Scientists Identify Key Factor That Controls HIV Latency

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ScienceDaily (June 27, 2009) — Scientists at the Gladstone Institutes of Virology and Immunology (GIVI) have found another clue that may lead to eradication of HIV from infected patients who have been on antiretroviral therapy. A real cure for HIV has been elusive because the virus can "hide" in a latent form in resting CD4-T cells. By understanding this "latency" effect, researchers can identify ways to reactivate the virus and enable complete clearance by current or future therapies.

Researchers in the laboratory of GIVI Associate Director Eric Verdin, MD have found that methylation of cytosine in the DNA of infected cells is associated with HIV latency and that inhibition of DNA methylation causes the reactivation of latent HIV. These observations offer a potential new strategy for inhibiting HIV latency and reactivating the virus. The discovery was reported in the current edition of PLoS Pathogens.
"While HIV-1 latency is likely to be a multifactorial process, we have shown that inhibiting the methylation of the provirus contributes to an almost complete reactivation of latent HIV-1," said lead author Steven E. Kauder.
The research team, which also included scientists from the University of Utah and Stockholm's Karolinska Institute, developed in vitro models of HIV-1 latency in T cells that harbor a full-length HIV genome. The provirus in the cell lines also encoded a fluorescent marker to illuminate HIV-1 transcriptional activity.
In addition to finding that DNA methylation is a mechanism of latency, the scientists also discovered that a host protein, called methlyl-CpG binding domain protein 2 (MBD2) binds to the methylated HIV DNA and is an important mediator of latency.
"Interfering with methylation greatly potentiates the reactivation of HIV," Kauder said. In this study, the researchers found that the drug 5-aza-2'deoxycytidine (aza-CdR) can inhibit HIV methylation and cause the virus to reactivate.
"Combined with other areas of our investigation into HIV latency, this research provides important new knowledge about the process and opens many new pathways for future study," said Dr. Verdin, senior author of the study.
The research team included Alberto Bosque and Vicente Planelles of the University of Utah and Annica Lindqvist of Karolinska University. The study was supported by the National Institutes of Health
Eric Verdin's primary affiliation is with the Gladstone Institute of Virology and Immunology, where his laboratory is located and all his research is conducted. He is also professor of medicine at the University of California, San Francisco.
Adapted from materials provided by Gladstone Institutes, via EurekAlert!, a service of AAAS.

TRAPping Proteins That Work Together Inside Living Cells

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ScienceDaily (June 18, 2009) — DNA might be the blueprint for living things, but proteins are the builders. Researchers trying to understand how and which proteins work together have developed a new crosslinking tool that is small and unobtrusive enough to use in live cells. Using the new tool, the scientists have discovered new details about a well-studied complex of proteins known as RNA polymerase. The results suggest the method might uncover collaborations between proteins that are too brief for other techniques to pinpoint.

"Conventional methods used to find interacting proteins have limitations that we are trying to circumvent," said biochemist Uljana Mayer of the Department of Energy's Pacific Northwest National Laboratory. "They also create conditions that are different from those inside cells, so you can't find all the interactions that proteins would normally engage in."
Proteins are the workhorses in an organism's cells. Whole fields of research are dedicated to teasing out which proteins work together to make cells function. For example, drug researchers seek chemicals that disrupt or otherwise change how proteins interact to combat diseases; environmental scientists need to understand how proteins collaborate in ecosystems to make them thrive or fail.
To learn about protein networks, scientists start with a familiar one and use it as bait to find others that work alongside it. To pin down the collaborators, researchers make physical connections between old and new proteins with chemicals called crosslinkers. The sticky crosslinkers will only connect proteins close enough to work together, the thinking goes. But most crosslinkers are too large to squeeze into living cells, are harmful to cells, or link proteins that are neighbors but not coworkers.
To address these issues, Mayer and her PNNL colleagues developed a crosslinking method that uses small crosslinkers whose stickiness can be carefully controlled. To find coworkers of a protein of interest, Mayer and her colleagues build a tiny molecule called a tag into the initial protein. They then add a small molecule called TRAP to the living cell, which finds and fits into the tag like two pieces in a puzzle. TRAP waves around, bumping into nearby proteins. The scientists control TRAP with a flash of light, causing it to stick to coworkers it bumps into. The researchers then identify the new "TRAPped" proteins in subsequent analyses.
To demonstrate how well this method works, Mayer and colleagues tested it out on RNA polymerase, a well-studied machine in cells. The polymerase is made up of many proteins that cooperate to translate DNA. One of the polymerase proteins has a tail that is known to touch the DNA and some helper proteins just before the polymerase starts translating. No one knew if this tail -- also known as the C-terminus of the alpha subunit -- touches anything else in the core of the RNA polymerase complex.
The team engineered a tag in the C-terminus and cultured bacteria with the tagged RNA polymerase. After adding TRAP to the cells and giving it time to find the C-terminus tag, the team shined a light on the cultures.
The team then identified the proteins marked with TRAP using instruments in EMSL, DOE's Environmental Molecular Sciences Laboratory on the PNNL campus. They found that the tagged protein, as expected, interacts with many other proteins, for example previously identified helper proteins, so-called transcription factors. But they also found it on another core protein called the beta subunit, suggesting the tail of the alpha subunit makes contact with the beta subunit as it plugs along. This interaction had never been seen before.
"No one knows what the polymerase looks like when it is running," said Uljana Mayer. "Here we see the C-terminus swings back to grab the beta subunit once the polymerase starts working."
The team report their results June 15 in the journal ChemBioChem. The tag in their unique method is made up of a "tetracysteine motif" -- two pairs of the amino acid cysteine separated by two other amino acids that doesn't interfere with the normal function of the protein of interest. TRAP includes a small "biarsenical" probe, which fluoresces so the team can find the proteins to which it has become attached. TRAP can also be easily unlinked from the tag with a simple biochemical treatment, allowing researchers to piece out the coworker from their original protein of interest.
The team also tested the method on other proteins, such as those found in young muscle cells. Mayer said they will use the method in the future to understand how environmental conditions affect how proteins work together in large networks.
Journal reference:
P. Yan, T. Wang, G.J. Newton, T.V. Knyushko, Y. Xiong, D. J. Bigelow, T.C. Squier, and M.U. Mayer. A Targeted Releasable Affinity Probe (TRAP) for In Vivo Photocrosslinking. ChemBioChem, 2009; 10: 1507-1518 DOI: 10.1002/cbic.200900029
Adapted from materials provided by DOE/Pacific Northwest National Laboratory.

Scientists Show Bacteria Can 'Learn' And Plan Ahead

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ScienceDaily (June 18, 2009) — Bacteria can anticipate a future event and prepare for it, according to new research at the Weizmann Institute of Science. In a paper that appeared June 17 in Nature, Prof. Yitzhak Pilpel, doctoral student Amir Mitchell and research associate Dr. Orna Dahan of the Institute's Molecular Genetics Department, together with Prof. Martin Kupiec and Gal Romano of Tel Aviv University, examined microorganisms living in environments that change in predictable ways.

Their findings show that these microorganisms' genetic networks are hard-wired to 'foresee' what comes next in the sequence of events and begin responding to the new state of affairs before its onset.
E. coli bacteria, for instance, which normally cruise harmlessly down the digestive tract, encounter a number of different environments on their way. In particular, they find that one type of sugar – lactose – is invariably followed by a second sugar – maltose – soon afterward. Pilpel and his team of the Molecular Genetics Department, checked the bacterium's genetic response to lactose, and found that, in addition to the genes that enable it to digest lactose, the gene network for utilizing maltose was partially activated. When they switched the order of the sugars, giving the bacteria maltose first, there was no corresponding activation of lactose genes, implying that bacteria have naturally 'learned' to get ready for a serving of maltose after a lactose appetizer.
Another microorganism that experiences consistent changes is wine yeast. As fermentation progresses, sugar and acidity levels change, alcohol levels rise, and the yeast's environment heats up. Although the system was somewhat more complicated that that of E. coli, the scientists found that when the wine yeast feel the heat, they begin activating genes for dealing with the stresses of the next stage. Further analysis showed that this anticipation and early response is an evolutionary adaptation that increases the organism's chances of survival.
Ivan Pavlov first demonstrated this type of adaptive anticipation, known as a conditioned response, in dogs in the 1890s. He trained the dogs to salivate in response to a stimulus by repeatedly ringing a bell before giving them food. In the microorganisms, says Pilpel, 'evolution over many generations replaces conditioned learning, but the end result is similar.' 'In both evolution and learning,' says Mitchell, 'the organism adapts its responses to environmental cues, improving its ability to survive.' Romano: 'This is not a generalized stress response, but one that is precisely geared to an anticipated event.'
To see whether the microorganisms were truly exhibiting a conditioned response, Pilpel and Mitchell devised a further test for the E. coli based on another of Pavlov's experiments. When Pavlov stopped giving the dogs food after ringing the bell, the conditioned response faded until they eventually ceased salivating at its sound. The scientists did something similar, using bacteria grown by Dr. Erez Dekel, in the lab of Prof. Uri Alon of the Molecular Cell Biology Department, in an environment containing the first sugar, lactose, but not following it up with maltose. After several months, the bacteria had evolved to stop activating their maltose genes at the taste of lactose, only turning them on when maltose was actually available.
'This showed us that there is a cost to advanced preparation, but that the benefits to the organism outweigh the costs in the right circumstances,' says Pilpel. What are those circumstances? Based on the experimental evidence, the research team created a sort of cost/benefit model to predict the types of situations in which an organism could increase its chances of survival by evolving to anticipate future events. They are already planning a number of new tests for their model, as well as different avenues of experimentation based on the insights they have gained.
Pilpel and his team believe that genetic conditioned response may be a widespread means of evolutionary adaptation that enhances survival in many organisms – one that may also take place in the cells of higher organisms, including humans. These findings could have practical implications, as well. Genetically engineered microorganisms for fermenting plant materials to produce biofuels, for example, might work more efficiently if they gained the genetic ability to prepare themselves for the next step in the process.
Prof. Yitzhak Pilpel's research is supported by the Ben May Charitable Trust and Madame Huguette Nazez, Paris, France.
Adapted from materials provided by Weizmann Institute of Science, via EurekAlert!, a service of AAAS.

Silver nanoparticles show 'immense potential' in prevention of blood clots

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Silver nanoparticles (shown) could help prevent blood clots. Credit: The American Chemical Society.
Scientists are reporting discovery of a potential new alternative to aspirin, ReoPro, and other anti-platelet agents used widely to prevent blood clots in coronary artery disease, heart attack and stroke. Their study, scheduled for the June 23 issue of ACS Nano, a monthly journal, involves particles of silver -- 1/50,000th the diameter of a human hair -- that are injected into the bloodstream.

Debabrata Dash and colleagues point out that patients urgently need new anti-thrombotic agents because traditionally prescribed medications too-often cause dangerous bleeding. At the same time, aging of the population, sedentary lifestyle and spiraling rates of certain diseases have increased the use of these drugs. Researchers are seeking treatments that more gently orchestrate activity of platelets, disk-shaped particles in the blood that form clots.
The scientists describe development and lab testing of silver nanoparticles that seem to keep platelets in an inactive state. Low levels of the nanosilver, injected into mice, reduced the ability of platelets to clump together by as much as 40 percent with no apparent harmful side effects.
The nanoparticles “hold immense potential to be promoted as an antiplatelet agent,” the researchers note. “Nanosilver appears to possess dual significant properties critically helpful to the health of mankind — antibacterial and antiplatelet — which together can have unique utilities, for example in coronary stents.”
More information: ACS Nano, Journal Article: “Characterization of Antiplatelet Properties of Silver Nanoparticles”
Provided by American Chemical Society (news : web)

Breakthrough in the treatment of bacterial meningitis

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It can take just hours after the symptoms appear for someone to die from bacterial meningitis. Now, after years of research, experts at The University of Nottingham have finally discovered how the deadly meningococcal bacteria is able to break through the body's natural defence mechanism and attack the brain.

The discovery could lead to better treatment and vaccines for meningitis and could save the lives of hundreds of children.
Bacterial meningitis in childhood is almost exclusively caused by the respiratory tract pathogens Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae. The mechanism used by these lethal germs to break through the blood brain barrier (BBB) has, until now, been unknown.
The team led by Dlawer Ala'Aldeen, Professor of Clinical Microbiology and Head of the Molecular Bacteriology and Immunology Group at the Centre for Biomolecular Sciences, recently discovered that all three pathogens target the same receptor on human cerebrovascular endothelial cells — the specialised filtering system that protects our brain from disease — enabling the organisms to cross the blood-brain barrier.
Their findings, published today in The Journal of Clinical Investigation, suggest that disruption or modulation of this interaction of bacterial adhesins with the receptor might offer unexpectedly broad protection against bacterial meningitis and may provide a therapeutic target for the prevention and treatment of disease.
Professor Ala'Aldeen, who has been studying meningitis and its causes for over 20 years, said: "This is a significant breakthrough which will help us design novel strategies for the prevention and treatment of bacterial meningitis. Identification of the human receptor and bacterial ligands is like identifying a mysterious key and its lock, which will open new doors and pave the way for new discoveries."
The research, carried out in collaboration with the Department of Infectious Diseases at St. Jude Children's Research Hospital in Memphis Tennessee, also involved students from the University who have been regular and willing volunteers in the research programme.
Professor Ala'Aldeen said: "The ultimate aim is to save lives by protecting the healthy and curing the sick. We are one step closer to new breakthroughs that would prevent disease or its complications. There still is a long way to go before we have the ultimate vaccine and the ultimate treatment of bacterial meningitis."
Source: University of Nottingham (news : web)

Developed a human monoclonal antibody that neutralizes the Hepatitis C virus (HCV).

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ScienceDaily (May 11, 2009) — aking aim at a leading cause of liver failure in the United States, a team of scientists at the Massachusetts Biologic Laboratories (MBL) of the University of Massachusetts Medical School (UMMS) has developed a human monoclonal antibody that neutralizes the Hepatitis C virus (HCV). The new antibody effectively neutralized the virus in culture, and then prevented infection by the virus in a pre-clinical animal model of the disease.

Details of the research were presented April 23 in Copenhagen, Denmark at the 44th Annual Meeting of the European Association for the Study of the Liver (EASL). "We are pleased with the progress of this program," said Donna Ambrosino, MD, executive director of the MBL and a professor of pediatrics at the Medical School. "This antibody shows significant efficacy against the virus."
In the current study, MBL scientists injected transgenic mice (HuMAb Mouse® technology, Medarex, Inc.) with elements of HCV and then painstakingly searched for individual human antibodies produced in the mice that would recognize and bind to the HCV's outer coat, known as the glycoprotein. Once they found human antibodies that looked promising, they evaluated in vitro the ability of those antibodies to neutralize the virus and selected a lead candidate antibody for further characterization. Collaborative work with clinical researchers from the Department of Medicine at the Medical School's Worcester campus demonstrated that this antibody, now known as MBL-HCV1, was able to bind tightly with all genotypes of HCV tested from infected patient samples.
MBL-HCV1 was then tested off-site on three non-human primates. In that study, one animal received no antibody, one a low dose of the new antibody, and one a higher dose. Then all three animals were exposed to HCV. The animals with low or no antibody dosages developed HCV infections, but the animal with the higher dose was protected. Subsequently, researchers gave the high-dose of the antibody to the animal that originally received no antibody, and in that case the HCV was cleared from that animal's system. "These results are encouraging as a possible treatment for HCV infected patients, but more work needs to be done before we know how effective it will be in people," Dr. Ambrosino noted.
HCV attacks the liver and can eventually lead to liver failure. According to the U.S. Centers for Disease Control and Prevention, 3.2 million Americans are chronically infected with HCV and some 10,000 die annually of the disease. Globally, as many as 170 million people are estimated to suffer from HCV infection. For the most serious cases of HCV that do not respond to antiviral drugs, liver transplantation is the only option.
Typically 2,000 to 4,000 liver transplants are done each year in the United States (far less than the number of people on the waiting list for available organs). Transplantation can be a life saving treatment; however, in nearly all cases the patient's new liver is eventually infected by HCV because the virus remains in the patient's bloodstream during surgery. The powerful antiviral drugs now used to attack HCV prior to end-stage liver failure are not routinely used during surgery due to the patients' weakened condition and because of the strong medication used to avoid rejection of the new liver. After re-infection with HCV, nearly 40 percent of patients suffer rapid liver failure.
To close that clinical gap, the new antibody developed at MBL is designed to be a therapy shortly before and after transplant surgery. By giving a patient the new antibody before and during the time when the donor liver is implanted, researchers hope the HCV virus left in the bloodstream will be neutralized and rendered unable to infect the new liver. Then, because monoclonal antibodies are highly specific and typically have little or no side-effects, additional dosages of the new antibody could, theoretically, be given immediately after transplant surgery to continue neutralizing any remaining virus.
It is also possible, researchers theorize, that the antibody could be used in combination with new antiviral drugs for treatment in patients with newly diagnosed HCV infection. Use of the new antibody for both liver transplant patients and in newly diagnosed HCV patients will now be further evaluated. A Phase 1 human clinical trial of MBL-HCV1 in healthy subjects is expected to begin later this year.
Adapted from materials provided by University of Massachusetts Medical School, via EurekAlert!, a service of AAAS.

How Cells Move: Cooperative Forces Boost Collective Mobility Of Cells

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ScienceDaily (May 11, 2009) — Research by scientists in Spain and their colleagues offers for the first time an experimental answer to the question of how cells move during biological processes as diverse as the development, metastasis, or regeneration of tissues.

The work addresses the issue of collective mobility of cells, that is to say, how cells are moved within tissues, and what is the prevalent form of movement inside living organisms.
"Research into collective cell mobility is very active due to the direct implications it has on fields such as embryologic development, organ regeneration, and cancer. For example, if we could find a way to control cell mobility during metastasis, cancer would be a curable disease in the majority of cases," says Dr. Xavier Trepat, senior researcher of the cellular and respiratory biomechanics group and researcher in the Department of Physiological Sciences at the University of Barcelona, and in the Networking Biomedical Research Centre for respiratory diseases (CIBER).
Up until now, scientists had proposed various mechanisms to explain collective cell migration. One hypothesis for example, suggests that the cells move collectively due to the existence of “leader” cells, which stretch out in the rest of the group, like a train pulls carriages behind it. Another hypothesis suggests that each cell moves independently to those around it, like cars on the motorway during a traffic jam, or like soldiers in a military parade. “We have rejected both these possibilities,” says Trepat.
According to his research, collective cell mobility is the result of a cooperative process in which each cell contributes to the movement of the group, stretching to those around it. “It is a mechanism similar to a tug-of-war game, in which two teams pull a rope by its extremes and the team that pulls the hardest wins. During the game, each player generates force and transmits it to the rope, so that the tension in the rope is the sum of the forces generated by each member of the team. Cells do the same. Each cell generates force to stretch to its neighbours in the direction of the movement» explains the researcher.
Journal reference:
Xavier Trepat, Michael R. Wasserman, Thomas E. Angelini, Emil Millet, David A. Weitz, James P. Butler & Jeffrey J. Fredberg. Physical forces during collective cell migration. Nature Physics, 2009; DOI: 10.1038/nphys1269
Adapted from materials provided by Universidad de Barcelona, via AlphaGalileo.

Deep in the Red: Using Infrared to Watch What Goes On in a Living Body

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An infrared version of the Nobel Prize-winning green fluorescent protein could make the technique even more powerful.

Fluorescent proteins, which are compounds that can absorb and then emit light, have become a powerful instrument in the cell biologist's toolkit—so powerful, in fact, that the discovery and development of green fluorescent proteins from jellyfish earned the 2008 Nobel Prize in Chemistry. (Here's a Q&A with one of the winners, Columbia University's Martin Chalfie, about his work.) These proteins have limitations, however: They need to be excited with the blue to orange part of the visible spectrum, at wavelengths of 495 to 570 nanometers. These wavelengths of light are too short to penetrate tissue very well, and so green fluorescent proteins are mainly used in test tube studies to watch cell division or to label certain cell types.
But one of the 2008 Nobelists, Roger Y. Tsien of the University of California, San Diego, and his U.C.S.D. colleagues report in today's issue of Science that they have developed a new fluorescent protein that could enable scientists to tag and visualize cellular activity as it happens inside a live animal. The protein, after absorbing light from the far-red part of the spectrum, shines in the near-infrared, at wavelengths of around 700 nanometers.
These longer wavelengths can penetrate mammalian tissue and even pass through bone. "Say you label a tumor with a green fluorescent protein, and if this labeled tumor is buried inside the animal, then you barely can get green fluorescence out," says lead researcher Xiaokun Shu. "But if you label this deeply buried tumor by infrared fluorescent proteins, you will get a stronger signal because infrared penetrates tissue more efficiently."
Tsien's group derived the infrared fluorescent protein from a hardy bacterium called Deinococcus radiodurans, famous for its ability to survive extreme environments. Bacteria do not actually use this class of proteins, called bacteriophytochromes, to emit light. "They use these bacteriophytochromes to control gene expression,” Shu says—the proteins convert absorbed light into energy to signal certain genes to turn on or off.
The initial challenge for researchers was to re-engineer the protein so that absorbed light would be re-emitted instead of being used as a source of power. They accomplished the feat by deleting the part of the protein that converts the absorbed light into chemical energy; as a result, this truncated and mutant form gave up its absorbed energy as an infrared glow. The scientists incorporated the engineered bacterial protein into mammalian cells—specifically, into the liver of a live mouse, which lit up with infrared light.
The finding paves the way for in vivo visualization of a wide range of biochemical processes and internal organs in animals. (Its use in humans is unlikely, as it would require gene therapy and the ethically dubious transplantation of bacterial genes into humans.)
“This is so important," comments David James, a cell biologist from Australia's Garvan Institute in Sydney, "because a lot of knowledge at the moment is confined to individual cells grown on a glass coverslip," leaving open the question of whether that knowledge "is transferable to an animal." The infrared version could also solve the problem of naturally occuring fluorescence from other biological molecules, which tend to glow at wavelengths similar to conventional fluorescent protein markers and thereby create a lot of “background noise," James says.

But even greater potential lies in harnessing the bacteriophytochrome's original function, namely, powering gene expression. It should be feasible, Tsien thinks, to put back in the signal-controlling properties of the phytochrome. Then it could be possible with animals to “switch on genes and control biochemistry" with light, he says.
For example, you want to explore the effects on mouse behavior of switching on a particular gene that controls some aspect of brain function, but, thankfully for the mouse, you do not want to open up its skull or stick a needle in its brain. "If the infrared fluorescent protein can be made to turn back into an infrared phytochrome, you could have the switch all ready and just waiting for enough infrared light," Tsien speculates. Because infrared light can penetrate the skull, it can reach the phytochrome and remotely switch the gene on, resulting in observable changes in the mouse's behavior.
It's the next evolutionary step for fluorescent proteins, remarks Tsien, who believes that phytochromes represent a class of proteins with enormous potential. If he's correct, then in the coming years, expect more scientists to see the (infrared) light.

By Bianca Nogrady

System To Build Transplant Tissue Created

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http://www.sciencedaily.com/releases/2007/10/071008161609.htm

Science Daily — One day soon, laboratories may grow synthetically engineered tissues such as muscle or cartilage needed for transplants. In a major step forward, Cornell engineers describe in the journal Nature Materials a microvascular system they have developed that can nourish growing tissues.

The researchers have engineered tiny channels within a water-based gel that mimic a vascular system at the cellular scale and can supply oxygen, essential nutrients and growth factors to feed individual cells. The so-called gel scaffold can hold tens of millions of living cells per milliliter in a 3-D arrangement, such as in the shape of a knee meniscus, to create a template for tissue to form.
In theory, the system could accommodate many kinds of tissue.
"A significant impediment to building engineered tissues is that you can't feed the core," said Abraham Stroock, Cornell assistant professor of chemical and biomolecular engineering and one of the paper's senior authors. "Simply embedding this mimic of a microvascular system allows you to maintain the core of the tissue during culture." Gel scaffolds, he said, "are the culture flasks of the future."
The embedded microchannels allow fluid with oxygen, sugar and proteins to travel through the system. The researchers can control the distributions of these solutes over both time and space within the developing tissue, allowing the fine-tuning of the biochemical environment of the cells while the tissue develops. For example, the tissue may need to develop into bone on one side and cartilage on the other. Now the researchers can supply the right nutrients and proteins to certain parts of the growing tissue to ensure an intended outcome.
The research provides solutions to the physical engineering aspects of growing tissues synthetically. Still, many biological challenges remain, such as finding a source of cells that can be harvested from a patient and grown without changing the cell's characteristics. Co-author Lawrence Bonassar, a Cornell associate professor of biomedical engineering who was instrumental in developing the gel for tissue growth and in determining the proper biological requirements for cell growth, is also among those trying to direct stem cells to produce desired tissue types. Currently, stem cell-derived cartilage has been made but is not functional.
As new tools develop, researchers hope to use these engineered tissues in non-clinical applications, such as replacements for animals in the testing of pharmaceuticals and chemicals. The technology, researchers believe, also offers the hope of growing implants from the patient's own cells to replace damaged or diseased tissue.
The research was funded by the Office of Naval Research, Cornell's Nanobiotechnology Center, Beckman Foundation, the Center for Life Science Enterprise at Cornell and the Cornell Center for Materials Research.
Note: This story has been adapted from material provided by Cornell University.

Fausto Intilla

www.oloscience.com

The Petri Dish Is Taken To New Dimensions

Source:

http://www.sciencedaily.com/releases/2007/09/070919115955.htm

Science Daily — A team of Brown University biomedical engineers has invented a 3-D Petri dish that can grow cells in three dimensions, a method that promises to quickly and cheaply produce more realistic cells for drug development and tissue transplantation.

The technique employs a new dish – cleverly crafted from a sugary substance long used in science laboratories – that allows cells to self-assemble naturally and form “microtissues.” A description of how the 3-D dish works appears in the journal Tissue Engineering.
“It’s a new technology with a lot of promise to improve biomedical research,” said Jeffrey Morgan, a Brown professor of medical science and engineering.
Morgan conceived and created the 3-D Petri dish with a team of Brown students led by Anthony Napolitano, a Ph.D. candidate in the biomedical engineering program. Napolitano spent two years perfecting the new dish and recently won a $15,000 award from the National Collegiate Inventors and Innovators Alliance to develop the patent-pending technology into a commercially viable product.
“This technology is an inexpensive and easy-to-use alternative to current 3-D cell culture methods,” Napolitano said. “It’s the next generation.”
The technology tackles a topic of increasing interest to scientists: creating hothouse cells that look and behave more like cells grown in the human body. Since 1877, scientists have relied on the Petri dish to grow, or culture, cells. The cells stick to the bottom of the dishes and spread out as they multiply. In the body, however, cells don’t grow that way. They are surrounded by other cells in three dimensions, forming tissues such as skin, muscle, and bone. This is what happens in Morgan’s 3-D dish.
The clear, rubbery dish is the size of a silver dollar. It is made from a water-based gel made of agarose, a complex carbohydrate long used in molecular biology. This gel has a few benefits. It is porous, allowing nutrients and waste to circulate. And it is non-adhesive, so cells won’t stick to it. At the bottom of the dish sit 820 tiny recesses or wells. When cells are added to the dish –about 1 million at a time – roughly 1,000 sink to the bottom of each well and form a pile. These close quarters allow cells to self-assemble, or form natural cell-to-cell connections, a process not possible in traditional Petri dishes.
The result: microtissues consisting of hundreds of cells, even of different types. In Tissue Engineering, the Brown team describes how they combined human fibroblasts, which make connective tissue, and endothelial cells, which line the heart and blood vessels. The cells came together to form spheres and doughnut-shaped clusters. The process was quick – self-assembly took place in less than 24 hours.
“These microtissues have several potential uses,” Morgan said. “They can be used to test new cancer compounds and other drugs. And they can be transplanted into the body to regenerate tissue, such as pancreatic cells for diabetics. While there are other methods out there for making microtissues, our 3-D technology is fast, easy and inexpensive. It can make hundreds of thousands of microtissues in a single step.”
Differences in culture techniques matter in biomedicine, according to a growing body of research. Studies show sometimes dramatic differences in the shape, function and growth patterns of cells cultured in 2-D compared with cells cultured in 3-D. For example, a recent Brown study found that nerve cells grown in 3-D environments grew faster, had a more realistic shape and deployed hundreds of different genes compared to cells grown in 2-D environments.
That’s why several laboratories are pursuing 3-D cell culture methods. Brown Technology Partnerships has filed a patent application based on the technology developed in the Morgan lab and is actively pursuing licensing partners.
Napolitano was lead author of the Tissue Engineering article, and Morgan was senior author. Other members of the research team included Peter Chai, a student at The Warren Alpert Medical School of Brown University and Dylan Dean, an M.D./Ph.D. graduate student in the molecular pharmacology and physiology program.
The National Science Foundation funded the research.
Note: This story has been adapted from a news release issued by Brown University.

Fausto Intilla

www.oloscience.com