10.4319/lo.2013.58.1.0325

Thank you to the National Science Foundation and College of Charleston’s Grice Marine Laboratory for funding my project. And, special thanks to Dr. Bob Podolsky for being a wonderful and supportive mentor!

 

Hiding in plain sight

Brian Wuertz, Warren Wilson College

Headshot

How much do we really know about all the chemicals that we are exposed to every day? Do we even know when we come into contact with them? How much do we know about what is in homogenized milk, soda, stool softeners, baby formula, and personal care products such as eyeliner? The answer may be “not enough” for one compound found in all of those products, dioctyl sodium sulfosuccinate, or DOSS. DOSS has recently been identified by my mentor, Dr. Spyropoulos and his Ph.D. student, Alexis Temkin, as a probable obesogen. Obesogens are a class of compounds that promote obesity by interfering with the body’s hormone signalling pathways related to energy use, fat cell regulation, and inflammation. These pathways are especially important in the developing fetus, where hormone signals influence development and may have long lasting effects on the health of the child after birth (Holder 2016).

File_000

I am working on a High Performance Liquid Chromatography  (HPLC) system, in the early stages of developing a method to measure the amount of DOSS in cell extracts. (More to come in future posts!)

We are especially concerned with regards to the developing fetus and child because stool softeners containing DOSS are are commonly taken by pregnant women. Approximately 35% of over 20,000 women who gave birth at MUSC in recent years reported taking a stool softener containing DOSS during their pregnancy. I am working to help understand the biochemical pathways DOSS may follow to affect changes in the  developing fetus through a mother’s exposure to DOSS. I am also working on a method to measure the amount of DOSS in cells so that we can learn where in the body DOSS goes and how much of it there actually is.

You might be wondering how this fits into the theme of marine organism health at this point since all I have talked about is human health and a compound found in products we put in our bodies, DOSS. A red flag was raised about DOSS through research on COREXIT, one of the agents used to clean up the Deepwater Horizon oil spill. Over 40 million gallons of COREXIT was dumped into the ocean as a part of the cleanup effort and DOSS is one of the major components (Temkin 2016).  DOSS was flagged as a potential human health hazard because of the research done on marine environmental degradation. It amazes me how a perhaps seemingly unrelated topic can end up having human health implications. I am excited to keep working on this puzzle to learn more about DOSS and how it interacts with the systems in our bodies!

Funding for this REU program is generously provided by the National Science Foundation and hosted by the College of Charleston. Dr Demetri Spyropoulos at the Medical University of South Carolina is graciously hosting my research project and providing mentorship.

CofClogo-300x139nsf1

Sources:

Holder, B., Jones, T., Sancho Shimizu, V., Rice, T.F., Donaldson, B., Bouqueau, M., Forbes, K., and Kampmann, B. (2016). Macrophage Exosomes Induce Placental Inflammatory Cytokines: A Novel Mode of Maternal–Placental Messaging. Traffic 17, 168–178.
Temkin, A.M., Bowers, R.R., Magaletta, M.E., Holshouser, S., Maggi, A., Ciana, P., Guillette, L.J., Bowden, J.A., Kucklick, J.R., Baatz, J.E., et al. (2016). Effects of Crude Oil/Dispersant Mixture and Dispersant Components on PPARγ Activity in Vitro and in Vivo: Identification of Dioctyl Sodium Sulfosuccinate (DOSS; CAS #577-11-7) as a Probable Obesogen. Environ Health Perspect 124, 112–119.

 

 

 

What’s living in the sand?

Jessie Lowry, Coker College

DSC01976

Visible microalgae seen on the surface of wet sand at Folly Beach.

Next time you go to the beach this summer, I want you to think about the sand that you are walking on. Did you know that there are tons of microscopic photosynthetic organisms, aka microalgae, that live on the surface of sand? Before this summer, I didn’t know about these organisms either. Here is a picture of visible microalgae on the surface of the sand. Look for this next time you’re at the beach!

Microalgae communities in sand are made up of single-celled eukaryotic algae and cyanobacteria living in the top several millimeters of the sand (Miller et al., 1996). These organisms play important roles in ecosystem productivity and food chain dynamics, as well as in sediment properties, such as erodibility (Miller et al., 1996).

IMG_8018

Dr. Craig Plante and Jessie Lowry collect samples of sediment from Folly Beach. Photo credit: Kristy Hill-Spanik.

I am studying these microalgal communities and what factors influence community structure. For example, does pH, salinity, nutrients, or grain size shape microalgal community structure? Or does geographic distance shape communities? To answer these questions, I am collecting samples from Kiawah Island, Folly Beach, Isle of Palms, and Pawley’s Island, SC. We are measuring environmental variables at each location, and using molecular tools to study microalgal community structure.

I am extracting the DNA from samples collected, amplifying specific regions from these samples using polymerase chain reaction (PCR), and then we will be getting these regions sequenced using Ion Torrent technology. We will then use QIIME to determine how similar these benthic microalgal communities are.

IMG_7918

Jessie Lowry preparing samples for PCR, or polymerase chain reaction, which is used to make millions of copies of a piece of DNA.

Diatoms, a group of microalgae, have been proposed as bioindicators of environmental health (Desrosiers et al., 2013). Bioindicators are really cool because instead of telling a snapshot of an environmental condition, such as pH, temperature, or amount of oxygen in an environment, biological indicators reflect those changes and can give an idea of how the ecosystem is being affected. This research will further our knowledge of what factors shape benthic microalgal communities, and give a better understanding of these organisms as a potential bioindicator. In addition, this research will add to knowledge about the distribution of microorganisms, which is also not fully understood.

Learn more:

http://web.vims.edu/bio/shallowwater/benthic_community/benthic_microalgae.html

http://www.aims.gov.au/docs/research/water-quality/runoff/bioindicators.html

References

Desrosiers, C., Leflaive, J., Eulin, A., Ten-Hage, L. (2013). Bioindicators in marine waters: benthic diatoms as a tool to assess water quality from eutrophic to oligotrophic coastal ecosystems. Ecological Indicators, 32, 25-34.

Miller, D.C., Geider, R.J., MacIntyre, H.L. Microphytobethos: The ecological role of the “Secret Garden” of unvegetated, shallow-water marine habitats. Estuaries, 19(2A): 186-212.

Acknowledgements

Thank you so much to my mentors Dr. Craig Plante, and Kristy Hill-Spanik. This research is funded through the National Science Foundation and College of Charleston’s Grice Marine Lab.

DSC01972Unknown-4Unknown-3