Breathing in diesel exhaust leads to changes 'deep under the hood'

A student participates in the study while seated in a booth.
Credit: Image courtesy of University of British Columbia

Just two hours of exposure to diesel exhaust fumes can lead to fundamental health-related changes in biology by switching some genes on, while switching others off, according to researchers at the University of British Columbia and Vancouver Coastal Health.

The study involved putting volunteers in a polycarbonate-enclosed booth -- about the size of a standard bathroom -- while breathing in diluted and aged exhaust fumes that are about equal to the air quality along a Beijing highway, or a busy port in British Columbia.

The researchers examined how such exposure affected the chemical "coating" that attaches to many parts of a person's DNA. That carbon-hydrogen coating, called methylation, can silence or dampen a gene, preventing it from producing a protein -- sometimes to a person's benefit, sometimes not. Methylation is one of several mechanisms for controlling gene expression, which is the focus of a rapidly growing field of study called epigenetics.

The study, published this month in Particle and Fibre Toxicology, found that diesel exhaust caused changes in methylation at about 2,800 different points on people's DNA, affecting about 400 genes. In some places it led to more methylation; in more cases, it decreased methylation.

How these changes in gene expression translate to health is the next step for researchers. But this study shows how vulnerable our genetic machinery can be to air pollution, and that changes are taking place even if there are no obvious symptoms.

"Usually when we look at the effects of air pollution, we measure things that are clinically obvious -- air flow, blood pressure, heart rhythm," said senior author Dr. Chris Carlsten, an associate professor in the Division of Respiratory Medicine. "But asthma, higher blood pressure or arrhythmia might just be the gradual accumulation of epigenetic changes. So we've revealed a window into how these long-term problems arise. We're looking at changes 'deep under the hood.'"

The fact that DNA methylation was affected after only two hours of exposure has positive implications, said Carlsten, the AstraZeneca Chair in Occupational and Environmental Lung Disease.

"Any time you can show something happens that quickly, it means you can probably reverse it -- either through a therapy, a change in environment, or even diet," he said.

Carlsten's team, having catalogued the changes along the entire human genome, is now sharing its data with scientists who are further exploring the function of specific genes.

Source: University of British Columbia

Sharing that crowded holiday flight with countless hitchhiking dust mites

Predicted structure of the group 1 allergen protein from an American house dust mite. Arrow points to the location of a novel mutation discovered by the University of Michigan-led team.
Credit: Rubaba Hamid

As if holiday travel isn't stressful enough. Now University of Michigan researchers say we're likely sharing that already overcrowded airline cabin with countless tiny creatures including house dust mites.

"What people might not realize when they board a plane is that they can share the flight with a myriad of microscopic passengers-- including house dust mites--that take advantage of humanity's technological progress for their own benefit," said U-M biologist Pavel Klimov.

"House dust mites can easily travel on an airline passenger's clothes, skin, food and baggage," said Klimov, an assistant research scientist in the U-M Department of Ecology and Evolutionary Biology. "Like humans, they use air travel to visit new places, where they establish new populations, expand their ranges and interact with other organisms through various means."

Air travel likely explains some of the findings of a new genetic study conducted by Klimov and U-M visiting scholar Rubaba Hamid that looked at the connections between house dust mite populations in the United States and South Asia.

They found genetic mutations shared by mites in the U.S. and Pakistan that demonstrate the eight-legged creatures' propensity for intercontinental dispersal, according to a research paper scheduled for online publication Dec. 10 in the journal PLOS ONE.

"What we found suggests that mite populations are indeed linked through migration across continents, though geographic differences still can be detected," Hamid said. "Every time a mite successfully migrates to a new place, it brings its own genetic signature that can be detected in the resident population a long time after the migration event."

The study focused on two medically important mite species, the American and European house dust mite. Both species have global distributions, though the former is more abundant in the U.S.

Ancestors of the two species probably separated from each other nearly 81 million years ago--long before the origin of humans--when they inhabited bird nests. Today, house dust mites are blamed for causing allergic reactions in more than 65 million people worldwide and thrive in the mattresses, sofas and carpets of even the cleanest homes.

Hamid, Klimov and their colleagues examined genetic variation in the group 1 allergen gene from samples of the two mite species collected in the U.S. and Pakistan. The group 1 allergen gene encodes for the most important allergy-causing protein in house dust mites.

An inactive form of this protein is used in clinics worldwide as part of the standard skin-prick test for allergies. Though the test can be inaccurate if it does not include local genetic variants of the allergy-causing protein, geographical variation in group 1 allergen proteins has not been extensively studied in the U.S., Klimov said.

"We need to have a better idea about the diversity of allergenic proteins around the world, and particularly in the United States," he said.

In genetic sequences from American house dust mites (Dermatophagoides farinae), the 
researchers observed mutations at 14 positions along the length of the group 1 allergen gene.

All but one of the mutations are "silent," meaning they occur at the DNA level without changing the amino acid structure of the protein. Only mutations at the protein level have medical significance because they can change allergenic properties.

"The most unexpected result was the finding that a previously unknown mutation occurred at the active site of the protein at position 197," Klimov said. "This was a rare mutation, found in only a single population of house dust mite in South Asia.

"Our analysis indicates that this mutation might alter the enzyme activity of the protein. But allergenic properties, immune response and cross-reactivity of the protein are unknown at this time," he said. "Follow-up experiments to elucidate these issues are underway in our lab."

Source: University of Michigan

Charged graphene gives DNA a stage to perform molecular gymnastics

DNA interacts with charged graphene and contorts into sequence-specific shapes when the charge is changed. Credit: Photo courtesy Alek Aksimentiev

When Illinois researchers set out to investigate a method to control how DNA moves through a tiny sequencing device, they did not know they were about to witness a display of molecular gymnastics.

Fast, accurate and affordable DNA sequencing is the first step toward personalized medicine. Threading a DNA molecule through a tiny hole, called a nanopore, in a sheet of graphene allows researchers to read the DNA sequence; however, they have limited control over how fast the DNA moves through the pore. In a new study published in the journal Nature Communications, University of Illinois physics professor Aleksei Aksimentiev and graduate student Manish Shankla applied an electric charge to the graphene sheet, hoping that the DNA would react to the charge in a way that would let them control its movement down to each individual link, or nucleotide, in the DNA chain.

"Ideally, you would want to step the DNA through the nanopore one nucleotide at a time," said Aksimentiev. "Take a measurement and then have another nucleotide in the sensing hole. That's the goal, and it hasn't been realized yet. We show that, to some degree, we can control the process by charging the graphene."

The researchers found that a positive charge in the graphene speeds up DNA movement through the nanopore, while a negative charge stops the DNA in its tracks. However, as they watched, the DNA seemed to dance across the graphene surface, pirouetting into shapes they had never seen, specific to the sequence of the DNA nucleotides.

"It reminds me of Swan Lake," Aksimentiev said. "It's very acrobatic. We were very surprised by the variety of DNA conformations that we can observe at the surface of graphene when we charge it. There is one sequence that starts out laying down on the surface, and when we change the charge, they all tilt on the side like they are doing a one-armed push-up. Then we also have nucleotides that would lay back, or go up like a ballerina en pointe."

Aksimentiev hypothesizes that the conformations are so different and so specific to the sequence because each nucleotide has a slightly different distribution of electrons, the negatively charged parts of the atoms. There is even a visible difference when a nucleotide is methylated, a tiny chemical change that can turn a gene on or off.

By switching the charge in the graphene, the researchers can control not only the DNA's motion through the pore, but also the shape the DNA contorts into.

"Because it's reversible, we can force it to adopt one conformation and then force it to go back. That's why we call it gymnastics," Aksimentiev said.

The researchers extensively used the Blue Waters supercomputer at the National Center for Supercomputing Applications, housed at the University of Illinois. They mapped each individual atom in the complex DNA molecule and ran numerous simulations of many different DNA sequences. Supercomputing power was essential to carrying out the work, Aksimentiev said.

"This is a really computationally intensive project," he said. "Having access to Blue Waters was essential because with the sheer number of simulations, we would not have been able to finish them. It would have taken too long."

The next step is to combine a charged nanopore setup with a sensor to build a DNA sequencing device that would incorporate both motion control and nucleotide recognition. The researchers also hope to explore the unexpected conformational changes for insights into epigenetics, the field that studies how genes are expressed and moderated.

"DNA is much more complicated than just a double helix. It's a complex molecule that has many properties, and we are still uncovering them," Aksimentiev said.

Video animation of DNA dancing as the graphene charge changes:


Source: University of Illinois at Urbana-Champaign

The simplest element: Turning hydrogen into 'graphene'

This image is a comparison of the carbon compound graphene with a similar hydrogen-based structure synthesized by Carnegie scientists. Credit: Carneige's Ivan Naumov and Russell Hemley

New work from Carnegie's Ivan Naumov and Russell Hemley delves into the chemistry underlying some surprising recent observations about hydrogen, and reveals remarkable parallels between hydrogen and graphene under extreme pressures. Their work is the cover story in the December issue of Accounts of Chemical Research.

Hydrogen is the most-abundant element in the cosmos. With only a single electron per atom, it is deceptively simple. As a result, hydrogen has been a testing ground for theories of the chemical bond since the birth of quantum mechanics a century ago. Understanding the nature of chemical bonding in extreme environments is crucial for expanding our understanding of matter over the broad range of conditions found in the universe.

Observing hydrogen's behavior under very high pressures has been a great challenge for researchers. But recently teams have been able to observe that at pressures of 2-to-3.5 million times normal atmospheric pressure it transforms into an unexpected structure consisting of layered sheets, rather than a close-packed metal as had been predicted many years ago.

These hydrogen sheets resemble the carbon compound graphene. Graphene's layers are each constructed of a honeycomb structure made of six-atom carbon rings. This conventional carbon graphene, first synthesized about a decade ago, is very light, but incredibly strong, and conducts heat and electricity very efficiently. These properties promise revolutionary technology, including advanced optical electronics for screens, high-functioning photovoltaic cells, and enhanced batteries and other energy storage devices.

The new work from Naumov and Hemley shows that the stability of the unusual hydrogen structure arises from the intrinsic stability of its hydrogen rings. These rings form because of so-called aromaticity, which is well understood in carbon-containing molecules such as benzene, as well as in graphene. Aromatic structures take on a ring-like shape that can be thought of as alternating single and double bonded carbons. But what actually happens is that the electrons that make up these theoretically alternating bonds become delocalized and float in a shared circle around the inside of the ring, increasing stability.

Naumov and Hemley's study also indicates that hydrogen initially becomes a dark poorly conducting metal like graphite instead of a conventional shiny metal and a good conductor, as was originally suggested in theoretical calculations going back to the 1930's using early quantum mechanical models for solids.

Though the discovery of this layered sheet character of dense hydrogen has come as a surprise to many, chemists 30 years ago--before the discovery of graphene--predicted the structure based on simple chemical considerations. Their work is validated and extended by the new findings.

"Overall, our results indicate that chemical bonding occurs over a much broader range of conditions than people had previously considered. However, the structural effects of that chemical bonding under extreme conditions can be very different than that observed under the ordinary conditions that are familiar to us," Hemley said.

Source: Carnegie Institution

Simple textiles can be used with catalysts to enable complex chemical reactions

To attach the "chemical tools" to the nylon fibers the chemist simply irradiate the soaked textile with UV light.

In future, it will be much easier to produce some active pharmaceutical substances and chemical compounds than was the case to date. An international team working with chemists from the Max-Planck-Institut für Kohlenforschung in Mülheim an der Ruhr have immobilised various catalysts on nylon in a very simple way. Catalysts mediate between the reagents in a chemical reaction and control the process leading to the desired end product. When textile material is used as a support for the chemical auxiliaries, the reaction can proceed on a large surface thereby increasing its efficiency.

One of the catalysts that the researchers used in this way plays an important role in the synthesis of a pharmaceutical agent which could only be used previously in dissolved form, making the production process very complicated and expensive. Immobilising this catalyst on fabric simplifies production considerably. This process may be expected to yield similar advantages for other chemical processes.

Functional textiles are usually understood as the textiles used to make windproof jackets, breathable footwear and particularly effective thermal underwear. However, the term could soon refer to something else -- textiles which are "functionalised" with the help of organic catalysts. Working in collaboration with scientists from the Deutsches 

Textilforschungszentrum in Krefeld and Sungkyunkwan University in Suwon, Korea, researchers at the Max-Planck-Institut für Kohlenforschung in Mülheim an der Ruhr have developed a process for immobilising different organic catalysts on textiles with the help of ultraviolet light. The fabric thereby acts as a support for the substances on which a chemical reaction occurs.

Up to now, science has focused more on the macroscopic functionality of textiles, for example clothing, explains Dr. Ji-Woong Lee, who recently completed his doctorate at the Max-Planck-Institut für Kohlenforschung under the supervision of Professor Benjamin List, head of the Institute's Homogenous Catalysis Group. "As opposed to this, our method can give simple textiles microscopic functionalities," explains the Korean scientist. Together with his colleagues, Dr. Lee armed pieces of nylon with catalysts. The latter can be imagined as chemical tools which fulfil various tasks during chemical reactions.

Excellent yields, little wear and tear

For their tests, the Mühlheim-based researchers used three organic catalysts: a base (dimethylaminopyridine, DMAP), a sulfonic acid and a catalyst which functions as both an acid and a base. The latter is used in the pharmaceuticals industry to steer a reaction to one of two products, which are chemically completely identical. The two forms have mirror-image structures, like a left and right hand, but only one variant has the desired medical effect. Up to now, the catalyst that generates this variant could only be used in dissolved form and then had to be separated again. The complicated separation process could be avoided using a catalyst immobilised on fabric.

To attach the catalysts to the nylon fibres, the chemists irradiated the textile to which a catalyst was applied with UV light for five minutes -- but no longer, as this would impede the activity of the catalyst and its immobilisation on the nylon. A comparable process did not exist up to now.

The catalysts, which were practically interwoven with the fabric, displayed all of the characteristics that the chemists expect from such a system: the result of the chemical reactions which the scientists undertook with the catalyst-loaded nylon strips is impressive. 

All three catalysts converted around 90 percent of the source materials to the desired products. And the catalyst which is used in the pharmaceutical industry and only generates one out of two mirror-image molecules, achieved a success rate of over 95 percent without showing any major signs of wear and tear. Ji-Woong Lee carried out several hundred test-runs and observed that the catalysts relinquished little of their functionality.

A large surface makes chemical reactions more efficient

Compared with other ways of immobilising catalysts, "organotextile catalysis" has several advantages: in particular, it provides the reagents with a larger surface than other supports, for example plastic spheres or foils -- the larger the surface, the more efficiently a reaction proceeds. Moreover, nylon is flexible and very inexpensive. Dry textiles loaded with catalysts are easy to transport, which means that it is simpler to meet the requirements for some chemical processes where it is practically impossible to set up sophisticated chemical systems. For example, organotextile catalysis could help in the treatment of water in locations where people are cut off from the water supply.

"Our method enables the low-cost production of long-term functionalised textiles without causing any pollution," says Ji-Woong Lee. He is entirely convinced that the process can be applied in several scientific areas -- and industrial processes. "In addition to chemistry, these could include biology, the materials science and pharmaceutics."

Source: Max-Planck-Institut für Kohlenforschung

Mode of action of protein channelrhodopsin-2 decoded: Findings facilitate manufacture of optogenetic tools

The pore of the ion channel is opened by removing the amino acid E90. Water molecules enter and tilt Helix H2, thus opening the continuous channel. Credit: © RUB, graphics: Eisenhauer

Researchers have shed light upon the mode of action of the light-controlled channelrhodopsin-2 with high spatiotemporal resolution. This biomolecule is used in optogenetic applications, which is deployed to control the activity of living cells with light.

"The model we developed makes it possible to create customised optogenetic tools for individual applications," says Prof Dr Klaus Gerwert from the Department of Biophysics at the Ruhr-Universität Bochum. Together with colleagues at the Humboldt Universität zu Berlin from the team headed by Prof Dr Peter Hegemann, the Bochum researchers report about their finding in the magazine "Angewandte Chemie."

Channelrhodopsin-2 has revolutionised optogenetics

Discovered by Peter Hegemann in green algae, channelrhodopsin-2 is the central light-activated channel protein in optogenetics. If this ion channel is applied to nerve cells, the channels can be opened by light, thus activating the cell. "The application of channelrhodopsin-2 in optogenetics has revolutionised neurobiology in the recent years," says Klaus Gerwert. The magazine "Nature Methods" awarded this process as "Method of the Year" in 2010. "However, scientists had not been aware of what is actually happening inside a protein and thus ultimately triggers its activation," continues the Bochum researcher. But it is the understanding of processes on the atomic level that is essential for optimising the protein specifically for its applications.

"EHT" model describes the mode of action of channelrhodopsin-2

With time-resolved vibrational spectroscopy and bio-molecular simulations, the Bochum-Berlin team has now closed that gap. The EHT (E90-Helix2-tilt) model describes the mode of action of channelrhodopsin-2 as follows: the light-sensitive group of the protein, i.e. the retinal, is twistedunder incidence of light. This twist then continues in the protein and opens a pore ultra fast, which is closed by amino acid E90 in the dark. E90 marks the narrowest place in the pore and opens it through a downward move , similar to the motion of a swing door, so that water can enter an empty vestibule above the narrowest place in the pore. The entering water then tilts the protein helix H2, which eventually triggers a protein-traversing open ion channel. When forming this model, the Bochum researchers benefitted from their comprehensive experience that they had gained resolving the mechanism of light-driven proton pump bacteriorhodopsin in detail.

"Protein engineering": pioneering novel optogenetic tools

"With this structural model, the next step, i.e. protein engineering, will become possible," explains Klaus Gerwert. Through mutation of the amino acid E90, the protein's properties can be controlled in a targeted manner. The conductivity or the selectivity for certain ions could be customised for specific applications, and the protein could be specifically activated with different wavelengths.

Source: Ruhr-Universitaet-Bochum

Synthetic biology for space exploration

Microbial-based biomanufacturing could be transformative once explorers arrive at an extraterrestrial site. Credit: Image courtesy of Royal Academy Interface

Does synthetic biology hold the key to manned space exploration of the Moon and Mars? Berkeley Lab researchers have used synthetic biology to produce an inexpensive and reliable microbial-based alternative to the world's most effective anti-malaria drug, and to develop clean, green and sustainable alternatives to gasoline, diesel and jet fuels. In the future, synthetic biology could also be used to make manned space missions more practical.

"Not only does synthetic biology promise to make the travel to extraterrestrial locations more practical and bearable, it could also be transformative once explorers arrive at their destination," says Adam Arkin, director of Berkeley Lab's Physical Biosciences Division (PBD) and a leading authority on synthetic and systems biology.

"During flight, the ability to augment fuel and other energy needs, to provide small amounts of needed materials, plus renewable, nutritional and taste-engineered food, and drugs-on-demand can save costs and increase astronaut health and welfare," Arkin says. "At an extraterrestrial base, synthetic biology could make even more effective use of the catalytic activities of diverse organisms."

Arkin is the senior author of a paper in the Journal of the Royal Society Interface that reports on a techno-economic analysis demonstrating "the significant utility of deploying non-traditional biological techniques to harness available volatiles and waste resources on manned long-duration space missions." The paper is titled "Towards Synthetic Biological Approaches to Resource Utilization on Space Missions." The lead and corresponding author is Amor Menezes, a postdoctoral scholar in Arkin's research group at the University of California (UC) Berkeley. Other co-authors are John Cumbers and John Hogan with the NASA Ames Research Center.

One of the biggest challenges to manned space missions is the expense. The NASA rule-of-thumb is that every unit mass of payload launched requires the support of an additional 99 units of mass, with "support" encompassing everything from fuel to oxygen to food and medicine for the astronauts, etc. Most of the current technologies now deployed or under development for providing this support are abiotic, meaning non-biological. Arkin, Menezes and their collaborators have shown that providing this support with technologies based on existing biological processes is a more than viable alternative.

"Because synthetic biology allows us to engineer biological processes to our advantage, we found in our analysis that technologies, when using common space metrics such as mass, power and volume, have the potential to provide substantial cost savings, especially in mass," Menezes says.

In their study, the authors looked at four target areas: fuel generation, food production, biopolymer synthesis, and pharmaceutical manufacture. They showed that for a 916 day manned mission to Mars, the use of microbial biomanufacturing capabilities could reduce the mass of fuel manufacturing by 56-percent, the mass of food-shipments by 38-percent, and the shipped mass to 3D-print a habitat for six by a whopping 85-percent. In addition, microbes could also completely replenish expired or irradiated stocks of pharmaceuticals, which would provide independence from unmanned re-supply spacecraft that take up to 210 days to arrive.

"Space has always provided a wonderful test of whether technology can meet strict engineering standards for both effect and safety," Arkin says. "NASA has worked decades to ensure that the specifications that new technologies must meet are rigorous and realistic, which allowed us to perform up-front techno-economic analysis."

The big advantage biological manufacturing holds over abiotic manufacturing is the remarkable ability of natural and engineered microbes to transform very simple starting substrates, such as carbon dioxide, water biomass or minerals, into materials that astronauts on long-term missions will need. This capability should prove especially useful for future extraterrestrial settlements.

"The mineral and carbon composition of other celestial bodies is different from the bulk of Earth, but the earth is diverse with many extreme environments that have some relationship to those that might be found at possible bases on the Moon or Mars," Arkin says. "Microbes could be used to greatly augment the materials available at a landing site, enable the biomanufacturing of food and pharmaceuticals, and possibly even modify and enrich local soils for agriculture in controlled environments."

The authors acknowledge that much of their analysis is speculative and that their calculations show a number of significant challenges to making biomanufacturing a feasible augmentation and replacement for abiotic technologies. However, they argue that the investment to overcome these barriers offers dramatic potential payoff for future space programs.

"We've got a long way to go since experimental proof-of-concept work in synthetic biology for space applications is just beginning, but long-duration manned missions are also a ways off," says Menezes. "Abiotic technologies were developed for many, many decades before they were successfully utilized in space, so of course biological technologies have some catching-up to do. However, this catching-up may not be that much, and in some cases, the biological technologies may already be superior to their abiotic counterparts."

This research was supported by the National Aeronautics and Space Administration (NASA) and the University of California, Santa Cruz.

Source: DOE/Lawrence Berkeley National Laboratory

Squid supplies blueprint for printable thermoplastics

This is a whimsical image of a squid creating 3-D printed devices. Credit: Adriás Bago

Squid, what is it good for? You can eat it and you can make ink or dye from it, and now a Penn State team of researchers is using it to make a thermoplastic that can be used in 3-D printing.

"Most of the companies looking into this type of material have focused on synthetic plastics," said Melik C. Demirel, professor of engineering science and mechanics. "Synthetic plastics are not rapidly deployable for field applications, and more importantly, they are not eco-friendly."

Demirel and his team looked at the protein complex that exists in the squid ring teeth (SRT). The naturally made material is a thermoplastic, but obtaining it requires a large amount of effort and many squid.

"We have the genetic sequence for six squid collected around the world, but we started with the European common squid," said Demirel, who with his team collected the cephalopods.

The researchers looked at the genetic sequence for the protein complex molecule and tried synthesizing a variety of proteins from the complex. Some were not thermoplastics, but others show stable thermal response, for example, the smallest known molecular weight SRT protein was a thermoplastic. The results of their work were published in today's (Dec. 17) issue of Advanced Functional Materials and illustrates the cover.

Most plastics are currently manufactured from fossil fuel sources like crude oil. Some high-end plastics are made from synthetic oils. Thermoplastics are polymer materials that can melt, be formed and then solidify as the same material without degrading materials properties.

This particular thermoplastic can be fabricated either as a thermoplastic, heated and extruded or molded, or the plastic can be dissolved in a simple solvent like acetic acid and used in film casting. The material can also be used in 3D printing machines as the source material to create complicated geometric structures.

To manufacture this small, synthetic SRT molecule, the researchers used recombinant techniques. They inserted SRT protein genes into E. coli, so that this common, harmless bacteria could produce the plastic molecules as part of their normal activity and the thermoplastic was then removed from the media where the E. coli lived. Wayne Curtis, professor of chemical engineering and Demirel collaborating on this project together with their students worked on this aspect of the project.

"The next generation of materials will be governed by molecular composition -- sequence, structure and properties," said Demirel.

The thermoplastic the researchers created is semi-crystalline and can be rigid or soft. It has a very high tensile strength and is a wet adhesive; it will stick to things even if it is wet.

This thermoplastic protein has a variety of tunable properties, which can be adjusted to individual requirements of manufacturing. Because it is a protein, it can be used for medical or cosmetic applications.

"Direct extraction or recombinant expression of protein based thermoplastics opens up new avenues for materials fabrication and synthesis, which will eventually be competitive with the high-end synthetic oil based plastics," the researchers report.

Source: Penn State

Visualizing DNA double-strand break process for the first time

The enzyme I-DmoI (purple) is specifically associated to the double strand of DNA (yellow and green). Credit: CNIO

Scientists from the Spanish National Cancer Research Centre (CNIO), led by Guillermo Montoya, have developed a method for producing biological crystals that has allowed scientists to observe --for the first time-- DNA double chain breaks. They have also developed a computer simulation that makes this process, which lasts in the order of millionths of a second, visible to the human eye. The study is published today by the journal Nature Structural & Molecular Biology.

"We knew that enzymes, or proteins, endonucleases, are responsible for these double strand breaks, but we didn't know exactly how it worked until now," said Montoya. "In our study, we describe in detail the dynamics of this basic biological reaction mediated by the enzyme I-Dmol. Our observations can be extrapolated to many other families of endonucleases that behave identically."

DNA breaks occur in several natural processes that are vital for life: mutagenesis, synthesis, recombination and repair. In the molecular biology field, they can also be generated synthetically. Once the exact mechanism that produces these breaks has been uncovered, this knowledge can be used in multiple biotechnological applications: from the correction of mutations to treat rare and genetic diseases, to the development of genetically modified organisms.

Slow-motion reaction

Enzymes are highly specialised dynamic systems. Their nicking function could be compared, said Montoya, to a specially designed fabric-cutting machine that "it would only make a cut when a piece of clothing with a specific combination of colours passed under the blade."

In this case, researchers concentrated on observing the conformational changes that occurred in the I-Dmol active site; the area that contains the amino acids that act as a blade and produces DNA breaks.

By altering the temperature and pH balance, the CNIO team has managed to delay a chemical reaction that typically occurs in microseconds by up to ten days. Under those conditions, they have created a slow-motion film of the whole process.

"By introducing a magnesium cation we were able to trigger the enzyme reaction and subsequently to produce biological crystals and freeze them at -200ºC," said Montoya. "In that way, we were able to collect up to 185 crystal structures that represent all of the conformational changes taking place at each step of the reaction."

Finally, using computational analysis, the researchers illustrated the seven intermediate stages of the DNA chain separation process. "It is very exciting, because the elucidation of this mechanism will give us the information we need to redesign these enzymes and provide precise molecular scissors, which are essential tools for modifying the genome," he concluded.

Source: Centro Nacional de Investigaciones Oncologicas (CNIO)

Nitrogen fingerprint in biomolecules could be from early sun

Ultraviolet light splits more readily splits molecules of nitrogen gas if one or both are the heavier isotope, nitrogen-15. Free nitrogen atoms combine with hydrogen to form ammonia molecules enriched in nitrogen-15. Credit: Image courtesy of University of California - San Diego

Chemical fingerprints of the element nitrogen vary by extremes in materials from the molecules of life to the solar wind to interstellar dust. Ideas for how this great variety came about have included alien molecules shuttled in by icy comets from beyond our solar system and complex chemical scenarios.

New experiments using a powerful source of ultraviolet light have shown that no extra-solar explanation is needed and the chemistry is straight forward, scientists from the University of California, San Diego, Hebrew University and UCLA report in the early online edition of the Proceedings of the National Academy of Sciences the week of September 29.

Nitrogen occurs in two stable forms. Nitrogen-14, with an equal number of protons and neutrons in its nucleus, is most abundant. Nitrogen-15, with an extra neutron, is far more rare, but biological molecules like proteins have larger proportions; they're enriched in nitrogen-15 relative to the nitrogen gas in Earth's atmosphere. And Earth's atmosphere itself has relatively more nitrogen-15 than other sources, such as the solar wind and Jupiter's atmosphere.

Samples from the icy comet Wild 2, retrieved by NASA's Stardust mission, and several kinds of meteorites also have relatively more nitrogen-15, and within the inhomogenous mix that makes up stony meteorites are inclusions, individual crystals that can have extremely high proportions of nitrogen-15. These observations led to the idea that the building blocks of life could have been 'seeded' on Earth, delivered perhaps by comet.

The new experiments render that hypothesis unnecessary. "We can generate this nitrogen enrichment inside the solar system. You can form all these building blocks of life inside our solar system. You don't have to bring the pieces in from outside," said Subrata Chakraborty, a project scientist in chemistry at UC San Diego and lead author of the report.

By shining a bright beam of very short wave ultraviolet light through nitrogen and hydrogen gas, Chakraborty and colleagues generated ammonia with drastically skewed ratios of nitrogen-15 relative to that found in the initial gas, which matched that of Earth's atmosphere. Pairs of nitrogen atoms -- the molecules of the gas -- were more likely to be split by UV photons if one or both atoms are the heavier version. Those freed nitrogen atoms recombined with hydrogen to form ammonia.

Ammonia molecules, a nitrogen atom bound to three hydrogen atoms, makes up a fundamental chemical group, the 'amines' the characterize amino acids, which link up in long chains to form proteins. They also join aromatic rings of carbon to form nitrogenous bases, the information carrying components of DNA and RNA. And RNA is how many think life got its start.

Light like this, with wavelengths this short, doesn't make it to Earth anymore. It's deflected by Earth's atmosphere. In fact, the experiments took place in a custom-engineered vacuum chamber aligned with the Lawrence Berkeley National Laboratory's Advanced Light Source synchrotron and were a challenge to pull off.

The chemical events that produced amines with extra nitrogen-15 would have happened long ago, probably in the icy outer reaches of the early solar nebula said Mark Thiemens, professor of chemistry at UC San Diego who directed the work. "It's the right time for this to have happened: before planets, before life."

Additional authors include Harel Muskatel of Hebrew University in Jerusalem, Teresa Jackson of UC San Diego, Musahid Ahmed of Lawrence Berkeley National Laboratory and R.D. Levine of Hebrew University and UCLA. NASA's Cosmochemistry and Origins of Solar System programs funded this work. The U.S. Department of Energy supports the operation of the Advanced Light Source.

Source:  University of California - San Diego

New conversion process turns biomass 'waste' into lucrative chemical products

Purdue's R.B. Wetherill Professor of Chemistry, holds a small vial containing results of a new catalytic process that can convert the lignin in wood into high-value chemical products for use in fragrances and flavoring.
Credit: Purdue University photo/Mark Simons

A new catalytic process is able to convert what was once considered biomass waste into lucrative chemical products that can be used in fragrances, flavorings or to create high-octane fuel for racecars and jets.

A team of researchers from Purdue University's Center for Direct Catalytic Conversion of Biomass to Biofuels, or C3Bio, has developed a process that uses a chemical catalyst and heat to spur reactions that convert lignin into valuable chemical commodities. Lignin is a tough and highly complex molecule that gives the plant cell wall its rigid structure.

Mahdi Abu-Omar, the R.B. Wetherill Professor of Chemistry and Professor of Chemical Engineering and associate director of C3Bio, led the team.

"We are able to take lignin -- which most biorefineries consider waste to be burned for its heat -- and turn it into high-value molecules that have applications in fragrance, flavoring and high-octane jet fuels," Abu-Omar said. "We can do this while simultaneously producing from the biomass lignin-free cellulose, which is the basis of ethanol and other liquid fuels. We do all of this in a one-step process."

Plant biomass is made up primarily of lignin and cellulose, a long chain of sugar molecules that is the bulk material of plant cell walls. In standard production of ethanol, enzymes are used to break down the biomass and release sugars. Yeast then feast on the sugars and create ethanol.

Lignin acts as a physical barrier that makes it difficult to extract sugars from biomass and acts as a chemical barrier that poisons the enzymes. Many refining processes include harsh pretreatment steps to break down and remove lignin, he said.

"Lignin is far more than just a tough barrier preventing us from getting the good stuff out of biomass, and we need to look at the problem differently," Abu-Omar said. "While lignin accounts for approximately 25 percent of the biomass by weight, it accounts for approximately 37 percent of the carbon in biomass. As a carbon source lignin can be very valuable, we just need a way to tap into it without jeopardizing the sugars we need for biofuels."

The Purdue team developed a process that starts with untreated chipped and milled wood from sustainable poplar, eucalyptus or birch trees. A catalyst is added to initiate and speed the desired chemical reactions, but is not consumed by them and can be recycled and used again. A solvent is added to the mix to help dissolve and loosen up the materials. The mixture is contained in a pressurized reactor and heated for several hours. The process breaks up the lignin molecules and results in lignin-free cellulose and a liquid stream that contains two additional chemical products, Abu-Omar said.

The liquid stream contains the solvent, which is easily evaporated and recycled, and two phenols, a class of aromatic hydrocarbon compounds used in perfumes and flavorings. A commonly used artificial vanilla flavoring is currently produced using a phenol that comes from petroleum, he said.

The team also developed an additional process that uses another catalyst to convert the two phenol products into high-octane hydrocarbon fuel suitable for use as drop-in gasoline. The fuel produced has a research octane rating greater than100, whereas the average gas we put into our cars has an octane rating in the eighties, he said.

The processes and resulting products are detailed in a paper published online in the Royal Society of Chemistry journal Green Chemistry. The U.S. Department of Energy funded the research.

In addition to Abu-Omar, co-authors include Trenton Parsell, a visiting scholar in the Department of Chemistry; chemical engineering graduate students Sara Yohe, John Degenstein, Emre Gencer, and Harshavardhan Choudhari; chemistry graduate students Ian Klein, Tiffany Jarrell, and Matt Hurt; agricultural and biological engineering graduate student Barron Hewetson; Jeong Im Kim, associate research scientist in biochemistry; Basudeb Saha, associate research scientist in chemistry; Richard Meilan, professor of forestry and natural reserouces; Nathan Mosier, associate professor of agricultural and biological engineering; Fabio Ribeiro, the R. Norris and Eleanor Shreve Professor of Chemical Engineering; W. Nicholas Delgass, the Maxine S. Nichols Emeritus Professor of Chemical Engineering; Clint Chapple, the head and distinguished professor of biochemistry; Hilkka I. Kenttamaa, professor of chemistry; and Rakesh Agrawal, the Winthrop E. Stone Distinguished Professor of Chemical Engineering.

The catalyst is expensive, and the team plans to further study efficient ways to recycle it, along with ways to scale up the entire process, Abu-Omar said.

"A biorefinery that focuses not only on ethanol, but on other products that can be made from the biomass is more efficient and profitable overall," he said. "It is possible that lignin could turn out to be more valuable than cellulose and could subsidize the production of ethanol from sustainable biomass."

The U.S. Department of Energy-funded C3Bio center is an Energy Frontier Research Center. It is part of Discovery Park's Energy Center and the Bindley Bioscience Center at Purdue.

Purdue Research Foundation has filed patent applications and launched a startup company, Spero Energy, which was founded by Abu-Omar.

Watch Video

Source:  Purdue University