Researchers create living human gut-on-a-chip

Building on the Wyss Institute's breakthrough "Organ-on-Chip" technology that uses microfabrication techniques to build living organ mimics, the gut-on-a-chip is a silicon polymer device about the size of a computer memory stick. Wyss Founding Director, Donald Ingber, M.D., Ph.D., led the research team, which included Postdoctoral Fellow, Hyun Jung Kim, Ph.D; Technology Development Fellow, Dan Huh, Ph.D.; and Senior Staff Scientist, Geraldine Hamilton, Ph.D. Ingber is also the Judah Folkman Professor of Vascular Biology at Harvard Medical School and the Vascular Biology Program at Children's Hospital Boston, and Professor of Bioengineering at Harvard's School of Engineering and Applied Sciences.

The new device mimics complex 3D features of the intestine in a miniaturized form. Inside a central chamber, a single layer of human intestinal epithelial cells grows on a flexible, porous membrane, recreating the intestinal barrier. The membrane attaches to side walls that stretch and recoil with the aid of an attached vacuum controller. This cyclic mechanical deformation mimics the wave-like peristaltic motions that move food along the digestive tract. The design also recapitulates the intestinal tissue-tissue interface, which allows fluids to flow above and below the intestinal cell layer, mimicking the luminal microenvironment on one side of the device and the flow of blood through capillary vessels on the other.

In addition, the researchers were able to grow and sustain common intestinal microbes on the surface of the cultured intestinal cells, thereby simulating some of the physiological features important to understanding many diseases. These combined capabilities suggest that gut-on-a-chip has the potential to become a valuable in vitro diagnostic tool to better understand the cause and progression of a variety of intestinal disorders and to help develop safe and effective new therapeutics, as well as probiotics. The gut-on-a-chip could also be used to test the metabolism and oral absorption of drugs and nutrients.

"Because the models most often available to us today do not recapitulate human disease, we can't fully understand the mechanisms behind many intestinal disorders, which means that the drugs and therapies we validate in animal models often fail to be effective when tested in humans," said Ingber. "Having better, more accurate in vitro disease models, such as the gut-on-a-chip, can therefore significantly accelerate our ability to develop effective new drugs that will help people who suffer from these disorders."

Gut-on-a-chip represents the most recent advance in the Wyss Institute's portfolio of engineered organ models. The platform technology was first reported on in Science in June 2010, where a living, breathing, human lung-on-a-chip was described. That same year, the Wyss received funding from the National Institutes of Health and the U.S. Food and Drug Administration to develop a heart-lung micromachine to test the safety and efficacy of inhaled drugs on the integrated heart and lung function. In September 2011, the Wyss was awarded a four-year grant from the Defense Advanced Research Projects Agency to develop a spleen-on-a-chip to treat sepsis, a commonly fatal bloodstream infection.

Provided by Harvard University (news : web)

Scientists X-ray key enzyme of common pathogen crystallized in living cells

The three-dimensional structure of a biomolecule gives biologists clues about its function, and in the case of a pathogen it also offers the perspective to block a harmful protein with a tailor-made artificial molecule. For example, if the enzyme cathepsin B of Trypanosoma brucei is blocked, the parasite will die. However, the structure analysis of biomolecules is a difficult and time-consuming process. Normally, a sufficiently large crystal of the protein in question has to be grown in the lab before it can be investigated with X-ray light of a synchrotron radiation source.

Crystal growing is complicated and often takes weeks or even months. Therefore, the team of scientists – among them scientists from the universities of Tübingen, Hamburg and Lübeck, and from Deutsches Elektronen-Synchrotron DESY in Hamburg – chose another approach. With the help of a virus, they inserted the genetic blueprint for cathepsin B into living insect cells. The infected cells started to produce the enzyme incessantly, and with the steadily increasing concentration, the enzyme eventually crystallised. After about 70 hours, the micrometre-small crystals became visible in the microscope, some of them even sticking out of the cells.

At the US accelerator centre SLAC in California, the scientists bombarded these crystals with the world’s strongest X-ray free-electron laser LCLS. Although its intensive X-ray flash completely vaporises the crystals in less than a billionth of a second, it is bright enough to previously take a detailed diffraction image of the crystal, making it possible to calculate the structure of the crystallised enzyme. However, to gain the complete structural information the experiment must be repeated very often with a large number of crystals, which was not part of the study.

But the result shows that with the new technology it is possible to generate high-quality data of the protein structure of nanocrystals. "Our experiments have shown that the promise of X-ray lasers to revolutionize structural biology is indeed becoming true," said DESY scientist Prof. Henry Chapman from the Center for Free-Electron Laser Science (CFEL). "We have shown that previous limitations to protein crystallography can be overcome by using pulses of X-rays so intense, that they transform the proteins into a dense plasma similar to the conditions inside the sun. Yet the pulses are so short that fine details are seen before destroying the sample," Dr. Anton Barty from CFEL added.

Apart from the Federal Ministry of Education and Research-funded young investigators group "Structural Infection Biology Using new Radiation Sources (SIAS)" of the universities of Hamburg and Lübeck, and the Hamburg School for Structure and Dynamics in Infection (SDI) of the State of Hamburg Excellence Initiative, the research was done with the participation of a team of scientists headed by professor Michael Duszenko from the University of Tübingen, a CFEL-group headed by professor Henry Chapman as well as other DESY scientists and international collaboraters. CFEL is a cooperation of DESY, the Max Planck Society and the University of Hamburg.

“Our result shows that the super lasers offer completely new possibilities for the structure determination of biological macromolecules, and perhaps the days will be over soon when we needed months or even years to grow crystals of certain proteins being large enough for X-ray radiation sources at synchrotrons” said SIAS leader Dr. Lars Redecke, one of the main authors of this study.

As from 2010, SIAS - an initiative of the structural research scientists professor Christian Betzel, University of Hamburg, and professor Rolf Hilgenfeld, University of Lübeck - investigates the use of innovative radiation sources for structural determination of proteins and other biological molecules.

The European X-ray laser European XFEL, currently being built in Hamburg, will open a unique opportunity for biomolecule investigation. Already today, DESY operates the free-electron laser FLASH for soft X-ray radiation.

More information: "In vivo protein crystallization opens new routes in structural biology"; Michael Duszenko et al.; "Nature Methods", Advance Online Publication; DOI: 10.1038/nmeth.1859

Provided by DESY

New information on the waste-disposal units of living cells

"Using electron microscopy and a revolutionary new system for protein expression, we have determined at a subnanometer scale the complete architecture, including the relative positions of all its protein components, of the proteasome regulatory particle," says biophysicist Eva Nogales, the research team's co-principal investigator. "This provides a structural basis for the ability of the proteasome to recognize and degrade unwanted proteins and thereby regulate the amount of any one type of protein that is present in the cell."

Says the team's other co-principal investigator and corresponding author, biochemist Andreas Martin, "While the biochemical function of many of the proteasome components have been determined, and some subnanometer structures have been identified, it was unclear before now which component goes where and which components interact with one another. Now we have a much better understanding as to how the proteasome machinery works to control cellular processes and this opens the possibility of manipulating proteasome activity for the treatment of cancer and other diseases."

Nogales, who holds appointments with Berkeley Lab, UC Berkeley, and the Howard Hughes Medical Institute, and Martin, who holds appointments with UC Berkeley and the QB3 Institute, are the senior authors of a paper describing this work in the journal Nature. The paper is titled "Complete subunit architecture of the proteasome regulatory particle." Other co-authors were Gabriel Lander, Eric Estrin, Mary Matyskiela and Charlene Bashore.

At any given moment, a human cell typically contains about 100,000 different proteins, with certain proteins being manufactured and others being discarded as needed for the cell's continued prosperity. Unwanted proteins are tagged with a "kiss-of-death" label in the form of a polypeptide called "ubiquitin." A protein marked with ubiquitin is delivered to any one of the some 30,000 proteasomes in the cell – barrel-shaped complexes which act as waste disposal units that rapidly break-down or degrade the protein. The 2004 Nobel Prize in chemistry was awarded to a trio of scientists who first described the proteasome process, but a lack of structural information has limited the scientific understanding of the mechanics behind this process.

Nogales, an expert on electron microscopy and image analysis, and Martin, who developed the new protein expression system used in this work, combined the expertise of their respective research groups to study the proteasome regulatory particle in yeast. The particle features 19 sub-units that are organized into two sub-complexes, a "lid" and a "base." The lid contains the regulatory elements that identify the ubiquitin tag marking a protein for destruction, and the base features a hexameric ring that pulls the tagged protein inside the chamber of the proteasome barrel where it is degraded.

"The lid consists of nine non-ATPase proteins including ubiquitin receptors that accept properly tagged proteins but prevent a protein not marked for degradation from engaging with the proteasome," Nogales says. "Since degradation is irreversible, it is critical that only ubiquitin-tagged proteins engage the proteasome. Interestingly, the ubiquitin tag has to be removed before the protein can be translocated into the proteasome's destruction chamber, so the lid also contains de-ubiquitination enzymes that remove the tags after the protein has engaged with the proteasome."

The proteasome regulatory particle's base contains six distinct AAA+ ATPases that form the hetero-hexameric ring, which serves as the molecular motor of the proteasome.

"We predict that the ATPases use the energy of ATP binding and hydrolysis to exert a pulling force on engaged proteins, unfolding and translocating them through a narrow central pore and into the degradation chamber," Martin says. "The steps in the proteasome process – from protein recognition to de-ubiquitination and degradation have to be very highly coordinated in time and space. Locating all of these components and identifying their relative orientations has been very telling about how the processes are coordinated with each other."

Nogales credits the protein expression system developed by Martin and his research group, in which proteins are expressed and assembled in bacteria, as being critical to the success of this research.

"Until now researchers had to work with purified protein complexes from the cell, which could not be manipulated or modified in any way," she says. "Andy Martin's new heterologous expression system allows for the manipulation and dissection of protein functions. For our studies it was crucial to generate lid sub-complexes that had one marker at a time in each of the subunits so that we could determine the position of each protein within the lid. With this new system we generated truncations, deletions and fusion constructs that were used to localize individual subunits and delineate their boundaries within the lid."

Provided by Lawrence Berkeley National Laboratory (news : web)

Researchers show how iron activates oxygen in living things

The team used high-powered X-rays from the Stanford Synchrotron Radiation Lightsource to capture the fine details of how these enzymes work. The results could have applications in medicine, energy production and industrial processes.

Led by Edward Solomon, a professor of photon science at SLAC, Wonwoo Nam, of Ewha Womans University in South Korea, and Joan Valentine of the University of California, Los Angeles, the team reported its findings in the Oct. 27 issue of Nature.

“It’s what we’re all after: How does nature make these metal sites do this chemistry better than scientists can do in the labs?” Solomon said.

In its most abundant form, oxygen exists as a two-atom molecule, O2. Electronically speaking, O2 is “forbidden” from reacting with other biological molecules until it is split into two separate oxygen atoms.

Specialized enzymes, containing metallic elements like iron, drive this important preparatory step. The precise mechanism of oxygen activation by iron complexes has long eluded researchers, in part because the reaction—which proceeds through multiple intermediate stages—happens in mere fractions of a second.

Researchers recently captured all three of the intermediate structures that one iron complex morphs into as it cleaves the O2 bond – including one wily intermediate that exists for less than 2 milliseconds before converting into a different form.

They used two specialized instruments at the SSRL to determine the electronic and geometric structure of each intermediate stage. Chemical tests at Ewha Womans University further revealed that these iron-based intermediates are versatile chemical catalysts, able to react with both electron-rich and electron-deficient molecules.

This is the first time researchers have so fully characterized this type of iron- and oxygen-containing molecule — called non-heme iron because it lacks the heme group for which the iron-containing molecule hemoglobin that carries oxygen in red blood cells is named.

Solomon says the newly discovered reaction mechanism could help scientists better understand diseases of non-heme iron enzymes, such as phenylketonuria. The disease, which prevents breakdown of the amino acid phenylalanine, can cause developmental defects in babies and lasting health problems for adults.

Other more distant applications may affect energy production and industrial processes that use similar chemistry. One day, Solomon says, industrial chemists may find a way to co-opt the non-heme iron enzyme reaction to drive reactions more quickly and cheaply.

Other co-authors on the paper, "Structure and reactivity of a mononuclear non-haem iron(III)–peroxo complex," include Stanford photon science professors Britt Hedman, who is also deputy director of SSRL, and Keith Hodgson, SLAC's chief research officer.

Provided by SLAC National Accelerator Laboratory (news : web)

New tool developed for the study of spatial patterns in living cells

 Football has often been called "a game of inches," but biology is a game of nanometers, where spatial differences of only a few nanometers can determine the fate of a cell -- whether it lives or dies, remains normal or turns cancerous. Scientists with the U.S. Department of Energy (DOE)'s Lawrence Berkeley National Laboratory (Berkeley Lab) have developed a new and better way to study the impact of spatial patterns on living cells.

Berkeley Lab chemist Jay Groves led a study in which artificial membranes made up of a fluid bilayer of lipid molecules were embedded with fixed arrays of gold nanoparticles to control the spacing of proteins and other cellular molecules placed on the membranes. This provided the researchers with an unprecedented opportunity to study how the spatial patterns of chemical and physical properties on membrane surfaces influence the behavior of cells.

"The gold nanoparticles are similar to the size of a single protein molecule, which gets us to a scale we couldn't really access before," says Groves. "As the first example of a biological membrane platform that combines fixed nanopatterning with the mobility of fluid lipid bilayers, our technique represents an important improvement over previous patterning methods."

Groves holds joint appointments with Berkeley Lab's Physical Biosciences Division and the University of California (UC) Berkeley's Chemistry Department, and is a Howard Hughes Medical Institute (HHMI) investigator. He is the corresponding author of a paper that reports these results in the journal Nano Letters.

Spatial patterning of chemical and physical properties on artificial membranes of lipid bilayers is a time-tested way to study the behavior of cultured biological cells. Natural lipid bilayer membranes surround virtually all living cells as well as many of the structures inside the cell including the nucleus. These membranes provide a barrier that restrains the movement of proteins and other cellular molecules, penning them into their proper locations and preventing them from moving into areas where they do not belong. Past spatial patterning efforts on artificial membranes have been done on an all-or-nothing basis -- proteins placed on a membrane either had complete mobility or were fixed in a static position.

"Immobile patterning intrinsically defeats any cellular process that naturally involves movement," Groves says. "On the other hand we need to be able to impose some fixed barriers in order to manipulate membranes in really novel ways."

Groves is a recognized leader in the development of unique "supported" synthetic membranes that are constructed out of lipids and assembled onto a substrate of solid silica. He and his group have used these supported membranes to demonstrate that living cells not only interact with their environment through chemical signals but also through physical force.

"We call our approach the spatial mutation strategy because molecules in a cell can be spatially re-arranged without altering the cell in any other way," he says.

However, until now Groves and his group were unable to get to the tens of nanometers length-scales that they can now reach by embedding their supported membranes with gold nanoparticles.

"Our new membranes provide a hybrid interface consisting of mobile and immobile components with controlled geometry," Groves says. "Proteins or other cellular molecules can be associated with the fluid lipid component, the fixed nanoparticle component, or both."

The gold nanoparticle arrays were patterned through a self-assembly process that provides controllable spacing between particles in the array in the important range of 50 to 150 nanometers. The gold nanoparticles themselves measure about five to seven nanometers in diameter.

Groves and his team successfully tested their hybrid membranes on a line of breast cancer cells known as MDA-MB-231 that is highly invasive. With their hybrid membranes, the team demonstrated that in the absence of cell adhesion molecules, the membrane remained essentially free of the cancer cells, but when both the nanoparticles and the lipid were functionalized with molecules that promote cell adhesion, the cancer cells were found all over the surface.

Groves and his research group are now using their gold nanoparticle membranes to study both cancer metastasis and T cell immunology. They expect to report their results soon.

Co-authoring the Nano Letters paper with Groves were Theobald Lohmuller, Sara Triffo, Geoff O'Donoghue, Qian Xu and Michael Coyle. This research was supported by the DOE Office of Science.

Story Source:

The above story is reprinted from materials provided by DOE/Lawrence Berkeley National Laboratory.

Note: Materials may be edited for content and length. For further information, please contact the source cited above.

Journal Reference:

Theobald Lohmüller, Sara Triffo, Geoff P. O’Donoghue, Qian Xu, Michael P. Coyle, Jay T. Groves. Supported Membranes Embedded with Fixed Arrays of Gold Nanoparticles. Nano Letters, 2011 DOI: 10.1021/nl202847t

Imaging inflammation in the living brain

Inflammation occurs in the human brain during illnesses such as Alzheimer's disease, Parkinson’s disease, stroke and traumatic brain injury. Now, a research team in Japan has developed a probe that can bind to the pro-inflammatory enzyme cyclooxygenase (COX). The probe, 11C-ketoprofen methyl ester, enables researchers to observe when and where the enzyme is acting in the brains of living animals using positron emission tomography (PET) imaging.

In PET imaging, a radioactive tracer that binds specifically to a specific molecule in the body is injected into a living organism. Images are then taken with a PET scanner, indicating where in the body that tracer is found.

Led by Hirotaka Onoe at the RIKEN Center for Molecular Imaging Science in Kobe, the researchers had previously discovered that 11C-ketoprofen methyl ester could recognize COX, but not which of its two forms. To determine which isoform is responsible for binding their molecular probe, Miho Shukuri, a young member of Onoe’s team, utilized a series of mice lacking the genes for either COX-1 or COX-2. She found that the PET probe could bind to the brains of COX-2-deficient mice, but not to those lacking COX-1. According to the researchers, 11C-ketoprofen methyl ester is therefore the first PET probe that is specific to COX-1 in living animals.

When Shukuri injected bacterial antigens into the brain of rats to induce inflammation, she saw the PET probe build up in the brain within six hours to one day after antigen injection. The levels dropped a week later. Because COX-1 is rapidly activated by brain injury, this may mean that administration of drugs that block COX-1 soon after injury could prevent the progression of brain damage. “COX-1 could therefore be a promising target for the neurodegenerative diseases that exhibit neuro-inflammation,” explains Onoe.

Microglia are immune cells in the brain that proliferate in response to injury, while macrophages are immune cells normally found within the blood that invade the brain after injury. The researchers observed that the injury-induced increase in brain COX-1 seemed to occur within microglia and macrophages (Fig. 1), which also became more numerous in the brain after exposure to bacterial antigens. Other research groups have found COX-1-expressing microglia in diseases such as Alzheimer's disease, Parkinson’s disease and multiple sclerosis. This suggests to Onoe and colleagues that 11C-ketoprofen methyl ester could be used to track the time course and localization of increased COX-1 expression in living organisms, including humans, suffering from diseases linked to neuro-inflammation.

More information: Shukuri, M., et al. In vivo expression of cyclooxygenase-1 in activated microglia and macrophages during neuroinflammation visualized by PET with 11C-ketoprofen methyl ester. The Journal of Nuclear Medicine published online 1 July, 2011 (doi: 10.2967/jnumed.110.084046).

Takashima-Hirano, M., et al. General method for the 11C-labeling of 2-arylpropionic acids and their esters: construction of a PET tracer library for a study of biological events involved in COXs expression. Chemistry 16, 4250–4258 (2010).

Provided by RIKEN (news : web)

Proton-based transistor could let machines communicate with living things

Human devices, from light bulbs to iPods, send information using electrons. Human bodies and all other living things, on the other hand, send signals and perform work using ions or protons.

Materials scientists at the University of Washington have built a novel transistor that uses protons, creating a key piece for devices that can communicate directly with living things. The study is published online in the interdisciplinary journal Nature Communications.

Devices that connect with the human body's processes are being explored for biological sensing or for prosthetics, but they typically communicate using electrons, which are negatively charged particles, rather than protons, which are positively charged hydrogen atoms, or ions, which are atoms with positive or negative charge.

"So there's always this issue, a challenge, at the interface -- how does an electronic signal translate into an ionic signal, or vice versa?" said lead author Marco Rolandi, a UW assistant professor of materials science and engineering. "We found a biomaterial that is very good at conducting protons, and allows the potential to interface with living systems."

In the body, protons activate "on" and "off" switches and are key players in biological energy transfer. Ions open and close channels in the cell membrane to pump things in and out of the cell. Animals including humans use ions to flex their muscles and transmit brain signals. A machine that was compatible with a living system in this way could, in the short term, monitor such processes. Someday it could generate proton currents to control certain functions directly.

A first step toward this type of control is a transistor that can send pulses of proton current. The prototype device is a field-effect transistor, a basic type of transistor that includes a gate, a drain and a source terminal for the current. The UW prototype is the first such device to use protons. It measures about 5 microns wide, roughly a twentieth the width of a human hair.

"In our device large bioinspired molecules can move protons, and a proton current can be switched on and off, in a way that's completely analogous to an electronic current in any other field effect transistor," Rolandi said.

The device uses a modified form of the compound chitosan originally extracted from squid pen, a structure that survives from when squids had shells. The material is compatible with living things, is easily manufactured, and can be recycled from crab shells and squid pen discarded by the food industry.

First author Chao Zhong, a UW postdoctoral researcher, and second author Yingxin Deng, a UW graduate student, discovered that this form of chitosan works remarkably well at moving protons. The chitosan absorbs water and forms many hydrogen bonds; protons are then able to hop from one hydrogen bond to the next.

Computer models of charge transport developed by co-authors M.P. Anantram, a UW professor of electrical engineering, and Anita Fadavi Roudsari at Canada's University of Waterloo, were a good match for the experimental results.

"So we now have a protonic parallel to electronic circuitry that we actually start to understand rather well," Rolandi said.

Applications in the next decade or so, Rolandi said, would likely be for direct sensing of cells in a laboratory. The current prototype has a silicon base and could not be used in a human body. Longer term, however, a biocompatible version could be implanted directly in living things to monitor, or even control, certain biological processes directly.

The other co-author is UW materials science and engineering graduate student Adnan Kapetanovic. The research was funded by the University of Washington, a 3M Untenured Faculty Grant, a National Cancer Institute fellowship and the UW's Center for Nanotechnology, which is funded by the National Science Foundation.

Story Source:

The above story is reprinted (with editorial adaptations ) from materials provided by University of Washington. The original article was written by Hannah Hickey.

Journal Reference:

Chao Zhong, Yingxin Deng, Anita Fadavi Roudsari, Adnan Kapetanovic, M.P. Anantram, Marco Rolandi. A polysaccharide bioprotonic field-effect transistor. Nature Communications, 2011; 2: 476 DOI: 10.1038/ncomms1489

Genetic code used to engineer a living protein

Yale University researchers have successfully re-engineered the protein-making machinery in bacteria, a technical tour de force that promises to revolutionize the study and treatment of a variety of diseases.

"Essentially, we have expanded the genetic code of E. coli, which allows us synthesize special forms of proteins that can mimic natural or disease states," said Jesse Rinehart of the Department of Cellular and Molecular Physiology and co-corresponding author of the research published in the August 26 issue of the journal Science.

Since the structure of DNA was revealed in the 1950s, scientists have been working hard to understand the nature of the genetic code. Decades of research and recent advances in the field of synthetic biology have given researchers the tools to modify the natural genetic code within organisms and even rewrite the universal recipe for life.

"What we have done is taken synthetic biology and turned it around to give us real biology that has been synthesized," Rinehart explained.

The Yale team — under the direction of Dieter Söll, Sterling Professor of Molecular Biophysics and Biochemistry, professor of chemistry and corresponding author of the paper — developed a new way to influence the behavior of proteins, which carry out almost all of life's functions. Instead of creating something new in nature, the researchers essentially induced phosphorylation, a fundamental process that occurs in all forms of life and can dramatically change a protein's function. The rules for protein phosphorylation are not directly coded in the DNA but instead occur after the protein is made. The Yale researchers fundamentally rewrote these rules by expanding the E. coli genetic code to include phosphoserine, and for the first time directed protein phosphorylation via DNA.

This new technology now enables the production of human proteins with their naturally occurring phosphorylation sites, a state crucial to understanding disease processes. Previously, scientists lacked the ability to study proteins in their phosphorylated or active state. This has hindered research in diseases such as cancer, which is marked by damagingly high levels of protein activation.

"What we are doing is playing with biological switches — turning proteins on or off — which will give us a completely new way to study disease states and hopefully guide the discovery of new drugs," Rinehart said.

"We had to give some very ancient proteins a few modern upgrades," Söll said.

Söll and Rinehart now are attempting to create proteins in states known to be linked to cancer, type 2 diabetes, and hypertension. Both men, however, stressed the technique can be done for any type of protein.

"Dr. Söll and his colleagues have provided researchers with a powerful new tool to use in uncovering how cells regulate a broad range of processes, including cell division, differentiation and metabolism," said Michael Bender, who oversees protein synthesis grants at the National Institute of General Medical Sciences of the National Institutes of Health.

Provided by Yale University (news : web)

Nano-thermometers show first temperature response differences within living cells

 Using a modern version of open-wide-and-keep-this-under-your-tongue, scientists have reported taking the temperature of individual cells in the human body, and finding for the first time that temperatures inside do not adhere to the familiar 98.6 degree Fahrenheit norm. They presented the research at the 242nd National Meeting & Exposition of the American Chemical Society (ACS), being held in Denver.

Haw Yang and Liwei Lin, who collaborated on the research, did not use a familiar fever thermometer to check the temperature of cells, the 100 trillion or so microscopic packages of skin, nerve, heart, liver and other material that make up the human body. Cells are so small that almost 60,000 would fit on the head of a common pin. Yang is with Princeton University and Lin is with the University California-Berkeley.

"We used 'nano-thermometers'," Yang explained. "They are quantum dots, semiconductor crystals small enough to go right into an individual cell, where they change color as the temperature changes. We used quantum dots of cadmium and selenium that emit different colors (wavelengths) of light that correspond to temperature, and we can see that as a color change with our instruments."

Yang said that information about the temperatures inside cells is important, but surprisingly lacking among the uncountable terabytes of scientific data available today.

"The inside of a cell is so complicated, and we know very little about it," he pointed out. "When one thinks about chemistry, temperature is one of the most important physical factors that can change in a chemical reaction. So, we really wanted to know more about the chemistry inside a cell, which can tell us more about how the chemistry of life occurs."

Scientists long have suspected that temperatures vary inside individual cells. Yang explained that thousands of biochemical reactions at the basis of life are constantly underway inside cells. Some of those reactions produce energy and heat. But some cells are more active than others, and the unused energy is discharged as heat. Parts of individual cells also may be warmer because they harbor biochemical power plants termed mitochondria for producing energy.

The researchers got that information by inserting the nano-thermometers into mouse cells growing in laboratory dishes. They found temperature differences of a few degrees Fahrenheit between one part of some cells and another, with parts of cells both warmer and cooler than others. Their temperature measurements are not yet accurate enough to give an exact numerical figure. Yang's team also intentionally stimulated cells in ways that boosted the biochemical activity inside cells and observed temperature changes.

Yang says that those temperature changes may have body-wide impacts in determining health and disease. Increases in temperature inside a cell, for instance, may change the way that the genetic material called DNA works, and thus the way that the genes, which are made from DNA, work. Changing the temperature will also change how protein molecular machines operate. At higher temperatures, some proteins may become denatured, shutting down production.

"With these nano thermometer experiments, I believe we are the first to show that the temperature responses inside individual living cells are heterogeneous -- or different," said Yang. "This leads us to our next hypothesis, which is that cells may use differences in temperature as a way to communicate."

Yang's team is now conducting experiments to determine what regulates the temperature inside individual cells. One goal is to apply the information in improving prevention, diagnosis and treatment of diseases.

Story Source:

The above story is reprinted (with editorial adaptations ) from materials provided by American Chemical Society, via EurekAlert!, a service of AAAS.

Engineers discover nanoscale balancing act that mirrors forces at work in living systems

 A delicate balance of atomic forces can be exploited to make nanoparticle superclusters that are uniform in size -- an attribute that's important for many nanotech applications but hard to accomplish, University of Michigan researchers say.

The same type of forces are at work bringing the building blocks of viruses together, and the inorganic supercluster structures in this research are in many ways similar to viruses.

U-M chemical engineering professors Nicholas Kotov and Sharon Glotzer led the research. The findings are newly published online in Nature Nanotechnology.

In another instance of forces behaving in unexpected ways at the nanoscale, they discovered that if you start with small nanoscale building blocks that are varied enough in size, the electrostatic repulsion force and van der Waals attraction force will balance each other and limit the growth of the clusters. This equilibrium enables the formation of clusters that are uniform in size.

"The breakthrough here is that we've discovered a generic mechanism that causes these nanoparticles to assemble into near perfect structures," Glotzer said. "The physics that we see is not special to this system, and could be exploited with other materials. Now that we know how it works, we can design new building blocks that will assemble the same way."

The inorganic superclusters -- technically called "supraparticles" -- that the researchers created out of red, powdery cadmium selenide are not artificial viruses. But they do share many attributes with the simplest forms of life, including size, shape, core-shell structure and the abilities to both assemble and dissemble, Kotov said.

"Having these functionalities in totally inorganic system is quite remarkable," Kotov said. "There is the potential to combine them with the beneficial properties of inorganic materials such as environmental resilience, light adsorption and electrical conductivity."

Zhiyong Tang, a collaborating professor at the National Center of Nanoscience and Technology in China, said, "It is also very impressive that such supraparticles can be further used as the building blocks to fabricate three-dimensional ordered assemblies. This secondary self-assembly behavior provides a feasible way to obtain large-scale nanostructures that are important for practical application."

Kotov is currently working on "breeding" these supraparticles to produce synthetic fuels from carbon dioxide. The work also has applications in drug delivery and solar cell research and it could dramatically reduce the cost of manufacturing large quantities of supraparticles.

"By replicating the self-assembly processes that allow living organisms to grow and heal, we can simplify the production of many useful nanostructured systems from semiconductors and metals so much so that they can be made in any high school laboratory," Kotov said.

This research is funded by the Department of Defense, the National Science Foundation and the U.S. Army Research Office.

Story Source:

The above story is reprinted (with editorial adaptations ) from materials provided by University of Michigan.

Journal Reference:

Yunsheng Xia, Trung Dac Nguyen, Ming Yang, Byeongdu Lee, Aaron Santos, Paul Podsiadlo, Zhiyong Tang, Sharon C. Glotzer, Nicholas A. Kotov. Self-assembly of self-limiting monodisperse supraparticles from polydisperse nanoparticles. Nature Nanotechnology, 2011; DOI: 10.1038/nnano.2011.121

Mussel adhesive inspires tough coating for living cells

Inspired by Mother Nature, scientists are reporting development of a protective coating with the potential to enable living cells to survive in a dormant condition for long periods despite intense heat, dryness and other hostile conditions. In a report in Journal of the American Chemical Society, they liken the coating to the armor that encloses the spores that protect anthrax and certain other bacterial cells, making those microbes difficult to kill.

Insung S. Choi and colleagues say their simple method for coating the yeast cells could "serve as a new strategy for controlling cell division and protection of artificial spore like structures in a designed way." The technique could be used to encapsulate individual cells for a variety of purposes, including the creation of tiny chemical probes, single-cell chemical factories, and perhaps armor for transplanted cells used in anti-cancer therapies.

The new coating is an organic material called polydopamine, chemically similar to mussel adhesive. In laboratory experiments, the coating slowed down cell division in the yeast, while protecting them from cell-digesting chemicals. "We believe that polydopamine encapsulation would be a good starting point for both fundamental research and applications based on artificial spores," Choi and colleagues note in their study, "as it endows living cells with durability against harsh environments, controllability in cell cycles, and reactivity for cell-surface modification."

Provided by American Chemical Society (news : web)

Scientists use light to move molecules within living cells

Using a light-triggered chemical tool, Johns Hopkins scientists report that they have refined a means of moving individual molecules around inside living cells and sending them to exact locations at precise times.

This new tool, they say, gives scientists greater command than ever in manipulating single molecules, allowing them to see how molecules in certain cell locations can influence cell behavior and to determine whether cells will grow, die, move or divide. A report on the work was published online December 13 in the Journal of the American Chemical Society.

Studying how just one signaling molecule communicates in various parts of a living cell has posed a challenge for scientists investigating how different interactions influence cell behavior, such as the decision to move, change shape or divide.

"By using one magical chemical set off by light, we modified our previous technique for moving molecules around and gained much more control," says Takanari Inoue, Ph.D., assistant professor of cell biology and member of the Center for Cell Dynamics in the Institute for Basic Biomedical Sciences. "The advantage of using light is that it is very controllable, and by confining the light, we can manipulate communication of molecules in only a tiny region of the cell," he says.

Specifically, the Hopkins team designed a way to initiate and spatially restrict the molecular interactions to a small portion of the cell by attaching a light-triggered chemical to a bulky molecule, the bond between which would break when researchers shined a defined beam of ultraviolet light on it. This enabled the chemical to enter the cell and force two different and specific proteins in that cell to mingle when they otherwise wouldn't. Normally, these proteins would have nothing to do with each other without the presence of the light-triggered chemical, but researchers decided to take advantage of this mingling to explore how certain proteins in a cell behave when transported to precise locations.

Next, researchers modified the two mingling proteins by attaching special molecules to them -- one sent one of the proteins to the edge of the cell and another caused ripples to form on the edge of the cell -- so that if ripples form on the edge of the cell, they would know that the proteins were interacting there.

The researchers put both modified proteins inside human skin cells and bathed the cells in the light-triggered chemical tool. Then, they shone a tiny UV beam directed on approximately ten percent of the edge of a skin cell. Ripples appeared only on the region of the cell near where the light was beamed, demonstrating that the tool could limit cell activity to a precise location in the cell.

The tool can be used in larger cells, Inoue says, to monitor as little as one percent of a specific molecule if the beam intensity is varied. That in turn could reveal in even more detail the secret affairs of proteins in cellular cubbyholes.

"With this technique, we can get a finer understanding of cell function on the molecular level," says Inoue. "Our technique allows us to monitor whatever molecule we choose in whichever tiny space we choose so that we can understand how a molecule functions in a specific part of a live cell."

This study was funded by the National Institutes of Health and fellowships from the Japan Society for the Promotion of Science.

Other authors on this manuscript are Nobuhiro Umeda, Tasuku Ueno and Christopher Pohlmeyer, and Tetsuo Nagano of The University of Tokyo.

Story Source:

The above story is reprinted (with editorial adaptations) from materials provided by Johns Hopkins Medical Institutions.

Journal Reference:

Nobuhiro Umeda, Tasuku Ueno, Christopher Pohlmeyer, Tetsuo Nagano, Takanari Inoue. A Photocleavable Rapamycin Conjugate for Spatiotemporal Control of Small GTPase Activity. Journal of the American Chemical Society, 2011; 133 (1): 12 DOI: 10.1021/ja108258d

Glowing spirals: Chemical scaffolds guide living cells into precisely defined three-dimensional patterns

To find our way, we use maps. Cells use "chemical maps" to find the way: they orient themselves by following concentration gradients of attractants or repellants. David H. Gracias and a team at Johns Hopkins University (Baltimore, USA) have now developed a clever new method to produce three-dimensional patterns of chemical concentration gradients in vitro -- with previously unattainable versatility and precision in both space and time.

As the scientists report in the journal Angewandte Chemie, they use tiny containers of different shapes and patterned with different arrangements of slits through which chemical messenger substances can diffuse. They were thus able to induce fluorescing cells to organize themselves into a glowing green spiral.

Concentration gradients not only can guide bacteria, fungi, and amoebae; they are also very important in the early stages of embryogenesis because the development of seed leaves (cotyledon) is controlled through concentration gradients of messenger molecules. Three-dimensional chemical patterns play a role in many physiological and pathological processes, including the growth of blood vessels, regulation of blood pressure and heart rate, and tumor metastasis. Our immune cells also follow concentration gradients to find the spot where they are needed.

In order to examine these processes more closely, scientists want to imitate such chemical gradients in vitro. Making a three-dimensional chemical pattern and maintaining it long enough is not so easy. Previous microfluidic methods only allowed for the generation of two-dimensional patterns of limited size. An alternative technique discussed here is the diffusion of chemicals through precisely formed porous containers in stationary media. Variation of the container geometry and pore pattern in the walls makes it possible to realize a wide variety of three-dimensional concentration patterns.

The special trick: Gracias and his co-workers “build” their containers from two-dimensional surfaces held together with tiny hinges. These were designed so that the containers fold up on their own when heated and then stay tightly closed on cooling. In this way, they are able to make containers ranging in size from 100 nm to a few millimeters for potential applications at the sub-cellular to tissue scale. Before being folded, established lithographic methods can be used to perforate each surface with a well-defined arrangement of slits or holes with nano-microscale precision.

With an offset arrangement of slits on four surfaces of a cube shaped container, the researchers were able to release an attractant to generate a concentration gradient in the form of a spiral winding around the container. Fluorescing bacteria followed this pattern and arranged themselves into a glowing spiral.

More information: David Gracias, Direction of Cellular Self-Organization by the Generation of Three- Dimensional Chemical Patterns, Angewandte Chemie International Edition 2011, 50, No. 11, 2549–2553, http://dx.doi.org/ … ie.201007107

Provided by Wiley (news : web)