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  • richardmitnick 6:24 pm on October 21, 2014 Permalink | Reply
    Tags: , Biology, Brain Studies,   

    From Princeton: “Immune proteins moonlight to regulate brain-cell connections” 

    Princeton University
    Princeton University

    October 21, 2014
    Morgan Kelly, Office of Communications

    When it comes to the brain, “more is better” seems like an obvious assumption. But in the case of synapses, which are the connections between brain cells, too many or too few can both disrupt brain function.

    Researchers from Princeton University and the University of California-San Diego (UCSD) recently found that an immune-system protein called MHCI, or major histocompatibility complex class I, moonlights in the nervous system to help regulate the number of synapses, which transmit chemical and electrical signals between neurons. The researchers report in the Journal of Neuroscience that in the brain MHCI could play an unexpected role in conditions such as Alzheimer’s disease, type II diabetes and autism.

    MHCI proteins are known for their role in the immune system where they present protein fragments from pathogens and cancerous cells to T cells, which are white blood cells with a central role in the body’s response to infection. This presentation allows T cells to recognize and kill infected and cancerous cells.

    In the brain, however, the researchers found that MHCI immune molecules are one of the only known factors that limit the density of synapses, ensuring that synapses form in the appropriate numbers necessary to support healthy brain function. MHCI limits synapse density by inhibiting insulin receptors, which regulate the body’s sugar metabolism and, in the brain, promote synapse formation.

    Tangled web

    web
    Researchers from Princeton University and the University of California-San Diego recently found that an immune-system protein called MHCI, or major histocompatibility complex class I, moonlights in the nervous system to help regulate the number of synapses, which transmit chemical and electrical signals between neurons. Pictured is a mouse hippocampal neuron studded with thousands of synaptic connections (yellow). The number and location of synapses — not too many or too few — is critical to healthy brain function. The researchers found that MHCI proteins, known for their role in the immune system, also are one of the only known factors that ensure synapse density is not too high. The protein does so by inhibiting insulin receptors, which promote synapse formation. (Image courtesy of Lisa Boulanger, Department of Molecular Biology)

    Senior author Lisa Boulanger, an assistant professor in the Department of Molecular Biology and the Princeton Neuroscience Institute (PNI), said that MHCI’s role in ensuring appropriate insulin signaling and synapse density raises the possibility that changes in the protein’s activity could contribute to conditions such Alzheimer’s disease, type II diabetes and autism. These conditions have all been associated with a complex combination of disrupted insulin-signaling pathways, changes in synapse density, and inflammation, which activates immune-system molecules such as MHCI.

    Patients with type II diabetes develop “insulin resistance” in which insulin receptors become incapable of responding to insulin, the reason for which is unknown, Boulanger said. Similarly, patients with Alzheimer’s disease develop insulin resistance in the brain that is so pronounced some have dubbed the disease “type III diabetes,” Boulanger said.

    “Our results suggest that changes in MHCI immune proteins could contribute to disorders of insulin resistance,” Boulanger said. “For example, chronic inflammation is associated with type II diabetes, but the reason for this link has remained a mystery. Our results suggest that inflammation-induced changes in MHCI could have consequences for insulin signaling in neurons and maybe elsewhere.”

    green
    This image of a neuron from a mouse hippocampus shows insulin receptors (green) and the protein calbindin (red). In this area of the brain, calbindin is present in dentate granule cells, which form synapses on MHCI-expressing cells. The extensive overlap (yellow) suggests that this neuron, which expresses insulin receptors, is a dentate granule cell neuron. (Image courtesy of Lisa Boulanger, Department of Molecular Biology)

    MHCI levels also are “dramatically altered” in the brains of people with Alzheimer’s disease, Boulanger said. Normal memory depends on appropriate levels of MHCI. Boulanger was senior author on a 2013 paper in the journal Learning and Memory that found that mice bred to produce less functional MHCI proteins exhibited striking changes in the function of the hippocampus, a part of the brain where some memories are formed, and had severe memory impairments.

    “MHCI levels are altered in the Alzheimer’s brain, and altering MHCI levels in mice disrupts memory, reduces synapse number and causes neuronal insulin resistance, all of which are core features of Alzheimer’s disease,” Boulanger said.

    Links between MHCI and autism also are emerging, Boulanger said. People with autism have more synapses than usual in specific brain regions. In addition, several autism-associated genes regulate synapse number, often via a signaling protein known as mTOR (mammalian target of rapamycin). In their study, Boulanger and her co-authors found that mice with reduced levels of MHCI had increased insulin-receptor signaling via the mTOR pathway, and, consequently, more synapses. When elevated mTOR signaling was reduced in MHCI-deficient mice, normal synapse density was restored.

    Thus, Boulanger said, MHCI and autism-associated genes appear to converge on the mTOR-synapse regulation pathway. This is intriguing given that inflammation during pregnancy, which alters MHCI levels in the fetal brain, may slightly increase the risk of autism in genetically predisposed individuals, she said.

    “Up-regulating MHCI is essential for the maternal immune response, but changing MHCI activity in the fetal brain when synaptic connections are being formed could potentially affect synapse density,” Boulanger said.

    Ben Barres, a professor of neurobiology, developmental biology and neurology at the Stanford University School of Medicine, said that while it is known that both insulin-receptor signaling increases synapse density, and MHCI signaling decreases it, the researchers are the first to show that MHCI actually affects insulin receptors to control synapse density.

    “The idea that there could be a direct interaction between these two signaling systems comes as a great surprise,” said Barres, who was not involved in the research. “This discovery not only will lead to new insight into how brain circuitry develops but to new insight into declining brain function that occurs with aging.”

    cer
    This section of adult mouse cerebellum shows insulin receptors (green) and calbindin (red), which in this case is present in the cerebellar neurons known as Purkinje cells. Insulin receptors are highly expressed in fibers that form synapses onto Purkinje cells, which express MHCI. Thus both in the cerebellum and hippocampus (previous image), insulin receptors are highly expressed in cells that form synapses onto MHCI-expressing neurons, which suggests MHCI and insulin receptors could interact, either directly or indirectly, in the living brain. (Image courtesy of Lisa Boulanger, Department of Molecular Biology)

    Particularly, the research suggests a possible functional connection between type II diabetes and Alzheimer’s disease, Barres said.

    “Type II diabetes has recently emerged as a risk factor for Alzheimer’s disease but it has not been clear what the connection is to the synapse loss experienced with Alzheimer’s disease,” he said. “Given that type II diabetes is accompanied by decreased insulin responsiveness, it may be that the MHCI signaling becomes able to overcome normal insulin signaling and contribute to synapse decline in this disease.”

    Research during the past 15 years has shown that MHCI lives a prolific double-life in the brain, Boulanger said. The brain is “immune privileged,” meaning the immune system doesn’t respond as rapidly or effectively to perceived threats in the brain. Dozens of studies have shown, however, that MHCI is not only present throughout the healthy brain, but is essential for normal brain development and function, Boulanger said. A 2013 paper from her lab published in the journal Molecular and Cellular Neuroscience showed that MHCI is even present in the fetal-mouse brain, at a stage when the immune system is not yet mature.

    “Many people thought that immune molecules like MHCI must be missing from the brain,” Boulanger said. “It turns out that MHCI immune proteins do operate in the brain — they just do something completely different. The dual roles of these proteins in the immune system and nervous system may allow them to mediate both harmful and beneficial interactions between the two systems.”

    The paper, MHC Class I Limits Hippocampal Synapse Density by Inhibiting Neuronal Insulin Receptor Signaling, was published Aug. 27 in the Journal of Neuroscience. Boulanger worked with Carolyn Tyler, a postdoctoral research fellow in PNI; Julianna Poole, who received her master’s degree in molecular biology from Princeton in 2014; Princeton senior Joseph Park; and Lawrence Fourgeaud and Tracy Dixon-Salazar, both at UCSD. The work was supported by the Whitehall Foundation; the Sloan Foundation; Cure Autism Now; the Princeton Neuroscience Institute Innovation Fund; the Silvio Varon Chair in Neuroregeneration at UCSD; Autism Speaks; and the National Science Foundation.

    See the full article here.

    About Princeton: Overview

    Princeton University is a vibrant community of scholarship and learning that stands in the nation’s service and in the service of all nations. Chartered in 1746, Princeton is the fourth-oldest college in the United States. Princeton is an independent, coeducational, nondenominational institution that provides undergraduate and graduate instruction in the humanities, social sciences, natural sciences and engineering.

    As a world-renowned research university, Princeton seeks to achieve the highest levels of distinction in the discovery and transmission of knowledge and understanding. At the same time, Princeton is distinctive among research universities in its commitment to undergraduate teaching.

    Today, more than 1,100 faculty members instruct approximately 5,200 undergraduate students and 2,600 graduate students. The University’s generous financial aid program ensures that talented students from all economic backgrounds can afford a Princeton education.

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  • richardmitnick 4:42 pm on October 21, 2014 Permalink | Reply
    Tags: , , , Biology,   

    From astrobio.net: “Scientists create possible precursor to life” 

    Astrobiology Magazine

    Astrobiology Magazine

    Oct 21, 2014
    University of Southern Denmark
    Contact Professor, Head of FLINT Center, Steen Rasmussen. Email: steen@sdu.dk. Mobile: +45 60112507

    How did life originate? And can scientists create life? These questions not only occupy the minds of scientists interested in the origin of life, but also researchers working with technology of the future. If we can create artificial living systems, we may not only understand the origin of life – we can also revolutionize the future of technology.

    pro
    Model of a protocell. Image by Janet Iwasa

    Protocells are the simplest, most primitive living systems, you can think of. The oldest ancestor of life on Earth was a protocell, and when we see, what it eventually managed to evolve into, we understand why science is so fascinated with protocells. If science can create an artificial protocell, we get a very basic ingredient for creating more advanced artificial life.

    However, creating an artificial protocell is far from simple, and so far no one has managed to do that. One of the challenges is to create the information strings that can be inherited by cell offspring, including protocells. Such information strings are like modern DNA or RNA strings, and they are needed to control cell metabolism and provide the cell with instructions about how to divide.

    Essential for life

    If one daughter cell after a division has a slightly altered information (maybe it provides a slightly faster metabolism), they may be more fit to survive. Therefore it may be selected and an evolution has started.

    Now researchers from the Center for Fundamental Living Technology (FLINT), Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, describe in the journal Europhysics Letters, how they, in a virtual computer experiment, have discovered information strings with peculiar properties.

    Professor and head of FLINT, Steen Rasmussen, says: “Finding mechanisms to create information strings are essential for researchers working with artificial life.”

    auto
    An autocatalytic network is a network of molecules, which catalyze each other’s production. Each molecule can be formed by at least one chemical reaction in the network, and each reaction can be catalyzed by at least one other molecule in the network. This process will create a network that exhibits a primitive form of metabolism and an information system that replicates itself from generation to generation. Credit University of Southern Denmark.

    Steen Rasmussen and his colleagues know they face two problems:

    Firstly long molecular strings are decomposed in water. This means that long information strings “break” quickly in water and turn into many short strings. Thus it is very difficult to maintain a population of long strings over time.

    Secondly, it is difficult to make these molecules replicate without the use of modern enzymes, whereas it is easier to make a so-called ligation. A ligation is to connect any combination of two shorter strings into a longer string, assisted by another matching longer string. Ligation is the mechanism used by the SDU-researchers.

    “In our computer simulation – our virtual molecular laboratory – information strings began to replicate quickly and efficiently as expected. However, we were struck to see that the system quickly developed an equal number of short and long information strings and further that a strong pattern selection on the strings had occurred. We could see that only very specific information patterns on the strings were to be seen in the surviving strings. We were puzzled: How could such a coordinated selection of strings occur, when we knew that we had not programmed it. The explanation had to be found in the way the strings interacted with each other”, explains Steen Rasmussen.

    It is like society

    According to Steen Rasmussen, a so-called self-organizing autocatalytic network was created in the virtual pot, into which he and his colleagues poured the ingredients for information strings.

    “An autocatalytic network works like a community; each molecule is a citizen who interacts with other citizens and together they help create a society”, explains Steen Rasmussen.

    This autocatalytic set quickly evolved into a state where strings of all lengths existed in equal concentrations, which is not what is usually found. Further, the selected strings had strikingly similar patterns, which is also unusual.

    “We might have discovered a process similar to the processes that initially sparked the first life. We of course don’t know if life actually was created this way – but it could have been one of the steps. Perhaps a similar process created sufficiently high concentrations of longer information strings when the first protocell was created”, explains Steen Rasmussen.

    Basis for new technology

    The mechanisms underlying the formation and selection of effective information strings are not only interesting for the researchers who are working to create protocells. They also have value to researchers working with tomorrow’s technology, like they do at the FLINT Center.

    “We seek ways to develop technology that’s based on living and life-like processes. If we succeed, we will have a world where technological devices can repair themselves, develop new properties and be re-used. For example a computer made of biological materials poses very different – and less environmentally stressful – requirements for production and disposal”, says Steen Rasmussen.

    Ref: http://epljournal.edpsciences.org/articles/epl/abs/2014/14/epl16388/epl16388.html

    See the full article here.

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  • richardmitnick 3:04 pm on October 20, 2014 Permalink | Reply
    Tags: , Biology, ,   

    From LLNL: “Supercomputers link proteins to drug side effects” 


    Lawrence Livermore National Laboratory

    10/20/2014
    Kenneth K Ma, LLNL, (925) 423-7602, ma28@llnl.gov

    New medications created by pharmaceutical companies have helped millions of Americans alleviate pain and suffering from their medical conditions. However, the drug creation process often misses many side effects that kill at least 100,000 patients a year, according to the journal Nature.

    Lawrence Livermore National Laboratory researchers have discovered a high-tech method of using supercomputers to identify proteins that cause medications to have certain adverse drug reactions (ADR) or side effects. They are using high-performance computers (HPC) to process proteins and drug compounds in an algorithm that produces reliable data outside of a laboratory setting for drug discovery.

    The team recently published its findings in the journal PLOS ONE, titled Adverse Drug Reaction Prediction Using Scores Produced by Large-Scale Drug-Protein Target Docking on High-Performance Computer Machines.

    “We need to do something to identify these side effects earlier in the drug development cycle to save lives and reduce costs,” said Monte LaBute, a researcher from LLNL’s Computational Engineering Division and the paper’s lead author.

    It takes pharmaceutical companies roughly 15 years to bring a new drug to the market, at an average cost of $2 billion. A new drug compound entering Phase I (early stage) testing is estimated to have an 8 percent chance of reaching the market, according to the Food and Drug Administration (FDA).

    A typical drug discovery process begins with identifying which proteins are associated with a specific disease. Candidate drug compounds are combined with target proteins in a process known as binding to determine the drug’s effectiveness (efficacy) and/or harmful side effects (toxicity). Target proteins are proteins known to bind with drug compounds in order for the pharmaceutical to work.

    While this method is able to identify side effects with many target proteins, there are myriad unknown “off-target” proteins that may bind to the candidate drug and could cause unanticipated side effects.

    Because it is cost prohibitive to experimentally test a drug candidate against a potentially large set of proteins — and the list of possible off-targets is not known ahead of time — pharmaceutical companies usually only test a minimal set of off-target proteins during the early stages of drug discovery. This results in ADRs remaining undetected through the later stages of drug development, such as clinical trials, and possibly making it to the marketplace.

    There have been several highly publicized medications with off-target protein side effects that have reached the marketplace. For example, Avandia, an anti-diabetic drug, caused heart attacks in some patients; and Vioxx, an anti-inflammatory medication, caused heart attacks and strokes among certain patient populations. Both therapeutics were recalled because of their side effects.

    “There were no indications of side effects of these medications in early testing or clinical trials,” LaBute said. “We need a way to determine the safety of such therapeutics before they reach patients. Our work can help direct such drugs to patients who will benefit the most from them with the least amount of side effects.”

    LaBute and the LLNL research team tackled the problem by using supercomputers and information from public databases of drug compounds and proteins. The latter included protein databases of DrugBank, UniProt and Protein Data Bank (PDB), along with drug databases from the FDA and SIDER, which contain FDA-approved drugs with ADRs.

    The team examined 4,020 off-target proteins from DrugBank and UniProt. Those proteins were indexed against the PDB, which whittled the number down to 409 off-proteins that have high-quality 3D crystallographic X-ray diffraction structures essential for analysis in a computational setting.

    mp

    The 409 off-target proteins were fed into a Livermore HPC software known as VinaLC along with 906 FDA-approved drug compounds. VinaLC used a molecular docking matrix that bound the drugs to the proteins. A score was given to each combination to assess whether effective binding occurred.

    The binding scores were fed into another computer program and combined with 560 FDA-approved drugs with known side effects. An algorithm was used to determine which proteins were associated with certain side effects.

    The Lab team showed that in two categories of disorders — vascular disorders and neoplasms — their computational model of predicting side effects in the early stages of drug discovery using off-target proteins was more predictive than current statistical methods that do not include binding scores.

    In addition to LLNL ADR prediction methods performing better than current prediction methods, the team’s calculations also predicted new potential side effects. For example, they predicted a connection between a protein normally associated with cancer metastasis to vascular disorders like aneurysms. Their ADR predictions were validated by a thorough review of existing scientific data.

    “We have discovered a very viable way to find off-target proteins that are important for side effects,” LaBute said. “This approach using HPC and molecular docking to find ADRs never really existed before.”

    The team’s findings provide drug companies with a cost-effective and reliable method to screen for side effects, according to LaBute. Their goal is to expand their computational pharmaceutical research to include more off-target proteins for testing and eventually screen every protein in the body.

    “If we can do that, the drugs of tomorrow will have less side effects that can potentially lead to fatalities,” Labute said. “Optimistically, we could be a decade away from our ultimate goal. However, we need help from pharmaceutical companies, health care providers and the FDA to provide us with patient and therapeutic data.”

    two
    LLNL researchers Monte LaBute (left) and Felice Lightstone (right) were part of a Lab team that recently published an article in PLOS ONE detailing the use of supercomputers to link proteins to drug side effects. Photo by Julie Russell/LLNL

    The LLNL team also includes Felice Lightstone, Xiaohua Zhang, Jason Lenderman, Brian Bennion and Sergio Wong.

    See the full article here.

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  • richardmitnick 1:45 pm on October 17, 2014 Permalink | Reply
    Tags: , Biology,   

    From AAAS: “Would-be drug mimics ‘good’ cholesterol” 

    AAAS

    AAAS

    16 October 2014
    Robert F. Service

    A new drug candidate designed to mimic the body’s “good” cholesterol shows a striking ability in mice to lower cholesterol levels in the blood and dissolve artery-clogging plaques. What’s more, the compound works when given orally, rather than as an injection. If the results hold true in humans—a big if, given past failures at transferring promising treatments from mice—it could provide a new way to combat atherosclerosis, the biggest killer in developed countries.

    Although doctors already have effective cholesterol-lowering agents, such as statins, at their disposal, there’s room for improvement. Statins have significant side effects in some people and don’t always reduce cholesterol enough in others. “There is still plenty of heart disease out there even among people who take statins,” says Godfrey Getz, an experimental pathologist at the University of Chicago in Illinois.

    For that reason, researchers around the globe are searching for novel drugs that affect cholesterol levels in one of two ways. The first has been to reduce levels of low-density lipoprotein (LDL), commonly known as bad cholesterol, which has been associated with higher heart disease risk. This is the goal of statins, which block an enzyme involved in cholesterol production. The second strategy is to increase levels of good cholesterol, or high-density lipoprotein (HDL), which seems to boost heart health in people who have a lot of it. But producing HDL-raising drugs that prevent heart disease has proven difficult. In the body, a large protein called apolipoprotein A-I (apoA-I) wraps around fatty lipid molecules to create HDL particles that sop up LDL and ferry it to the liver where it is eliminated. So for several decades researchers have been designing and testing small protein fragments called peptides to see if they could mimic the behavior of apoA-I. One such peptide, known as 4F, did not reduce serum cholesterol levels, but it did shrink arterial plaques in mice, rabbits, and monkeys. And in an early clinical trial by researchers at Bruin Pharma Inc. in Beverly Hills, California, that was designed only to measure its safety in people, 4F didn’t appear to show any beneficial effect.

    pro
    Multiple copies of a four-armed peptide wrap around lipids to create particles that mimic the behavior of HDL, the “good” cholesterol.
    Y.Zhao et al., J. Am. Chem. Soc

    M. Reza Ghadiri, a chemist at the Scripps Research Institute in San Diego, California, and his colleagues took a slightly different tack, creating a peptide that mimics another part of the apoA-I protein than 4F does. Initial in vitro studies suggested the peptide formed HDL-like particles and sopped up LDL, an encouraging result that prompted them to push it further. Ghadiri and his Scripps colleagues have now tested their compound in mice that develop artery clogging plaques when fed a Western-style high-fat diet. One group of animals received the peptide intravenously. For another group, the researchers simply added the compound to the animals’ water, a strategy they considered unlikely to work, because the gut contains high amounts of proteases designed to chop proteins apart. To their surprise, in both groups, serum cholesterol levels dropped 40% from their previous levels within 2 weeks of starting to take the drug. And by 10 weeks, the number of artery-clogging lesions had been reduced by half, the team reports in the October issue of the Journal of Lipid Research. What remains puzzling, however, is that Ghadiri and his colleagues did not detect their peptides in the blood of their test animal. Ghadiri says this suggests that the new peptide may work by removing cholesterol precursors in the gut before they enter the bloodstream.

    “It’s a very interesting result,” Getz says. But he cautions that the work has been tested only in animals, and many therapies—including the closely related 4F peptide—fail to transfer to humans. That said, Getz notes that some of the initial promising results with this peptide and other apoA-I mimics offer hope that researchers may soon come up with novel drugs capable of dissolving artery-clogging plaques before they can wreak their havoc.

    See the full article here.

    The American Association for the Advancement of Science is an international non-profit organization dedicated to advancing science for the benefit of all people.

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  • richardmitnick 8:38 am on October 17, 2014 Permalink | Reply
    Tags: , Biology, , ,   

    From UC Berkeley: “New front in war on Alzheimer’s, other protein-folding diseases” 

    UC Berkeley

    UC Berkeley

    October 16, 2014
    Robert Sanders

    A surprise discovery that overturns decades of thinking about how the body fixes proteins that come unraveled greatly expands opportunities for therapies to prevent diseases such as Alzheimer’s and Parkinson’s, which have been linked to the accumulation of improperly folded proteins in the brain.

    “This finding provides a whole other outlook on protein-folding diseases; a new way to go after them,” said Andrew Dillin, the Thomas and Stacey Siebel Distinguished Chair of Stem Cell Research in the Department of Molecular and Cell Biology and Howard Hughes Medical Institute investigator at the University of California, Berkeley.

    br
    A cell suffering heat shock is like a country besieged, where attackers first sever lines of communications. The pat-10 gene helps repair communication to allow chaperones to treat misfolded proteins. (Andrew Dillin graphic)

    Dillin, UC Berkeley postdoctoral fellows Nathan A. Baird and Peter M. Douglas and their colleagues at the University of Michigan, The Scripps Research Institute and Genentech Inc., will publish their results in the Oct. 17 issue of the journal Science.

    Cells put a lot of effort into preventing proteins – which are like a string of beads arranged in a precise three-dimensional shape – from unraveling, since a protein’s activity as an enzyme or structural component depends on being properly shaped and folded. There are at least 350 separate molecular chaperones constantly patrolling the cell to refold misfolded proteins. Heat is one of the major threats to proteins, as can be demonstrated when frying an egg – the clear white albumen turns opaque as the proteins unfold and then tangle like spaghetti.

    Heat shock

    For 35 years, researchers have worked under the assumption that when cells undergo heat shock, as with a fever, they produce a protein that triggers a cascade of events that field even more chaperones to refold unraveling proteins that could kill the cell. The protein, HSF-1 (heat shock factor-1), does this by binding to promoters upstream of the 350-plus chaperone genes, upping the genes’ activity and launching the army of chaperones, which originally were called “heat shock proteins.”

    Injecting animals with HSF-1 has been shown not only to increase their tolerance of heat stress, but to increase lifespan.

    Because an accumulation of misfolded proteins has been implicated in aging and in neurodegenerative diseases such as Alzheimer’s, Parkinson’s and Huntington’s diseases, scientists have sought ways to artificially boost HSF-1 in order to reduce the protein plaques and tangles that eventually kill brain cells. To date, such boosters have extended lifespan in lab animals, including mice, but greatly increased the incidence of cancer.

    Dillin’s team found in experiments on the nematode worm C. elegans that HSF-1 does a whole lot more than trigger release of chaperones. An equal if not more important function is to stabilize the cell’s cytoskeleton, which is the highway that transports essential supplies – healing chaperones included – around the cell.

    “We are suggesting that, rather than making more of HSF-1 to prevent diseases like Huntington’s, we should be looking for ways to make the actin cytoskeleton better,” Dillin said. Such tactics might avoid the carcinogenic side effects of upping HSF-1.

    Dillin is codirector of the Paul F. Glenn Center for Aging Research, a new collaboration between UC Berkeley and UC San Francisco supported by the Glenn Foundation for Medical Research. Center investigators will study the many ways that proteins malfunction within cells, ideally paving the way for novel treatments for neurodegenerative diseases.

    A cell at war

    Dillin compares a cell experiencing heat shock to a country under attack. In a war, an aggressor first cuts off all communications, such as roads, train and bridges, which prevents the doctors from treating the wounded. Similarly, heat shock disrupts the cytoskeletal highway, preventing the chaperone “doctors” from reaching the patients, the misfolded proteins.

    chap
    Chaperones help newborn proteins (polypeptides) fold properly, but also fix misfolded proteins.

    “We think HSF-1 not only makes more chaperones, more doctors, but also insures that the roadways stay intact to keep everything functional and make sure the chaperones can get to the sick and wounded warriors,” he said.

    The researchers found specifically that HSF-1 up-regulates another gene, pat-10, that produces a protein that stabilizes actin, the building blocks of the cytoskeleton.

    By boosting pat-10 activity, they were able to cure worms that had been altered to express the Huntington’s disease gene, and also extend the lifespan of normal worms.

    Dillin suspects that HSF-1’s main function is, in fact, to protect the actin cytoskeleton. He and his team mutated HSF-1 so that it no longer boosted chaperones, demonstrating, he said, that “you can survive heat shock with the normal level of heat shock proteins, as long as you make your cytoskeleton work better.”

    He noted that the team’s results – that boosting chaperones is not essential to surviving heat stress – were so contradictory to current thinking that “I made my post-docs’ lives hell for three years” insisting on more experiments to rule out errors. Yet, when Dillin presented the results recently to members of the protein-folding community, he said the first reaction of many was, “That makes perfect sense.”

    Dillin’s colleagues include Milos S. Simic and Suzanne C. Wolff of UC Berkeley, Ana R. Grant of the University of Michigan in Ann Arbor, James J. Moresco and John R. Yates III of Scripps in La Jolla, Calif., and Gerard Manning of Genentech, South San Francisco, Calif. The work is funded by the Howard Hughes Medical Institute as well as by the National Institute of General Medical Sciences (8 P41 GM103533-17) and National Institute on Aging (R01AG027463-04) of the National Institutes of Health.

    See the full article here.

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  • richardmitnick 9:04 pm on October 16, 2014 Permalink | Reply
    Tags: , Biology, , ,   

    From LLNL: “Lab, UC Davis partner to personalize cancer medications” 


    Lawrence Livermore National Laboratory

    10/16/2014
    Stephen P Wampler, LLNL, (925) 423-3107, wampler1@llnl.gov

    Buoyed by several dramatic advances, Lawrence Livermore National Laboratory scientists think they can tackle biological science in a way that couldn’t be done before.

    Over the past two years, Lab researchers have expedited accelerator mass spectrometer sample preparation and analysis time from days to minutes and moved a complex scientific process requiring accelerator physicists into routine laboratory usage.

    Ken Turteltaub, the leader of the Lab’s Biosciences and Biotechnology Division, sees the bio AMS advances as allowing researchers to undertake quantitative assessments of complex biological pathways.

    “We are hopeful that we’ll be able to quantify the individual steps in a metabolic pathway and be able to measure indicators of disease processes and factors important to why people differ in responses to therapeutics, to diet and other factors,” Turteltaub said.

    Graham Bench, the director of the Lab’s Center for Accelerator Mass Spectrometry, anticipates the upgrades will enable Lab researchers “to produce high-density data sets and tackle novel biomedical problems that in the past couldn’t be addressed.”

    Ted Ognibene, a chemist who has worked on AMS for 15 years and who co-developed the technique that accommodates liquid samples, also envisions new scientific work coming forth.

    two
    Ted Ognibene (left), a chemist who co-developed the technique that accommodates liquid samples for accelerator mass spectrometry, peers with biomedical scientist Mike Malfatti at the new biological AMS instrument that has been installed in the Laboratory’s biomedical building. Photo by George Kitrinos

    “We previously had the capability to detect metabolites, but now with the ability to see our results almost immediately for a fraction of the cost, it’s going to enable a lot more fundamental and new science to be done,” Ognibene said.

    Biological AMS is a technique in which carbon-14 is used as a tag to study with extreme precision and sensitivity complex biological processes, such as cancer, molecular damage, drug and toxin behavior, nutrition and other areas.

    Among the biomedical studies that will be funded through the five-year, $7.8 million National Institutes of Health grant for biological AMS work is one to try to develop a test to predict how people will respond to chemotherapeutic drugs.

    Another research project seeks to create an assay that is so sensitive that it can detect one cancer cell among one million healthy cells. If this work is successful, it could be possible to evaluate the metastasis potential of different primary human cancer cells.

    Lab biomedical scientist Mike Malfatti and two researchers - Paul Henderson, an associate professor, and Chong-Xian Pan, a medical oncologist — from the University of California, Davis Comprehensive Cancer Center, are using the AMS in a human trial with 50 patients to see how cancer patients respond or don’t respond to the chemotherapeutic drug carboplatin. This drug kills cancer cells by binding to DNA, and is toxic to rapidly dividing cells.

    The three researchers have the patients take a microdose of carboplatin — about 1/100th of a therapeutic dose — that has no toxicity or therapeutic value to evaluate how effectively the drug will bind to a person’s DNA during full dose treatment.

    Within a few days of patients receiving the microdose, the degree of drug binding is checked by blood sample, in which the DNA is isolated from white blood cells, or by tumor biopsy, in which the DNA is isolated from the tumor cells.

    The carboplatin dose is prepared with a carbon-14 tag. The DNA sample is analyzed using AMS and the instrument quantifies the carbon-14 level, with a high level of carbon-14 indicating a high level of drug binding to the DNA.

    “A high degree of binding indicates that you have a high probability of a favorable response to the drug,” Malfatti said. “Conversely, a low degree of binding means it is likely the person’s body won’t respond to the treatment.

    “If we can identify which people will respond to which chemotherapeutic drug, we can tailor the treatment to the individual.

    “There are many negative side effects associated with chemotherapy, such as nausea, loss of appetite, loss of hair and even death. We don’t want someone to receive chemotherapy that’s not going to help them, yet leave them with these negative side effects,” he added.

    Malfatti, Henderson and Pan also are using the AMS in pre-clinical studies to investigate the resistance or receptivity of other commonly used chemotherapeutic agents such as cisplatin, oxaliplatin and gemcitabine.

    Another team of researchers, led by Gaby Loots, a Lab biomedical scientist and an associate professor at the University of California, Merced, wants to use AMS to measure cancer cells labeled with carbon-14 to study the cancer cells’ migration to healthy tissues to determine how likely they are to form metastatic tumors.

    While today’s standard methods can detect tumors that are comprised of thousands of cells, the team would like to develop an assay with a thousand-fold better resolution – to detect one cancer cell among one million healthy ones.

    “The sensitivity of AMS allows us to develop more accurate, quantitative assays with single-cell resolution. Is the cancer completely gone, or do we see one cell worth of cancer DNA?” Loots noted.

    Some of the questions the team would like to answer are: 1) why certain cells metastasize? 2) how do cells metastasize? 3) what new methods can be developed to prevent metastasis?

    “Tumors shed cells all the time that enter our circulation. We would like to find ways to prevent the circulating tumor cells from forming metastatic tumors,” Loots continued.

    As a part of their research, the team members hope to determine whether cancer cells with stem-cell-like properties form more aggressive tumors.

    “We’re going to separate the cancer cells into stem-cell-like and non-stem-cell-like populations and seek to determine if they behave differently,” said Loots, who is working with fellow Lab biomedical scientists Nick Hum and Nicole Collette.

    See the full article here.

    LLNL Campus

    Operated by Lawrence Livermore National Security, LLC, for the Department of Energy’s National Nuclear Security
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  • richardmitnick 6:09 pm on October 16, 2014 Permalink | Reply
    Tags: , , Biology, ,   

    From Johns Hopkins: “Chemical derived from broccoli sprouts shows promise in treating autism” 

    Johns Hopkins
    Johns Hopkins University

    October 13, 2014
    Catherine Kolf

    Many trial participants who received daily dose of sulforaphane show improvements in social interaction, verbal communication, researchers say

    Results of a small clinical trial suggest that a chemical derived from broccoli sprouts—and best known for claims that it can help prevent certain cancers—may ease classic behavioral symptoms in those with autism spectrum disorders.

    bro

    The study, a joint effort by scientists at MassGeneral Hospital for Children and the Johns Hopkins University School of Medicine, involved 40 teenage boys and young men, ages 13 to 27, with moderate to severe autism.

    In a report published online in the journal Proceedings of the National Academy of Sciences, the researchers say that many of those who received a daily dose of the chemical sulforaphane experienced substantial improvements in their social interaction and verbal communication, along with decreases in repetitive, ritualistic behaviors, compared to those who received a placebo.

    “We believe that this may be preliminary evidence for the first treatment for autism that improves symptoms by apparently correcting some of the underlying cellular problems,” says Paul Talalay, a professor of pharmacology and molecular sciences at the Johns Hopkins University School of Medicine who has researched these vegetable compounds for the past 25 years.

    “We are far from being able to declare a victory over autism, but this gives us important insights into what might help,” says co-investigator Andrew Zimmerman, a professor of pediatric neurology at UMass Memorial Medical Center.

    Autism experts estimate that the group of disorders affects 1 to 2 percent of the world’s population, with a much higher incidence in boys than in girls. Its behavioral symptoms, such as poor social interaction and verbal communication, are well known and were first described 70 years ago by Leo Kanner, the founder of pediatric psychiatry at Johns Hopkins University.

    Unfortunately, the root causes of autism remain elusive, though progress has been made, Talalay says, in describing some of the biochemical and molecular abnormalities that tend to accompany the disorders. Many of these are related to the efficiency of energy generation in cells. He says that studies show that the cells of those on the autism spectrum often have high levels of oxidative stress, the buildup of harmful, unintended byproducts from the cell’s use of oxygen that can cause inflammation, damage DNA, and lead to cancer and other chronic diseases.

    In 1992, Talalay’s research group discovered that sulforaphane has some ability to bolster the body’s natural defenses against oxidative stress, inflammation, and DNA damage. In addition, the chemical later turned out to improve the body’s heat-shock response—a cascade of events used to protect cells from the stress caused by high temperatures, including those experienced when people have fever.

    Intriguingly, he says, about 50% of parents report that their children’s autistic behavior improves noticeably when they have a fever, then reverts back when the fever is gone. In 2007, Zimmerman, a principal collaborator in the current study, tested this anecdotal trend clinically and found it to be true, though a mechanism for the fever effect was not identified.

    Because fevers, like sulforaphane, initiate the body’s heat-shock response, Zimmerman and Talalay wondered if sulforaphane could cause the same temporary improvement in autism that fevers do. The current study was designed to find out.

    Before the start of the trial, the patients’ caregivers and physicians filled out three standard behavioral assessments: the Aberrant Behavior Checklist (ABC), the Social Responsiveness Scale (SRS), and the Clinical Global Impressions-Improvement scale (CGI-I). The assessments measure sensory sensitivities, ability to relate to others, verbal communication skills, social interactions, and other behaviors related to autism.

    Twenty-six of the subjects were randomly selected to receive, based on their weight, 9 to 27 milligrams of sulforaphane daily, and 14 received placebos. Behavioral assessments were again completed at four, 10, and 18 weeks while treatment continued. A final assessment was completed for most of the participants four weeks after the treatment had stopped.

    Most of those who responded to sulforaphane showed significant improvements by the first measurement at four weeks and continued to improve during the rest of the treatment. After 18 weeks of treatment, the average ABC and SRS scores of those who received sulforaphane had decreased 34 and 17 percent, respectively, with improvements in bouts of irritability, lethargy, repetitive movements, hyperactivity, awareness, communication, motivation, and mannerisms.

    After 18 weeks of treatment, according to the CGI-I scale, sulforaphane recipients experienced noticeable improvements in social interaction (46%), aberrant behaviors (54%), and verbal communication (42%).

    Talalay notes that the scores of those who took sulforaphane trended back toward their original values after they stopped taking the chemical, just like what happens to those who experience improvements during a fever. “It seems like sulforaphane is temporarily helping cells to cope with their handicaps,” he says.

    Zimmerman adds that before they learned which subjects got the sulforaphane or placebo, the impressions of the clinical team—including parents—were that 13 of the participants noticeably improved. For example, some treated subjects looked them in the eye and shook their hands, which they had not done before. They found out later that all 13—half of the treatment group—had been taking sulforaphane.

    Talalay cautions that the levels of sulforaphane precursors present in different varieties of broccoli are highly variable. Furthermore, the capacity of individuals to convert these precursors to active sulforaphane also varies greatly. It would be very difficult to achieve the levels of sulforaphane used in this study by eating large amounts of broccoli or other cruciferous vegetables, he notes.

    See the full article here.

    Johns Hopkins Campus

    The Johns Hopkins University opened in 1876, with the inauguration of its first president, Daniel Coit Gilman. “What are we aiming at?” Gilman asked in his installation address. “The encouragement of research … and the advancement of individual scholars, who by their excellence will advance the sciences they pursue, and the society where they dwell.”

    The mission laid out by Gilman remains the university’s mission today, summed up in a simple but powerful restatement of Gilman’s own words: “Knowledge for the world.”

    What Gilman created was a research university, dedicated to advancing both students’ knowledge and the state of human knowledge through research and scholarship. Gilman believed that teaching and research are interdependent, that success in one depends on success in the other. A modern university, he believed, must do both well. The realization of Gilman’s philosophy at Johns Hopkins, and at other institutions that later attracted Johns Hopkins-trained scholars, revolutionized higher education in America, leading to the research university system as it exists today.

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  • richardmitnick 5:55 pm on October 16, 2014 Permalink | Reply
    Tags: , Biology, , ,   

    From BNL: “Scientists Map Key Moment in Assembly of DNA-Splitting Molecular Machine” 

    Brookhaven Lab

    October 15, 2014
    Justin Eure, (631) 344-2347 or Peter Genzer, (631) 344-3174

    The proteins that drive DNA replication—the force behind cellular growth and reproduction—are some of the most complex machines on Earth. The multistep replication process involves hundreds of atomic-scale moving parts that rapidly interact and transform. Mapping that dense molecular machinery is one of the most promising and challenging frontiers in medicine and biology.

    Now, scientists have pinpointed crucial steps in the beginning of the replication process, including surprising structural details about the enzyme that “unzips” and splits the DNA double helix so the two halves can serve as templates for DNA duplication.

    The research combined electron microscopy, perfectly distilled proteins, and a method of chemical freezing to isolate specific moments at the start of replication. The study—authored by scientists from the U.S. Department of Energy’s Brookhaven National Laboratory, Stony Brook University, Cold Spring Harbor Laboratory, and Imperial College, London—published on Oct. 15, 2014, in the journal Genes and Development.

    “The genesis of the DNA-unwinding machinery is wonderfully complex and surprising,” said study coauthor Huilin Li, a biologist at Brookhaven Lab and Stony Brook University. “Seeing this helicase enzyme prepare to surround and unwind the DNA at the molecular level helps us understand the most fundamental process of life and how that process might go wrong. Errors in copying DNA are found in certain cancers, and this work could one day help develop new treatment methods that stall or break dangerous runaway machinery.”

    The research picks up where two previous studies by Li and colleagues left off. They first determined the structure of the “Origin Recognition Complex” (ORC), a protein that identifies and attaches to specific DNA sites to initiate the entire replication process. The second study revealed how the ORC recruits, cracks open, and installs a crucial ring-shaped protein structure (Mcm2-7) that lies at the core of the helicase enzyme.

    But DNA replication is a bi-directional process with two helicases moving in opposite directions. The key question, then, was how does a second helicase core get recruited and loaded onto the DNA in the opposite orientation of the first?

    dr
    Three-dimensional model (based on electron microscopy data) of the double-ring structure loaded onto a DNA helix.

    “To our surprise, we found an intermediate structure with one ORC binding two rings,” said Brookhaven Lab biologist and lead author Jingchuan Sun. “This discovery suggests that a single ORC, rather than the commonly believed two-ORC system, loads both helicase rings.”

    One step further along, the researchers also determined the molecular architecture of the final double-ring structure left behind after the ORC leaves the system, offering a number of key biological insights.

    “We now have clues to how that double-ring structure stably lingers until the cell enters the DNA-synthesis phase much later on in replication,” said study coauthor Christian Speck of Imperial College, London. “This study revealed key regulatory principles that explain how the helicase activity is initially suppressed and then becomes reactivated to begin its work splitting the DNA.”

    three
    Precision methods, close collaboration
    Collaborating scientists and study coauthors Zuanning Yuan of Stony Brook University (standing), Huilin Li of Stony Brook and Brookhaven Lab (seated, back), and Jingchuan Sun of Brookhaven Lab (seated, front) examining protein structures.

    Examining these fleeting molecular structures required mastery of biology, chemistry, and electron microscopy techniques.

    “This three-way collaboration took advantage of each lab’s long standing collaboration and expertise,” said study coauthor Bruce Stillman of Cold Spring Harbor. “Imperial College and Cold Spring Harbor handled the challenging material preparation and functional characterization, while Brookhaven and Stony Brook led the sophisticated molecular imaging and three-dimensional image reconstruction.”

    The researchers used proteins from baker’s yeast—a model organism for the more complex systems found in animals. The scientists isolated the protein mechanisms involved in replication and removed structures that might otherwise complicate the images.

    Once the isolated proteins were mixed with DNA, the scientists injected chemicals to “freeze” the binding and recruitment process at intervals of 2, 7, and 30 minutes.

    They then used an electron microscope at Brookhaven to pin down the exact structures at each targeted moment in a kind of molecular time-lapse. Rather than the light used in a traditional microscope, this technique uses focused beams of electrons to illuminate a sample and form images with atomic resolution. The instrument produces a large number of two-dimensional electron beam images, which a computer then reconstructs into three-dimensional structure.

    “This technique is ideal because we’re imaging relatively massive proteins here,” Li said. “A typical protein contains three hundred amino acids, but these DNA replication mechanisms consist of tens of thousands of amino acids. The entire structure is about 20-nanometers across, compared to 4 nanometers for an average protein.”

    Unraveling the DNA processes at the most fundamental level, the focus of this team’s work, could have far-reaching implications.

    “The structural knowledge may help others engineer small molecules that inhibit DNA replication at specific moments, leading to new disease prevention or treatment techniques,” Li said.

    Additional collaborators on this research include Alejandra Fernandez, Alberto Riera, and Silvia Tognetti of the MRC Clinical Science Centre of Imperial College, London; and Zuanning Yuan of Stony Brook University.

    The research was funded by the National Institutes of Health (GM45436, GM74985) and the United Kingdom Medical Research Council.

    See the full article here.

    BNL Campus

    One of ten national laboratories overseen and primarily funded by the Office of Science of the U.S. Department of Energy (DOE), Brookhaven National Laboratory conducts research in the physical, biomedical, and environmental sciences, as well as in energy technologies and national security. Brookhaven Lab also builds and operates major scientific facilities available to university, industry and government researchers. The Laboratory’s almost 3,000 scientists, engineers, and support staff are joined each year by more than 5,000 visiting researchers from around the world.Brookhaven is operated and managed for DOE’s Office of Science by Brookhaven Science Associates, a limited-liability company founded by Stony Brook University, the largest academic user of Laboratory facilities, and Battelle, a nonprofit, applied science and technology organization.
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  • richardmitnick 4:12 pm on October 16, 2014 Permalink | Reply
    Tags: , Biology,   

    From Caltech: “Improving The View Through Tissues and Organs” 

    Caltech Logo
    Caltech

    10/16/2014
    Kimm Fesenmaier

    This summer, several undergraduate students at Caltech had the opportunity to help optimize a promising technique that can make tissues and organs—even entire organisms—transparent for study. As part of the Summer Undergraduate Research Fellowship (SURF) program, these students worked in the lab of Assistant Professor of Biology Viviana Gradinaru, where researchers are developing such so-called clearing techniques that make it possible to peer straight through normally opaque tissues rather than seeing them only as thinly sectioned slices that have been pieced back together.

    tissue
    Credit: iStock

    Gradinaru’s group recently published a paper in the journal Cell describing a new approach to tissue clearing. The method they have created builds on a technique called CLARITY that Gradinaru helped develop while she was a research associate at Stanford. CLARITY allowed researchers to, for the first time, create a transparent whole-brain specimen that could then be imaged with its structural and genetic information intact.

    CLARITY was specifically developed for studying the brain. But the new approach developed in Gradinaru’s lab, which the team has dubbed PARS (perfusion-assisted agent release in situ), can also clear other organs, such as the kidney, as well as tissue samples, such as tumor biopsies. It can even be applied to entire organisms.

    Like CLARITY, PARS involves removing the light-scattering lipids in the tissue to make samples transparent without losing the structural integrity that lipids typically provide. First the sample is infused with acrylamide monomers that are then polymerized into a hydrogel that provides structural support. Next, this tissue–hydrogel hybrid is immersed in a detergent that removes the lipids. Then the sample can be stained, often with antibodies that specifically mark cells of interest, and then immersed in RIMS (refractive index matching solution) for imaging using various optical techniques such as confocal or lightsheet microscopy.

    Over the summer, Sam Wie, a junior biology major at Caltech, spent 10 weeks in the Gradinaru lab working to find a polymer that would perform better than acrylamide, which has been used in the CLARITY hydrogel. “One of the limitations of CLARITY is that when you put the hydrogel tissue into the detergent, the higher solute concentration in the tissue causes liquid to rush into the cell. That causes the sample to swell, which could potentially damage the structure of the tissue,” Wie explains. “So I tried different polymers to try to limit that swelling.”

    Wie was able to identify a polymer that produces, over a similar amount of time, about one-sixth of the swelling in the tissue.

    “The SURF experience has been very rewarding,” Wie says. “I’ve learned a lot of new techniques, and it’s really exciting to be part of, and to try to improve, CLARITY, a method that will probably change the way that we image tissues from now on.”

    At another bench in Gradinaru’s lab, sophomore bioengineering major Andy Kim spent the summer focusing on a different aspect of the PARS technique. While antibodies have been the most common markers used to tag cells of interest within cleared tissues, they are too large for some studies—for example, those that aim to image deeper parts of the brain, requiring them to cross the blood–brain barrier. Kim’s project involved identifying smaller proteins, such as nanobodies, which target and bind to specific parts of proteins in tissues.

    “While PARS is a huge improvement over CLARITY, using antibodies to stain is very expensive,” Kim says. “However, some of these nanobodies can be produced easily, so if we can get them to work, it would not only help image the interior of the brain, it would also be a lot less costly.”

    During his SURF, Kim worked with others in the lab to identify about 30 of these smaller candidate binding proteins and tested them on PARS-cleared samples.

    While Wie and Kim worked on improving the PARS technique itself, Donghun Ryu, a third SURFer in Gradinaru’s lab, investigated different methods for imaging the cleared samples. Ryu is a senior electrical engineering and computer science major at the Gwangju Institute of Science and Technology (GIST) in the Republic of Korea.

    Last summer Ryu completed a SURF as part of the Caltech–GIST Summer Undergraduate Research Exchange Program in the lab of Changhuei Yang, professor of electrical engineering, bioengineering, and medical engineering at Caltech. While completing that project, Ryu became interested in optogenetics, the use of light to control genes. Since optogenetics is one of Gradinaru’s specialties, Yang suggested that he try a SURF in Gradinaru’s lab.

    This summer, Ryu was able to work with both Yang and Gradinaru, investigating a technique called Talbot microscopy to see whether it would be better for imaging thick, cleared tissues than more common techniques. Ryu was able to work on the optical system in Yang’s lab while testing the samples cleared in Gradinaru’s lab.

    “It was a wonderful experience,” Ryu says. “It was special to have the opportunity to work for two labs this summer. I remember one day when I had a meeting with both Professor Yang and Professor Gradinaru; it was really amazing to get to meet with two Caltech professors.”

    Gradinaru says that the SURF projects provided a learning opportunity not only for the participating students but also for her lab. “For example,” she says, “Ryu strengthened the collaboration that we have with the Yang group for the BRAIN Initiative. And my lab members benefited from the chance to serve as mentors—to see what works and what can be improved when transferring scientific knowledge. These are very important skills in addition to the experimental know-how that they master.”

    See the full article here.

    The California Institute of Technology (commonly referred to as Caltech) is a private research university located in Pasadena, California, United States. Caltech has six academic divisions with strong emphases on science and engineering. Its 124-acre (50 ha) primary campus is located approximately 11 mi (18 km) northeast of downtown Los Angeles. “The mission of the California Institute of Technology is to expand human knowledge and benefit society through research integrated with education. We investigate the most challenging, fundamental problems in science and technology in a singularly collegial, interdisciplinary atmosphere, while educating outstanding students to become creative members of society.”
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  • richardmitnick 1:55 pm on October 13, 2014 Permalink | Reply
    Tags: , Biology,   

    From ORNL: “Unlocking enzyme synthesis of rare sugars to create drugs with fewer side effects” 

    i1

    Oak Ridge National Laboratory

    September 26, 2014
    Katie Bethea, 865.576.8039

    A team led by the U.S. Department of Energy’s Oak Ridge National Laboratory has unlocked the enzymatic synthesis process of rare sugars, which are useful in developing drugs with low side effects using a process more friendly to the environment.

    In a paper published in Structure, the research team reported the pioneering use of neutron and X-ray crystallography and high performance computing to study how the enzyme D-xylose isomerase, or XI, can cause a biochemical reaction in natural sugar to produce rare sugars. Unlike drugs made from natural sugar compounds, drugs made from rare sugars do not interfere with cellular processes. As a result, rare sugars have important commercial and biomedical applications as precursors for the synthesis of different antiviral and anti-cancer drugs with fewer side effects.

    poly
    An artist’s rendering of the enzyme D-xylose isomerase as it isomerizes L-arabinose into rare sugars not found in nature. The enzyme acts as a filter by capturing and performing catalysis only on the high-energy 5S1 conformation of L-arabinose, while remaining inactive on other more abundant sugar conformations. Neutron macromolecular crystallography has unequivocally demonstrated how this high-energy conformer of L-arabinose binds in the enzyme active site and is converted to the linear intermediate form. Simulations provide evidence for the experimental results. Image credit: Genevieve Martin/ORNL

    “The goal of this study is to dramatically improve the performance of enzymes that can be used by the pharmaceutical industry to synthesize drug precursors,” said ORNL’s Andrey Kovalevsky, the lead author of the study. “We’re trying to find a new way to do enzyme design – neutron studies combined with high performance computing could be an elegant means to do that.”

    Enzymes speed up reactions in organisms, ultimately making life itself possible, and are increasingly used by industry to synthesize value-added compounds. Biotechnological syntheses are “greener” than other techniques that use heavy metal chemical catalysts and large amounts of organic solvents. However, many natural enzymes are not very well suited for industrial processes. XI, for example, is used effectively for the production of high-fructose corn syrup from starch in the food industry, but its applications in the pharmaceutical industry are limited by its performance. Researchers in the pharmaceutical industry want to engineer mutations in enzymes to improve reactions. But first, they have to understand how the enzymes work.

    “We had no idea how the enzyme, D-xylose isomerase, binds its non-physiological substrate – natural sugar L-arabinose,” said Kovalevsky. “You have to know how an enzyme binds its substrate to engineer mutations to improve binding and reaction.”

    Using X-ray and neutron crystallography combined with theoretical calculations, the team figured out how the enzyme isomerizes L-arabinose into the rare sugar L-ribulose and then epimerizes the latter into another rare sugar L-ribose. Importantly, L-ribose is the enantiomer, a mirror image, of the ubiquitous D-ribose that is a building block of DNA and RNA.

    “We found, completely unexpectedly, that the enzyme binds the substrate L-arabinose –an abundant natural sugar found in plants– in a very high energy geometry in the active site, which explained the xylose isomerase’s poor efficiency with the substrate and provided us with clues on how we can re-engineer it to improve its activity,” said Kovalevsky.

    Combining crystallographic observations and computation, the team saw the XI enzyme isomerize the sugar L-arabinose when bound to the active site. is the process in which the sugar changes its configuration through a chemical reaction. An enzyme’s active site is the binding place where catalysis is performed on substrates or where inhibitors dock to hinder catalysis. Binding a substrate in a high energy geometry means the efficiency of catalysis would be low, something researchers would like to improve, explained Kovalevsky.

    This is the first time researchers have looked at enzymatic synthesis by combining neutrons, X-rays and high performance computing.

    “Neutron crystallography gives the location of hydrogen atoms, which is important in enzyme reactions where there’s a lot of shuffling of hydrogen around,” said Kovalevsky. “X-rays can’t see those reactions. But once you have the neutron structures and know the hydrogen positions, then your calculations and theoretical models are much more correct.”

    In the past, researchers had to infer the hydrogen atom location from chemical knowledge, which, as experience shows, may be wrong. Now, neutrons show the exact location of the hydrogen atoms so they do not have to guess.

    Calculations can be misleading if hydrogens are placed incorrectly, leading in many cases to the wrong inference from calculations about how enzymes function. Combining neutrons, calculations and simulations gives a more thorough view of the enzymes’ mechanisms and a complete look at how enzymes work.

    Kovalevsky said future simulations will explore the possibility of tailoring the XI active site to bind lower-energy conformations of L-arabinose to improve catalytic activity.

    This research was partially funded through a National Institutes of HealthNational Institute of General Medical Sciences consortium between ORNL and DOE’s Lawrence Berkeley National Laboratory (LBNL). The work was conducted in part at the Los Alamos Neutron Science Center, a National Nuclear Security Administration User Facility at DOE’s Los Alamos National Laboratory., and at the National Energy Research Scientific Computing Center, a DOE Office of Science User Facility at LBNL.

    See the full article here.

    ORNL is managed by UT-Battelle for the Department of Energy’s Office of Science. DOE’s Office of Science is the single largest supporter of basic research in the physical sciences in the United States, and is working to address some of the most pressing challenges of our time.

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