Team shapes cell behavior research

A team led by James Henderson, assistant professor of biomedical and chemical engineering in Syracuse University's L.C. Smith College of Engineering and Computer Science (LCS) and researcher in the Syracuse Biomaterials Institute, has used shape memory polymers to provide greater insight into how cells sense and respond to their physical environment.

Most cell biomechanics research has examined cell behavior on unchanging, flat surfaces. "Living cells are remarkably complex, dynamic and versatile systems, but the material substrates currently used to culture them are not," says Henderson (at right in photo). "What motivated our work was the need for cell culture technologies that would allow dynamic control of cell-material interactions. We wanted to give a powerful new tool to biologists and bioengineers."

The goal of the current research was to develop a temperature-sensitive shape memory polymer substrate that could be programmed to change shape under cell-compatible conditions. Shape memory polymers (SMPs) are a class of "smart" materials that can switch between two shapes on command, from a fixed (temporary) shape to a pre-determined permanent shape, via a trigger such as a temperature change.

The breakthrough needed to achieve the research goal was made by Kevin Davis, a third-year Ph.D. student in the Henderson lab. Davis was able to develop a SMP with a transition temperature that worked within the limited range required for cells to live. He observed greater than 95 percent cell viability before and after topography and temperature change. This is the first demonstration of this type of cell-compatible, programmable topography change. Davis' and Henderson's work collaboration with Kelly Burke of Case Western Reserve University and Patrick T. Mather, Milton and Ann Stevenson Professor of Biomedical and Chemical Engineering at Syracuse University, is highlighted in the January issue of the journal Biomaterials, the leading journal in biomaterials research.

After confirming that cells remained viable on the substrate, Davis then investigated the changes in cell alignment on the surface that results from topography change. Davis programmed a SMP substrate that transitioned from a micron-scale grooved surface to a smooth surface. When the cells were seeded on the grooved sample at 30oC, the cells lined up along the grooves of the surface. The substrates were then placed in a 37oC incubator, which was the transition temperature for the substrate to recover to a smooth surface. Following shape memory recovery, the cells were observed to be randomly oriented on the substrate.

This research project aimed to determine if cells could remain viable with a change in substrate topography and determine whether cells responded to the change. The next phase of this research is to move from a 2D substrate to a 3D substrate and examine cell viability. Additionally, Henderson's team will be looking at what is going on inside the cells as a result of topography changes.

The application of shape memory principles offers potential solutions for current limitations of static substrate research in bioengineering research, such as medical devices and tissue engineering scaffolds. "For the first time, we've shown that this general concept can be used successfully with cells, which suggests that it can be extended to a number of biomaterials that could be used for scaffolds and many other applications," says Davis. Since most scaffolding is made out of polymers, Henderson envisions one day using SMPs to create scaffolds that can expand inside the body, allowing for less invasive surgical procedures.

Provided by Syracuse University

The role of metal ions in amyotrophic lateral sclerosis

Infrared and x-ray fluorescence microscopy (XFM) images of half a spinal cord cross section from a normal mouse (non-transgenic), a healthy mouse expressing normal SOD (wild-type), and diseased ALS mice with SOD mutations (G93A, G37R, and H46R/H48Q). The top row contains infrared data showing the lipid-rich white matter around the protein-rich gray matter. The second and third rows show the copper and zinc content from x-ray fluorescence microprobe X27A. Copper was decreased in gray matter of the H46R/H48Q mutations, which does not bind copper. All of the spinal cords from the sick mice contain more zinc in the white matter compared to the healthy mice. The white scale bar represents 0.1 mm, and the XFM scale bars are in concentration units of mM.

(PhysOrg.com) -- Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease, is a progressive neurodegenerative disease that affects motor neurons in the spinal cord, leading to muscle weakness, paralysis, and death within two to five years. With a lifetime risk of 1 in 2,000, ALS is the most common motor neuron disease.

Approximately 90 percent of all ALS cases are sporadic in nature, with no known cause. However, the remaining 10 percent are inherited. Of these genetic cases, 20 percent are linked to mutations in the metal-containing protein superoxide dismutase (SOD1), which is an important antioxidant enzyme that requires zinc for structural stability and copper for its detoxifying function. Interestingly, over 145 different mutations in SOD1 have been identified in patients with ALS.

In a study recently published in the Journal of Biological Chemistry, our team at BNL and collaborators from UCLA, the University of Florida, and Stony Brook University used mouse models of ALS to understand how metal ions and SOD1 mutations play a role in the disease process. These mice were genetically predisposed to develop ALS-like disease by over-producing human forms of mutated SOD1. Similar to human patients, they develop aggregates of SOD1 in the spinal cord and undergo progressive paralysis. To explore the role of metal ions in aggregation and disease, we analyzed copper and zinc in the spinal cord tissue in two different ways. First, we determined whether the SOD1 protein molecules were fully "metallated," which makes the protein functional. Second, we examined the overall distributions of copper and zinc in the spinal cord.

For the first part of the study, SOD1 was gently extracted from the spinal cord and separated into soluble (non-aggregated) and insoluble (aggregated) fractions, which were analyzed for metal content. The results showed that the aggregates did not contain the necessary metal ions for the proteins to be functional. In contrast, the soluble SOD1 contained the expected amount of copper and zinc needed for the protein to function. Since SOD1 is highly stable once it is fully metallated, these findings support the hypothesis that the aggregates of mutated SOD1 are derived from immature protein before it has a chance to acquire the necessary copper and zinc ions.

In the second part of the study, NSLS beamline X27A was used to image the distribution of copper and zinc in cross-sections of the spinal cords. For those mutations where SOD1 was able to bind copper ions, results showed that copper was redistributed to regions with high SOD1, leaving other regions of tissue copper-deficient. The zinc distribution followed a different trend, where a high concentration of zinc was found in the spinal cord's "white matter" for all mutations, regardless of the mutation's ability to bind metal ions. Since the white matter is the region of the spinal cord where nerve transmission occurs, high zinc content may indicate short-circuiting or death of neurons.

As a result of these studies, we have new information about the progression of ALS. For example, it is likely the aggregates in ALS arise from newly formed SOD1, prior to metallation. Once copper and zinc bind to SOD1, it becomes very stable and is no longer susceptible to aggregation. Thus, one treatment approach to ALS may involve methods for metallating SOD1 prior to aggregate formation. In addition, the disease process induces a redistribution of metal ions in the spinal cord, further compromising the tissue metabolism. Interestingly, the change in zinc content may provide a diagnostic marker of the disease process, and future studies at earlier stages of the disease will investigate this possibility.

More information: H.L. Lelie, et al., "Copper and Zinc Metallation Status of Copper-Zinc Superoxide Dismutase from Amyotrophic Lateral Sclerosis Transgenic Mice," Journal of Biological Chemistry, 286, 2795 (2011).

An advance toward blood transfusions that require no typing

Scientists are reporting an "important step" toward development of a universal blood product that would eliminate the need to "type" blood to match donor and recipient before transfusions. A report on the "immunocamouflage" technique, which hides blood cells from antibodies that could trigger a potentially fatal immune reaction that occurs when blood types do not match, appears in the ACS journal, Biomacromolecules.

Maryam Tabrizian and colleagues note that blood transfusions require a correct match between a donor and the recipient's blood. This can be a tricky proposition given that there are 29 different red blood cells types, including the familiar ABO and Rh types. The wrong blood type can provoke serious immune reactions that result in organ failure or death, so scientists have long sought a way to create an all-purpose red blood cell for transfusions that doesn't rely on costly blood typing or donations of a specific blood type.

To develop this "universal" red blood cell, the scientists discovered a way to encase living, individual red blood cells within a multilayered polymer shell. The shell serves as a cloaking device, they found, making the cell invisible to a person's immune system and able to evade detection and rejection. Oxygen can still penetrate the polymer shell, however, so the red blood cells can carry on their main business of supplying oxygen to the body. "The results of this study mark an important step toward the production of universal RBCs," the study states.

More information: "Investigation of Layer-by-Layer Assembly of Polyelectrolytes on Fully Functional Human Red Blood Cells in Suspension for Attenuated Immune Response", Biomacromolecules.

Provided by American Chemical Society (news : web)

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)

Greenhouse gas-producing enzyme may yield insights into earliest oxygen-breathing ancestor evolution

 Every year, nitrogen-metabolizing bacteria in the soil and seas churn out more than ten billion kilograms of nitrous oxide (N2O) gas as they respire in these oxygen-deficient environments.

Nitric oxide reductase (NOR) enzymes are the powerhouse underlying production of this gas, taking pairs of nitric oxide (NO) molecules and transforming them into N2O and water via a chemical reaction known as ‘reduction’. These enzymes also help pathogenic bacteria to evade destruction by the immune system, as some T cells use NO as a chemical weapon against infectious agents.

More generally, scientists are interested in the potential to employ these enzymes as a tool for synthesizing useful, customized molecules for a variety of applications. “The nitrogen-oxygen bond cleavage and nitrogen–nitrogen bond formation reactions executed by these enzymes are the essence of chemistry,” says Yoshitsugu Shiro of the RIKEN SPring-8 Center in Harima.

Although a great deal is known about the biochemical properties of these proteins, scientists have found it challenging to determine the structure of bacterial NOR in fine detail. Now, after seven years of hard work, Shiro and colleagues have finally obtained the first such structure for NOR from Pseudomonas aeruginosa, a pathogenic bacterium associated with opportunistic infections in immune-compromised patients (Fig. 1).

Not-so-distant relations

Although all NOR enzymes execute essentially the same chemical reaction to produce N2O, they can be subdivided into three major classes: cNOR, qNOR and qCuNOR. The enzyme crystallized by Shiro and colleagues (Fig. 2) belongs to the cNOR family, and contains a subunit known as cytochrome c that also enables bacteria to engage in aerobic (oxygen-driven) respiration. This ability to switch from oxygen-based to nitrogen-based respiration is highly beneficial for survival in the oxygen-poor conditions deep in the soil or beneath the waves.

 

Accordingly, cNORs are thought to be closely related to the cytochrome oxidases (COX), enzymes that play a central role in aerobic respiration, and these new findings have revealed a number of structural parallels between the two. “There is a long history of research into these respiratory enzymes, COX and NOR, and a lot of knowledge on NOR has been accumulated by biochemical, chemical, molecular biology and microbiological studies,” says Shiro. “From these points of view, our NOR structure is not surprising, but seeing is believing!”

The reduction process is dependent on the directional transport of electrons and protons, and COX and cNOR appear to closely resemble one another in terms of the structure of their electron-transfer networks. Both enzymes depend on precisely positioned metal ions to enable electron transport, and the four iron atoms contained within cNOR are arranged in a configuration that closely resembles COX, maintained via interactions between these positively charged iron atoms and a set of evolutionarily conserved, negatively charged histidine and glutamate amino acids.

On the other hand, the researchers observed some notable differences with regard to the movement of protons. Both COX and cNOR are bound within membranes, but COX contains channels that are believed to direct the flow of protons across the membrane from the interior of the cell. This flow helps generate electrical potential that subsequently powers a variety of cellular motors. However, cNOR lacks such membrane-spanning channels, and protons entering the enzyme from the exterior of the cell only make it as far as the membrane interior, where the reductase catalytic site is located.

Back to the beginning

Even with a structure in hand for this well-studied enzyme, a number of mysteries remain to be addressed. For example, these data are insufficient to resolve an ongoing debate over the fine details of the N2O production mechanism. Shiro and colleagues were readily able to identify the two iron atoms involved in catalysis, but their structure reveals insufficient space at this ‘active site’ to accommodate the two molecules of NO believed to be required for this reaction.

“The NOR active site is tightly packed and very crowded,” says Shiro. “This observation suggests that some conformational change [is] needed to achieve catalytic turnover, but no one knows of any such conformational change so far.” Resolving this issue will require the acquisition of additional, high-resolution structures that might offer clear snapshots of the enzyme at intermediate stages in the catalytic process.

This structure offers tentative support for the hypothesis that the COX aerobic respiratory machinery originally evolved from NOR enzymes, although additional work will clearly be required to confirm this. Unlike NOR, which exclusively employs iron ions, COX makes use of both copper and iron for catalysis, and the researchers have tentatively identified a few amino acid changes that might have enabled this transition to take place. In addition, although NOR lacks the ‘K-channel’ that allows COX to deliver protons from the cytoplasm, Shiro’s team has identified some structural elements that could potentially represent early evolutionary precursors in the formation of this channel.

In future studies, Shiro plans to develop experimental tests for some of these still-speculative models. “We want to follow the molecular evolution of the respiratory enzymes from anaerobic to aerobic conditions on Earth, from NOR to COX,” he says. “Using mutagenesis, based on our structural comparisons, we are hoping to convert NO-reducing NOR into oxygen-reducing COX.” For the present, though, he is optimistic that this structure will give a boost to researchers seeking to understand and manipulate this enzymatic process. “Scientists worldwide who are interested in NO reduction can enter a new stage of NOR research with this structure,” he says.

More information: Hino, T., et al. Structural basis of biological N2O generation by bacterial nitric oxide reductase. Science 330, 1666–1670 (2010). http://www.science … 666.abstract

Provided by RIKEN (news : web)

New method for studying molecule reactions a breakthrough in organic chemistry

 Good chemists are passive-aggressive -- they manipulate molecules without actually touching them.

In a feat of manipulating substances at the nanoscale, UCLA researchers and colleagues demonstrated a method for isolating two molecules together on a substrate and controlling how those two molecules react when excited with ultraviolet light, making detailed observations both before and after the reaction.

Their research is published today in the journal Science.

"This is one step in measuring and understanding the interactions between light and molecules, which we hope will eventually lead to more efficient conversion of sunlight to electrical and other usable forms of energy," said lead study author Paul S. Weiss, a distinguished professor of chemistry and biochemistry who holds UCLA's Fred Kavli Chair in Nanosystems Sciences. "Here, we used the energy from the light to induce a chemical reaction in a way that would not happen for molecules free to move in solution; they were held in place by their attachment to a surface and by the unreactive matrix of molecules around them."

Weiss is also director of UCLA's California NanoSystems Institute (CNSI) and a professor of materials science and engineering at the UCLA Henry Samueli School of Engineering and Applied Science.

Two molecules are placed in proximity in "cutouts" in self-assembled monolayers. When excited with ultraviolet light, they are constrained to react along a pathway different than they would if they could reorient in solution.

Controlling exactly how molecules combine in order to study the resulting reactions is called regioselectivity. It is important because there are a variety of ways that molecules can combine, with varying chemical products. One way to direct a reaction is to isolate molecules and to hold them together to get regioselective reactions; this is the strategy used by enzymes in many biochemical reactions.

"The specialized scanning tunneling microscope used for these studies can also measure the absorption of light and charge separation in molecules designed for solar cells," Weiss said. "This gives us a new way to optimize these molecules, in collaboration with synthetic chemists. This is what first brought us together with our collaborators at the University of Washington, led by Prof. Alex Jen."

Alex K-Y. Jen holds the Boeing-Johnson Chair at the University of Washington, where he is a professor of materials science and engineering and of chemistry. The theoretical aspects of the study were led by Kendall Houk, a UCLA professor of chemistry and biochemistry who holds the Saul Winstein Chair in Organic Chemistry. Houk is a CNSI researcher.

The study's first author, Moonhee Kim, a graduate student in Weiss' lab, managed to isolate and control the reactions of pairs of molecules by creating nanostructures tailored to allow only two molecules fit in place. The molecules used in the study are photosensitive and are used in organic solar cells; similar techniques could be used to study a wide variety of molecules. Manipulating the way molecules in organic solar cells come together may also ultimately lead to greater efficiency.

To isolate the two molecules and align them in the desired — but unnatural — way, Kim utilized a concept similar to that of toddler's toys that feature cutouts in which only certain shapes will fit.

She created a defect, or cutout, in a self-assembled monolayer, or SAM, a single layer of molecules on a flat surface — in this case, gold. The defect in the SAM was sized so that only two organic reactant molecules would fit and would only attach with the desired alignment. As a guide to attach the molecules to the SAM in the correct orientation, sulfur was attached to the bottoms of the molecules, as sulfur binds readily to gold.

"The standard procedure for this type of chemistry is to combine a bunch of molecules in solution and let them react together, but through random combinations, only 3 percent of molecules might react in this way," UCLA's Houk said. "Our method is much more targeted. Instead of doing one measurement on thousands of molecules, we are doing a range of measurements on just two molecules."

After the molecules were isolated and trapped on the substrate, they still needed to be excited with light to react. In this case, the energy was supplied by ultraviolet light, which triggered the reaction. The researchers were able to verify the proper alignment and the reaction of the molecules using the special microscope developed by Kim and Weiss.

The work was funded by the U.S. Department of Energy, the National Science Foundation, the Air Force Office of Scientific Research and the Kavli Foundation.

Provided by University of California - Los Angeles

Synthetic biology: German researchers develop novel kind of fluorescent protein

Since the 1990s a green fluorescent protein known as GFP has been used in research labs worldwide. Protein designers at Technische Universitaet Muenchen have now taken it a step further: They have managed to incorporate a synthetic amino acid into the natural GFP and thus to create a new kind of chimeric fluorescent bio-molecule by means of synthetic biology. By exploiting a special physical effect, the fluorescent protein glows in turquoise and displays unmatched properties.

Proteins are the most important functional biomolecules in nature with numerous applications in life science research, biotechnology and medicine. So how can they be modified in the most effective way to attain certain desired properties? In the past, the modifications were usually carried out either chemically or via genetic engineering. The team of Professor Arne Skerra from the TUM Chair of Biological Chemistry has now developed a more elegant combined solution: By extending the otherwise universal genetic code, the scientists are able to coerce bacterial cells to produce tailored proteins with synthetic functional groups. To put their idea to the test, they set out to crack a particularly hard nut: The scientists wanted to incorporate a non-natural amino acid at a specific site into a widely used natural protein.

In bioresearch this protein is commonly known as "GFP" (= green fluorescent protein). It emits a bright green glow and stems originally from a jellyfish that uses the protein to make itself visible in the darkness of the deep sea. The team chose a pale lavender coumarin pigment, serving as side chain of a non-natural amino acid, as the synthetic group. The scientists "fed" this artificial amino acid to a laboratory culture of Escherichia coli bacteria – the microorganism workhorses of genetic engineering, whose natural siblings are also found in the human intestine. Since the team had transferred the modified genetic blueprints for the GFP to the bacteria – including the necessary biosynthesis machinery – it incorporated the coumarin amino acid at a very specific site into the fluorescent protein.

This spot in the GFP was carefully chosen, explains Professor Skerra: "We positioned the synthetic amino acid at a very close distance from the fluorescence center of the natural protein." The scientists employed the principle of the so-called Foerster resonance energy transfer, or FRET for short. Under favorable conditions, this process of physical energy transfer, named after the German physical chemist Theodor Foerster, allows energy to be conveyed from one stimulated pigment to another in a radiation-less manner.

It was precisely this FRET effect that the scientists implemented very elegantly in the new fluorescent protein. They defined the distance between the imported chemical pigment and the biological blue-green (cyan, to be more precise) pigment of the jellyfish protein in such a way that the interplay between the two dyes resulted in a completely novel kind of fluorescent chimeric biomolecule. Because of the extreme proximity of the two luminescent groups the pale lavender of the synthetic amino acid can no longer be detected; instead, the typical blue-green color of the fluorescent protein dominates. "What is special here, and different from the natural GFP, is that, thanks to the synthetically incorporated amino acid, the fluorescence can be excited with a commercially available black-light lamp in place of an expensive dedicated LASER apparatus," explains Sebastian Kuhn, who conducted these groundbreaking experiments as part of his doctoral thesis.

According to Skerra, the design principle of the novel bio-molecule, which is characterized by a particularly large and hard to achieve wavelength difference between excitation and emitted light, should open numerous interesting applications: "We have now demonstrated that the technology works. Our strategy will enable the preparation of customized fluorescent proteins in various colors for manifold future purposes." This research project was financially supported by the German Research Foundation (DFG) as part of the Excellence Cluster "Munich Center for Integrated Protein Science" (CIPS-M).

More information: Sebastian M. Kuhn, Marina Rubini, Michael A. Müller und Arne Skerra (2011): Biosynthesis of a fluorescent protein with extreme pseudo-Stokes shift by introducing a genetically encoded non-natural amino acid outside the fluorophore. Journal of the American Chemical Society 133, 3708-3711. Advanced online publication at DOI:10.1021/ja1099787