3-D Printer Can Build Synthetic Tissues

3-D Printer Can Build Synthetic Tissues

  4:26:00 PM    Science Lover

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A custom-built programmable 3D printer can create materials with several of the properties of living tissues, Oxford University scientists have demonstrated.

A custom-built programmable 3D printer can create materials with several of the properties of living tissues, Oxford University scientists have demonstrated: Droplet network c.500 microns across with electrically conductive pathway between electrodes mimicking nerve. (Credit: Oxford University/G Villar)

The new type of material consists of thousands of connected water droplets, encapsulated within lipid films, which can perform some of the functions of the cells inside our bodies.

These printed 'droplet networks' could be the building blocks of a new kind of technology for delivering drugs to places where they are needed and potentially one day replacing or interfacing with damaged human tissues. Because droplet networks are entirely synthetic, have no genome and do not replicate, they avoid some of the problems associated with other approaches to creating artificial tissues -- such as those that use stem cells.

The team report their findings in this week's Science.

'We aren't trying to make materials that faithfully resemble tissues but rather structures that can carry out the functions of tissues,' said Professor Hagan Bayley of Oxford University's Department of Chemistry, who led the research. 'We've shown that it is possible to create networks of tens of thousands connected droplets. The droplets can be printed with protein pores to form pathways through the network that mimic nerves and are able to transmit electrical signals from one side of a network to the other.'

Each droplet is an aqueous compartment about 50 microns in diameter. Although this is around five times larger than living cells the researchers believe there is no reason why they could not be made smaller. The networks remain stable for weeks.

'Conventional 3D printers aren't up to the job of creating these droplet networks, so we custom built one in our Oxford lab to do it,' said Professor Bayley. 'At the moment we've created networks of up to 35,000 droplets but the size of network we can make is really only limited by time and money. For our experiments we used two different types of droplet, but there's no reason why you couldn't use 50 or more different kinds.'

The unique 3D printer was built by Gabriel Villar, a DPhil student in Professor Bayley's group and the lead author of the paper.

The droplet networks can be designed to fold themselves into different shapes after printing -- so, for example, a flat shape that resembles the petals of a flower is 'programmed' to fold itself into a hollow ball, which cannot be obtained by direct printing. The folding, which resembles muscle movement, is powered by osmolarity differences that generate water transfer between droplets.

Gabriel Villar of Oxford University's Department of Chemistry said: 'We have created a scalable way of producing a new type of soft material. The printed structures could in principle employ much of the biological machinery that enables the sophisticated behaviour of living cells and tissues.'

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Biological Transistor Enables Computing Within Living Cells

Biological Transistor Enables Computing Within Living Cells

  10:14:00 AM    Science Lover

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When Charles Babbage prototyped the first computing machine in the 19th century, he imagined using mechanical gears and latches to control information. ENIAC, the first modern computer developed in the 1940s, used vacuum tubes and electricity. Today, computers use transistors made from highly engineered semiconducting materials to carry out their logical operations.

Artist's rendering of cells. (Credit: © Jezper / Fotolia)

And now a team of Stanford University bioengineers has taken computing beyond mechanics and electronics into the living realm of biology. In a paper to be published March 28 in Science, the team details a biological transistor made from genetic material -- DNA and RNA -- in place of gears or electrons. The team calls its biological transistor the "transcriptor."

"Transcriptors are the key component behind amplifying genetic logic -- akin to the transistor and electronics," said Jerome Bonnet, PhD, a postdoctoral scholar in bioengineering and the paper's lead author.

The creation of the transcriptor allows engineers to compute inside living cells to record, for instance, when cells have been exposed to certain external stimuli or environmental factors, or even to turn on and off cell reproduction as needed.

"Biological computers can be used to study and reprogram living systems, monitor environments and improve cellular therapeutics," said Drew Endy, PhD, assistant professor of bioengineering and the paper's senior author.

The biological computer

In electronics, a transistor controls the flow of electrons along a circuit. Similarly, in biologics, a transcriptor controls the flow of a specific protein, RNA polymerase, as it travels along a strand of DNA.

"We have repurposed a group of natural proteins, called integrases, to realize digital control over the flow of RNA polymerase along DNA, which in turn allowed us to engineer amplifying genetic logic," said Endy.

Using transcriptors, the team has created what are known in electrical engineering as logic gates that can derive true-false answers to virtually any biochemical question that might be posed within a cell.

They refer to their transcriptor-based logic gates as "Boolean Integrase Logic," or "BIL gates" for short.

Transcriptor-based gates alone do not constitute a computer, but they are the third and final component of a biological computer that could operate within individual living cells.

Despite their outward differences, all modern computers, from ENIAC to Apple, share three basic functions: storing, transmitting and performing logical operations on information.

Last year, Endy and his team made news in delivering the other two core components of a fully functional genetic computer. The first was a type of rewritable digital data storage within DNA. They also developed a mechanism for transmitting genetic information from cell to cell, a sort of biological Internet.

It all adds up to creating a computer inside a living cell.

Boole's gold

Digital logic is often referred to as "Boolean logic," after George Boole, the mathematician who proposed the system in 1854. Today, Boolean logic typically takes the form of 1s and 0s within a computer. Answer true, gate open; answer false, gate closed. Open. Closed. On. Off. 1. 0. It's that basic. But it turns out that with just these simple tools and ways of thinking you can accomplish quite a lot.

"AND" and "OR" are just two of the most basic Boolean logic gates. An "AND" gate, for instance, is "true" when both of its inputs are true -- when "a" and "b" are true. An "OR" gate, on the other hand, is true when either or both of its inputs are true.

In a biological setting, the possibilities for logic are as limitless as in electronics, Bonnet explained. "You could test whether a given cell had been exposed to any number of external stimuli -- the presence of glucose and caffeine, for instance. BIL gates would allow you to make that determination and to store that information so you could easily identify those which had been exposed and which had not," he said.

By the same token, you could tell the cell to start or stop reproducing if certain factors were present. And, by coupling BIL gates with the team's biological Internet, it is possible to communicate genetic information from cell to cell to orchestrate the behavior of a group of cells.

"The potential applications are limited only by the imagination of the researcher," said co-author Monica Ortiz, a PhD candidate in bioengineering who demonstrated autonomous cell-to-cell communication of DNA encoding various BIL gates.

Building a transcriptor

To create transcriptors and logic gates, the team used carefully calibrated combinations of enzymes -- the integrases mentioned earlier -- that control the flow of RNA polymerase along strands of DNA. If this were electronics, DNA is the wire and RNA polymerase is the electron.

"The choice of enzymes is important," Bonnet said. "We have been careful to select enzymes that function in bacteria, fungi, plants and animals, so that bio-computers can be engineered within a variety of organisms."

On the technical side, the transcriptor achieves a key similarity between the biological transistor and its semiconducting cousin: signal amplification.

With transcriptors, a very small change in the expression of an integrase can create a very large change in the expression of any two other genes.

To understand the importance of amplification, consider that the transistor was first conceived as a way to replace expensive, inefficient and unreliable vacuum tubes in the amplification of telephone signals for transcontinental phone calls. Electrical signals traveling along wires get weaker the farther they travel, but if you put an amplifier every so often along the way, you can relay the signal across a great distance. The same would hold in biological systems as signals get transmitted among a group of cells.

"It is a concept similar to transistor radios," said Pakpoom Subsoontorn, a PhD candidate in bioengineering and co-author of the study who developed theoretical models to predict the behavior of BIL gates. "Relatively weak radio waves traveling through the air can get amplified into sound."

Public-domain biotechnology

To bring the age of the biological computer to a much speedier reality, Endy and his team have contributed all of BIL gates to the public domain so that others can immediately harness and improve upon the tools.

"Most of biotechnology has not yet been imagined, let alone made true. By freely sharing important basic tools everyone can work better together," Bonnet said.

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Using 3-D Printing and Injectable Molds, Bioengineered Ears Look and Act Like the Real Thing

Using 3-D Printing and Injectable Molds, Bioengineered Ears Look and Act Like the Real Thing

  9:17:00 AM    Science Lover

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Cornell bioengineers and physicians have created an artificial ear -- using 3-D printing and injectable molds -- that looks and acts like a natural ear, giving new hope to thousands of children born with a congenital deformity called microtia.

A 3-D printer in Weill Hall deposits cells encapsulated in a hydrogel that will develop into new ear tissue. The printer takes instructions from a file built from 3-D photographs of human ears taken with a scanner in Rhodes Hall. (Credit: Lindsay France/University Photography)

In a study published online Feb. 20 in PLOS One, Cornell biomedical engineers and Weill Cornell Medical College physicians described how 3-D printing and injectable gels made of living cells can fashion ears that are practically identical to a human ear. Over a three-month period, these flexible ears grew cartilage to replace the collagen that was used to mold them.

"This is such a win-win for both medicine and basic science, demonstrating what we can achieve when we work together," said co-lead author Lawrence Bonassar, associate professor of biomedical engineering.

The novel ear may be the solution reconstructive surgeons have long wished for to help children born with ear deformity, said co-lead author Dr. Jason Spector, director of the Laboratory for Bioregenerative Medicine and Surgery and associate professor of plastic surgery at Weill Cornell in New York City.

"A bioengineered ear replacement like this would also help individuals who have lost part or all of their external ear in an accident or from cancer," Spector said. Replacement ears are usually constructed with materials that have a Styrofoam-like consistency, or sometimes, surgeons build ears from a patient's harvested rib. This option is challenging and painful for children, and the ears rarely look completely natural or perform well, Spector said.

To make the ears, Bonassar and colleagues started with a digitized 3-D image of a human subject's ear, and converted the image into a digitized "solid" ear using a 3-D printer to assemble a mold.

This Cornell-developed, high-density gel is similar to the consistency of Jell-o when the mold is removed. The collagen served as a scaffold upon which cartilage could grow.

The process is also fast, Bonassar added: "It takes half a day to design the mold, a day or so to print it, 30 minutes to inject the gel, and we can remove the ear 15 minutes later. We trim the ear and then let it culture for several days in nourishing cell culture media before it is implanted."

The incidence of microtia, which is when the external ear is not fully developed, varies from almost 1 to more than 4 per 10,000 births each year. Many children born with microtia have an intact inner ear, but experience hearing loss due to the missing external structure.

Spector and Bonassar have been collaborating on bioengineered human replacement parts since 2007. The researchers specifically work on replacement human structures that are primarily made of cartilage -- joints, trachea, spine, nose -- because cartilage does not need to be vascularized with a blood supply in order to survive.

"Using human cells, specifically those from the same patient, would reduce any possibility of rejection," Spector said.

He added that the best time to implant a bioengineered ear on a child would be when they are about 5 or six 6 old. At that age, ears are 80 percent of their adult size. If all future safety and efficacy tests work out, it might be possible to try the first human implant of a Cornell bioengineered ear in as little as three years, Spector said.

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Strange Behavior: New Study Exposes Living Cells to Synthetic Protein

Strange Behavior: New Study Exposes Living Cells to Synthetic Protein

  10:22:00 AM    Science Lover

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One approach to understanding components in living organisms is to attempt to create them artificially, using principles of chemistry, engineering and genetics. A suite of powerful techniques -- collectively referred to as synthetic biology -- have been used to produce self-replicating molecules, artificial pathways in living systems and organisms bearing synthetic genomes.

The depletion of ATP in cells of the bacterium Escherichia coli causes them to transition to a filamentous state and form dense lipid structures known as endoliposomes. The structures can be clearly observed in these transmission electron micrographs of increasing magnification. (Credit: Image courtesy of Arizona State University)

In a new twist, John Chaput, a researcher at Arizona State University's Biodesign Institute and colleagues at the Department of Pharmacology, Midwestern University, Glendale, AZ have fabricated an artificial protein in the laboratory and examined the surprising ways living cells respond to it.

"If you take a protein that was created in a test tube and put it inside a cell, does it still function," Chaput asks. "Does the cell recognize it? Does the cell just chew it up and spit it out?" This unexplored area represents a new domain for synthetic biology and may ultimately lead to the development of novel therapeutic agents.

The research results, reported in the advanced online edition of the journal ACS Chemical Biology, describe a peculiar set of adaptations exhibited by Escherichia coli bacterial cells exposed to a synthetic protein, dubbed DX. Inside the cell, DX proteins bind with molecules of ATP, the energy source required by all biological entities.

"ATP is the energy currency of life," Chaput says. The phosphodiester bonds of ATP contain the energy necessary to drive reactions in living systems, giving up their stored energy when these bonds are chemically cleaved. The depletion of available intracellular ATP by DX binding disrupts normal metabolic activity in the cells, preventing them from dividing, (though they continue to grow).

After exposure to DX, the normally spherical E. coli bacteria develop into elongated filaments. Within the filamentous bacteria, dense intracellular lipid structures act to partition the cell at regular intervals along its length. These unusual structures, which the authors call endoliposomes, are an unprecedented phenomenon in such cells.

"Somewhere along the line of this filamentation, other processes begin to happen that we haven't fully understood at the genetic level, but we can see the results phenotypically," Chaput says. "These dense lipid structures are forming at very regular regions along the filamented cell and it looks like it could be a defense mechanism, allowing the cell to compartmentalize itself." This peculiar adaptation has never been observed in bacterial cells and appears unique for a single-celled organism.

Producing a synthetic protein like DX, which can mimic the elaborate folding characteristics of naturally occurring proteins and bind with a key metabolite like ATP is no easy task. As Chaput explains, a clever strategy known as mRNA display was used to produce, fine-tune and amplify synthetic proteins capable of binding ATP with high affinity and specificity, much as a naturally occurring ATP-binding protein would.

First, large libraries of random sequence peptides are formed from the four nucleic acids making up DNA, with each strand measuring around 80 nucleotides in length. These sequences are then transcribed into RNA with the help of an enzyme -- RNA polymerase. If a natural ribosome is then introduced, it attaches to the strand and reads the random sequence RNA as though it was a naturally-occurring RNA, generating a synthetic protein as it migrates along the strand. In this way, synthetic proteins based on random RNA sequences can be generated.

Exposing the batch of synthetic proteins to the target molecule and extracting those that bind can then select for ATP-binding proteins. But as Chaput explains, there's a problem: "The big question is how do you recover that genetic information? You can't reverse transcribe a protein back into DNA. You can't PCR amplify a protein. So we have to do all these molecular biology tricks."

The main trick involves an earlier step in the process. A molecular linker is chemically attached to the RNA templates, such that each RNA strand forms a bond with its newly translated protein. The mRNA-protein hybrids are exposed to selection targets (like ATP) over consecutive rounds of increasing stringency. After each round of selection, those library members that remain bound to the target are reverse-transcribed into cDNA (using their conveniently attached RNA messages), and then PCR amplified.

In the current study, E. coli cells exposed to DX transitioned into a filamentous form, which can occur naturally when such cells are subject to conditions of stress. The cells display low metabolic activity and limited cell division, presumably owing to their ATP-starved condition.

The study also examined the ability of E. coli to recover following DX exposure. The cells were found to enter a quiescent state known as viable but non-culturable (VBNC), meaning that they survived ATP sequestration and returned to their non-filamentous state after 48 hours, but lost their reproductive capacity. Further, this condition was difficult to reverse and seems to involve a fundamental reprogramming of the cell.

In an additional response to DX, the filamentous cells form previously undocumented structures, which the authors refer to as endoliposomes. These dense lipid concentrations, spanning the full width of the filamented E. coli, segment the cells into distinct compartments, giving the cells a stringbean-like appearance under the microscope.

The authors speculate that this adaptation may be an effort to maintain homeostasis in regions of the filamentous cell, which have essentially been walled off from the intrusion of ATP-depleting DX. They liken endoliposomes to the series of water-tight compartments found in submarines which are used to isolate damaged sections of the ship and speculate that DX-exposed cells are partitioning their genetic information into regions where it can be safely quarantined. Such self-compartmentalization is known to occur in some eukaryotic cells, but has not been previously observed in prokaryotes like E. coli.

The research indicates that there is still a great deal to learn about bacterial behavior and the repertoire of responses available when such cells encounter novel situations, such as an unfamiliar, synthetic protein. The study also notes that many infectious agents rely on a dormant state, (similar to the VBNC condition observed in the DX-exposed E. coli), to elude detection by antibiotics. A better understanding of the mechanisms driving this behavior could provide a new approach to targeting such pathogens.

The relative safety of E. coli as a model organism for study may provide a fruitful tool for more in-depth investigation of VBNC states in pathogenic organisms. Further, given ATP's central importance for living organisms, its suppression may provide another avenue for combating disease. One example would be an engineered bacteriophage capable of delivering DX genes to pathogenic organisms.

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Scientists Create Nanoscale Window to Biological World

Scientists Create Nanoscale Window to Biological World

  9:13:00 AM    Science Lover

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If the key to winning battles is knowing both your enemy and yourself, then scientists are now well on their way toward becoming the Sun Tzus of medicine by taking a giant step toward a priceless advantage -- the ability to see the soldiers in action on the battlefield.

A novel microfluidics platform allowed viewing of structural 
details of rotavirus double-layered particles; the 3-D graphic 
of the virus, in purple, was reconstructed from data gathered by 
the new technique. (Credit: Virginia Tech)

Investigators at the Virginia Tech Carilion Research Institute have invented a way to directly image biological structures at their most fundamental level and in their natural habitats. The technique is a major advancement toward the ultimate goal of imaging biological processes in action at the atomic level.

"It's sort of like the difference between seeing Han Solo frozen in carbonite and watching him walk around blasting stormtroopers," said Deborah Kelly, an assistant professor at the VTC Research Institute and a lead author on the paper describing the first successful test of the new technique. "Seeing viruses, for example, in action in their natural environment is invaluable."

The technique involves taking two silicon-nitride microchips with windows etched in their centers and pressing them together until only a 150-nanometer space between them remains. The researchers then fill this pocket with a liquid resembling the natural environment of the biological structure to be imaged, creating a microfluidic chamber.

Then, because free-floating structures yield images with poor resolution, the researchers coat the microchip's interior surface with a layer of natural biological tethers, such as antibodies, which naturally grab onto a virus and hold it in place.

In a recent study in Lab on a Chip, Kelly joined Sarah McDonald, also an assistant professor at the VTC Research Institute, to prove that the technique works. McDonald provided a pure sample of rotavirus double-layered particles for the study.

"What's missing in the field of structural biology right now is dynamics -- how things move in time," said McDonald. "Debbie is developing technologies to bridge that gap, because that's clearly the next big breakthrough that structural biology needs."

Rotavirus is the most common cause of severe diarrhea among infants and children. By the age of 5, nearly every child in the world has been infected at least once. And although the disease tends to be easily managed in the developed world, in developing countries rotavirus kills more than 450,000 children a year.

At the second step in the pathogen's life cycle, rotavirus sheds its outer layer, which allows it to enter a cell, and becomes what is called a double-layered particle. Once its second layer is exposed, the virus is ready to begin using the cell's own infrastructure to produce more viruses. It was the viral structure at this stage that the researchers imaged in the new study.

Kelly and McDonald coated the interior window of the microchip with antibodies to the virus. The antibodies, in turn, latched onto the rotaviruses that were injected into the microfluidic chamber and held them in place. The researchers then used a transmission electron microscope to image the prepared slide.

The technique worked perfectly.

The experiment gave results that resembled those achieved using traditional freezing methods to prepare rotavirus for electron microscopy, proving that the new technique can deliver accurate results.

"It's the first time scientists have imaged anything on this scale in liquid," said Kelly.

The next step is to continue to develop the technique with an eye toward imaging biological structures dynamically in action. Specifically, McDonald is looking to understand how rotavirus assembles, so as to better know and develop tools to combat this particular enemy of children's health.

The researchers said their ongoing collaboration is an example of the cross-disciplinary work that is becoming a hallmark of the VTC Research Institute.

"It's an ideal collaboration because Sarah provides a phenomenal model system by which we can develop new technologies to move the field of microstructural biology forward," said Kelly.

"It's very win-win," McDonald added. "While the virus is a great tool for Debbie to develop her techniques, her technology is critical for allowing me to understand how this deadly virus assembles and changes dynamically over time."

The paper "Visualizing viral assemblies in a nanoscale biosphere" was published online and will appear in a 2013 edition of Lab on a Chip.

The authors are Brian Gilmore, a research associate at the VTC Research Institute; Shannon Showalter, a research assistant at the VTC Research Institute; Madeline Dukes, an applications scientist at Protochips; Justin Tanner, a postdoctoral associate at the VTC Research Institute; Andrew Demmert, a student at the Virginia Tech Carilion School of Medicine; McDonald, in addition to her position at the VTC Research Institute, is an assistant professor of biomedical sciences and pathobiology in the Virginia-Maryland Regional College of Veterinary Medicine; and Kelly, in addition to her position at the VTC Research Institute, is an assistant professor of biological sciences in Virginia Tech's College of Science.

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Researchers Watch Tiny Living Machines Self-Assemble

Researchers Watch Tiny Living Machines Self-Assemble

  1:05:00 PM    Science Lover

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Enabling bioengineers to design new molecular machines for nanotechnology applications is one of the possible outcomes of a study by University of Montreal researchers that was published in Nature Structural and Molecular Biology June 10. The scientists have developed a new approach to visualize how proteins assemble, which may also significantly aid our understanding of diseases such as Alzheimer's and Parkinson's, which are caused by errors in assembly.

Vallée-Bélisle and Michnick have developed a new 
approach to visualize how proteins assemble, which may 
also significantly aid our understanding of diseases such 
as Alzheimer’s and Parkinson’s, which are caused by errors 
in assembly. Here shown are two different assembly stages 
(purple and red) of the protein ubiquitin and the fluorescent 
probe used to visualize these stage (tryptophan: see yellow).

"In order to survive, all creatures, from bacteria to humans, monitor and transform their environments using small protein nanomachines made of thousands of atoms," explained the senior author of the study, Prof. Stephen Michnick of the university's department of biochemistry. "For example, in our sinuses, there are complex receptor proteins that are activated in the presence of different odor molecules. Some of those scents warn us of danger; others tell us that food is nearby." Proteins are made of long linear chains of amino acids, which have evolved over millions of years to self-assemble extremely rapidly -- often within thousandths of a split second -- into a working nanomachine. "One of the main challenges for biochemists is to understand how these linear chains assemble into their correct structure given an astronomically large number of other possible forms," Michnick said.

"To understand how a protein goes from a linear chain to a unique assembled structure, we need to capture snapshots of its shape at each stage of assembly said Dr. Alexis Vallée-Bélisle, first author of the study. "The problem is that each step exists for a fleetingly short time and no available technique enables us to obtain precise structural information on these states within such a small time frame. We developed a strategy to monitor protein assembly by integrating fluorescent probes throughout the linear protein chain so that we could detect the structure of each stage of protein assembly, step by step to its final structure."

The protein assembly process is not the end of its journey, as a protein can change, through chemical modifications or with age, to take on different forms and functions. "Understanding how a protein goes from being one thing to becoming another is the first step towards understanding and designing protein nanomachines for biotechnologies such as medical and environmental diagnostic sensors, drug synthesis of delivery," Vallée-Bélisle said.

This research was supported by the Natural Sciences and Engineering Research Council of Canada and Le fond de recherché du Québec, Nature et Technologie. The article, "Visualizing transient protein folding intermediates by tryptophan scanning mutagenesis," published in Nature Structural & Molecular Biology, was coauthored by Alexis Vallée-Bélisle and Stephen W. Michnick of the Département de Biochimie de l'Université de Montréal. The University of Montreal is known officially as Université de Montréal.

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Building Molecular 'Cages' to Fight Disease

Building Molecular 'Cages' to Fight Disease

  8:30:00 PM    Science Lover

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UCLA biochemists have designed specialized proteins that assemble themselves to form tiny molecular cages hundreds of times smaller than a single cell. The creation of these miniature structures may be the first step toward developing new methods of drug delivery or even designing artificial vaccines.

This is a molecular cage created by designing 
specialized protein puzzle pieces. Every color 
represents a separate protein, where cylindrical 
segments indicate rigid parts and ribbon-like 
segments indicate flexible parts of each protein 
chain. The grey sphere in the protein cage was 
placed there to indicate the empty space in the 
middle of the container and is not part of the 
molecular structure. (Credit: Todd Yeates, Yen-
Ting Lai/UCLA Chemistry and Biochemistry)

"This is the first decisive demonstration of an approach that can be used to combine protein molecules together to create a whole array of nanoscale materials," said Todd Yeates, a UCLA professor of chemistry and biochemistry and a member of the UCLA-DOE Institute of Genomics and Proteomics and the California NanoSystems Institute at UCLA.

Published June 1 in the journal Science, the research could be utilized to create cages from any number of different proteins, with potential applications across the fields of medicine and molecular biology.

UCLA graduate student Yen-Ting Lai, lead author of the study, used computer models to identify two proteins that could be combined to form perfectly shaped three-dimensional puzzle pieces. Twelve of these specialized pieces fit together to create a molecular cage a mere fraction of the size of a virus.

"If you just connect two random proteins together, you expect to get an irregular network," said Yeates, senior author of the study. "In order to control the geometry, the idea was to make a rigid link holding the two proteins in place as if they were parts of a toy puzzle."

The specifically designed proteins intermesh to form a hollow lattice that could act as a vessel for drug delivery, he said.

"In principle, it would be possible to attach a recognition sequence for cancer cells on the outside of the cage, with a toxin or some other 'magic bullet' contained inside," said Yeates. "That way, the drug could be delivered directly to certain targets like tumor cells."

At this stage, the assembled protein cages are porous enough that a drug placed inside would likely leak out during the delivery process, Lai said. His next project will involve constructing a new molecular cage with an interior that will be better sealed.

Another use for the versatile protein structures might be as artificial vaccines. Some traditional vaccines use an inactive surface protein from a virus to trick the body's immune system into thinking it is under attack. This method isn't always effective, because sometimes the protein in question doesn't look enough like the virus to trigger a strong response from the body's defenders.

However, by decorating the surface of a molecular cage with segments of virus-derived proteins, the tiny structures might better mimic a virus, stimulating an immune response even stronger than a traditional vaccine and better protecting the human recipient from illness.

Before these protein structures can be used in medical applications, the molecular containers themselves must be constructed from human-like proteins, rather than the currently employed bacterial proteins that the human body might immediately clear from circulation, Yeates said.

"Our first challenge will be repeating these kinds of designs with molecules that are less likely to generate a host immune response," he said. "Generally, we want to use proteins that look like human proteins so the body does not recognize them as foreign."

The idea of building complex, self-assembled protein structures has been Yeates' ambition since he published a paper outlining preliminary work on this method in 2001. Yet the concept remained on the back burner for 10 years until Yen-Ting Lai joined Yeates' research group. With three master's degrees -- in structural biology, bioinformatics and biomedical engineering -- Lai had the right combination of skills to bring the research to fruition, Yeates said.

This project is federally funded by the National Science Foundation. Other co-authors include UCLA senior staff scientist Duilio Cascio.

A second breakthrough

A second paper co-authored by Yeates creates similarly designed molecular cages using multiple copies of the same protein as building blocks. The scientists control the shape of the cage by computing the sequence of amino acids necessary to link the proteins together at the correct angles. The research, also published May 31 in Science, resulted from a collaboration between the UCLA team and professor David Baker at the University of Washington.

This alternative method represents a more versatile approach because it requires only one type of protein to form a structure, Yeates said. However, devising different kinds of links between the identical proteins remains a major challenge. Lead author Neil King, a postdoctoral scholar at the University of Washington and a former student of Yeates, took the numerous computer-generated possibilities and tested each version experimentally until he found one which produced the right behavior.

This research is federally funded by the National Institutes of Health and the U.S. Department of Energy. Other UCLA co-authors include senior staff researcher Michael Sawaya.

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Editing the Genome: Scientists Unveil New Tools for Rewriting the Code of Life

Editing the Genome: Scientists Unveil New Tools for Rewriting the Code of Life

  2:50:00 AM    Science Lover

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The power to edit genes is as revolutionary, immediately useful and unlimited in its potential as was Johannes Gutenberg's printing press. And like Gutenberg's invention, most DNA editing tools are slow, expensive, and hard to use -- a brilliant technology in its infancy. Now, Harvard researchers developing genome-scale editing tools as fast and easy as word processing have rewritten the genome of living cells using the genetic equivalent of search and replace -- and combined those rewrites in novel cell strains, strikingly different from their forebears.

Researchers have unveiled genome-engineering technologies capable of fundamentally re-engineering genomes from the nucleotide to the megabase scale. Treating the chromosome as both an editable and an evolvable template, the researchers have demonstrated methods to rewrite a cell's genome through powerful new tools for biotechnology, energy and agriculture. (Credit: iStockphoto/Zmeel Photography)

"The payoff doesn't really come from making a copy of something that already exists," said George Church, a professor of genetics at Harvard Medical School who led the research effort in collaboration with Joe Jacobson, an associate professor at the Media Lab at the Massachusetts Institute of Technology. "You have to change it -- functionally and radically."

Such change, Church said, serves three goals. The first is to add functionality to a cell by encoding for useful new amino acids. The second is to introduce safeguards that prevent cross-contamination between modified organisms and the wild. A third, related aim, is to establish multi-viral resistance by rewriting code hijacked by viruses. In industries that cultivate bacteria, including pharmaceuticals and energy, such viruses affect up to 20 percent of cultures. A notable example afflicted the biotech company Genzyme, where estimates of losses due to viral contamination range from a few hundred million dollars to more than $1 billion.

In a paper scheduled for publication July 15 in Science, the researchers describe how they replaced instances of a codon -- a DNA "word" of three nucleotide letters -- in 32 strains of E. coli, and then coaxed those partially-edited strains along an evolutionary path toward a single cell line in which all 314 instances of the codon had been replaced. That many edits surpasses current methods by two orders of magnitude, said Harris Wang, a research fellow in Church's lab at the Wyss Institute for Biologically Inspired Engineering who shares lead-author credit on the paper with Farren Isaacs, an assistant professor of molecular, cellular and developmental biology at Yale University and former Harvard research fellow, and Peter Carr, a research scientist at the MIT Media Lab.



In the genetic code, most codons specify an amino acid, a protein building block. But a few codons tell the cell when to stop adding amino acids to a protein chain, and it was one of these "stop" codons that the Harvard researchers targeted. With just 314 occurrences, the TAG stop codon is the rarest word in the E. coli genome, making it a prime target for replacement. Using a platform called multiplex automated genome engineering, or MAGE, the team replaced instances of the TAG codon with another stop codon, TAA, in living E. coli cells. (Unveiled by the team in 2009, the MAGE process has been called an evolution machine for its ability to accelerate targeted genetic change in living cells.)

While MAGE, a small-scale engineering process, yielded cells in which TAA codons replaced some but not all TAG codons, the team constructed 32 strains that, taken together, included every possible TAA replacement. Then, using bacteria's innate ability to trade genes through a process called conjugation, the researchers induced the cells to transfer genes containing TAA codons at increasingly larger scales. The new method, called conjugative assembly genome engineering, or CAGE, resembles a playoff bracket -- a hierarchy that winnows 16 pairs to eight to four to two to one -- with each round's winner possessing more TAA codons and fewer TAG, explains Isaacs, who invokes "March Madness."

"We're testing decades-old theories on the conservation of the genetic code," Isaacs said. "And we're showing on a genome-wide scale that we're able to make these changes."

Eager to share their enabling technology, the team published their results as CAGE reached the semifinal round. Results suggested that the final four strains were healthy, even as the team assembled four groups of 80 engineered alterations into stretches of the chromosome surpassing 1 million DNA base pairs. "We encountered a great deal of skepticism early on that we could make so many changes and preserve the health of these cells," Carr said. "But that's what we've seen."

The researchers are confident that they will create a single strain in which TAG codons are completely eliminated. The next step, they say, is to delete the cell's machinery that reads the TAG gene -- freeing up the codon for a completely new purpose, such as encoding a novel amino acid.

"We're trying to challenge people," Wang said, "to think about the genome as something that's highly malleable, highly editable."

This research was funded by U.S. Department of Energy and the National Science Foundation.

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U of T researchers build an antenna for light

U of T researchers build an antenna for light

  9:53:00 AM    Science Lover

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University of Toronto researchers have derived inspiration from the photosynthetic apparatus in plants to engineer a new generation of nanomaterials that control and direct the energy absorbed from light.

Their findings are reported in a forthcoming issue of Nature Nanotechnology, which will be released on July 10, 2011.

The U of T researchers, led by Professors Shana Kelley and Ted Sargent, report the construction of what they term "artificial molecules."

"Nanotechnologists have for many years been captivated by quantum dots – particles of semiconductor that can absorb and emit light efficiently, and at custom-chosen wavelengths," explained co-author Kelley, a Professor at the Leslie Dan Faculty of Pharmacy, the Department of Biochemistry in the Faculty of Medicine, and the Department of Chemistry in the Faculty of Arts & Science. "What the community has lacked – until now – is a strategy to build higher-order structures, or complexes, out of multiple different types of quantum dots. This discovery fills that gap."

The team combined its expertise in DNA and in semiconductors to invent a generalized strategy to bind certain classes of nanoparticles to one another.

"The credit for this remarkable result actually goes to DNA: its high degree of specificity – its willingness to bind only to a complementary sequence – enabled us to build rationally-engineered, designer structures out of nanomaterials," said Sargent, a Professor in The Edward S. Rogers Sr. Department of Electrical & Computer Engineering at the University of Toronto, who is also the Canada Research Chair in Nanotechnology. "The amazing thing is that our antennas built themselves – we coated different classes of nanoparticles with selected sequences of DNA, combined the different families in one beaker, and nature took its course. The result is a beautiful new set of self-assembled materials with exciting properties."



Traditional antennas increase the amount of an electromagnetic wave – such as a radio frequency – that is absorbed, and then funnel that energy to a circuit. The U of T nanoantennas instead increased the amount of light that is absorbed and funneled it to a single site within their molecule-like complexes. This concept is already used in nature in light harvesting antennas, constituents of leaves that make photosynthesis efficient. "Like the antennas in radios and mobile phones, our complexes captured dispersed energy and concentrated it to a desired location. Like the light harvesting antennas in the leaves of a tree, our complexes do so using wavelengths found in sunlight," explained Sargent.

"Professors Kelley and Sargent have invented a novel class of materials with entirely new properties. Their insight and innovative research demonstrates why the University of Toronto leads in the field of nanotechnology," said Professor Henry Mann, Dean of the Leslie Dan Faculty of Pharmacy.

"This is a terrific piece of work that demonstrates our growing ability to assemble precise structures, to tailor their properties, and to build in the capability to control these properties using external stimuli," noted Paul S. Weiss, Fred Kavli Chair in NanoSystems Sciences at UCLA and Director of the California NanoSystems Institute.

Kelley explained that the concept published in today's Nature Nanotechnology paper is a broad one that goes beyond light antennas alone.

"What this work shows is that our capacity to manipulate materials at the nanoscale is limited only by human imagination. If semiconductor quantum dots are artificial atoms, then we have rationally synthesized artificial molecules from these versatile building blocks."

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Brain's Master Switch Is Verified

Brain's Master Switch Is Verified

  9:20:00 PM    Science Lover

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The protein that has long been suspected by scientists of being the master switch allowing brains to function has now been verified by an Iowa State University researcher.

Yeon-Kyun Shin, professor of biochemistry, 
biophysics and molecular biology at ISU, has 
shown that the protein called synaptotagmin1 
(Syt1) is the sole trigger for the release of 
neurotransmitters in the brain using this 
instrument that allows a new technique called 
single vesicle fusion method. 
(Credit: ISU photo by Bob Elbert)

Yeon-Kyun Shin, professor of biochemistry, biophysics and molecular biology at ISU, has shown that the protein called synaptotagmin1 (Syt1) is the sole trigger for the release of neurotransmitters in the brain.

Prior to this research, Syt1 was thought to be a part of the protein structure (not the sole protein) that triggered the release of neurotransmitters at 10 parts per million of calcium.

Shin's research is published in the current issue of the journal Science.

"Syt1 was a suspect previously, but people were not able to pinpoint that it's the real one, even though there were lots and lots of different trials," said Shin.

"In this case, we are trying to show in the laboratory that it's the real one. So we excluded everything else, and included SNARE proteins -- that's the machinery of the release, and the Syt1 is a calcium-sensing timer."

Syt1 senses, at 10 ppm of calcium, and tells the SNARE complex to open the pore to allow the movement of the neurotransmitters.

Brain activity occurs when neurotransmitters move into a fusion pore.

"We are showing that this Syt1 senses the calcium at 10 ppm, and sends the signal to the SNARE complex to open the fusion pore. That is the process that we are showing right now," Shin said.

Shin and his researchers were able to pinpoint the protein using a new technique called single vesicle fusion method. Using this method, they were able to create and monitor a single fusion event.

Previous research didn't allow scientists to look at single events, and instead required detecting many events and then taking an average of those events, Shin says.

Shin, who has been looking at this brain activity for 15 years, is happy about the discovery.

"We are quite excited that for the first time we are showing that Syt1 is really what triggers the signal in the brain," he said. "This is a really important thing in terms of neurosciences. This is the heart of the molecular part of the brain function."

Shin believes his discovery may be useful in understanding brain malfunctions such as autism, epilepsy and others.

While researching brain function, Shin has previously shown that taking statin drugs to lower cholesterol may actually inhibit some brain function.

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Synthetic Red Blood Cells Developed: Red-Blood-Cell-Like Particles Carry Oxygen, Drugs, and More

Synthetic Red Blood Cells Developed: Red-Blood-Cell-Like Particles Carry Oxygen, Drugs, and More

  12:54:00 AM    Science Lover

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Scientists at UC Santa Barbara, in collaboration with scientists at University of Michigan, have developed synthetic particles that closely mimic the characteristics and key functions of natural red blood cells, including softness, flexibility, and the ability to carry oxygen.

Biocompatible synthetic red blood cells (sRBCs) synthesized by the UCSB team, 

where the shell is composed of alternate layers of hemoglobin and BSA. 

(Scale bar, 5 microns). (Credit: Image courtesy of)

The primary function of natural red blood cells is to carry oxygen, and the synthetic red blood cells (sRBCs) do that very well, retaining 90% of their oxygen-binding capacity after a week. The sRBCs also, however, have been shown to deliver therapeutic drugs effectively and with controlled release, and to carry well-distributed contrast agents for enhanced resolution in diagnostic imaging.

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