Duke Research Blog

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

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

Immerse Yourself in Virtual Reality on the Quad

Open since September 2016, the Virtual Reality Room on the first floor lounge of Edens 1C allows students to experience virtual reality using the HTC Vive headset and controllers.

DURHAM, N.C. — The virtual reality headset looked like something out of a science fiction film. It was tethered by a long cable to a glass-encased PC, which in turn was connected to thick hoses filled with glowing blue coolant.

I slipped the mask over my head and was literally transported to another world.

In real life, I was in the lower level of Edens residence hall testing out the recently opened BoltVR gaming room during an event hosted by the Duke Digital Initiative (DDI). Virtual reality is one of the technologies that DDI is exploring for its potential in teaching and learning.

Rebekkah Huss shoots invaders with a virtual bow and arrow in Duke's newest virtual reality space.

Rebekkah Huss shoots invaders with a virtual bow and arrow in Duke’s newest virtual reality space. Open to students 4 p.m. to 10 p.m. on weekdays, noon to midnight on weekends.

BoltVR is a virtual reality space outfitted with the immersive room-scale technology of the HTC Vive, an $800 gaming system consisting of the headset, hand-held controllers and motion sensors in the room. The VR experience is a new addition to the Bolt gaming suite that opened in 2015 for Duke students.

Once I had the headset on, suddenly the bare walls and carpet were replaced by the yellow lined grid of the Holodeck from Star Trek. It was like nothing I’d ever seen. This is like the home screen for the gaming system, explained  Mark-Everett McGill the designer of the BoltVR game room, as he scrolled through the more than 70 downloaded VR experiences on the BoltVR online account at Steam.

McGill chose a story experience so that I could adjust to being able to move around physical objects in a virtual space.

It was like the floor melted away. On a tiny asteroid in front of me The Little Prince and his rose played out their drama from the cover of the classic children’s book. The stars surrounded me and I tilted my head back to watch a giant planet fly over.

I could walk around the prince’s tiny asteroid and inspect the little world from all angles, but I found it disorienting to walk with normal stability while my eyes told me that I was floating in space. The HTC Vive has a built-in  guidance system called the Chaperone that used a map of the room to keep me from crashing into the walls, I still somehow managed to bump a spectator.

“A lot of people get motion sickness when they use VR because your eyes are sensing the movement but your ears are telling you, you aren’t doing anything.” said, McGill.

Lucky for me, I have a strong stomach and suffered no ill effects while wearing the headset. The HTC Vive also helps counteract motion sickness because is room scale design allows for normal walking and movement.

There was however, one part of the experience that felt very odd, and that was the handheld controllers. The controllers  are tracked by wall-mounted sensors so they show up really well in the VR headset. The problem was that in the titles I played my hands and body were invisible to me.

The headset and controller themselves are incredibly sensitive and accurate. I think most people would intuitively understand how to use them, especially if they have a gaming background, but I missed having the comfort of my own arms. So while the VR worlds are visually believable and the technology powering them is absolutely fascinating, there is still lots of room for new innovations.

Once I started playing games though, I no longer cared about the limitations of the tech because I was having so much fun!

The most popular student choice in the BoltVR is a subgame of The Lab by Valve, it’s a simple tower defense game where the player uses a bow and arrow to shoot little 2D stickmen and stop their attack.

Everything about using the bow felt pretty realistic like loading arrows, and using angles to control the trajectory of a shot. There was even a torch that I used to light my arrow on fire before launching it at an attacker. With unlimited ammunition, I happily guarded my tower from waves of baddies until I finally had to let someone else have a turn.

To learn more about VR experiences for teaching and learning at Duke, join the listserv at https://lists.duke.edu/sympa/subscribe/vr2learn.

Post by Rebekkah Huss

Post by Rebekkah Huss

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

Cooking Up “Frustrated” Magnets in Search of Superconductivity

Sara Haravifard

A simplified version of Sara Haravifard’s recipe for new superconductors, by the National High Magnetic Field Laboratory

Duke physics professor Sara Haravifard is mixing, cooking, squishing and freezing “frustrated” magnetic crystals in search of the origins of superconductivity.

Superconductivity refers to the ability of electrons to travel endlessly through certain materials, called superconductors, without adding any energy — think of a car that can drive forever with no gas or electricity. And just the way gas-less, charge-less cars would make travel vastly cheaper, superconductivity has the potential to revolutionize electronics and energy industry.

But superconductors are extremely rare, and are usually only superconductive at extremely cold temperatures — too cold for any but a few highly specialized applications. A few “high-temperature” superconductors have been discovered, but scientists are still flummoxed at why and how these superconductors exist.

Haravifard hopes that her magnet experiments will reveal the origins of high-temperature superconductivity so that researchers can design and build new materials with this amazing property. In the process, her team may also discover materials that are useful in quantum computing, or even entirely new states of matter.

Learn more about their journey on this fascinating infographic by The National High Magnetic Field Laboratory.

Infographic describing magnetic crystal research

Infographic courtesy of the National High Magnetic Field Laboratory

Kara J. Manke, PhD

Post by Kara Manke

Durham Students Give Themselves a Hand Up

Picture this: a group of young middle schoolers are gathered trying to get a “hand” they’ve built out of drinking straws, thread and clay to grasp a small container. What could such a scene possibly have to do with encouraging kids to stay in school and pursue science? It turns out, quite a lot!

brothers keeper

Angelo Moreno (right), a graduate student in molecular genetics and microbiology, helps students with their soda straw hand.

This scene was part of an event designed just for boys from Durham schools that took place one March evening at the Durham Marriot and Convention Center. It was hosted by Made in Durham, a local non-profit focused on helping Durham’s young people graduate from high school, go to college, and ultimately be prepared for their careers, and My Brother’s Keeper Durham, the local branch of former President Obama’s mentoring initiative for young men of color.

The first evening of a convention centered on building equity in education and was geared toward career exploration. Each of the boys got to choose from a series of workshops that highlighted careers in science, technology, engineering, art, and mathematics — also known as STEAM. The workshops ranged from architectural design to building body parts, which was where they learned to build the artificial hands.

Sharlini Sankaran, the executive director of Duke’s Regeneration Next Initiative, who heard about my outreach activities from earlier this year, contacted me, and together we drummed up a group of scientists for the event.

With the help of Victor Ruthig in Cell Biology, Angelo Moreno in Molecular Genetics and Microbiology, Ashley Williams in Biomedical Engineering, and Devon Lewis, an undergraduate in the Biology program, we dove into the world of prosthetics and tissue engineering with the young men who came to our workshop.

Biology undergrad Devon Lewis (top) worked with several of the students.

After some discussion on what it takes to build an artificial body part, we let the boys try their hand at building their own. We asked them what the different parts of the hand were that allowed us to bend them and move them in certain ways, and from there, they developed ideas for how to turn our household materials into fully functioning hands. We used string as tendons and straws as finger bones, cutting notches where we wanted to create joints.

There was a lot of laughter in the room, but also a lot of collaboration between the different groups of kids. When one team figured out how to make a multi-jointed finger, they would share that knowledge with other groups. Similar knowledge sharing happened when one group figured out how to use the clay to assemble all their fingers into a hand. Seeing these young men work together, problem solve, and be creative was amazing to watch and be a part of!

According to feedback from event organizers, “ours was the most popular session!” Sharlini said. When we reached the end of our session, the kids didn’t want to leave, and instead wanted to keep tinkering with their hands to see what they could accomplish.

The boys had a lot of fun, asked a lot of good questions, and got to pick our brains for advice on staying in school and using it to propel them towards career success. I have distilled some of the best pieces of advice from that night, since they’re good for everyone to hear:

  • Don’t be afraid to ask a lot of questions.
  • Don’t be discouraged when someone tells you no. Go for it anyways.
  • Don’t be afraid of failure.
  • And don’t think you have to fit a particular mold to succeed at something.

“I left feeling really inspired about our future generation of scientists and engineers,” Sharlini said. ”It’s good to know there are so many Duke students with the genuine and selfless desire to help others.”

It was a joy to participate in this event. We all had fun, and left having learned a lot — even the parents who came with their sons!

Outreach like this is incredibly important. Being mentors for young people with a budding interest in science can make the difference between them pursuing it further or dropping it altogether. Engaging with them to show them the passion we have for our work and that we were kids just like they are allows them to see that they can do it too.

Guest Post by Ariana Eily

Duke Scientists Visit Raleigh to Share Their Work

This post by graduate student Dan Keeley originally appeared on Regeneration NEXT. It is a followup to one of our earlier posts.

As a scientist, it is easy to get caught up in the day-to-day workflow of research and lose sight of the bigger picture. We are often so focused on generating and reporting solid, exciting data that we neglect another major aspect of our job; sharing our work and its impacts with the broader community. On Tuesday May 23rd, a group of graduate students from Duke went to the North Carolina legislative building to do just that.

L-R: Andrew George, Representative Marcia Morey (Durham County), Senator Terry Van Duyn (Buncombe County), Sharlini Sankaran, Dan Keeley, and Will Barclay at the NC legislative building.

Dr. Sharlini Sankaran, Executive Director of Duke’s Regeneration Next Initiative, organized a group of graduate students to attend the North Carolina Hospital Associations (NCHA) “Partnering for a Healthier Tomorrow!” advocacy day at the state legislature in Raleigh. The event gave representatives from various hospital systems an opportunity to interact with state legislators about the work they do and issues affecting healthcare in the state. Andrew George, a graduate student in the McClay Lab, Will Barclay, a graduate student in the Shinohara Lab, and I joined Dr. Sankaran to share some of the great tissue regeneration-related research going on at Duke.

Our morning was busy as elected officials, legislative staff, executive branch agency officials, and staff from other hospital systems stopped by our booth to hear what Regeneration Next is all about. We talked about the focus on harnessing Duke’s strengths in fundamental research on molecular mechanisms underlying regeneration and development, then pairing that with the expertise of our engineers and clinicians. We discussed topics including spine and heart regeneration mechanisms from the Poss Lab, advances in engineering skeletal muscle from the Bursac Lab, and clinical trials of bioengineered blood vessels for patients undergoing dialysis from Duke faculty Dr. Jeffrey Lawson.

It was remarkable to hear how engaged everyone was, we got great questions like ‘what is a zebrafish and why do you use them?’ and ‘why would a bioengineered ligament be better than one from an animal model or cadaver?’.  Every person who stopped by was supportive and many had a personal story to share about a health issue experienced by friends, family, or even themselves. As a graduate student who does basic research, it really underscored how important these personal connections are to our work, even though it may be far removed from the clinic.

Communicating our research to legislators and others at NCHA advocacy day was a great and encouraging experience. Health issues affect all of us. Our visit to the legislature on Tuesday was a reminder that there is support for the work that we do in hopes it will help lead to a healthier tomorrow.

Guest post by Dan Keeley, graduate student in BiologyDan Keeley

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

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