Artificial Liver For Drug Tests.

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ScienceDaily (June 25, 2009) — If you have hay fever, headaches or a cold, it’s only a short way to the nearest chemist. The drugs, on the other hand, can take eight to ten years to develop. Until now animal experiments have been an essential step, yet they continue to raise ethical issues. “Our artificial organ systems are aimed at offering an alternative to animal experiments,” says Professor Heike Mertsching of the Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB in Stuttgart.

“Particularly as humans and animals have different metabolisms. 30 per cent of all side effects come to light in clinical trials.” The test system, which Professor Mertsching has developed jointly with Dr. Johanna Schanz, should in future give pharmaceutical companies greater security and shorten the path to new drugs. Both researchers received the “Human-centered Technology” prize for their work.
“The special feature, in our liver model for example, is a functioning system of blood vessels,” says Dr. Schanz. “This creates a natural environment for cells.” Traditional models do not have this, and the cells become inactive. “We don’t build artificial blood vessels for this, but use existing ones – from a piece of pig’s intestine.” All of the pig cells are removed, but the blood vessels are preserved. Human cells are then seeded onto this structure – hepatocytes, which, as in the body, are responsible for transforming and breaking down drugs, and endothelial cells, which act as a barrier between blood and tissue cells.
In order to simulate blood and circulation, the researchers put the model into a computer-controlled bioreactor with flexible tube pump, developed by the IGB. This enables the nutrient solution to be fed in and carried away in the same way as in veins and arteries in humans. “The cells were active for up to three weeks,” says Dr. Schanz. “This time was sufficient to analyze and evaluate the functions. A longer period of activity is possible, however.”
The researchers established that the cells work in a similar way to those in the body. They detoxify, break down drugs and build up proteins. These are important pre-conditions for drug tests or transplants, as the effect of a substance can change when transformed or broken down – many drugs are only metabolized into their therapeutic active form in the liver, while others can develop poisonous substances. The researchers have demonstrated the basic possibilities for use of the tissue models – liver, skin, intestine and windpipe. At the moment, the test system is being examined. Within two years it could provide a safer alternative to animal experiments.
Adapted from materials provided by Fraunhofer-Gesellschaft.

Structural biology scores with protein snapshot.

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Surface-filled representation of diacylglycerol kinase. The "porch-like" structure of the enzyme is highlighted, and the substrate diacylglycerol is depicted bound to the active site. Investigators at the Vanderbilt Center for Structural Biology used NMR methods to determine the structure of diacylglycerol kinase, the largest membrane-spanning protein studied by NMR to date. Credit: Charles Sanders, Ph.D., Vanderbilt University Center for Structural Biology.

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In a landmark technical achievement, investigators in the Vanderbilt Center for Structural Biology have used nuclear magnetic resonance (NMR) methods to determine the structure of the largest membrane-spanning protein to date.

Although NMR methods are routinely used to "take molecular pictures" of small proteins, large proteins - and particularly those that reside within the cell membrane - have been reluctant to smile for the camera.
In the June 26 issue of Science, Charles Sanders, Ph.D., professor of Biochemistry, and colleagues report the NMR structure of the large bacterial protein diacylglycerol kinase (DAGK), a complex of three subunits that each cross the membrane three times (for a total of nine membrane spans).
The group's ability to determine the NMR structure of DAGK suggests that similar methods can now be used to study the structures of other membrane proteins.
"We're taking the methods that we used for diacylglycerol kinase and applying them to high value targets such as G protein-coupled receptors," Sanders said.
G protein-coupled receptors - the largest family of cell signaling proteins - are targets for about half of all pharmaceuticals. Sanders is collaborating with other Vanderbilt investigators to tackle G protein-coupled receptor structure using both NMR and a complementary structural approach, X-ray crystallography.
DAGK may be a therapeutic target for certain types of bacterial infections. It is a virulence factor in the bacteria Streptococcus mutans, which causes tooth decay.
Sanders selected DAGK as a model for studying membrane enzymes when he started his own research lab 17 years ago. DAGK is the smallest known kinase (a protein that adds chemical groups called phosphates onto other molecules), and it is not similar to any other known proteins.
The DAGK structure, Sanders said, "confirmed that this is a really strange kinase." The enzyme has a porch-like structure, with a wide opening for its substrate diacylglycerol and the active site at the top of the porch.
"The active site looks nothing like any other kinase active site - it's a unique architecture," Sanders said.

The researchers also performed exhaustive mutagenesis studies in which they characterized mutations at each amino acid in DAGK and used the data to map the active site of the enzyme onto the structure. They identified two sets of mutations that resulted in non-functional DAGK. One set altered the active site so that it no longer did its job, and the second set caused the protein to fold incorrectly (misfolding).
Sanders said the team was surprised to find that nearly all of the mutations that caused misfolding were in the active site. The expectation, he explained, is that mutations in the active site would cause a loss of function but would not usually affect protein folding, whereas key residues for folding would be located elsewhere in the protein to underpin the scaffold for the active site.
"Our study shows that you can't make that assumption," he said.
Sanders cautions that investigators cannot simply predict the impact of a mutation based on it being located in the active site. The finding has implications for personalized medicine, which aims to use the predicted impact of disease-causing mutations to make therapy decisions.
"The therapeutic strategy for addressing catastrophic misfolding versus simple loss of function may be very different," Sanders said.
Sanders and his team, who got interested in protein folding because of their work with DAGK, are now pursuing structural studies of misfolded membrane proteins that cause diseases including peripheral neuropathy (Charcot-Marie-Tooth Disease), diabetes insipidus and Alzheimer's disease.
"For proteins that misfold because of mutations, we're using NMR tools to understand exactly what the mutations do to the proteins in terms of structure and stability," Sanders said. "We believe that understanding will lead to predictions about how to intervene and avoid misfolding."
Source: Vanderbilt University Medical Center (news : web)

TRAPping Proteins That Work Together Inside Living Cells

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ScienceDaily (June 18, 2009) — DNA might be the blueprint for living things, but proteins are the builders. Researchers trying to understand how and which proteins work together have developed a new crosslinking tool that is small and unobtrusive enough to use in live cells. Using the new tool, the scientists have discovered new details about a well-studied complex of proteins known as RNA polymerase. The results suggest the method might uncover collaborations between proteins that are too brief for other techniques to pinpoint.

"Conventional methods used to find interacting proteins have limitations that we are trying to circumvent," said biochemist Uljana Mayer of the Department of Energy's Pacific Northwest National Laboratory. "They also create conditions that are different from those inside cells, so you can't find all the interactions that proteins would normally engage in."
Proteins are the workhorses in an organism's cells. Whole fields of research are dedicated to teasing out which proteins work together to make cells function. For example, drug researchers seek chemicals that disrupt or otherwise change how proteins interact to combat diseases; environmental scientists need to understand how proteins collaborate in ecosystems to make them thrive or fail.
To learn about protein networks, scientists start with a familiar one and use it as bait to find others that work alongside it. To pin down the collaborators, researchers make physical connections between old and new proteins with chemicals called crosslinkers. The sticky crosslinkers will only connect proteins close enough to work together, the thinking goes. But most crosslinkers are too large to squeeze into living cells, are harmful to cells, or link proteins that are neighbors but not coworkers.
To address these issues, Mayer and her PNNL colleagues developed a crosslinking method that uses small crosslinkers whose stickiness can be carefully controlled. To find coworkers of a protein of interest, Mayer and her colleagues build a tiny molecule called a tag into the initial protein. They then add a small molecule called TRAP to the living cell, which finds and fits into the tag like two pieces in a puzzle. TRAP waves around, bumping into nearby proteins. The scientists control TRAP with a flash of light, causing it to stick to coworkers it bumps into. The researchers then identify the new "TRAPped" proteins in subsequent analyses.
To demonstrate how well this method works, Mayer and colleagues tested it out on RNA polymerase, a well-studied machine in cells. The polymerase is made up of many proteins that cooperate to translate DNA. One of the polymerase proteins has a tail that is known to touch the DNA and some helper proteins just before the polymerase starts translating. No one knew if this tail -- also known as the C-terminus of the alpha subunit -- touches anything else in the core of the RNA polymerase complex.
The team engineered a tag in the C-terminus and cultured bacteria with the tagged RNA polymerase. After adding TRAP to the cells and giving it time to find the C-terminus tag, the team shined a light on the cultures.
The team then identified the proteins marked with TRAP using instruments in EMSL, DOE's Environmental Molecular Sciences Laboratory on the PNNL campus. They found that the tagged protein, as expected, interacts with many other proteins, for example previously identified helper proteins, so-called transcription factors. But they also found it on another core protein called the beta subunit, suggesting the tail of the alpha subunit makes contact with the beta subunit as it plugs along. This interaction had never been seen before.
"No one knows what the polymerase looks like when it is running," said Uljana Mayer. "Here we see the C-terminus swings back to grab the beta subunit once the polymerase starts working."
The team report their results June 15 in the journal ChemBioChem. The tag in their unique method is made up of a "tetracysteine motif" -- two pairs of the amino acid cysteine separated by two other amino acids that doesn't interfere with the normal function of the protein of interest. TRAP includes a small "biarsenical" probe, which fluoresces so the team can find the proteins to which it has become attached. TRAP can also be easily unlinked from the tag with a simple biochemical treatment, allowing researchers to piece out the coworker from their original protein of interest.
The team also tested the method on other proteins, such as those found in young muscle cells. Mayer said they will use the method in the future to understand how environmental conditions affect how proteins work together in large networks.
Journal reference:
P. Yan, T. Wang, G.J. Newton, T.V. Knyushko, Y. Xiong, D. J. Bigelow, T.C. Squier, and M.U. Mayer. A Targeted Releasable Affinity Probe (TRAP) for In Vivo Photocrosslinking. ChemBioChem, 2009; 10: 1507-1518 DOI: 10.1002/cbic.200900029
Adapted from materials provided by DOE/Pacific Northwest National Laboratory.

Nanocrystals Reveal Activity Within Cells

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ScienceDaily (June 18, 2009) — Researchers at the U.S. Department of Energy’s (DOE) Lawrence Berkeley National Laboratory have created bright, stable and bio-friendly nanocrystals that act as individual investigators of activity within a cell. These ideal light emitting probes represent a significant step in scrutinizing the behaviors of proteins and other components in complex systems such as a living cell.

Labeling a given cellular component and tracking it through a typical biological environment is fraught with issues: the probe can randomly turn on and off, competes with light emitting from the cell, and often requires such intense laser excitation, it eventually destroys the probe, muddling anything you’d be interested in seeing.
“The nanoparticles we’ve designed can be used to study biomolecules one at a time,” said Bruce Cohen, a staff scientist in the Biological Nanostructures Facility at Berkeley Lab’s nanoscience research center, the Molecular Foundry. “These single-molecule probes will allow us to track proteins in a cell or around its surface, and to look for changes in activity when we add drugs or other bioactive compounds.”
Molecular Foundry post-doctoral researchers Shiwei Wu and Gang Han, led by Cohen, Imaging and Manipulation of Nanostructures staff scientist Jim Schuck and Inorganic Nanostructures Facility Director Delia Milliron, worked to develop nanocrystals containing rare earth elements that absorb low-energy infrared light and transform it into visible light through a series of energy transfers when they are struck by a continuous wave, near-infrared laser. Biological tissues are more transparent to near-infrared light, making these nanocrystals well suited for imaging living systems with minimal damage or light scatter.
“Rare earths have been known to show phosphorescent behavior, like how the old-style television screen glows green after you shut it off. These nanocrystals draw on this property, and are a million times more efficient than traditional dyes,” said Schuck. “No probe with ideal single-molecule imaging properties had been identified to date—our results show a single nanocrystal is stable and bright enough that you can go out to lunch, come back, and the intensity remains constant.”
To study how these probes might behave in a real biological system, the Molecular Foundry team incubated the nanocrystals with embryonic mouse fibroblasts, cells crucial to the development of connective tissue, allowing the nanocrystals to be taken up into the interior of the cell. Live-cell imaging using the same near-infrared laser showed similarly strong luminescence from the nanocrystals within the mouse cell, without any measurable background signal.
“While these types of particles have existed in one form or another for some time, our discovery of the unprecedented ’single-molecule’ properties these individual nanocrystals possess opens a wide range of applications that were previously inaccessible,” Schuck adds.
“Non-blinking and photostable upconverted luminescence from single lanthanide-doped nanocrystals,” by Shiwei Wu, Gang Han, Delia J. Milliron, Shaul Aloni, Virginia Altoe, Dmitri Talapin, Bruce E. Cohen and P. James Schuck, appears in Proceedings of the National Academy of Sciences and is available in Proceedings of the National Academy of Sciences online.
Work at the Molecular Foundry was supported by the Office of Basic Energy Sciences within the DOE Office of Science.
Adapted from materials provided by DOE/Lawrence Berkeley National Laboratory.

Discovery Of Facial Malformation Gene

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ScienceDaily (June 19, 2009) — The first specific genetic mutation which can cause a potentially serious facial disfigurement has been identified by researchers at Oxford University. The finding, published online in the American Journal of Human Genetics, offers the promise of improved genetic counselling for parents at risk.

Formation of the human face is a complex and exquisitely orchestrated developmental process that occurs between four and eight weeks of embryonic development. Disturbance to this development can lead to malformations of the head and face, including abnormal nasal configuration, cleft lip, and widely spaced eyes.
Most cases of disfigurement are caused by damage to the developing embryo early in pregnancy; genetic causes are thought to be responsible for only a minority of cases, and these usually also involve other parts of the body. No mutation of a single gene has previously been identified that leads specifically to facial malformations.
Researchers, led by Professor Andrew Wilkie from the Weatherall Institute for Molecular Medicine at the University of Oxford and Dr Irene Mathijssen from the Erasmus Medical Centre in the Netherlands and funded by the Wellcome Trust, identified individuals from seven families who shared a similar, distinctive facial appearance, including an abnormally large distance between the eyes and a wide, malformed nose. They termed this condition ‘frontorhiny’.
Genetic analysis showed that each of the individuals carried two copies of a mutation in the gene ALX3. Mouse models have previously highlighted the involvement of the equivalent gene in the production of a protein which regulates other genes involved in facial development – in other words, switching them on and off. However, while the absence of the protein produced by this gene does not disrupt facial development in mice, Professor Wilkie and colleagues found that in humans it leads to frontorhiny.
‘Frontorhiny can be a very distressing condition,’ says Professor Wilkie. ‘It causes facial disfigurement and other health problems, such as breathing difficulties and dermoids (benign cysts under the skin). The cosmetic surgery can be very challenging, requiring multiple operations.’
By identifying and naming the condition, the researchers believe that they will be able to diagnose more cases and provide improved genetic counselling. Because this is a recessive genetic disorder, a parent with the condition is very unlikely to have a similarly affected child. However, where unaffected parents have a child with the condition, they have a one in four chance of each future child being affected.
‘This finding is very important from the point of view of genetic counselling and offers hope to those families considered to be at risk,’ explains Professor Wilkie. ‘For example, by correctly diagnosing the condition in an adult, we can reassure them that their children are unlikely be affected.’
Professor Wilkie believes that the research also highlights the power of genetics to identify the origins of genetic disorders.
‘This study illustrates the tremendous power of genetics to identify the origins of rare disorders such as frontorhiny, even when working with very small numbers of individuals. In this research, just three affected individuals helped us to narrow the search for the particular genetic mutation responsible to around one three thousandth of the human genome. The previous mouse genetic work then helped finish the job for us.’
Adapted from materials provided by University of Oxford.

Scientists Show Bacteria Can 'Learn' And Plan Ahead

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ScienceDaily (June 18, 2009) — Bacteria can anticipate a future event and prepare for it, according to new research at the Weizmann Institute of Science. In a paper that appeared June 17 in Nature, Prof. Yitzhak Pilpel, doctoral student Amir Mitchell and research associate Dr. Orna Dahan of the Institute's Molecular Genetics Department, together with Prof. Martin Kupiec and Gal Romano of Tel Aviv University, examined microorganisms living in environments that change in predictable ways.

Their findings show that these microorganisms' genetic networks are hard-wired to 'foresee' what comes next in the sequence of events and begin responding to the new state of affairs before its onset.
E. coli bacteria, for instance, which normally cruise harmlessly down the digestive tract, encounter a number of different environments on their way. In particular, they find that one type of sugar – lactose – is invariably followed by a second sugar – maltose – soon afterward. Pilpel and his team of the Molecular Genetics Department, checked the bacterium's genetic response to lactose, and found that, in addition to the genes that enable it to digest lactose, the gene network for utilizing maltose was partially activated. When they switched the order of the sugars, giving the bacteria maltose first, there was no corresponding activation of lactose genes, implying that bacteria have naturally 'learned' to get ready for a serving of maltose after a lactose appetizer.
Another microorganism that experiences consistent changes is wine yeast. As fermentation progresses, sugar and acidity levels change, alcohol levels rise, and the yeast's environment heats up. Although the system was somewhat more complicated that that of E. coli, the scientists found that when the wine yeast feel the heat, they begin activating genes for dealing with the stresses of the next stage. Further analysis showed that this anticipation and early response is an evolutionary adaptation that increases the organism's chances of survival.
Ivan Pavlov first demonstrated this type of adaptive anticipation, known as a conditioned response, in dogs in the 1890s. He trained the dogs to salivate in response to a stimulus by repeatedly ringing a bell before giving them food. In the microorganisms, says Pilpel, 'evolution over many generations replaces conditioned learning, but the end result is similar.' 'In both evolution and learning,' says Mitchell, 'the organism adapts its responses to environmental cues, improving its ability to survive.' Romano: 'This is not a generalized stress response, but one that is precisely geared to an anticipated event.'
To see whether the microorganisms were truly exhibiting a conditioned response, Pilpel and Mitchell devised a further test for the E. coli based on another of Pavlov's experiments. When Pavlov stopped giving the dogs food after ringing the bell, the conditioned response faded until they eventually ceased salivating at its sound. The scientists did something similar, using bacteria grown by Dr. Erez Dekel, in the lab of Prof. Uri Alon of the Molecular Cell Biology Department, in an environment containing the first sugar, lactose, but not following it up with maltose. After several months, the bacteria had evolved to stop activating their maltose genes at the taste of lactose, only turning them on when maltose was actually available.
'This showed us that there is a cost to advanced preparation, but that the benefits to the organism outweigh the costs in the right circumstances,' says Pilpel. What are those circumstances? Based on the experimental evidence, the research team created a sort of cost/benefit model to predict the types of situations in which an organism could increase its chances of survival by evolving to anticipate future events. They are already planning a number of new tests for their model, as well as different avenues of experimentation based on the insights they have gained.
Pilpel and his team believe that genetic conditioned response may be a widespread means of evolutionary adaptation that enhances survival in many organisms – one that may also take place in the cells of higher organisms, including humans. These findings could have practical implications, as well. Genetically engineered microorganisms for fermenting plant materials to produce biofuels, for example, might work more efficiently if they gained the genetic ability to prepare themselves for the next step in the process.
Prof. Yitzhak Pilpel's research is supported by the Ben May Charitable Trust and Madame Huguette Nazez, Paris, France.
Adapted from materials provided by Weizmann Institute of Science, via EurekAlert!, a service of AAAS.

Long-standing Mystery Of How Plants Make Eggs Solved

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ScienceDaily (June 4, 2009) — A long-standing mystery surrounding a fundamental process in plant biology has been solved by a team of scientists at the University of California, Davis.

The group’s groundbreaking discovery that a plant hormone called auxin is responsible for egg production has several major implications.
First, this is the first definitive report of a plant hormone acting as a morphogen, that is, a substance that directs the pattern of development of cells based on its concentration.
Also, the study’s results provide tantalizing new insights into the evolutionary pathway that flowering plants took 135 million years ago when they split off from gymnosperms, the “naked-seeded” plant group that includes conifers, cycads and ginkgo trees.
Finally, the group used their discovery to make additional egg cells within plant reproductive structures, raising the prospects that these techniques may someday be used for enhancing the reproduction and fertility of crop plants.
“So the sequence becomes clear now,” said Venkatesan Sundaresan, the UC Davis professor of plant biology and plant sciences who led the study. “The plant triggers auxin synthesis at one end of the female reproductive unit called the embryo sac, creating an auxin gradient. The eight nuclei in the sac are then exposed to different levels of auxin, but only the nucleus in the correct position in the gradient becomes an egg cell. And that cell is subsequently fertilized to make the next generation.”
A paper describing the study was published June 4 in the journal Science’s online site, Science Express, in advance of its publication in the journal later this month.
Development of sperm and egg cells in plants
In humans and other animals, the germ cells for production of eggs and sperm are established at birth. But cells in flowering plants are assigned more or less randomly to become reproductive units when the plant reaches sexual maturity. Within the flower, sperm cells are produced by pollen at the tips of stamens, while egg cells develop in ovules, tiny structures embedded in the ovary at the base of the pistil.
At the start of the process of egg-cell development, a “mother cell” in the ovule divides several times, in a sequence involving both meiosis and mitotic divisions. These divisions result in the creation of an oblong, cell-like structure called the embryo sac, which contains eight nuclei, three of which are clustered near the open end of the ovule.
Within hours cell membranes start forming, eventually, creating seven cells: the all-important egg cell near the ovule opening where pollen will enter, and six other supporting cells, with essential functions for seed formation.
“The big question in our field for the past 50 years or more has been: How does this process happen in such a beautifully orchestrated pattern?” Sundaresan said. “It’s been clear that there’s a program here telling the plants exactly what to do, and that it is working not on cells, but on nuclei.”
Auxin concentrations determine fate of nuclei
Two years ago Sundaresan and a postdoctoral fellow in his laboratory, Gabriela Pagnussat, used genetic tools to shift the position of a single nucleus at one end of an embryo sac in the plant Arabidopsis. When they examined the mature sac, they found that it had produced two egg cells instead of one.
Sundaresan recognized that a pattern shift like this was similar to the response that had been reported two decades earlier in Drosophila fruit flies in experiments that provided the first direct evidence for the existence of morphogens.
This prompted him to begin searching for a substance in Arabadopsis that might be acting as a morphogen. When the group discovered that auxin was accumulating at the open end of the ovule, they turned their attention to this ubiquitous hormone, which is known to play myriad signaling roles in plant growth and behavioral processes. (The hormone’s existence was first guessed by Charles Darwin when he was studying how plants grow towards light.)
After many tests, Sundaresan and his group found that during embryo sac formation, auxin concentrations did indeed follow a gradient, with the highest levels occurring in the ovule at the end of the embryo sac where the pollen enters and lowest levels occurring at the opposite end of the sac.
To test the theory that this gradient was determining the fate of nuclei in the sac, Sundaresan and his group created a series of genetically manipulated Arabadopsis plants. In some plants they ratcheted up production of auxin in the embryo sac, and in others they decreased the sac’s sensitivity to auxin, creating the same effect that a decline in auxin would make.
When they examined these experimental plants, their hypothesis was confirmed: Auxin concentrations determined the fate of the nuclei. Knowing whether auxin levels were high or low, it became possible to predict the appearance or disappearance of egg cells at different positions within the embryo sac.
Finally, the group employed a long series of bio-manipulative techniques to determine that the auxin gradient they had discovered within the embryo sac was due to on-site synthesis rather than transport from a source outside the sac.
“What we have found about the way auxin works here is amazing,” Sundaresan said. “The idea that you can have a small molecule like this being maintained in a gradient within this eight-nucleate structure through synthesis alone is mind-boggling.”
Implications for flowering plant evolution
Development of the embryo sac is arguably the key element in the evolution from gymnosperms to flowering plants, also known as angiosperms.
Yet the fossil record reveals very little about the stages that led from gymnosperm seed production to angiosperm seed production when the transition occurred around 135 million years ago. The rapid expansion of flowering plants and their eventual domination of the Earth’s vegetation was called “an abominable mystery” by Darwin.
By elucidating the mechanism of embryo sac development, Sundaresan and his team have opened the door to new work into the evolutionary pathway between these two major plant groups. The discovery supports what is known as the modular theory, which posits that the first angiosperms underwent a drastic reduction of their female reproductive unit compared to the gymnosperms, allowing flowering plants to reproduce more efficiently and eventually supplant their naked-seeded forebears.
Most remarkably, perhaps, the new work suggests that the eight nuclei of the angiosperm embryo sac have retained developmental plasticity in their evolution from gymnosperms. “It’s amazing that even though the split supposedly happened over a hundred million years ago,” Sundaresan said, “all these nuclei still have the capacity to become egg cells.”
Collaborators in the study are lead author Gabriela Pagnussat and Monica Alandete-Saez, who were postdoctoral researchers with Sundaresan when they did the work, and John L. Bowman, a professor of plant biology at UC Davis at the time of the study, now at Monash University in Melbourne, Australia.
The work was supported by grants from the National Science Foundation.
Adapted from materials provided by University of California - Davis.

Silver nanoparticles show 'immense potential' in prevention of blood clots

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Silver nanoparticles (shown) could help prevent blood clots. Credit: The American Chemical Society.
Scientists are reporting discovery of a potential new alternative to aspirin, ReoPro, and other anti-platelet agents used widely to prevent blood clots in coronary artery disease, heart attack and stroke. Their study, scheduled for the June 23 issue of ACS Nano, a monthly journal, involves particles of silver -- 1/50,000th the diameter of a human hair -- that are injected into the bloodstream.

Debabrata Dash and colleagues point out that patients urgently need new anti-thrombotic agents because traditionally prescribed medications too-often cause dangerous bleeding. At the same time, aging of the population, sedentary lifestyle and spiraling rates of certain diseases have increased the use of these drugs. Researchers are seeking treatments that more gently orchestrate activity of platelets, disk-shaped particles in the blood that form clots.
The scientists describe development and lab testing of silver nanoparticles that seem to keep platelets in an inactive state. Low levels of the nanosilver, injected into mice, reduced the ability of platelets to clump together by as much as 40 percent with no apparent harmful side effects.
The nanoparticles “hold immense potential to be promoted as an antiplatelet agent,” the researchers note. “Nanosilver appears to possess dual significant properties critically helpful to the health of mankind — antibacterial and antiplatelet — which together can have unique utilities, for example in coronary stents.”
More information: ACS Nano, Journal Article: “Characterization of Antiplatelet Properties of Silver Nanoparticles”
Provided by American Chemical Society (news : web)

Chemists see first building blocks to life on Earth

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British scientists said on Wednesday that they had figured out key steps in the process by which life on Earth may have emerged from a seething soup of simple chemicals.

Genetic information in living organisms today is held in deoxyribonucleic acid (DNA), the famous "double helix" molecule of sugar, phosphate and a base.
But DNA is too sophisticated to have popped up in an instant, and one avenue of thought says its single-stranded cousin, ribonucleic acid, or RNA, came first.
RNA plays a key role in making proteins and, in viruses, is used to store genetic code.
It is chemically similar to DNA but is simpler and tougher in structure, and thus looks like a good candidate for Earth's first information-coding nucleic acid.
But for all its allure, the "RNA first" theory has run into practical problems.
Its three ingredients -- the base, ribose sugar and phosphate -- must have formed separately and then combined to form the molecule, according to conventional thinking.
Critics, though, say that RNA, while somewhat simpler than DNA, is still a complex molecule and could not have been assembled spontaneously.
These doubters have been comforted by the failure to find any feasible chain of chemical events to explain how the three components all came together.
But a paper published in the British journal Nature by University of Manchester chemists puts forward a different explanation.
The team, led by Professor John Sutherland, venture that an RNA-like synthesis took place through a series of chemical reactions and an important intermediate substance.
Their lab model uses starting materials and environmental conditions that are believed to have been around in early Earth and are also used in the standard "RNA first" scenario.
Their theory starts with a simple sugar called glycolaldehyde, which reacts with cyanmide (a compound of cyanide and ammonia) and phosphate to produce an intermediate compound called 2-aminooxazole.
Gentle warming from the Sun and cooling at night help purify the 2-aminooxazole, turning it into a plentiful precursor which contributes the sugar and base portions of the new ribonucleotide molecule.
The presence of phosphate and ultraviolet light from the Sun complete the synthesis.
In a commentary also published by Nature, US molecular biologist Jack Szostak hailed the research as an elegant explanation as to why the sugar and base would not have to form separately before forming the new molecule.
"It will stand for years as one of the great advances in prebiotic chemistry," the term for the study of the chemical processes that led to life on Earth, he enthused.
Opinions vary as to when the first organisms appeared on Earth.
One estimate, based on fossilised mats of bacteria found in Australia, is that this happened around 3.8 billion years ago, around 700 million years after the planet was formed.
(c) 2009 AFP

Breakthrough in the treatment of bacterial meningitis

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It can take just hours after the symptoms appear for someone to die from bacterial meningitis. Now, after years of research, experts at The University of Nottingham have finally discovered how the deadly meningococcal bacteria is able to break through the body's natural defence mechanism and attack the brain.

The discovery could lead to better treatment and vaccines for meningitis and could save the lives of hundreds of children.
Bacterial meningitis in childhood is almost exclusively caused by the respiratory tract pathogens Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae. The mechanism used by these lethal germs to break through the blood brain barrier (BBB) has, until now, been unknown.
The team led by Dlawer Ala'Aldeen, Professor of Clinical Microbiology and Head of the Molecular Bacteriology and Immunology Group at the Centre for Biomolecular Sciences, recently discovered that all three pathogens target the same receptor on human cerebrovascular endothelial cells — the specialised filtering system that protects our brain from disease — enabling the organisms to cross the blood-brain barrier.
Their findings, published today in The Journal of Clinical Investigation, suggest that disruption or modulation of this interaction of bacterial adhesins with the receptor might offer unexpectedly broad protection against bacterial meningitis and may provide a therapeutic target for the prevention and treatment of disease.
Professor Ala'Aldeen, who has been studying meningitis and its causes for over 20 years, said: "This is a significant breakthrough which will help us design novel strategies for the prevention and treatment of bacterial meningitis. Identification of the human receptor and bacterial ligands is like identifying a mysterious key and its lock, which will open new doors and pave the way for new discoveries."
The research, carried out in collaboration with the Department of Infectious Diseases at St. Jude Children's Research Hospital in Memphis Tennessee, also involved students from the University who have been regular and willing volunteers in the research programme.
Professor Ala'Aldeen said: "The ultimate aim is to save lives by protecting the healthy and curing the sick. We are one step closer to new breakthroughs that would prevent disease or its complications. There still is a long way to go before we have the ultimate vaccine and the ultimate treatment of bacterial meningitis."
Source: University of Nottingham (news : web)

Developed a human monoclonal antibody that neutralizes the Hepatitis C virus (HCV).

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ScienceDaily (May 11, 2009) — aking aim at a leading cause of liver failure in the United States, a team of scientists at the Massachusetts Biologic Laboratories (MBL) of the University of Massachusetts Medical School (UMMS) has developed a human monoclonal antibody that neutralizes the Hepatitis C virus (HCV). The new antibody effectively neutralized the virus in culture, and then prevented infection by the virus in a pre-clinical animal model of the disease.

Details of the research were presented April 23 in Copenhagen, Denmark at the 44th Annual Meeting of the European Association for the Study of the Liver (EASL). "We are pleased with the progress of this program," said Donna Ambrosino, MD, executive director of the MBL and a professor of pediatrics at the Medical School. "This antibody shows significant efficacy against the virus."
In the current study, MBL scientists injected transgenic mice (HuMAb Mouse® technology, Medarex, Inc.) with elements of HCV and then painstakingly searched for individual human antibodies produced in the mice that would recognize and bind to the HCV's outer coat, known as the glycoprotein. Once they found human antibodies that looked promising, they evaluated in vitro the ability of those antibodies to neutralize the virus and selected a lead candidate antibody for further characterization. Collaborative work with clinical researchers from the Department of Medicine at the Medical School's Worcester campus demonstrated that this antibody, now known as MBL-HCV1, was able to bind tightly with all genotypes of HCV tested from infected patient samples.
MBL-HCV1 was then tested off-site on three non-human primates. In that study, one animal received no antibody, one a low dose of the new antibody, and one a higher dose. Then all three animals were exposed to HCV. The animals with low or no antibody dosages developed HCV infections, but the animal with the higher dose was protected. Subsequently, researchers gave the high-dose of the antibody to the animal that originally received no antibody, and in that case the HCV was cleared from that animal's system. "These results are encouraging as a possible treatment for HCV infected patients, but more work needs to be done before we know how effective it will be in people," Dr. Ambrosino noted.
HCV attacks the liver and can eventually lead to liver failure. According to the U.S. Centers for Disease Control and Prevention, 3.2 million Americans are chronically infected with HCV and some 10,000 die annually of the disease. Globally, as many as 170 million people are estimated to suffer from HCV infection. For the most serious cases of HCV that do not respond to antiviral drugs, liver transplantation is the only option.
Typically 2,000 to 4,000 liver transplants are done each year in the United States (far less than the number of people on the waiting list for available organs). Transplantation can be a life saving treatment; however, in nearly all cases the patient's new liver is eventually infected by HCV because the virus remains in the patient's bloodstream during surgery. The powerful antiviral drugs now used to attack HCV prior to end-stage liver failure are not routinely used during surgery due to the patients' weakened condition and because of the strong medication used to avoid rejection of the new liver. After re-infection with HCV, nearly 40 percent of patients suffer rapid liver failure.
To close that clinical gap, the new antibody developed at MBL is designed to be a therapy shortly before and after transplant surgery. By giving a patient the new antibody before and during the time when the donor liver is implanted, researchers hope the HCV virus left in the bloodstream will be neutralized and rendered unable to infect the new liver. Then, because monoclonal antibodies are highly specific and typically have little or no side-effects, additional dosages of the new antibody could, theoretically, be given immediately after transplant surgery to continue neutralizing any remaining virus.
It is also possible, researchers theorize, that the antibody could be used in combination with new antiviral drugs for treatment in patients with newly diagnosed HCV infection. Use of the new antibody for both liver transplant patients and in newly diagnosed HCV patients will now be further evaluated. A Phase 1 human clinical trial of MBL-HCV1 in healthy subjects is expected to begin later this year.
Adapted from materials provided by University of Massachusetts Medical School, via EurekAlert!, a service of AAAS.

Sexually Transmitted Infections: Transistors Used To Detect Fungus Candida Albicans

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ScienceDaily (May 11, 2009) — The Nanosensors group from the Universidad Rovira i Virgili has created a biosensor, an electrical and biological device, which is able to selectively detect the Candida albicans yeast in very small quantities of only 50 cfu/ml (colony-forming units per millilitre).

"The technique uses field-effect transistors (electronic devices that contain an electrode source and a draining electrode connected to a transducer) based on carbon nanotubes and with Candida albicans-specific antibodies", Raquel A. Villamizar, lead author of the study said.
The Candida samples, which can be obtained from blood, serum or vaginal secretions, are placed directly on the biosensor, where the interaction between antigens and antibodies changes the electric current of the devices. This change is recorded and makes it possible to measure the amount of yeast present in a sample.
"Thanks to the extraordinary charge transference properties of the carbon nanotubes, the fungus detection process is direct, fast, and does not require the use of any marker", remarks Villamizar, who is co-author of a study that provides details of the biosensor and was published recently in the journal Sensors and Actuators B: Chemical.
To date, conventional diagnosis of Candida has been carried out using microbial cultures, serological tests, PCR molecular biology techniques (polymerase chain reactions used to amplify DNA), or immunoassays such as ELISA (Enzyme Linked Inmunoabsorbent Assay).
These techniques require long analysis times and sometimes give rise to false positives and negatives. ELISA also requires the use of markers (compounds that must be added to detect the presence of yeast by fluorescence and other techniques).
The new carbon nanotubes biosensor, however, "makes it possible to improve some of the quality parameters of the traditional methods, for example the speed and simplicity of measurements, and it is an alternative tool that could be used in routine sample analysis", explains Villamizar.
The researcher adds that by using this biosensor "it will be possible in future to obtain a rapid diagnosis of infection with this pathogen, which will help to ensure administration of the correct prophylactic treatments".
The Candida albicans fungus exists naturally in the skin, mouth, the mucous membranes lining the digestive tract, and the respiratory and genitourinary systems. This yeast can cause anything from simple mycosis of the skin to complicated cases of candidiasis. It is much more commonly found in patients suffering from immunodeficiency, tumours, diabetes and lymphomas, among other diseases.
Journal reference:
Villamizar et al. Improved detection of Candida albicans with carbon nanotube field-effect transistors. Sensors and Actuators B Chemical, 2009; 136 (2): 451 DOI: 10.1016/j.snb.2008.10.013
Adapted from materials provided by Plataforma SINC.

In the Genome Race, the Sequel Is Personal

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http://www.nytimes.com/2007/09/04/science/04vent.html?ref=science

By NICHOLAS WADE
Published: September 4, 2007

The race to decode the human genome may not be entirely over: the loser has come up with a new approach that may let him prevail in the end.

In 2003, a government-financed consortium of academic centers announced it had completed the human genome, fending off a determined challenge from the biologist J. Craig Venter. The consortium’s genome comprised just half the DNA contained in a normal cell, and the DNA used in the project came from a group of people from different racial and ethnic backgrounds.
But the loser in the race, Dr. Venter, could still have the last word. In a paper published today, his research team is announcing that it has decoded a new version of the human genome that some experts believe may be better than the consortium’s.
Called a full, or diploid genome, it consists of the DNA in both sets of chromosomes, one from each parent, and it is the normal genome possessed by almost all the body’s cells. And the genome the team has decoded belongs to just one person: Dr. Venter.
The new genome, Dr. Venter’s team reports, makes clear that the variation in the genetic programming carried by an individual is much greater than expected. In at least 44 percent of Dr. Venter’s genes, the copies inherited from his mother differ from those inherited from his father, according to the analysis published in Tuesday’s issue of PLoS Biology.
Huntington F. Willard, a geneticist at Duke University who has had early access to Dr. Venter’s genome sequence, said that the quality of the new genome was “exceptionally high” and that “until the next genome comes along this is the gold standard right now.”
Dr. Willard said it was “hugely better” than the consortium’s sequence, at least for his particular research interest.
“I don’t want to fan the fires but I like this, it’s a really good genome,” said Edward M. Rubin, a genome expert at the Lawrence Berkeley National Laboratory.
Dr. Venter’s race with the consortium began in 1998 when he spotted a quicker method of decoding the human genome. He tried to wrest this rich scientific prize from his academic rivals by co-founding a genome-decoding company called Celera. By June 2000, the two sides were neck and neck preparing a draft sequence of the genome. But in January 2002, Dr. Venter was abruptly fired as president of Celera. The consortium went on to claim victory when it announced its completion of the genome the next year.
But the consortium’s genome, though immensely useful to biologists, was full of gaps and only complete in the sense that it was the best that could be done with existing technology.
Dr. Venter has spent the last five years and an extra $10 million of his institute’s money in improving the draft genome he prepared at Celera. That genome was based mostly on his own DNA, and the new diploid version is entirely so. His critics may accuse him of an egocentricity of considerable dimension, but by analyzing his own genome he has sidestepped the problems of privacy and consent that could have arisen with other people’s DNA when he made the whole sequence publicly available, as he is doing now.
Like James Watson, the co-discoverer of DNA, whose genome is also being decoded, Dr. Venter believes strongly in making individual DNA sequences public to advance knowledge and hasten the era of personalized genomic medicine.
If other experts find that Dr. Venter’s genome is the best available, could it be said that he won the human genome race after all?
“There is this long history of Craig’s vanity, which for much of the scientific community is irritating,” Dr. Rubin said, declining to give a direct answer.
Asked the same question, Dr. Venter replied: “I’m not sure I’d want to be the one to say that, but we’re not through racing yet. I’ll let you know when we’ve stopped.”
James Shreeve, author of “The Genome War,” said, “I think he already believes he’s the true winner of the genome race for what he did at Celera,” noting that the consortium, too, believed it had won.
Though there are now novel technologies for decoding DNA very cheaply, Dr. Venter’s genome sequence could set a high bar for a long time. It was decoded with an old method, known as Sanger sequencing, that is expensive but analyzes stretches of DNA up to 800 units in length. The cheaper new technologies at present analyze pieces of DNA only 200 units or so long, and the shorter lengths are much harder to assemble into a complete genome.
Dr. Watson’s genome is being decoded with a next-generation machine developed by 454 Life Sciences. But the company’s researchers are putting the pieces in correct order by matching them to the consortium’s genome sequence rather than by doing an independent assembly.

Dr. Venter’s genome could be the gold standard for many years, especially if he continues to improve it. Samuel Levy, who led the J. Craig Venter Institute team that decoded the genome, said that it was a work in progress and that new versions would be published as the remaining gaps were closed. There are 4,500 gaps where the sequence of DNA units is uncertain, and no technology yet exists for decoding the large amounts of DNA at the center and tips of the chromosomes.

Biologists studying variation in the human genome, whether to discover causes of disease or for other reasons, have mostly looked at what are called SNPs or “snips,” which are sites on the genome where a single unit of DNA is changed.
But there are other kinds of variation, all of which can have consequences for a person. One type is called indels, where a single DNA unit has either been inserted or deleted from the genome. Another is copy number variation, in which the same gene can exist in multiple copies. There are also inversions, in which a stretch of DNA has been knocked out of its chromosome and reinserted the wrong way around. Dr. Venter’s genome has four million variations compared with the consortium’s, including three million snips, nearly a million indels and 90 inversions.
“This is the first time that anyone has had an accurate representation of how much variation there is in a human genome,” said Stephen W. Scherer of the University of Toronto, a co-author of the study.
Biologists had estimated that two individuals would be identical in 99.9 percent of their DNA, but the true figure now emerges as much less, around 99.5 percent, Dr. Scherer said.
The genome is being made publicly available on the database operated by the National Center for Biotechnology Information and is free for any use. Dr. Venter said he would add phenotypic information to the version on his own Web site, meaning medical records and other data to help researchers correlate his bodily characteristics with his DNA.
What little is understood about the human genome at present consists mostly of medical variants that put people at risk of disease. So interpreting a genome brings mostly adverse news. Dr. Venter reports that he has variants that increase his risk of alcoholism, coronary artery disease, obesity, Alzheimer’s disease, antisocial behavior and conduct disorder.
But these predictions are far from certain. As more individual genomes are decoded, the information from them will become more valuable, Dr. Venter said, provided that people can overcome “irrational fears of even seeing their genetic code.”
Although Dr. Venter has decoded the DNA sequence inherited from both of his parents, he does not yet know which sequences are from his mother and which from his father. The issue could be resolved by analyzing DNA from his mother, who is alive and well, and the matter is under consideration, Dr. Levy said. Dr. Venter has traced his ancestry for three generations and found that his mother’s and father’s ancestors came from England.
Next month, Dr. Venter will publish an autobiography, “A Life Decoded.” The book describes the twists and turns that led him down the unlikely path into scientific research. “Rebellious and disobedient,” as he describes himself, he dedicated his teenage years to the pursuit of young women and the California surf, to the detriment of his academic career.
He was drafted at the time of the Vietnam war and enlisted in the Navy. Because of a high I.Q. score, he was given a choice of any Navy career, from nuclear engineering to electronics. He chose the hospital corps school, because it was the only course that did not require any further enlistment. Only too late did he discover the reason. Corpsmen in Vietnam did not usually survive long enough to re-enlist — the half-life of medics in the field was six weeks, he writes.
Learning how to manipulate the Navy bureaucracy, he got himself assigned to the Navy hospital in Da Nang, where chances of survival were better. But the work was harrowing. He witnessed several hundred soldiers die on his operating table, mostly when he was massaging their heart or trying to breathe life into them.
“I learned more than any 20-year-old should ever have to about triage, about sorting those you can salvage from those you cannot do anything for except ease their pain as they died,” Dr. Venter writes in the autobiography.
He escaped from Vietnam with his life and an interest in medical research. With his lack of academic skills, this was a hard field for him to break into, but by 1975 he had a Ph.D. By the late 1980s, he was starting to make his mark as one of the few scientists who could get useful results out of the first DNA sequencing machines that were then becoming available.
He was the first to sequence the genome of a bacterium, Hemophilus influenzae, even though his grant application was turned down by the National Institutes of Health on the advice of experts who said his method would not work. With the human genome, an even greater prize, the pace of competition was intense, especially when his approach turned out to be more efficient than the one his rivals had chosen.
In the book, Dr. Venter says that detractors badmouthed his work, pressured other scientists not to cooperate with him and tried strenuously to block publication of his report, of which they had earlier maneuvered to be made co-authors.
“Like most human endeavors, science is driven in no small part by envy,” he writes.
Dr. Venter has never fully lost his youthful disrespect for authority and establishments. His investment in himself — choosing his own genome to sequence, naming his laboratory the J. Craig Venter Institute — may come across as vainglorious, but it can also be seen as a signal of survival, defying the establishments he believes have sought to crush him. However nettlesome he may seem to some of his colleagues, he has the charm and the personal skills to have recruited many highly able researchers to his teams.
Another reason for his success has been his skill at raising private finances to achieve research goals after being denied support from the National Institutes of Health. That a scientist of his ability has been forced to work outside the N.I.H.’s peer-review system puts peer review in a strange light. If his diploid human genome should become a standard, the success is one that he will have earned by perseverance and defiance of long odds.

Fausto Intilla

www.oloscience.com