A hot species for cool structures: complex proteins in 3D, thanks to simple heat-loving fungus

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A fungus that lives at extremely high temperatures could help understand structures within our own cells.

Scientists at the European Molecular Biology Laboratory (EMBL) and Heidelberg University, both in Heidelberg, Germany, were the first to sequence and analyse the genome of a heat-loving fungus, and used that information to determine the long sought 3-dimensional structure of the inner ring of the nuclear pore. The study was published today in Cell.

Model of the inner ring (green) of the nuclear pore, showing its components. Credit: Heidelberg University Center of Biochemistry.

The fungus Chaetomium thermophilum lives in soil, dung and compost heaps, at temperatures up to 60ºC. This means its proteins – including some which are very similar to our own – have to be very stable, and the Heidelberg scientists saw this stability as an advantage.

“There are a number of structures that we couldn’t study before, because they are too unstable in organisms that live at more moderate temperatures,” explains Peer Bork, who led the genome analysis at EMBL. “Now with this heat-loving fungus, we can.”

The scientists compared the fungus’ genome and proteome to those of other eukaryotes – organisms whose cells have a nucleus – and identified the proteins that make up the innermost ring of the nuclear pore, a channel that controls what enters and exits a cell’s nucleus. Having identified the relevant building blocks, the scientists determined the complex 3D structure of that inner ring for the first time.

“This work shows the power of interdisciplinary collaborations,” says Ed Hurt, who led the structural and biochemical analyses at Heidelberg University: “the nuclear pore is an intricate biological puzzle, but by combining bioinformatics with biochemistry and structural biology, we were able to solve this piece of it for the first time.”

The scientists have made C. thermophilum’s genome and proteome publicly available, and are confident that these will prove valuable for studying other eukaryotic structures and their interactions, as well as general adaptations to life in hot places. Such knowledge could potentially lead to new biotechnology applications.

View the full study in PDF: A hot species for cool structures, a heat-loving fungus.

Reference

Amlacher, S., Sarges, P., Flemming, D., van Noort, V., Kunze, R., Devos, D.P., Arumugam, M., Bork, P. & Hurt, E. Insight into Structure and Assembly of the Nuclear Pore Complex by Utilizing the Genome of a Eukaryotic Thermophile. Cell, 22 July 2011.

Nanotechnology for water filter

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(“Biomechanism.com“) — Nanotechnology has developed tremendously in the past decade and was able to create many new materials with a vast range of potential applications.

Carbon nanotubes by scanning electron microscopy. (Credit: University of Vienna).

Carbon nanotubes are an example of these new materials and consist of cylindrical molecules of carbon with diameters of a few nanometers – one nanometer is one millionth of a millimeter. Carbon nanotubes possess exceptional electronic, mechanical and chemical properties, for example they can be used to clean polluted water. Scientists of the University of Vienna had recently published to this new research field in the well-known journal “Environmental Science & Technology”.”

Among many potential applications, carbon nanotubes are great candidate materials for cleaning polluted water. Many water pollutants have very high affinity for carbon nanotubes and pollutants could be removed from contaminated water by filters made of this nanomaterial, for example water soluble drugs which can hardly be separated from water by activated carbon. Problems due to filters’ saturation could be reduced as carbon nanotubes have a very large surface area (e.g. 500 m2 per gram of nanotube) and consequently a very high capacity to retain pollutants. “Maintenance and wastes related to water depollution could thus be reduced”, says Thilo Hofmann, Vice Dean of the Faculty of Earth Sciences, Geography and Astronomy of the University of Vienna.

Assessing carbon nanotubes’ environmental sustainabilityA lot of research has focussed on carbon nanotubes in the past decade. However, the exceptional properties of carbon nanotubes make them difficult to study. Standard methods give limited results and the behaviour of carbon nanotubes in realistic conditions is still poorly understood. “Innovative technologies always come with benefits and drawbacks for human and environmental quality and a good understanding of the interactions between contaminants and carbon nanotubes as well as how carbon nanotubes behave in the environment is essential before they can be used in filters”, explains Mélanie Kah, who does research on this project together with Xiaoran Zhang.

A team of researchers at the Department of Environmental Geosciences at the University of Vienna is currently carrying out research on the subject. They developed a method called “passive sampling”. Data produced by this new method are much more reliable for realistic applications as they include concentrations likely to occur in the environment (generally very low). This was not possible with classical methods that can only deal with elevated concentrations.

The experiments published now in the internationally recognised journal “Environmental Science & Technology” took more than a year. First, the “passive sampling method” was developed which allows measuring the affinity of a category of carcinogenic contaminants – i.e. Polycyclic Aromatic Hydrocarbons (PAHs) – to carbon nanotubes. “Series of tests which use analytical chemistry and electron microscopy were performed with collaborators from the University of Utrecht in the Netherlands, to ensure that the method is suitable, reliable and optimised for carbon nanotubes”, illustrates Thilo Hofmann. Once validated, the “passive sampling method” was used to measure the affinity (absorption and adsorption) of several contaminants (PAHs) to carbon nanotubes over a very wide range of concentrations.

Contaminants fight for a place on carbon nanotubesAnother aspect investigated by the scientists of the Department for Environmental Geosciences is the phenomenon of competition between contaminants. Many chemicals often co-exist in the environment, especially in polluted bodies of water. If competition occurs, it means that a contaminant may not attach to carbon nanotubes if better competitors co-exist. Competition is not acceptable for filter application as the efficacy of the filter will vary according to the quantity and type of contaminants present. Studying competition also provides information on the mechanisms of sorption.

Using classical techniques with relatively high concentrations showed that competition can be very strong when three PAHs co-exist with carbon nanotubes. Conversely, experiments with the “passive sampling method” at concentrations likely to occur in the environment showed that no competition occurs if 13 PAHs are considered together. This example highlights the importance of developing and using experimental methods to produce results relevant to environmental conditions.

There are still many questions to answer to fully evaluate the potential of carbon nanotubes to clean polluted water. “We keep on working on the subject and the results of our last experiments will be soon presented at international conferences”, concludes the environmental geoscientist, Thilo Hofmann.

Reference 

Measuring and Modeling Adsorption of PAHs to Carbon Nanotubes Over a Six Order of Magnitude Wide Concentration Range: Melanie Kah, Xiaoran Zhang, Michiel T.O. Jonker, and Thilo Hofmann. In: Environmental Science & Technology, 2011, 45 (14), pp 6011-6017. DOI: 10.1021/es2007726.

Software helps synthetic biologists customize protein production

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(“Biomechanism.com“) — A software program developed by a Penn State synthetic biologist could provide biotechnology companies with genetic plans to help them turn bacteria into molecular factories, capable of producing everything from biofuels to medicine.

“It’s similar to how an engineer designs a plane or a car,” said Howard M. Salis, assistant professor in agricultural and biological engineering, and chemical engineering. “When designing a biological organism, there are many combinations that the engineer must test to find the best combination. This technology allows us to quickly identify the best DNA sequence for a particular biotechnological application.”

The program, called a DNA compiler, designs synthetic DNA sequences to control protein production inside simple organisms. Salis said narrowing down the exact genetic plans from the billions of possible sequence combinations will save biotechnology companies money and time.

To produce proteins, which are integral for creating and maintaining cells, an organism’s DNA sequence controls the proteins that it makes and how much of each protein is produced.

DNA serves as a genetic template to create messenger RNA (mRNA). Another form of RNA, transfer RNA, carries amino acids, the components of proteins, as ingredients for the proteins.

The software predicts how fast an organism will produce a specific protein. It can also design new DNA sequences to increase or decrease protein production across a large scale and to find the best protein production rates.

Salis, whose work appears in a recent issue of Methods in Enzymology, said that synthetic DNA sequences will play a more important role in industries as diverse as medicine and manufacturing. The biofuel industry is particularly interested in maximizing the amount of proteins produced to optimize metabolism. To be profitable, companies have to produce large quantities of biofuels.

“We’re learning how to predict, control and design the behavior of biological organisms,” said Salis. “We can do it much faster than evolution.”

In one of the software’s modes, genetic engineers can type strings of letters A, T, G and C that represent adenine, thymine, guanine and cystosine — molecules in DNA– into the software, which then calculates which protein will be made and how much protein will be produced, said Salis. In another mode, engineers select a protein’s production rate inside the organism and the software optimizes a synthetic DNA sequence to achieve that rate.

Video: An interesting TED talk. Paul Rothemund discusses how genetic engineers can fold proteins by writing the sequence of the DNA and compiling it with a DNA compiler. The result? Well, you can come up with whatever you want – from simple figures to actual living organisms.

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Companies have obtained the licensed software to produce various chemicals, such as methyl ethyl ketone, a substance with numerous commercial and industrial uses, including inks, paints and industrial cements. A noncommercial version of the software, available on the web at http://salis.psu.edu/software/, designs over 300 sequences a month, Salis said.

The technology previously appeared in Nature Biotechnology. A Defense Advanced Research Projects Agency’s young faculty award and the Penn State Institutes of Energy and Environment support this research.

Gardening in the brain

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Specialist cells prune connections between neurons.

Caption: This is microglia (green) in a mouse brain. The nuclei of all cells in the brain are labeled blue. Credit: EMBL/R.Paolicelli

Gardeners know that some trees require regular pruning: some of their branches have to be cut so that others can grow stronger. The same is true of the developing brain: cells called microglia prune the connections between neurons, shaping how the brain is wired, scientists at the European Molecular Biology Laboratory (EMBL) in Monterotondo, Italy, discovered. Published online today in Science, the findings could one day help understand neurodevelopmental disorders like autism.

“We’re very excited, because our data shows microglia are critical to get the connectivity right in the brain,” says Cornelius Gross, who led the work: “they ‘eat up’ synapses to make space for the most effective contacts between neurons to grow strong.”

Microglia are related to the white blood cells that engulf pathogens and cellular debris, and scientists knew already that microglia perform that same clean-up task when the brain is injured, ‘swallowing up’ dead and dying neurons. Looking at the developing mouse brain under the microscope, Gross and colleagues found proteins from synapses – the connections between neurons – inside microglia, indicating that microglia are able to engulf synapses too.

To probe further, the scientists introduced a mutation that reduced the number of microglia in the developing mouse brain.

“What we saw was similar to what others have seen in at least some cases of autism in humans: many more connections between neurons,” Gross says. “So we should be aware that changes in how microglia work might be a major factor in neurodevelopmental disorders that have altered brain wiring.”

Caption: This is a 3-dimensional reconstruction of a single microglia cell. Credit: EMBL/ R.Paolicelli

The microglia-limiting mutation the EMBL scientists used has only temporary effects, so eventually the number of microglia increases and the mouse brain establishes the right connections. However, this happens later in development than it normally would, and Gross and colleagues would now like to find out if that delay has long-term consequences. Does it affect the behaviour of the mice behaviour, for example? At the same time, Gross and colleagues plan to investigate what microglia do in the healthy adult brain, where their role is essentially unknown.

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This work was carried out in collaboration with the groups of Davide Ragozzino at the University of Rome and Maurizio Giustetto and Patrizia Panzanelli at the University of Turin.

Repairing Our Inner Clock with a Two-Inch Zebrafish

A model organism is a non-human animal used in research.  Zebrafish have gained much popularity as a research organism for a variety of disciplines such as genetics, pharmacology and biological research.  This is due to a number of reasons including their high fecundity, production of transparent embryos, cost-effectiveness, similarities to humans, as well as an abundance of available data. In addition, the larvae and embryos of Zebrafish develop externally and at a rapid pace compared to some other model organisms. This allows for manipulation and observation of early life stages to be less difficult. The embryos of Zebrafish are able to absorb additives from the water, with a high tolerance to chemical mutagens, allowing for easier administration of chemicals at greater dosages.

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Circadian rhythms — the natural cycle that dictates our biological processes over a 24-hour day — does more than tell us when to sleep or wake. Disruptions in the cycle are also associated with depression, problems with weight control, jet lag and more. Now Prof. Yoav Gothilf of Tel Aviv University’s Department of Neurobiology at the George S. Wise Faculty of Life Sciences is looking to the common zebrafish to learn more about how the human circadian system functions.

Prof. Gothilf and his Ph.D. student Gad Vatine, in collaboration with Prof. Nicholas Foulkes of the Karlsruhe Institute for Technology in Germany and Dr. David Klein of the National Institute of Health in Maryland, has discovered that a mechanism that regulates the circadian system in zebrafish also has a hand in running its human counterpart.

The zebrafish discovery provides an excellent model for research that may help to develop new treatments for human ailments such as mental illness, metabolic diseases or sleep disorders. The research appears in the journals PLoS Biology and FEBS Letters.

A miniature model

Zebrafish may be small, but their circadian system is similar to those of human beings. And as test subjects, says Prof. Gothilf, zebrafish also have several distinct advantages: their embryos are transparent, allowing researchers to watch as they develop; their genetics can be easily manipulated, and their development is quick — eggs hatch in two days and the fish become sexually mature at three months old.

Previous research on zebrafish revealed that a gene called Period2, also present in humans, is associated with the fish’s circadian system and is activated by light. “When we knocked down the gene in our zebrafish models,” says Prof. Gothilf, “the circadian system was lost.” This identified the importance of the gene to the system, but the researchers had yet to discover how light-triggered gene activity.

The team subsequently identified a region called LRM (Light Responsive Model) within Period2 that explains the phenomenon. Within this region, there are short genetic sequences called Ebox, which mediate clock activity, and Dbox, which confer light-driven expression — the interplay between the two sequences is responsible for light activation. Based on this information, they identified the proteins which bind the Ebox and Dbox and trigger the light-induction of the Period2 gene, a process that is important for synchronization of the circadian system.

To determine whether a similar mechanism may exist in humans, Prof. Gothilf and his fellow researchers isolated and tested the human LRM and inserted it into zebrafish cells. In these fish cells, the human LRM behaved in exactly the same way, activating Period2 when exposed to light — and unveiling a fascinating connection between humans and the two-inch-long fish.

Shedding new light on circadian systems and the brain

Zebrafish and humans could have much more in common, Prof. Gothilf says, leading to breakthroughs in human medicine. Unlike rats and mice but like human beings, zebrafish are diurnal — awake during the day and asleep at night — and they have circadian systems that are active as early as two days after fertilization. This provides an opportunity to manipulate the circadian clock, testing different therapies and medications to advance our understanding of the circadian system and how disruptions, whether caused by biology or lifestyle, can best be treated.

Prof. Gothilf believes this model has further application to brain and biomedical research. Researchers can already manipulate the genetic makeup of zebrafish, for example, to make specific neurons and their synapses (the junctions between neurons in the brain) fluorescent — easy to see and track. “Synapses can be actually counted. This kind of accessible model can be used in research into degenerative brain disorders,” he notes, adding that several additional research groups at TAU are now using zebrafish to advance their work.