Implant Bacteria, Beware: Researchers Create Nano-sized Assassins

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ScienceDaily (June 28, 2009) — Staphylococcus epidermidis is quite an opportunist. Commonly found on human skin, the bacteria pose little danger. But S. epidermidis is a leading cause of infections in hospitals. From catheters to prosthetics, the bacteria are known to hitch a ride on a range of medical devices implanted into patients.

Inside the body, the bacteria multiply on the implant's surface and then build a slimy, protective film to shield the colony from antibiotics. According to a study in the journal Clinical Infectious Diseases, up to 2.5 percent of hip and knee implants alone in the United States become infected, affecting thousands of patients, sometimes fatally.
More ominously, there is no effective antidote for infected implants. The only way to get rid of the bacteria is to remove the implant. "There is no [easy] solution," said Thomas Webster, a biomedical engineer at Brown University.
Now, Webster and Brown graduate student Erik Taylor have created a nano-sized headhunter that zeroes in on the implant, penetrates S. epidermidis's defensive wall and kills the bacteria. The finding, published in the International Journal of Nanomedicine, is the first time iron-oxide nanoparticles have been shown to eliminate a bacterial infection on an implanted prosthetic device.
In lab tests, Taylor, the lead author, and Webster, associate professor of engineering and orthopaedics, noted that up to 28 percent of the bacteria on an implant had been eliminated after 48 hours by injecting 10 micrograms of the nanoparticle agents. The same dosage repeated three times over six days destroyed essentially all the bacteria, the experiments showed.
The tests show "there will be a continual killing of the bacteria until the film is gone," said Webster, who is editor-in-chief of the peer-reviewed journal in which the paper appears.
A surprising added benefit, the scientists learned, is the nanoparticles' magnetic properties appear to promote natural bone cell growth on the implant's surface, although this observation needs to be tested further.
To carry out the study, the researchers created iron-oxide particles (they call them "superparamagnetic") with an average diameter of eight nanometers. They chose iron oxide because the metallic properties mean the particles can be guided by a magnetic field to the implant, while its journey can be tracked using a simple magnetic technique, such as magnetic resonance imaging (MRI). Moreover, previous experiments showed that iron seemed to cause S. epidermidis to die, although researchers are unsure why. (Webster said it may be due to iron overload in the bacteria's cell.)
Once the nanoparticles arrive at the implant, they begin to penetrate the bacterial shield. The researchers are studying why this happens, but they believe it's due to magnetic horsepower. In the tests, the researchers positioned a magnet below the implant, producing a strong enough field to force the nanoparticles above to filter through the film and proceed to the implant, Webster explained.
The particles then penetrate the bacterial cells because of their super-small size. A micron-sized particle, a thousand times larger than a nanoparticle, would be too large to penetrate the bacterial cell wall.
The researchers plan to test the iron-oxide nanoparticles on other bacteria and then move on to evaluating the results on implants in animals. The research was funded by the private Hermann Foundation Inc. In addition, Taylor's tuition and stipend are funded through the National Science Foundation GK-12 program.
Adapted from materials provided by Brown University.

Contrary to predictions, males of high genetic quality are not very successful when it comes to fertilizing eggs.

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Contrary to predictions, males of high genetic quality are not very successful when it comes to fertilizing eggs. A new study on seed beetles by Swedish and Danish scientists Göran Arnqvist and Trine Bilde shows that when a female mates with several males, the males of low genetic quality are the most successful in fertilizing eggs. The study is published in this week's issue of Science.

In almost all animals, females mate with several different males, despite the fact that a single mating is often sufficient to fertilize her eggs. Multiple mating also carries costs to females, such as the risk of catching sexually transmitted diseases.
One commonly held belief is that this behaviour may allow females to choose the sperm of the male with highest genetic quality to fertilize her eggs. Professor Göran Arnqvist from the Department of Ecology and Evolution, Uppsala University and associate professor Trine Bilde from the Department of Biological Sciences, University of Aarhus, have tested this possibility directly for the first time and shown that it is not true.
Their study on seed beetles shows that, contrary to predictions, males of low genetic quality are more successful in fertilizing eggs. Males who gained the highest share of paternity were actually males with low genetic quality. These males also fathered offspring that did less well.
"The results support the suggestion that genes that are good for males may often be bad for their mates. Therefore, in beetles at least, multiple mating does not award females with genetic benefits," says Göran Arnqvist.
Source: Uppsala University (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.

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)

Biotechnology: Engineered Moss Can Produce Human Proteins

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ScienceDaily (May 11, 2009) — ETH Zurich researchers have shown that mosses and humans share unexpected common characteristics. These evolutionary relics could be useful in the production of therapeutic proteins.

At first glance, mosses and human beings have little in common. The moss Physcomitrella patens is small, pale green, immobile, and uses sunlight as its energy source. Humans are large, mobile, and need to obtain energy by eating vegetable or animal foods.
Transferring mammalian genes into moss
This made the result of the experiments carried out by researchers in the group led by Martin Fussenegger, Professor of Chemical and Bioengineering at ETH Zurich, all the more astonishing. In collaboration with researchers at the University of Freiburg im Breisgau, the PhD student Marc Gitzinger carried out tests to see what happens when unmodified human or mammalian genes are inserted into the moss genome. They transferred the foreign, unmodified genes into the moss and discovered that the moss was easily able to manufacture the proteins encoded therein.
This cannot be taken for granted, since the same process does not work when a mammalian gene is implanted into what are known as “higher” flowering plants. The reason is that sections of the start and finish sequences of the genes of animals, plants, fungi and bacteria are considerably different. They are responsible for ensuring that a gene in the organism is recognized as such, and the proteins encoded by it are produced in the correct amount and are released from the cell. The more remote the relationship between living organisms, the greater the difference between these sequences. This is why biotechnologists must normally adapt them to a foreign organism before transplanting a gene into it. The researchers were astonished to find that this was not necessary in the case of the moss.
Moss as a generalist
The explanation given for this by Ralf Reski, Professor of Plant Biotechnology at the University of Freiburg im Breisgau, is that the moss has remained a generalist. It underwent the last major modification about 450 million years ago when it changed from living in water to a life on land, adapting to the new living conditions and then remaining unaltered for millions of years, both in its appearance and at a genetic level.
The process used by the moss to produce its proteins is less sophisticated than in “higher” organisms. In contrast to the moss, these latter organisms underwent major further developments and specializations over the course of 450 million years. On the other hand, the moss clearly retained – for millions of years – the ability to read foreign genes such as those from mammals and thus also from humans, and to translate them into proteins, probably without ever having made any use of this capability during these 450 million years.
A cost-effective alternative to mammalian cells
Today, the moss Physcomitrella patens and its ability to manufacture mammalian proteins could help to satisfy the large worldwide demand for therapeutic proteins. One well-known example is insulin, which enables diabetics to control their blood sugar level.
Nowadays, therapeutic proteins are mainly manufactured in mammalian cells, which are very expensive to culture. They need to be maintained at body temperature with a continuous supply of nutrients and oxygen, and the production process is costly. At present, global production capacity cannot match the demand. Because of the difficulties involved in handling them, production is possible only in industrialized countries.
In contrast, the moss Physcomitrella patens is comparatively undemanding. It needs water, a couple of nutrient salts and some light to allow it to flourish and produce proteins. This makes it convenient and simple to handle in a bioreactor, and, in the future, it might enable even less developed nations to satisfy their requirement for therapeutic proteins. However, further research will be needed before the moss can be used to produce therapeutic proteins on an industrial scale.
Journal reference:
Gitzinger et al. Functional cross-kingdom conservation of mammalian and moss (Physcomitrella patens) transcription, translation and secretion machineries. Plant Biotechnology Journal, 2009; 7 (1): 73 DOI: 10.1111/j.1467-7652.2008.00376.x
Adapted from materials provided by ETH Zurich.

How Cells Move: Cooperative Forces Boost Collective Mobility Of Cells

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ScienceDaily (May 11, 2009) — Research by scientists in Spain and their colleagues offers for the first time an experimental answer to the question of how cells move during biological processes as diverse as the development, metastasis, or regeneration of tissues.

The work addresses the issue of collective mobility of cells, that is to say, how cells are moved within tissues, and what is the prevalent form of movement inside living organisms.
"Research into collective cell mobility is very active due to the direct implications it has on fields such as embryologic development, organ regeneration, and cancer. For example, if we could find a way to control cell mobility during metastasis, cancer would be a curable disease in the majority of cases," says Dr. Xavier Trepat, senior researcher of the cellular and respiratory biomechanics group and researcher in the Department of Physiological Sciences at the University of Barcelona, and in the Networking Biomedical Research Centre for respiratory diseases (CIBER).
Up until now, scientists had proposed various mechanisms to explain collective cell migration. One hypothesis for example, suggests that the cells move collectively due to the existence of “leader” cells, which stretch out in the rest of the group, like a train pulls carriages behind it. Another hypothesis suggests that each cell moves independently to those around it, like cars on the motorway during a traffic jam, or like soldiers in a military parade. “We have rejected both these possibilities,” says Trepat.
According to his research, collective cell mobility is the result of a cooperative process in which each cell contributes to the movement of the group, stretching to those around it. “It is a mechanism similar to a tug-of-war game, in which two teams pull a rope by its extremes and the team that pulls the hardest wins. During the game, each player generates force and transmits it to the rope, so that the tension in the rope is the sum of the forces generated by each member of the team. Cells do the same. Each cell generates force to stretch to its neighbours in the direction of the movement» explains the researcher.
Journal reference:
Xavier Trepat, Michael R. Wasserman, Thomas E. Angelini, Emil Millet, David A. Weitz, James P. Butler & Jeffrey J. Fredberg. Physical forces during collective cell migration. Nature Physics, 2009; DOI: 10.1038/nphys1269
Adapted from materials provided by Universidad de Barcelona, via AlphaGalileo.

Deep in the Red: Using Infrared to Watch What Goes On in a Living Body

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An infrared version of the Nobel Prize-winning green fluorescent protein could make the technique even more powerful.

Fluorescent proteins, which are compounds that can absorb and then emit light, have become a powerful instrument in the cell biologist's toolkit—so powerful, in fact, that the discovery and development of green fluorescent proteins from jellyfish earned the 2008 Nobel Prize in Chemistry. (Here's a Q&A with one of the winners, Columbia University's Martin Chalfie, about his work.) These proteins have limitations, however: They need to be excited with the blue to orange part of the visible spectrum, at wavelengths of 495 to 570 nanometers. These wavelengths of light are too short to penetrate tissue very well, and so green fluorescent proteins are mainly used in test tube studies to watch cell division or to label certain cell types.
But one of the 2008 Nobelists, Roger Y. Tsien of the University of California, San Diego, and his U.C.S.D. colleagues report in today's issue of Science that they have developed a new fluorescent protein that could enable scientists to tag and visualize cellular activity as it happens inside a live animal. The protein, after absorbing light from the far-red part of the spectrum, shines in the near-infrared, at wavelengths of around 700 nanometers.
These longer wavelengths can penetrate mammalian tissue and even pass through bone. "Say you label a tumor with a green fluorescent protein, and if this labeled tumor is buried inside the animal, then you barely can get green fluorescence out," says lead researcher Xiaokun Shu. "But if you label this deeply buried tumor by infrared fluorescent proteins, you will get a stronger signal because infrared penetrates tissue more efficiently."
Tsien's group derived the infrared fluorescent protein from a hardy bacterium called Deinococcus radiodurans, famous for its ability to survive extreme environments. Bacteria do not actually use this class of proteins, called bacteriophytochromes, to emit light. "They use these bacteriophytochromes to control gene expression,” Shu says—the proteins convert absorbed light into energy to signal certain genes to turn on or off.
The initial challenge for researchers was to re-engineer the protein so that absorbed light would be re-emitted instead of being used as a source of power. They accomplished the feat by deleting the part of the protein that converts the absorbed light into chemical energy; as a result, this truncated and mutant form gave up its absorbed energy as an infrared glow. The scientists incorporated the engineered bacterial protein into mammalian cells—specifically, into the liver of a live mouse, which lit up with infrared light.
The finding paves the way for in vivo visualization of a wide range of biochemical processes and internal organs in animals. (Its use in humans is unlikely, as it would require gene therapy and the ethically dubious transplantation of bacterial genes into humans.)
“This is so important," comments David James, a cell biologist from Australia's Garvan Institute in Sydney, "because a lot of knowledge at the moment is confined to individual cells grown on a glass coverslip," leaving open the question of whether that knowledge "is transferable to an animal." The infrared version could also solve the problem of naturally occuring fluorescence from other biological molecules, which tend to glow at wavelengths similar to conventional fluorescent protein markers and thereby create a lot of “background noise," James says.

But even greater potential lies in harnessing the bacteriophytochrome's original function, namely, powering gene expression. It should be feasible, Tsien thinks, to put back in the signal-controlling properties of the phytochrome. Then it could be possible with animals to “switch on genes and control biochemistry" with light, he says.
For example, you want to explore the effects on mouse behavior of switching on a particular gene that controls some aspect of brain function, but, thankfully for the mouse, you do not want to open up its skull or stick a needle in its brain. "If the infrared fluorescent protein can be made to turn back into an infrared phytochrome, you could have the switch all ready and just waiting for enough infrared light," Tsien speculates. Because infrared light can penetrate the skull, it can reach the phytochrome and remotely switch the gene on, resulting in observable changes in the mouse's behavior.
It's the next evolutionary step for fluorescent proteins, remarks Tsien, who believes that phytochromes represent a class of proteins with enormous potential. If he's correct, then in the coming years, expect more scientists to see the (infrared) light.

By Bianca Nogrady

How Do Cells Sense And Respond To Messages? Major Signal Transduction Discovery Made

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http://www.sciencedaily.com/releases/2007/10/071004121038.htm

Science Daily — The chemical process known as acetylation plays a central role in cytokine receptor signal transduction – a fundamental biochemical cascade inside cells that controls the activity of antiviral and tumor-suppressing genes.
A team of cell biologists led by Eugene Chin, M.D., a research professor at The Warren Alpert Medical School of Brown University and a staff researcher at Rhode Island Hospital, reports its findings in the journal Cell. Their results are surprising.
Scientists have long known that phosphorylation, an amino acid modifying process in proteins, is critical for switching receptors on and off on the surface of cells. Chin and his team studied how type 1 interferon binds to a receptor complex, known as the IFN-α receptor, on the cell surface to trigger an immune response. Chin and his team found that acetylation, another chemical process that modifies amino acids, plays a central role in activating interferon receptors.
Interferons play a crucial role in the body’s defense against infection and uncontrolled cell growth. Type 1 interferon is widely used to treat hepatitis B and C and cancers such as melanoma and leukemia.
“This is a major discovery in the field of signal transduction,” Chin said. “Tyrosine phosphorylation has so far been considered the major player in signal transduction. But what we discovered challenges this concept. We found another player – acetylation – in the process.”
In their experiments, Chin and his team looked at how cells respond to type 1 interferon, a protein produced in response to a viral infection or other immune trigger. The researchers found that type 1 interferon receptors, which are found in every cell in the body, call up cytoplasmic CREB-binding protein, or CBP, to move up to the cell surface. CPB acetylates these receptors. That, in turn, sparks a biochemical cascade that attracts more proteins to create a complex called ISGF3. To activate this protein complex, Chin found, acetylation is required. Once that occurs, the complex travels to the cell nucleus to switch on anti-viral or tumor-suppressing genes.
The discovery of the acetylation of cytokine receptors marks a milestone in the study of signal transduction, the process of how cells receive and respond to chemical messages.
Many diseases, such as diabetes, cancer and heart disease, occur when signal transduction goes awry. That is why some drugs either inhibit or amplify signaling inside cells by targeting tyrosine phosphorylation. By showing that another chemical process is critical to signal transduction, Chin’s findings may explain why some anti-cancer or anti-viral drugs do not work for everyone. The findings provide an important new target for therapies that fight cancer and viral infectious diseases.
The Brown research team also included Xiaoli Tang, Jin-Song Gao, and Ying-jie Guan, all post-doctoral research associates in Chin’s Rhode Island Hospital laboratory. Bharat Ramratnam, associate professor of medicine at Brown, also assisted with the research along with Katya McLane, a scientist with Upstate/Chemicon International Inc.
The National Cancer Institute funded the work.
Note: This story has been adapted from material provided by Brown University.

Fausto Intilla
www.oloscience.com

Sizing Cells Up: Researchers Pinpoint When A Cell Is Ready To Reproduce

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http://www.blogger.com/post-create.g?blogID=4305283407805626298

Science Daily — For more than 100 years, scientists have tried to figure out the cell size problem: How does a cell know when it is big enough to divide?

In research conducted in budding yeast (Saccharomyces cerevisiae), scientists at Rockefeller University have now identified the cellular event that marks the moment when a cell knows it is big enough to commit to cell division and spawn genetic replicas of itself. The findings provide a precise and quantitative framework for studying the possible mechanisms that allow cells to monitor and sense their size.
During the first phase of the cell cycle, known as G1, budding yeast grows and begins to form a bud; in the final stage, the cell splits into two — one bigger than the other. Although researchers have identified several key proteins that regulate and play a role in coordinating cell growth and division during G1, they have not been able to get to the core mechanism that senses whether a cell possesses enough resources to divide. Scientists needed a way to organize and confidently sort out molecular candidates involved in cell size control from those that played other roles.
Graduate student Stefano Di Talia, a biophysicist, and postdoc Jan Skotheim, an applied mathematician, provided just that. Working with Eric Siggia, head of the Laboratory of Theoretical Condensed Matter Physics, and Fred Cross, head of the Laboratory of Yeast Molecular Genetics, Di Talia and Skotheim showed that a unique cellular event, the exiting of the protein Whi5 from the nucleus, separates G1 into two independent steps: one controlled by a sizer (T1) and one controlled by a timer (T2). T1 begins when the mother and daughter cells have completely separated from each other; T2 starts in G1 once Whi5 has exited the nucleus and lasts until the new daughter cell forms its own bud.
“You need some way to know how big you are,” says first author Di Talia, whose work appears in the August 23 issue of Nature. “This precise quantitative framework allows us to narrow down the possibility of events that are involved in size control.”
By measuring the sizes of budding yeast and how long they spend in G1 and in T1, Di Talia saw that daughter cells, which are much smaller than their mother cells, need to spend more time in T1 growing. Once daughter cells reach the required size for division, they spend as much time as their mothers in T2, subsequently replicating their DNA and producing daughter cells of their own. Di Talia and his colleagues used genetics to show that a different medley of proteins coordinate cell growth and division during T1 and T2, a crucial finding highlighting that these two parts of G1 are independent from each other and are regulated by different mechanisms.
“If we continue to identify the molecular events that change how T1 is regulated,” says Di Talia, “we can really hope to get to the core of what the size-sensing machinery is.”
Nature 448(7156): 947-951 (August 23, 2007)
Note: This story has been adapted from material provided by Rockefeller University.

Fausto Intilla

www.oloscience.com

First-ever Atomic-detail Computer Simulation Of How Proteins May Vibrate In A Crystal Completed

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http://www.sciencedaily.com/releases/2007/09/070927113146.htm

Science Daily — An international collaboration directed by an Oak Ridge National Laboratory researcher has performed the first-ever atomic-detail computer simulation of how proteins vibrate in a crystal.

Jeremy Smith, who leads ORNL's Center for Molecular Biophysics, said experimental testing of the theoretical work will require the capabilities of the Office of Science's recently completed Spallation Neutron Source at ORNL.
The study is a collaboration between Smith, who also holds a University of Tennessee-ORNL Governor's Chair, and researchers from the California Institute of Technology and the National Institute of Chemistry, Ljubljana, Slovenia.
Understanding how proteins--life's worker molecules--interact with each other is a major goal of biological sciences. The simulation, which was made possible by recent advances in scientific computing, describes the forces and vibrations involved in protein crystals, which provide an environment in which the proteins are ordered and thus lend themselves to detailed study.
According to Smith, lattice dynamics describe how the repeating units of a crystal vibrate relative to each other. The resulting "phonon dispersion relations" relate the frequencies to the wavelengths of the oscillations.
Phonon dispersion relations provide information on how proteins interact with each other that could be useful for understanding protein-protein interactions in the living cell. Until now, researchers have lacked the computing power to allow atomic-detail lattice dynamical calculations.
Smith said the PRL paper predicts the existence and forms of the protein crystal lattice modes.
"In doing so it throws out a challenge to next-generation neutron science to finally make the breakthrough and determine the forms and frequencies of the vibrations experimentally," he said.
In other words, having overcome their computational hurdle, the lattice dynamics team is now ready for the SNS to test the simulation work and see if what is predicted is really there.
"Atomic-detail crystal dynamics calculations have not been possible before, and now we also have an experimental tool--the SNS--that will have the capability to test our simulations. We are looking forward to seeing the next generation of instruments at SNS demonstrate their talents." Smith said, humbly adding, "Hopefully, the calculations won't be too painfully off the mark."
Smith believes the SNS and its arsenal of specialized analytical instruments will be able to confirm--or contradict--what the simulations indicate.
"We appreciate that examining complicated proteins in this way will not be easy, even for SNS. However, with SNS instruments expected to be in some cases hundreds of times improved over currently existing facilities, we are confident that the neutron breakthrough is within reach," Smith said.
The work was recently published in Physical Review Letters.
Note: This story has been adapted from material provided by DOE, Oak Ridge National Laboratory.

Fausto Intilla

www.oloscience.com

Mystery Behind How Nuclear Membrane Forms During Mitosis Solved

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http://www.sciencedaily.com/releases/2007/09/070915100302.htm

Science Daily — Just how a dividing cell rebuilds the nuclear envelope, the protective, functional wrapping that encases both the original and newly copied genetic material, has been a source of controversy for the last 20 years.

The answer matters because the architecture established during formation of the envelope is regarded as key to future regulation of gene expression.
Now scientists at the Salk Institute of Biological Studies, reporting in the September 9 advanced online edition of Nature Cell Biology, lay the debate to rest. Studying frog eggs, they discovered that the endoplasmic reticulum (ER), a key cellular organelle shaped like a network of tubes, flattens part of itself during mitosis to form a double-sided sheet, which then bends around what will become the nucleus, the control hub of the cell.
“The process is simple and elegant,” says Martin W. Hetzer, Ph.D., an assistant professor in the Molecular and Cell Biology Laboratory, who explains, “the membrane tubules are flattened as they associate with chromatin. Our model does not involve vesicle fusion.”
The most dramatic event during nuclear assembly following replication and separation of chromosomes is the reformation of the nuclear envelope, a highly structured barrier that separates the nuclear interior from the rest of the cell, say Hetzer and Daniel J. Anderson, a graduate student in Hetzer’s lab and co-author of the study. The envelope is the gateway into the nucleus and, thus, restricts access to the genome; it is composed of a concentric double membrane that is penetrated by nuclear pores, which serve as transport channels between the nucleus and the cytoplasm.
Just as chromosomes duplicate, the cell’s organelles, including the ER, also reproduce themselves. “The ER is always there and remains an intact network of tubes,” says Hetzer. In a mature cell the ER works closely with the genome, synthesizing and transporting the proteins produced under the direction of genes housed inside the nucleus.
Some scientists have believed that the ER in the sister cells help form the nuclear envelope, although proof of the process has been lacking. Others have argued that the new membrane is resurrected from bits of the “old” nuclear membrane that disintegrates when nuclear chromosomes duplicate and pull apart during mitosis.
“The problem with this theory, however, is that these fragments would then have to be fused together in the newly produced cells, and that would require massive membrane fusion and a dedicated protein machinery,” Hetzer says. “But no one has ever found it. Our data suggests that the search for this fusion machinery is now obsolete,” he says.
Hetzer and Anderson used a popular scientific model of mitosis, the eggs of Xenopus, an African frog, to determine how the nuclear envelope is restored. They found that the tubules of the ER are connected to each other in a three-way junction that allows them to constantly move and change position relative to each other.
During the early phases of mitosis, the end of the tubes bind directly to DNA found at the surface of the chromatin, the tightly bundled coil of genetic material and proteins that form chromosomes after DNA replication. Then, as mitosis proceeds, extra DNA binding proteins that reside in the ER are employed to progressively immobilize some of the tubules, flattening them out to create the nuclear membrane
“The tubules are squeezed into flat sheets that merge with each other, forming large membrane sheets that cover the entire surface of the chromatin,” Hetzer says. Why this matters, according to Hetzer, is because it is becoming increasingly clear that the nuclear envelope plays an important role in cell function.
“Anchorage of chromatin at the nuclear periphery and its three-dimensional organization within the nuclear interior helps regulate gene expression, and we know that mutations in nuclear envelope proteins cause a variety of different human diseases,” he says. “So knowing how the nuclear membrane is assembled will help us understand nuclear architecture and, in the long run, gene expression.”
The study was funded by NIH and a Pew Scholar Award.
Animation available: http://www.salk.edu/video/Sec61-U2OS-1/Sec61-U2OS-1.html
Note: This story has been adapted from a news release issued by Salk Institute for Biological Studies.

Fausto Intilla

www.oloscience.com

Microfluidic Chambers Advance The Science Of Growing Neurons

Source:

http://www.sciencedaily.com/releases/2007/08/070830160803.htm

Science Daily — Researchers at the University of Illinois have developed a method for culturing mammalian neurons in chambers not much larger than the neurons themselves. The new approach extends the lifespan of the neurons at very low densities, an essential step toward developing a method for studying the growth and behavior of individual brain cells.

The technique is described this month in the journal of the Royal Society of Chemistry – Lab on a Chip.
“This finding will be very positively greeted by the neuroscience community,” said Martha Gillette, who is an author on the study and the head of the cell and developmental biology department at Illinois. “This is pushing the limits of what you can do with neurons in culture.”
Growing viable mammalian neurons at low density in an artificial environment is no easy task. Using postnatal neurons only adds to the challenge, Gillette said, because these cells are extremely sensitive to environmental conditions.
All neurons rely on a steady supply of proteins and other “trophic factors” present in the extracellular fluid. These factors are secreted by the neurons themselves or by support cells, such as the glia. This is why neurons tend to do best when grown at high density and in the presence of other brain cells. But a dense or complex mixture of cells complicates the task of characterizing the behavior of individual neurons.
One technique for keeping neural cultures alive is to grow the cells in a medium that contains serum, or blood plasma. This increases the viability of cells grown at low density, but it also “contaminates” the culture, making it difficult to determine which substances were produced by the cells and which came from the serum.
Those hoping to understand the cellular origins of trophic factors in the brain would benefit from a technique that allows them to measure the chemical outputs of individual cells. The research team made progress toward this goal by addressing a few key obstacles.
First, the researchers scaled down the size of the fluid-filled chambers used to hold the cells. Chemistry graduate student Matthew Stewart made the small chambers out of a molded gel of polydimethylsiloxane (PDMS). The reduced chamber size also reduced – by several orders of magnitude – the amount of fluid around the cells, said Biotechnology Center director Jonathan Sweedler, an author on the study. This “miniaturization of experimental architectures” will make it easier to identify and measure the substances released by the cells, because these “releasates” are less dilute.
“If you bring the walls in and you make an environment that’s cell-sized, the channels now are such that you’re constraining the releasates to physiological concentrations, even at the level of a single cell,” Sweedler said.
Second, the researchers increased the purity of the material used to form the chambers. Cell and developmental biology graduate student Larry Millet exposed the PDMS to a series of chemical baths to extract impurities that were killing the cells.
Millet also developed a method for gradually perfusing the neurons with serum-free media, a technique that resupplies depleted nutrients and removes cellular waste products. The perfusion technique also allows the researchers to collect and analyze other cellular secretions – a key to identifying the biochemical contributions of individual cells.
“We know there are factors that are communicated in the media between the cells,” Millet said. “The question is what are they, and how can we get at those?”
This combination of techniques enabled the research team to grow postnatal primary hippocampal neurons from rats for up to 11 days at extremely low densities. Prior to this work, cultured neurons in closed-channel devices made of untreated, native PDMS remained viable for two days at best.
The cultured neurons also developed more axons and dendrites, the neural tendrils that communicate with other cells, than those grown at low densities with conventional techniques, Gillette said.
“Not only have we increased the cells’ viability, we’ve also increased their ability to differentiate into what looks much more like a mature neuron,” she said.
Sweedler noted that the team’s successes are the result of a unique collaboration among scientists with very different backgrounds.
“(Materials science and engineering professor) Ralph Nuzzo is one of the pioneers in self-assembled monolayers and surface chemistry,” Sweedler said. “Martha Gillette’s expertise is in understanding how these neurons grow, and in imaging them. My lab does measurement science on a very small scale. It’s almost impossible for any one lab to do all that.”
Nuzzo and Sweedler are William H. and Janet Lycan professors of chemistry. Gillette is Alumni Professor of Cell and Developmental Biology. All are appointed in the Institute for Genomic Biology. Sweedler and Gillette are affiliates of the Beckman Institute and the Neuroscience Program. Sweedler is a professor in the Bioengineering Program and Gillette in the College of Medicine.
Note: This story has been adapted from a news release issued by University of Illinois at Urbana-Champaign.

Fausto Intilla

www.oloscience.com