Your entire viral infection history from a single drop of blood

Systematic viral epitope scanning (VirScan). This method allows comprehensive analysis of antiviral antibodies in human sera. VirScan combines DNA microarray synthesis and bacteriophage display to create a uniform, synthetic representation of peptide epitopes comprising the human virome. Immunoprecipitation and high-throughput DNA sequencing reveal the peptides recognized by antibodies in the sample. The color of each cell in the heatmap depicts the relative number of antigenic epitopes detected for a virus (rows) in each sample (columns). (credit: George J. Xu et al./Science).

New technology called called VirScan developed by Howard Hughes Medical Institute (HHMI) researchers makes it possible to test for current and past infections with any known human virus by analyzing a single drop of a person’s blood.

With VirScan, scientists can run a single test to determine which viruses have infected an individual, rather than limiting their analysis to particular viruses. That unbiased approach could uncover unexpected factors affecting individual patients’ health, and also expands opportunities to analyze and compare viral infections in large populations.

The comprehensive analysis can be performed for about $25 per blood sample, but the test is currently being used only as a research tool and is not yet commercially available.

Stephen J. Elledge, an HHMI investigator at Brigham and Women’s Hospital, led the development of VirScan. He and his colleagues have already used VirScan to screen the blood of 569 people in the United States, South Africa, Thailand, and Peru. The scientists described the new technology and reported their findings in the June 5, 2015, issue of the journal Science.

Virus antibodies: clues that last decades

Bacteriophage (credit: Wikimedia Commons)

VirScan works by screening the blood for antibodies against any of the 206 species of viruses known to infect humans*. The immune system ramps up production of pathogen-specific antibodies when it encounters a virus for the first time, and it can continue to produce those antibodies for years or decades after it clears an infection.

That means VirScan not only identifies viral infections that the immune system is actively fighting, but also provides a history of an individual’s past infections.

To develop the new test, Elledge and his colleagues synthesized more than 93,000 short pieces of DNA encoding different segments of viral proteins. They introduced those pieces of DNA into bacteria-infecting viruses called bacteriophage.

Each bacteriophage manufactured one of the protein segments — known as a peptide — and displayed the peptide on its surface. As a group, the bacteriophage displayed all of the protein sequences found in the more than 1,000 known strains of human viruses.

To test the method, the team used it to analyze blood samples from patients known to be infected with particular viruses, including HIV and hepatitis C. “It turns out that it works really well,” Elledge says. “We were in the sensitivity range of 95 to 100 percent for those, and the specificity was good—we  didn’t falsely identify people who were negative. That gave us confidence that we could detect other viruses, and when we did see them we would know they were real.”

Harvard Medical School | Viral History in a Drop of Blood

International study

Elledge and his colleagues used VirScan to analyze the antibodies in 569 people from four countries, examining about 100 million potential antibody/epitope interactions. They found that on average, each person had antibodies to ten different species of viruses. As expected, antibodies against certain viruses were common among adults but not in children, suggesting that children had not yet been exposed to those viruses. Individuals residing South Africa, Peru, and Thailand, tended to have antibodies against more viruses than people in the United States. The researchers also found that people infected with HIV had antibodies against many more viruses than did people without HIV.

Elledge says the team was surprised to find that antibody responses against specific viruses were surprisingly similar between individuals, with different people’s antibodies recognizing identical  amino acids in the viral peptides. “In this paper alone we identified more antibody/peptide interactions to viral proteins than had been identified in the previous history of all viral exploration,” he says. The surprising reproducibility of those interactions allowed the team to refine their analysis and improve the  sensitivity of VirScan, and Elledge says the method will continue to improve as his team analyzes more samples. Their findings on viral epitopes may also have important implications for vaccine design.

Elledge says the approach his team has developed is not limited to antiviral antibodies. His own lab is also using it to look for antibodies that attack a body’s own tissue in certain autoimmune diseases that are associated with cancer. A similar approach could also be used to screen for antibodies against other types of pathogens.

* Antibodies in the blood find their viral targets by recognizing unique features known as epitopes that are embedded in proteins on the virus surface. To perform the VirScan analysis, all of the peptide-displaying bacteriophage are allowed to mingle with a blood sample. Antiviral antibodies in the blood find and bind to their target epitopes within the displayed peptides. The scientists then retrieve the antibodies and wash away everything except for the few bacteriophage that cling to them. By sequencing the DNA of those bacteriophage, they can identify which viral protein pieces were grabbed onto by antibodies in the blood sample. That tells the scientists which viruses a person’s immune system has previously encountered, either through infection or through vaccination. Elledge estimates it would take about 2-3 days to process 100 samples, assuming sequencing is working optimally. He is optimistic the speed of the assay will increase with further development.

Abstract of Comprehensive serological profiling of human populations using a synthetic human virome

The human virome plays important roles in health and immunity. However, current methods for detecting viral infections and antiviral responses have limited throughput and coverage. Here, we present VirScan, a high-throughput method to comprehensively analyze antiviral antibodies using immunoprecipitation and massively parallel DNA sequencing of a bacteriophage library displaying proteome-wide peptides from all human viruses. We assayed over 108 antibody-peptide interactions in 569 humans across four continents, nearly doubling the number of previously established viral epitopes. We detected antibodies to an average of 10 viral species per person and 84 species in at least two individuals. Although rates of specific virus exposure were heterogeneous across populations, antibody responses targeted strongly conserved “public epitopes” for each virus, suggesting that they may elicit highly similar antibodies. VirScan is a powerful approach for studying interactions between the virome and the immune system.

Creating DNA-based nanostructures without water

Three different DNA nanostructures assembled at room temperature in water-free glycholine (left) and in 75 percent glycholine-water mixture (center and right). The structures are (from left to right) a tall rectangle two-dimensional DNA origami, a triangle made of single-stranded tails, and a six-helix bundle three-dimensional DNA origami (credit: Isaac Gállego).

Researchers at the Georgia Institute of Technology have discovered an new process for assembling DNA nanostructures in a water-free solvent, which may allow for fabricating more complex nanoscale structures — especially, nanoelectronic chips based on DNA.

Scientists have been using DNA to construct sophisticated new structures from nanoparticles (such as a recent development at Brookhaven National Labs reported by KurzweilAI May 26), but the use of DNA has required a water-based environment. That’s because DNA naturally functions inside the watery environment of living cells. However, the use of water limited the types of structures that are possible.

The viscosity of a new solvent used for assembling DNA nanostructures (credit: Rob Felt)

In addition, the Georgia Tech researchers discovered that, paradoxically, adding a small amount of water to their water-free solvent during the assembly process (and removing it later) increases the assembly rate. It could also allow for even more complex structures, by reducing the problem of DNA becoming trapped in unintended structures by aggregation (clumping).

The new solvent they used is known as glycholine, a mixture of glycerol (used for sweetening and preserving food) and choline chloride, but the researchers are exploring other materials.

The solvent system could improve the combined use of metallic nanoparticles and DNA based materials at room temperature. The solvent’s low volatility could also allow for storage of assembled DNA structures without the concern that a water-based medium would dry out.

The research on water-free solvents grew out of Georgia Tech researchers’ studies in the origins of life. They wondered if the molecules necessary for life, such as the ancestor of DNA, could have developed in a water-free solution. In some cases, they found, the chemistry necessary to make the molecules of life would be much easier without water being present.

Sponsored by the National Science Foundation and NASA, the research will be published as the cover story in Volume 54, Issue 23 of the journal Angewandte Chemie International Edition.

* The assembly rate of DNA nanostructures can be very slow, and depends strongly on temperature. Raising the temperature increases this rate, but temperatures that are too high can cause the DNA structures to fall apart. The solvent system developed at Georgia Tech adds a new level of control over DNA assembly. DNA structures assemble at lower temperatures in this solvent, and adding water can adjust the solvent’s viscosity (resistance to flow), which allows for faster assembly compared to the water-free version of the solvent.

Abstract of Folding and Imaging of DNA Nanostructures in Anhydrous and Hydrated Deep-Eutectic Solvents

There is great interest in DNA nanotechnology, but its use has been limited to aqueous or substantially hydrated media. The first assembly of a DNA nanostructure in a water-free solvent, namely a low-volatility biocompatible deep-eutectic solvent composed of a 4:1 mixture of glycerol and choline chloride (glycholine), is now described. Glycholine allows for the folding of a two-dimensional DNA origami at 20 °C in six days, whereas in hydrated glycholine, folding is accelerated (≤3 h). Moreover, a three-dimensional DNA origami and a DNA tail system can be folded in hydrated glycholine under isothermal conditions. Glycholine apparently reduces the kinetic traps encountered during folding in aqueous solvent. Furthermore, folded structures can be transferred between aqueous solvent and glycholine. It is anticipated that glycholine and similar solvents will allow for the creation of functional DNA structures of greater complexity by providing a milieu with tunable properties that can be optimized for a range of applications and nanostructures.

South Korean Team Kaist wins DARPA Robotics Challenge

DRC-Hubo robot turns valve 360 degrees in DARPA Robotics Challenge Final (credit: DARPA)

First place in the DARPA Robotics Challenge Finals this past weekend in Pomona, California went to Team Kaist of South Korea for its DRC-Hubo robot, winning $2 million in prize money.

Team IHMC Robotics of Pensacola, Fla., with its Running Man (Atlas) robot came in at second place ($1 million prize), followed by Tartan Rescue of Pittsburgh with its CHIMP robot ($500,000 prize).

DRC-Hubo, Running Man, and CHIMP (credit: DARPA)

The DARPA Robotics Challenge, with three increasingly demanding competitions over two years, was launched in response to a humanitarian need that became glaringly clear during the nuclear disaster at Fukushima, Japan, in 2011, DARPA said.

The goal was to “accelerate progress in robotics and hasten the day when robots have sufficient dexterity and robustness to enter areas too dangerous for humans and mitigate the impacts of natural or man-made disasters.”

The difficult course of eight tasks simulated Fukushima-like conditions, such as driving alone, walking through rubble, tripping circuit breakers, turning valves, and climbing stairs.

Representing some of the most advanced robotics research and development organizations in the world, a dozen teams from the United States and another eleven from Japan, Germany, Italy, Republic of Korea and Hong Kong competed.

DARPA | DARPA Robotics Challenge 2015 Proving the Possible

DARPA | A Celebration of Risk (a.k.a., Robots Take a Spill)

More DARPA Robotics Challenge videos

Super-resolution electron microscopy of soft materials like biomaterials

CLAIRE image of Al nanostructures with an inset that shows a cluster of six Al nanostructures (credit: Lawrence Berkeley National Laboratory)

Soft matter encompasses a broad swath of materials, including liquids, polymers, gels, foam and — most importantly — biomolecules. At the heart of soft materials, governing their overall properties and capabilities, are the interactions of nano-sized components.

Observing the dynamics behind these interactions is critical to understanding key biological processes, such as protein crystallization and metabolism, and could help accelerate the development of important new technologies, such as artificial photosynthesis or high-efficiency photovoltaic cells.

Observing these dynamics at sufficient resolution has been a major challenge, but this challenge is now being met with a new non-invasive nanoscale imaging technique that goes by the acronym of CLAIRE.

CLAIRE stands for “cathodoluminescence activated imaging by resonant energy transfer.” Invented by researchers with the U.S. Department of Energy (DOE)’s Lawrence Berkeley National Laboratory (Berkeley Lab) and the University of California (UC) Berkeley, CLAIRE extends the extremely high resolution of electron microscopy to the dynamic imaging of soft matter.

“Traditional electron microscopy damages soft materials and has therefore mainly been used to provide topographical or compositional information about robust inorganic solids or fixed sections of biological specimens,” says chemist Naomi Ginsberg, who leads CLAIRE’s development and holds appointments with Berkeley Lab’s Physical Biosciences Division and its Materials Sciences Division, as well as UC Berkeley’s departments of chemistry and physics.

“CLAIRE allows us to convert electron microscopy into a new non-invasive imaging modality for studying soft materials and providing spectrally specific information about them on the nanoscale.”

Ginsberg is also a member of the Kavli Energy NanoScience Institute (Kavli-ENSI) at Berkeley. She and her research group recently demonstrated CLAIRE’s imaging capabilities by applying the technique to aluminum nanostructures and polymer films that could not have been directly imaged with electron microscopy.

“What microscopic defects in molecular solids give rise to their functional optical and electronic properties? By what potentially controllable process do such solids form from their individual microscopic components, initially in the solution phase? The answers require observing the dynamics of electronic excitations or of molecules themselves as they explore spatially heterogeneous landscapes in condensed phase systems,” Ginsberg says.

“In our demonstration, we obtained optical images of aluminum nanostructures with 46 nanometer resolution, then validated the non-invasiveness of CLAIRE by imaging a conjugated polymer film. The high resolution, speed and non-invasiveness we demonstrated with CLAIRE positions us to transform our current understanding of key biomolecular interactions.”

How to avoid destroying soft matter with electron beams

CLAIRE works by essentially combining the best attributes of optical and scanning electron microscopy into a single imaging platform.

Scanning electron microscopes use beams of electrons rather than light for illumination and magnification. With much shorter wavelengths than photons of visible light, electron beams can be used to observe objects hundreds of times smaller than those that can be resolved with an optical microscope. However, these electron beams destroy most forms of soft matter and are incapable of spectrally specific molecular excitation.

Ginsberg and her colleagues get around these problems by employing a process called “cathodoluminescence,” in which an ultrathin scintillating film, about 20 nanometers thick, composed of cerium-doped yttrium aluminum perovskite, is inserted between the electron beam and the sample.

When the scintillating film is excited by a low-energy electron beam (about 1 KeV), it emits energy that is transferred to the sample, causing the sample to radiate. This luminescence is recorded and correlated to the electron beam position to form an image that is not restricted by the optical diffraction limit (which limits optical microscopy).

The CLAIRE imaging demonstration was carried out at the Molecular Foundry, a DOE Office of Science User Facility.

Observing biomolecular interactions, solar cells, and LEDs

While there is still more work to do to make CLAIRE widely accessible, Ginsberg and her group are moving forward with further refinements for several specific applications.

“We’re interested in non-invasively imaging soft functional materials like the active layers in solar cells and light-emitting devices,” she says. “It is especially true in organics and organic/inorganic hybrids that the morphology of these materials is complex and requires nanoscale resolution to correlate morphological features to functions.”

Ginsberg and her group are also working on the creation of liquid cells for observing biomolecular interactions under physiological conditions. Since electron microscopes can only operate in a high vacuum, as molecules in the air disrupt the electron beam, and since liquids evaporate in high vacuum, aqueous samples must either be freeze-dried or hermetically sealed in special cells.

“We need liquid cells for CLAIRE to study the dynamic organization of light-harvesting proteins in photosynthetic membranes,” Ginsberg says. “We should also be able to perform other studies in membrane biophysics to see how molecules diffuse in complex environments, and we’d like to be able to study molecular recognition at the single molecule level.”

In addition, Ginsberg and her group will be using CLAIRE to study the dynamics of nanoscale systems for soft materials in general. “We would love to be able to observe crystallization processes or to watch a material made of nanoscale components anneal or undergo a phase transition,” she says. “We would also love to be able to watch the electric double layer at a charged surface as it evolves, as this phenomenon is crucial to battery science.”

A paper describing the most recent work on CLAIRE has been published in the journal Nano Letters. This research was primarily supported by the DOE Office of Science and by the National Science Foundation.

Abstract of Cathodoluminescence-Activated Nanoimaging: Noninvasive Near-Field Optical Microscopy in an Electron Microscope

We demonstrate a new nanoimaging platform in which optical excitations generated by a low-energy electron beam in an ultrathin scintillator are used as a noninvasive, near-field optical scanning probe of an underlying sample. We obtain optical images of Al nanostructures with 46 nm resolution and validate the noninvasiveness of this approach by imaging a conjugated polymer film otherwise incompatible with electron microscopy due to electron-induced damage. The high resolution, speed, and noninvasiveness of this “cathodoluminescence-activated” platform also show promise for super-resolution bioimaging.

3-D printing tough biogel structures for tissue engineering or soft robots

Lasagna? No, an open lattice of 3-D printed material, with materials having different characteristics of strength and flexibility indicated by different colors (credit: the researchers)

Researchers at three universities have developed a new way of making tough — but soft and wet — biocompatible hydrogel materials into complex and intricately patterned shapes. The process might lead to scaffolds for repair or replacement of load-bearing tissues, such as cartilage. It could also allow for tough but flexible actuators for future robots, the researchers say.

The new process is described in a paper in the journal Advanced Materials, co-authored by MIT associate professor of mechanical engineering Xuanhe Zhao and colleagues at MIT, Duke University, and Columbia University.

Zhao says the process can produce complex hydrogel structures that are “extremely tough and robust,” but still allow for encapsulating cells in the structures. That could make it possible to 3D-print complex biostructures.

Biocompatible structures

Hydrogels are defined by water molecules encased in rubbery polymer networks that provide shape and structure. They are similar to natural tissues such as cartilage, which is used by the body as a natural shock absorber.

While synthetic hydrogels are commonly weak or brittle, a number of them that are tough and stretchable have been developed over the last decade. However, making tough hydrogels has usually involved “harsh chemical environments” that would kill living cells encapsulated in them, Zhao says.

The new hydrogel materials are generated by combining polyethylene glycol (PEG) and sodium alginate, which synergize to form a hydrogel tougher than natural cartilage. The materials are benign enough to synthesize together with living cells — such as stem cells — which could then allow high viability of the cells, says Zhao, who holds a joint appointment in MIT’s Department of Civil and Environmental Engineering.

3-D printing strong, flexible biomaterials

3-D printed tough, biocompatible PEG–alginate–nanoclay hydrogels in ear and nose shapes (credit: Sungmin Hong et al./ Advanced Materials)

Previous work was not able to produce complex 3-D structures with tough hydrogels, Zhao says. The new biocompatible tough hydrogel can be printed into diverse 3-D structures such as a hollow cube, hemisphere, pyramid, twisted bundle, multilayer mesh, or physiologically relevant shapes, such as a human nose or ear.

The new method uses a commercially available 3D-printing mechanism, Zhao explains. “The innovation is really about the material — a new ink for 3-D printing of biocompatible tough hydrogel,” he says, specifically, a composite of two different biopolymers.

“Each [material] individually is very weak and brittle, but once you put them together, it becomes very tough and strong. It’s like steel-reinforced concrete.”

The PEG material provides elasticity to the printed material, while sodium alginate allows it to dissipate energy under deformation without breaking. A third ingredient, a biocompatible “nanoclay,” makes it possible to fine-tune the viscosity (how easily it flows) of the material, improving the ability to control its flow through the 3D-printing nozzle.

The material can be made so flexible that a printed shape, such as a pyramid, can be compressed by 99 percent, and then spring back to its original shape, Sungmin Hong, a lead author of the paper and a former postdoc in Zhao’s group, says; it can also be stretched to five times its original size. Such resilience is a key feature of natural bodily tissues that need to withstand a variety of forces and impacts.

Such materials might eventually be used to custom-print shapes for the replacement of cartilaginous tissues in ears, noses, or load-bearing body joints, Zhao says. Lab tests have already shown that the material is even tougher than natural cartilage.

Enhancing resolution

The next step in the research will be to improve the resolution of the printer, which is currently limited to details about 500 micrometers (0.5 millimeters) in size, and to test the printed hydrogel structures in animal models. “We are enhancing the resolution,” Zhao says, “to be able to print more accurate structures for applications.”

The technique could also be applied to printing a variety of soft but tough structural materials, he says, such as actuators for soft robotic systems.

“This is really beautiful work that demonstrates major advances in the utilization of tough hydrogels,” says David Mooney, a professor of bioengineering at Harvard University who was not involved in this work. “This builds off earlier work using other polymer systems, with some of this earlier work done by Dr. Zhao, but the demonstration that one can achieve similar mechanical performance with a common biomedical polymer is a substantial advance.

“It is also quite exciting that these new tough gels can be used for 3-D printing, as this is new for these gels, to my knowledge.”

The work was supported by the National Institutes of Health, the Office of Naval Research, AOSpine Foundation, and the National Science Foundation.

Abstract of 3D Printing of Highly Stretchable and Tough Hydrogels into Complex, Cellularized Structures

A 3D printable and highly stretchable tough hydrogel is developed by combining poly(ethylene glycol) and sodium alginate, which synergize to form a hydrogel tougher than natural cartilage. Encapsulated cells maintain high viability over a 7 d culture period and are highly deformed together with the hydrogel. By adding biocompatible nanoclay, the tough hydrogel is 3D printed in various shapes without requiring support material.