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Week ending FRIDAY April 17, 2009---------------------------News Archive

Study Yields Clue to How Stem Cells Form
An Emory University study shows some of the first direct evidence of a process required for epigenetic reprogramming between generations – a finding that could shed more light on the mechanisms of fertilization, stem-cell formation and cloning

The journal Cell published the results of the study on the nematode C. elegans in its April 17 issue. "We believe that we have demonstrated one of the processes that erases the information in a fertilized egg, so that the offspring can begin life with a clean slate," says David Katz, lead author of the study. Katz is a post-doctoral fellow in the lab of William Kelly, associate professor of biology at Emory and a co-author of the study.

"One of the most fundamental mysteries in biology is how a sperm and egg create a new organism. By looking at the process at the molecular level, we're gaining understanding of this basic question of life," Katz says.

When a sperm cell fertilizes an egg cell, the specialized programming of each parent cell must be erased, in order to form a zygote that can give rise to a new organism. The process by which these two differentiated cells return to a developmental ground state in the zygote – the ultimate stem cell – is little understood.

The Emory researchers wanted to test the theory that removal of a particular histone protein modification involved in the packaging of DNA – dimethylation of histone H3 on lysine 4 – is involved in reprogramming the germ line.

They compared successive generations of a normal strain of C. elegans – a microscopic worm commonly used for studying cell differentiation – with a mutant strain. The mutants lacked an enzyme that test-tube experiments have previously shown appears to play an "erasing" role – demethylating histones to remove information from the packaging of DNA.

In the normal strain of the worms, the histone modification the Emory researchers had targeted was not passed on to the next generation, but in the mutant strain the modification continued through 30 generations, and each generation became progressively less fertile.

"That's an amazing phenotype," Katz says. "The organism gradually lost its ability to reproduce. We have shown that when this enzyme is missing, the worms can inherit the histone modification – not only from cell to cell, but from generation to generation."

When the researchers re-inserted the missing enzyme into the sterile generations of mutant worms, they were able to reverse the process: the worms no longer inherited the histone modification, and they regained fertility.

For years, it's been accepted that histone proteins help coil six-foot strands of DNA into tight balls, compact enough to fit inside the nucleus of a cell. Histone modifications have also been known to correlate with gene expression. More recently, researchers have theorized that a chemical change in the histone packaging of DNA, known as an epigenetic event, can be passed on – just as genes themselves can be inherited.

"This study is one of the first demonstrations in a living organism that this theory may be true – that every generation can be affected by an epigenetic event," Kelly says. "Our work provides some of the best, direct evidence that chemical modifications in the packaging of DNA can be inherited from cell to cell," Katz added. "That indicates that these chemical modifications are not just involved in packaging – they contain information."

A better understanding of the role of histones, and the enzymes involved in their modification, could lead to therapies for everything from cancer to infertility. "Stem-cell therapies are an incredibly promising technology for treating any problem that has to do with defective cells," Katz says. "We're hoping that our work will help this technology to develop."

Katz and his colleagues are now building on the results of the study, to see if a lack of the erasing enzyme shows a similar effect in mice.


The Story of X — Evolution of a Sex Chromosome
In the first evolutionary study of the chromosome associated with being female, University of California, Berkeley, biologist Doris Bachtrog and her colleagues show that the history of the X chromosome is every bit as interesting as the much-studied, male-determining Y chromosome, and offers important clues to the origins and benefits of sexual reproduction

The neo-X (top) and neo-Y chromosomes of the fruit fly Drosophila miranda, showing how the Y has shrunken slightly through loss of genes. The X has remained about the same size as the fly's other chromosomes, though its genes are in the process of adapting to the Y's degeneration. (Doris Bachtrog/UC Berkeley) "Contrary to the traditional view of being a passive player, the X chromosome has a very active role in the evolutionary process of sex chromosome differentiation," said Bachtrog, an assistant professor of integrative biology and a member of UC Berkeley's Center for Theoretical Evolutionary Genomics.

Bachtrog, UC Berkeley post-doctoral fellow Jeffrey D. Jensen and former UC San Diego post-doc Zhi Zhang, now at the University of Munich, detail their findings in this week's edition of the open-access journal PLoS Biology.

"In our manuscript, we demonstrate for the first time the flip side of the sex chromosome evolution puzzle: The X chromosome undergoes periods of intense adaptation in the evolutionary process of creating new sections of the genome that govern sexual differentiation in many species, including our own," she said.

Not all animals and plants employ genes to determine if an embryo becomes male or female. Many reptiles, for example, rely on environmental cues such as temperature to specify male or female.

But in life forms that do set aside a pair of chromosomes to specify sex - from fruit flies to mammals and some plants - the two X chromosomes inherited by females look nearly identical to the other non-sex chromosomes, so-called autosomes, Bachtrog said. The Y chromosome, however, which is inherited by males in concert with one X chromosome, is a withered version of the X, having lost many genes since it stopped recombining with the X chromosome.

In mammals, that probably took place about 150 million years ago, while in the fruit fly Drosophila melanogaster, a laboratory favorite, the sex chromosomes arose independently about 100 million years ago. In both humans and fruit flies, the Y chromosome has dwindled from a few thousand genes to a few dozen.

Hence the intense interest in why and how the Y chromosome lost genes once it stopped interacting with the X. Scientists have found that, as the only chromosome pair that doesn't break and recombine every time a cell divides, the XY pair in males is unable to take advantage of the main way deleterious genetic mutations are eliminated. The XX pair in females does recombine, but for the Y, the only way to get rid of a bad mutation in a gene is to inactivate or delete the entire gene. Over millions of years, inactive genes are lost, and the Y shrinks.

"If you have no recombination, natural selection is less effective at removing detrimental genes," said Bachtrog. "Y is an asexual chromosome, and it pays a price for that: It keeps losing genes."

Bachtrog, whose career has revolved mostly around the study of the degeneration of the Y chromosome, decided to focus on the X chromosome several years ago and went about searching for sex chromosome pairs that have arisen more recently - and thus might be in the process of adapting to their new role. Her paper centers around study of the three sex chromosomes in a rare western fruit fly, Drosophila miranda, a darker-colored cousin of D. melanogaster. (Many creatures have more than one pair of sex chromosomes; the platypus, for example, has five pairs, all inherited together.)

While one of D. miranda's sex chromosomes is descended from the original sex chromosome that appeared in Drosophila nearly 100 million years ago, a second originated perhaps 10 million years ago, and the third about a million years ago. The older two look much alike, Bachtrog said: The Y chromosome in each pair has lost genes to become a shadow of its former self, while the two X chromosomes are indistinguishable from each other.

The third and youngest sex chromosome is different. The Y is not yet shriveled, though it contains many non-functional genes - about half the total - that will eventually be lost. The X, which is dubbed neo-X, is undergoing rapid change, however, with about 10 times the normal amount of adaptation seen in the autosomes, according to the researchers.

By adaptation, Bachtrog means that the gene sequences in the X chromosome are becoming fixed as random mutations have finally settled on a few beneficial changes that accommodate the increasingly irrelevant Y chromosome. Between 10 and 15 percent of neo-X genes show adaptation, compared to only 1-3 percent of autosome genes.

"In hindsight, that is not surprising," Bachtrog said. "Neo-X is facing a much more challenging situation than the autosomes because its pair, the Y chromosome, is degenerating. Its genes are no longer producing proteins, so neo-X has to compensate by up-regulating its genes. We find a lot of genes on the X chromosome are involved in dosage compensation."

In humans, for example, all genes on the X chromosome are twice as active to account for the lack of genes on the Y. Women accommodate this by inactivating one entire X chromosome so as not to produce too much protein, Bachtrog said.

Another change in neo-X that Bachtrog suspects is taking place is the elimination of genes that are harmful to females. Biologists have realized recently that some genes have opposite effects in males and females, and evolution is a tug of war between males jettisoning genes that they find detrimental only to have females put them back, and vice versa.

"A good place to put sexually antagonistic genes that are beneficial to one sex but detrimental to the other is on the sex chromosomes," she said. The Y always ends up in the male, she said, so genes on the Y chromosome won't affect females.

"Conversely, the X chromosome becomes feminized with genes that are good for the female but detrimental to the male," said Bachtrog, adding that the X also becomes demasculinized, losing genes that are of use only in the male.

In search of more insights into the evolution of the X chromosome, Bachtrog said she is looking for fruit fly species with older and younger sex chromosomes "to study sex chromosome evolution in action." She said evidence suggests that adaptation to being a sex chromosome is most intense between 1 and 10 million years after it starts. Bachtrog also is completing assembly of the genome sequence for D. miranda, which is not among the 12 species of Drosophila currently targeted by the genome sequencing community. She hopes that the fly will become a model system like D. melanogaster.

"Now, finally, we are within reach of studying model systems like D. miranda that we couldn't think of several years ago," she said, predicting that "whole genome comparisons will revolutionize evolutionary biology, ecology and many other fields."

The research was funded by the National Institutes of Health, an Alfred P. Sloan Faculty Research Fellowship in Molecular and Computational Biology and a David and Lucile Packard Foundation Fellowship.

Worms Control Lifespan at High Temperatures
The common research worm, C. elegans, is able to use heat-sensing nerve cells to not only regulate its response to hotter environments, but also to control the pace of its aging as a result of that heat, according to new research at the University of California, San Francisco.

The new findings have turned upside down a widespread assumption about how cold-blooded animals respond to and regulate heat, the researchers say. The study is reported in the online early edition of the journal “Current Biology” and is available at http://www.cell.com/current-biology/home.

Researchers have known for years that cold-blooded animals, or ectotherms, go through life more quickly at high temperatures than at low temperatures, according to UCSF Professor Cynthia Kenyon, PhD, who was senior researcher on the paper.

At temperatures of 25 degrees Celsius and above, worms move, eat and digest food faster, mature faster and age faster than their counterparts at a more normal 20 degrees. The common assumption, she said, is that the accelerated aging process at higher temperatures occurred passively, in much the same way that a chemical reaction speeds up at higher temperatures.

“We’ve shown it’s not so simple,” said Kenyon, a professor in the UCSF Department of Biochemistry and Biophysics and director of the Larry L. Hillblom Center for the Biology of Aging at UCSF. She is renowned for her ongoing research on C. elegans (Caenorhabditis elegans) and aging.

Humans and other warm-blooded animals have a mechanism that enables us to maintain a constant temperature as our environment heats up or cools. Kenyon said most textbooks explain that worms and other ectotherms cannot do that.

“It’s true that worms don’t regulate their body temperature, but they do regulate their response to high temperature, slowing down processes that would otherwise go much faster. In fact, they even use steroid hormones to do this, just as we do to regulate our temperature,” she said, noting that this might have been a very early evolutionary link between cold- and warm-blooded animals.

C. elegans has been known to have thermosensory or heat-sensing neurons, which allow the worms to move towards temperatures they associate with food. If the “chemical reaction” theory were accurate, worms at a constant hotter temperature would age at the same fast rate, whether their thermosensory neurons perceived the heat or not.

The researchers found that when they either killed the heat-sensing neurons or deactivated the worm’s genes that produce steroids, the worms had an even greater response to the heat, and as a result aged and died much faster than their counterparts with active neurons. The authors conclude that these heat-sensing neurons actually help the worm regulate its response to increasing heat.

The data suggest that these thermosensory neurons affect lifespan at warm temperature by changing the activity of a steroid signaling pathway, which in turn affects longevity, according to the paper. Specifically, the authors suggest that at high temperature, the worm’s thermosensory neurons produce a signal that stimulates expression of the daf-9 gene, which produces a steroid hormone that extends lifespan.

The researchers propose that this thermosensory system allows C. elegans to reduce the effect that warm temperature would otherwise have on the processes that affect aging, which is something that warm-blooded animals do by controlling the temperature itself.

This system may allow the animal to maintain a more normal rate of aging even if the temperature rises, Kenyon said.

Previous research also has linked the rate of aging in mammals with temperature. If mice are tricked into thinking that they are in a hot climate, they lower their body temperature and live longer.
While similar steroid hormone pathways exist in humans, Kenyon stressed that, at least for now, this research is more relevant to our general understanding of biology than to any human biomedical connection.

“These findings probably won’t result in a new cure for cancer or Alzheimer’s,” she said. “But they may force us to rewrite the section on cold-blooded animals in high school biology textbooks.”


Gold ShedS Light on the Inner Workings of Cells
Researchers from Singapore’s Institute of Bioengineering and Nanotechnology (IBN) have achieved another biomedical breakthrough with highly fluorescent gold nanoclusters for sub-cellular imaging. Their new invention has broad implications for biolabeling and disease diagnosis.

Measuring less than 1 nanometer in diameter, IBN’s gold clusters are much smaller than currently available nanoscale imaging technologies such as semiconducting quantum dots, which are usually at least 3 nanometers in size.

Unlike quantum dots, the gold nanoclusters are suitable for use within the body as they do not contain toxic metals such as cadmium and lead. Their sub-nanometer size makes it easy to target the nucleus inside the cell for sub-cellular biolabeling and bioimaging. Tracking the cell nucleus can help scientists monitor the fundamental life processes of healthy DNA replication and any genomic changes. With improved bioimaging at the cell nucleus, scientists can also study the effectiveness of drug and gene therapies.

“Gold nanoclusters have promising characteristics for applications in vivo. Our materials are smaller, less toxic and more biocompatible than the existing inorganic fluorescent quantum dot tags. The red fluorescence of the nanoclusters enhances biomedical images of the body greatly as there is reduced background fluorescence and better tissue penetration,” said IBN Postdoctoral Fellow, Dr Jianping Xie.

Synthesized via a single-step reaction at body temperature (37°C), the gold nanoclusters are formed with a commercially available common protein such as bovine serum albumin (BSA). “The protein holds and interacts with gold ions in aqueous solution. We are able to use this protein to provide a scaffold for the formation of gold nanoclusters,” explained Dr Yuangang Zheng, IBN Senior Research Scientist.

IBN’s gold nanoclusters are stable in aqueous solution as well as in the solid form, which facilitates their storage and distribution. Besides the low cost of the required reagents, the preparation of the gold nanoclusters also adopts an environmentally friendly method that does not involve toxic chemicals or high temperatures. In addition, the simple synthesis technique can be scaled up easily for mass production. IBN’s research on gold nanoclusters has been recently published in the leading international chemistry journal, Journal of the American Chemical Society, 131 (2009) 888-889.

“We are inspired by nature’s ability to create elegant and functional materials. Our process is similar to biomineralization in nature that is found in the formation of bones and shells: where functional proteins mostly interact with sequestered inorganic ions to provide scaffolds for mineral formation,” Principal Investigator and IBN Executive Director Professor Jackie Y. Ying elaborated. “There is a significant potential for our technology to impact biological and medical research, where our gold nanoclusters can significantly enhance the details available for precision bioimaging in medical diagnosis and treatment.”

New Nucleotide Could Revolutionize Epigenetics
Anyone who studied a little genetics in high school has heard of adenine, thymine, guanine and cytosine - the A,T,G and C that make up the DNA code. But those are not the whole story. The rise of epigenetics in the past decade has drawn attention to a fifth nucleotide, 5-methylcytosine (5-mC), that sometimes replaces cytosine in the famous DNA double helix to regulate which genes are expressed. And now there’s a sixth. In experiments to be published online Thursday by Science, researchers reveal an additional character in the mammalian DNA code, opening an entirely new front in epigenetic research.

The work, conducted in Nathaniel Heintz’s Laboratory of Molecular Biology at The Rockefeller University, suggests that a new layer of complexity exists between our basic genetic blueprints and the creatures that grow out of them. “This is another mechanism for regulation of gene expression and nuclear structure that no one has had any insight into,” says Heintz, who is also a Howard Hughes Medical Institute investigator. “The results are discrete and crystalline and clear; there is no uncertainty. I think this finding will electrify the field of epigenetics.”

Genes alone cannot explain the vast differences in complexity among worms, mice, monkeys and humans, all of which have roughly the same amount of genetic material. Scientists have found that these differences arise in part from the dynamic regulation of gene expression rather than the genes themselves. Epigenetics, a relatively young and very hot field in biology, is the study of nongenetic factors that manage this regulation.

One key epigenetic player is DNA methylation, which targets sites where cytosine precedes guanine in the DNA code. An enzyme called DNA methyltransferase affixes a methyl group to cytosine, creating a different but stable nucleotide called 5-methylcytosine. This modification in the promoter region of a gene results in gene silencing.

Some regional DNA methylation occurs in the earliest stages of life, influencing differentiation of embryonic stem cells into the different cell types that constitute the diverse organs, tissues and systems of the body. Recent research has shown, however, that environmental factors and experiences, such as the type of care a rat pup receives from its mother, can also result in methylation patterns and corresponding behaviors that are heritable for several generations. Thousands upon thousands of scientific papers have focused on the role of 5-methylcytosine in development.

The discovery of a new nucleotide may make biologists rethink their approaches to investigating DNA methylation. Ironically, the latest addition to the DNA vocabulary was found by chance during investigations of the level of 5-methylcytosine in the very large nuclei of Purkinje cells, says Skirmantas Kriaucionis, a postdoctoral associate in the Heintz lab, who did the research. “We didn’t go looking for this modification,” he says. “We just found it.”

Kriaucionis was working to compare the levels of 5-methylcytosine in two very different but connected neurons in the mouse brain — Purkinje cells, the largest brain cells, and granule cells, the most numerous and among the smallest. Together, these two types of cells coordinate motor function in the cerebellum. After developing a new method to separate the nuclei of individual cell types from one another, Kriaucionis was analyzing the epigenetic makeup of the cells when he came across substantial amounts of an unexpected and anomalous nucleotide, which he labeled ‘x.’

The nucleotide accounted for roughly 40 percent of the methylated cytosine in Purkinje cells and 10 percent in granule neurons. He then performed a series of tests on ‘x,’ including mass spectrometry, which determines the elemental components of molecules by breaking them down into their constituent parts, charging the particles and measuring their mass-to-charge ratio.

He repeated the experiments more than 10 times and came up with the same result: x was 5-hydroxymethylcytosine, a stable nucleotide previously observed only in the simplest of life forms, bacterial viruses. A number of other tests showed that ‘x’ could not be a byproduct of age, DNA damage during the cell-type isolation procedure or RNA contamination. “It’s stable and it’s abundant in the mouse and human brain,” Kriaucionis says. “It’s really exciting.”

What this nucleotide does is not yet clear. Initial tests suggested that it may play a role in demethylating DNA, but Kriaucionis and Heintz believe it may have a positive role in regulating gene expression as well.

The reason that this nucleotide had not been seen before, the researchers say, is because of the methodologies used in most epigenetic experiments. Typically, scientists use a procedure called bisulfite sequencing to identify the sites of DNA methylation. But this test cannot distinguish between 5-hydroxymethylcytosine and 5-methylcytosine, a shortcoming that has kept the newly discovered nucleotide hidden for years, the researchers say. Its discovery may force investigators to revisit earlier work.

The Human Epigenome Project, for example, is in the process of mapping all of the sites of methylation using bisulfite sequencing. “If it turns out in the future that [5-hydroxymethylcytosine and 5-methylcytosine] have different stable biological meanings, which we believe very likely, then epigenome mapping experiments will have to be repeated with the help of new tools that would distinguish the two,” says Kriaucionis.

Providing further evidence for their case that 5-hydroxymethylcytosine is a serious epigenetic player, a second paper to be published in Science by an independent group at Harvard University reveals the discovery of genes that produce enzymes that specifically convert 5-methylcytosine into 5-hydroxymethylcytosine. These enzymes may work in a way analogous to DNA methyltransferase, suggesting a dynamic system for regulating gene expression through 5-hydroxymethylcytosine. Kriaucionis and Heintz did not know of the other group’s work, led by Anjana Rao, until earlier this month. “You look at our result, and the beautiful studies of the enzymology by Dr. Rao’s group, and realize that you are at the tip of an iceberg of interesting biology and experimentation,” says Heintz, a neuroscientist whose research has not focused on epigenetics in the past. “This finding of an enzyme that can convert 5-methylcytosine to 5-hydroxymethylcytosine establishes this new epigenetic mark as a central player in the field.”

Kriaucionis is now mapping the sites where 5-hydroxymethylcytosine is present in the genome, and the researchers plan to genetically modify mice to under- or overexpress the newfound nucleotide in specific cell types in order to study its effects. “This is a major discovery in the field, and it is certain to be tied to neural function in a way that we can decipher,” Heintz says.

Cold and Brown Fat Raise Prospect of New Method for Treating Obesity
Sven Enerbäck, Professor at the Institute of Biomedicine at the Sahlgrenska Academy, University of Gothenburg, Sweden, is one of the scientists who published their results in The New England Journal of Medicine this week. Studies carried out by Enerbäck and others show that adults use brown fat to convert energy to heat - a discovery that may provide new possibilities in treating overweight and obesity.

It has previously been believed that the brown fat found in infants disappears as we grow up, but the new study shows that this is not the case. Brown fat cells have been found in adults, in the lower part of the neck just above the collarbone.

The region of brown fat cells in the neck was tested by placing five volunteers, in thin clothing, in a chilly room for a couple of hours. The researchers then investigated this region by PET scanning and discovered that metabolism there was on average 15 times higher than in the neighbouring white fat tissue. The result suggests that the brown fat may play a significant role in metabolism.

Enerbäck believes that this discovery can lead to new and better ways of treating obesity. These would be based on an exciting treatment strategy that focuses on increasing the amount of fat burnt by the body rather than focusing solely on reducing the intake of energy.


THURSDAY April 16, 2009---------------------------News Archive

Novel Technique to Sequence Human Genome
Physicists at Brown University have developed a novel procedure to map a person’s genome

They report in the journal Nanotechnology the first experiment to move a DNA chain through a nanopore using magnets. The approach is promising because it allows multiple segments of a DNA strand to be read simultaneously and accurately.

Since the human genome was sequenced six years ago, the cost of producing a high-quality genome sequence has dropped precipitously. More recently, the National Institutes of Health called for cutting the cost to $1,000 or less, which may enable sequencing as part of routine medical care.


The obstacles to reaching that goal have been primarily technological: Scientists have struggled to figure out how to accurately read the 3 billion base pairs - the amount of DNA found in humans and other mammals - without time-consuming, inefficient methods.

Physicists at Brown University may have an answer. They introduce a novel procedure to vastly slow the DNA’s movement through openings that are used to read the code. In the journal Nanotechnology, the physicists report the first experiment to move DNA through a solid-state nanopore using magnets. The approach is promising because it allows multiple segments of a DNA strand to be threaded simultaneously through numerous tiny pores and for each fragment to move slowly enough through the opening so that the base pairs can be accurately read.

“When it comes to sequencing anyone’s genome, you need to do it cheaply, and you need to do it quickly,” explained Xinsheng Sean Ling, professor of physics, who joined the Brown faculty in 1996. “This is a step in that direction.”

The idea of reading DNA by threading strands through tiny openings is not new. Scientists have shown that an applied electric field can drive the DNA molecules through a nanopore, a tiny hole in a membrane. But in those experiments, the base pairs moved too quickly through the openings for the code to be read accurately. So, while a large electric field is needed to draw the DNA molecules into the pore, Ling explained, the same field moves the DNA too quickly, a classic scientific Catch-22.


The trick is to figure out how to slow the strands’ movement through the opening so the base pairs (A, T, C, and G) can be read. To solve that, Ling and Hongbo Peng, the lead author who performed the work as a graduate student at Brown and who now works at IBM, attached the DNA strand to a bead using a streptavidin-biotin bond. Like previous researchers, they used an electric field to drive the DNA strand toward the pore. But while the strand could pass through the pore, the bead, with a 2.8-micron diameter, was too large for the pore, which has a diameter of only 10 nanometers. So the bead was stuck in the hole with the attached DNA strand suspended on the other side of the membrane.

The Brown researchers then used magnets — they call them “magnetic tweezers” — to draw the iron-oxide bead away from the pore. As the bead moves toward the magnets, the attached DNA strand moves through the pore — slowly enough so that the base pairs can be read.

The scientists named their process “reverse DNA translocation” because, as Ling explained, “the DNA is essentially caught in a tug-of-war. And the speed of translocation will be controlled not solely by the electric field but by striking some balance between the magnetic and the electric fields. From there, we can tune it to dictate the speed.”

The scientists report their technique reduces the average speed of the DNA strand’s passage by more than 2,000-fold. “It can be slower even. There is no limit,” Ling said.

A similar experiment has been done using optical tweezers, Ling said, but it involves only one DNA strand at a time. The Brown method sends multiple strands through the nanopores simultaneously. “It is scalable,” Ling said.

The researchers expect to test their technique in experiments using bacterial DNA.

The research was funded by the National Human Genome Research Institute and the National Science Foundation's Nanoscale Interdisciplinary Research Teams.


Brain Mechanisms for Behavioral Flexibility
New research provides insight into how the brain can execute different actions in response to the same stimulus

The study, published by Cell Press in the April 16 issue of the journal Neuron, suggests that information from single brain cells cannot be interpreted differently within a short time period, a finding that is important for understanding both normal cognition and psychiatric disorders.

Humans exhibit incredible flexibility when it comes to adjusting to the demands of a particular task. For example, when the word "blue" is written in red ink, separate responses to the color or the meaning of the word can be elicited. "Although the roles played by the frontal cortex in this kind of switching behavior have been well documented, little is known about how neural pathways governing sensory and motor associations accomplish such a switch," explains senior study author, Dr. Takanori Uka from the Juntendo University School of Medicine in Tokyo.

Dr. Uka and coauthor Dr. Ryo Sasaki investigated where and how identical sensory signals are converted into distinct motor signals. The researchers examined the responses of middle temporal (MT) neurons and the associations between MT neurons and downstream functions in monkeys as they switched between direction and depth discrimination tasks. Previous work has shown that the MT area is critical for both direction and depth discrimination.

The monkeys were trained to view dots on a screen and to indicate whether dots moved up or down when they saw the color magenta or whether the dots were nearer or father away when they saw the color cyan. "We found that neuronal sensitivities were nearly identical during both the direction and depth discrimination tasks; that is, neural activity depended on the visual stimulus and not the task itself," says Dr. Uka. This finding suggests that inputs to the MT area were not directly responsible for task switching.

Importantly, the researchers went on to show that signals from different MT populations were read out to perform different tasks. "We suggest that task switching is accomplished via the communication of distinct populations of MT neurons into a downstream decision system," explains Dr. Uka. "We hypothesize that single neurons probably cannot switch outputs in a short period of time, so the brain realizes behavioral flexibility by preparing separate pathways for each task through learning, and then chooses the appropriate pathways, rather than switching outputs, in a given trial."

Gene Therapy for Muscular Dystrophy Shows Promise Beyond Safety
Researchers have cleared a safety hurdle in efforts to develop a gene therapy for a form of muscular dystrophy that disables patients by gradually weakening muscles near the hips and shoulders

Described as the first gene therapy trial in muscular dystrophy demonstrating promising findings, researchers from the University of Florida (UF), Nationwide Children's Hospital in Columbus, Ohio, and The Ohio State University report how they safely transferred a gene to produce a protein necessary for healthy muscle fiber growth into three teenagers with limb-girdle muscular dystrophy.


The findings, which have relevance to genetic disorders beyond muscular dystrophy as well as conditions in which muscles atrophy, were published online today in the Annals of Neurology.

"We think this is an important milestone in establishing the successful use of gene therapy in muscular dystrophy," said Jerry Mendell, MD, director of the Center for Gene Therapy in The Research Institute at Nationwide Children's Hospital and the lead author of the study. "This trial sets the stage for moving forward with treatment for this group of diseases and we are very pleased with these promising initial results. In subsequent steps we plan to deliver the gene through the circulation in hopes of reaching multiple muscles. We also want to extend the trials over longer time periods to be sure of the body's reaction." Mendell is also a professor of Pediatrics and Pathology at The Ohio State University College of Medicine.

Limb-girdle muscular dystrophy actually describes more than 19 disorders that occur because patients have a faulty alpha-sarcoglycan gene. In each of the disorders, the muscle fails to produce a protein essential for muscle fibers to thrive. It can occur in children or adults, and it causes their muscles to get weaker throughout their lifetimes. The trial evaluated the safety of a modified adeno-associated virus - an apparently harmless virus known as AAV that already exists in most people - as a vector to deliver the alpha-SG gene to muscle tissue.

"The safety data is accumulating because this is the same type of vector that we and other research groups have successfully used in gene therapy trials for other diseases," said Barry Byrne, MD, a UF pediatric cardiologist who is a member of the UF Genetics Institute and director of the Powell Gene Therapy Center. "In this effort, although proof of safety was the main endpoint, the added benefit was that this was an effective gene transfer. Even though we were dealing with a small area of muscle, the effect was long-lasting, and that has never been observed before."

Research subjects received a dose of the gene on one side of the body and saline on the opposite side. Neither the researchers nor the patients knew which of the foot muscles received the actual treatment until the end of the experiment.

The volunteers were evaluated at set intervals through 180 days, and therapy effectiveness was measured by assessing alpha-SG protein expression in the muscle, which was four to five times higher than in the muscles that received only the saline. The volunteers encountered no adverse health events, and the transferred genes continued to produce the needed protein for at least six months after treatment.

In addition, scientists actually saw that muscle-fiber size increased in the treated areas, suggesting that it may be possible to combat the so-called "dystrophic process" that causes muscles to waste away during the course of the disease.

Beyond muscular dystrophy, the discovery shows muscle tissue can be an effective avenue to deliver therapeutic genes for a variety of muscle disorders, including some that are resistant to treatment, such as inclusion body myositis, and in conditions where muscle is atrophied, such as in cancer and aging.

"These exciting results demonstrate the feasibility of gene therapy to treat limb-girdle muscular dystrophy," said Jane Larkindale, portfolio director with Muscular Dystrophy Association Venture Philanthropy, a program that moves basic research into treatment development. "The lack of adverse events seen in this trial not only supports gene therapy for this disease, but it also supports such therapies for many other diseases."


New Approach for Treatment of the AIDS Virus?
HIV dearms protective protein in cells

The AIDS-causing HIV specifically counteracts the mechanisms of human cells that protect these against viral infections – a special viral protein marks protective cellular proteins for their rapid destruction and thus diminishes the cell’s supply. A team of researchers in Heidelberg under supervision of virologist Dr. Oliver Keppler demonstrated this mechanism for the first time in cell cultures, thus discovering a target for a novel treatment strategy.

Another important discovery of the Heidelberg virologists – this strategy of the human HIV is not effective in a rat model for AIDS. The protective protein in rats is immune to HIV counteraction. Consequently, HIV cannot propagate itself as easily in the animal model as in humans – one limitation of the current rat model. However, this new knowledge may enable an improvement of the small animal model developed by the Heidelberg researchers. The study was published in the journal Cell Host & Microbe in March 2009.

Newly formed viruses are retained at the cell surface

In addition to the immune system, the body can activate other protective mechanisms to fight or stop virus infections – the infected cells themselves dispose of several proteins that inhibit various steps of virus reproduction. In the presence of the protective protein CD317, newly formed viruses are tethered to the cell surface when they are in the process of leaving the cell and this prevents them from infecting other cells of the body. HIV overcomes this restriction by its protein Vpu by specifically counteracting this protective mechanism, which, interestingly, is effective against many types of viruses.

Dr. Keppler’s team of virologists from the Department of Virology at the Hygiene Institute of Heidelberg University Hospital (Medical Director: Professor Dr. Hans-Georg Kräusslich) studied how Vpu disrupts protection by CD317. They determined that in human cells in which Vpu was formed after infection with HIV, the pool of CD317 was reduced to about one quarter of the original amount. “When Vpu is present, CD317 is rapidly degraded by a cellular system. Vpu presumably binds to the CD317 and marks it for rapid destruction,” explains Dr. Keppler.

The less CD317 is present in the cell, the more viruses can escape interception. “Disrupting this interaction between Vpu and CD317 to increase the cells’ own protective mechanisms could thus be a promising strategy for therapy,” says Dr. Keppler.

Rats and mice also have this protective protein; it has the same function and is able to block HIV. However, there is a significant difference – the Heidelberg virologists discovered that in rat cells, Vpu has no effect on CD317. “HIV is adapted to humans and the disruptive mechanism of Vpu does not affect protection against infection in animals,” said Dr. Christine Goffinet, first author of the study.

Rat model now to be improved

This detail is important when one wants to imitate and study HIV infection in rats in an animal model – the infection in rats does not follow the same course as in humans, since fewer viruses are released due to the intact protective mechanisms. Based on the new research results, the Heidelberg scientists now hope to improve the current transgenic rat model of HIV infection. The goal is to suppress CD317 in rats through genetic engineering and thus achieve a degree of HIV infection that is more similar to that in humans.

As early as 2007, the researchers in Heidelberg first succeeded in making rats susceptible to HIV infection by specifically modifying their genetic material. They successfully tested drugs against HIV infection in humans in these transgenic rats. Using this small animal model, it is possible to test the efficacy of medications against the AIDS virus HIV rapidly and on a larger scale prior to clinical studies in humans and thus to accelerate the further development of virostatics.

Alzheimer's: New Findings Resolve Long Dispute About How the Disease Might Kill Brain Cells
For a decade, Alzheimer's disease researchers have been entrenched in debate about one of the mechanisms believed to be responsible for brain cell death and memory loss in the illness

Now researchers at the University of Michigan and the University of California, San Diego have settled the dispute. Resolving this controversy improves understanding of the disease and could one day lead to better treatments.

Michael Mayer, an assistant professor in the U-M departments of Biomedical Engineering and Chemical Engineering, and Jerry Yang, an assistant professor in the Department of Chemistry and Biochemistry at UCSD, and their colleagues found a flaw in earlier studies supporting one side of the debate. Their findings are published online in the Journal of Neurotoxicity Research. They will appear in the May print edition.

Their results clarify how small proteins called amyloid-beta peptides damage brain cell membranes, allowing extra calcium ions to enter the neurons. An ion is an electrically-charged particle. An ion imbalance in a cell can trigger its suicide.

Amyloid-beta peptides are the prime suspects for causing cell death in Alzheimer's, although other mechanisms could also be to blame. The disease is not well understood.

The researchers confirmed evidence found by others that amyloid-beta peptides prick pores into brain cell membranes, opening channels where calcium ions can rush in. This was one mechanism the field had contemplated, but other evidence suggested a different scenario. Some researchers believed that the peptide caused a general thinning of the cell membranes and these thinned membranes lost their ability to keep calcium ions out of brain cells. Mayer and Yang disproved this latter theory.

"When you understand these mechanisms better, you have a better chance of being able to pharmaceutically counteract them as a possible treatment. For instance, if amyloid-beta thins membranes, this general effect might be difficult to treat. On the other hand, if it forms pores, this effect might be treatable with pore blockers. Ion channel blockers are medications sold today to treat a variety of diseases," Mayer said. He cautions that much research is needed before it is known whether such medications are effective and safe to treat Alzheimer's.

Mayer and Yang were able to explain the other experimental results that blamed cell membrane thinning for uncontrolled calcium ion fluctuations. It turns out that in these studies, trace amounts of residual solvent used to prepare the peptide had a dramatic effect. The Michigan- and UCSD-led team reproduced these experimental results using only the solvent, without the peptide. The solvent is called Hexafluoroisopropanol, or HFIP.

"HFIP is a good solvent used to break up clumps of the peptide to prepare for experiments, but it's toxic and membrane-active. What we found was that the reported preparation procedure did not remove the solvent effectively," Mayer said. "Our findings are watertight since we could reproduce the thinning effect in the absence of amyloid-beta peptides by this solvent alone."

Yang and Mayer carried out these experiments by examining how the electric current fluctuates across artificial membranes and live human cancer cell membranes in the presence of the amyloid-beta peptide. (Cancer cells are often used in biological experiments because they reproduce rapidly.) They also measured the fluctuation of ions in mouse brain cells and in genetically-modified mouse brain cells that produce human amyloid-beta peptide.

In all these trials, the electrodes measuring across the cell membrane registered spikes in electric current consistent with what researchers would expect from the formation of pores in the cell membrane and not from thinning of membranes.

"This ongoing controversy has slowed our own progress in Alzheimer's research as well as progress in other labs," Mayer said. "It is our hope that putting this disagreement to rest by showing that amyloid beta peptides do not thin membranes but instead form discrete pores in membrane can help the field move forward at a more rapid pace."


WEDNESDAY April 15, 2009---------------------------News Archive

Vaccine for E. coli Diarrheal Diseases Created
A Michigan State University researcher has developed a working vaccine for a strain of E. coli that kills 2 million to 3 million children each year in the developing world

Enterotoxigenic E. Coli, which is responsible for 60 percent to 70 percent of all E. coli diarrheal disease, also causes health problems for U.S. troops serving overseas and is responsible for what is commonly called traveler’s diarrhea.

A. Mahdi Saeed, professor of epidemiology and infectious disease in MSU’s colleges of Veterinary Medicine and Human Medicine, has applied for a patent for his discovery and has made contact with pharmaceutical companies for commercial production. Negotiations with several firms are ongoing.

“This strain of E. coli is an international health challenge that has a huge impact on humanity,” said Saeed, who has devoted four years to develop a working vaccine at MSU’s National Food Safety and Toxicology Center. “By creating a vaccine, we can save untold lives. The implications are massive.”

ETEC affects millions of adults and children across the globe, mainly in southern hemisphere countries throughout Africa and South America. It also poses a risk to U.S. troops serving in southern Asia and the Middle East.

Saeed’s breakthrough was discovering a way to overcome the miniscule molecular size of one of the illness-inducing toxins produced by the E. coli bug. Since the toxin was so small, it did not prompt the body’s defense system to develop immunity, allowing the same individual to repeatedly get sick, often with more severe health implications.

Saeed created a biological carrier to attach to the toxin that once introduced into the body induces a strong immune response. This was done by mapping the toxin’s biology and structure during the design of the vaccine. Saeed’s work was funded in part by a $510,000 grant from the National Institutes of Health.

After creating the carrier in a lab at MSU, Saeed and his team tested it on mice and found the biological activity of the toxin was enhanced by more than 40 percent, leading to its recognition by the body’s immune system. After immunizing a group of 10 rabbits, the vaccine led to the production of the highest neutralizing antibody ever reported for this type of the toxin.

Saeed hopes that human clinical trials could begin late in the year.

There also are several other human health implications for the vaccine, besides providing immunity against most E. coli disease, according to Saeed. Many patients who undergo anesthesia during a medical procedure surgery suffer from post-operative paralytic ileus, or an inability to have a bowel movement. A small oral dosage of the vaccine could act as a laxative, which often aren’t prescribed after a surgery for fear of side effects, Saeed said. A small dose also could help with urinary retention.

The vaccine will be available for animals as well, Saeed added. He pointed out the E. coli bug also is a major cause of sickness and death for newborn animals such as calves and piglets, which in the United States alone causes $300 million in loss of agricultural products each year.


Signaling Networks that Set Up Genetic Code
In a new study, researchers at the University of Illinois have identified and visualized the signaling pathways in protein-RNA complexes that help set the genetic code in all organisms. The genetic code allows information stored in DNA to be translated into proteins

The researchers report their findings in a paper accepted for publication in the Proceedings of the National Academy of Sciences, and posted on the journal’s Web site.

“Using molecular dynamics simulations and new visualization software, we can examine and compare different signaling pathways found in living organisms,” said Zaida Luthey-Schulten, a William and Janet Lycan Professor of Chemistry at the U. of I., and the paper’s corresponding author. “Our methodology is applicable to all protein-RNA complexes involved in translation.”

Translation is an essential process in living cells whereby messenger RNA (mRNA) serve as templates in the ribosome to produce specific proteins. How a signal moves through the ribosome during translation has not been understood.

In the first step of translation, an amino acid must be added onto the end of the correct transfer RNA (tRNA) containing the corresponding anticodon according to the genetic code. (The anticodon is a sequence of three nucleotides that can bind to a specific sequence of three nucleotides – the codon – in mRNA.)

From a pool of thousands of amino acids and tRNAs in the cell, each aminoacyl-tRNA synthetase must recognize and pair the correct amino acid and tRNA. After this pairing has occurred, the tRNA is transported to the ribosome where the messenger RNA obtained from the DNA sequence is translated into a protein.

How the signal is transmitted from the anticodon on the tRNA at one end of the protein-tRNA complex to the site of chemical reaction in the aminoacyl-tRNA synthetase at the other end has puzzled scientists for a long time. Now, using network analysis algorithms in combination with molecular dynamics simulations, the U. of I. researchers have been able to identify the network of interactions.

The network algorithms, developed originally by computer scientists to study the communication patterns in the World Wide Web, air travel, and the spread of disease, were adapted to find the optimal and suboptimal communication pathways.

“The signaling pathways in the protein-tRNA complexes are analogous to air travel, where passengers departing from small airports pass through major hubs on their way to distant destinations,” Luthey-Schulten said. “So too in biology, modules of amino acids and nucleotides communicate using paths that pass through a few important interconnecting links.”

To study those links using network analysis algorithms, the researchers represented each amino acid and nucleotide in the protein-RNA complex as a node. Edges connected pairs of nodes, and the network consisted of nodes with connecting edges.

Variations in the connectivity of the network give rise to modules, which can be viewed as local communities in the network, the researches report. Nodes within the same community are highly interconnected and can communicate through a large number of paths with a small difference in distance.

To further analyze the protein-tRNA network, the researchers “weighted” the edges by correlation values derived from molecular dynamics simulations.

“The optimal path is the shortest path, the most correlated path along which the information is transferred,” said graduate student and co-author John Eargle. “There are other paths nearby that are also highly correlated, so the optimal path is not the only path where communication is being taken through the protein.”

Visualization of the communication pathways allowed the researchers to study the redundancy of the pathways and the effects of mutations on the signaling pathways that set the genetic code.

“With our visualization software, we also can overlap the optimal paths from different proteins and compare networks,” Luthey-Schulten said. “The ability to rapidly visualize and compute the networks will accelerate the design efforts to expand the genetic code to include novel amino acids and allow researchers to apply the technique to much larger systems like the ribosome.”

The enhanced visualization software is part of VMD (Visual Molecular Dynamics), a program for visualizing and analyzing molecular dynamics simulations. Developed at the U. of I. and distributed free of charge, VMD is designed to efficiently handle large three-dimensional systems containing more than a million atoms.

A Way to Jumpstart Bone's Healing Process
In a new study, researchers at the University of Illinois have identified and visualized the signaling pathways in protein-RNA complexes that help set the genetic code in all organisms. The genetic code allows information stored in DNA to be translated into proteins

The researchers report their findings in a paper accepted for publication in the Proceedings of the National Academy of Sciences, and posted on the journal’s Web site.

“Using molecular dynamics simulations and new visualization software, we can examine and compare different signaling pathways found in living organisms,” said Zaida Luthey-Schulten, a William and Janet Lycan Professor of Chemistry at the U. of I., and the paper’s corresponding author. “Our methodology is applicable to all protein-RNA complexes involved in translation.”

Translation is an essential process in living cells whereby messenger RNA (mRNA) serve as templates in the ribosome to produce specific proteins. How a signal moves through the ribosome during translation has not been understood.

In the first step of translation, an amino acid must be added onto the end of the correct transfer RNA (tRNA) containing the corresponding anticodon according to the genetic code. (The anticodon is a sequence of three nucleotides that can bind to a specific sequence of three nucleotides – the codon – in mRNA.)

From a pool of thousands of amino acids and tRNAs in the cell, each aminoacyl-tRNA synthetase must recognize and pair the correct amino acid and tRNA. After this pairing has occurred, the tRNA is transported to the ribosome where the messenger RNA obtained from the DNA sequence is translated into a protein.

How the signal is transmitted from the anticodon on the tRNA at one end of the protein-tRNA complex to the site of chemical reaction in the aminoacyl-tRNA synthetase at the other end has puzzled scientists for a long time. Now, using network analysis algorithms in combination with molecular dynamics simulations, the U. of I. researchers have been able to identify the network of interactions.

The network algorithms, developed originally by computer scientists to study the communication patterns in the World Wide Web, air travel, and the spread of disease, were adapted to find the optimal and suboptimal communication pathways.

“The signaling pathways in the protein-tRNA complexes are analogous to air travel, where passengers departing from small airports pass through major hubs on their way to distant destinations,” Luthey-Schulten said. “So too in biology, modules of amino acids and nucleotides communicate using paths that pass through a few important interconnecting links.”

To study those links using network analysis algorithms, the researchers represented each amino acid and nucleotide in the protein-RNA complex as a node. Edges connected pairs of nodes, and the network consisted of nodes with connecting edges.

Variations in the connectivity of the network give rise to modules, which can be viewed as local communities in the network, the researches report. Nodes within the same community are highly interconnected and can communicate through a large number of paths with a small difference in distance.

To further analyze the protein-tRNA network, the researchers “weighted” the edges by correlation values derived from molecular dynamics simulations.

“The optimal path is the shortest path, the most correlated path along which the information is transferred,” said graduate student and co-author John Eargle. “There are other paths nearby that are also highly correlated, so the optimal path is not the only path where communication is being taken through the protein.”

Visualization of the communication pathways allowed the researchers to study the redundancy of the pathways and the effects of mutations on the signaling pathways that set the genetic code.

“With our visualization software, we also can overlap the optimal paths from different proteins and compare networks,” Luthey-Schulten said. “The ability to rapidly visualize and compute the networks will accelerate the design efforts to expand the genetic code to include novel amino acids and allow researchers to apply the technique to much larger systems like the ribosome.”

The enhanced visualization software is part of VMD (Visual Molecular Dynamics), a program for visualizing and analyzing molecular dynamics simulations. Developed at the U. of I. and distributed free of charge, VMD is designed to efficiently handle large three-dimensional systems containing more than a million atoms.


Using PET/CT Imaging, UCLA Researchers Can Tell After Single Treatment if Chemotherapy Working
Findings will allow oncologists to discontinue therapies that aren't effective

Oncologists often have to wait months before they can determine whether a treatment is working. Now, using a non-invasive method, researchers at UCLA's Jonsson Comprehensive Cancer Center have shown that they can determine after a single cycle of chemotherapy whether the toxic drugs are killing the cancer or not.

Using a combination Positron Emission Tomography (PET) and computed tomography (CT) scanner, researchers monitored 50 patients undergoing treatment for high-grade soft tissue sarcomas. The patients were receiving neoadjuvant chemotherapy treatments to shrink their tumors prior to surgery. The study found that response could be determined about a week after the first dose of chemotherapy drugs. Typically, patients are scanned at about three months into chemotherapy to determine whether the treatment is working.

"The question was, how early could we pick up a response? We wanted to see if we could determine response after a single administration of chemotherapy," said Dr. Fritz Eilber, an assistant professor of surgical oncology, director of the Sarcoma Program at UCLA's Jonsson Cancer Center and senior author of the study. "There's no point in giving a patient a treatment that isn't working. These treatments make patients very sick and have long-term serious side effects. "

The study appears in the April 15 issue of the journal Clinical Cancer Research.

PET scanning shows biochemical functions in real time, acting as a sort of molecular camera. For this study, Eilber and his team monitored the tumor's metabolic function, or how much sugar was being consumed by the cancer cells. Because they're growing out of control, cancer cells use much more sugar than do normal cells, making them light up under PET scanning using a glucose uptake probe called FDG. In order to identify an effective response to treatment, researchers needed to see a 35 percent decrease in the tumor's metabolic activity.

Of the 50 patients in the study, 28 did not respond and Eilber and his team knew within a week of their initial treatment. This allows the treatment course to be discontinued or changed to another more effective treatment, getting the patient to surgery more quickly.

"The significance of this study was that it identified people – more than half of those in the study – who were not going to benefit from the treatment early in the course of their therapy," Eilber said. "This information significantly helps guide patient care. Although this study was performed in patients scheduled for surgery, I think these findings will have an even greater impact on patients with inoperable tumors or metastatic disease as you get a much quicker evaluation of treatment effectiveness and can make decisions that will hugely impact quality of life."

Eilber said he was surprised how soon response to therapy could be determined.

"We had an idea that patients either respond or do not respond to treatment, but we weren't sure how early you could see that," he said. "I really was not sure we would be able to see effectiveness this early."

Eilber and his team will continue to follow the patients and a clinical trial currently is underway based on the results of this study. Eilber believes it will help personalize treatment for each patient and may one day become the standard of care.

Researchers also may use the non-invasive imaging method to gauge response to novel and targeted therapies. Eilber said that they are clinically testing new tracers as well. Instead of measuring glucose uptake, these probes look at cell growth. Response to therapy also may be tested using PET in other cancer types, he said.

The nearly two-year study represented a true multidisciplinary effort, Eilber said. Experts from surgery, medical oncology, molecular and medical pharmacology, radiology, pathology, orthopedics, nuclear medicine and biostatistics comprised the research team.

Findings Show Insulin - Not Genes - Linked to Obesity
Findings show insulin - not genes - linked to obesity

Researchers have uncovered new evidence suggesting factors other than genes could cause obesity, finding that genetically identical cells store widely differing amounts of fat depending on subtle variations in how cells process insulin.

Learning the precise mechanism responsible for fat storage in cells could lead to methods for controlling obesity.

"Insights from our study also will be important for understanding the precise roles of insulin in obesity or Type II diabetes, and to the design of effective intervention strategies," said Ji-Xin Cheng, an assistant professor in Purdue University's Weldon School of Biomedical Engineering and Department of Chemistry.

Findings indicate that the faster a cell processes insulin, the more fat it stores.

Other researchers have suggested that certain "fat genes" might be associated with excessive fat storage in cells. However, the Purdue researchers confirmed that these fat genes were expressed, or activated, in all of the cells, yet those cells varied drastically - from nearly zero in some cases to pervasive in others - in how much fat they stored.

The researchers examined a biological process called adipogenesis, using cultures of a cell line called 3T3-L1, which is often used to study fat cells. In adipogenesis, these cells turn into fat.

"This work supports an emerging viewpoint that not all biological information in cells is encoded in the genetic blueprint," said Thuc T. Le, a National Institutes of Health postdoctoral fellow at Purdue who is working with Cheng. "We found that the variability in fat storage is dependent on how 3T3-L1 cells process insulin, a hormone secreted by the pancreas after meals to trigger the uptake of glucose from the blood into the liver, muscle or fat cells."

The findings are detailed in a research paper appearing online in the journal PLoS ONE, published by the Public Library of Science, a non-profit organization of scientists and physicians.

"This varied capability to store fat among genetically identical cells is a well-observed but poorly understood phenomenon," Cheng said

The researchers determined that these differences in fat storage depend not on fat-gene expression but on variations in a cascade of events within an "insulin-signaling pathway." The pathway enables cells to take up glucose from the blood.

"Only one small variation at the beginning of the cascade can lead to a drastic variation in fat storage at the end of the cascade," Cheng said.

The researchers conducted "single cell profiling" using a combination of imaging techniques to precisely compare fat storage in cloned cells having the same fat genes expressed.

Single cell profiling allows researchers to precisely compare the inner workings of individual cells, whereas the conventional analytical approach in biochemistry measures entire populations of cells and then provides data representing an average.

"In this case, we don't want an average. We need to find out what causes fat storage at the single-cell level so that we can compare one cell to another, " Le said. "By profiling multiple events in single cells, we found that variability in fat storage is due to varied rates of insulin processing among cells."

The cell culture used in the research contains cloned mice fibroblast cells.

"This particular type of cell culture has been used to study the molecular control of obesity for the past 35 years," Cheng said. "Researchers have observed tremendous variability in how much fat is stored in cells with identical genes, but no one really knows why. Our findings have shed some light on this phenomenon."

The researchers used a specialized imaging method called coherent anti-Stokes Raman scattering, or CARS, combined with other techniques, including flow cytometry and fluorescence microscopy.

"This multimodal imaging system allows us to correlate different events, like fat storage, gene expression and insulin signaling," Le said. "We can monitor these different events at the same time, and that's why we can determine the mechanism at the single-cell level."

Insulin attaches to binding sites on cell membranes, signaling the cells to take up glucose from the blood. Cells that are said to be resistant to insulin fail to take up glucose, the primary cause of Type II diabetes, a medical condition affecting nearly 24 million Americans. About two-thirds of U.S. adults are overweight, and nearly one-third obese.

The research, which has been funded by the National Institutes of Health, is ongoing. Future work may seek to pinpoint specific events in the insulin-signaling cascade that are responsible for fat storage.


TUESDAY April 14, 2009---------------------------News Archive

How PCBs May Alter Neonatal Brain Development
In three new studies — including one appearing online today in the Public Library of Science - Biology (PLoS - Biology) - UC Davis researchers provide compelling evidence of how low levels of polychlorinated biphenyls (PCBs) alter the way brain cells develop.

The findings could explain at last - some 30 years after the toxic chemicals were banned in the United States - the associations between exposure of the developing nervous system to PCBs and behavioral deficits in children.

"We've never really understood the mechanism by which PCBs produce neurobehavioral problems in children," said Isaac N. Pessah, professor and chair of the Department of Molecular Biosciences at the UC Davis School of Veterinary Medicine, director of the UC Davis Center for Children's Environmental Health and co-author of all three studies. "With these studies we have now shown - from the whole animal level to the molecular level - how PCBs alter the development and excitability of brain cells. And that could explain why PCBs are associated with higher rates of neurodevelopmental and behavioral disorders," said Pessah, who is also a researcher with the UC Davis M.I.N.D. Institute.

Together, the studies - published within one month of each other - make a compelling case for the mechanism behind PCBs' harmful effects on human neurological development. "Not only will this help us deal with current exposures," Pessah said, "but we can also identify similar compounds that have come on line since PCBs were banned, and make better decisions about which ones we restrict and which new ones we allow to come to market."

PCBs have been implicated in epidemiological studies as an environmental cause of diverse neurodevelopmental disorders, including ADHD, learning disabilities, sensory deficits, developmental delays and mental retardation. "There is a large body of scientific literature in humans that points the finger at PCBs, linking them to neurodevelopmental problems we see in kids," said Pamela Lein, lead author of the Environmental Health Perspectives animal study and an associate professor in the Department of Molecular Biosciences at the UC Davis School of Veterinary Medicine. "The problem is that it has been difficult to establish a cause-and-effect relationship from the human epidemiological literature without a known mechanism," Lein said. "Now that we have a plausible biological mechanism that could account for neurodevelopmental deficits, we can use the information for diagnosis and for developing potential treatments for PCB exposure."

The study published in Environmental Health Sciences shows that exposure to PCBs in utero and through mothers' breast milk alters a characteristic of neuronal development called dendritic plasticity in young rats. Dendrites are the small, branch-like projections on a neuron that receive signals from other cells in the body. The shape of dendrites changes in response to signaling activity - a phenomenon known as dendritic plasticity. Lein performed the exposure and behavioral studies with colleagues while a researcher at the Johns Hopkins University.

In the study, researchers tried to mimic the low levels of PCB exposure that human children might experience. Experimental rats were fed PCB-laced cookies, while control rats ate normal cookies. Then, when rat pups were weaned from their mothers, they were trained in a water maze to test their ability to use visual cues to learn the location of a platform hidden under the surface of the water. The test has been used in other studies to stimulate dendritic growth, which makes it ideal for measuring effects of toxicants on dendritic plasticity.

The researchers looked at the pattern of dendritic growth in trained and untrained animals from both the control and experimental groups. They found that PCB exposure accelerated dendritic growth in the untrained experimental animals when compared to untrained controls. The trained PCB-exposed animals, however, took longer to learn the water maze and showed reversal of dendritic growth in response to water-maze training. This was in contrast to controls, which showed learning responses and increases in dendritic growth, as predicted by other published studies. "This tells us that PCBs are altering dendritic growth and plasticity," Lein said.

The results are important because problems in dendritic growth and plasticity have previously been implicated in many neurodevelopmental disorders, including autism, schizophrenia and mental retardation, she said. "Dendritic plasticity is important to how we process information and, when you perturb that, you interfere with complex behaviors like learning and memory," Lein said.

Pessah and his colleagues showed that brain tissue from untrained rats exposed to PCBs expressed higher levels of the ryanodine receptors. "We think PCBs are increasing the activity of these calcium channels, which we know generate the signals needed for the extension and branching of dendrites," Pessah said. He said he believes PCBs lead to overgrowth of dendrites and inhibition of neuronal pruning that takes place during gestational development. Brain cells exposed to PCBs cannot then respond properly to learning.

In the study appearing in Toxicology and Applied Pharmacology, Pessah and his colleagues examined the hippocampus, one region of the brain involved in water-maze learning. The researchers measured the excitability of neurons in hippocampal brain tissue of rats before and during exposure to two structurally different PCBs. "Neurons process and transmit information in the form of electrical signals. Their electrical excitability is due to the presence of voltage-sensitive ion channels that directly communicate with ryanodine receptors that reside inside the cell. When excitation is blocked, it is called inhibition. Normal information processing involves a complex balance between excitation and inhibition.

The researchers found that the two PCBs had different effects. The more potent, PCB95, enhanced the excitability of the brain cells. Researchers were able to decrease this effect by adding a chemical that dampens ryanodine signaling, again implicating the calcium channel as being the key to the disruptions caused by PCBs. The second compound, PCB170, first excited the circuitry, but then the signals returned to baseline because of enhanced inhibition. "These results are significant to the understanding of the potential impact of PCBs on human neurodevelopment, Pessah said. "We think that in autism, for example, at-risk children have deficient inhibitory circuits. So, if you have a PCB that promotes the excitatory side of the circuit, they would be much more at risk of developing the disorder," he said. "In fact, we chemically blocked inhibitory circuits that unmasked the purely excitotoxic properties of PCB170."

In the collaborative study between researchers at Davis and Harvard that appears in PLoS-Biology today, researchers showed that PCBs dramatically stabilize the ryanodine receptor in the "on" position, which could explain how PCBs alter brain cell development (as seen in the first study) and their excitability (as seen in the second). "We needed evidence that these compounds directly interact with what we believed to be the target of PCBs," Pessah said. To that end, the researchers exposed purified ryanodine receptors to PCBs and used electron microscopy to generate extremely high-resolution images of this interaction. "Our results show that PCB binds directly to ryanodine receptors and locks the channel in the open state, causing mayhem in calcium signaling," Pessah said. This, he added, would account for the effects seen in the first two studies.

"These channels are a target for PCBs, and they are contributing to brain cell dysfunction, even at the behavioral level." Pessah said that, as early as 1995, he and his colleagues suspected ryanodine receptors were one of the principal targets of PCBs. "In cellular studies, we couldn't find a way to block the effects of PCBs unless we blocked ryanodine receptors," he said.

Many studies used high doses of PCBs to find subtle or no changes from control. However, in the animal study, Lein used both high and low doses. She found that the low-dose group showed more pronounced effects on dendritic growth in the weanling rats than the higher dose.

According to Pessah, the brain has ways of dealing with high levels of toxicity. "We think that one major reason we have not seen effects in previous studies is that at higher doses PCBs become toxic to cells and the brain has defense mechanisms to deal with disposing of these damaged cells," he said. "These processes, like programmed cell death, would not necessarily be triggered if a neuron's shape is altered rather than damaged, he added. Both Lein and Pessah agreed that the current PCB studies have broader implications for the future study and regulation of PCBs and other environmental toxicants. "Future studies of PCBs and related compounds should be examined at lower doses more relevant to human exposures," Pessah said.

The researchers are planning to study PCB effects on mice that carry some of the same genetic variations of the ryanodine receptors that humans do. "These studies are important if we are to determine if some people are more susceptible to PCB toxicity than others," Lein said.

The team also will look at PCBs' effects on other areas of the brain that control behavior as well as testing compounds with structures similar to those of PCBs. We believe other PCB-like compounds in use today are also capable of changing the structure of protein targets that are contributing to neurobiological problems in humans," Pessah said, "and we hope to identify those and help get them off the market."

Creating Ideal Neural Cells for Clinical Use
New Protocol Quickly and Efficiently Differentiates Human Embryonic Stem Cells into Committed Neural Precursor Cells

Investigators at the Burnham Institute for Medical Research (Burnham) have developed a protocol to rapidly differentiate human embryonic stem cells (hESCs) into neural progenitor cells that may be ideal for transplantation. The research, conducted by Alexei Terskikh, Ph.D., and colleagues, outlines a method to create these committed neural precursor cells (C-NPCs) that is replicable, does not produce mutations in the cells and could be useful for clinical applications. The research was published on March 13 in the journal Cell Death and Differentiation.

When the C-NPCs created using the Terskikh protocol were transplanted into mice, they became active neurons and integrated into the cortex and olfactory bulb. The transplanted cells did not generate tumor outgrowth.

“The uniform conversion of embryonic stem cells into neural progenitors is the first step in the development of cell-based therapies for neurodegenerative disorders or spinal injuries,” said Dr. Terskikh. “Many of the methods used to generate neural precursor cells for research in the lab would never work in therapeutic applications. This protocol is very well suited for clinical application because it is robust, controllable and reproducible.”

Dr. Terskikh notes that the extensive passaging (moving cells from plate to plate) required by some protocols to expand the numbers of neural precursor cells limits the plasticity of the cells, can introduce mutations and may lead to the expression of oncogenes. The Terskikh protocol avoids this by using efficient conversion of hESCs into primary neuroepithelial cells without the extensive passaging.

The scientists were able to rapidly neuralize the hESCs by culturing them in small clusters in a liquid suspension. The cells developed the characteristic “rosettes” seen in neuroepithelial cells. The C-NPCs were then cultured in monolayers. Immunochemical and RT-PCR analysis of the cells demonstrated that they were uniformly C-NPCs. Whole-genome analysis confirmed this finding. Immunostaining and imaging showed that the cells could be differentiated into three distinct types of neural cells. The team then demonstrated that the C-NPCs differentiated into neurons after transplantation into the brains of neonatal mice.

This research received funding from the National Institutes of Health and the California Institute for Regenerative Medicine.


DNA Sensors that Identify Cancer Material Only One Atom Thick
Kansas State University engineers think the possibilities are deep for a very thin material

Vikas Berry, assistant professor of chemical engineering, is leading research combining biological materials with graphene, a recently developed carbon material that is only a single atom thick.

"The biological interfacing of graphene is taking this material to the next level," Berry said. "Discovered only four years ago, this material has already shown a large number of capabilities. K-Staters are the first to do bio-integrated research with graphene."

To study graphene, researchers rely on an atomic force microscope to help them observe and manipulate these single atom thick carbon sheets.

"It's a fascinating material to work with," Berry said. "The most significant feature of graphene is that the electrons can travel without interruptions at speeds close to that of light at room temperature. Usually you have to go near zero Kelvin -- that's about 450 degrees below zero Fahrenheit -- to get electrons to move at ultra high speeds."

One of Berry's developments is a graphene-based DNA sensor. When electrons flow on the graphene, they change speed if they encounter DNA. The researchers notice this change by measuring the electrical conductivity. The work was published in Nano-Letters.

"Most DNA sensors are optical, but this one is electrical," Berry said. "We are currently collaborating with researchers from Harvard Medical School to sense cancer cells in blood."

Another area he is exploring is loading graphene with antibodies and flowing bacteria across the surface.

"Most researchers focus on pristine graphene, but we're making it dirty," he said.

Berry and Nihar Mohanty, a graduate student in chemical engineering, used a type of bacteria commonly found in rice and interfaced it with graphene. They found that the graphene with tethered antibodies will wrap itself around an individual bacterium, which remains alive for 12 hours.

Berry said that possible applications include a high-efficiency bacteria-operated battery, where by using geobater, a type of bacteria known to produce electrons, can be wrapped with graphene to produce electricity. The research was presented at the annual American Physical Society conference in Pittsburgh and the American Institute for Chemical Engineers conference in Philadelphia.

"Materials science is an incredible field with several exploitable quantum effects occurring at molecular scale, and biology is a remarkable field with a variety of specific biochemical mechanisms," Berry said. "But for the most part the two fields are isolated. If you join these two fields, the possibilities are going to be immense. For example, one can think of a bacterium as a machine with molecular scale components and one can exploit the functioning of those components in a material device."

For his doctoral research, Berry used bacteria to make a humidity sensor.

"That was only possible through combining materials science with biological science," he said.

Another area of his current research is compressing and stretching molecular-junctions between nanoparticles. Berry said that his group has developed a molecular-spring device where they can compress and stretch molecules, which then act like springs, allowing researchers to study how they relax back. He said that this technology could be used to create molecular-timers in which the spring action from a decompressed molecule on a chip could trigger a circuit, for instance.

Berry said for stretching the molecules, Kabeer Jasuja, a doctoral student in chemical engineering, came up with the idea to place the device on a centrifuge to stretch the molecules with centrifugal force.

The work was published in the journal Small.

The New 'Epigenetics:' Poor Nutrition in the Womb Causes Permanent Genetic Changes in the Baby
The new science of epigenetics explains how genes can be modified by the environment, and a prime result of epigenetic inquiry has just been published online in The FASEB Journal (http://www.fasebj.org): You are what your mother did not eat during pregnancy

In the research report, scientists from the University of Utah show that rat fetuses receiving poor nutrition in the womb become genetically primed to be born into an environment lacking proper nutrition. As a result of this genetic adaptation, the rats were likely to grow to smaller sizes than their normal counterparts.

At the same time, they were also at higher risk for a host of health problems throughout their lives, such as diabetes, growth retardation, cardiovascular disease, obesity, and neurodevelopmental delays, among others. Although the study involved rats, the genes and cellular mechanisms involved are the same as those in humans.

“Our study emphasizes that maternal–fetal health influences multiple healthcare issues across generations,” said Robert Lane, professor of pediatric neonatology at the University of Utah, and one of the senior researchers involved in the study. “To reduce adult diseases such as diabetes, obesity, and cardiovascular disease, we need to understand how the maternal–fetal environment influences the health of offspring.”

The scientists made this discovery through experiments involving two groups of rats. The first group was normal. The second group had the delivery of nutrients from their mothers' placentas restricted in a way that is equivalent to preeclampsia.

The rats were examined right after birth and again at 21 days (21 days is essentially a preadolescent rat) to measure the amount of a protein, called IGF-1, that promotes normal development and growth in rats and humans. They found that the lack of nutrients caused the gene responsible for IGF-1 to significantly reduce the amount of IGF-1 produced in the body before and after birth.

“The new ‘epigenetics’ has taught us how nature is changed by nurture,” said Gerald Weissmann, M.D., Editor-in-Chief of The FASEB Journal. “The jury's in and, yes, expectant moms really are eating for two. This study shows not only that we need to address problems such as preeclampsia during pregnancy, but also that prenatal care is far more important than anyone could have imagined a decade ago.”


MONDAY April 13, 2009---------------------------News Archive

Key Point When Cells Either Repair Broken DNA -or Die
When cells undergo potentially catastrophic damage, for example as a result of exposure to ionizing radiation, they must make a decision: either to fix the damage or program themselves for death, a process called apoptosis

A team of biologists led by Professor Nicholas Tonks, Ph.D., F.R.S., of Cold Spring Harbor Laboratory (CSHL), this week revealed results of experiments suggesting at least one way in which cascades of intracellular signals are regulated at what they call a decision point, where cells decide whether to repair broken DNA strands or commit suicide following DNA damage.

In a report published online ahead of print in the Journal of Biological Chemistry, Tonks and colleagues identify a protein with the unlikely name Eyes Absent, or EYA, as performing a critical role in setting the damage-repair machinery in motion. Engaged within the larger context of a complex signaling cascade within the cell, EYA regulates the formation of specialized microenvironments on DNA, called gamma-H2A.X foci, which allow the cell to summon repair enzymes to the site of broken DNA strands. The team’s experiments, conducted by Navasona Krishnan, Ph.D., of the Tonks lab, showed that when Eyes Absent was not present in damaged cells grown in culture, such foci were not formed and the cells went the route of apoptosis—they programmed themselves to die.

‘A lovely moment’ of discovery
Tonks says the finding is “a powerful example of multiple lines of research and different kinds of expertise coming together” in what he describes as “a lovely moment” of synthesis and discovery. An important collaborator in the work, C. David Allis, Ph.D., of the Rockefeller University, had recently published a paper in Nature describing a critical component of the DNA damage-repair signaling cascade. The Tonks-Allis collaboration, along with contributions from Seung Jun Kim, Ph.D., a protein crystallographer from the Korea Research Institute of Bioscience and Biotechnology, led to the assembly of a puzzle from pieces whose precise relation was not previously understood.

The parts of the puzzle include a protein called H2A.X, one of a species of proteins called histones that form structures around which DNA is “spooled” for dense packing in the cell nucleus. “David Allis showed us that this particular histone, which is found at those critical decision points called gamma-H2A.X foci, has phosphate groups added to its structure at particular points at the end of the protein. David demonstrated that another kind of protein - a kinase called WSTF - attaches phosphate to a critical site, a tyrosine residue, at the extreme end of the protein,” Tonks explains.

“I was interested in the fact that when there is damage to double-stranded DNA – catastrophic damage, such as when the strand breaks in two – the phosphate molecule placed at the decision point by WSTF has to be removed in order for the cell to send out signals for DNA-repair enzymes to come to the scene.”

The removal of phosphate groups from proteins is accomplished by a family of enzymes called phosphatases – the focus of much research in Tonks’ CSHL laboratory. Tonks is well known for having characterized the first of what has come to be understood as a large superfamily of protein tyrosine phosphatases, or PTPs – enzymes that specifically remove phosphate molecules from amino acid residues called tyrosines. This function is critical in regulating cellular signaling in normal and disease conditions. In effect, important kinds of cellular signals can’t be sent without helper enzymes like PTPs that remove phosphate molecules from specific locations, and kinase enzymes that perform the reverse role – that of adding phosphates.

Another vital piece of the puzzle in the current work involves Dr. Kim, who recently spent 15 months in the Tonks lab at CSHL, working on growing crystals of protein phosphatases to determine their structure, including that oddly named protein, Eyes Absent. Why that one? As Tonks explains, “EYA is known from work on the fruit fly, or Drosophila, to be a very unusual protein: it’s the only one we know of that acts in the cell nucleus to regulate genes – what we call a transcription factor – while, in other contexts, is also known to act as a phosphatase – that is, it can remove phosphate groups from other proteins.”

The role of Eyes Absent in DNA damage repair
EYA was shown to be a phosphatase by three separate groups in 2003. Although it was thought to be a PTP - i.e., it was thought to take phosphate groups off tyrosine residues in proteins - the identity of its target proteins in the cell was unknown. Kim's structure was important because it revealed a particular distribution of charged residues on the surface of the protein that suggested to Tonks the possibility that basic proteins, such as histone H2A.X, may be one such critical substrate.

This led to the experiments in which Tonks’ team showed that Eyes Absent was in fact the protein that removed the critical phosphate group from the end of histone H2A.X, thereby allowing the formation of the so-called gamma-H2A.X foci, which set DNA repair in motion when double strands were broken. When EYA was experimentally “knocked out” via a technique called RNA interference, or RNAi, damaged cells with double-stranded DNA breaks did not repair themselves; instead, they simply died -- underwent apoptosis.

As is often the case in science, multiple labs are engaged on related subjects. Results similar to those reported by Tonks and colleagues recently have been obtained in Geoff Rosenfeld's lab at the University of California, San Diego. But there is more work to be done on the subject. It remains unclear if or how the role of Eyes Absent in the DNA repair machinery - in other words, its role as a tyrosine phosphatase, or remover of phosphate molecules - is related to its role, in other contexts, as a transcription factor (a regulator of gene expression). It is certainly a curiosity that a protein that can regulate genes in certain contexts can act in others as the fulcrum in a mechanism that repairs damaged genes. Tonks and colleagues expect to explore this in future work.

“Dephosphorylation of the C-terminal tyrosyl residue of the DNA damage-related histone H2A.X is mediated by the protein phosphatase Eyes Absent (EYA)” appeared online ahead of print April 7 in the Journal of Biological Chemistry (http://www.jbc.org/cgi/doi/10.1074/jbc.C900032200). The full citation is: Navasona Krishnan, Dae Gwin Jeong, Suk-kyeong Jung, Seong Eon Ryu, Andrew Xiao, C. David Allis, Seung Jun Kim, and Nicholas K. Tonks.

Genetics Alone Poor Indicator for Drug Response in Cancer
In certain respects, cells are less like machines and more like people

True, they have lots of components, but they also have lots of personality. For example, when specific groups of people are studied in aggregate (conservatives, liberals, atheists, evangelicals), they appear to be fairly uniform and predictable. But when looked at one person at a time, individuals often break the preconceptions.

Same with cells.

Researchers tend to identify characteristics of particular cells by looking at millions at a time. As a result, they'll find that, say, "group A" responds very well to a particular cancer treatment, whereas "group B" does not. They will then often compare group A to group B to find out why.

But often ignored is that not every cell in either group behaves in ways that the aggregate indicates. In a group of cells shown to be vulnerable to a particular cancer treatment, perhaps 10 percent resist it while 90 percent succumb. While researchers have offered various explanations for this, few have studied it.

Now a group of scientists in the lab of Harvard Medical School Professor of Systems Biology Peter Sorger have studied such "outlier" cells in the context of a new and highly touted cancer drug. They have found that vastly disparate reactions occur within genetically homogeneous cell groups. These discrepancies result from protein levels that vary from cell to cell, even among cells that are identical genetic twins. What's more, these protein levels and their subsequent traits can be passed down to daughter cells—a heritability that has nothing to do with genetics.

"Genetics are permanently heritable, while these protein levels are temporarily heritable," says Sorger. "But this temporary inheritance can make all the difference in the world when it comes to the effectiveness of certain medications."

These findings are published April 12 online in Nature.

In order to investigate this disparate behavior among cells, graduate student Sabrina Spencer and postdoctoral researcher Suzanne Gaudet, both in Sorger's lab, looked at a molecule called TRAIL, a protein that causes cells to, literally, commit suicide—a process scientists call apoptosis. While TRAIL is a natural cell product, drug makers have been investigating ways to harness its power so that it can directly target cancer cells.

While TRAIL continues to be a promising drug candidate, its success rate isn't 100 percent, and the researchers wanted to figure out why.

The researchers took both cancerous and non-cancerous cells and exposed them to varying doses of TRAIL. Although these cell lines were known to be vulnerable to the molecule, a fraction always managed to survive.

The researchers noticed that when this outlier group was isolated and once again exposed to TRAIL, the cells and their immediate progeny continued to remain highly resistant for a short time. An immediate explanation might be that this group had developed some sort of genetic defense. However, when this new "resistant" group was given several days to reproduce, the pattern soon reset to the original: 90 percent died, ten percent survived.

"We knew that there were clearly factors at work here that were not genetic," says Spencer. "Genetic resistance would remain uniform in subsequent generations. But the factors at work here were clearly more dynamic."

Using a variety of imaging techniques, the researchers soon discovered that even though these cells were genetically identical —the same cell in the same tissue doing the same thing, the actual numbers of proteins in each cell varied. Specifically, proteins involved in the cell-suicide mechanism triggered by TRAIL were affected. These protein levels altered the dynamics of the entire mechanism, sometimes making cells, for all intents and purposes, immune to TRAIL. While these protein levels were initially passed on to progeny, the heritability was transient. The scientists describe it as an extra layer of inheritance, one that is superimposed onto genetic inheritance.

As for what actually causes these protein levels to vary between identical cells, the researchers cited a simple explanation: It's completely random.

"For decades biologists have had this notion that cells produce proteins in orderly, uniform ways, like an assembly line, but they don't," says Sorger. "Rather, cells produce proteins in fits and starts, and the timing and degree varies from one cell to the next—even cells that are identical in every way. This randomness is something that we're just beginning to appreciate."

These findings also offer an alternative to the cancer stem-cell hypothesis. For that, scientists have posited that certain cancers survive standard treatments because a population of tumor-specific stem cells evades chemotherapy or radiation. This paper, however, offers an alternative explanation, namely, that purely through chance, certain cells produce quantities of proteins that fundamentally alter the cell's response to treatment.

Ultimately, Sorger and his group think that this new insight will make it possible to design anti-cancer treatments that are more effective than those available today.

Defining Neurons that Control Sociability in Worms
Ants colonize. Fish shoal. Flamingos flock and caribou herd. Earth is populated by inherently social beings. Even lowly worms seek out the benefits of companionship. New research at The Rockefeller University has dissected the social proclivities of a model worm, identifying a single type of neuron — RMG — that “decides” whether these worms will mingle with their fellows or keep to themselves.

“We can think of RMG neurons as the world’s simplest social brain, as the place where information relevant to the worm’s decision to hang out with other worms converges, and the decision is made,” says Cori Bargmann, head of the Laboratory of Neural Circuits and Behavior at Rockefeller, who led the research. The work was published this week in Nature.

Even the world’s simplest social brain, one pair of only 302 neurons in Caenorhabditis elegans’s compact nervous system, is rather complicated, it turns out. A host of genetic and environmental variables contribute to the decision-making. RMG is the central integrating hub of a network of sensory neurons that feeds the worm readings of its environment such as whether food is available, how much oxygen is in the air and

Social surgery. By using a laser to kill the RMG neurons in social strains of C. elegans, researchers showed that worms lacking this “social brain” failed to congregate (right) compared to those with it (left).

other factors known to influence gregarious behavior. Only when the right conditions are met will the animals congregate. Evan Macosko, an M.D.-Ph.D. student in the Bargmann lab, found that the crucial trigger for aggregation is a switch in the response to other worms’ pheromones. RMG and a pheromone-detecting neuron named ASK are the essential players in the “hub-and-spoke” circuit that drives social behavior.

Pheromones are known to bring creatures together in many species, from insects such as ants and moths to mammals including prairie voles and possibly even people. In social strains of C. elegans, a transparent, one-millimeter-long roundworm, the same is true. Bargmann determined that ASK, known to be involved in the attraction of the relatively rare males to the more common hermaphrodites, senses pheromones. ASK is relatively inactive in solitary hermaphrodites, which ignore or avoid pheromones. However, in social worms, RMG amplifies the signal from pheromone-sensitive ASK neurons, driving the worms toward each other and increasing sociability.

The hub-and-spoke circuit, Bargmann says, is a relatively rare but recurring theme in the map of C. elegans’s nervous system known as its wiring diagram. The neurons connected in such circuits primarily communicate electrically across gap junctions rather than through the more common connections of chemical synapses. Bargmann believes that hub-and-spoke circuits could be the integrative sites that coordinate different characteristic behaviors in worms and other species.

RMG is the hub of the worm’s social brain, and also a hub of genetic differences in social behavior. The activity of one receptor gene in the worm’s brain determines whether hermaphrodite worms will be relatively social, congregating at a moment’s notice, or relatively solitary. The receptor gene, npr-1, sets the activity level of RMG neurons, so genes and environment act on the same target when modifying social behavior.

“The decision to congregate gets made in certain environments and not in others and in certain genotypes and not in others,” says Bargmann, who is also a Howard Hughes Medical Institute investigator. “Aggregation is a true regulated behavior that’s an option, not a requirement. Behavior is all about choosing between your options based on your genes, your experience and your current situation.”

Human, Mouse Share Hearing Deficit Due to MicroRNA Mutation
In parallel studies in human and mouse, two groups of researchers have come to the same conclusion: that a new kind of gene is associated with progressive hearing loss. The new gene - called a microRNA - is a tiny fragment of RNA that affects the production of hundreds of other molecules within sensory hair cells of the inner ear

The research provides important new genetic understanding of a condition that is common in humans but remains poorly understood. One team, led by researchers from the Hospital Ramón y Cajal, Madrid, Spain, followed families who showed hearing loss. The second team, led by researchers from the Wellcome Trust Sanger Institute, Cambridge, UK, examined a new line of mice, called diminuendo, that showed progressive hearing loss from an early age. The two groups shared their emerging data.

"We were able quite quickly to show that if the mice carried one copy of the gene variant they suffered progressive hearing loss, if they carried two variants they were profoundly deaf," explains Professor Karen Steel, principal investigator of the programme at the Wellcome Trust Sanger Institute. "The important questions were could we determine what the variant is and how does it exert its effect on hearing?"

In their studies of families with progressive hearing loss, the Spanish team had proposed that the gene responsible lay on human chromosome 7. Both teams set about sequencing every gene in the equivalent genomic regions in human and mouse identified as implicated in hearing loss; the sequencing showed that most of the genes in the region could not be responsible for hearing loss.

However, they each found that a mutation in a microRNA gene called miR-96 was associated with the hearing loss.
"We know of a number of genes involved in deafness in humans and mice but, to our great surprise, this was one of a new class of genes called microRNAs," explains Professor Miguel Angel Moreno-Pelayo, senior author on the human study, from Hospital Ramón y Cajal and Centro de Investigación Biomédica en Red de Enfermedades Raras, Madrid, Spain.

MicroRNAs are tiny snippets of genetic information that, it is increasingly clear, can have dramatic consequences. Only five years ago, their role in human biology and disease was unknown. Today, it is becoming clear that these little molecules can control the activity of many genes. MicroRNAs can bind to the messengers involved in protein production in cells, disrupting this process.

"No one has seen a disease-causing mutation in the mature sequence of a microRNA," says Dr Moreno-Pelayo. "This is the first microRNA gene associated with hearing impairment and, remarkably, it is the first to be associated with an inherited disorder."

In the mouse, the precise role of the mutation can be examined. Mutation of the miR-96 gene seemed to disrupt development of intricate sensory hair cells in the mutant mice. Mice with two copies of the mutant gene had malformed hair cells from birth and the cells degenerated from an early age. In mice with one copy of the mutant gene, the effects were less severe, but became worse with age.

"The mutation - a change of a single letter of genetic code from A to T - in this tiny stretch of sequence is enough to lead to dramatic loss of hearing in these mice," explains Dr Morag Lewis, a Sanger Institute scientist, who found this mutation. "We wondered if this single change was preventing the miR-96 from binding to the sites it would normally target to influence gene activity, and looked at ways to determine if this was the case."

The scientists looked at many thousands of messengers to determine which of them seemed altered in the mice with the miR-96 mutation. Significantly, a handful of these seemed to play vital roles in the working of the ear.
"We had gone from one amazing result - that this variant microRNA was causing these dramatic effects - to another - that miR-96 does affect genes important for normal hearing and a clear path was laid."

"Finding that these targets are affected by the mutation in miR-96 was a real landmark in our studies," says Karen Steel. "Any one of the strongest candidates could have explained the hearing loss effects on its own. It was a really remarkable result."

In the human studies, two families showed mutations in miR-96 - but they each carried the mutation at different locations in the miR-96 gene. Intriguingly, neither mutation in humans is the same letter as in the mouse, but all three are close to one another in the miR-96 sequence.
"The mutation in the second family is just one letter away from the mutation in the first and just one away from the mutation in the mouse gene. All three sit in a vital region of seven letters in the mature sequence of miR96" says Dr Angeles Mencía, the Spanish team member who found the human mutations.

Remarkably, then, cases of deafness in two different organisms are both tied to equivalent microRNAs and to the equivalent region within the microRNA - just seven letters that are known to be important for interacting with the messenger targets.
"The human variants of miR-96 identified in the affected families were found to alter activity of other genes in experiments in test tubes," explains Dr Moreno-Pelayo.

The team also looked to see whether the mutations altered the production or stability of miR-96 and to see whether they affected the normal workings of miR-96. Both the genesis and function of miR-96 were impaired by these human mutations in the lab studies.

Researchers are using models of hearing in the mouse to understand human hearing deficits: by the age of ten, one in 500 children has suffered significant hearing impairment and the majority of over-70s are affected. The same genes have often been shown to be involved in deafness in both the mouse and humans.

The research was part funded by the UK's medical charity for deaf people, Deafness Research UK. The charity's Chief Executive, Vivienne Michael says "Hearing is an exquisitely complex process, involving the intricate interaction of genes and our environment. These exciting studies have opened new avenues to explore to understand better the processes that lead to deafness, with the hope that we will develop new tests and new treatments."

The research team expect that understanding the mechanism by which miR-96 leads to progressive hearing loss will give us clues to help develop therapies to ameliorate the effects of progressive deafness, whatever the trigger.

New Common Pathway in Neurodegenerative Disease Possible Door to "Point of No Return"
A just-out study suggests that what keeps chronic nervous system diseases such as Alzheimer’s, Huntington’s and ALS going — until they overcome the internal protective mechanisms a body can throw at them — may largely come down to poor conversational skills.

In the current issue of the journal Neuron, a team of Johns Hopkins scientists reports uncovering a much-sought molecular path that nerve cells (neurons) use to communicate with their neighboring cells, the astrocytes.

The team also shows how failure of this system could leave the brain and spinal cord vulnerable in disease.

Astrocytes are the most plentiful central nervous system cells. And while scientists have known for some time that they’re critical for neurons’ normal activity and even for their survival, precisely how the two cell types communicate hasn’t been clear.

“This new work shows that neurons dynamically direct astroglia,” says team leader Jeffrey Rothstein, M.D., Ph.D., “but more important to medicine, it defines how neurological disease may spread throughout the nervous system.” Rothstein directs The Robert Packard Center for ALS Research at Johns Hopkins.

Most exciting, Rothstein says, “is that any number of neurodegenerative diseases appear to hold this downhill process in common, once the disease has started.” And it apparently begins early in disease. “Even when neurons look OK,” says Rothstein, “the conversation between neurons and astrocytes has fallen off.

“Although many other processes go wrong in the diseases, this common mechanism appears key to keeping the disease going, to create further injury,” Rothstein adds.

The focus of the study is on the plentiful neurons that communicate with each other through the neurotransmitter glutamate. While glutamate is a necessary excitatory substance in the nervous system, in excess, it overstimulates and becomes toxic — excitotoxic — to neurons. Fortunately, neighboring astrocytes can mop up the excess via molecular transporters embedded in their outer membranes. The chief transporter is a protein called EAAT2.

Earlier Rothstein’s group showed that astroglia — and their EAAT2 protein — are critical for normal neuron activity. In test rats whose astroglia lack the EAAT2 equivalent there’s not only a flood of toxic glutamate but a resulting neuron death that leads to paralysis.

Post-mortem studies of patients with ALS and animal models of that disease frequently reveal a severe loss of EAAT2.

What the new study shows is that neurons themselves direct the creation of EAAT2 in nearby astrocytes.

Here, the scientists devised a microscopic platform containing two tiny chambers: One held neurons, another astrocytes. In this system, some neurons could send out their long, thin axon processes through microscopic channels that ended in astrocytes. Where axons reached close to astrocytes or touched them — and only there — the astrocytes quickly turned on their genes for the EAAT2 glutamate transporters, the very protein that could protect them from glutamate excess.

A second elegant but more intricate part of the work revealed that as neurons sidle up to astrocytes, they very specifically stimulate a tiny part of the astrocyte gene that turns on EAAT2. This stimulating molecule, called KBBP, highly regulates the right astrocyte genes that ultimately can keep neurons operating.

In the study, astrocytes whose KBBP was high bloomed with transporters. This didn’t occur if neurons in the chamber were poisoned. It also didn’t occur if production of KBBP was blocked.

The researchers next wanted to see if the pathway they’d uncovered was important in real injuries to the spinal cord or brain. They showed, in rodent models, that injuring the spinal cord neurons that control movement, whether by trauma (like spinal cord injury) or poison, plays havoc with nearby astrocytes. When astrocytes lose the connection with neurons, KBBP drops, they don’t make transporters, there’s a flood of glutamate and they themselves begin to sicken.

This accelerates the ongoing injury to neighboring neurons.

And last, animal models of familial ALS proved the principle of neuron-directs-astrocyte-to-mop-up-glutamate. The models carry a gene that causes the disease, and as the neurons deterioriate, the astrocytes follow. “The loss of the glutamate transporter in these animal models follows the path of neuron injury; it spreads through the spinal cord,” says Rothstein.

“Understanding this biology gives us new clues to the ways a neuron’s “neighborhood” forces disease to accelerate,” says Rothstein. “Fortunately, it also gives us ideas for roadblocks to slow the process down.”

This study was supported by The Robert Packard Center for ALS Research, the National Institutes of Health and the Muscular Dystrophy Association.


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