Dr. Richard Flavell (Yale University) and colleagues identify the c-Cbl protein as a critical repressor of hematopoietic stem cell (HSC) self-renewal in the April 15th issue of G&D,.  In addition to establishing a key role for protein ubiquitylation in HSC development, this finding posits c-Cbl as a potential target in research into stem cell engineering as well as cell-based leukemia treatments.

Dr. Flavell describes the work as elucidating “a novel dimension in our understanding the self-renewal of Hematopoietic stem cells.”

Like all stem cell populations, HSC reply upon asymmetric cell division to generate two different daughter cells: one future stem cell, and another cell that will further differentiate into a more specialized cell type.  Thus, a balance is struck between the production of new cell types and the renewal of the stem cell pool.  However, imbalances between HSC self-renewal and differentiation can lead to hematologic malignancies like leukemia.

Dr. Flavell’s group discovered that the E3 ubiquitin ligase, c-Cbl, suppresses HSC self-renewal.  The researchers generated transgenic mice deficient in c-Cbl, and demonstrated that these c-Cbl-mutant mice display an increased number of HSCs.

Lead author, Dr. Chozhavendan Rathinam, is confident that “our findings may facilitate the expansion and manipulation of hematopoietic stem cells for tissue engineering and stem cell based therapies.”

Migraine is the most common cause of episodic headache, and by far the most common neurological cause of a doctor’s visit.  It affects some 15% of the population, including some 41 million people in Europe, and places a considerable burden on healthcare in both the developed and the developing world.

During the last few years, great strides have been made in discovering common genes influencing the susceptibility to common diseases, such as diabetes, Crohn’s disease and schizophrenia.  However, no genes have yet been convincingly associated with migraine susceptibility, probably due to the high degree of variability of the disease phenotype combined with the lack of viable laboratory tests.

”To address this problem, we developed a new analysis technique concentrating on different symptoms of migraine”, says Professor Aarno Palotie (University of Helsinki, Finland, and the Sanger Institute, Cambridge, UK).  The new technique was used in the large international study including 1700 migraine patients and their close relatives from 210 Finnish and Australian migraine families.  The Finnish families had been ascertained through neurology clinics, while the Australian families had been collected through a twin study.  An initial genome-wide microsatellite study was followed up by an independent targeted replication study.

Researchers identified one gene locus on chromosome 10q23, which showed significant evidence of genetic linkage in both populations studied as well as in the replication study.  The gene locus was especially strongly linked to female migraineurs.  “In a further analysis, two independent previous studies, one Finnish and one Australian, had detected the same locus, but in those studies the level of evidence had been just below significance, and thus the connection had so far been missed”, tells researcher Verneri Anttila from Palotie’s group.

This locus is thus linked to migraine in a total of 4000 migraineurs or their close relatives.  “All of these findings depended on the newly discovered aspect of migraine genetics: different types of pain – such as pain that pulsates or pain that is unilateral – are more closely linked to specific genetic loci than general pain”, Palotie states.

In this study, researchers were able for the first time to convincingly demonstrate a genomic locus to be linked to migraine susceptibility in two diverse populations.  This is especially interesting as Finland and Australia are genetically distant, and also as it tied together previous research, resulting in very robust evidence for pinpointing the susceptibility region.

“This study is the first international collaboration as well as the largest linkage study in migraine to date.  It successfully applied new analysis strategies in detecting the locus and thus paved the way for subsequent large association studies”, Palotie and Anttila say.  According them, this study gives new hope to deciphering the migraine pathways and therefore discovering targets for future treatments, as well as discovering the first migraine gene variants.

A team of researchers at the National Institute of Standards and Technology (NIST) have demonstrated a new device that creates nanodroplet “test tubes” for studying individual proteins under conditions that mimic the crowded confines of a living cell. “By confining individual proteins in nanodroplets of water, researchers can directly observe the dynamics and structural changes of these biomolecules,” says physicist Lori Goldner, a coauthor of the paper* published in Langmuir.

With the flip of a switch: Nanodrop “test tubes” are created by an electronic switch that causes a micropipette to jerk back and leave behind a droplet less that 1 micron in diameter for study.
Credit: NIST

Researchers recently have turned their attention to the role that crowding plays in the behavior of proteins and other biomolecules—there is not much extra space in a cell. NIST’s nanodroplets can mimic the crowded environment in cells where the proteins live while providing advantages over other techniques to confine or immobilize proteins for study that may interfere with or damage the protein. This more realistic setting can help researchers study the molecular basis of disease and supply information for developing new pharmaceuticals. For example, misfolded proteins play a role in many illnesses including Type 2 diabetes, Alzheimer’s and Parkinson’s diseases. By seeing how proteins fold in these nanodroplets, researchers may gain new insight into these ailments and may find new therapies.

The NIST nanodroplet delivery system uses tiny glass micropipettes to create tiny water droplets suspended in an oily fluid for study under a microscope. An applied pressure forces the water solution containing protein test subjects to the tip of the micropipette as it sits immersed in a small drop of oil on the microscope stage. Then, like a magician whipping a tablecloth off a table while leaving the dinnerware behind, an electronic switch causes the pipette to jerk back, leaving behind a small droplet typically less than a micrometer in diameter.

The droplet is held in place with a laser “optical tweezer,” and another laser is used to excite fluorescence from the molecule or molecules in the droplet. In one set of fluorescence experiments, explains Goldner, “The molecules seem unperturbed by their confinement—they do not stick to the walls or leave the container—important facts to know for doing nanochemistry or single-molecule biophysics.” Similar to a previous work (see “‘Micro-boxes’ of Water Used to Study Single Molecules”, Tech Beat July 20, 2006), researchers also demonstrated that single fluorescent protein molecules could be detected inside the droplets.

Fluorescence can reveal the number of molecules within the nanodroplet and can show the motion or structural changes of the confined molecule or molecules, allowing researchers to study how two or more proteins interact. By using only a few molecules and tiny amounts of reagents, the technique also minimizes the need for expensive or toxic chemicals.

Reference:

* J. Tang, A.M. Jofre, G.M. Lowman, R.B. Kishore, J.E. Reiner, K. Helmerson, L.S. Goldner and M.E. Greene. Green fluorescent protein in inertially injected aqueous nanodroplets. Published in Langmuir, ASAP Article, Web release date: March 27, 2008.

University of Illinois researchers have developed a technique for imaging cells under an electron microscope that yields a sharper image of the structure of chromatin, the tightly wound bundle of genetic material and proteins that makes up the chromosomes. The findings appeared in Nature Methods.

Scientists have known for more than a century that proteins, such as histones, aid in packing DNA into the nucleus of a cell.  Human cells contain 2 to 3 meters of DNA, which must be kinked and coiled enough to fit into a region 1/10 the width of a human hair.

Despite the use of powerful, high-resolution imaging techniques such as electron microscopy, the mechanism by which this chromatin packing occurs remains a mystery.  The densely coiled chromatin fibers are very difficult to visualize, and little is known about how they condense during cell division, or unwind to allow gene expression.

In developing their method, the Illinois team tackled a key difficulty in imaging cells using electron microscopy.  Traditional studies “fix” the cells with potent chemicals (called fixatives) to preserve their structure for viewing under a microscope.  But standard fixation methods interfere with another step in the imaging process: the use of tagged antibodies to label key components of the cells.

These antibodies, which target and latch on to specific proteins in the cell, can be tagged with fluorescent labels for detection in light microscopy, or with metal particles (gold, in this case) for electron microscopy.

“If you fix the cells first, you have a dramatic drop in the efficiency of these immunochemical reactions,” said Igor Kireev, a visiting scientist in the department of cell and developmental biology and lead author of the paper.  Electron microscopy image Click photo to enlarge Image courtesy of Andrew Belmont and Igor Kireev The new technique exposes living cells to labeled antibodies, an approach that yields a much stronger signal for electron microscopy.

“And if your target is inside the condensed chromatin, the antibodies have no way to penetrate.”

Instead of fixing the cells before staining with antibodies, the researchers first exposed living animal cells to the labeled antibodies.  This allowed the antibodies to penetrate more deeply into the chromatin structure, and boosted the number of gold particles adhering to regions of interest.  The signal was enhanced by adding a silver solution that precipitated (solidified) upon contact with the gold.

“We are interested in chromatin structure, so our targets are mostly chromatin-bound proteins,” Kireev said.

The researchers had inserted several copies of a bacterial DNA, called the Lac operator, into the chromosomes.  A bacterial protein, the Lac repressor, recognizes and binds to the Lac operator in living cells.

The researchers combined a Lac repressor protein with another protein that fluoresces green under blue light.  This engineered protein adhered to the chromosomes in regions containing the Lac operator sequences.  Under blue light, these regions fluoresced.  A gold-tagged antibody targeted against green fluorescent protein (GFP) was then microinjected into the nucleus of a living cell, which added a metallic signal that could be boosted with silver.

“All this combined gives us a much better signal, a much stronger signal, with the very best structural preservation,” Kireev said.

The fluorescing protein helped the researchers find the regions of interest in the cells.  These areas were then “immunogold” labeled and targeted for electron microscopy.

In the resulting micrographs the researchers saw enhanced staining of the chromosomes.

“We can now apply this same live-cell labeling method to study at high resolution many different GFP-tagged proteins in the cell cytoplasm or nucleus,” said Andrew Belmont, a professor of cell and developmental biology and senior author of the paper.

“In trying to understand chromosomes, people have largely been limited to low resolution visualization of specific chromosomal proteins using light microscopy,” Belmost said.  “This meant everyone has had to do a lot of guessing of how things are put together, leading in many cases to vague, cartoon models of what are likely to be complicated chromosomal structures carrying out DNA functions such as replication and transcription.”

“Now we hope we can simply look and see the real structure using the more than 10-fold higher resolution of electron microscopy,” Belmont said.  “We are really excited to see what we will find using our new method”

CAMBRIDGE, Mass.  (April 18, 2008) – A team of researchers have demonstrated that fully mature, differentiated B cells can be reprogrammed to an embryonic-stem-cell-like state, without the use of an egg according to a study published in the April 18 issue of Cell.

In previous research, induced pluripotent stem (IPS) cells have been created from fibroblasts, a specific type of skin cells that may differentiate into other types of skin cells.  Because there is no way to tell if the fibroblasts were fully differentiated, the cells used in earlier experiments may have been less differentiated and therefore easier to convert to the embryonic-stem-cell-like state of IPS cells.

B cells are immune cells that can bind to specific antigens, such as proteins from bacteria, viruses or microorganisms.  Unlike fibroblasts, mature B cells have a specific part of their DNA cut out as a final maturation step.  “Once that piece of DNA is cut out, it can’t come back,” says Jacob Hanna, first author on the paper and a postdoctoral fellow in Whitehead Member Rudolf Jaenisch’s lab.  “Checking the genome give us a way to make sure the resulting IPS cells were not from immature cells.”

Hanna and his colleagues began the experiment by generating IPS cells from immature B cells.  Similar to the process used to create IPS cells from fibroblast cells, Hanna successfully reprogrammed the immature B cells into IPS cells by using retroviruses to transfer four genes (Oct4, Sox2, c-Myc and Klf4) into the cells’ DNA.

However, an additional factor, CCAAT/enhancer-binding-protein-?  (C/EBP?), was needed to nudge mature B cells to be reprogrammed as IPS cells.

Like IPS cells from earlier fibroblast studies, the IPS cells from both the mature and immature B cells could be used to create mice.  The mice grown from the reprogrammed mature B cells were missing the same part of their DNA as the mature B cells, demonstrating that Hanna and his colleagues had successfully reprogrammed fully differentiated cells.

In addition to demonstrating the power of reprogramming, this work offers the promise of powerful new mouse models for autoimmune diseases such as multiple sclerosis and type 1 diabetes, in which the body attacks certain types of its own cells.  For example, mature B or T cells specific for nerve cells called glia could be reprogrammed to IPS cells and then used to create mice with an entire immune system that is primed to only attack the glia cells, thereby creating a mouse model for studying multiple sclerosis.

Eventually, researchers will be able to study diseases by following a similar process with human cells, predicts Jaenisch, who is also a professor of biology at Massachusetts Institute of Technology.  “In principle, this will allow you to transfer a complex genetic human disease into a Petri dish, and study it,” he says.  “That could be the first step to analyze the disease and to define a therapy.”

Reference:

Cell, April 18, 2008 134(2). “Direct reprogramming of terminally differentiated mature B lymphocytes to pluripotency”

Jacob Hanna (1), Styliani Markoulaki (1), Patrick Schorderet (1), Caroline Beard (1), Bryce W. Carey (1), Marius Wernig (1), Menno P. Creyghton (1), Eveline J. Steine (1), (1), John P. Cassady (1), Christopher J. Lengner (1), Jessica A. Dausman (1), Rudolf Jaenisch (1,2)

1. Whitehead Institute for Biomedical Research, Cambridge, MA 02142 USA

2. Department of Biology, MIT, Cambridge, MA 02142 USA

UC San Diego study finds mice can sense oxygen through skin

Biologists at the University of California, San Diego have discovered that the skin of mice can sense low levels of oxygen and regulate the production of erythropoietin, or EPO, the hormone that stimulates our bodies to produce red blood cells and allows us to adapt to high-altitude, low-oxygen environments.

Their surprising finding, published in the April 18th issue of the journal Cell, contradicts the notion of mammalian skin as an envelope around our bodies with little connection to the respiratory system.

If found to apply to humans, the discovery could radically change the way physicians treat anemia and other diseases that require boosting our bodies’ ability to produce red blood cells.  It also could be used to improve the performance of endurance athletes competing in this summer’s Olympic Games.

“What we found in this study is really something quite unusual,” said Randall Johnson, a professor of biology at UC San Diego who headed the research study.  “We discovered that mammalian skin, at least in mice, responds to how much oxygen is above it and, by virtue of that response, changes blood flow through the skin.  This, in turn, changes one of the most basic responses to low oxygen that we have, which is the production of erythropoietin.”

Those responses, the researchers suspect, could be ancient traits retained as mammals evolved from lower forms of vertebrates, such as amphibians, that possess the same sorts of ion channels to promote oxygen diffusion in their extremely permeable skins as mammals have in their lungs.

“Amphibians—frogs most notably—breathe through their skin and are able to sense and respond to how much oxygen is in the air or water around their skin,” Johnson added.  “But nobody had ever thought about asking those questions about the skin of mammals.”

“From an evolutionary point of view, the results make sense, considering the important role of the skin for oxygen uptake in amphibians,” said Frank Powell, a professor of medicine at UCSD and expert in human and animal adaptations to high-altitude environments who was part of the team.  “It will be very interesting to see how these mechanisms work in humans and if, for example, different oxygen levels at the skin could affect how rapidly and how well one adapts to low oxygen in the intensive care unit of a hospital or at high altitude.”

The UC San Diego team found no evidence that mice could breathe through their skin.  But if their discovery that mice sense low oxygen through their skin and trigger EPO production is found to apply to humans, it would have dramatic implications for the training and testing of endurance athletes during the Summer Olympic Games in Beijing.

Besides training at altitude and in low-oxygen tents—the two generally accepted legal methods of boosting red blood cell production–runners, swimmers, cyclists and other endurance athletes seeking better performances by increasing the oxygen-carrying capacity of their blood may now have another legitimate way to increase their red blood counts.  Blood doping, the injection of additional red blood cells into the body, and the injection of synthetic recombinant EPO to boost red blood cell production are illegal in the Olympics and banned by most sports governing bodies.  But what if athletes could boost their own EPO and red blood cell counts by exposing their bodies to low levels of oxygen” Or, to obtain the same effect, by merely increasing blood flow through their skin”

“We’ve discovered a potent physiological trigger that can be enacted or enabled without exogenous sources of EPO,” said Johnson.  “We show in this paper that breathing in one level of oxygen and exposing your body to another level of oxygen is really a potent trigger for the body to produce its own EPO.  It’s not hard to foresee people taking what we’ve learned in mice and applying it to humans.”

If human skin is found to be sensitive to oxygen levels, it could revive the debate over the “Goldfinger Syndrome.” This idea, perpetuated by the famous James Bond movie in which the villain’s girlfriend is killed after being painted gold, has been the focus of urban legends and internet discussions about the possible negative health effects of painting the skin.  It has been the subject of two investigations by the Discovery Channel show “MythBusters.”

The team’s discovery—aided by collaborators in Sweden, Germany and the University of Pennsylvania—came after two years of trying to determine why certain mice the researchers had genetically engineered for experiments exhibited high levels of EPO.  In 2004, Johnson and his students published a paper in the journal Plos Biology, detailing how they had transformed ordinary laboratory mice into the rodent equivalent of Olympic endurance athletes.  They did this by deleting a gene that allows mammalian muscles to switch from aerobic to anaerobic metabolism when oxygen levels in the muscle run low.

Most of our daily activities are performed aerobically, through biochemical mechanisms in our muscles that make full use of oxygen.  But when the demands of our muscular system exceed its available supply of oxygen, as in sprinting for a bus or lifting a heavy object, a protein known as hypoxia inducible transcription factor-1, or HIF-1, is activated.  This protein enables the muscle to switch to the more energetically explosive, but expensive anaerobic process, which does not use oxygen and generates lactic acid as its byproduct.

When Johnson and students knocked out the negative regulator of the HIF-1 gene, they produced tiny mice with skin that look red and flushed.  These mice have trouble retaining body heat because a larger proportion of their blood is sent to their skin and cooled, much like a person sitting in a hot sauna or Jacuzzi.  But the most puzzling aspect of these mutant mice is their extremely high EPO levels—so high that 90 percent of their blood plasma is composed of red blood cells, compared to 40 to 50 percent for normal individuals.

“Their blood is basically paste and their hearts are enlarged as a result,” Johnson said.  “We could not understand why the skin was exerting this effect.  It just didn’t make sense to us.  We could figure out every other aspect of why this mutant mouse looked an acted the way it did, but this one thing was really bothersome to us, so that sent us down this road.  When we found that the EPO was coming from internal organs, not the skin of these mice, we thought there must be some kind of signal from the skin to the internal organs.”

Johnson and others in his laboratory—graduate student Adam Boutin, postdoctoral fellow Alexander Weidemann and undergraduate Lernik Mesropian—verified that the HIF-1 gene was responsible by genetically engineering mutant mice without the gene in their skin cells.  These mice were unable to signal the production of extra EPO when their skin was exposed a chamber filled with 10 percent oxygen—about the level found at Mount Everest.  The concentration of oxygen at sea level is about 21 percent.  Normal mice were able to increase the amount of EPO production at this 10 percent level.

This occurred, the researchers found, when more blood rushed into the skin.  By putting on the mouse’s skin a nitroglycerine patch, which increases blood flow through the skin, the researchers found that mice could dramatically increase their production of EPO and red blood cells.

“EPO administration is a multi-billion dollar drug market for the treatment of all sorts of diseases involving low red blood cell counts,” said Johnson.  “So the ability to manipulate red blood cell production just by changing blood flow through certain parts of the skin could be profound.  We show in this study that by just putting a little nitroglycerine patch we were able to trigger very big increases in EPO.  Whether this turns out to be true for humans, we don’t know yet.  But potentially this could be a very interesting way to manipulate this pathway.”

Johnson and his team, which included UCSD assistant professor of biology Colin Jamora, found that having mice breathe in a chamber with their entire bodies exposed to low levels of oxygen had the greatest response and produced the most EPO.  When the mice were allowed to breathe 10 percent oxygen in one chamber, but had the skin from their neck down exposed to 21 percent, or sea-level oxygen, in another chamber built by Powell, more than one-half of their adaptation to low oxygen was lost.

“If we put mice that lack a hypoxic response in their skin in a low oxygen chamber more than half of their hypoxic response goes away and that was surprising to us,” Johnson said.  “The skin really is a big contributor to the way the mouse responds to low oxygen.”

“All of the important responses to hypoxia, or low oxygen, were thought to be triggered by oxygen-sensitive nerves and molecules in the blood and internal organs,” said Powell.  “However, these experiments clearly show that the skin directly responds to changes in oxygen in the environment with changes in blood flow.  These changes in skin blood flow are highly significant by causing changes in the levels of hypoxic inducible factor, which is a sort of ‘master switch’ for adapting to low oxygen that activates multiple genes to enhance oxygen delivery throughout the body.”

Johnson said that because people with skin inflammations such as psoriasis and eczema can have low red blood cell counts, he and his team are interested in extending their study to investigate anemia caused by skin inflammations in their mutant mice.

“In people with anemia of inflammation it seems as if the EPO isn’t having an effect,” he added.  “We actually have mutant mice with skin inflammation that show this same effect.  They have high EPO levels, but they don’t have a high red blood cell count.  The mutants we used in our study have high EPO levels and high red blood cell counts.  But they don’t have inflammation.  The next step for us is going to be trying to figure out why these inflammatory diseases trigger EPO.  Is there something about inflammation that we can trigger so these people can be treated without suffering this kind of anemia””

The scientists said in their paper that their discovery also might explain why people in some parts of Nepal, India and Pakistan massage newborn babies in mustard oil, a mild irritant that promotes blood flow through the skin.

“We show in this study that if you paint the skin of a mouse with this mild irritant, mustard oil, it will also trigger EPO release at a somewhat lower level,” Johnson said.  “In India and Pakistan babies are in some communities massaged in mustard oil at birth; and some health workers have been trying to get them to stop this folk tradition.  But we show that in mice this increases EPO levels.  And since increased EPO levels contribute to increased red blood cell counts one could imagine it being beneficial.”

A group of Duke University researchers have made a major advance in understanding how bacteria divide.  These results could lead to new antibiotic treatments that prevent dangerous bacteria from multiplying.

Normally, bacteria divide by forming a ring that pinches the cell in two.  The ring is called a “Z ring” after the protein FtsZ, which forms a ring-shaped scaffold and then squeezes it smaller.  In bacteria, the Z ring also contains a dozen other proteins, all believed to be essential for division.

The Z ring normally pulls in on the cell membrane by binding to another protein, FtsA, which has one end attached to the inner cell membrane and the other end connected to FtsZ.  When the Z ring constricts, it completely pulls in the membrane and nips the bacterium in two.

But cell biology research scientist Masaki Osawa, Ph.D., cut FtsA out of the system by making an FtsZ that could bind directly to the membrane, and called it “membrane targeted FtsZ” or FtsZ-mts.

First, Osawa demonstrated that the new protein, FtsZ-mts, assembled Z rings in bacteria.

Then he constructed a greatly simplified cell-division machine in microscopic oil droplets, called liposomes, that demonstrated the important role of FtsZ in the division process.  He was able to assemble Z rings in this completely artificial system, the liposome, a tiny hollow sphere of fat that mimics natural cell membranes.

To do this, Osawa mixed the liposomes with FtsZ and GTP, a molecule that provides energy.  On a microscope slide the liposomes fused and stretched into tubes that mimicked the shape of E. coli and other rod-shaped bacteria.

“It was a happy coincidence that the size and shape of the liposomes was similar to that of rod-shaped bacteria,” says co-author Harold Erickson, professor of cell biology.  “These tubular liposomes are a new micro-structure, and their formation is still a mystery.”

During the experiment, fluorescently labeled FtsZ-mts was initially on the outside of the liposomes, but some of the tubular liposomes ended up with FtsZ on the inside.  “We don’t know how this happens, but it is a key to the discovery,” Osawa said.

Inside the liposome the FtsZ formed multiple closed rings that aligned perpendicular to the length of the tube, just as Z rings form in bacteria.  They also slid back and forth, and where they collided, they stayed together and formed brighter Z rings.  And as the Z rings grew in brightness, they visibly pulled the wall of the liposome inward.

“The Z rings are clearly generating force and causing the constriction,” Osawa said.  A movie the team made shows several constrictions in the wall occurring at the sites of the bright Z rings.  When the GTP in the liposome is used up, the tube eases out of its constrictions into its original shape.

“We believe our simple system may recreate the mechanism that the earliest bacteria used to divide.  They probably had FtsZ alone,” Erickson said.  “Osawa’s experiments show that FtsZ, a membrane tether, and the inside surface of a tubular membrane are all that’s needed to assemble the Z ring and generate a constriction force.”

The artificial Z rings were not sufficient to pinch the liposomes in half, “probably because their walls are much thicker than the membrane of a bacterium,” Osawa noted.  “We are now working to make thinner liposomes, so that we can achieve complete division.”

Erickson said that FtsZ is the bacterial ancestor of tubulin, the protein that makes the microtubules in animal cells and is the target of a number of anti-cancer drugs like taxol.  Although FtsZ is not sensitive to taxol, anything learned about the bacterial ancestor will help us understand microtubules, which help animal cells to keep their shape and control their movements, he explained.

(PITTSBURGH)

A team of University of Pittsburgh researchers have discovered that certain organic—or carbon-based—molecules exhibit the properties of atoms under certain circumstances and, in turn, conduct electricity as well as metal.  This allows a newfound ability of organic molecules to conduct electricity opens door to smaller, cheaper and more powerful technologies.

The work was detailed in the April 18 edition of Science, the finding is a breakthrough in developing nanotechnology that provides a new strategy for designing electronic materials, including inexpensive and multifunctional organic conductors that have long been considered the key to smaller, cheaper, and faster technologies.

The Pitt team found that the hollow, soccer-ball-shaped carbon molecules known as fullerenes can hold and transfer an electrical charge much like the most highly conductive atoms, explained project head Hrvoje Petek, a professor of physics and chemistry in Pitt’s School of Arts and Sciences and codirector of Pitt’s Petersen Institute for NanoScience and Engineering.  The research was performed by Pitt post-doctoral associates Min Feng and Jin Zhao.

When an electron was introduced into a fullerene molecule, the shape of the electron distribution mimicked that of a hydrogen atom or an atom from the alkali metal group, which includes lithium, sodium, and potassium.  Moreover, when two fullerenes were placed next to each other on a copper surface, they showed the electron distribution of their chemical bond and appeared as H2, a hydrogen molecule.  The assembly exhibited metal-like conductivity when the team extended it to a wire 1-molecule-wide.

“Our work provides a new perspective on what determines the electronic properties of materials,” Petek said.  “The realization that hollow molecules can have metal-like conductivity opens the way to develop novel materials with electronic and chemical properties that can be tailored by shape and size.”

Although the team worked with fullerenes, the team’s results apply to all hollow molecules, Petek added, including carbon nanotubes—rolled, 1-atom-thick sheets of graphite 100,000 times smaller than a human hair.

The team’s research shows promise for the future of electronics based on molecular conductors.  These molecule-based devices surpass the semiconductor and metal conductors of today in terms of lower cost, flexibility, and the ability to meld the speed and power of optics and electronics.  Plus, unlike such inorganic conductors as silicon, molecule-based electronics can be miniaturized to a 1-dimensional scale (1-molecule-wide), which may enable them to conduct electricity with minimal loss and thus improve the performance of an electronic device.

Traditionally, the problem has been that organic conductors have not conducted electrical current very well, Petek said.  The Pitt team’s discovery could enable scientists to finally overcome that problem, he added.

“Metal-like behavior in a molecular material—as we have found—is highly surprising and desirable in the emerging field of molecular electronics,” he said.

“Our work is a unique example of how nanoscale materials can be used as atom-sized building blocks for molecular materials that could replace silicon and copper in electronic devices, luminescent displays, photovoltaic cells, and other technologies.”

(LA JOLLA, CA) Salk researchers zoom in on genome-wide DNA methylation and transcriptomes at single base resolution. Until quite recently, the chemical marks littering the DNA inside our cells like trees dotting a landscape could only be studied one gene at a time.  But new high-throughput DNA sequencing technology has enabled researchers at the Salk Institute for Biological Studies to map the precise position of these individual DNA modifications throughout the genome of the plant Arabidopsis thaliana, and chart its effect on the activity of any of Arabidopsis’ roughly 26,000 genes.

“For a long time the prevailing view held that individual modifications are not critical,” says Joseph Ecker, Ph.D., a professor in the Plant Biology laboratory and director of the Salk Institute Genomic Analysis Laboratory.  “The genomes of higher eukaryotes are peppered with modifications but unless you can take a detailed look at a large scale there is no way of knowing whether a particular mark is critical or not.”

The Salk study, which appears today in the online issue of Cell, paints a detailed picture of a dynamic and ever-changing, yet highly controlled, epigenome, the layer of genetic control beyond the regulation inherent in the sequence of the genes themselves.

Being able to study the epigenome in great detail and in its entirety will provide researchers with a better understanding of plant productivity and stress resistance, the dynamics of the human genome, stem cells’ capacity to self-renew and how epigenetic factors contribute to the development of tumors and disease.

Discoveries in recent years made it increasingly clear that there is far more to genetics than the sequence of building blocks that make up our genes.  Adding molecules such as methyl groups to the backbone of DNA without altering the letters of the DNA alphabet can change how genes interact with the cell’s transcribing machinery and hand cells an additional tool to fine-tune gene expression.

“The goal of our study was to integrate multiple levels of epigenetic information since we still have a very poor understanding of the genome-wide regulation of methylation and its effect on the transcriptome,” explains postdoctoral researcher and co-first author Ryan Lister, Ph.D.

The transcriptome encompasses all RNA copies or transcripts made from DNA.  The bulk of transcripts consists of messenger RNAs, or mRNAs, that serve as templates for the manufacture of proteins but also includes regulatory small RNAs, or smRNAs.  The latter wield their power over gene expression by literally cutting short the lives of mRNAs or tagging specific sequences in the genome for methylation.

But before Lister could start to unravel the multiple layers of epigenetic regulation that control gene expression, he had to pioneer new technologies that allowed him to look at genome-wide methylation at single-base resolution and to sequence the complete transcriptome within a reasonable timeframe.

Collaborating scientists at the ARC Centre of Excellence in Plant Energy Biology at the University of Western Australia in Perth developed a powerful, web-based genome browser, which played a crucial role in unlocking the information hidden in the massive datasets.

Cells employ a whole army of enzymes that add methyl groups at specific sites, maintain established patterns or remove undesirable methyl groups.  When Lister and his colleagues compared normal cells with cells lacking different combination of enzymes they discovered that cells put a lot of effort in keeping certain areas of the genome methylation-free.

On the flipside, the Salk researchers found that when they knocked out a whole class of methylases, a different type of methylase would step into the breach for the missing ones.  This finding is relevant for a new class of cancer drugs that work by changing the methylation pattern in tumor cells.

“You might succeed in removing one type of methylation but end up with increasing a different type,” says Ecker.  “But very soon we will be able to look and see what kind of compensatory changes are happening and avoid unintended consequences.”

Previous studies had found that a subset of smRNAs could direct methylation enzymes to the region of genomic DNA to which they aligned.  Overlaying genome-wide methylome and smRNA datasets confirmed increased methylation precisely within the stretch of DNA that matched the sequence of the smRNA.  Conversely, heavily methylated smRNA loci tended to spawn more smRNAs.

“We looked at a plant genome but our method can be applied to any system, including humans,” says Lister.  Although the human genome is about 20 times bigger than the genome of Arabidopsis – plant biologists’ favorite model system not least because of its compact genome – Ecker predicts that within a year or so, sequencing technology will have advanced far enough to put the 3 billion base pairs of the human genome and their methyl buddies within reach.

“This really is just the beginning of unmasking the role of these powerful epigenetic regulatory mechanisms in eukaryotes,” says Ecker.

A novel type of drug can shrink primary breast cancer tumors significantly in just 6 weeks Research provides leads to a new target in cancer treatment -the cancer stem cell.

(Berlin, Germany) A drug that targets the cell surface receptors that play an important role in many types of cancer can bring about significant tumour regression in breast cancer after only six weeks of use, a scientist told the 6th European Breast Cancer Conference (EBCC-6) today (Thursday 17 April).  Dr. Angel Rodriguez, from the Lester and Sue Smith Breast Center, Baylor College of Medicine, Houston, USA, said that the work demonstrated for the first time that the tyrosine kinase inhibitor lapatinib could decrease tumour-causing breast cancer stem cells in the primary breast cancers of women receiving neoadjuvant treatment (treatment given before the primary surgery for the disease).

Dr. Rodriguez and colleagues studied 45 patients with locally advanced breast cancer in which the gene HER-2 was over-expressed.  The patients received lapatinib for six weeks, followed by a combination of weekly trastuzumab and three-weekly docetaxel, given over 12 weeks, before primary surgery.  Biopsies were performed at the time of diagnosis and also after six weeks of lapatinib and cells from the tumours were obtained and analyzed.

“We saw significant tumour regression after six weeks of single agent lapatinib,” said Dr. Rodriguez.  “Bi-dimensional tumour measurements showed a median decrease of minus 60.8%. We had previously showed that tumour-causing breast cancer stem cells were resistant to conventional preoperative chemotherapy; indeed, residual cancers that were exposed to such chemotherapy showed an increase in tumour-causing cells and enhanced tumour initiation by the formation of mammospheres, small tumours that form when tumour-causing cells are cultured in a test tube, which reflect the capacity of the cells to self-renew.  So we were excited to see that the results with lapatinib were different.”

Dr. Rodriguez’s results suggest that specific signalling inhibitors of the pathways responsible for stem cell self-renewal could provide a possible therapy for eliminating tumour-causing cells in order to achieve the long-term eradication of cancer.

Cancer stem cells help maintain the malignant tissue in the tumour by regenerating the tumour after attack from chemotherapy drugs.  “This indicates that the stem cells themselves should be the specific target of chemotherapy drugs,” said Dr: Rodriguez.  “Rather than the broad brush approach, in which cells are killed indiscriminately, targeting the stem cells may be more effective and also prevent some of the unpleasant side effects associated with conventional chemotherapy treatment.”

Scientists believe that cancer stem cells come into being through damage to their own DNA, which affects the regulation of their self-renewal.  Other cells divide into two ‘daughter’ cells, but a stem cell can divide into a new stem cell and a ‘progenitor’ cell.  The progenitor cell loses the power of self-renewal, but can still change into the cell type of the tissue served by the stem cell.  The stem cell population then continues to renew itself as it generates new cells for the tissue.  “This means that, unlike other cells, the stem cell has lost control over its own population size,” said Dr. Rodriguez.

Lapatinib has few side effects, and those that exist are minimal, including diarrhoea and acne.  But it is expensive.  “In the US it costs between $2000 and $3000 a month,” he said.

“This is an exciting finding, and we will be starting further studies on stem cells in order to confirm it.  We will also look into its applicability in testing novel agents targeting tumour-initiating cells.  This finding should also apply to other types of cancers and research of tumour-initiating stem cells in other cancers is ongoing,” said Dr. Rodriguez.

“International studies are currently underway looking at the effect of lapatinib in lung, colon, head and neck, gastric, oesophageal, and bladder cancer and lymphoma, among others,” he said.

Note:  Lapatinib has not yet been licensed for use in the EU, although it has been approved in Switzerland and received a positive opinion regarding a conditional marketing authorisation from the European Medicines Agency in December.  This conditional authorisation refers to its use in patients with advanced or metastatic breast cancer with HER-2 over-expression in the tumours.

Catalogue no: 204, Thursday 18 April, 17.15 hrs CEST (Hall 1)

A new mouse model for the study of Inclusion Body Myositis (IBM), a type of muscular dystrophy, has been developed by Dr. Ze’ev Ronai and a worldwide team of researchers.  The protein RNF5 is over-produced in the mice, resulting in extensive muscle damage similar to that seen in IBM patients.  The IBM mouse model will allow researchers to further study the mechanisms underlying development of the disease, as well as test potential new therapies.

Researchers have uncovered details about how dietary restriction slows down aging. A team of University of Washington scientists have uncovered details about the mechanisms through which dietary restriction slows the aging process.  Working in yeast cells, the researchers have linked ribosomes, the protein-making factories in living cells, and Gcn4, a specialized protein that aids in the expression of genetic information, to the pathways related to dietary response and aging.  The study, which was led by UW faculty members Brian Kennedy and Matt Kaeberlein, appears in the April 18 issue of the journal Cell.

Previous research has shown that the lifespan-extending properties of dietary restriction are mediated in part by reduced signaling through TOR, an enzyme involved in many vital operations in a cell.  When an organism has less TOR signaling in response to dietary restriction, one side effect is that the organism also decreases the rate at which it makes new proteins, a process called translation.

In this project, the UW researchers studied many different strains of yeast cells that had lower protein production.  They found that mutations to the ribosome, the cell’s protein factory, sometimes led to increased life span.  Ribosomes are made up of two parts -the large and small subunits -and the researchers tried to isolate the life-span-related mutation to one of those parts.

“What we noticed right away was that the long-lived strains always had mutations in the large ribosomal subunit and never in the small subunit,” said the study’s lead author, Kristan Steffen, a graduate student in the UW Department of Biochemistry.

The researchers also tested a drug called diazaborine, which specifically interferes with synthesis of the ribosomes’ large subunits, but not small subunits, and found that treating cells with the drug made them live about 50 percent longer than untreated cells.  Using a series of genetic tests, the scientists then showed that depletion of the ribosomes’ large subunits was likely to be increasing life span by a mechanism related to dietary restriction -the TOR signaling pathway.

“We knew that dietary restriction decreased TOR signaling, and that decreased TOR signaling reduced translation or protein production, but this was the first direct evidence that all three were acting in the same genetic pathway,” said Kennedy, an associate professor of biochemistry.

“The big question then became what’s happening in these translation-deficient cells to slow aging,” added Kaeberlein, an assistant professor of pathology.  “That’s when Vivian MacKay, a co-author on the study, had the idea to look at Gcn4.”

Gcn4 is a specialized protein called a transcription factor, which helps transfer genetic information during cell growth.  The protein is activated when a cell is starving for amino acids.  What made Gcn4 interesting to the UW team was its unique mode of regulation.

“When ribosomes aren’t working at 100 percent capacity, most proteins are made less efficiently, but Gcn4 is different,” explained Dr. MacKay, a research professor of biochemistry.  “Sometimes, you actually get more Gcn4 produced even when everything else is going down.  That’s precisely what we found in the longer-lived yeast strains with mutations in the large subunit of the ribosome.”

To make the link between Gcn4 and longevity, the scientists then asked whether preventing the increase of Gcn4 would block life span extension.  In every case, cells lacking Gcn4 did not respond as strongly as Gcn4-positive cells.

“The increased production of Gcn4 in long-lived yeast strains, combined with the requirement of Gcn4 for full life-span extension, makes a compelling case for Gcn4 as an important downstream factor in this longevity pathway,” Kaeberlein said.

Although scientists don’t yet know whether Gcn4 plays a similar role in organisms other than yeast, Kennedy points out that worms, flies, mice and humans all have Gcn4-like proteins that appear to be regulated in a similar way.

“The role of TOR and translation in aging is known to be conserved across many different species, so it’s plausible that this function of Gcn4 is conserved as well,” Kennedy said.  Future research will be aimed at testing this hypothesis.

“Clearly TOR signaling is one component, and perhaps the major component, of the beneficial health effects associated with dietary restriction,” said Kaeberlein.  “The difficulty with TOR as a therapeutic target, however, is the potential for negative side effects.  As we learn more of the mechanistic details behind how TOR regulates aging, we will hopefully be able to identify even better targets for treating age-associated diseases in people.”

A team of researchers at Yale School of Medicine have identified, characterized and cloned ovarian cancer stem cells and have shown that these stem cells may be the source of ovarian cancer’s recurrence and its resistance to chemotherapy.

“These results bring us closer to more effective and targeted treatment for epithelial ovarian cancer, one of the most lethal forms of cancer,” said Gil Mor, M.D., associate professor in the Department of Obstetrics, Gynecology & Reproductive Sciences at Yale School of Medicine.

Mor presented his findings recently at the annual meeting of the American Association for Cancer Research (AACR) Meeting in San Diego, California.

Cancerous tumors are made up of cells that are both cancerous and non-cancerous.  Within cancerous cells, there is a further subclass referred to as cancer stem cells, which can replicate indefinitely.

“Present chemotherapy modalities eliminate the bulk of the tumor cells, but cannot eliminate a core of these cancer stem cells that have a high capacity for renewal,” said Mor, who is also a member of the Yale Cancer Center.  “Identification of these cells, as we have done here, is the first step in the development of therapeutic modalities.”

Mor and colleagues isolated cells from 80 human samples of either peritoneal fluid or solid tumors.  The cancer stem cells that were identified were positive for traditional cancer stem cell markers including CD44 and MyD88.  These cells also showed a high capacity for repair and self-renewal.

The isolated cells formed tumors 100 percent of the time.  Within those tumors, 10 percent of the cells were positive for cancer stem cell marker CD44, while 90 percent were CD44 negative.

Mor and his team were able to isolate and clone the ovarian cancer stem cells.  They found that these cells were highly resistant to conventional chemotherapy while the non-cancer stem cells responded to treatment.  “Isolating and cloning these cells will lead to development of new treatments to target and eliminate the cancer stem cells and hopefully prevent recurrence,” said Mor.

A new drug compound leads to the death of ovarian cancer cells resistant to chemotherapy.

Dr. Gil Mor, Associate Professor in the Department of Obstetrics, Gynecology & Reproductive Sciences at Yale School of Medicine.

Credit: Yale University

In a discovery that may be useful for maintaining remission in chemo-resistant ovarian cancer, Yale scientists report that pre-clinical studies have shown the drug compound NV-128 can induce the death of ovarian cancer cells by halting the activation of a protein pathway called mTOR.

Gil Mor, M.D., associate professor in the Department of Obstetrics, Gynecology & Reproductive Sciences at Yale School of Medicine, and associate research scientist Ayesha Alvero, M.D. presented the data April 15 during an oral presentation at the annual meeting of the American Association for Cancer Research.

In cancer cells, mTOR signals enhance tumor growth and may be associated with resistance to conventional therapies.  Inhibition of mTOR could shut down many of these survival pathways, including proteins that protect the mitochondria of cancer cells.

NV-128, developed by Novogen Limited, holds promise as a more targeted therapy for ovarian cancer because it works differently from traditional therapies that are dependent on enzymes known as caspases to trigger cell death.  Therapies using caspases to kill cancer cells can be ineffective in chemo-resistant cancer cells due to mutations that short-circuit signals that trigger cancer cell death.

“We consider that the capacity of NV-128 to trigger caspase-independent cell death, in otherwise chemoresistant ovarian cancer cells, opens new possibilities for the use of NV-128 as a potential addition to conventional chemotherapy targeting ovarian cancer cells,” said Mor.

In the context of developing therapies for late stage ovarian cancer, Mor said, the finding may be “a key step to the development of alternative targeted therapy for patients with cancer recurrence.”

Breast cancers behave differently before and after the age of 70 Do the immune defense mechanisms play a role?

Berlin, Germany: Researchers in Belgium have discovered that increasing age affects the way breast cancer behaves.  As women approach the age of 70, they become less likely to be diagnosed with aggressive tumours that have spread to the lymph nodes.  But after 70, the cancer is increasingly likely to spread, particularly if the tumours are small.

Until now, there has been conflicting evidence on aging and lymph node involvement and this study is the first to show clearly how the link between the two changes before and after the age of 70.

Professor Hans Wildiers told the 6th European Breast Cancer Conference (EBCC-6) in Berlin today (Friday), that he suspects that women older than 70 have decreased immune defence mechanisms, which are less able to deal with tumours that are likely to metastasise to other sites in the body.

“The effect of age of lymph node positivity is not straightforward.  There seems to be a different effect between women aged up to 70 years and women older than 70.  For the younger group of women, age appears to have a negative effect on lymph node status – the older they become, the less likely the cancer is to have spread to the lymph nodes.  For the older group of women (aged over 70), age appears to influence lymph node status in the opposite way – the older they become, the more likely they are to have cancer cells in the lymph nodes if the tumour is small,” said Prof Wildiers, who is adjunct head of clinic in the department of general medical oncology at the Multidisciplinary Breast Centre, University Hospitals Leuven, Belgium.

“There is an interaction between age and tumour size, suggesting that, up to the age of 70, age mainly has a positive effect on lymph node status for older women with small tumours.  A likely explanation is that breast tumours metastasise less frequently to lymph nodes with increasing age due to the decreased biological aggressiveness in these tumours.  On the other hand, over the age of 70, if the tumours have the potential to metastasise to lymph nodes, this occurs more rapidly in smaller tumours and this might be related to decreased immune defence mechanisms in elderly patients.”

Prof Wildiers and his colleagues investigated 2,227 women who had been treated for breast cancer between 2000 and 2006 at the University Hospitals Leuven.  Then they compared the results with a separate database of over 11,000 breast cancer patients on the Eindhoven Cancer Registry.

They found that for women aged 70 or younger, increasing age was associated with a decreased prevalence of cancer spreading to the lymph nodes.  The women’s risk of having positive lymph nodes decreased by 13% for every decade they aged, up to age 70.

Once aged 70 and over, the odds of lymph node involvement doubled with every 10-year increase in age for women who had tumours that were no bigger than 15mm across.  If the tumours were larger than 42-43 mm, then risk of lymph node involvement continued to decrease.

Prof Wildiers said: “We know that the elderly have depressed immune defences, and, therefore, it is possible that these decreased defences are unable to prevent invasion of the lymph nodes by metastases in a subset of breast tumours in elderly women.  Although breast cancer survival in older women appears to be similar to survival in the general population irrespective of disease status, it might well be that there is a balance in the elderly between, on the one hand, a less aggressive type of tumour, and, on the other hand, their decreased immunological defences.”

He said the findings supported the idea that there are two types of tumour in elderly women: ones that are slow-growing and don’t invade the lymph nodes even if the tumours are larger, and ones that are aggressive and metastasise very early to the lymph nodes.  Women with slow-growing tumours might benefit from less aggressive treatment, while the smaller tumours in the women aged over 70 might need to be treated more aggressively.

“Further research now needs to be conducted into the role the immune system plays in lymph node invasion,” he concluded.

Reference:
Catalogue no: 401, Friday, Poster discussion session, 14.30 hrs CEST (Hall 2)