Duke Research Blog

Following the people and events that make up the research community at Duke.

Category: Biology (Page 1 of 28)

Not Your Basic Bench: Zebrafish Reveal Secrets of the Developing Gut

Our intestine is a highly complex organ – a tortuous, rugged channel built of many specialized cell-types and coated with a protective, slimy matrix. Yet the intestine begins as a simple tube consisting of a central lumen lined by a sheet of epithelial cells, which are smooth cells that lie on the surface of the lumen. These intestinal epithelial cells are central players in many human diseases.

A portrait of Daniel Levic

Daniel Levic, a postdoctoral research associate in the department of cell biology at the Duke University Medical Center.

Daniel Levic of the Bagnat Lab is using zebrafish as experimental models to understand how intestines are formed in hopes of finding new ways to combat disease. He wants to learn how the intestinal lumen forms during early development, and how intestinal epithelial cells take on their physiological functions.

Levic, a postdoctoral research associate in the department of cell biology at the Duke University Medical Center, focuses on projects in both basic and translational science. Daniel uses zebrafish to analyze the formation of the lumen and the polarity of epithelial cells — how specialized they are for carrying out different functions —  at the genetic and cellular level. He focuses on how membrane proteins are sorted into different, specialized domains of the cell surface and how this process affects intestinal formation. Additionally, Daniel studies how inflammation is evaded in intestinal epithelial cells in Crohn’s disease using a combination of patient biopsy samples and animal studies in zebrafish. This project is a collaborative effort aided by clinicians and human geneticists at the Duke University Medical Center.

A microscope image of a zebrafish gut

The developing gut of a zebrafish, magnified.

Though complex human diseases can’t be fully mimicked in animal models like zebrafish, this type of research can be extremely useful. These model organisms can be used to study the basic, fundamental cellular mechanisms that ultimately underlie disease. An example is Daniel’s work on Crohn’s disease, where he is trying to understand how inflammatory signaling networks become activated, specifically in intestinal epithelial cells. This problem is difficult, if not impossible, to address using exclusively human biopsy samples.

Overall, Daniel hopes that his translational research will provide new knowledge of the role of intestinal epithelial cells in Crohn’s disease and provide biomarkers that will aid clinicians in predicting how patients will respond to therapeutic interventions. Daniel’s research and basic science research are rapidly changing the way we diagnose disease, treat patients, and interact with the world around us.

Guest post by Vaishnavi Siripurapu

Science on the Trail

Duke launches free two-week girls science camp in Pisgah National Forest.

Duke launches free two-week girls science camp in Pisgah National Forest.

DURHAM, N.C. — To listen to Destoni Carter from Raleigh’s Garner High School, you’d never know she had a phobia of snails. At least until her first backpacking trip, when a friend convinced her to let one glide over her outstretched palm.

Destoni Carter

Destoni Carter from Raleigh’s Garner High School was among eight high schoolers in a new two-week camp that combines science and backpacking.

Soon she started picking them up along the trail. She would collect a couple of snails, put them on a bed of rocks or soil or leaves, and watch to see whether they were speedier on one surface versus another, or at night versus the day.

The experiment was part of a not-so-typical science class.

From June 11-23, 2017, eight high school girls from across North Carolina and four Duke Ph.D. students left hot showers and clean sheets behind, strapped on their boots and packs, and ventured into Pisgah National Forest.

For the high schoolers, it was their first overnight hike. They experienced a lot of things you might expect on such a trip: Hefty packs. Sore muscles. Greasy hair. Crusty socks. But they also did research.

The girls, ages 15-17, were part of a new free summer science program, called Girls on outdoor Adventure for Leadership and Science, or GALS. Over the course of 13 days, they learned ecology, earth science and chemistry while backpacking with Duke scientists.

Duke ecology Ph.D. student Jacqueline Gerson came up with the idea for the program. “Backpacking is a great way to get people out of their comfort zones, and work on leadership development and teambuilding,” said Gerson, who also teamed up with co-instructors Emily Ury, Alice Carter and Emily Levy, all Ph.D. students in ecology or biology at Duke.

Marwa Hassan of Riverside High School in Durham studying stream ecology as part of a two-week summer science program in Pisgah National Forest. Photo by Savannah Midgette.

Marwa Hassan of Riverside High School in Durham studying stream ecology as part of a two-week summer science program in Pisgah National Forest. Photo by Savannah Midgette.

The students hauled 30- to 40-pound loads on their backs for up to five miles a day, through all types of weather. For the first week and a half they covered different themes each day: evolution, geology, soil formation, aquatic chemistry, contaminants. Then on the final leg they chose an independent project. Armed with hand lenses, water chemistry test strips, measuring tapes and other gear, each girl came up with a research question, and had two days to collect and analyze the data.

Briyete Garcia-Diaz of Kings Mountain High School surveyed rhododendrons and other trees at different distances from streambanks to see which species prefer wet soils.

Marwa Hassan of Riverside High School in Durham waded into creeks to net mayfly nymphs and caddisfly larvae to diagnose the health of the watershed.

Savannah Midgette of Manteo High School counted mosses and lichens on the sides of trees, but she also learned something about the secret of slug slime.

“If you lick a slug it makes your tongue go numb. It’s because of the protective coating they have,” Midgette said.

High schoolers head to the backcountry to learn the secret of slug slime and other discoveries of science and self in new girls camp

High schoolers head to the backcountry to learn the secret of slug slime and other discoveries of science and self in new girls camp

The hiking wasn’t always easy. On their second day they were still hours from camp when a thunderstorm rolled in. “We were still sore from the previous day. It started pouring. We were soaking wet and freezing. We did workouts to keep warm,” Midgette said.

At camp they took turns cooking. They stir fried chicken and vegetables and cooked pasta for dinner, and somebody even baked brownies for breakfast. Samantha Cardenas of Charlotte Country Day School discovered that meals that seem so-so at home taste heavenly in the backcountry.

“She would be like, ugh, chicken in a can? And then eat it and say: ‘That’s the most amazing thing I’ve ever had,’” said co-instructor Emily Ury.

Savannah Midgette and Briyete Garcia-Diaz drawing interactions within terrestrial systems as part of a new free summer science program called Girls on outdoor Adventure for Leadership and Science, or GALS. Learn more at https://sites.duke.edu/gals/.

Savannah Midgette and Briyete Garcia-Diaz drawing interactions within terrestrial systems as part of a new free summer science program called Girls on outdoor Adventure for Leadership and Science, or GALS. Learn more at https://sites.duke.edu/gals/.

The students were chosen from a pool of over 90 applicants, said co-instructor Emily Levy. There was no fee to participate in the program. Thanks to donations from Duke Outdoor Adventures, Project WILD and others, the girls were able to borrow all the necessary camping gear, including raincoats, rain pants, backpacks, tents, sleeping bags, sleeping pads and stoves.

The students presented their projects on Friday, June 23 in Environment Hall on Duke’s West Campus. Standing in front of her poster in a crisp summer dress, Destoni Carter said going up and down steep hills was hard on her knees. But she’s proud to have made it to the summit of Shining Rock Mountain to see the stunning vistas from the white quartz outcrop near the top.

“I even have a little bit of calf muscle now,” Carter said.

Funding and support for GALS was provided by Duke’s Nicholas School of the Environment, Duke ecologist Nicolette Cagle, the Duke Graduate School and private donors via GoFundMe.

2017 GALS participants (left to right): Emily Levy of Duke, Destoni Carter of Garner High School, Zyrehia Polk of East Mecklenburg High School, Rose DeConto of Durham School of the Arts, Briyete Garcia-Diaz of Kings Mountain High School, Marwa Hassan of Riverside High School, Jackie Gerson of Duke, Daiana Mendoza of Harnett Central High School, Savannah Midgette of Manteo High School, Samantha Cardenas of Charlotte Country Day School and Alice Carter of Duke.

2017 GALS participants (left to right): Emily Levy of Duke, Destoni Carter of Garner High School, Zyrehia Polk of East Mecklenburg High School, Rose DeConto of Durham School of the Arts, Briyete Garcia-Diaz of Kings Mountain High School, Marwa Hassan of Riverside High School, Jackie Gerson of Duke, Daiana Mendoza of Harnett Central High School, Savannah Midgette of Manteo High School, Samantha Cardenas of Charlotte Country Day School and Alice Carter of Duke.

 

Marine Parasites — Little Guys That Make a Big Difference

If you’re anything like me, the first images that come to mind when you hear the words “marine biology” are singing whales, dolphins racing each other, sharks flying out of the water, maybe a swordfish brawl or two — all the big, flashy stuff.

Of all the things “marine biology” invokes, parasites are probably at the very bottom of my list.

Not so for Joe Morton, a PhD student at the Nicholas School of the Environment and self-taught expert on the parasites that inhabit marine organisms. In fact, Morton posits that parasites play one of the most important roles in all of ecology, by modifying the behavior of ecologically influential host species. And he’s got the research to back it up.

Once back at the lab, Morton takes his place behind the microscope to study his research subjects: marine parasites. Courtesy: Joe Morton.

Morton’s academic quest into the world of marine parasites began about six years ago when he was a master’s student at UNC’s Institute of Marine Sciences — just down the road from Duke’s own Marine Lab, where he’s now stationed. Having just read Carl Zimmer’s pop-science book Parasite Rex, Morton wondered whether the marsh periwinkle snails (Littoraria irrorata) he was studying could be infected.

“In my spare time, I would go into the lab at night with a hammer and crack open a bunch of snails to see what I would find,” Morton said. “I didn’t find anything in the literature at the time about Littoraria harboring parasites, which I thought was really unusual because they’re really well-known, important marsh gastropod.”

Morton began to systematically collect Littoraria from local salt marshes, determine their infection status, then examine how the parasites affected the behavior of infected individuals and, in turn, how these behavioral changes affected the ecological health of the salt marsh. This way, Morton figured out that Littoraria infected with digenean trematodes (a class of parasite) climbed and grazed on marsh grass less often than uninfected Littoraria. He also noticed that infected Littoraria congregated at salt marsh “die-off borders,” the edges where marsh grasses stop growing sparsely and start growing in healthy amounts.

A microsopic view of digenean trematodes, the parasites that infect marsh periwinkle snails. Courtesy: Joe Morton.

Based on these observations, Morton designed an experiment to test whether the prevalence of infection among Littoraria correlated with marsh grass health.

“I found that, even under drought stress conditions, parasites could effectively slow the rate at which the marsh died off and help maintain marsh ecosystem structure,” Morton said. “More structure means more nursery habitat for fish. It means more nursery habitat for fiddler crabs. Increased filtration rate of water into the sediment because of crab burrows. The point is, parasites help to increase ecosystem resistance to drought stress.”

Joe Morton traipses through the salt marsh on a windy day. Courtesy: Joe Morton.

Morton was the first to demonstrate this relationship between parasites and marsh health in a behavioral experiment. It’s been a major focus of his research ever since.

“Parasites constitute more than half the life on the planet, but until very recently, parasites were somewhat ignored by ecologists,” Morton said.

Indeed, Morton’s former advisor once told him “never study anything smaller than your thumb.” According to Morton, this was a very widely-held view in ecology up until the last few decades.

“That was very much the idea at the time: these are small things; they probably mean a lot to individual organisms, but they’re may not be important to ecosystems. And now we know that’s just not the case,” Morton said. “Almost everywhere we look, parasites are there; they’re ubiquitous. And they have an important role to play.”

Though parasites are a hot topic in ecology nowadays, Morton, a self-declared “lifelong contrarian,” has a very distinct memory of a childhood moment foreshadowing his current research focus.

“I remember sitting in a barber shop and reading Popular Science magazine, which has an annual list of the ten worst jobs in science. I remember right at the top of the list was ‘parasitic worm biologist.’ And something in my head was just like ‘yeah, I’ll do that,’” Morton said.

Post by Maya Iskandarani

3D Virus Cam Catches Germs Red-Handed

A 3D plot of a virus wiggling around

The Duke team used their 3D virus cam to spy on this small lentivirus as it danced through a salt water solution.

Before germs like viruses can make you sick, they first have to make a landing on one of your cells — Mars Rover style — and then punch their way inside.

A team of physical chemists at Duke is building a microscope so powerful that it can spot these minuscule germs in the act of infection.

The team has created a new 3D “virus cam” that can spy on tiny viral germs as they wriggle around in real time. In a video caught by the microscope, you can watch as a lentivirus bounces and jitters through an area a little wider that a human hair.

Next, they hope to develop this technique into a multi-functional “magic camera” that will let them see not only the dancing viruses, but also the much larger cell membranes they are trying breech.

“Really what we are trying to investigate is the very first contacts of the virus with the cell surface — how it calls receptors, and how it sheds its envelope,” said group leader Kevin Welsher, assistant professor of chemistry at Duke. “We want to watch that process in real time, and to do that, we need to be able to lock on to the virus right from the first moment.”

A 3D plot spells out the name "Duke"

To test out the microscope, the team attached a fluorescent bead to a motion controller and tracked its movements as it spelled out a familiar name.

This isn’t the first microscope that can track real-time, 3D motions of individual particles. In fact, as a postdoctoral researcher at Princeton, Welsher built an earlier model and used it to track a bright fluorescent bead as it gets stuck in the membrane of a cell.

But the new virus cam, built by Duke postdoc Shangguo Hou, can track particles that are faster-moving and dimmer compared to earlier microscopes. “We were trying to overcome a speed limit, and we were trying to do so with the fewest number of photons collected possible,” Welsher said.

The ability to spot dimmer particles is particularly important when tracking viruses, Welsher said. These small bundles of proteins and DNA don’t naturally give off any light, so to see them under a microscope, researchers first have to stick something fluorescent on them. But many bright fluorescent particles, such as quantum dots, are pretty big compared to the size of most viruses. Attaching one is kind of like sticking a baseball onto a basketball – there is a good chance it might affect how the virus moves and interacts with cells.

The new microscope can detect the fainter light given off by much smaller fluorescent proteins – which, if the virus is a basketball, are approximately the size of a pea. Fluorescent proteins can also be inserted to the viral genome, which allows them to be incorporated into the virus as it is being assembled.

“That was the big move for us,” Welsher said, “We didn’t need to use a quantum dot, we didn’t need to use an artificial fluorescent bead. As long as the fluorescent protein was somewhere in the virus, we could spot it.” To create their viral video, Welsher’s team enlisted Duke’s Viral Vector Core to insert a yellow fluorescent protein into their lentivirus.

Now that the virus-tracking microscope is up-and-running, the team is busy building a laser scanning microscope that will also be able to map cell surfaces nearby. “So if we know where the particle is, we can also image around it and reconstruct where the particle is going,” Welsher said. “We hope to adapt this to capturing viral infection in real time.”

Robust real-time 3D single-particle tracking using a dynamically moving laser spot,” Shangguo Hou, Xiaoqi Lang and Kevin Welsher. Optics Letters, June 15, 2017. DOI: 10.1364/OL.42.002390

Kara J. Manke, PhDPost by Kara Manke

Lemur Research Gets a Gut Check

Baby Coquerel’s sifaka

Clinging to her mom, this baby Coquerel’s sifaka represents the only lemur species at the Duke Lemur Center known to fall prey to cryptosporidium, a microscopic parasite that causes diarrhea that can last for a week or more. The illness wipes out much of the animals’ gut microbiome, researchers report, but fecal transplants can help them recover. Photo by David Haring, Duke Lemur Center.

DURHAM, N.C. — “Stool sample collector” is not a glamorous way to introduce oneself at a party. But in the course of their research, gut microbiologists Erin McKenney and Lydia Greene have spent a lot of time waiting for animals to relieve themselves.

They estimate they have hundreds of vials of the stuff, from a dozen primate species including lemurs, baboons and gorillas, sitting in freezers on the Duke University campus.

The researchers aren’t interested in the poop per se, but in the trillions of bacteria inhabiting the gastrointestinal tract, where the bugs help break down food, produce vitamins and prevent infection.

A few years ago, McKenney and Greene started collecting stool samples at the Duke Lemur Center to see how the microbial makeup of lemurs’ guts varies from birth to weaning, and as their diets change over the seasons. And what happens when they get sick?

Illustration of Cryptosporidium, a widespread intestinal parasite that causes diarrhea in people, pets, livestock and wildlife worldwide. Courtesy of the U.S. Centers for Disease Control.

Illustration of Cryptosporidium, a widespread intestinal parasite that causes diarrhea in people, pets, livestock and wildlife worldwide. Courtesy of the U.S. Centers for Disease Control.

Between 2013 and 2016, ten of the lemurs they were studying contracted cryptosporidium, or “crypto” for short, a waterborne parasite that causes diarrhea in people, pets, livestock and wildlife worldwide.

All of the infected animals were Coquerel’s sifakas — the only lemur species out of roughly 20 at the Duke Lemur Center known to fall prey to the parasite — and most of them were under five years old when they fell ill.

Animals that tested positive were moved into separate holding areas away from other animals and visitors. Keepers wore protective suits, gloves, face masks and booties while working in the animals’ enclosures to prevent infection.

All of the animals eventually recovered. Along the way, six of the affected animals were treated with antibiotics, and three were also fed a slurry of saline and feces from a healthy relative.

McKenney and Greene collected stool samples before, during and after infection for up to two months. They used a technique called 16S ribosomal RNA sequencing to identify the types of bacteria in the samples based on their genes, and compared the results with those of 35 unaffected individuals.

In a healthy gut microbiome, “good” bacteria in the gut compete with “bad” microbes for space and nutrients, and secrete substances that inhibit their growth.

The guts of sick and recovering sifakas are host to a very different assortment of microbes than those of unaffected animals, the researchers found.

Not surprisingly, both crypto infection, and antibiotic treatment, wiped out much of the animals’ gut flora — particularly the bacterial groups Bifidobacterium, Akkermansia, Succinivibrio and Lachnospiraceae.

Even after the infections cleared, most animals took another several weeks to stabilize and return to normal levels of gut biodiversity, with younger animals taking longer to recover.

The only animals that made a full comeback within the study period were those that received a fecal transplant, suggesting that the treatment can help restore gut bacterial diversity and speed recovery.

The patterns of gut recolonization following crypto infection mirrored those seen from birth to weaning, said McKenney, now a postdoctoral researcher at North Carolina State University.

The researchers hope their findings will help control and prevent crypto outbreaks in captive primates. Because lemurs are more closely related to humans than lab mice are, the research could also help scientists understand how the gut microbiome protects humans from similar infections and facilitates recovery.

“Thanks to bioinformatics and advances in sequencing, the microbiome gives us a window into the health of these animals that we’ve never had before,” said Greene, a graduate student in ecology at Duke.

They published their findings June 15, 2017, in the journal Microbial Ecology in Health and Disease.

Duke evolutionary anthropology professors Christine Drea and Anne Yoder were senior authors on this study. This research was supported by the National Science Foundation (1455848) and the Duke Lemur Center Directors Fund.

CITATION:  “Down for the Count: Cryptosporidium Infection Depletes Gut Microbiota in Coquerel’s Sifakas,” Erin McKenney, Lydia Greene, Christine Drea and Anne Yoder. Microbial Ecology in Health and Disease, June 15, 2017. http://dx.doi.org/10.1080/16512235.2017.1335165

Post by Robin Smith, science writer, Office of News & Communications

Scientists Engineer Disease-Resistant Rice Without Sacrificing Yield

Researchers have developed a way to make rice more resistant to bacterial blight and other diseases without reducing yield. Photo by Max Pixel.

Researchers have successfully developed a novel method that allows for increased disease resistance in rice without decreasing yield. A team at Duke University, working in collaboration with scientists at Huazhong Agricultural University in China, describe the findings in a paper published May 17, 2017 in the journal Nature.

Rice is one of the most important staple crops, responsible for providing over one-fifth of the calories consumed by humans worldwide. Diseases caused by bacterial or fungal pathogens present a significant problem, and can result in the loss of 80 percent or more of a rice crop.

Decades of research into the plant immune response have identified components that can be used to engineer disease-resistant plants. However, their practical application to crops is limited due to the decreased yield associated with a constantly active defense response.

“Immunity is a double-edged sword, ” said study co-author Xinnian Dong, professor of biology at Duke and lead investigator of the study. “There is often a tradeoff between growth and defense because defense proteins are not only toxic to pathogens but also harmful to self when overexpressed,” Dong said. “This is a major challenge in engineering disease resistance for agricultural use because the ultimate goal is to protect the yield.”

Previous studies have focused on altering the coding sequence or upstream DNA sequence elements of a gene. These upstream DNA elements are known as promoters, and they act as switches that turn on or off a gene’s expression. This is the first step of a gene’s synthesis into its protein product, known as transcription.

By attaching a promoter that gives an “on” signal to a defense gene, a plant can be engineered to be highly resistant to pathogens, though at a cost to growth and yield. These costs can be partially alleviated by attaching the defense gene to a “pathogen specific” promoter that turns on in the presence of pathogen attack.

To further alleviate the negative effects of active defense, the Dong group sought to add an additional layer of control. They turned newly discovered sequence elements, called upstream open reading frames (uORFs), to help address this problem. These sequence elements act on the intermediate of a gene, or messenger (RNA, a molecule similar to DNA) to govern its “translation” into the final protein product. A recent study by the Dong lab in an accompanying paper in Nature has identified many of these elements that respond in a pathogen-inducible manner.

The Dong group hypothesized that adding this pathogen-inducible translational regulation would result in a tighter control of defense protein expression and minimize the lost yield associated with enhanced disease resistance.

To test this hypothesis, the researchers started with Arabidopsis, a flowering plant commonly used in laboratory research. They created a DNA sequence that contains both the transcriptional and translational elements (uORFs) and fused them upstream of the potent “immune activator” gene called snc1. This hybrid sequence was called a “transcriptional/translational cassette” and was inserted into Arabidopsis plants.

When plants have snc1 constitutively active, they are highly resistant to pathogens, but have severely stunted growth. Strikingly, plants with the transcriptional/translational cassette not only have increased resistance, but they also lacked growth defects and resembled healthy wild-type plants. These results show the benefits of adding translational control in engineering plants that have increased resistance without significant costs.

The Dong group then sought to apply these findings to engineer disease-resistant rice, as it is one of the world’s most important crops. They created transgenic rice lines containing the transcriptional/translational cassette driving expression of another potent “immune activator” gene called AtNPR1. This gene was chosen as it has been found to confer broad spectrum pathogen resistance in a wide variety of crop species, including rice, citrus, apple and wheat.

The dry yellowish leaves on these rice plants are a classic symptom of bacterial blight, a devastating disease that affects rice fields worldwide. Photo by Meng Yuan.

The transgenic rice lines containing the transcriptional/translational cassette were infected with bacterial/fungal pathogens that cause three major rice diseases — rice  blight, leaf streak, and fungal blast. These showed high resistance to all three pathogens, indicating broad spectrum resistance could be achieved. Importantly, when grown in the field, their yield — both in terms of grain quantity and quality per plant — was almost unaffected. These results indicate a great potential for agricultural applications.

This strategy is the first known use of adding translational control for the engineering of disease-resistant crops with minimal yield costs. It has many advantages, as it is broadly applicable to a variety of crop species against many pathogens. Since this strategy involves activating the plants’ endogenous defenses, it may also reduce the use of pesticides on crops and hence protect the environment.

Additionally, these findings may be broadly applicable to other systems as well. These upstream elements (uORFs) are widely present in organisms from yeast to humans, with nearly half of all human transcripts containing them. “The great potential in using these elements in controlling protein translation during specific biological processes has yet to be realized,” Dong said.

Corresponding author Xinnian Dong can be reached at xdong@duke.edu or (919) 613-8176.

CITATION:  “uORF-Mediated Translation Allows Engineered Plant Disease Resistance Without Fitness Costs,” Guoyong Xu, Meng Yuan,   Chaoren Ai, Lijing Liu, Edward Zhuang, Sargis Karapetyan, Shiping Wang and Xinnian Dong. Nature, May 17, 2017. DOI: 10.1038/nature22372

 

Guest post by Jonathan Motley

Page 1 of 28

Powered by WordPress & Theme by Anders Norén