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  • richardmitnick 9:43 am on September 14, 2014 Permalink | Reply
    Tags: , Biochemistry, , , ,   

    From M.I.T Tech Review: “Gene-Silencing Drugs Finally Show Promise” 

    MIT Technology Review
    M.I.T Technology Review

    September 14, 2014
    Kevin Bullis

    After more than a decade of disappointment, a startup leads the development of a powerful new class of drugs based on a Nobel-winning idea.

    The disease starts with a feeling of increased clumsiness. Spilling a cup of coffee. Stumbling on the stairs. Having accidents that are easy to dismiss—everyone trips now and then.

    But it inevitably gets worse. Known as familial amyloid polyneuropathy, or FAP, it can go misdiagnosed for years as patients lose the ability to walk or perform delicate tasks with their hands. Most patients die within 10 to 15 years of the first symptoms.

    There is no cure. The disease is caused by malformed proteins produced in the liver, so one treatment is a liver transplant. But few patients can get one—and it only slows the disease down.

    Now, after years of false starts and disappointment, it looks like an audacious idea for helping these patients finally could work.

    In 1998, researchers at the Carnegie Institution and the University of Massachusetts made a surprising discovery about how cells regulate which proteins they produce. They found that certain kinds of RNA—which is what DNA makes to create proteins—can turn off specific genes. The finding, called RNA interference (RNAi), was exciting because it suggested a way to shut down the production of any protein in the body, including those connected with diseases that couldn’t be touched with ordinary drugs. It was so promising that its discoverers won the Nobel Prize just eight years later.

    Inspired by the discovery, another group of researchers—including the former thesis supervisor of one of the Nobel laureates—founded Alnylam in Cambridge, Massachusetts, in 2002. Their goal: fight diseases like FAP by using RNAi to eliminate bad proteins (see “The Prize of RNAi” and “Prescription RNA”). Never mind that no one knew how to make a drug that could trigger RNAi. In fact, that challenge would bedevil the researchers for the better part of a decade. Along the way, the company lost the support of major drug companies that had signed on in a first wave of enthusiasm. At one point the idea of RNAi therapy was on the verge of being discredited.

    But now Alnylam is testing a drug to treat FAP in advanced human trials. It’s the last hurdle before the company will seek regulatory approval to put the drug on the market. Although it’s too early to tell how well the drug will alleviate symptoms, it’s doing what the researchers hoped it would: it can decrease the production of the protein that causes FAP by more than 80 percent.

    This could be just the beginning for RNAi. Alnylam has more than 11 drugs, including ones for hemophilia, hepatitis B, and even high cholesterol, in its development pipeline, and has three in human trials —progress that led the pharmaceutical company Sanofi to make a $700 million investment in the company last winter. Last month, the pharmaceutical giant Roche, an early Alnylam supporter that had given up on RNAi, reversed its opinion of the technology as well, announcing a $450 million deal to acquire the RNAi startup Santaris. All told, there are about 15 RNAi-based drugs in clinical trials from several research groups and companies.

    “The world went from believing RNAi would change everything to thinking it wouldn’t work, to now thinking it will,” says Robert Langer, a professor at MIT, and one of Alnylam’s advisors.

    Delivering Drugs

    Alnylam started with high hopes. Its founders, among them the Nobel laureate and MIT biologist Philip Sharp, had solved one of the biggest challenges facing the idea of RNAi therapies. When RNAi was discovered, the process was triggered by introducing a type of RNA, called double stranded RNA, into cells. This worked well in worms and fruit flies. But the immune system in mammals reacted violently to the RNA, causing cells to die and making the approach useless except as a research tool. The Alnylam founders figured out that shorter strands, called siRNA, could slip into mammalian cells without triggering an immune reaction, suggesting a way around this problem.

    Yet another huge problem remained. RNA interference depends upon delivering RNA to cells, tricking the cells into allowing it through the protective cell membrane, and then getting the cells to incorporate it into molecular machinery that regulates proteins. Scientists could do this in petri dishes but not in animals.

    Alnylam looked everywhere for solutions, scouring the scientific literature, collaborating with other companies, considering novel approaches of its own. It focused on two options. One was encasing RNA in bubbles of fat-like nanoparticles of lipids. They are made with the same materials that make up cell membranes—the thought was that the cell would respond well to the familiar substance. The other approach was attaching a molecule to the RNA that cells like to ingest, tricking the cell into eating it.

    And both approaches worked, sort of. Researchers were able to block protein production in lab animals. But getting the delivery system right wasn’t easy. The early mechanisms were too toxic at the doses required to be used as drugs.

    As a result, delivering RNA through the bloodstream like a conventional drug seemed a far-off prospect. The company tried a shortcut of injecting chemically modified RNA directly into diseased tissue —for example, into the retina to treat eye diseases. That approach even got to clinical trials. But it was shelved because it didn’t perform as well as up-and-coming drugs from other companies.

    By 2010, some of the major drug companies that were working with and investing in Alnylam lost patience. Novartis decided not to extend a partnership with Alnylam; Roche gave up on RNAi altogether. Alnylam laid off about a quarter of its workers, and by mid-2011, its stock price had plunged by 80 percent from its peak.

    But Alnylam and partner companies, notably the Canadian startup Tekmira, were making steady progress in the lab. Researchers identified one part of the lipid nanoparticles that was keeping them from delivering its cargo of RNA to the right part of a cell. That was “the real eureka moment,” says Rachel Meyers, Alnylam’s vice president of research. Better nanoparticles improved the potency of a drug a hundredfold and its safety by about five times, clearing the way for clinical trials for FAP—a crucial event that kept the company alive.

    Even with that progress, Alnylam needed more. The nanoparticle delivery mechanism is costly to make and requires frequent visits to the hospital for hour-long IV infusions—something patients desperate to stay alive will put up with, but likely not millions of people with high cholesterol.

    So Alnylam turned to its second delivery approach—attaching molecules to RNA to trick cells into ingesting it. Researchers found just the right inducement—attaching a type of sugar molecule. This approach allows for the drug to be administered with a simple injection that patients could give themselves at home.

    In addition to being easier to administer, the new sugar-based drugs are potentially cheaper to make. The combination of low cost and ease-of-use is allowing Alnylam to go after more common diseases—not just the rare ones that patients will go to great lengths to treat. “Because we’ve made incredible improvements in the delivery strategy,” Meyers says, “we can now go after big diseases where we can treat millions of patients potentially.”

    The Next Frontier

    In a sixth-floor lab on the MIT campus, postdoctoral researcher James Dahlman takes down boxes from a high shelf. They contain hundreds of vials, each containing a unique type of nanoparticle that Dahlman synthesized painstakingly, one at a time. “It turns out we have a robot in the lab that can do that,” he says. “But I didn’t know about it at the time.”

    Dahlman doesn’t work for Alnylam; he had been searching for the next great delivery mechanism, one that could greatly expand the diseases that can be treated by RNAi. Some of the materials look like clear liquids. Some are waxy, some like salt crystals. He points to a gap in the rows of vials, where a vial is conspicuously missing. “That’s the one that worked. That’s the miracle material,” he says.

    For all of their benefits, the drug delivery mechanisms Alnylam uses have one flaw—they’re effective only for delivering drugs to liver cells. For a number of reasons, the liver is a relatively easy target—that’s where all kinds of nanoparticles tend to end up. Alnylam sees the potential for billions of dollars in revenue from liver-related diseases. Yet most diseases involve other tissues in the body.

    Dahlman and his colleagues at MIT are some of the leaders in the next generation of RNAi delivery—targeting delivery to places throughout the body. Last month, in two separate articles, they published the results of studies showing that Dahlman’s new nanoparticles are a powerful way to deliver RNAi to blood vessel cells, which are associated with a wide variety of diseases. The studies showed that the method could be used to reduce tumor growth in lung cancer, for example.

    Treating cancer is one area where RNAi’s particular advantages are expected to shine. Conventional chemotherapy affects more than just the target cancer cells—it also hurts healthy tissue, which is why it makes people feel miserable. But RNAi can be extremely precise, potentially shutting down only proteins found in cancer cells. And Dahlman’s latest delivery system makes it possible to simultaneously target up to 10 proteins at once, which could make cancer treatments far more effective. Lab work like this is far from fruition, but if it maintains its momentum, the drugs currently in clinical trials could represent just a small portion of the benefits of the discovery of RNAi.

    See the full article here.

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  • richardmitnick 1:49 pm on September 3, 2014 Permalink | Reply
    Tags: , Biochemistry, , , , Peptoids   

    From LBL: “Peptoid Nanosheets at the Oil/Water Interface” 

    Berkeley Logo

    Berkeley Lab

    September 3, 2014
    Lynn Yarris (510) 486-5375

    From the people who brought us peptoid nanosheets that form at the interface between air and water, now come peptoid nanosheets that form at the interface between oil and water. Scientists at the U.S. Department of Energy (DOE)’s Lawrence Berkeley National Laboratory (Berkeley Lab) have developed peptoid nanosheets – two-dimensional biomimetic materials with customizable properties – that self-assemble at an oil-water interface. This new development opens the door to designing peptoid nanosheets of increasing structural complexity and chemical functionality for a broad range of applications, including improved chemical sensors and separators, and safer, more effective drug delivery vehicles.

    Supramolecular assembly at an oil-water interface is an effective way to produce 2D nanomaterials from peptoids because that interface helps pre-organize the peptoid chains to facilitate their self-interaction,” says Ron Zuckermann, a senior scientist at the Molecular Foundry, a DOE nanoscience center hosted at Berkeley Lab. “This increased understanding of the peptoid assembly mechanism should enable us to scale-up to produce large quantities, or scale- down to screen many different nanosheets for novel functions.”

    nano
    Peptoid nanosheets are among the largest and thinnest free-floating organic crystals ever made, with an area-to-thickness equivalent of a plastic sheet covering a football field. Peptoid nanosheets can be engineered to carry out a wide variety of functions.
    two
    Ron Zuckerman and Geraldine Richmond led the development of peptoid nanosheets that form at the interface between oil and water, opening the door to increased structural complexity and chemical functionality for a broad range of applications.

    Zuckermann, who directs the Molecular Foundry’s Biological Nanostructures Facility, and Geraldine Richmond of the University of Oregon are the corresponding authors of a paper reporting these results in the Proceedings of the National Academy of Sciences (PNAS). The paper is titled Assembly and molecular order of two-dimensional peptoid nanosheets at the oil-water interface. Co-authors are Ellen Robertson, Gloria Olivier, Menglu Qian and Caroline Proulx.

    Peptoids are synthetic versions of proteins. Like their natural counterparts, peptoids fold and twist into distinct conformations that enable them to carry out a wide variety of specific functions. In 2010, Zuckermann and his group at the Molecular Foundry discovered a technique to synthesize peptoids into sheets that were just a few nanometers thick but up to 100 micrometers in length. These were among the largest and thinnest free-floating organic crystals ever made, with an area-to-thickness equivalent of a plastic sheet covering a football field. Just as the properties of peptoids can be chemically customized through robotic synthesis, the properties of peptoid nanosheets can also be engineered for specific functions.

    “Peptoid nanosheet properties can be tailored with great precision,” Zuckermann says, “and since peptoids are less vulnerable to chemical or metabolic breakdown than proteins, they are a highly promising platform for self-assembling bio-inspired nanomaterials.”

    In this latest effort, Zuckermann, Richmond and their co-authors used vibrational sum frequency spectroscopy to probe the molecular interactions between the peptoids as they assembled at the oil-water interface. These measurements revealed that peptoid polymers adsorbed to the interface are highly ordered, and that this order is greatly influenced by interactions between neighboring molecules.

    “We can literally see the polymer chains become more organized the closer they get to one another,” Zuckermann says.

    ft
    Peptoid polymers adsorbed to the oil-water interface are highly ordered thanks to interactions between neighboring molecules.

    The substitution of oil in place of air creates a raft of new opportunities for the engineering and production of peptoid nanosheets. For example, the oil phase could contain chemical reagents, serve to minimize evaporation of the aqueous phase, or enable microfluidic production.

    “The production of peptoid nanosheets in microfluidic devices means that we should soon be able to make combinatorial libraries of different functionalized nanosheets and screen them on a very small scale,” Zuckermann says. “This would be advantageous in the search for peptoid nanosheets with the molecular recognition and catalytic functions of proteins.”

    Zuckermann and his group at the Molecular Foundry are now investigating the addition of chemical reagents or cargo to the oil phase, and exploring their interactions with the peptoid monolayers that form during the nanosheet assembly process.

    “In the future we may be able to produce nanosheets with drugs, dyes, nanoparticles or other solutes trapped in the interior,” he says. “These new nanosheets could have a host of interesting biomedical, mechanical and optical properties.”

    This work was primarily funded by the DOE Office of Science and the Defense Threat Reduction Agency. Part of the research was performed at the Molecular Foundry and the Advanced Light Source, which are DOE Office of Science User Facilities.

    See the full article here.

    A U.S. Department of Energy National Laboratory Operated by the University of California

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  • richardmitnick 1:41 pm on August 29, 2014 Permalink | Reply
    Tags: , Biochemistry, , , ,   

    From LBL: “Going to Extremes for Enzymes” 

    Berkeley Logo

    Berkeley Lab

    August 29, 2014
    Lynn Yarris (510) 486-5375

    In the age-old nature versus nurture debate, Douglas Clark, a faculty scientist with Berkeley Lab and the University of California (UC) Berkeley, is not taking sides. In the search for enzymes that can break lignocellulose down into biofuel sugars under the extreme conditions of a refinery, he has prospected for extremophilic microbes and engineered his own cellulases.

    ext
    Extremophiles thriving in thermal springs where the water temperature can be close to boiling can be a rich source of enzymes for the deconstruction of lignocellulose.

    Speaking at the national meeting of the American Chemical Society (ACS) in San Francisco, Clark discussed research for the Energy Biosciences Institute (EBI) in which he and his collaborators are investigating ways to release plant sugars from lignin for the production of liquid transportation fuels. Sugars can be fermented into fuels once the woody matter comprised of cellulose, hemicellulose, and lignin is broken down, but lignocellulose is naturally recalcitrant.

    “Lignocellulose is designed by nature to stand tall and resist being broken down, and lignin in particular acts like a molecular glue to help hold it together” said Clark, who holds appointments with Berkeley Lab’s Physical Biosciences Division and UC Berkeley’s Chemical and Biomolecular Engineering Department where he currently serves as dean of the College of Chemistry. “Consequently, lignocellulosic biomass must undergo either chemical or enzymatic deconstruction to release the sugars that can be fermented to biofuels.”

    dc
    Douglas Clark holds joint appointments with Berkeley Lab and UC Berkeley and is a principal investigator with the Energy Biosciences Institute. (Photo by Roy Kaltschmidt)

    For various chemical reasons, all of which add up to cost-competitiveness, biorefineries could benefit if the production of biofuels from lignocellulosic biomass is carried out at temperatures between 65 and 70 degrees Celsius. The search by Clark and his EBI colleagues for cellulases that can tolerate these and even harsher conditions led them to thermal springs near Gerlach, Nevada, where the water temperature can be close to boiling. There they discovered a consortium of three hyperthermophilic Archaea that could grow on crystalline cellulose at 90 degrees Celsius.

    “This consortium represents the first instance of Archaea able to deconstruct lignocellulose optimally above 90°C,” Clark said.

    Following metagenomic studies on the consortium, the most active high-temperature cellulase was identified and named EBI-244.

    “The EBI-244 cellulase is active at temperatures as high as 108 degrees Celsius, the most extremely heat-tolerant enzyme ever found in any cellulose-digesting microbe,” Clark said.

    The most recent expedition of Clark and his colleagues was to thermal hot springs in Lassen Volcanic National Park, where they found an enzyme active on cellulose up to 100°C under highly acidic conditions – pH approximately 2.2.

    “The Lassen enzyme is the most acidothermophilic cellulase yet discovered,” Clark said. “The final products that it forms are similar to those produced by EBI244.”

    three
    A consortium of three hyperthermophilic Archaea that could grow on crystalline cellulose at 90 degrees Celsius yielded EBI-244, the most active high-temperature cellulase ever identified.

    In addition to bioprospecting for heat tolerant enzymes, Clark and his colleagues have developed a simple and effective mutagenesis method to enhance the properties of natural enzymes. Most recently they used this technique to increase the optimal temperature and enhance the thermostability of Ce17A, a fungal cellulase that is present in high concentrations in commercial cellulase cocktails. They engineered yeast to produce this enzyme with encouraging results.

    “The yeast Saccharomyces cerevisiae has often been used both in the engineering and basic study of Cel7A; however, Cel7A enzymes recombinantly expressed in yeast are often less active and less stable than their native counterparts,” Clark said. “We discovered that an important post-translational modification that was sometimes absent in the yeast-expressed enzyme was the underlying cause of this disparity and successfully carried out the post-translational modification in vitro. After this treatment, the properties of Cel7A recombinantly expressed in yeast were improved to match those of the native enzyme.”

    Collaborators in this research include Harvey Blanch, who also holds joint appointments with Berkeley Lab and UC Berkeley, and Frank Robb from the University of Maryland.

    EBI, which provided the funding for this research, is a collaborative partnership between BP, the funding agency, UC Berkeley, Berkeley Lab and the University of Illinois at Urbana-Champaign.

    See the full article here.

    A U.S. Department of Energy National Laboratory Operated by the University of California

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  • richardmitnick 11:20 am on August 19, 2014 Permalink | Reply
    Tags: , , Biochemistry, ,   

    From Astrobiology: “Study Reveals Immune System is Dazed and Confused During Spaceflight” 

    Astrobiology Magazine

    Astrobiology Magazine

    Aug 19, 2014
    No Writer Credit

    There is nothing like a head cold to make us feel a little dazed. We get things like colds and the flu because of changes in our immune system. Researchers have a good idea what causes immune system changes on Earth—think stress, inadequate sleep and improper nutrition.

    But the results of two NASA collaborative investigations—Validation of Procedures for Monitoring Crewmember Immune Function (Integrated Immune) and Clinical Nutrition Assessment of ISS Astronauts, SMO-016E (Clinical Nutrition Assessment)—recently published in the Journal of Interferon & Cytokine Research suggest that spaceflight may temporarily alter the immune system of crew members flying long duration missions aboard the International Space Station. This is of concern as NASA looks ahead to six-month and multiple-year missions to asteroids, the moon and Mars because something as simple as a cold or the flu can be risky business in space.

    ISS
    ISS

    Data generated early in NASA’s Integrated Immune study indicated that the distribution of immune cells in the blood of crew members aboard the space station is relatively unchanged during flight. However, they also revealed that some cell function is significantly lower than normal, or depressed, and some cell activity is heightened. In a sense, the immune systems of crew members are confused.

    ak
    European Space Agency astronaut Andre Kuipers, Expedition 30 flight engineer, prepares vials in the Columbus laboratory of the International Space Station for venous blood sample draws during an immune system investigation. Image Credit: NASA

    When cell activity is depressed, the immune system is not generating appropriate responses to threats. This may also lead to the asymptomatic viral shedding observed in some crew members, which means latent, or dormant, viruses in the body reawaken, but without symptoms of illness. When activity heightens, the immune system reacts excessively, resulting in things like increased allergy symptoms and persistent rashes, which have been reported by some crew members.

    “Prior to the Integrated Immune study, little immune system in-flight data had been collected,” said Brian Crucian, Ph.D. and NASA biological studies and immunology expert. “Previous post-flight studies were not enough to make any determination about spaceflight’s effect on the immune system. This in-flight data provided the information we needed to determine that immune dysregulation does occur and actually persists during long-duration spaceflight.”

    Recently, in a collaboration between NASA’s Integrated Immune and Clinical Nutrition Assessment flight studies, researchers examined the blood plasma of 28 crew members before, during and after their missions. They were measuring for the concentration of cytokines – the proteins that regulate immunity. Cytokines recruit immune cells to the infected or injured body site, facilitate cell-to-cell communication, and signal immune cells to activate and mount a defense against invaders. This process is usually referred to as inflammation. The data indicated that, like the changes in cell function indicated in the Integrated Immune study, crew members also have changes in blood cytokines that persist during flight. This gives researchers an idea of what areas of a crew member’s immune system may be confused during flight.

    ah
    Japan Aerospace Exploration Agency astronaut Akihiko Hoshide, Expedition 32 flight engineer, poses for a photo after undergoing a generic blood draw in the European Laboratory/Columbus Orbital Facility. International Space Station crew members routinely perform blood draws for investigations. Image Credit: NASA

    According to Crucian, the immune system is likely being altered by many factors associated with the overall spaceflight environment. “Things like radiation, microbes, stress, microgravity, altered sleep cycles and isolation could all have an effect on crew member immune systems,” said Crucian. “If this situation persisted for longer deep space missions, it could possibly increase risk of infection, hypersensitivity, or autoimmune issues for exploration astronauts.”

    Despite these immune system changes, it has yet to be determined whether these alterations increase crew risk for medical issues during spaceflight. According to Crucian, further investigations are required to precisely assess whether there is increased clinical risk to crew members on longer duration missions.

    NASA Human Research Program Chief Scientist Mark Shelhamer says continued study of the immune system is critical. “These studies tell us that this is an important issue and that we are measuring the right things,” said Shelhamer. “They also tell us there is no place during spaceflight where we see stabilization of the immune system. This is critical as we pursue longer duration missions and why we are studying this further during the upcoming one-year mission.”

    Once these investigations are complete, Crucian expects the agency will have a decision point for establishing countermeasures that it must then decide how to implement. If deemed necessary, countermeasures for immunity could include new types of radiation shielding, nutritional supplementation, pharmaceuticals and more.

    Studies of how space flight affects the immune system may provide benefits to Earth-based medicine. This includes information on how stress causes immune system changes in healthy adults, changes that may precede disease.

    In the end, NASA may just shift the immune system during spaceflight from dazed to unfazed.

    See the full article here.
    Astrobiology Magazine is a NASA-sponsored online popular science magazine. Our stories profile the latest and most exciting news across the wide and interdisciplinary field of astrobiology — the study of life in the universe. In addition to original content, Astrobiology Magazine also runs content from non-NASA sources in order to provide our readers with a broad knowledge of developments in astrobiology, and from institutions both nationally and internationally. Publication of press-releases or other out-sourced content does not signify endorsement or affiliation of any kind.
    Established in the year 2000, Astrobiology Magazine now has a vast archive of stories covering a broad array of topics.

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  • richardmitnick 7:50 am on August 14, 2014 Permalink | Reply
    Tags: , Biochemistry, , ,   

    From The New York Times: “Our Microbiome May Be Looking Out for Itself” 

    New York Times

    The New York Times

    AUG. 14, 2014
    Carl Zimmer

    Your body is home to about 100 trillion bacteria and other microbes, collectively known as your microbiome. Naturalists first became aware of our invisible lodgers in the 1600s, but it wasn’t until the past few years that we’ve become really familiar with them.

    This recent research has given the microbiome a cuddly kind of fame. We’ve come to appreciate how beneficial our microbes are — breaking down our food, fighting off infections and nurturing our immune system. It’s a lovely, invisible garden we should be tending for our own well-being.

    microbe
    A highly magnified view of Enterococcus faecalis, a bacterium that lives in the human gut. Microbes may affect our cravings, new research suggests. Credit Centers for Disease Control and Prevention

    But in the journal Bioessays, a team of scientists has raised a creepier possibility. Perhaps our menagerie of germs is also influencing our behavior in order to advance its own evolutionary success — giving us cravings for certain foods, for example.

    Maybe the microbiome is our puppet master.

    “One of the ways we started thinking about this was in a crime-novel perspective,” said Carlo C. Maley, an evolutionary biologist at the University of California, San Francisco, and a co-author of the new paper. What are the means, motives and opportunity for the microbes to manipulate us? They have all three.

    The idea that a simple organism could control a complex animal may sound like science fiction. In fact, there are many well-documented examples of parasites controlling their hosts.

    Some species of fungi, for example, infiltrate the brains of ants and coax them to climb plants and clamp onto the underside of leaves. The fungi then sprout out of the ants and send spores showering onto uninfected ants below.

    How parasites control their hosts remains mysterious. But it looks as if they release molecules that directly or indirectly can influence their brains.

    Our microbiome has the biochemical potential to do the same thing. In our guts, bacteria make some of the same chemicals that our neurons use to communicate with one another, such as dopamine and serotonin. And the microbes can deliver these neurological molecules to the dense web of nerve endings that line the gastrointestinal tract.

    A number of recent studies have shown that gut bacteria can use these signals to alter the biochemistry of the brain. Compared with ordinary mice, those raised free of germs behave differently in a number of ways. They are more anxious, for example, and have impaired memory.

    Adding certain species of bacteria to a normal mouse’s microbiome can reveal other ways in which they can influence behavior. Some bacteria lower stress levels in the mouse. When scientists sever the nerve relaying signals from the gut to the brain, this stress-reducing effect disappears.

    Some experiments suggest that bacteria also can influence the way their hosts eat. Germ-free mice develop more receptors for sweet flavors in their intestines, for example. They also prefer to drink sweeter drinks than normal mice do.

    Scientists have also found that bacteria can alter levels of hormones that govern appetite in mice.

    Dr. Maley and his colleagues argue that our eating habits create a strong motive for microbes to manipulate us. “From the microbe’s perspective, what we eat is a matter of life and death,” Dr. Maley said.

    Different species of microbes thrive on different kinds of food. If they can prompt us to eat more of the food they depend on, they can multiply.

    Microbial manipulations might fill in some of the puzzling holes in our understandings about food cravings, Dr. Maley said. Scientists have tried to explain food cravings as the body’s way to build up a supply of nutrients after deprivation, or as addictions, much like those for drugs like tobacco and cocaine.

    But both explanations fall short. Take chocolate: Many people crave it fiercely, but it isn’t an essential nutrient. And chocolate doesn’t drive people to increase their dose to get the same high. “You don’t need more chocolate at every sitting to enjoy it,” Dr. Maley said.

    Perhaps, he suggests, the certain kinds of bacteria that thrive on chocolate are coaxing us to feed them.

    John F. Cryan, a neuroscientist at University College Cork in Ireland who was not involved in the new study, suggested that microbes might also manipulate us in ways that benefited both them and us. “It’s probably not a simple parasitic scenario,” he said.

    Research by Dr. Cryan and others suggests that a healthy microbiome helps mammals develop socially. Germ-free mice, for example, tend to avoid contact with other mice.

    That social bonding is good for the mammals. But it may also be good for the bacteria.

    “When mammals are in social groups, they’re more likely to pass on microbes from one to the other,” Dr. Cryan said.

    “I think it’s a very interesting and compelling idea,” said Rob Knight, a microbiologist at the University of Colorado, who was also not involved in the new study.

    If microbes do in fact manipulate us, Dr. Knight said, we might be able to manipulate them for our own benefit — for example, by eating yogurt laced with bacteria that would make use crave healthy foods.

    “It would obviously be of tremendous practical importance,” Dr. Knight said. But he warned that research on the microbiome’s effects on behavior was “still in its early stages.”

    The most important thing to do now, Dr. Knight and other scientists said, was to run experiments to see if microbes really are manipulating us.

    Mark Lyte, a microbiologist at the Texas Tech University Health Sciences Center who pioneered this line of research in the 1990s, is now conducting some of those experiments. He’s investigating whether particular species of bacteria can change the preferences mice have for certain foods.

    “This is not a for-sure thing,” Dr. Lyte said. “It needs scientific, hard-core demonstration.”

    See the full article here.

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  • richardmitnick 6:28 pm on August 13, 2014 Permalink | Reply
    Tags: , Biochemistry, , ,   

    From Stanford via NPR: “Biologists Choose Sides In Safety Debate Over Lab-Made Pathogens” 

    NPR

    National Public Radio (NPR)

    August 13, 2014
    Nell Greenfieldboyce

    A smoldering debate about whether researchers should ever deliberately create superflu strains and other risky germs in the interest of science has flared once again.
    Some scientists think new types of bird flus should arise only in chickens, not in labs.

    bird
    Here a worker collects poultry on a farm in Kathmandu, Nepal, where the H5N1 virus was infecting animals in October 2011.

    Proponents of the work say that in order to protect the public from the next naturally occurring pandemic, they have to understand what risky infectious agents are capable of — and that means altering the microbes in experiments. Critics argue that the knowledge gained from making new strains of these germs isn’t worth the risk, because a lab-made pathogen might escape the laboratory and start spreading among people.

    Now, as scientists on both sides of the dispute have formed groups that have issued manifestos and amassed lists of supporters, it looks like the prestigious will step in to weigh the risks and benefits.

    “ I don’t think we have adequately involved the public so that they understand the possible consequences of mistakes, or errors, or misadventures in performing this kind of science.

    A representative of the National Institutes of Health, which funds this research, says that NIH, too, is “giving deep consideration to the many views expressed by various highly respected parties” about the best way forward.

    In a recent editorial in “mBio,” the journal’s editor-in-chief, Arturo Casadevall, M.D., Ph.D. , urged his colleagues to “lower the level of rhetoric and focus on the scientific questions at hand.”

    Scientists have passionate debates all the time, but it’s usually about the meaning of some experimental result, says Casadevall, a microbiologist at the Albert Einstein College of Medicine in New York.

    “What is different here is that we are facing a set of intangibles,” he says. “And because they involve judgment calls at this point, people are often weighing the risks and the benefits very differently.”

    Dr. David Rellman, a microbiologist at Stanford University, thinks the risks of making a new strain of flu virus that has the potential to cause a pandemic are very real.

    “I don’t think we have adequately involved the public,” Relman says, “so that they understand the possible consequences of mistakes, or errors, or misadventures in performing this kind of science — the kinds of consequences that would result in many, many people becoming ill or dying.”

    “ These viruses are out there. They cause disease; they have killed many, many people in the past. We bring them to the laboratory to work with them.

    • Paul Duprex, Boston University microbiologist

    Controversial work on lab-altered bird flu was halted for more than a year in a , voluntary moratorium after two labs generated new, more contagious forms of the bird flu virus H5N1. Eventually, after federal officials promised more oversight, the experiments started back up and the controversy quieted down. But key questions were never answered, Relman says.

    “One of the big issues that has not been advanced over the last two years is a discussion about whether there are experiments that ought not to be undertaken and, if so, what they look like,” he says, noting that scientists keep publishing more studies that involve genetically altered flu viruses. “You know, every time that one of these experiments comes up, it just ups the ante a bit. It creates additional levels of risk that force the question: Do we accept all of this?”

    Last month, Relman met in Massachusetts with others who are worried. They formed the Cambridge Working Group and issued a statement saying that researchers should curtail any experiments that would lead to new pathogens with pandemic potential, until there’s a better assessment of the dangers and benefits.

    By coincidence, they released their official statement just as the public started hearing news reports of various , such as a forgotten vial of smallpox found in an old freezer, and mishaps involving anthrax and bird flu at the Centers for Disease Control and Prevention.

    What’s more, the unprecedented Ebola outbreak has reminded the public what it looks like when a deadly virus .

    All of this led a different band of scientists to also form a group — to publicly defend research on dangerous pathogens.

    “There are multiple events that have come together in a rather unusual convergence,” says Paul Duprexa microbiologist at Boston University.

    He sees the recent reports of lab mistakes as exceptions — they don’t mean you should shut down basic science that’s essential to protecting public health, he says.

    “These viruses are out there. They cause disease; they have killed many, many people in the past,” Duprex says. “We bring them to the laboratory to work with them.”

    Duprex helped form a group that calls itself Scientists for Science. The group’s position statement emphasizes that studies on already are subject to extensive regulations. It says focusing on lab safety is the best defense — not limiting the types of experiments that can be done.

    Whenever questions about safety are raised, Duprex says, scientists have one of two options. They can keep their heads down, do their experiments and hope it will all go away. Or, he says, they can proactively engage the public and provide an informed opinion.

    His group has taken the latter approach, “because ultimately we’re the people working with these things.”

    Each of these two groups of scientists now has a website, and each website features its own list of more than a hundred supporters, including Nobel Prize winners and other scientific superstars.

    One thing that almost everyone seems to agree on is that, to move forward, there needs to be some sort of independent, respected forum for discussing the key issues.

    The American Society for Microbiology has called on the prestigious National Academy of Sciences to take the lead. A representative of the Academy says NAS does plan to hold a symposium, later this year. The details are still being worked out.

    Tim Donohue, a microbiologist at the University of Wisconsin, Madison who is president of ASM, says a similar kind of debate happened back in the mid-1970s, when brand-new technologies for manipulating DNA forced scientists and the public to tackle thorny questions.

    “And I think that is a productive exercise,” Donohue says, “to have scientists and the public, sitting around the table, making sure each one understands what the benefits and risks are, and putting in place policies that allow these types of experiments to go on so that they are safe and so that society can benefit from the knowledge and innovation that comes out of that work.”

    See the full article, with links, here.

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  • richardmitnick 4:12 pm on August 13, 2014 Permalink | Reply
    Tags: , Biochemistry, , ,   

    From PBS NOVA: “Teaching the Nervous System to Forget Chronic Pain” 

    PBS NOVA

    NOVA

    13 Aug 2014
    Eleanor Nelsen

    “It was an emergency situation,” she says. The horse Sally was riding was barreling straight towards another, younger horse, and the only way to stop him was to pull back on one rein, hard. She felt a pop in her wrist. Heat shot up her arm, excruciating pain fast on its heels.

    That was four years ago. No one knows quite what happened to her wrist that day, but whatever it was has left her with constant pain that stretches from her fingertips to her neck, and sometimes creeps into her ribs. On the really bad days, even a hug is unbearably painful.

    Sally is my youngest sister, and she is one of an unlucky fraction of people for whom an injury catapults their nervous system into a state of chronic pain. The injury itself heals, but like an insidious memory, the pain lingers. We don’t know why. “The whole issue of the transition from acute pain to chronic pain—why some individuals develop that chronic pain and many don’t—is a major, major question,” says Allan Basbaum, a professor at the University of California, San Francisco. Genetics may play a role. So can the severity of the original injury.

    pills
    Today’s painkillers are based on well-known compounds like morphine and are often highly addictive.

    But what we do know is that once that pain has gotten a foothold, doctors and patients don’t have very many choices. “The irony is that morphine, the 2,000-year-old drug, still remains the number-one weapon against pain,” says Yves De Koninck, a professor of neuroscience at Université Laval in Canada.

    And it’s not a weapon that anyone enjoys using. Opioids like morphine and oxycodone are famously addictive, and the numbers of people who abuse them are climbing. Painkiller overdoses now kill more people than cocaine and heroin combined. And while opioids are invaluable for acute pain, they’re less effective for persistent, chronic pain. In fact—in a particularly cruel irony—long-term opioid treatment can actually make pain worse. Non-opioid pain medications exist, but they don’t work for the majority of patients, and even then they are only partly effective.
    Chronic pain is like “a maladaptive memory.”

    Opioids work so well for acute pain because they bind to the receptors the body has designed for its own painkillers—molecules like endorphins and dynorphins that blunt the pain response. Finding good alternatives to opioids for treating chronic pain will mean finding different neurological mechanisms to target—mechanisms that explain not just why people hurt, but why some people hurt for so long.

    De Koninck has found such a mechanism. One of the keys to understanding chronic pain, he believes, is to pay attention to the similarities between long-lasting pain and another, very familiar, neurological process that makes some connections stick around longer than others: memory.

    Chronic pain is like “a maladaptive memory,” Basbaum explains. Both constitute patterns etched in your brain and nervous system that quicken the connections between “snake” and “poison” or between “bump” and “ouch.” Evidence has been piling up that chronic pain and memory share some of the same cellular mechanisms—and now, De Koninck’s work has shown that a neurochemical trick used to erase memory may be able to turn off chronic pain, too.

    An Unmet Need

    The number of people struggling with chronic pain has been hotly debated, and the fact that chronic pain is broadly defined and difficult to quantify doesn’t help. But even conservative estimates suggest that about 20% of the population have had at least one episode of serious, chronic pain. In the United States alone, that’s more than 60 million people. “It’s a major unmet need,” De Koninck says.

    Pain is physically and psychologically debilitating in way that few other conditions are. “In fact, it’s often the most debilitating component of many diseases,” De Koninck notes. And it sharply circumscribes the lives of people who suffer from it. People can find a way to live with the other challenges of painful conditions like arthritis, cancer, even paralysis, he says, but “if you actually ask the patient, their number-one concern, and the one thing that they want us to cure, is the pain.”
    When pain pathways are functioning properly, they play a protective role.

    When chronic pain gets severe, many patients withdraw, sometimes even from their families. Sally says that she’s constantly nervous, afraid to accept invitations or do things that she loves—like riding horses—in case it makes her arm even worse. The ride that day, Sally says, “changed my life.” For some patients, chronic pain can lead to serious mental health problems—it’s strongly correlated with depression and suicide risk.

    When pain pathways are functioning properly, they play a protective role. They are a relay of chemical and electrical signals that move from nerve endings to our brains. Pain teaches us to avoid things that are sharp, prickly, or hot. It’s the way our nervous system has adapted to living in a hazardous world. People who can’t feel any pain typically don’t live very long.

    Our skin is packed with millions of specialized nerve endings, programmed to detect dangerous conditions like heat or pressure. When one of these pathways is activated, the neuron sends an electrical current shooting up its long, thin axon towards the spinal cord. When it reaches the end of the neuron, that electrical signal prompts the release of chemicals called neurotransmitters into the synapse, or the gap between the first neuron and the next. The neurotransmitters dock in receptors on the next neuron, triggering pores to open in the cell’s membrane. Charged particles rush in through these open pores, creating a new electrical current that carries the signal farther up the nervous system.

    nerves
    Nerve cells, like these seen here from a mouse’s spinal cord, send impulses along their axons and connect over synapses.

    The first handoff occurs in a region of the spinal cord known as the dorsal horn, a column of grey matter that looks, in cross-section, like a butterfly. From this first relay point, the signal travels to the thalamus, one of the brain’s switchboards, and eventually to the cerebral cortex, where the signal is processed and decoded.

    After an injury, it’s normal for the damage sensors near the trauma site to be touchy for a little while. During that time, your nervous system is encouraging you to protect the damaged tissue while it’s healing. But sometimes that extra sensitivity, called “hyperalgesia,” sticks around long after it’s useful. Hyperalgesia is often a major component of chronic pain, and it means that people with chronic pain have to be unceasingly alert. For example, Sally says, before she hurt her arm, hot coffee sloshing onto her hand might have hurt for a few seconds. Now, a careless moment like that means days of burning pain.

    Symptoms like this suggest that changes in the nervous system have migrated to the spinal cord. De Koninck believes that a major factor is the number of receptors on the signal-receiving neurons in the dorsal horn. If those neurons synthesize too many receptors, they’ll pick up too many neurotransmitter molecules. Then the neurons’ pores will flutter open to let charged particles in more often than they should, sending electrical signals shooting up to the brain at too high a frequency. The result is a pain signal that’s much stronger than it should be. De Koninck’s work gives us a new window into how it happens, and how to stop it.

    Recall, Then Erase

    The key lies in a study about memory that was published nearly 15 years ago. Long-term memories seem to depend on the synthesis of extra receptors, too, and scientists knew that blocking the synthesis of those receptors during a memorable event could keep memories from forming.

    But what a group of researchers at New York University discovered was that there is a brief period when interrupting receptor synthesis can actually erase old memories. Memories are reinforced when they’re retrieved, but, paradoxically, during that process, even well-established memories have a brief window of vulnerability—like jewelry in a safe deposit box, memories are useless when they’re stored but accessible to thieves when they’re being used. A chemical called anisomycin blocks the production of receptors that neurons need to form memories. When the researchers injected anisomycin into rats’ brains right after triggering a particular memory, that memory didn’t just fail to get reinforced—it was erased altogether.

    The right chemical injected at just the right place at just the right time could erase the physiological “memory” of pain.

    Accumulating evidence that pain and memory use similar mechanisms led De Koninck to wonder if this same neurochemical trick could erase chronic hyperalgesia. De Koninck and his colleagues made mice hypersensitive to pain by injecting their paws with capsaicin, the chemical responsible for chili peppers’ fiery bite. Capsaicin activates the same pain sensors that respond to extreme heat and can turn on hyperalgesia without the tissue damage that an actual burn would cause. After their capsaicin injection, the mice’s paws were more sensitive to pressure for hours afterward.

    Before that sensitivity had had a chance to wear off, the team gave the mice a second capsaicin injection—and this time, they added an injection of anisomycin. What happened after this second injection is “like magic,” De Koninck says. When the second injection initiated the same flurry of neurotransmitters and electrical signals that encoded the hyperalgesia the first time—the pain analogue of recalling a memory—anisomycin shut down the pain-amplifying mechanism by keeping the spinal cord neurons from making extra receptors. “It’s in the process of reorganizing itself,” De Koninck explains, “and there there’s that window of opportunity to actually shut it back down.” The mice lost seventy percent of their hypersensitivity to pain.

    The theory that overdeveloped connections other than memories could be attenuated by retriggering them “is not a new idea,” Basbaum says, “but the fact is, there really has been very little evidence that it’s doable.” De Koninck’s results suggest that the right chemical injected at just the right place at just the right time, can erase the physiological “memory” of pain. Ted Price, a professor at the University of Texas-Dallas, says that this “ paves the road to disease modification instead of just palliatively treating people with these terrible drugs like opioids, which everybody, everybody in the field wants to get away from.”
    New Options

    For now, there are a few other types of treatment doctors can turn to besides opioids. Antidepressants help some people, as do certain antiseizure medications. A controversial technique called “transcutaneous electrical nerve stimulation” may work by making sure that there are plenty of receptors in the dorsal horn for the body’s natural opioid chemicals; a wearable device using this technology was just approved for over-the-counter sale by the FDA.

    Treatments based on De Konick’s capsaicin-anisomycin model would constitute an entirely new category of drugs. “When you find a new mechanism,” De Koninck says, “boy, it opens a whole new array of things.” But finding the right combination of chemicals won’t be easy. Capsaicin patches are already sold over the counter at drugstores, but anisomycin is far too indiscriminate for clinical use. Brian Wainger, a physician and researcher at Massachusetts General Hospital, says, “It’s obviously going to be a long time for a discovery like this to work towards a clinical approach, but I think it sort of sets a framework for some options.”

    “Options” is a word that seems to come up a lot among pain specialists. One of the reasons chronic pain is so difficult to treat is because “there’s a lot of different forms of chronic pain,” De Koninck says. “But the arsenal that we have so far to treat it is still quite meager.” And the weapons we do have are woefully inadequate.

    Still, discovering that this retrigger-and-erase phenomenon works for hyperalgesia, as well as for memory, suggests that it may be useful in other parts of the nervous system. If that’s true, these kinds of treatments could help with pain syndromes more complicated than hyperalgesia—conditions that are so severe that even light touches become painful, or in cases where patients experience pain with no stimulus at all.

    One big advantage of De Koninck’s strategy is that it isn’t just an incremental improvement, a way to make a slightly more effective or slightly less addictive analgesic. It’s a totally different angle on the problem. It targets the “chronic” part of chronic pain. “What the field I think really needs is options,” Price says. “And more importantly, patients need options.” For millions of people, and their doctors, a totally different angle is exactly what they’ve been looking for.

    See the full article here.

    NOVA is the highest rated science series on television and the most watched documentary series on public television. It is also one of television’s most acclaimed series, having won every major television award, most of them many times over.

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  • richardmitnick 12:05 pm on July 28, 2014 Permalink | Reply
    Tags: , Biochemistry, Bioenergy, , , ,   

    From Berkeley Lab: “How Sweet It Is: New Tool for Characterizing Plant Sugar Transporters Developed at Joint BioEnergy Institute” 


    Berkeley Lab

    July 28, 2014
    Lynn Yarris

    A powerful new tool that can help advance the genetic engineering of “fuel” crops for clean, green and renewable bioenergy, has been developed by researchers with the U.S. Department of Energy (DOE)’s Joint BioEnergy Institute (JBEI), a multi-institutional partnership led by Lawrence Berkeley National Laboratory (Berkeley Lab). The JBEI researchers have developed an assay that enables scientists to identify and characterize the function of nucleotide sugar transporters, critical components in the biosynthesis of plant cell walls.

    build
    The Joint BioEnergy Institute (JBEI) is one of three Bioenergy Research Centers established by DOE’s Office of Science to accelerate the development of advanced, next-generation biofuels. (Photo by Roy Kaltschmidt)

    plants
    A family of six nucleotide sugar transporters never before described have been characterized in Arabidopsis, a model plant for research in advanced biofuels. (Photo by Roy Kaltschmidt)

    “Our unique assay enabled us to analyze nucleotide sugar transporter activities in Arabidopsis and characterize a family of six nucleotide sugar transporters that has never before been described,” says Henrik Scheller, the leader of JBEI’s Feedstocks Division and a leading authority on cell wall biosynthesis. “Our method should enable rapid progress to be made in determining the functional role of nucleotide sugar transporters in plants and other organisms, which is very important for the metabolic engineering of cell walls.”

    Scheller is the corresponding author, along with Ariel Orellana at the Universidad Andrés Bello, Santiago, Chile, of a paper describing this research in the Proceedings of the National Academy of Sciences (PNAS). The paper is titled The Golgi localized bifunctional UDP-rhamnose/UDP-galactose transporter family of Arabidopsis. The lead authors are Carsten Rautengarten and Berit Ebert, both of whom hold appointments with JBEI, and both of whom, like Scheller, also hold appointments with Berkeley Lab’s Physical Biosciences Division. (See below for the full list of co-authors.)

    The sugars in plant biomass represent an enormous potential source of environmentally benign energy if they can be converted into transportation fuels – gasoline, diesel and jet fuel – in a manner that is economically competitive with petroleum-based fuels. One of the keys to success in this effort will be to engineer fuel crops whose cells walls have been optimized for sugar content.

    With the exception of cellulose and callose, the complex polysaccharide sugars in plant cell walls are synthesized in the Golgi apparatus by enzymes called glycosyltransferases. These polysaccharides are assembled from substrates of simple nucleotide sugars which are transported into the Golgi apparatus from the cytosol, the gel-like liquid that fills a plant cell’s cytoplasm. Despite their importance, few plant nucleotide sugar transporters have been functionally characterized at the molecular level. A big part of the holdup has been a lack of substrates that are necessary to carry out such characterizations.

    “Substrates of mammalian nucleotide sugar transporters are commercially available because of the medical interest but have not been available for plants, which made it difficult to study both nucleotide sugar transporters and glycosyltransferases,” Scheller says.

    For their assay, Scheller, Rautengarten, Ebert and their collaborators, created several artificial substrates for nucleotide sugar transporters, then reconstituted the transporters into liposomes for analysis with mass spectrometry. The researchers used this technique to characterize the functions of the six new nucleotide sugar transporters they identified in Arabidopsis, a relative of mustard that serves as a model plant for research in advanced biofuels.

    “We found that these six new nucleotide sugar transporters are bispecific, which is a surprise since the two substrates are not very similar from a physical standpoint to the human eye,” Scheller says. “We also found that limiting substrate availability has different effects on different polysaccharide products, which suggests that cell wall polysaccharide biosynthesis in the Golgi apparatus of plants is also regulated by substrate transport mechanisms.”

    In addition to these six nucleotide sugar transporters, the assay was used to characterize the functions of 20 other transporters, the details of which will soon be published.

    “Thanks largely to the efforts these past two years of Carsten Rautengarten and Berit Ebert, we now know the activity of three times more nucleotide sugar transporters than are known in humans, and we have determined the function of two-thirds of the plant transporters as compared to one-quarter of the human ones,” Scheller says. “This is a tremendous accomplishment and we are already using this information at JBEI to improve biomass sugar composition for biofuel production.”

    Other co-authors of the PNAS paper reporting this research were Ignacio Moreno, Henry Temple, Thomas Herter, Bruce Link, Daniela Doñas-Cofré, Adrián Moreno, Susana Saéz-Aguayo, Francisca Blanco, Jennifer Mortimer, Alex Schultink, Wolf-Dieter Reiter, Paul Dupre, Markus Pauly and Joshua Heazlewood.

    people
    (From left) Berit Ebert, Carsten Rautengarten and Henrik Scheller at JBEI have developed an assay for characterizing the functions of nucleotide sugar transporters in plant cell walls. (Photo by Irina Silva, JBEI)

    This research was supported by the DOE Office of Science.

    See the full article here.

    A U.S. Department of Energy National Laboratory Operated by the University of California

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  • richardmitnick 3:10 pm on April 24, 2014 Permalink | Reply
    Tags: , Biochemistry, , , , ,   

    From PNNL Lab: “How a plant beckons the bacteria that will do it harm” 


    PNNL Lab

    April 24, 2014
    Tom Rickey, PNNL, (509) 375-3732

    Work on microbial signaling offers a window into better biofuels, human health

    A common plant puts out a welcome mat to bacteria seeking to invade, and scientists have discovered the mat’s molecular mix.

    The study published this week in the Proceedings of the National Academy of Sciences reveals new targets during the battle between microbe and host that researchers can exploit to protect plants.

    The team showed that the humble and oft-studied plant Arabidopsis puts out a molecular signal that invites an attack from a pathogen. It’s as if a hostile army were unknowingly passing by a castle, and the sentry stood up and yelled, “Over here!” — focusing the attackers on a target they would have otherwise simply passed by.

    “This signaling system triggers a structure in bacteria that actually looks a lot like a syringe, which is used to inject virulence proteins into its target. It’s exciting to learn that metabolites excreted by the host can play a role in triggering this system in bacteria,” said Thomas Metz, an author of the paper and a chemist at the Department of Energy’s Pacific Northwest National Laboratory.

    The findings come from a collaboration of scientists led by Scott Peck of the University of Missouri that includes researchers from Missouri, the Biological Sciences Division at PNNL, and EMSL, DOE’s Environmental Molecular Sciences Laboratory.

    The research examines a key moment in the relationship between microbe and host, when a microbe recognizes a host as a potential target and employs its molecular machinery to pierce it, injecting its contents into the plant’s cells — a crucial step in infecting an organism.

    The work focused on bacteria known as Pseudomonas syringae pv. tomato DC3000, which can ruin tomatoes as well as Arabidopsis. The bacteria employ a molecular system known as the Type 3 Secretion System, or T3SS, to infect plants. In tomatoes, the infection leads to unsightly brown spots.

    rot
    Infection of tomatoes by Pseudomonas syringae
    Image courtesy of Cornell University.

    Peck’s team at the University of Missouri had discovered a mutant type of the plant, known as Arabidopsis mkp1, which is resistant to infection by Pseudomonas syringae. The Missouri and PNNL groups compared levels of metabolites in Arabidopsis to those in the mutant mkp1 form of the plant. Peck’s group used those findings as a guide to find the compounds that had the biggest effect — a combination that invites infection.

    The researchers discovered a group of five acids that collectively had the biggest effect on turning on the bacteria’s T3SS: pyroglutamic, citric, shikimic, 4-hydroxybenzoic, and aspartic acids.

    They found that the mutant has a much lower level of these cellular products on the surface of the plant than found in normal plants. Since the resistant plants don’t have high levels of these acids, it stops the bacteria from unfurling the “syringe” in the presence of the plant. But when the combination of acids is introduced onto mkp1, it quickly becomes a target for infection.

    “We know that microbes can disguise themselves by altering the proteins or molecules that the plant uses to recognize the bacteria, as a strategy for evading detection,” said Peck, associate professor of biochemistry at the University of Missouri and lead author of the PNAS paper. “Our results now show that the plant can also disguise itself from pathogen recognition by removing the signals needed by the pathogen to become fully virulent.”

    While Peck’s study focused on bacteria known mostly for damaging tomatoes, the findings also could have implications for people. The same molecular machinery employed by Pseudomonas syringae is also used by a host of microbes to cause diseases that afflict people, including salmonella, the plague, respiratory disease, and chlamydia.

    On the energy front, the findings will help scientists grow plants that can serve as an energy source and are more resistant to infection. Also, a better understanding of the signals that microbes use helps scientists who rely on such organisms for converting materials like switchgrass and wood chips into useable fuel.

    The work opens the door to new ways to rendering harmful bacteria harmless, by modifying plants so they don’t become invasive.

    “There isn’t a single solution for disease resistance in the field, which is part of the reason these findings are important,” said Peck. “The concept of another layer of interaction between host and microbe provides an additional conceptual strategy for how resistance might be manipulated. Rather than trying to kill the bacteria, eliminating the recognition signals in the plant makes the bacteria fairly innocuous, giving the natural immune system more time to defend itself.”

    The research was funded by the National Science Foundation, and some of the research took place at EMSL. PNNL authors of the paper include Metz, Young-Mo Kim, and Ljiljana Pasa-Tolic. Missouri authors include Peck, Ying Wan, and first author Jeffrey C. Anderson.

    See the full article here.

    Pacific Northwest National Laboratory (PNNL) is one of the United States Department of Energy National Laboratories, managed by the Department of Energy’s Office of Science. The main campus of the laboratory is in Richland, Washington.

    PNNL scientists conduct basic and applied research and development to strengthen U.S. scientific foundations for fundamental research and innovation; prevent and counter acts of terrorism through applied research in information analysis, cyber security, and the nonproliferation of weapons of mass destruction; increase the U.S. energy capacity and reduce dependence on imported oil; and reduce the effects of human activity on the environment. PNNL has been operated by Battelle Memorial Institute since 1965.

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  • richardmitnick 12:37 pm on April 22, 2014 Permalink | Reply
    Tags: , Biochemistry, ,   

    From Berkeley Lab: “Berkeley Lab Researchers Demonstrate First Size-based Chromatography Technique for the Study of Living Cells” 


    Berkeley Lab

    April 21, 2014
    Lynn Yarris (510) 486-5375 lcyarris@lbl.gov

    Using nanodot technology, Berkeley Lab researchers have demonstrated the first size-based form of chromatography that can be used to study the membranes of living cells. This unique physical approach to probing cellular membrane structures can reveal information critical to whether a cell lives or dies, remains normal or turns cancerous, that can’t be obtained through conventional microscopy.

    “We’ve developed membrane-embedded nanodot array platforms that provide a physical means to both probe and manipulate membrane assemblies, including signaling clusters, while they are functioning in the membrane of a living cell,” says Jay Groves, a chemist with Berkeley Lab’s Physical Biosciences Division, who led this research.

    t
    With size-based chromatography, a hexagonally ordered array of gold nanoparticles is fabricated onto a hybrid live cell-supported membrane. Membrane components move freely through the array provided they don’t exceed its physical dimensions. This reveals organizational aspects of the membrane environment unobservable by other techniques.

    Groves, who is also a professor with the University of California (UC) Berkeley’s Chemistry Department, and a Howard Hughes Medical Institute (HHMI) investigator, is a recognized leader in developing techniques for studying the impact of spatial patterns on living cells. The live-cell supported synthetic membranes he and his group have been developing are constructed out of lipids and assembled onto a substrate of solid silica. These membranes are being used to determine how living cells not only interact with their environment through chemical signals but also through physical force and spatial patterns.

    “We call our approach the spatial mutation strategy because molecules in a cell can be spatially re-arranged without altering the cell in any other way,” Groves says. “Our live cell-supported membranes provide a hybrid interface consisting of mobile and immobile components with controlled geometry that allows us to utilize solid-state nanotechnology to manipulate and control molecular systems inside living cells.”

    jg
    Jay Groves (photo by Roy Kaltschmidt)

    While the work of Groves and others in recent years has demonstrated the importance of protein and lipid spatial organization within cellular membranes, details as to how spatial organization is tied to function are scarce primarily because of the limitations of optical microscopy at length scales below the 250 nanometer diffraction limit. The size-based chromatography technique developed by Groves and his group allows them to probe supramolecular structures in a cell membrane at the needed nanometer length-scales.

    “We now have a way to translate nano-sized structures that approach molecular dimensions into geometric constraints on the movement of molecules inside a living cell,” Groves says.

    For their size-based chromatography technique, the spacing of proteins and other cellular molecules is controlled by a hexagonal or honeycomb array of gold nanoparticles that is fabricated into the membrane. The spacing between nanoparticles in each array can be controlled, with accessible sizes ranging from 30 to nearly 200 nanometers.

    “Individual membrane components move freely throughout the array, but movement of larger assemblies is impeded if they exceed the physical dimensions of the array, Groves says.

    Groves and his colleagues tested their size-based chromatography technique on T cell receptor (TCR) microclusters in T cell membranes, which is the functional module for antigen recognition by T cells (lymphocytes from the thymus) in the body’s immune system. These TCR signaling clusters occupy a size regime ranging from tens to a few hundred nanometers, which is typically below the diffraction limit of conventional optical microscopy. Size-based chromatography was used to probe the physical properties of TCR signaling clusters as a function of antigen density. The results revealed that TCR signaling cluster is distinctly dependent on the amount of antigen encountered by the cell.

    “This is something we did not know before about the TCR microcluster signaling system, which has been well-studied using conventional optical microscopy,” Groves says. “It is a proof-of-principle demonstration that represents another step in the direction of interfacing living cells with synthetic materials to achieve molecular level control of the cell.”

    A paper on this research has been published in NANO Letters. The paper is titled Size-based chromatography of signaling clusters in a living cell membrane. Groves is the corresponding author. Others authors are Niña Caculitan, Hiroyuki Kai, Eulanca Liu, Nicole Fay, Yan Yu, Theobald Lohmüller and Geoff O’Donoghue.

    See the full article here.

    A U.S. Department of Energy National Laboratory Operated by the University of California

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