Friday, April 30, 2010

CERN Global Network

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Forging a global community for science and innovation

This week, CERN is launching the CERN Global Network, which responds to a real need for us to keep in touch, to share our knowledge and expertise, and to build on the fantastic resource of the CERN community broadly defined.

Here at CERN, we pride ourselves on the cross fertilization of ideas that occurs when people from around the world come together for a common goal. The Network extends that to our alumni and to our partners in academia, commerce and industry, allowing expertise to be shared among all its members. The CERN Global Network is open to anyone who works or has worked at or with CERN at any time. You don’t get much more inclusive than that.

In an increasingly competitive world, knowledge transfer is vitally important for an organization like CERN. The primary outcome of our basic science is knowledge, but what use is knowledge if it’s confined to a select few? The people who drew up the CERN Convention over half a century ago saw the importance of transferring knowledge when they wrote that CERN should do its utmost to make the results of its research as widely known as possible. That spirit has always driven CERN’s open and transparent approach to communication, and the Network is the next logical step. It will help us to disseminate knowledge as far as possible, and to share expertise within a wide constituency.

Furthermore, the nature of our knowledge and expertise is not confined to scientific and technical domains, but also encompasses experience with how people of very differing cultures and backgrounds can work harmoniously together.

As of this week, the Network is open to all current and former members of the CERN personnel, who are invited to join and create their profiles on the Network’s website. As of the summer, once the website is populated, the Network will go live to a wider community encompassing research institutes the world over and companies in CERN’s Member States. In short, it’s time to get better connected at


Saturday, April 24, 2010

Micro-RNA Can Move, New Evidence in Plants Shows

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ScienceDaily (Apr. 24, 2010) — Ever since tiny bits of genetic material known as microRNA were first characterized in the early 1990s, scientists have been discovering just how important they are to regulating the activity of genes within cells.

A new study now shows that microRNAs don't just control the activity of genes within a given cell -- they also can move from one cell to another to send signals that influence gene expression on a broader scale.

Researchers at the Duke Institute for Genome Sciences & Policy (IGSP), in collaboration with groups at the Universities of Helsinki and Uppsala and the Boyce Thompson Institute for Plant Research at Cornell University, made the discovery while working out the intricate details of plant root development in Arabidopsis, a highly-studied mustard plant. Although they still don't know exactly how the microRNAs travel, it appears that this mobility allows them to play an important developmental role in sharpening the boundaries that define one plant tissue from another.

"To our knowledge, this is the first solid evidence that microRNAs can move from one cell to another," said Philip Benfey, director of the Duke IGSP Center for Systems Biology.

The findings, which appear in an advanced online publication of the journal Nature on April 21, add microRNA to the list of mobile molecules, including hormones, proteins and other forms of small RNA, that allow for essential communication between cells in the process of organ development.

They also add a new element to the already complex interplay in Arabidopsis roots between two proteins, known as Scarecrow and Short-root, that Benfey's team had described in earlier work. Those proteins interact and restrain one another to allow the assembly of a waterproofing layer of cells that ultimately enables plants to control precisely how much water and nutrients they take in.

The researchers now show that Short-root moves from cells in the plant's inner vasculature out into the waterproofing endodermis that surrounds it to activate Scarecrow. Together, those two transcription factors (genes that control other genes) activate microRNAs, known as MIR165a and 166b. Those microRNAs then head back toward the vascular cells, meeting and degrading another transcription factor (called Phabulosa) as well as other regulatory factors along the way.

"Dr. Benfey and his colleagues have shown how two modes of gene regulation work together across cellular borders to ensure the proper patterning of plant root tissues," said Susan Haynes of the NIH's National Institute of General Medical Sciences, which partially funded the study. "This study provides important insight into how cells communicate positional information to orchestrate the complex process of tissue and organ development."

"Now we know that microRNAs can and do move to form gradients in the context of plant development," Benfey added. "It adds a new dimension to gene regulation."

According to Benfey, history suggests this kind of two-way communication involving microRNAs in the developing root is likely to be a more general phenomenon. "Just about everything in biology that once seemed particular sooner or later proves to be more general," he said.

He said there's also reason to think that the specific regulatory interactions they've uncovered were key in the evolutionary transition from single-celled algae to land plants.

"Formation of vascular tissue with a surrounding endodermal layer that acts as waterproofing was a key milestone in the evolution of land plants," Benfey said. "Without a tube to conduct water, you can't grow a tree or a sunflower."

Key collaborators on the study include Yrjo Helariutta of the University of Helsinki; Ji-Young Lee of the Boyce Thompson Institute and Annelie Carlsbecker of Uppsala University.

Journal Reference:
annelie Carlsbecker, Ji-Young Lee, Christina J. Roberts, Jan Dettmer, Satu Lehesranta, Jing Zhou, Ove Lindgren, Miguel A. Moreno-Risueno, Anne Vatén, Siripong Thitamadee, Ana Campilho, Jose Sebastian, John L. Bowman, Ykä Helariutta & Philip N. Benfey. Cell signalling by microRNA165/6 directs gene dose-dependent root cell fate. Nature, 2010;
DOI: 10.1038/nature08977


Friday, April 23, 2010

Simplifying Complexity – New Insights Into How Genomes Work

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ScienceDaily (Apr. 22, 2010) — A genome is a complex system of genes and factors that regulate them. A European research team has clarified how such dynamic systems work, leading to a new way to predict genetic regulators.

As an organism develops and interacts with its environment, suites of genes are constantly being turned on and off, orchestrating every aspect of life. Researchers worldwide are trying to understand transcriptional networks, the intricate webs of genes and regulatory agents that control the growth and functioning of every organism from Escherichia coli to Homo sapiens.

The EU-funded research project GENNETEC (for GENetic NETworks Emergence and Complexity -- set itself the ambitious goal of developing a deeper understanding of all complex systems and then applying those insights to living organisms, including humans.

Among GENNETEC's accomplishments is a new way to predict which transcription factors -- molecules that turn genes on or off -- regulate particular genes. Their findings promise to boost research into the functioning of genetic networks in general, and into the dynamics of the human genetic system in health and disease.

"We're now in a better position to understand genetic regulation in human cells, for a lower cost and in a shorter time," says François Képès, coordinator of the GENNETEC project.

Much like a pianist fingering particular keys or chords to play a melody, transcription factors bind to particular sites along a chromosome to turn nearby genes on or off. Decades of research have shown that the resulting patterns of gene expression direct a cell or organism's development, normal functioning, and responses to environmental challenges.

In addition, malfunctions in the genetic system can cause various diseases including cancer. "A disease might sometimes be considered an improper change in the dynamics of a network of interactions," says Képès. "So understanding their properties and how to correct or control their dynamics is essential."

Trolling for transcription factors

Until now, the most effective way researchers had to try to match genes and provisional transcription factors was to look for short DNA sequences that were known to bind to specific regulatory molecules. This approach remains useful, says Képès, but produces many false positives -- potential regulatory relationships that prove false.

Following up on those false leads is wasteful. "Doing it with a pipette takes a long time and costs a lot of money," says Képès.

The GENNETEC team decided to address that problem by studying a new and independent way to predict whether a gene is controlled by a particular factor.

In earlier research, Képès and his colleagues discovered that genes that respond to the same transcription factor are often spaced regularly along a chromosome. They suspected that this periodic spacing is related to the way that DNA coils up inside the nucleus of a cell, and serves to optimise the functioning of related genes and transcription factors by grouping them geographically.

Scientists are always more comfortable if they understand the mechanism that produces an observed regularity. The GENNETEC researchers used sophisticated numerical simulations of DNA folding to prove that the presence of those periodically spaced genes helps determine the structure of the folded or condensed strand of DNA.

They also found that the final shape, which brings related genes close together physically, is important for gene expression.

"What we discovered is that there is a clear link between chromosome structure and gene expression," says Képès, "a link that we can now predict in a very precise and workable way."

Faster, more focused search

When the GENNETEC team combined their new positional predictor with the standard sequence predictor, they found that they could identify new gene-regulator relationships far more efficiently.

"Combining the two predictors allows us to predict the regulators of a particular gene much better, by cutting down on the false hits," says Képès. "We typically double the specificity of the prediction."

One of the consortium partners, NorayBio, based in northern Spain, is developing a commercial software package that will allow researchers worldwide to apply this more powerful approach to deciphering genetic networks.

The consortium is also making a functional, but less sophisticated, version of the software available for free.

While Képès is pleased with this new research tool, he emphasises that the consortium's fundamental research on complex systems is equally important. Their findings can be applied in fields as diverse as designing software that does only what it's supposed to do and engineering systems that, like cells, can respond optimally to a wide variety of situations.

"Cells have just one genome, but with that one genome they can cope with multiple challenges," says Képès. "We can use this biological solution as inspiration to make a new generation of algorithms to address complex problems better than before."

The GENNETEC project received funding from the IST FET Proactive Initiative 'Simulating Emergent Properties in Complex Systems' of the EU's Sixth Framework Programme for research.


A Little Less Force: Making Atomic Force Microscopy Work for Cells

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ScienceDaily (Apr. 22, 2010) — Atomic force microscopy, a tactile-based probe technique, provides a three-dimensional nanoscale image of a material by gliding a needle-like arm across the material's surface. The core of this AFM imaging workhorse is a cantilever with a sharp tip that deflects as it encounters undulations across a surface. Due to a minimum force required for imaging, conventional AFM cantilevers can deform or even tear apart living cells and other biological materials.

While scientists have made strides in reducing this minimum force by making smaller cantilevers, the force is still too great to image cells with high resolution. Indeed, for imaging objects smaller than the diffraction limit of light -- that is, nanometer dimensions -- this approach hits a roadblock as the instrument can no longer sense minute forces.

Now, however, scientists with the Molecular Foundry, a U.S. Department of Energy User Facility located at Berkeley Lab, have developed nano-sized cantilevers whose gentle touch could help discern the workings of living cells and other soft materials in their natural, liquid environment. Used in combination with a revolutionary detection mechanism, this new imaging tool is sensitive enough to investigate soft materials without the limitations present in other cantilevers.

"Whether we are considering biological systems or other complex, self-assembling nanostructures, this organization will be done in a liquid," says Paul Ashby, a Molecular Foundry staff scientist who led this research in the Foundry's Imaging and Manipulation of Nanostructures Facility. "If we have an investigative probe that excels in this environment, we could image individual proteins as they function on the cell surface."

Says Babak Sanii, a post-doctoral researcher in the Foundry, "Shrinking the cantilever down to nanoscale dimensions dramatically reduces the force it applies, but to monitor the movements of such a small cantilever, we needed a new detection scheme."

Rather than measuring the cantilever's deflection by bouncing a laser off it, Ashby and Sanii place the nanowire cantilever in the focus of a laser beam and detect the resulting light pattern, pinpointing the nanowire's position with high resolution. The duo say this work provides a launching pad for building a nanowire-based atomic force microscopes that could be used to study biological cells and model cellular components such as vesicles or bilayers. In particular, Ashby and Sanii hope to learn more about integrins, proteins found on the surface of cells that mediate adhesion and are part of signaling pathways linked to cell growth and migration.

"No present technique probes the assembly and dynamics of protein complexes in the cell membrane," adds Ashby. "A dynamic probe is the holy grail of soft matter imaging, and would help determine how protein complexes associate and disassociate."

"High sensitivity deflection detection of nanowires," by Babak Sanii and Paul D. Ashby, appears in Physical Review Letters.

This work at the Molecular Foundry was supported by the DOE's Office of Science.

The Molecular Foundry is one of the five DOE Nanoscale Science Research Centers (NSRCs), premier national user facilities for interdisciplinary research at the nanoscale. Together the NSRCs comprise a suite of complementary facilities that provide researchers with state-of-the-art capabilities to fabricate, process, characterize and model nanoscale materials, and constitute the largest infrastructure investment of the National Nanotechnology Initiative. The NSRCs are located at DOE's Argonne, Brookhaven, Lawrence Berkeley, Oak Ridge and Sandia and Los Alamos National Laboratories. For more information about the DOE NSRCs, please visit

Journal Reference:
Babak Sanii, Paul D. Ashby. High Sensitivity Deflection Detection of Nanowires. Physical Review Letters, 2010; 104 (14): 147203 DOI: 10.1103/PhysRevLett.104.147203


Designer Threads: New Insight Into Protein Fiber Assembly

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ScienceDaily (Apr. 21, 2010) — Understanding how mixtures of proteins assemble and how to manipulate them in the laboratory has many exciting biomedical applications, such as providing scaffolds for the engineering of tissues that can replace diseased or damaged human tissues. Now, research published by Cell Press in the April 20th issue of Biophysical Journal, reveals new information about the kinetics of protein assembly and demonstrates how to manipulate conditions in order to provide different distributions of protein fiber lengths.

"Developing a good understanding of the relationship between the sequence of a protein fiber and its structure, stability and how it folds up and assembles together with other proteins is key and underpins our ability to design new protein-based materials for bioengineering," explains senior study author, Professor Derek N. Woolfson from the School of Chemistry at the University of Bristol in the United Kingdom. "It is also critical to determine the timescale of protein assembly so that the process can be fully controlled and accurately manipulated."

In previous work, Prof. Woolfson and colleagues designed two short peptides that, when mixed together, assembled to form fibers. These peptides were engineered to have "sticky ends" that interacted to form long fibers which exhibited a natural protein structural motif called the "alpha-helical coiled-coil"; a structure where fibrous proteins coil up like the strands of a rope. In the current study, the researchers used multiple sophisticated and complementary biophysical tools along with peptide engineering to gain further insight into the molecular process and timing of going from the small nanoscale peptides to large micron-length fibers.

Using these techniques, the researchers were able to build a specific descriptive mathematical model for the self-assembly of the alpha-helical protein fibers. Prof. Woolfson's group was also able to demonstrate that they could intervene in the assembly process to manipulate the resulting fibrous structures with some precision. "This study and the resulting mechanism we propose present a potential route to temporal control of the assembly of fibers with future applications in biotechnology and nanoscale science and medicine," proposes Prof. Woolfson.

Researchers include Elizabeth H. C. Bromley, University of Bristol, Bristol, UK; Kevin J. Channon, University of Bristol, Bristol, UK; Patrick J. S. King, University of Bristol, Bristol, UK; Zahra N. Mahmoud, University of Bristol, Bristol, UK; Eleanor F. Banwell, University of Bristol, Bristol, UK; Michael F. Butler, Unilever Corporate Research, Colworth Science Park, Sharnbrook, Bedford, UK; Matthew P. Crump, University of Bristol, Bristol, UK; Timothy R. Dafforn, University of Birmingham, Birmingham, UK; Matthew R. Hicks, University of Warwick, Coventry, UK; Jonathan D. Hirst, University of Nottingham, Nottingham, UK; Alison Rodger, University of Warwick, Coventry, UK; and Derek N. Woolfson, University of Bristol, Bristol, UK.

Journal Reference:
Elizabeth H.C. Bromley, Kevin J. Channon, Patrick J.S. King, Zahra N. Mahmoud, Eleanor F. Banwell, Michael F. Butler, Matthew P. Crump, Timothy R. Dafforn, Matthew R. Hicks, Jonathan D. Hirst. Assembly Pathway of a Designed %u03B1-Helical Protein Fiber. Biophysical Journal, 2010; 98 (8): 1668 DOI: 10.1016/j.bpj.2009.12.4309


Assembly of Protein Strands Into Fibrils

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ScienceDaily (Apr. 11, 2010) — Researchers have detailed a basic general mechanism describing how filamentous proteins assemble into ribbon like structures, the so-called amyloid fibrils. Combining experiments and theory, they can explain how denatured milk proteins assemble into ribbon like structures composed of up to five filaments. These findings are shining a surprisingly new light on the assembly of these proteins.

The atomic force microscope depicts on its screen the few nanometer thick and few micrometer long fibers as white flexible sticks, crisscrossing the surface on which they are deposited. The very peculiar property of these proteins lies in fact that they can self assemble into complex ribbon-like twisted fibers.

Researchers at ETH Zürich, EPF Lausanne and University of Fribourg have teamed up to take atomic force microscopy images of the fibers and to analyze them using concepts from polymer physics and theoretical modeling. This combination of expertise has lead them to propose a set of general rules governing the assembly of filaments into thicker and twisted ribbon like fibers. Their results are published in the current issue of the scientific journal Nature Nanotechnology. "The model that we propose is extremely precise in its predictions," says Raffaele Mezzenga, Professor of Food and Soft Materials Sciences at the ETH Zürich. "Up to now there was no such exact and general model for the formation of amyloid fibers," continues Giovanni Dietler, Professor of Physics of Living Matter, at the EPF Lausanne.

The structure of the amyloid fibers as it was unveiled by the experiments, surprised the researchers. Single proteins build the long filaments and subsequently the filaments assemble side by side to form the ribbon-like twisted fibers.

Mezzenga explains that the ribbon-like structure is the logic consequence of the strong charge carried by the building blocks of the fibers. In fact, the single proteins feel a strong mutual repulsion preventing them to pack and the ribbon structure is the only one that permits to limit this repulsion. Presently one missing information in the present model, is the exact nature of the short range attraction between the building blocks that drives in the first place the assembly among the protein filaments. Scientists agree that along the filaments there are charge-less domains of hydrophobic character (water repellent) that are the source of the short-range attraction. So there is a balance between attractive and repulsive interactions and the results is the ribbon like twisted conformation.

Self-organizing proteins are common in living matter and they are found in large aggregates for example in blood coagulation. Spherical like proteins are used in food industry as emulsifiers, gelling and foaming agents and in vitro they form amyloid like structures. These latter fibers have properties (elasticity, solubility, etc) favorable for food texturing or to produce special structures. The milk protein beta-lactoglubulin studied by Mezzenga and his colleagues is at the beginning spherical and by a heat treatment accompanied by acid environment it aggregates into the filamentous structures. Beta-lactoglobulin is an important component of the milk serum and therefore very relevant for food industry.

The knowledge gained by the scientists on this food protein can potentially benefit medical sciences. In fact amyloid-like fibers appear in humans affected by neurodegenerative diseases, like Alzheimer- or Creutzfeldt-Jakob disease. These human fibers, although made out of a very different proteins, are also ribbon-like and twisted and their assembly into long aggregates is presently under intense scrutiny. The model proposed by the team could also help to understand the genesis and development of theses diseases.

Journal Reference:
Adamcik J, Jung J-M, Flakowski J, De Los Rios P, Dietler G, Mezzenga R. Understanding amyloid aggregation by statistical analysis of atomic force microscopy images. Nature Nanotechnology, 2010; DOI: 10.1038/nnano.2010.59


Your Fat May Help You Heal: Researcher Extracts Natural Scaffold for Tissue Growth

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ScienceDaily (Mar. 26, 2010) — It frequently happens in science that what you throw away turns out to be most valuable. It happened to Deepak Nagrath, but not for long.

The Rice assistant professor in chemical and biomolecular engineering was looking for ways to grow cells in a scaffold, and he discarded the sticky substance secreted by the cells.

"I thought it was contamination, so I threw the plates away," said Nagrath, then a research associate at Harvard Medical School.

That substance, derived from adipose cells -- aka body fat -- turned out to be a natural extracellular matrix, the very thing he was looking for.

Nagrath, who joined Rice in 2009, and his co-authors have since built a biological scaffold that allows cells to grow and mature. He hopes the new material, when suffused with stem cells, will someday be injected into the human body, where it can repair tissues of many types without fear of rejection.

The research by Nagrath and his co-authors appeared last week in the Federation of American Societies for Experimental Biology (FASEB) Journal.

The basic idea is simple: Prompt fat cells to secrete what bioengineers call "basement membrane." This membrane mimics the architecture tissues naturally use in cell growth, literally a framework to which cells attach while they form a network. When the cells have matured into the desired tissue, they secrete another substance that breaks down and destroys the scaffold.

Structures that support the growth of living cells into tissues are highly valuable to pharmaceutical companies for testing drugs in vitro. Companies commonly use Matrigel, a protein mixture secreted by mouse cancer cells, but for that reason it can't be injected into patients.

"Fat is one thing that is in excess in the body. We can always lose it," Nagrath said. The substance derived from the secretions, called Adipogel, has proven effective for growing hepatocytes, the primary liver cells often used for pharmaceutical testing.

"My approach is to force the cells to secrete a natural matrix," he said. That matrix is a honey-like gel that retains the natural growth factors, cytokines (substances that carry signals between cells) and hormones in the original tissue.

Nagrath's strategy for growing cells isn't the only approach being pursued, even at Rice: Another method reported last week in Nature Nanotechnology uses magnetic levitation to grow three-dimensional cell cultures.

But Nagrath is convinced his strategy is ultimately the most practical for rebuilding tissue in vivo, and not only because it may cost significantly less than Matrigel. "The short-term goal is to use this as a feeder layer for human embryonic stem cells. It's very hard to maintain them in the pluripotent state, where they keep dividing and are self-renewing," he said.

Once that goal is achieved, Adipogel may be just the ticket for transplanting cells to repair organs. "You can use this matrix as an adipogenic scaffold for stem cells and transplant it into the body where an organ is damaged. Then, we hope, these cells and the Adipogel can take over and improve their functionality."

Nagrath's co-authors are Nripen S. Sharma, a research associate at Rutgers University, and Martin Yarmush, the Helen Andrus Benedict Professor of Surgery and Bioengineering at Harvard Medical School.

The National Institutes of Health and the Shriners Hospitals for Children supported their research.

Journal Reference:
Sharma et al. Adipocyte-derived basement membrane extract with biological activity: applications in hepatocyte functional augmentation in vitro. The FASEB Journal, 2010;
DOI: 10.1096/fj.09-135095


New Model Tracks the Immune Response to a T

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ScienceDaily (Apr. 10, 2010) — Using T cells primed for the infectious disease toxoplasmosis, Whitehead Institute researchers have created novel mouse models of the immune system that more accurately reflect how immune cells actually respond to pathogens in their presence.

"These models have a lot of potential," says Oktay Kirak, who is a postdoctoral researcher in the labs of Whitehead Members Hidde Ploegh and Rudolf Jaenisch. "It allows us to study both the biology of T cells as well as their role in toxoplasmosis."

Toxoplasma gondii, the single-celled parasite that causes toxoplasmosis with more than 30% of humans infected, the symptoms are generally minor, although toxoplasmosis can have serious or fatal effects for an immunocompromised person or a fetus, if the mother is infected during pregnancy.

In the April 9 issue of Science, Kirak and his colleagues describe how he used somatic cell nuclear transfer (SCNT) to create mice from single T cells that recognize the Toxoplasma gondii parasite.

In SCNT, a cell's nucleus is inserted into a de-nucleated, unfertilized egg cell to generate embryonic stem cells and animals with the same genetic code. In this study, Kirak, who is first author of the Science paper, transferred the nucleus of a T cell primed for toxoplasmosis into an enucleated, unfertilized mouse egg to create mice.

Kirak used T cells -- a class of white blood cells that include several types of cells including cytotoxic or so-called killer T cells -- because they have been difficult to study in current immune system models. Each cytotoxic T cell has its genetic material rearranged, so that it produces properly activated a receptor that can identify an antigen. An antigen is a particular part of an infectious agent, such as a virus, bacterium, or parasite, that the immune system can recognize. Once properly activated by the relevant antigen, the cytotoxic T cell will then kill any cell that is infected with that pathogen.

Many T cells may respond to a particular infectious agent, such as Toxoplasma gondii and they all may recognize different antigens on the parasite's surface. This multiple-pronged attack, though highly beneficial in eliminating infectious agents, complicates the study of specific T cell's interactions with the antigen and infectious agent. Kirak's method solves this problem by creating a mouse with identical T cells and activating them in the course of infection.

"Kirak's work was a really imaginative application of the nuclear transfer technique: to create a mouse with defined and predictable immunological properties, which will be useful for studying infectious diseases," says Jaenisch, who is also a professor at MIT.

This is a unique approach for immune system models. Some earlier models rely on transgenic mice in a process that leaves indelible marks created by the experiment itself and depend on a researcher subjectively choosing the mouse with the "best" immune response. Other models trick the infectious agent into producing a surrogate protein, not usually produced by that pathogen, like a protein found in egg whites. A researcher then documents how immune cells that recognize the egg white protein "respond" to the infectious agent. This model has serious drawbacks because T cells can respond at different times to different antigens during a natural infection.

In contrast to these models, SCNT generates T cell mouse models, with minimal tampering by the researcher and that depend entirely on the natural course of events. And that is an important advance for immune system models.

"The opportunity to look at true pathogen-derived antigens in the course of a natural infection provides us with a new window on how the immune system operates," says Ploegh, who is also a professor at MIT. "There's no current model that even comes close to this, by my reckoning."

This research was supported by a donation from Landon Clay.

Journal Reference:
Oktay Kirak, Eva-Maria Frickel, Gijsbert M. Grotenbreg, Heikyung Suh, Rudolf Jaenisch, and Hidde L. Ploegh. Transnuclear Mice with Predefined T Cell Receptor Specificities Against Toxoplasma gondii Obtained via SCNT. Science, 2010; 328 (5975): 243
DOI: 10.1126/science.1178590


Cancer Drug Effectiveness Substantially Advanced: Co-Administered Peptide Directs Medicines Deep Into Tumor Tissue

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ScienceDaily (Apr. 11, 2010) — Researchers have shown that a peptide (a chain of amino acids) called iRGD helps co-administered drugs penetrate deeply into tumor tissue. The peptide has been shown to substantially increase treatment efficacy against human breast, prostate and pancreatic cancers in mice, achieving the same therapeutic effect as a normal dose with one-third as much of the drug.

In a transformative paper published in the online edition of the journal Science, Erkki Ruoslahti, M.D., Ph.D., distinguished professor at Sanford-Burnham Medical Research Institute and founding member of the UC Santa Barbara-Sanford|Burnham Center for Nanomedicine, Kazuki N. Sugahara, M.D., Ph.D., Tambet Teesalu, Ph.D., and fellow researchers at the Center for Nanomedicine and the Cancer Center of Santa Barbara, announced this significant advance in cancer therapy.

"Drugs generally have difficulty penetrating tumors beyond a few cell diameters from a blood vessel," said Dr. Ruoslahti. "This leaves some tumor cells with a suboptimal dose, increasing the risk of both recurrence and drug resistance. The iRGD peptide solves this problem by activating a transport system in tumors that distributes co-injected drugs into the entire tumor and increases drug accumulation in the tumor."

Dr. Ruoslahti showed in the 1980s that a 3 amino-acid peptide motif (RGD -- Arginine- Glycine-Aspartic Acid) serves as a highly selective identifier of malignant tissue, binding to unique re-ceptors in the vasculature of cancers. The RGD peptide's ability to home to tumors has been used to design new compounds for cancer diagnosis and treatment.

The new variant of RGD (iRGD -- internalizing RGD) combines the RGD motif with a tissue penetration element called CendR. Like the earlier RGD peptides, iRGD homes to tumors, but exposure of the CendR motif when the iRGD is enzymatically cleaved activates a transport system through tumor blood vessel walls into the tumor core. In a paper published in Cancer Cell late last year, the research team showed that coupling iRGD to anti-cancer drugs allowed them to penetrate deep into tumors, effectively increasing the activity of the drugs.

The research reported in this latest Science paper adds a new and important twist to the story: The researchers made the unanticipated discovery that anti-cancer drugs do not need to be chemically attached to the iRGD peptide for iRGD to boost their efficacy. Simply co-administering iRGD with a drug enhances the drug's anti-cancer properties. Co -administration could be even more effective at delivering therapeutic agents inside tumors than conjugating the agents with the peptide. This new paradigm means that iRGD has the potential to enhance the efficacy of already approved drugs without creating new chemical entities, which would complicate the path to approval for clinical use.

In addition to being effective against human breast, prostate and pancreatic cancers grown in mice, iRGD can penetrate other tumor types and could possibly be used to treat most, if not all, solid tumors. The iRGD peptide was also shown to enhance the therapeutic effects of multiple types of anti-cancer drugs, including a small molecule drug, a monoclonal antibody and two nanoparticle drugs. Tumors essentially resistant to a particular drug showed good responses when the drug was combined with iRGD, and tumors partially responsive to another drug were eradicated by the combination.

"We are really excited about the potential of iRGD, and I'd like to thank my colleagues, Kazuki Sugahara and Tambet Teesalu in particular, who made this all happen," said Dr. Ruoslahti. "These results with human tumors in mice are very promising, but we still have to demonstrate the value of iRGD in treating cancers in humans."

Journal Reference:
Kazuki N. Sugahara, Tambet Teesalu, Priya Prakash Karmali, Venkata Ramana Kotamraju, Lilach Agemy, Daniel R. Greenwald, and Erkki Ruoslahti. Coadministration of a Tumor- Penetrating Peptide Enhances the Efficacy of Cancer Drugs. Science, 2010;
DOI: 10.1126/science.1183057


Thursday, April 22, 2010

How Cells Recognize Viral Toxins

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ScienceDaily (Mar. 26, 2010) — For many years it's been known that the fever, achiness and other symptoms you feel during the flu are triggered by a viral molecule that travels through the body acting like a toxin.

But what scientists haven't understood is how this molecule -- known as double-stranded RNA -- is recognized and taken up by cells.

New research from McMaster University has identified how specific proteins on the surface of cells, known as class A scavenger receptors, bind to double-stranded RNA and bring it into the cell, jumpstarting the immune response to a virus.

This finding, published in the March 26 issue of the journal PLoS Pathogens, could lead to the development of new antiviral therapies.

"Since the 1950s and '60s, it's been known that double-stranded RNA is a viral toxin," said Karen Mossman, an associate professor in the Department of Pathology and Molecular Medicine in the Michael G. DeGroote School of Medicine. "But what we haven't known is how cells recognize double-stranded RNA outside of the cell. We know how they respond to it. We know they take it up. But we've never appreciated how that happens."

Mossman, an investigator with the Michael G. DeGroote Institute for Infectious Disease Research at McMaster University, led a research team to investigate the "gatekeeper" function of scavenger receptors in both human and mouse cells. Until now, it was thought that the role of scavenger receptors was limited to the removal of foreign substances and waste materials from the body.

The researchers examined the five members of the class A scavenger receptor family and discovered that they all had overlapping functions in mediating the response of a cell to double-stranded RNA. They also found that no matter what type of cell they looked at -- including those not thought to express scavenger receptors -- all had at least two or three scavenger receptor family members.

"We found that they are ubiquitously expressed," Mossman said. "But that make sense to us because nearly every cell type responds to double-stranded RNA."

Previously, scavenger receptors were thought to be found only on white blood cells, or macrophages, and a small population of other blood cells. The McMaster research has shown that scavenger receptors are also expressed on fibroblast cells, which play an important role in healing wounds and maintaining the structural framework of tissue.

By identifying these receptors, the researchers have uncovered an ideal target for antiviral drug therapies which could potentially decrease the side effects associated with viral infections.

"Now that we know what to manipulate, we can start looking at how we can manipulate it to be beneficial during a viral infection," Mossman said. "Since all viruses make double-stranded RNA, targeting these receptors should be effective against many different viral infections including influenza and other pandemic viruses."

The research was supported with funds from the National Institutes of Health (NIH) and the Canadian Institutes of Health Research (CIHR).

"Since double-stranded RNA is produced by many different viruses, Dr. Mossman's findings may have a significant impact on the treatment of a wide variety of infections," said Dr. Marc Ouellette, scientific director of the Institute of Infection and Immunity at the Canadian Institutes of Health Research. "Understanding the mechanism employed by viruses to infect surrounding cells is important if we are to develop more effective antiviral therapies, and prevent the spread of viral infections to a wider population."

Stephanie DeWitte-Orr, a postdoctoral fellow in the Mossman lab, was the lead author of the study. The research also involved other collaborators in the Michael G. DeGroote Institute for Infectious Disease Research at McMaster University.

Journal Reference:
DeWitte-Orr et al. An Accessory to the ‘Trinity’: SR-As Are Essential Pathogen Sensors of Extracellular dsRNA, Mediating Entry and Leading to Subsequent Type I IFN Responses. PLoS Pathogens, 2010; 6 (3): e1000829 DOI: 10.1371/journal.ppat.1000829


Significant Findings About Protein Architecture May Aid in Drug Design, Generation of Nanomaterials

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ScienceDaily (Apr. 9, 2010) — Researchers in Singapore are reporting this week that they have gleaned key insights into the architecture of a protein that controls iron levels in almost all organisms. Their study culminated in one of the first successful attempts to take apart a complex biological nanostructure and isolate the rules that govern its natural formation.

The Nanyang Technological University team's work on the protein ferritin, the results of which appear in this week's issue of the Journal of Biological Chemistry, is expected to have significant ramifications on the fields of drug design and nanomaterials.

"Engineering the structure of a protein is one of the ultimate dreams of structural biologists," wrote one of the journal's peer reviewers, "and approaching that dream is greatly enabled through studies aimed at finding out what governs the nanoarchitecture of the protein."

Brendan P. Orner, the assistant professor who oversaw the team's work, described the protein ferritin as a potential model for explaining complicated protein structure in general.

Across the biological kingdoms, ferritin regulates the distribution of iron, which is necessary for a number of cellular functions but also forms reactive ions that can be lethal to cells. Shaped like a spherical nanocage, ferritin is made up of 24 proteins, and it sequesters the reactive iron ions in its hollow interior. In humans, ferritin prevents iron deficiency and overload.

"The rules that govern self-assembling nanosystems, like the ferritin model, are poorly understood," Orner explained. "We systematically analyzed the interactions between the 24 ferritin units that make up the nanocage and identified the hot spots that are crucial to the cage's formation."

Their goal was to discover which amino acids are responsible for assembling the cage, and they found that it is possible to both disassemble ferritin by removing single side chains of amino acids and, surprisingly, to stabilize the structure by removing other side chains.

Understanding the assembly of the nanocage could open the door to drug design that will disrupt the structure and function of defective proteins that cause or contribute to disease. It also may aid in the creation of biological nanostructures in which scientists can grow special particles and materials with a variety of properties and applications.

"Cell biology provides many structures that are on the nanoscale and have amazing complexity and symmetry," Orner said. "The problem is that many of these structures are, like ferritin, self-assembled proteins, and, if we are going to use them for nanomaterials applications, we need to understand the fundamentals that make them form this way naturally."

Orner and his team members are particularly interested in growing nanoparticles of precise dimensions inside ferritin shells. Already, they have developed a new method to grow gold nanoparticles in them.

"Slight deviations in size or shape can radically change nanoparticles' properties, particularly in the case of metals and semiconductors," Orner said. "Our ferritin proteins are hollow, so, when we grow mineral or metal clusters inside them, the growth stops when the nanoparticles reach the limits of the protein shell."

By studying the rules that control the folding and assembly of such a protein in nature, Orner said, the investigators hope to be able to manipulate them one day to create new proteins with novel sizes and shapes and, therefore, generate nanoparticles of novel sizes and shapes inside them.

"Those nanoparticles could be used for in-vitro assays to do high-throughput drug screening of some protein-protein interactions involved in virus infection and cancer, for example," he said.

Orner's team included doctoral students Yu Zhang and Rongli Fan, undergraduate students Siti Raudah, Huihian Teo and Gwenda Teo, and scholar Xioming Sun. Their research was funded by the Singapore Ministry of Education and Nanyang Technological University.

Their resulting article has been named a "Paper of the Week" by the Journal of Biological Chemistry, putting it in the top 1 percent of papers reviewed by the editorial board in terms of significance and overall importance.

Journal Reference:
Zhang et al. Alanine-shaving Mutagenesis to Determine Key Interfacial Residues Governing the Assembly of a Nano-cage Maxi-ferritin. Journal of Biological Chemistry, 2010; 285 (16): 12078 DOI: 10.1074/jbc.M109.092445


Scientists Put Proteins Right Where They Want Them

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ScienceDaily (Apr. 19, 2010) — Using a method they developed to watch moment to moment as they move a molecule to precise sites inside live human cells, Johns Hopkins scientists are closer to understanding why and how a protein at one location may signal division and growth, and the same protein at another location, death.

Their research, published Feb. 14 in Nature Methods, expands on a more limited method using a chemical tool to move proteins inside of cells to the periphery, a locale known as the plasma membrane.

"Where a particular protein is activated and the timing of that activation influence how a cell responds to outside stimulus," says Takanari Inoue, Ph.D., an assistant professor of cell biology at Johns Hopkins University School of Medicine. "Our goal with this newly expanded tool is to manipulate protein activities in many places in cells on a rapid timescale."

Cells cleverly have resolved the predicament of needing to respond to a near infinite array of external stimuli -- temperature, for instance -- even though they employ only a limited number of molecular players. The notion is that a single protein assumes multiple roles by changing its location or altering the speed and duration of activation.

Chemical signaling inside cells connects protein molecules through complex feedback loops and crosstalk, Inoue says, so knowing exactly how each protein contributes to which signals at what locations requires the ability to rapidly move proteins of interest to specific organelles found in cells. These include mitochondria (the power generators of cells) and Golgi bodies (the delivery systems of cells).

The Hopkins team chose the signaling protein Ras as the molecule it would attempt to send packing throughout a cell's interior. A regulator of cell growth that's often implicated in cancer, Ras has been long studied and it's known to be a molecular switch. However, no one has had the ability to discern what Ras does at different locations such as Golgi bodies and mitrochondria, much less what happens when Ras is activated simultaneously at any combination of these and other organelles.

Working with live human HeLa cells and Ras under a microscope, the team used a dimerization probe consisting of a special small molecule that simultaneously attracts two proteins that wouldn't normally have an affinity for each other and binds them together. In this system, one of the partner proteins is anchored to an organelle and the other is free floating inside the cell. Adding a chemical dimerizer induces the free protein to join the tethered one.

Using scissor-like enzymes, the team sliced and diced the DNA of the paired proteins to change the molecular address of its destination. They cut out the "mailing address" -- known as a targeting sequence -- that formerly delivered the protein unit to the plasma membrane and replaced it with new addresses (targeting sequences) that shipped it instead to specific organelles.

"We were able to manipulate protein activities in situ and very rapidly on each individual organelle," Inoue said. "Ultimately, this will help us to better understand protein function at these critical cellular components."

This study was funded by the National Institutes of Health.

In addition to Inoue, authors of this paper are Toru Komatsu, Igor Kukelyansky, J. Michael McCaffery, Tasuku Ueno and Lidenys C. Varela, all of Johns Hopkins.

Journal Reference:
Komatsu et al. Organelle-specific, rapid induction of molecular activities and membrane tethering. Nature Methods, 2010; 7 (3): 206 DOI: 10.1038/nmeth.1428


'Start/stop Switch' for Retroviruses Found

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ScienceDaily (Apr. 10, 2010) — A University of British Columbia doctoral candidate has discovered a previously unknown mechanism for silencing retroviruses, segments of genetic material that can lead to fatal mutations in a cell's DNA.

The findings, published in the journal Nature, could lead to new cancer treatments that kill only tumour cells and leave healthy surrounding tissue unharmed.

Danny Leung, a 27-year-old graduate student in the laboratory of Asst. Prof. Matthew Lorincz in the Dept. of Medical Genetics, UBC Faculty of Medicine, found that a protein called ESET is crucial to preventing the activity of endogenous retroviruses in mouse embryonic stem cells. Distant relatives of such retroviruses are more active in the cells of testicular, breast and skin cancers in humans.

If ESET can be blocked, retroviruses would become dramatically more active, thus either killing the cancer cells hosting them or flagging them as targets for the immune system.

Leung, who was co-lead author with a graduate student at Kyoto University in Japan, has devoted his studies at UBC to the growing field of epigenetics -- changes to the genome that do not involve changes to the underlying genetic code. Such changes determine whether or not a gene is expressed.

The common method for silencing certain genes is DNA methylation, in which a chemical group attaches to the DNA structure. But Leung and his collaborators at UBC and Kyoto University found that the activity of ESET is far more potent than DNA methylation in silencing retroviruses in embryonic stem cells of mice. This indicates an independent parallel pathway of silencing the retroviruses.

Their research has direct bearing on cancer treatments because cancer cells are stem-like -- they can differentiate into other types of cells. Also, for unknown reasons, cancer cells have significantly less DNA methylation than normal cells. So blocking ESET holds the promise of affecting only cancer cells, allowing retroviruses to flourish to the detriment of their hosts. Normal, differentiated cells, which still have DNA methylation to keep retroviruses in check, would be unaffected.

"Inhibiting ESET may affect just the cancer cells, allowing further expression of retroviruses, which in turn would kill the cancer cells," says Leung, who is in his third year of graduate studies at UBC. His co-lead author on the paper, Toshiyuki Matsui, is a student in the lab of Yoichi Shinkai at Kyoto University.

Journal Reference:
Toshiyuki Matsui, Danny Leung, Hiroki Miyashita, Irina A. Maksakova, Hitoshi Miyachi, Hiroshi Kimura, Makoto Tachibana, Matthew C. Lorincz, Yoichi Shinkai. Proviral silencing in embryonic stem cells requires the histone methyltransferase ESET. Nature, 2010; 464 (7290): 927
DOI: 10.1038/nature08858


Key Protein Aids in DNA Repair

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ScienceDaily (Apr. 12, 2010) — Scientists have shown in multiple contexts that DNA damage over our lifetimes is a key mechanism behind the development of cancer and other age-related diseases. Not everyone gets these diseases, because the body has multiple mechanisms for repairing the damage caused to DNA by aging, the environment and other human behaviors -- but the mechanisms behind certain kinds of DNA repair have not been well-understood.

In a paper published in the journal Nature, researchers at the University of North Carolina at Chapel Hill's Lineberger Comprehensive Cancer Center have shown that a particular protein -- called Ku -- is particularly adept at healing damaged strands of DNA.

According to Dale Ramsden, PhD, associate professor in the department of biochemistry and biophysics and a member of the curriculum in genetics and molecular biology, Ku is a very exciting protein because it employs a unique mechanism to repair a particularly drastic form of DNA damage.

"Damage to DNA in the form of a broken chromosome, or double strand break, can be very difficult to repair -- it is not a clean break and areas along the strand may be damaged at the level of the fundamental building blocks of DNA -- called nucleotides," he notes.

Broken chromosomes can be compared to a break in a strand of yarn made up of several different threads or plies. Unless scissors are used to cut the yarn, the strand frays and may break or be damaged at several different places up and down the length of the yarn. These rough ends get "dirty" -- making them harder to repair.

"It has been assumed in the past that double strand breaks are the most difficult class of DNA damage to repair and it is often presumed that they simply can not be repaired accurately," says Ramsden.

The team found that the protein Ku, which has long been appreciated for its ability to find chromosome breaks along a strand of DNA, actually removes the "dirt" at broken chromosome ends, allowing for much more accurate repair than believed possible.

"This protein actually heals at the nucleotide level as well as the level of the chromosome," says Ramsden, comparing its action to washing and disinfecting a cut before trying to sew it up to promote healing.

The team is hopeful that the discovery of this mechanism for DNA repair may lead to a target for treatment of age-related diseases caused by chromosome damage in the future.

Other team members include Steven Roberts, Natasha Strande, Martin Burkhalter, Christina Strom and Jody Havener from UNC and Paul Hasty from the University of Texas Health Science Center at San Antonio.

Journal Reference:
Roberts et al. Ku is a 5′-dRP/AP lyase that excises nucleotide damage near broken ends. Nature, 2010; DOI: 10.1038/nature08926


A Push Makes Neuron Longer

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ScienceDaily (Mar. 30, 2010) — Some neurons from spinal cord have quite long neurites, but the molecular mechanism of long-neurite outgrowth has been still mysterious. The research team led by Assistant Professor Koji Shibasaki in Gumma University and Professor Makoto Tominaga in National Institute for Physiological Sciences (NIPS) in Japan, reported that TRPV2 receptor can act as mechanical stretch-sensor in developing neurons to help their neurites grow much longer.

They report their finding in Journal of Neuroscience published on March 31, 2010.

TRPV2 receptor has been known as noxious heat-sensor (activated by >52°C). The research group found that, during early embryonic stages, TRPV2 had been already expressed in restricted neurons (spinal motor neurons and DRG sensory neurons), although embryos don't have any situation to be exposed to such high temperature. Thus, these results strongly indicate that TRPV2 has a distinct role to contribute neuronal development except for its thermo-sensitive role. The research group found that activation of TRPV2 in developing neurons caused further axon outgrowth. Endogenous TRPV2 was activated in a membrane-stretch dependent manner in developing neurons. Thus, for the first time, Dr. Shibasaki and the research group elucidated that TRPV2 is an important regulator for axon outgrowth through its activation by mechanical membrane stretch during development.

"We revealed the molecular mechanism why spinal motor and DRG sensory axons can extend such long neuritis toward peripheral tissues. It is really important finding that extending axon can convert mechanical power to electrical energy. I hypothesized that axon outgrowth is regulated by the positive feedback mechanisms through membrane stretch. Now, we can explain why rehabilitation is necessary to improve severe neuronal damage (ex. after traffic accident). The answer could be the activation of TRPV2 by movement of damaged tissue. This molecular mechanism can be applied to neuronal repair, if we can synthesize TRPV2 targeted medication," said Dr Shibasaki.


Novel Genetic Pathway Responsible for Triggering Vascular Growth

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ScienceDaily (Apr. 5, 2010) — Most solid cancers can't grow beyond a limited size without an adequate blood supply and supporting vascular network. Because of this, cancer researchers have sought to understand how a tumor's vascular network develops -- and, more importantly, how to prevent it from developing: If the vascular network never develops, the theory goes, the tumor cannot grow.

Researchers at the University of Massachusetts Medical School have discovered a critical step for blood vessel growth in zebrafish embryos, providing new insight into how vascular systems develop and offering a potential therapeutic target for preventing tumor growth. UMMS Associate Professor of Molecular Medicine and the Program in Gene Function and Expression Nathan Lawson, PhD, and colleagues have identified a novel microRNA-mediated genetic pathway responsible for new blood vessel growth, or angiogenesis, in zebrafish embryos.

Published online by Nature, Dr. Lawson's work provides new insights into how vascular systems use the forces of existing blood flow to initiate the growth of new vessels.

Focusing on the development of the fifth and sixth aortic arches in the zebrafish, Dr. Lawson describes how the forces exerted by blood flow on endothelial cells are a critical component for expressing a microRNA that triggers new vessel development. In the early stages of development, when blood flow is present in the aortic vessels, but the vascular linkages between the two arches have yet to be established, the stimulus provided by active blood flow leads to expression of an endothelial-cell specific microRNA (mir-126). In turn, this microRNA turns on vascular endothelial growth factor (VEGF), a chemical signal produced by surrounding cells that normally stimulates angiogenesis. Thus, blood flow allows the endothelial cells to respond to VEGF by growing into new blood vessels. However, when blood flow in the aortic arches was restricted, mir-126 failed to be expressed. In the absence of this microRNA, new blood vessels were unable to develop due to a block in VEGF signaling.

"We have known for over a hundred years that blood flow makes new vessels grow," said Dr. Lawson. "But we never really knew how cells in a growing vessel interpreted this signal. Our results show that miR-126 is the crucial switch that allows flow to turn on VEGF signaling and drive blood vessel growth. Since VEGF is crucial for tumor progression, not to mention a number of other vascular diseases, our findings may provide new ways to modify this pathway in these settings."

In his research, Dr. Lawson identifies the microRNA as a key facilitator in the integration of the physiological stimulus of blood flow with the activation of VEGF signaling, which guides angiogenesis, in endothelial cells. As a result, regulation of the microRNA, mir-126, could be a potential therapeutic target in limiting blood vessel development in solid cancers.

Journal Reference:
Stefania Nicoli, Clive Standley, Paul Walker, Adam Hurlstone, Kevin E. Fogarty & Nathan D. Lawson. MicroRNA-mediated integration of haemodynamics and Vegf signalling during angiogenesis. Nature, 2010; DOI: 10.1038/nature08889


Zebrafish Study With Human Heart Implications: Cellular Grown-Ups Outperform Stem Cells in Cardiac Repair

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ScienceDaily (Mar. 28, 2010) — Bony fish like the tiny zebrafish have a remarkable ability that mammals can only dream of: if you lop off a chunk of their heart they swim sluggishly for a few days but within a month appear perfectly normal. How they accomplish this -- or, more importantly, why we can't -- is one of the significant questions in regenerative medicine today.

In a paper published in the March 25, 2010 issue of Nature, researchers working at the Salk Institute for Biological Studies and the Center of Regenerative Medicine in Barcelona (CMRB) identified a fish heart cell population that is the source of this astonishing healing feat, a finding that could provide insight into how mammalian hearts might be coaxed into repairing themselves after injury brought on by heart attack.

Juan Carlos Izpisúa Belmonte, Ph.D., professor in the Gene Expression Laboratory, and colleagues report that it is not stem cells -- those "usual" regeneration suspects -- that patch up an injured fish heart. Instead, repair is accomplished by differentiated cardiac muscle cells known as cardiomyocytes, those cellular grown-ups whose normal job is to supply the contractile force of the heart.

"What the results of our study show is that mother nature utilizes other ways besides going all the way back to pluripotent stem cells to regenerate tissues and organs," says Izpisúa Belmonte, noting that at least in fish, the body may have evolved surprising repair strategies driven by cell types more seasoned than stem cells.

To identify which cells actually filled in excised zebrafish heart muscle, Izpisúa Belmonte's team first employed some genetic engineering to only make cardiomyocytes "transgenic" by inserting into them a tracer gene that made them glow green under a microscope.

They then literally chopped off about 20% of each fish ventricle and waited a couple of weeks for the hearts to regenerate: if regenerated heart muscle didn't glow, it would mean that cells other than cardiomyocytes, such as a cardiac stem cell population, had replaced the damaged muscle.

But in a striking finding, all regenerated heart muscle cells glowed green, indicating that well established cardiomyocytes remaining after injury had likely regressed to a more "youthful" state, started dividing again to replenish lost cells, and then matured a second time into new heart muscle. The group also showed cardiomyocytes recaptured lost youth in part by re-activating the production of proteins associated with cell proliferation, factors typically expressed in immature progenitors.

Human hearts cannot undergo these types of regenerative changes on their own. When damaged by heart attack, our heart muscle is replaced by scar tissue incapable of contracting. However, prior to heart failure, damaged mammalian heart muscle cells enter a save-yourself state known as "hibernation," in which they cease contracting in an effort to survive.

Chris Jopling, Ph.D., a postdoctoral fellow of Izpisúa Belmonte's at CMRB and first author of the study, sees human heart "hibernation" as significant. "During heart regeneration in the zebrafish we found that cardiomyocytes displayed structural changes similar to those observed in hibernating cardiomyocytes," he said, noting that those changes were actually necessary before the fish cardiomyoctes could start dividing. "Because of these similarities, we hypothesize that hibernating mammalian cardiomyocytes may represent cells that are attempting to proliferate."

So the good news is that mammalian hearts can undergo a kind of metabolic "downsizing" that is a prelude to cell division. "This idea fits nicely with the findings from a number of groups -- that forced expression of cell cycle regulators can induce cardiomyocyte proliferation in mammals," says Jopling. "Maybe all they need is a bit of a push in the right direction."

A search is on for factors that could supply that "push." Although he is optimistic about the outcome, Izpisúa Belmonte also feels that the study should caution researchers not to overlook potential contributions that mature cells might make to regeneration. "We can no longer view differentiated cells as being a static endpoint of the differentiation process," says Izpisúa Belmonte, who also directs the CMRB. "If we could mimic in mammalian cells what happens in zebrafish, perhaps we could be in a position to understand why regeneration does not occur in humans."

Also contributing to this work were Merce Marti, Ph.D., Angel Raya, M.D., Ph.D., Edward Sleep, and Marina Raya, all of the CMRB in Spain. The study was funded in part by Fundacion Cellex, the Ipsen Foundation, the G. Harold and Leila Y. Mathers Charitable Foundation, the National Institutes of Health, and Sanofi-Aventis.

Journal Reference:
Jopling et al. Zebrafish heart regeneration occurs by cardiomyocyte dedifferentiation and proliferation. Nature, 2010; 464 (7288): 606 DOI: 10.1038/nature08899


Wednesday, April 21, 2010

Abusing Internet Explorer 8's XSS Filters

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Internet Explorer 8 introduced a new type of defense against Cross-site Scripting (XSS) attacks.The idea was to build filters into the browser which can detect and prevent certain types of malicious XSS attacks. Most filter based XSS approaches are implemented on the server side inside a web application or as part of a Web Application Firewall.This made the Microsoft approach a somewhat novel approach but one which other browser vendors have begun to follow. Although the filters do not protect against all types of XSS attacks, nor do they attempt to, they do attempt to raise the bar for a would-be attacker by making certain commonly attack scenarios non-exploitable.

Download PDF


OWASP Top 10 for 2010

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The OWASP Top Ten provides a powerful awareness document for web application security. The OWASP Top Ten represents a broad consensus about what the most critical web application security flaws are. Project members include a variety of security experts from around the world who have shared their expertise to produce this list. Versions of the 2007 were translated into English, French, Spanish, Japanese, Korean and Turkish and other languages. Translation efforts for the 2010 version are underway and they will be posted as they become available.

This version was updated based on numerous comments received during the comment period after the release candidate was released in Nov. 2009.

The OWASP Top 10 Web Application Security Risks for 2010 are:
A1: Injection
A2: Cross-Site Scripting (XSS)
A3: Broken Authentication and Session Management
A4: Insecure Direct Object References
A5: Cross-Site Request Forgery (CSRF)
A6: Security Misconfiguration
A7: Insecure Cryptographic Storage
A8: Failure to Restrict URL Access
A9: Insufficient Transport Layer Protection
A10: Unvalidated Redirects and Forwards

Download and more info:


Tor Exit Node + sslstrip

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sslstrip, hijacking SSL in network

posted Feb 23rd 2009 7:25pm by Eliot Phillips

Last week at Black Hat DC, [Moxie Marlinspike] presented a novel way to hijack SSL. You can read about it in this Forbes article, but we highly recommend you watch the video. sslstrip can rewrite all https links as http, but it goes far beyond that. Using unicode characters that look similar to / and ? it can construct URLs with a valid certificate and then redirect the user to the original site after stealing their credentials. The attack can be very difficult for even above average users to notice. This attack requires access to the client’s network, but [Moxie] successfully ran it on a Tor exit node.

This is a hacker’s hack – a really cool proof of concept. But in the real world, someone would have to hack your bank, your ISP or your home network. If they root the bank (e.g. Heartland) why bother with SSL traffic, just get the raw data. If they get your PC, they can grab keystrokes regardless of how good the network security is. And let’s face it, there are a lot of people who can be fooled by a site that just looks the same, never mind the URL or certificate. Although, just maybe, a wireless hotspot at a hotel or cafe might be a candidate for sslstrip. I think it would be hard – diverting traffic through a PC instead of going straight to a switch – but it’s probably easier than hacking an ISP or bank.

The paper is worth a read:
.. hey, maybe combine it with the BGP attack: (that was amazing – stole all the DEFCON traffic for an hour or so..)


Tool to check for bad nodes:


Read also:

Monday, April 19, 2010

Scientists Track Variant of Gene-Regulating Protein in Embryonic Stem Cells

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ScienceDaily (Apr. 16, 2010) — The journey from embryonic stem cell to a fully developed liver, heart or muscle cell requires not only the right genes, but genes that are turned on and off at the right time -- a job that is handled in part by DNA-packaging proteins known as histones. But it turns out that not all histones are created equally. New research from Rockefeller University shows that minute variations between histones play an important role in determining how and when genes are read.

The findings, recently reported in the journal Cell, hint at an unimagined complexity of the genome and may open a new avenue of investigation regarding the mysterious causes of the human genetic disease known as ATR-X syndrome.

"Our work shows that the regulation of histone variant localization -- the shape of the so-called epigenetic landscape at different regions of the genome -- is more complex than previously thought," says first author Aaron Goldberg, an M.D.-Ph.D. student in the Laboratory of Chromatin Biology and Epigenetics at Rockefeller.

Cells use a number of mechanisms to establish and maintain the activation or silencing of specific genes. Among these is the chemical modification of histones. But in addition, histone variants, which differ from other histone proteins by just a handful of amino acids, can be inserted at specific locations in the genome to provide a cell with another mechanism for fine-tuning gene regulation.

Previous work had identified a number of histone variants, including one known as H3.3. Studies in fruit flies established that histone H3.3 is prevalent in regions of the genome where active genes are found as well as at the ends of chromosomes, in telomeres.

To track histone H3.3 and distinguish it from other histone proteins, Goldberg and C. David Allis, Joy and Jack Fishman Professor and head of the Laboratory of Chromatin Biology and Epigenetics, collaborated with researchers at Sangamo Biosciences. Together, they designed and used a DNA-cutting enzyme called a zinc finger nuclease to chemically tag histone H3.3 and distinguish it from other histone proteins in mouse embryonic stem cells. They then used a technique called ChIP sequencing to produce the first genome-wide maps of H3.3 localization first in mammalian embryonic stem cells and then again after the cells had differentiated to become neurons. In collaboration with bioinformatics experts at the Albert Einstein College of Medicine, they found that the location of histone H3.3 throughout the genome changed with stem cell differentiation.

Most scientists in the field believed that a factor known as HIRA was responsible for controlling the localization of histone H3.3. To test this idea, Goldberg and Allis used ChIP sequencing to track H3.3 in normal embryonic stem cells and in genetically modified embryonic stem cells that lack HIRA, generated by colleagues in the United Kingdom. The researchers compared the genome-wide localization of H3.3 in the presence and absence of HIRA and, as expected, found that HIRA is required for H3.3 localization at genes. Without HIRA, H3.3 was mostly gone from genes.

But that wasn't the whole story. Even without HIRA, H3.3 was still present in many other specific areas of the genome. The researchers went on to identify several additional proteins associated with H3.3.

Two of them, ATRX and Daxx, had never before been linked to H3.3. ATRX is particularly interesting, because mutations in the gene that codes for this protein in humans causes a genetic disease known as the α-thalassemia and X-linked mental retardation (ATR-X) syndrome.

"Instead of one universal factor for a particular histone variant, different factors are used to localize the same histone variant (H3.3) to different regions of the genome," says Allis. "We now know that genes, transcription factor binding sites and telomeres all have their own dedicated series of proteins to properly localize histone H3.3."

"Our work also demonstrates an important new function of the ATRX protein: the proper localization of histone H3.3 to telomeres," says Goldberg. "This finding may provide a clue as to how mutations in the ATRX gene lead to the human genetic disease of α-thalassemia and X-linked mental retardation."

Journal Reference:
Goldberg et al. Distinct Factors Control Histone Variant H3.3 Localization at Specific Genomic Regions. Cell, 2010; 140 (5): 678 DOI: 10.1016/j.cell.2010.01.003


Chinese Scientists Discover Marker Indicating the Developmental Potential of Stem Cells

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ScienceDaily (Apr. 18, 2010) — Researchers in China are reporting that they have found a way to determine which somatic cells -- or differentiated body cells -- that have been reprogrammed into a primordial, embryonic-like state are the most viable for therapeutic applications.

In a paper published online by the Journal of Biological Chemistry, two collaborating teams from institutes at the Chinese Academy of Sciences point to a marker they found in induced-pluripotent stem cells, or iPS cells, taken from mice. That marker is a cluster of small RNA whose expression appears strictly correlated with levels of pluripotency, or "stemness." (The more pluripotent, the more likely a stem cell will develop into the desired tissue, organ or being.)

"We identified a genomic region encoding several genes and a large cluster of microRNAs in the mouse genome whose expression is high in fully pluripotent embryonic stem cells and iPS cells but significantly reduced in partially pluripotent iPS cells, indicating that the Dlk1-Dio3 region may serve as a marker," said Qi Zhou, a researcher at the CAS Institute of Zoology and co-author of the paper. "No other genomic regions were found to exhibit such clear expression changes between cell lines with different pluripotent levels."

After the creation of the first iPS cells in Japan in 2006, Zhou and others set out to determine whether the reprogrammed adult cells are versatile enough to generate an entire mammalian body, as embryonic stem cells can.

Then, last summer, Zhou announced that his team had reprogrammed somatic cells of mice, injected them into embryos and created 27 live offspring, which clearly demonstrated that iPS cells can, like embryonic stem cells, produce healthy adults. Though lauded as a huge step forward, they also found not all iPS cells were perfect: Many of the iPS cell lines used did not produce mice, and some of the mice that were produced had abnormalities.

"The success rate of obtaining iPS cells with full pluripotency was still extremely low, which significantly hindered the application of iPS cells in therapeutics and other aspects," Zhou said.

Believing that there might be some intrinsic gene expression difference between the lines of iPS cells with varying levels of pluripotency that could be identified at early culture stages, so that less viable lines could be abandoned and more viable lines focused on, Zhou teamed up with bioinformatics specialist Xiu-Jie Wang, who works at the Chinese academy's Institute of Genetics and Developmental Biology.

Together, their groups profiled the small RNA expression patterns of ES and iPS cell lines from different genetic backgrounds and with different pluripotent levels using Solexa technology.

"There are nearly 50 miRNAs encoded in this region, and those expressed miRNAs all exhibited consistent and significant expression differences between stem-cell lines with different pluripotency levels," Wang said. "With this discovery, iPS cells with different pluripotency can be distinguished in their early phases, which will, thus, significantly improve the production of full pluripotent iPS cells and promote their application in disease therapy," Wang said.

As stem cells can be applied in the treatment of many diseases related to tissue replacement or organ implantation, Zhou said, if the team's findings also are true for humans, "it will cause a revolution in stem-cell research and the application of it in the very near future."

The team's work is supported by China's National High-Technology Research and Development Program, Ministry of Science and Technology and National Natural Science Foundation. Their Journal of Biological Chemistry paper went online April 9 and will appear in a forthcoming print edition.

Other co-authors included Lei Liu, Guan-Zheng Luo, Wei Yang, Xiaoyang Zhao, Qinyuan Zheng, Zhuo Lv, Wei Li, Hua-Jun Wu and Liu Wang.

Journal Reference:
L. Liu, G. Z. Luo, W. Yang, X. Zhao, Q. Zheng, Z. Lv, W. Li, H. J. Wu, L. Wang, X. J. Wang, Q. Zhou. Activation of the imprinted Dlk1-Dio3 region correlates with pluripotency levels of mouse stem cells. Journal of Biological Chemistry, 2010;
DOI: 10.1074/jbc.M110.131995


Dividing Cells 'Feel' Their Way Out Of Warp

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ScienceDaily (Sep. 11, 2009) — Every moment, millions of a body's cells flawlessly divvy up their genes and pinch perfectly in half to form two identical progeny for the replenishment of tissues and organs — even as they collide, get stuck, and squeeze through infinitesimally small spaces that distort their shapes.

Now Johns Hopkins scientists, working with the simplest of organisms, have discovered the molecular sensor that lets cells not only "feel" changes to their neat shapes, but also to remodel themselves back into ready-to-split symmetry. In a study published September 15 in Current Biology, the researchers show that two force-sensitive proteins accumulate at the sites of cell-shape disturbances and cooperate first to sense the changes and then to resculpt the cells. The proteins — myosin II and cortexillin I — monitor and correct shape changes in order to ensure smooth division.

"What we found is an exquisitely tuned mechanosensory system that keeps the cells shipshape so they can divide properly," says Douglas N. Robinson, Ph.D., an associate professor of Cell Biology, Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine.

Faulty cell division can put organisms, including people, on the pathway to diseases such as cancer, Robinson notes, and a better understanding of how cells respond to mechanical stress on their shapes could present new targets for both diagnosing and treating such diseases.

Working with hardy, single-celled protozoa that move and divide similarly to human cells, the scientists watched through microscopes while they deformed the cells' shapes with a tiny instrument that, like a soda straw, sucks in on the cell surface and creates distorted shapes.

"This particular method, based on a very old principle that dates back to Archimedes, enables us to deform cells without killing them, much in the same way that natural processes in the body constantly assault them, Robinson says."

Once the cells were warped, the scientists monitored the movements of fluorescent-tagged myosin II and cortexillin I. Myosin, which normally accumulates in the middles of cells during division to help power that process, collected instead at the sites of disturbances made by the micropipette. Also amassing with myosin was cortexillin I, a so-called actin-crosslinking protein that, like glue, holds the toothpick-like filaments of a cell's housing together.

In the experiments, as soon as the two proteins accumulated to a certain level, the cells contracted, escaping the pipettes and assuming their original shapes. After the cells righted themselves, the proteins realigned along the cells' midlines and pinched to divide symmetrically into two daughter cells.

The researchers repeated the experiment using cells engineered to lack myosin II and then again with cells lacking cortexillin I. They discovered that cortexillin I responded to deformations except when myosin II was removed, and myosin II responded to deformations except when cortexillin I was removed.

"It's clear that the two need each other to operate as a cellular mechanosensor," Robinson says.

The research was funded by grants from the National Institutes of Health, the American Cancer Society and the National Science Foundation.

In addition to Robinson, authors of the paper are Yixin Ren, Janet C. Effler, Pablo A. Iglesias and Tianzhi Luo, all of Johns Hopkins; Melanie Norstrom and Ronald S. Rock, both of the University of Chicago; and Richard A. Firtel, University of California San Diego.

Watch dividing cells "feel" their way out of warp: