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Institute of Bioengineering and Nanotechnology (IBN) researchers have engineered an artificial human liver that mimics the natural tissue environment closely. The development makes it possible for companies to predict the toxicity of new drugs earlier, potentially speeding up the drug development process and reducing the cost of manufacturing
Via Ray and Terry's
Vall d’Hebron Institute of Oncology (VHIO) scientists eradicate lung tumours in a pre-clinical mouse model. Previous studies had already shown that Myc was a key protein in tumour development and had established how to inhibit Myc through gene therapy. The protein Myc is involved in the development of diverse tumours and so Myc-targeted therapy could make a positive contribution to the therapeutic options for different types of cancer. The study has managed to eliminate mouse lung tumours by inhibiting Myc, a protein that plays a key role in the development of many different tumours. The results, published in the journal Genes & Development, confirm that repeated, long-term treatment does not cause side effects. Even more importantly, no resistance to treatment has been encountered, which is one of the biggest concerns with anticancer therapies. These results show that anticancer therapies based on Myc inhibition are a safe, effective therapeutic option in new drug development. Myc is a protein that plays a big role in regulating gene transcription and it is involved in cell processes such as proliferation, differentiation and apoptosis (programmed cell death - an essential part of regenerating tissues and eliminating damaged cells). It acts as a regulator gene that controls the expression of some 15% of human genes. However, imbalances in this protein bring about uncontrolled cell growth which in turn can lead to the onset of cancer in different tissues. In fact, deregulated Myc is found in most tumours, including cervical, breast, colon, lung and stomach cancer. The work conducted by the Mouse Models of Cancer Therapy group at the VHIO, led by Dr Laura Soucek, shows that Myc can be controlled and inhibited through a mutant called Omomyc that hijacks Myc and prevents it from acting. “Even if we clearly identify a mechanism behind tumour development, it is still extremely complex to pinpoint how to intervene in cells' internal machinery or modify genetic processes,” explained Dr Soucek. “We have found a way to inhibit Myc through Omomyc,” she continued. “We induced Omomyc expression in mice through gene therapy and managed to activate and deactivate it by administering an antibiotic to the mice in their drinking water.”
In the study, multiple lung tumours were induced in the mouse (up to 200 tumours in each individual) and Myc inhibition episodes were achieved by activating Omomyc expression for 4-weeks, followed by 4-week rest periods. This therapy - known as metronomic therapy - was maintained for more than a year, regularly checking tumour progress in each mouse. All mice became tumour free after the first inhibition period, but 63% of cases then relapsed. After the second Myc inhibition period, only 11% of the initial tumours reappeared. According to Dr Soucek, “the most important finding was that there were no signs of resistance to treatment. This is one of the biggest disadvantages of many anticancer therapies: the disease develops resistance and can return even more aggressively.” Finally, only two remaining tumours were found after more than one year of treatment among the mice that received eight inhibition and rest cycles. Dr Soucek found that Omomyc expression had been suppressed in these tumours, and this was the only adaptive mechanism that mice developed to treatment. “These results are hugely positive for us, because one year of life in a mouse is equivalent to almost 40 human years. The fact that the results are maintained over time, that there is no tumour relapse and no resistance, suggests that Myc-targeted therapy may offer an unprecedented way forward."
These encouraging results provide sufficient scientific evidence to consider taking the next step: inhibiting Myc in patients. “Now our challenge for the future is to make Myc inhibition feasible from a pharmacological point of view, so that it can be administered, and done so safely. This will be the last step before designing clinical trials with Myc inhibitors,” explained Dr Soucek. “We're so excited about reaching this turning point and I am quite certain that it will change the course of cancer therapy, despite there being a long road ahead.”
New technique could give conventional immunoassays a run for their money Carbon-nanotube transistors could be used to detect minute quantities of disease biomarkers, such as the proteins implicated in prostate cancer, according to new experiments by researchers in the US. The technique could rival conventional methods when it comes to sensitivity, cost and speed. Conventional techniques to detect proteins are typically based on some form of "immunoassay", with the most famous of these being enzyme-linked immunosorbent assay (ELISA). This technique involves introducing an enzyme-modified antibody protein to an unknown amount of target molecule or protein, known as an antigen, and allowing them to bind together. Unreacted antibodies are washed away, leaving behind only antibody–antigen pairs. The reaction can usually be detected by a colour change in the solution or by a fluorescent signal. The degree of colour change or fluorescence depends upon the number of enzyme-modified antibodies present, which in turn depends on the initial concentration of antigen in the sample. Although such tests are routinely used in hospitals and clinics, they are quite long, taking several days or even weeks to complete. They are also costly, complicated to perform and can only detect single proteins at a time. "Our new nanotube sensors are relatively simple compared to these ELISA tests," team member Mitchell Lerner, at the University of Pennsylvania, told physicsworld.com. "Detection occurs in just minutes, not days, and even at the laboratory scale, the cost of an array of 2000 such sensors is roughly $50 or 2.5 cents per sensor." More importantly still, the sensors are much more sensitive to the target proteins in question. Indeed the Pennsylvania researchers showed that they could detect a prostate-cancer biomarker called osteopontin (OPN) at 1 pg/mL, which is roughly 1000 times lower than that possible with clinical ELISA measurements.
Detecting Lyme disease: The team, which is led by A T Charlie Johnson of Penn's Department of Physics and Astronomy, made its nanotube sensors by attaching OPN-binding antibodies to carbon-nanotube transistors on a silicon chip. Many proteins in the body bind very strongly to specific target molecules or proteins, and OPN is no exception. When the chip is immersed in a test sample, the OPN binds to the antibodies, something that changes the electronic characteristics of the transistor. Measuring the voltage and current through each device thus allows the researchers to accurately measure how much OPN there is in the sample.
Arrays of carbon-nanotube transistors can detect prostate cancer with a much higher sensitivity than conventional techniques. The early detection of many diseases dramatically improves prognoses. Prostate cancer is a case in point, with a 60 to 90 percent long-term survival rate in patients who are diagnosed at an early stage. The problem, of course, is making such a diagnosis quickly and accurately. The standard test is for prostate-specific antigen, which is produced by a cancerous prostate in relatively high quantities. However, the trouble with PSA tests is they produce a significant number of false positives and false negatives. So some healthy men end up undergoing invasive tests while others with the disease go undetected. There is another biomarker of the disease known as osteopontin, or OPN. The state-of-the art technique for spotting this is known as ELISA. In this test, OPN from a sample is attached to a surface. The surface is washed with OPN-binding antibodies, which are themselves attached to colour-changing enzymes. If these antibodies bond with the OPN, the colour change can be easily detected. But while this technique is sensitive, it is relatively time-consuming and difficult to use to quantify the amount of OPN. So more sensitive and accurate techniques are highly sought after. Today, Mitchell Lerner at the University of Pennsylvania and a few buddies reveal just such a technique that uses an array of carbon nanotube transistors on a silicon chip to detect antigens such as OPN. The trick they’ve perfected is a way of attaching an OPN-binding antibody to the carbon nanotube in each transistor. The electronic characteristics of the antibody-nanotube transistor can then be easily measured by the on-chip electronics. When the chip is immersed in a sample, the OPN binds to the antibodies connected to the nanotubes, and this changes the electronic characteristics of the transistor. So measuring the current and voltage through each transistor is an accurate way of measuring how much OPN there is in the sample. Lerner and co say their device can detect OPN at concentrations of 1 picogram per millilitre. That’s a concentration three orders of magnitude weaker than ELISA can manage. And by replacing the OPN-binding antibody with molecules sensitive to other antigens, the carbon nanotube transistors can be made sensitive to other diseases. Lerner and co say they have successfully used the technique to detect Lyme disease and salmonella. That’s impressive work that has the potential to improve the diagnosis and detection of many diseases and pathogens. Of course, more work is needed to characterise the behaviour of these chips and in particular the circumstances in which they might give false positives or negatives. But in the meantime, expect to hear more about them. Refs: • arxiv.org/abs/1302.2961: Detecting Lyme Disease Using Antibody-Functionalized SingleWalled Carbon Nanotube Transistors • arxiv.org/abs/1302.2959: A Carbon Nanotube Immunosensor for Salmonella • arxiv.org/abs/1302.2958: Hybrids of a Genetically Engineered Antibody and a Carbon Nanotube Transistor for Detection of Prostate Cancer
If engineers at Stanford have their way, biological research may soon be transformed by a new class of light-emitting probes small enough to be injected into individual cells without harm to the host. Welcome to biophotonics, a discipline at the confluence of engineering, biology and medicine in which light-based devices – lasers and light-emitting diodes (LEDs) – are opening up new avenues in the study and influence of living cells. The team described their probe in a paper published online Feb. 13, 2013 by the journal Nano Letters. It is the first study to demonstrate that tiny, sophisticated devices known as light resonators can be inserted inside cells without damaging the cell. Even with a resonator embedded inside, a cell is able to function, migrate and reproduce as normal. The researchers call their device a "nanobeam," because it resembles a steel I-beam with a series of round holes etched through the center. This beam, however, is not massive, but measure only a few microns in length and just a few hundred nanometers in width and thickness. It looks a bit like a piece from an erector set of old. The holes through the beam act like a nanoscale hall of mirrors, focusing and amplifying light at the center of the beam in what are known as photonic cavities.
Structurally, the new device is a sandwich of extremely thin layers of the semiconductor gallium arsenide alternated with similarly thin layers of light-emitting crystal, a sort of photonic fuel known as quantum dots. The structure is carved out of chips or wafers, much like sculptures are chiseled out of rock. Once sculpted, the devices remain tethered to the thick substrate. For biological applications, the thick, heavy substrate presents a serious hurdle for interfacing with single cells. The underlying and all-important nanocavities are locked in position on the rigid material and unable to penetrate cell walls. Shambat's breakthrough came when he was able to peel away the photonic nanobeams. He then glued the ultrathin photonic device to a fiberoptic cable with which he steers the needle-like probe toward and into the cell.
Once inserted in the cell, the probe emits light, which can be observed from outside. For engineers, it means that almost any application of these powerful photonic devices can be translated into the previously off-limits environment of the cell interior. In one finding that the authors describe as stunning, they loaded their nanobeams into cells and watched as the cells grew, migrated around the research environment and reproduced. Each time a cell divided, one of the daughter cells inherited the nanobeam from the parent and the beam continued to function as expected. This inheritability frees researchers to study living cells over long periods of time, a research advantage not possible with existing detection techniques, which require cells be either dead or fixed in place. "Our nanoscale probes can reside in cells for long periods of time, potentially providing sensor feedback or giving control signals to the cells down the road," said Shambat. "We tracked one cell for eight days. That's a long time for a single-cell study."
New imaging technology has revealed how the molecular machines that remodel genetic material inside cells 'grab onto' DNA like a rock climber looking for a handhold.
The experiments, reported in this week's Science, use laser light to generate very bright patches close to single cells. When coupled with fluorescent tags this 'spotlight' makes it possible to image the inner workings of cells fast enough to see how the molecular machines inside change size, shape, and composition in the presence of DNA. The Oxford team built their own light microscopy technology for the study, which is a collaboration between the research groups of Mark Leake in Oxford University's Department of Physics and David Sherratt in Oxford University's Department of Biochemistry. The molecular machines in question are called Structural Maintenance of Chromosome (SMC) complexes: they remodel the genetic material inside every living cell and work along similar principles to a large family of molecules that act as very small motors performing functions as diverse as trafficking vital material inside cells to allowing muscles to contract. The researchers studied a particular SMC, MukBEF (which is made from several different protein molecules), inside the bacterium E.coli. David Sheratt and his team found a way to fuse 'fluorescent proteins' directly to the DNA coding for MukBEF, effectively creating a single dye tag for each component of these machines. Up until now conventional techniques of biological physics or biochemistry have not been sufficiently fast or precise to monitor such tiny machines inside living cells at the level of single molecules. 'Each machine functions in much the same way as rock-climber clinging to a cliff face,' says Mark Leake of Oxford University’s Department of Physics, 'it has one end anchored to a portion of cellular DNA while the other end opens and closes randomly by using chemical energy stored in a ubiquitous bio-molecule called adenosine triphosphate, or 'ATP': the universal molecular fuel for all living cells. 'This opening and closing action of the machine is essentially a process of mechanical 'grabbing', in which it attempts to seize more free DNA, like the rock-climber searching for a new handhold.' It is hoped that pioneering biophysics experiments such as this will give fresh insights into the complex processes which are vital to life, and pave the way for a whole new approach to biomedical research at the very tiny length scale for understanding the causes of many diseases in humans, and how to devise new strategies to combat them.
Synthetic biologists have developed DNA modules that perform logic operations in living cells. These ‘genetic circuits’ could be used to track key moments in a cell’s life or, at the flick of a chemical switch, change a cell’s fate, the researchers say. Synthetic biology seeks to bring concepts from electronic engineering to cell biology, treating gene functions as components in a circuit. To that end, researchers at the Massachusetts Institute of Technology (MIT) in Cambridge have devised a set of simple genetic modules that respond to inputs much like the Boolean logic gates used in computers. “These developments will more readily enable one to create programmable cells with decision-making capabilities for a variety of applications,” says James Collins, a synthetic biologist at Boston University in Massachusetts who was not involved in the study. Lu’s logic modules are based on plasmids, circular strings of DNA, that are inserted intoEscherichia coli cells. He and his colleagues devised 16 plasmids — one for each of the binary logic functions allowable in computation. Each variant comprises promoter and terminator DNA sequences, which start or halt gene transcription, and an ‘output gene’ that encodes a green fluorescent protein. The key to the system is the use of recombinase enzymes, which cut and rearrange promoter and terminator DNA sequences to turn them on or off. In other words, recombinase enzymes are the inputs that determine whether the output gene is transcribed. An electronic ‘AND’ gate, for example, gives a positive output only when voltage is applied to both of its inputs. In the genetic version, the output gene is transcribed only when both terminator sequences between it and the promoter sequence are neutralized by two inputs in the form of recombinase enzymes. Lu says that although recombinases have been used similarly in the past — to write data into a DNA memory, for example — the latest work takes the idea a step further by making the DNA part of the computation itself. “If the DNA that you alter is a regulatory element, like a promoter sequence or a terminator, then that gives you the ability to control something inside the cell. And it’s that control that gives you the logic.” The advantage to this system is that the change is permanent. After the cells die, the information can still be retrieved from the DNA.” Indeed, the researchers found that the altered plasmids are passed down through at least 90 cell generations — which would be important for a biologist wanting to record key moments in a cell’s ancestry. Lu says that the approach could also be useful in biotechnology. Using simple forms of these addressable switches, manufacturers could grow cell cultures in which key genes are turned off until activated by a signal compound, permanently turning on production of a drug, for example, when the system is ready. Other switches could halt production, he says, when some threshold has been reached.
A drug-like molecule has been found to let researchers control movements in mice and fish with flashes of light. Unlike similar experiments using a light-based technique known as optogenetics, the new method doesn’t require researchers to genetically engineer animals in order to achieve the neural control. A study published online in today’s Nature Chemical Biology describes a novel approach for controlling neurons and behaviors with light. Such techniques are powerful research tools for understanding the brain, and may one day be used therapeutically. Today’s report describes a method for using light to control neuronal activity in unmodified animals. Fish given a small molecule called “optovin” will move around very quickly in response to a flash of light, report Massachusetts General Hospital’s David Kokel and colleagues. The response is not dependent on the fish perceiving the light—embryonic fish treated with the chemical react to light even before their eyes develop, and decapitated adults respond as well. The compound instead binds to pain sensation receptors on the fish’s body, and when activated by light, it elicits fast movements. The team screened through 10,000 different compounds—each dissolved in a small well with not-yet-hatched zebrafish—before they found one that drastically changed the animals’ behavior in response to light. The compound also works on mice—if optovin is rubbed onto the ears of mice, a flash of light will cause the mice to shake their heads. The team determined that optovin docks onto a specific kind of protein channel that sits in the membrane of nerve cells that are the first to respond to pain. Researchers could use optovin in experiments to study pain; they also think it could be useful in treating pain, says Kokel. “If you over-activate these channels, they become desensitized,” he says. But optovin cannot control the behavior of other kinds of neurons, which is a disadvantage compared to optogenetics. Ed Boyden, a neuroscientist at MIT who has developed optogenetics tools, points out that the genetic engineering-based method gives researchers more flexibility. “A chief utility of our … optogenetic tools is that we can target them to practically any class of neurons, enabling them specifically to be activated and silenced by light,” Boyden says. However, it’s possible, says Kokel, that researchers could identify compounds other than optovin that could regulate the protein channels that control neuron behavior. “You could have a whole tool box of compounds that activate different channels,” he says.
Scientists do not fully understand how nature uses proteins to develop new materials and minerals, but learning more about the natural processes could lead to bioengineering methods such as the biological synthesis of solid-state materials for electronics applications. Now researchers in the US have designed a synthetic biological platform to facilitate the study of these processes and genetically engineer new materials.
The scientists, led by Professor Emeritus Daniel E. Morse of the University of California, Santa Barbara, created synthetic cells containing a polystyrene micro-bead as a nucleus. They then created DNA segments containing genes from two related silicateins along with random mutations and attached a piece of this DNA to each plastic bead. They soaked each bead in a mixture of bacterial proteins required by the synthetic cells to manufacture silicateins, and surrounded the beads with oil to act as the cell membrane.
Silicateins are biomineralizing proteins found in marine sponges that synthesize silica (silicon dioxide) and titania (titanium dioxide) materials. The marine sponge Tethya aurantia, for example, produces silica spicules that make up 75 percent of its dry weight. Silica is commonly used in computer chips, while titania is used in photovoltaic solar cells.
The synthetic cells manufactured silicateins, which appeared on the nuclear bead’s surface attached to antibodies. The researchers then ruptured the artificial cells to release the silicateins, and soaked them in a solution containing the silica or titania precursors. The resulting minerals formed a coating on the beads.
The researchers then set out to direct the evolution of the synthetic cells. They first sorted the beads to identify those with DNA coding for proteins making particularly strong minerals. They sorted them by size, with those having the thickest layers of minerals being selected. They then shook the selected beads to break up the minerals, and selected only those beads that survived this process.
Thirty genes randomly selected from the DNA for either silica- or titania-forming enzymes in the selected beads were then sequenced. They found that the genes contained sequences common to the two original silicatein genes, but they also identified new genes that were completely different from the initial genes. The original genes coded for silicatein alpha, which manufactures silica in clumps of particles. The new genes coded for proteins that produced silica and titania in a dispersed nanoparticle form. One of the new proteins, silicatein X1, manufactured silica in the form of folded sheets of silica-protein fibers.
The Motley Fool - 50 amazing statistics that will amuse you and entice you to dig deeper into the health-care sector.
Via Carla Gentry CSPO
Last year's drought took a big bite out of the two most prodigious US crops, corn and soy. But it apparently didn't slow down the spread of weeds that have developed resistance to Monsanto's herbicide Roundup (glyphosate), used on crops engineered by Monsanto to resist it. More than 70 percent of all the the corn, soy, and cotton grown in the US is now genetically modified to withstand glyphosate. Back in 2011, such weeds were already spreading fast. "Monsanto's 'Superweeds' Gallop Through Midwest," declared the headline of a post I wrote then. What's the word you use when an already-galloping horse speeds up? Because that's what's happening. Let's try this: "Monsanto's 'Superweeds' Stampede Through Midwest." That pretty much describes the situation last year, according to a new report from the agribusiness research consultancy Stratus. Since the 2010 growing season, the group has been polling "thousands of US farmers" across 31 states about herbicide resistance. Here's what they found in the 2012 season: • Nearly half (49 percent) of all US farmers surveyed said they have glyphosate-resistant weeds on their farm in 2012, up from 34 percent of farmers in 2011.
• Resistance is still worst in the South. For example, 92 percent of growers in Georgia said they have glyphosate-resistant weeds.
• But the mid-South and Midwest states are catching up. From 2011 to 2012 the acres with resistance almost doubled in Nebraska, Iowa, and Indiana.
• It's spreading at a faster pace each year: Total resistant acres increased by 25 percent in 2011 and 51 percent in 2012.
• And the problem is getting more complicated. More and more farms have at least two resistant species on their farm. In 2010 that was just 12 percent of farms, but two short years later 27 percent had more than one.
UC Irvine biologists, chemists and computer scientists have identified an elusive pocket on the surface of the p53 protein that can be targeted by cancer-fighting drugs. The finding heralds a new treatment approach, as mutant forms of this protein are implicated in nearly 40 percent of diagnosed cases of cancer, which kills more than half a million Americans each year. In an open-source study published online this week in Nature Communications, the UC Irvine researchers describe how they employed a computational method to capture the various shapes of the p53 protein. In its regular form, p53 helps repair damaged DNA in cells or triggers cell death if the damage is too great; it has been called the “guardian of the genome.” Mutant p53, however, does not function properly, allowing the cancer cells it normally would target to slip through control mechanisms and proliferate. For this reason, the protein is a key target of research on cancer therapeutics. Within cells, p53 proteins undulate constantly, much like a seaweed bed in the ocean, making binding sites for potential drug compounds difficult to locate. But through a computational method called molecular dynamics, the UC Irvine team created a computer simulation of these physical movements and identified an elusive binding pocket that’s open only 5 percent of the time. After using a computer to screen a library of 2,298 small molecules, the researchers selected the 45 most promising to undergo biological assays. Among these 45 compounds, they found one, called stictic acid, that fits into the protein pocket and triggers tumor-suppressing abilities in mutant p53s. While stictic acid cannot be developed into a viable drug, noted study co-leader Peter Kaiser, professor of biological chemistry, the work suggests that a comprehensive screening of small molecules with similar traits may uncover a usable compound that binds to this specific p53 pocket. “The discovery and pharmaceutical development of such a compound could have a profound impact on cancer treatments,” Kaiser said. “Instead of focusing on a specific form of the disease, oncologists could treat a wide spectrum of cancers, including those of the lung and breast.” He added that there is currently one group of experimental drugs – called Nutlins – that stop p53 degradation, but they don’t target protein mutations as would a drug binding to the newly discovered pocket. The results are the culmination of years of labor by researchers with UC Irvine’s Institute for Genomics & Bioinformatics and the Chao Family Comprehensive Cancer Center.
Flow cytometry (FCM) can now be officially used for the quantification of microbial cells in drinking water. The new analytical method – developed at Eawag and extensively tested both in Switzerland and abroad – has been incorporated into the Swiss Food Compendium (SLMB) by the Federal Office of Public Health (FOPH). FCM provides much more realistic results than the conventional method, in which bacterial colonies are grown on agar plates. The results demonstrate that even good-quality drinking water harbours 100 to 10,000 times more living cells than the conventional plate count method would suggest.
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At some point in the next decade, if advances in biotechnology continue on their current path, clones of extinct species such as the passenger pigeon, Tasmanian tiger and wooly mammoth could once again live among us. But cloning lost species—or “de-extinction” as some scientists call it—presents us with myriad ethical, legal and regulatory questions that must be answered, such as which (if any) species should be brought back and whether or not such creatures could be allowed to return to the wild. Such questions are set to be addressed at the TEDx DeExtinction conference, a day-long event in Washington, D.C., organized by Stewart Brand’s Revive & Restore project. Brand previewed the topics for discussion last week at the TED2013 conference in Long Beach, Calif. Scientists are actively working on methods and procedures for bringing extinct species back to life, says Ryan Phelan, executive director of Revive & Restore and co-organizer of the TEDx event. “The technology is moving fast. What Stewart and I are trying to do with this meeting is for the first time to allow the public to start thinking about this. We’re going to hear from people who take it quite seriously. De-extinction is going to happen, and the questions are how does it get applied, when does it get used, what are the criteria which are going to be set?” Cloning extinct species has been tried before—with moderate success. An extinct Pyrenean ibex, or bucardo, (Capra pyrenaica pyrenaica) was born to a surrogate mother goat in 2009, nine years after the last member of its species was killed by a falling tree. The cloned animal lived for just seven minutes. Revive & Restore itself has launched a project to try to resurrect the passenger pigeon, which went extinct in 1914. More: http://www.wired.com/wiredscience/2013/03/passenger-pigeon-de-extinction/
Researchers in Japan successfully cloned 581 healthy mice from the same furry source. Remember Dolly the Sheep? Having started her life in a test tube in 1996, she was the first animal cloned by scientists using a somatic cell (as distinct, say from a germline cell, or “gamete,” like sperm and eggs). Dolly was beautiful. She was Scottish. Her mere existence was profound. It was also unusually short, at just six years. But scientists in Japan have now succeeded in cloning mice using the same technique that created Dolly with more or less perfect results: The mice are healthy, they live just as long as regular mice, and they’ve been flawlessly cloned and recloned from the same source to the 25th generation. Researchers claim it's the first example of seamless, repeat cloning using the Dolly method—known as “somatic cell nuclear transfer” (SCNT)—in which the nucleus from an adult source animal is transferred to an egg with its nucleus removed. Until recently, the process was fraught with failures and mutations. But the team led by Teruhiko Wakayama, whose results were published today in the journal Cell Stem Cell, was able to create 581 clones from the same original mouse. Scientists, including Dolly’s creator, have long felt the process was still too unstable—and too wasteful of precious eggs, given the failure rate—to be used on humans any time soon. But perhaps it's not so far off, after all. Cue the Clone Wars fantasies. Cloning of this kind has been fraught with trouble since the beginning, though to be fair Dolly was an unequivocal success story, whose early demise was relatively inconclusive. To be sure, Dolly developed arthritis at the young age of four and died of a kind of lung cancer when she was not yet six. Most sheep like her live to about 11 or 12. But the cancer that killed her was caused by a common, contagious virus that is deadly to any sheep, her creators concluded. In other words, they claimed, Dolly’s death wasn’t specifically linked to her having been cloned. But Dolly had certain abnormalities that were indicative of the sorts of problems researchers would face using the SCNT method. With Dolly, the telomeres in her cells—which act a bit like “molecular clocks” for the length of time cells can effectively renew—were abnormally short. They were identical in length to the telomeres of the source sheep from which Dolly was cloned, which was six years-old. In other words, there’s a good chance she was never long for this world, with or without the cancer. The early-onset arthritis hinted as much, though according to the scientists who created her, the arthritis was never explained. Cloning experiments since then have produced varying results. In experiments with cattle, for example, some scientists found their clones’ telomeres had been restored to their original lengths. What seemed consistent, however, was that cloning successive generations from the same source—creating a clone of a clone, and so on—always led to some kind genetic degradation, worsening from one copy to the next. It’s thought that the genetic abnormalities we all possess simply worsened with each repeat. Cloning clones was like dubbing copies of copies of cassettes. Eventually, the copies were useless. In the case of animals, they always failed after just a few replications. Beginning in 2005, however, Wakayama and his team began adding trichostatin, a histone deacetylase inhibitor, to the medium used to facilitate the cell-cloning process. Doing so seems to have inhibited what the study’s authors describe as “accumulations of epigenetic or genetic abnormalities in the mice, even after repeated cloning.” We don’t know yet if the process holds up indefinitely, but the 25 generations the Japanese researchers have created so far is a pretty good start.
Macrophages — literally, "big eaters" — are a main part of the body's innate immune system . These cells find and engulf invaders, like bacteria, viruses, splinters and dirt. Unfortunately, macrophages also eat helpful foreigners, including nanoparticles that deliver drugs or help image tumors.
Along with members of his lab, Dennis Discher, professor of chemical and biomolecular engineering in the School of Engineering and Applied Science, has developed a "passport" that could be attached to therapeutic particles and devices, tricking macrophages into leaving them alone.
Taking a cue from a membrane protein that the body's own cells use to tell macrophages not to eat them, the researchers engineered a the simplest functional version of that protein and attached it to plastic nanoparticles. These passport-carrying nanoparticles remained in circulation significantly longer than ones without the peptide, when tested in a mouse model.
In 2008, Discher’s group showed that the human protein CD47, found on almost all mammalian cell membranes, binds to a macrophage receptor known as SIRPa in humans. Like a patrolling border guard inspecting a passport, if a macrophage’s SIRPa binds to a cell’s CD47, it tells the macrophage that the cell isn’t an invader and should be allowed to proceed on. “There may be other molecules that help quell the macrophage response,” Discher said. “But human CD47 is clearly one that says, ‘Don’t eat me’.” Since the publication of that study, other researchers determined the combined structure of CD47 and SIRPa together. Using this information, Discher’s group was able to computationally design the smallest sequence of amino acids that would act like CD47. This “minimal peptide” would have to fold and fit well enough into the receptor of SIRPa to serve as a valid passport. After chemically synthesizing this minimal peptide, Discher’s team attached it to conventional nanoparticles that could be used in a variety of experiments. “Now, anyone can make the peptide and put it on whatever they want,” Rodriguez said. The research team’s experiments used a mouse model to demonstrate better imaging of tumors and as well as improved efficacy of an anti-cancer drug-delivery particle. As this minimal peptide might one day be attached to a wide range of drug-delivery vehicles, the researchers also attached antibodies of the type that could be used in targeting cancer cells or other kinds of diseased tissue. Beyond a proof of concept for therapeutics, these antibodies also served to attract the macrophages’ attention and ensure the minimal peptide’s passport was being checked and approved.
Video is here: http://tinyurl.com/b6dthgb
Zinc-finger nucleases (ZFNs) are a powerful tool that can be used to edit the human genome ad libitum. The technology has experienced remarkable development in the last few years with regard to both the target site specificity and the engineering platforms used to generate zinc-finger proteins. As a result, two phase I clinical trials aimed at knocking out the CCR5 receptor in T cells isolated from HIV patients to protect these lymphocytes from infection with the virus have been initiated. Moreover, ZFNs have been successfully employed to knockout or correct disease-related genes in human stem cells, including hematopoietic precursor cells and induced pluripotent stem cells. Targeted genome engineering approaches in multipotent and pluripotent stem cells hold great promise for future strategies geared toward correcting inborn mutations for personalized cell replacement therapies. Short-lived ZFN expression from episomal DNA-based expression vectors—such as plasmid DNA, integrase-deficient lentiviral vectors, adenoviral vectors, and vectors based on adeno-associated virus—can only be achieved in mitotic cells, which ensures rapid dilution of the vectors during cell divisions. Because DNA-based vector systems have a tendency to integrate into the host genome, it will be important to closely follow the fate of the ZFN expression vectors in the target cells. An alternative way of delivering ZFNs is the transfer of ZFN-encoding mRNA, which ensures rapid but transient ZFN expression and avoids the issue of illegitimate integration. Microinjection of ZFN-encoding mRNA has been performed in zebrafish and rat single-cell embryos, and the ZFN-mediated gene disruption frequency was comparable to plasmid DNA delivery. Moreover, delivery of ZFNs by mRNA transfection has been used to target the integration of a transgene into the AAVS1 locus in human iPSCs. If direct in situ correction of a disease locus is not an option, an important consideration will be to determine where to integrate a therapeutic transgene cassette into the human genome. The AAVS1 site on chromosome 19 is thus far the most promising candidate for such a safe harbor, as a native insulator region appears to both protect transgene expression from position-effect variegation and silencing and prevent the transgene promoter from affecting the host transcriptome. The fact that ZFNs can be used to create knockout animals is especially encouraging and emphasizes the high specificity the technology has reached in the last 3 years. Moreover, the development of alternative designer nucleases, such as TALENs and meganucleases, has further spurred interest in targeted genome engineering approaches. Conversely, studies reporting ZFN off-target activities in zebrafish and human cells must not be overlooked and should serve as the basis for further improvement of the technology. The employment of highly specific designer nucleases is especially important when DSB-based genome engineering is applied to multipotent or pluripotent stem cells, such as HSCs or iPSCs, with their high proliferative potential. Even so, the remarkable progress achieved in the last few years demonstrates that ZFNs represent a tool that allows researchers and clinicians for the first time to rationally edit the genome of human cells and to take this technology from the bench to the bedside for therapeutic applications.
Molecular imprinting (MI) is a technique for preparing polymer scaffolds that function as synthetic receptors, and imprinted polymers that can selectively recognize organic compounds have been proven useful for sensor development. Although creating synthetic MI polymers (MIPs) that recognize proteins remains challenging, nanodevices and nanomaterials show promise for protein recognition into sensor architectures. Arrays of carbon nanotube (nanotube) tips imprinted with a non-conducting polymer coating can be used to recognize proteins with subpicogram per liter sensitivity using electrochemical impedance spectroscopy. Specific MI sensors for human ferritin and human papillomavirus derived E7 protein were developed by one research group. The MI-based nanosensor can also discriminate between Ca2+-induced conformational changes in calmodulin. This ultrasensitive, label-free electrochemical detection of proteins offers an alternative to biosensors based on biomolecule recognition. MI technology offers considerable potential as a cost-effective alternative to the use of biomolecule-based recognition in a variety of sensor applications. MIPs afford the creation of specific recognition sites in synthetic polymers by a process that involves co-polymerization of functional monomers and cross-linkers around template molecules. The molecules are removed from the polymer, rendering complementary binding sites capable of subsequent template molecule recognition. Although deposition of MIPs onto the surface of nanostructures may improve sensitivity for recognition of a range of organic compounds, electronic nanosensors capable of recognizing proteins continue to be a challenge to implement, in part, because: 1) the MIP film may attenuate signals generated in response to template binding (due to the large thickness); 2) the detection mechanisms do not readily allow for effective signal conversion of template molecule binding; and 3) the sensor platforms do not support highly sensitive detection. In conclusion, these types of nanosensors should prove highly useful in diagnosis of human disease, such as detection of cancer biomarkers, and in a host of proteomic applications.
A genetically-engineered virus tested in 30 terminally-ill liver cancer patients significantly prolonged their lives, killing tumors and inhibiting the growth of new ones, scientists reported on Sunday. Sixteen patients given a high dose of the therapy survived for 14.1 months on average, compared to 6.7 months for the 14 who got the low dose. "For the first time in medical history we have shown that a genetically-engineered virus can improve survival of cancer patients," study co-author David Kirn told AFP. The four-week trial with the vaccine Pexa-Vec or JX-594, reported in the journal Nature Medicine, may hold promise for the treatment of advanced solid tumors. "Despite advances in cancer treatment over the past 30 years with chemotherapy and biologics, the majority of solid tumours remain incurable once they are metastatic (have spread to other organs)," the authors wrote. There was a need for the development of "more potent active immunotherapies", they noted.
Pexa-Vec "is designed to multiply in and subsequently destroy cancer cells, while at the same time making the patients' own immune defence system attack cancer cells also," said Kirn from California-based biotherapy company Jennerex. "The results demonstrated that Pexa-Vec treatment at both doses resulted in a reduction of tumour size and decreased blood flow to tumours," said a Jennerex statement. "The data further demonstrates that Pexa-Vec treatment induced an immune response against the tumor."
MIT engineers have created genetic circuits in bacterial cells that not only perform logic functions, but also remember the results, which are encoded in the cell’s DNA and passed on for dozens of generations.
The circuits could be used as long-term environmental sensors, efficient controls for biomanufacturing, or to program stem cells to differentiate into other cell types.
“Almost all of the previous work in synthetic biology that we’re aware of has either focused on logic components or on memory modules that just encode memory. We think complex computation will involve combining both logic and memory, and that’s why we built this particular framework to do so,” says Timothy Lu, an MIT assistant professor of electrical engineering and computer science and biological engineering. Synthetic biologists use interchangeable genetic parts to design circuits that perform a specific function, such as detecting a chemical in the environment. In that type of circuit, the target chemical would generate a specific response, such as production of green fluorescent protein (GFP).
Circuits can also be designed for any type of Boolean logic function, such as AND gates and OR gates. Using those kinds of gates, circuits can detect multiple inputs. In most of the previously engineered cellular logic circuits, the end product is generated only as long as the original stimuli are present: Once they disappear, the circuit shuts off until another stimulus comes along.
Lu and his colleagues set out to design a circuit that would be irreversibly altered by the original stimulus, creating a permanent memory of the event. To do this, they drew on memory circuits that Lu and colleagues designed in 2009. Those circuits depend on enzymes known as recombinases, which can cut out stretches of DNA, flip them, or insert them. Sequential activation of those enzymes allows the circuits to count events happening inside a cell.
Lu designed the new circuits so that the memory function is built into the logic gate itself. With a typical cellular AND gate, the two necessary inputs activate proteins that together turn on expression of an output gene. However, in the new circuits, the inputs stably alter regions of DNA that control GFP production. These regions, known as promoters, recruit the cellular proteins responsible for transcribing the GFP gene into messenger RNA, which then directs protein assembly.
For example, in one circuit described in the paper, two DNA sequences called terminators are interposed between the promoter and the output gene (GFP, in this case). Each of these terminators inhibits the transcription of the output gene and can be flipped by a different recombinase enzyme, making the terminator inactive.
Each of the circuit’s two inputs turns on production of one of the recombinase enzymes needed to flip a terminator. In the absence of either input, GFP production is blocked. If both are present, both terminators are flipped, resulting in their inactivation and subsequent production of GFP.
Once the DNA terminator sequences are flipped, they can’t return to their original state — the memory of the logic gate activation is permanently stored in the DNA sequence. The sequence also gets passed on for at least 90 generations. Scientists wanting to read the cell’s history can either measure its GFP output, which will stay on continuously, or if the cell has died, they can retrieve the memory by sequencing its DNA.
Using this design strategy, the researchers can create all two-input logic gates and implement sequential logic systems. “It’s really easy to swap things in and out,” says Lu, who is also a member of MIT’s Synthetic Biology Center. “If you start off with a standard parts library, you can use a one-step reaction to assemble any kind of function that you want.”
Cognitive decline in old age is linked to decreasing production of new neurons. Scientists from the German Cancer Research Center have discovered in mice that significantly more neurons are generated in the brains of older animals if a signaling molecule called Dickkopf-1 is turned off. In tests for spatial orientation and memory, mice in advanced adult age whose Dickkopf gene had been silenced reached an equal mental performance as young animals. The hippocampus – a structure of the brain whose shape resembles that of a seahorse – is also called the "gateway" to memory. This is where information is stored and retrieved. Its performance relies on new neurons being continually formed in the hippocampus over the entire lifetime. "However, in old age, production of new neurons dramatically decreases. This is considered to be among the causes of declining memory and learning ability", Prof. Dr. Ana Martin-Villalba, a neuroscientist, explains. Martin-Villalba, who heads a research department at the German Cancer Research Center (DKFZ), and her team are trying to find the molecular causes for this decrease in new neuron production (neurogenesis). Neural stem cells in the hippocampus are responsible for continuous supply of new neurons. Specific molecules in the immediate environment of these stem cells determine their fate: They may remain dormant, renew themselves, or differentiate into one of two types of specialized brain cells, astrocytes or neurons. One of these factors is the Wnt signaling molecule, which promotes the formation of young neurons. However, its molecular counterpart, called Dickkopf-1, can prevent this. "We find considerably more Dickkopf-1 protein in the brains of older mice than in those of young animals. We therefore suspected this signaling molecule to be responsible for the fact that hardly any young neurons are generated any more in old age." The scientists tested their assumption in mice whose Dickkopf-1 gene is permanently silenced. Professor Christof Niehrs had developed these animals at DKFZ. The term "Dickkopf" (from German "dick" = thick, "Kopf" = head) also goes back to Niehrs, who had found in 1998 that this signaling molecule regulates head development during embryogenesis. Martin-Villalba's team discovered that stem cells in the hippocampus of Dickkopf knockout mice renew themselves more often and generate significantly more young neurons. The difference was particularly obvious in two-year old mice: In the knockout mice of this age, the researchers counted 80 percent more young neurons than in control animals of the same age. Moreover, the newly formed cells in the adult Dickkopf-1 mutant mice matured into potent neurons with multiple branches. In contrast, neurons in control animals of the same age were found to be more rudimentary already. Blocking Dickkopf improves spatial orientation and memory. Several years ago, Ana Martin-Villalba had shown that mice lose their spatial orientation when neurogenesis in the hippocampus is blocked. Now, is it possible that the young neurons in Dickkopf-deficient mice improve the animals' cognitive performance? The DKFZ researchers used standardized tests to study how the mice orient themselves in a maze. While in the control animals, the younger ones (3 months) performed much better in orienting themselves than the older ones (18 months), the Dickkopf-1-deficient mice showed no age-related decline in spatial orientation capabilities. Older Dickkopf-1 mutant mice also outperformed normal animals in tests determining spatial memory. "Our result proves that Dickkopf-1 promotes age-related decline of specific cognitive abilities," says Ana Martin-Villalba. "Although we had expected silencing of Dickkopf-1 to improve spatial orientation and memory of adult mice, we were surprised and impressed that animals in advanced adult age actually reach the performance levels of young animals." These results give rise to the question whether the function of Dickkopf-1 may be turned off using drugs. Antibodies blocking the Dickkopf protein are already being tested in clinical trials for treating a completely different condition. "It is fascinating to speculate that such a substance may also slow down age-related cognitive decline. But this is still a dream of the future, since we have only just started first experiments in mice to explore this question."
The development of stimuli-responsive, nano-scale therapeutics that selectively target and attack tumors is a major research focus in cancer nanotechnology. A potent therapeutic option is to directly arming the cancer cells with apoptotic-inducing proteins that are not affected by tumoral anti-apoptotic maneuvers. The avian virus-derived apoptin forms a high-molecular weight protein complex that selectively accumulates in the nucleus of cancer cell to induce apoptotic cell death. To achieve the efficient intracellular delivery of this tumor-selective protein in functional form, we synthesized degradable, sub-100 nm, core–shell protein nanocapsules containing the 2.4 MDa apoptin complexes. Recombinant apoptin is reversibly encapsulated in a positively charged, water soluble polymer shell and is released in native form in response to reducing conditions such as the cytoplasm. As characterized by confocal microscopy, the nanocapsules are efficiently internalized by mammalian cells lines, with accumulation of rhodamine-labeled apoptin in the nuclei of cancer cells only. Intracellularly released apoptin induced tumor-specific apoptosis in several cancer cell lines and inhibited tumor growth in vivo, demonstrating the potential of this polymer–protein combination as an anticancer therapeutic. The process does not present the risk of genetic mutation posed by gene therapies for cancer, or the risk to healthy cells caused by chemotherapy, which does not effectively discriminate between healthy and cancerous cells, Tang said.
"This approach is potentially a new way to treat cancer," said Tang. "It is a difficult problem to deliver the protein if we don't use this vehicle. This is a unique way to treat cancer cells and leave healthy cells untouched."
The cell-destroying material, apoptin, is a protein complex derived from an anemia virus in birds. This protein cargo accumulates in the nucleus of cancer cells and signals to the cell to undergo programmed self-destruction.
The polymer shells are developed under mild physiological conditions so as not to alter the chemical structure of the proteins or cause them to clump, preserving their effectiveness on the cancer cells.
Tests done on human breast cancer cell lines in laboratory mice showed significant reduction in tumor growth.
"Delivering a large protein complex such as apoptin to the innermost compartment of tumor cells was a challenge, but the reversible polymer encapsulation strategy was very effective in protecting and escorting the cargo in its functional form," said Muxun Zhao, lead author of the research and a graduate student in chemical and biomolecular engineering at UCLA.
Help may be on the way for people with compromised immune systems, severe allergies, or who otherwise have to be wary of airborne nasties. A team of scientists have created something known as a soft x-ray electrostatic precipitator, or an SXC ESP for short. It filters all manner of bacteria, allergens, viruses, and ultrafine particles from the air – plus, it kills everything it catches.
Via Ray and Terry's
Biochemical trick could aid in recovery of gold from metal waste. Gold prospectors may one day use Petri dishes to help with their quests. A species of bacterium forms nanoscale gold nuggets to help it to grow in toxic solutions of the precious metal, reports a paper published online today in Nature Chemical Biology. The molecule with which the bacteria create the particles could one day be used to collect gold from mine waste, says Frank Reith, an environmental microbiologist at the University of Adelaide in Australia, who works on gold-processing bacteria but was not involved in the latest study. Reith found some of the first convincing evidence that bacteria thrive on gold particles about ten years ago. At multiple sites, thousands of kilometres apart, he and his team found the bacterium Cupriavidus metallidurans living in biofilms on gold nuggets. The bacteria detoxify dissolved gold by accumulating it in inert nanoparticles inside their cells; Reith and his colleagues have spent the past decade working out how, but have not yet published their complete conclusions. Some biofilms also contained a second species of bacterium:Delftia acidovarans. Nathan Magarvey, a biochemist at McMaster University in Hamilton, Canada, and his team grew this species in the presence of a gold solution and discovered that the bacterial colonies were surrounded by dark haloes of gold nanoparticles. The researchers concluded that D. acidovarans was somehow creating gold particles outside its cell wall, instead of inside as C. metallidurans does.Using biochemical and genome analysis, the researchers discovered a set of genes and a chemical metabolite that were responsible for precipitating the gold. Bacteria engineered to lack the genes no longer formed dark haloes, and their growth was stunted in the presence of gold. The team also isolated a chemical produced by the unengineered bacteria that caused gold particles to precipitate out of a solution. The chemical was dubbed delftibactin. The researchers suggest that the genes they identified are involved in producing delftibactin and shunting it outside the cell. By precipitating gold, D. acidovarans may keep the metal from entering its cells in solution. But Magarvey says that it is possible that D. acidovarans also uses other mechanisms to detoxify gold that breaches its cell walls. Margarvey's work “complements ours really well”, says Reith. The two bacterial species might live in symbiosis, with D. acidovarans using delftibactin to diminish the soluble gold to levels that both species can cope with. A microbe-assisted gold rush might yet happen, says Reith. Delftibactin could be used to produce gold-nanoparticle catalysts for many chemical reactions, or to precipitate gold from waste water produced at mines. “The idea could be to use a bacterium or metabolite to seed these waste-drop piles, leave them standing for years, and see if bigger particles form,” says Reith.
One of the big challenges in treating AIDS is that the virus is notorious for mutating, so patients must be treated with a cocktail of drugs — known as highly active antiretroviral therapy or HAART — which hit it at various stages of the replication process. The researchers were able to get around that problem with a new, multi-pronged genetic attack that blocks HIV on several fronts. Essentially, they hope to mimic HAART through genetic manipulation. The technique hinges on the fact that the virus typically enters T cells by latching onto one of two surface proteins known as CCR5 and CXCR4. Some of the latest drugs now used in treatment work by interfering with these receptors’ activity. A small number of people carry a mutation in CCR5 that makes them naturally resistant to HIV. One AIDS patient with leukemia, now famously known as the Berlin patient, was cured of HIV when he received a bone marrow transplant from a donor who had the resistant CCR5 gene. Scientists at Sangamo BioSciences in Richmond, Calif., have developed a technique using a protein that recognizes and binds to the CCR5 receptor gene, genetically modifying it to mimic the naturally resistant version. The technique uses a zinc finger nuclease, a protein that can break up pieces of DNA, to effectively inactivate the receptor gene. The company is now testing its CCR5-resistant genes in phase-1 and -2 trials with AIDS patients at the University of Pennsylvania. The Stanford scientists used a similar approach but with an added twist. They used the same nuclease to zero in on an undamaged section of the CCR5 receptor’s DNA. They created a break in the sequence and, in a feat of genetic editing, pasted in three genes known to confer resistance to HIV, Porteus said. This technique of placing several useful genes at a particular site is known as “stacking.” Incorporating the three resistant genes helped shield the cells from HIV entry via both the CCR5 and CXCR4 receptors. The disabling of the CCR5 gene by the nuclease, as well as the addition of the anti-HIV genes, created multiple layers of protection. Blocking HIV infection through both the CCR5 and CXCR4 receptors is important, Porteus said, as it hasn’t been achieved before by genome editing. To test the T cells’ protective abilities, the scientists created versions in which they inserted one, two and all three of the genes and then exposed the T cells to HIV. Though the T cells with the single- and double-gene modifications were somewhat protected against an onslaught of HIV, the triplets were by far the most resistant to infection. These triplet cells had more than 1,200-fold protection against HIV carrying the CCR5 receptor and more than 1,700-fold protection against those with the CXCR4 receptor, the researchers reported. The T cells that hadn’t been altered succumbed to infection with 25 days.
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