Shiny new tool for imaging biomolecules

 At the heart of the immune system that protects our bodies from disease and foreign invaders is a vast and complex communications network involving millions of cells, sending and receiving chemical signals that can mean life or death. At the heart of this vast cellular signaling network are interactions between billions of proteins and other biomolecules. These interactions, in turn, are greatly influenced by the spatial patterning of signaling and receptor molecules. The ability to observe signaling spatial patterns in the immune and other cellular systems as they evolve, and to study the impact on molecular interactions and, ultimately, cellular communication, would be a critical tool in the fight against immunological and other disorders that lead to a broad range of health problems including cancer. Such a tool is now at hand.

Researchers with the U.S. Department of Energy (DOE)'s Lawrence Berkeley National Laboratory (Berkeley Lab) and the University of California (UC) Berkeley, have developed the first practical application of optical nanoantennas in cell membrane biology. A scientific team led by chemist Jay Groves has developed a technique for lacing artificial lipid membranes with billions of gold "bowtie" nanoantennas. Through the phenomenon known as "plasmonics," these nanoantennas can boost the intensity of a fluorescent or Raman optical signal from a protein passing through a plasmonic "hot-spot" tens of thousands of times without the protein ever being touched.

"Our technique is minimally invasive since enhancement of optical signals is achieved without requiring the molecules to directly interact with the nanoantenna," Groves says. "This is an important improvement over methods that rely on adsorption of molecules directly onto antennas where their structure, orientation, and behavior can all be altered."

Groves holds joint appointments with Berkeley Lab's Physical Biosciences Division and UC Berkeley's Chemistry Department, and is also a Howard Hughes Medical Institute investigator. He is the corresponding author of a paper that reports these results in the journal NanoLetters. The paper is titled "Single Molecule Tracking on Supported Membranes with Arrays of Optical Nanoantennas." Co-authoring the paper were Theo Lohmuller, Lars Iversen, Mark Schmidt, Christopher Rhodes, Hsiung-Lin Tu and Wan-Chen Lin.

Fluorescent emissions, in which biomolecules of interest are tagged with dyes that fluoresce when stimulated by light, and Raman spectroscopy, in which the scattering of light by molecular vibrations is used to identify and locate biomolecules, are work-horse optical imaging techniques whose value has been further enhanced by the emergence of plasmonics. In plasmonics, light waves are squeezed into areas with dimensions smaller than half-the-wavelength of the incident photons, making it possible to apply optical imaging techniques to nanoscale objects such as biomolecules. Nano-sized gold particles in the shape of triangles that are paired in a tip-to-tip formation, like a bow-tie, can serve as optical antennas, capturing and concentrating light waves into well-defined hot spots, where the plasmonic effect is greatly amplified. Although the concept is well-established, applying it to biomolecular studies has been a challenge because gold particle arrays must be fabricated with well-defined nanometer spacing, and molecules of interest must be delivered to plasmonic hot-spots.

"We're able to fabricate billions of gold nanoantennas in an artificial membrane through a combination of colloid lithography and plasma processing," Groves says. "Controlled spacing of the nanoantenna gaps is achieved by taking advantage of the fact that polystyrene particles melt together at their contact point during plasma processing. The result is well-defined spacing between each pair of gold triangles in the final array with a tip-to-tip distance between neighboring gold nanotriangles measuring in the 5-to-100 nanometer range."

Until now, Groves says, it has not been possible to decouple the size of the gold nanotriangles, which determines their surface plasmon resonance frequency, from the tip-to-tip distance between the individual nanoparticle features, which is responsible for enhancing the plasmonic effect. With their colloidal lithography approach, a self-assembling hexagonal monolayer of polymer spheres is used to shadow mask a substrate for subsequent deposition of the gold nanoparticles. When the colloidal mask is removed, what remains are large arrays of gold nanoparticles and triangles over which the artificial membrane can be formed.

The unique artificial membranes, which Groves and his research group developed earlier, are another key to the success of this latest achievement. Made from a fluid bilayer of lipid molecules, these membranes are the first biological platforms that can combine fixed nanopatterning with the mobility of fluid bilayers. They provide an unprecedented capability for the study of how the spatial patterns of chemical and physical properties on membrane surfaces influence the behavior of cells.

"When we embed our artificial membranes with gold nanoantennas we can trace the trajectories of freely diffusing individual proteins as they sequentially pass through and are enhanced by the multiple gaps between the triangles," Groves says. "This allows us to study a realistic system, like a cell, which can involve billions of molecules, without the static entrapment of the molecules."

As molecules in living cells are generally in a state of perpetual motion, it is often their movement and interactions with other molecules rather than static positions that determine their functions within the cell. Groves says that any technique requiring direct adsorption of a molecule of interest onto a nanoantenna intrinsically removes that molecule from the functioning ensemble that is the essence of its natural behavior. The technique he and his co-authors have developed allows them to look at individual biomolecules but within the context of their surrounding community.

"The idea that optical nanoantennas can produce the kinds of enhanced signals we are observing has been known for years but this is the first time that nanoantennas have been fabricated into a fluid membrane so that we can observe every molecule in the system as it passes through the antenna array," Groves says. "This is more than a proof-of-concept we've shown that we now have a useful new tool to add to our repertoire."

This research was primarily supported by the DOE Office of Science.

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:

T. Lohmüller, L. Iversen, M. Schmidt, C. Rhodes, H.-L. Tu, W.-C. Lin, J. T. Groves. Single Molecule Tracking on Supported Membranes with Arrays of Optical Nanoantennas. Nano Letters, 2012; 12 (3): 1717 DOI: 10.1021/nl300294b

Protein 'jailbreak' helps breast cancer cells live

All four proteins were already under suspicion. Researchers, for example, have already tried to assess what levels of HDAC6 in patients with estrogen-receptor positive breast cancer may mean for their prognosis. The results have been inconclusive. The new research suggests that measuring overall levels may not be enough, said the study's senior author Dr. Rachel Altura, associate professor of pediatrics in The Warren Alpert Medical School of Brown University and a pediatric oncologist at Hasbro Children's Hospital.

"We need to look not only at the levels, but also where is it in the cell," she said.

Altura's emphasis on location comes from what her research team found as they tracked and tweaked the comings and goings of survivin in cells. Inside the nucleus, survivin is no problem. Outside the nucleus, but within the cell, it can prevent normal cell death, allowing cancer cells to persist.

In previous work, Altura and her collaborators established that under normal circumstances, CBP chemically regulates survivin, a process called acetylation, and keeps it in the nucleus. The question in the new work was how survivin gets out.

In a series of experiments, what they observed was that in human and mouse breast cancer cells, HDAC6 gathers at the boundary between the nucleus and the rest of the cell, becomes activated by CBP, then binds survivin and undoes its acetylation. This deacetylation allows survivin to then be shuttled out of the nucleus by CRM1.

In the classic jailbreak, CBP is a corrupt guard who looks the other way as HDAC6, the shovel, is smuggled in. The final accomplice, CRM1, is the tunnel with a getaway car on the other end.

Working the new leads

Altura said the research suggests a clear strategy — to keep survivin in the nucleus — and two leads to pursue it, both of which she has already begun working on with collaborators in academia and in the pharmaceutical industry.

One idea is to inhibit HDAC6 in an attempt to prevent it from misregulating the acetylation of survivin. While general HDAC inhibitors are in clinical trials, Altura is optimistic that blocking just HDAC6, using specific inhibitors developed by a colleague in Japan, would have fewer complications.

"You always have to worry about all the things you don't know that you are targeting," she said. "If we can target HDAC6, we can maybe block survivin from coming out of the nucleus and maintain it in its good state."

The other strategy is to block CRM1, Altura said, an idea she is pursuing with a pharmaceutical company in breast cancer cells in the lab. She said preliminary experiments look promising in keeping survivin inside the nucleus and making cancer cells more susceptible to dying.

Provided by Brown University (news : web)

Unexpected behaviour of microdroplets

Physicists agree that laminar flow of liquids has been well understood and described in detail from the theoretical point of view. Researchers at the Institute of Physical Chemistry of the Polish Academy of Sciences in Warsaw have, however, observed that droplets of chemical substances flowing in a carrier liquid inside microchannels -- although presenting laminar flow inside them -- present ultiple mysteries.

Researchers from the Institute of Physical Chemistry of the Polish Academy of Sciences (IPC PAS) in Warsaw discovered a new phenomenon related to the fluid dynamics. It occurs when minute droplets translate through microfluidic channels. "The effect observed by our group is related to changes in swirls inside microdroplets and as yet has not been predicted by existing theoretical models," says Dr Sławomir Jakieła from the IPC PAS. The results of the research pursued thanks to a TEAM grant from the Foundation for Polish Science, have just been published in the journal Physical Review Letters.

Microfluidic systems are miniature chemical reactors of a credit card in size, or even less. Inside these systems, microchannels with diameters of tenths or hundredths of a milimeter provide a path for laminar flow of a carrier fluid (commonly oil) with floating microdroplets of appropriate chemical compounds.

"Using a single microfluidic system, even today one can carry out as much as a few tens of thousands of different chemical reactions a day. In future, these systems will become for chemistry what integrated circuits turned out to be for electronics. Yet before we build chemical devices as revolutionary as silicon microprocessors, we have to reach a comprehensive understanding of all physical phenomena occurring in flows of microdroplets," continues Dr Jakieła.

The flows that we experience at the macroscale are often dominated by inertia and turbulences. With small volumes that are typical for microfluidic systems, the flow of a liquid is laminar and subject to viscosity-related effects.

The speed of oil flowing in microchannels is not uniform. The layers close to the walls move with the lowest speed, whereas those near the middle of a channel -- with the highest speed. "If a microdroplet is distinctly smaller than the channel diameter, it can find a place in the middle part of the flow, reaching the speed even twice as high as the average oil speed. This is nothing surprising. Similar effect can be observed for instance in rivers: the current near the banks is much slower than in the middle of the river," explains Sylwia Makulska, a PhD student at the IPC PAS.

If a sufficiently large droplet flows in a circular channel, it occupies almost the entire lumen of the channel. The droplet speed is then almost identical as that of the oil flow. The situation gets much more interesting when the droplet translates in rectangular channels that are typical to microfluidic systems. Due to interfacial tension the cross-section of a microdroplet remains rounded leaving the corners of the channel free for the flow of oil.

The team from the IPC PAS produced microdroplets from aqueous solutions of glycerine of different concentrations, and therefore of different viscosities. They translated in oil (hexadecane) through a 10 cm long rectangular channel. The researchers measured the speed of microdroplets relative to the oil as a function of their volume (length in a microchannel), droplet and oil viscosities and the flow speed of the carrier liquid.

When the viscosity of microdroplets was less than or comparable to that of the carrier liquid, their speed relative to the oil turned out to decrease with increasing droplet length, but in a certain range only. The droplets were translating with the lowest speed when their length was two, three times greater than the channel width. "Every time we observed the minimum speed relative to oil. Everything seemed to be in line with what the theoreticians would expect," says Jakieła.

But what was really interesting were things that happened when the researchers started to change the rate of oil flow. It turned out that the minimum of the droplet speed relative to oil was disappearing with increasing flow rate. Further increase in the oil flow rate resulted, however, in reappearance of the minimum -- but this time deeper and wider. "To make the long story short: we discovered that, depending on the oil flow rate, a droplet of specific length can translate under some conditions faster and under other conditions slower relative to oil," concludes Jakieła.

To find out what is the reason for the surprising behaviour of the droplets, the researchers from the IPC PAS introduced to microdroplets fluorescent markers of a few micrometers in size. When the droplets were moving along the microchannel, they were irradiated with laser light to excite fluorescence of the markers, which allowed for observation of fluid movements inside the droplets.

The measurements revealed that the distribution of swirls inside a droplet changes with increasing flow rate of the carrier liquid. "We expected changes, but the existing theories suggested that the number of swirls in microdroplets decreases with increasing oil flow rate. We observed, meanwhile, an opposite phenomenon: the faster was the oil flow, the more swirls were in a droplet. The Nature played again a trick on theoreticians," sums up Prof. Piotr Garstecki (IPC PAS).

At the Institute of Physical Chemistry PAS a work has started to make use of the new phenomenon in processes related to mixing the contents of microdroplets in microfluidic systems.

Story Source:

The above story is reprinted from materials provided by Institute of Physical Chemistry of the Polish Academy of Sciences, via AlphaGalileo.

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

Journal Reference:

Slawomir Jakiela, Piotr Korczyk, Sylwia Makulska, Olgierd Cybulski, Piotr Garstecki. Discontinuous Transition in a Laminar Fluid Flow: A Change of Flow Topology inside a Droplet Moving in a Micron-Size Channel. Physical Review Letters, 2012; 108 (13) DOI: 10.1103/PhysRevLett.108.134501

Sweet success in hunt for honey's healing factor

The ground-breaking research, carried out at Industrial Research Ltd (IRL), Plant & Food Research and Massey University, found that different varieties of New Zealand honey appear to trigger different immune responses.
IRL's role was to provide its world-class expertise in the extraction, analysis, and purification of complex molecules that play an important role in biological systems.

Comvita’s Chief Technology Officer Dr. Ralf Schlothauer says the research provides the tools for understanding why honey stimulates healing of stalled wounds.

“We know a lot about the anti-microbial properties of manuka honey but had much less scientific information about the immune system-related effects of honey in wound healing.

“The findings suggest there could be a number of honeys to consider if you want to stimulate the immune system. Ultimately, it might mean we produce medical honey products that are specifically tailored for certain treatments or that we select a range of honeys for their particular properties to include in a specific blend.”

Headquartered in Paengaroa in the Bay of Plenty, Comvita is the world’s largest manufacturer and marketer of Manuka honey and produces natural health products for the wound care, health care, personal care and functional foods markets. It also produces Medihoney™ wound care products that are sold through a global licensing deal with US-based Derma Sciences.

Prior to the latest work, Dr. Schlothauer says published research had shown there were big carbohydrate molecules in honey that stimulated immune cells but their structure had not been analysed.

Comvita put two students, Swapna Gannabathula and Gregor Steinhorn, onto the task and their discoveries eventually led the company to Crown Research Institute IRL.

“We started separating the molecule but were puzzled about what it was. Initially we thought it was a glycan and sought appropriate analysis but they put us on to Dr. Ian Sims in the Carbohydrate Chemistry group at IRL, who is a leading expert in analysing complex molecules that play an important role in biological systems,” says Dr. Schlothauer.

IRL has one of only three laboratories world-wide with the capability and expertise required to carry out complex research into the extraction, purification and analysis of oligo- and poly-saccharides, and glycoconjugates.

Dr. Sims began his work with small-scale analyses that were conducted on Manuka, Kanuka and Clover honeys. Starting with five grams of honey, separation of high molecular weight polymers from small sugars yielded just a few milligrams of sample for analysis. 

After Dr Sims completed an initial, detailed analysis of the sugars Gregor Steinhorn, who now works full-time for Comvita, spent many hours purifying buckets of honey and identified its exact nature under the supervision of Dr. Sims and Dr. Alistair Carr (Massey University).

Comvita is determining the commercial value of this discovery and has a range of new products under development.

The findings from the research have been published in Food Chemistry, an international, peer-reviewed publication that reports on the chemistry and biochemistry of foods and raw materials.

Dr. Schlothauer says the next challenge is to better understand how and why honey promotes healing, with Comvita planning to do more research with the University of Auckland and IRL.

“The work is helping us ensure there is much better information about natural medicines,” says Dr. Schlothauer. "We need to be able to talk about the immune relevance of honey and have proof of its scientific efficacy to ensure natural medicines can sit alongside conventional health products.”

Provided by Industrial Research