Neutrons Provide First Sub-Nanoscale Snapshots of Huntington's Disease Protein

Huntington's disease is caused by a renegade protein"huntingtin" that destroys neurons in areas of the brain concerned with the emotions, intellect and movement. All humans have the normal huntingtin protein, which is known to be essential to human life, although its true biological functions remain unclear.

Christopher Stanley, a Shull Fellow in the Neutron Scattering Science Division at ORNL, and Valerie Berthelier, a UT Graduate School of Medicine researcher who studies protein folding and misfolding in Huntington's, have used a small-angle neutron scattering instrument, called Bio-SANS, at ORNL's High Flux Isotope Reactor to explore the earliest aggregate species of the protein that are believed to be the most toxic.

Stanley and Berthelier, in research published inBiophysical Journal, were able to determine the size and mass of the mutant protein structures―from the earliest small, spherical precursor species composed of two (dimers) and three (trimers) peptides―along the aggregation pathway to the development of the resulting, later-stage fibrils. They were also able to see inside the later-stage fibrils and determine their internal structure, which provides additional insight into how the peptides aggregate.

"Bio-SANS is a great instrument for taking time-resolved snapshots. You can look at how this stuff changes as a function of time and be able to catch the structures at the earliest of times," Stanley said."When you study several of these types of systems with different glutamines or different conditions, you begin to learn more and more about the nature of these aggregates and how they begin forming."

Normal huntingtin contains a region of 10 to 20 glutamine amino acids in succession. However, the DNA of Huntington's disease patients encodes for 37 or more glutamines, causing instability in huntingtin fragments that contain this abnormally long glutamine repeat. Consequentially, the mutant protein fragment cannot be degraded normally and instead forms deposits of fibrils in neurons.

Those deposits, or clumps, were originally seen as the cause of the devastation that ensues in the brain. More recently researchers think the clumping may actually be a kind of biological housecleaning, an attempt by the brain cells to clean out these toxic proteins from places where they are destructive. Stanley and Berthelier set out to learn through neutron scattering what the toxic proteins were and when and where they occurred.

At the HFIR Bio-SANS instrument, the neutron beam comes through a series of mirrors that focus it on the sample. The neutrons interact with the sample, providing data on its atomic structure, and then the neutrons scatter, to be picked up by a detector. From the data the detector sends of the scattering pattern, researchers can deduce at a scale of less than billionths of a meter the size and shape of the diseased, aggregating protein, at each time-step along its growth pathway.

SANS was able to distinguish the small peptide aggregates in the sample solution from the rapidly forming and growing larger aggregates that are simultaneously present. In separate experiments, they were able to monitor the disappearance of the single peptides, as well as the formation of the mature fibrils.

Now that they know the structures, the hope is to develop drugs that can counteract the toxic properties in the early stages, or dissuade them from taking the path to toxicity."The next step would be, let's take drug molecules and see how they can interact and affect these structures," Stanley said.

For now, the researchers believes Bio-SANS will be useful in the further study of Huntington's disease aggregates and applicable for the study of other protein aggregation processes, such as those involved in Alzheimer's and Parkinson's diseases.

"That is the future hope. Right now, we feel like we are making a positive contribution towards that goal," Stanley said.

The research was supported by the National Institutes of Health. HFIR and Bio-SANS are supported by the DOE Office of Science.

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Controling Robotic Arms Is Child's Play

"The input device contains various movement sensors, also called inertial sensors," says Bernhard Kleiner of the Fraunhofer Institute for Manufacturing Engineering and Automation IPA in Stuttgart, who leads the project. The individual micro-electromechanical systems themselves are not expensive. What the scientists have spent time developing is how these sensors interact."We have developed special algorithms that fuse the data of individual sensors and identify a pattern of movement. That means we can detect movements in free space," summarizes Kleiner.

What may at first appear to be a trade show gimmick, is in fact a technology that offers numerous advantages in industrial production and logistical processes. The system could be used to simplify the programming of industrial robots, for example. To date, this has been done with the aid of laser tracking systems: An employee demonstrates the desired motion with a hand-held baton that features a white marker point. The system records this motion by analyzing the light reflected from a laser beam aimed at the marker. Configuring and calibrating the system takes a lot of time. The new input device should eliminate the need for these steps in the future -- instead, employees need only pick up the device and show the robot what it is supposed to do.

The system has numerous applications in medicine, as well. Take, for example, gait analysis. Until now, cameras have made precise recordings of patients as they walk back and forth along a specified path. The films reveal to the physician such things as how the joints behave while walking, or whether incorrect posture in the knees has been improved by physical therapy. Installing the cameras, however, is complex and costly, and patients are restricted to a predetermined path. The new sensor system can simplify this procedure: Attached to the patient's upper thigh, it measures the sequences and patterns of movement -- without limiting the patient's motion in any way.

"With the inertial sensor system, gait analysis can be performed without a frame of reference and with no need for a complex camera system," explains Kleiner. In another project, scientists are already working on comparisons of patients' gait patterns with those patterns appearing in connection with such diseases as Parkinson's.

Another medical application for the new technology is the control of active prostheses containing numerous small actuators. Whenever the patient moves, the prosthesis in turn also moves; this makes it possible for a leg prosthesis to roll the foot while walking. Here, too, the sensor could be attached to the patient's upper thigh and could analyze the movement, helping to regulate the motors of the prosthesis. Research scientists are currently working on combining the inertial sensor system with an electromyographic (EMG) sensor. Electromyography is based on the principle that when a muscle tenses, it produces an electrical voltage which a sensor can then measure by way of an electrode. If the sensor is placed, for example, on the muscle responsible for lifting the patient's foot, the sensor registers when the patient tenses this muscle -- and the prosthetic foot lifts itself. EMG sensors like this are already available but are difficult to position.

"While standard EMG sensors consist of individual electrodes that have to be positioned precisely on the muscle, our system is made up of many small electrodes that attach to a surface area. This enables us to sense muscle movements much more reliably," says Kleiner.

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Scientists Achieve Guiding of Electrons by Purely Electric Fields

The research is published online inPhysical Review Letters.

This new technique of electron guiding -- which resembles the guiding of light waves in optical fibres -- promises a variety of applications, from guided matter-wave experiments to non-invasive electron microscopy.

Electrons have been the first elementary particles revealing their wave-like properties and have therefore been of great importance in the development of the theory of quantum mechanics. Even now the observation of electrons leads to new insight into the fundamental laws of physics. Measurements involving confined electrons have so far mainly been performed in so-called Penning traps, which combine a static magnetic field with an oscillating electric field.

For a number of experiments with propagating electrons, like interferometry with slow electrons, it would be advantageous to confine the electrons by a purely electric field. This can be done in an alternating quadrupole potential similar to the standard technique that is used for ion trapping. These so-called Paul traps are based on four electrodes to which a radiofrequency voltage is applied. The resulting field evokes a driving force which keeps the particle in the centre of the trap. Wolfgang Paul received the Nobel Prize in physics for the invention of these traps in 1989.

For several years by now scientists realize Paul traps with micro structured electrodes on planar substrates, using standard microelectronic chip technology. Dr. Hommelhoff and his group have now applied this method for the first time to electrons. Since the mass of these point-like particles is only a tenth of a thousandth of the mass of an ion, electrons react much faster to electric fields than the rather heavy ions. Hence, in order to guide electrons, the frequency of the alternating voltage applied to the electrodes has to be much higher than for the confinement of ions and is in the microwave range, at around 1 GHz.

In the experiment electrons are generated in a thermal source (in which a tungsten wire is heated like in a light bulb) and the emitted electrons are collimated to a parallel beam of a few electron volts. From there the electrons are injected into the"wave-guide." It is being generated by five electrodes on a planar substrate to which an alternating voltage with a frequency of about 1 GHz is applied. This introduces an oscillating quadrupole field in a distance of half a millimetre above the electrodes, which confines the electrons in the radial direction. In the longitudinal direction there is no force acting on the particles so that they are free to travel along the"guide tube." As the confinement in the radial direction is very strong the electrons are forced to follow even small directional changes of the electrodes.

In order to make this effect more visible the 37mm long electrodes are bent to a curve of 30 degrees opening angle and with a bending radius of 40mm. At the end of the structure the guided electrons are ejected and registered by a detector. A bright spot caused by guided electrons appears on the detector right at the exit of the guide tube, which is situated in the left part of the picture. When the alternating field is switched off a more diffusively illuminated area shows up on the right side. It is caused by electrons spreading out from the source and propagating on straight trajectories over the substrate.

"With this fundamental experiment we were able to show that electrons can be efficiently guided be purely electric fields," says Dr. Hommelhoff."However, as our electron source yields a rather poorly collimated electron beam we still lose many electrons." In the future the researchers plan to combine the new microwave guide with an electron source based on field emission from an atomically sharp metal tip. These devices deliver electron beams with such a strong collimation that their transverse component is limited by the Heisenberg uncertainty principle only.

Under these conditions it should be feasible to investigate the individual quantum mechanical oscillations of the electrons in the radial potential of the guide."The strong confinement of electrons observed in our experiment means that a"jump" from one quantum state to the neighbouring higher state requires a lot of energy and is therefore not very likely to happen," explains Johannes Hoffrogge, doctoral student at the experiment."Once a single quantum state is populated it will remain so for an extended period of time and can be used for quantum experiments." This would make it possible to conduct quantum physics experiments such as interferometry with guided slow electrons. Here the wave function of an electron is first split up; later on, its two components are brought together again whereby characteristic superpositions of quantum states of the electron can be generated. But the new method could also be applied for a new form of electron microscopy.

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Antibody-Based Biosensor Can Guide Environmental Clean-Ups, Provide Early Warning System for Spills

Testing of the biosensor in the Elizabeth River and Yorktown Creek, which both drain into lower Chesapeake Bay, shows that the instrument can process samples in less than 10 minutes, detect pollutants at levels as low as just a few parts per billion, and do so at a cost of just pennies per sample. Current technology requires hours of lab work, with a per-sample cost of up to$1,000.

"Our biosensor combines the power of the immune system with the sensitivity of cutting-edge electronics," says Dr. Mike Unger of VIMS."It holds great promise for real-time detection and monitoring of oil spills and other releases of contaminants into the marine environment."

The biosensor was developed and tested by Unger, fellow VIMS professor Steve Kaattari, and their doctoral student Candace Spier, with assistance from marine scientist George Vadas. The team's report of field tests with the sensor appears in this month's issue ofEnvironmental Toxicology and Chemistry.

The instrument was developed in conjunction with Sapidyne Instruments, Inc., with funding from the state of Virginia, the Office of Naval Research, and the Cooperative Institute for Coastal and Estuarine Environmental Technology, a partnership between NOAA and the University of New Hampshire.

The tests in the Elizabeth River took place during clean up of a site contaminated by polycyclic aromatic hydrocarbons (PAHs), byproducts of decades of industrial use of creosote to treat marine pilings. The U.S. Environmental Protection Agency considers PAHs highly toxic and lists 17 as suspected carcinogens.

The biosensor allowed the researchers to quantify PAH concentrations while the Elizabeth River remediation was taking place, gaining on-site knowledge about water quality surrounding the remediation site. Spier says the test was"the first use of an antibody-based biosensor to guide sampling efforts through near real-time evaluation of environmental contamination."

In the Yorktown Creek study, the researchers used the biosensor to track the runoff of PAHs from roadways and soils during a rainstorm.

Biosensor development

Kaattari says"Our basic idea was to fuse two different kinds of technologies -- monoclonal antibodies and electronic sensors -- in order to detect contaminants."

Antibodies are proteins produced by the immune system of humans and other mammals. They are particularly well suited for detecting contaminants because they have, as Kaattari puts it, an"almost an infinite power to recognize the 3-dimensional shape of any molecule."

Mammals produce antibodies that recognize and bind with large organic molecules such as proteins or with viruses. The VIMS team took this process one step further, linking proteins to PAHs and other contaminants, then exposing mice to these paired compounds in a manner very similar to a regular vaccination.

"Just like you get vaccinated against the flu, we in essence are vaccinating our mice against contaminants," says Kaattari."The mouse's lymphatic system then produces antibodies to PAHs, TNT, tributyl tin {TBT, the active ingredient in anti-fouling paints for boats}, or other compounds."

Once a mouse has produced an antibody to a particular contaminant, the VIMS team applies standard clinical techniques to produce"monoclonal antibodies" in sufficiently large quantities for use in a biosensor.

"This technology allows you to immortalize a lymphocyte that produces only a very specific antibody," says Kaattari."You grow the lymphocytes in culture and can produce large quantities of antibodies within a couple of weeks. You can preserve the antibody-producing lymphocyte forever, which means you don't have to go to a new animal every time you need to produce new antibodies."

From antibody to electrical signal

The team's next step was to develop a sensor that can recognize when an antibody binds with a contaminant and translate that recognition into an electrical signal. The Sapidyne^®sensor used by the VIMS team works via what Kaattari calls a"fluorescence-inhibitory, spectroscopic kind of assay."

In the sensor used on the Elizabeth River and Yorktown Creek, antibodies designed to recognize a specific class of PAHs were joined with a dye that glows when exposed to fluorescent light. The intensity of that light is in turn recorded as a voltage. The sensor also houses tiny plastic beads that are coated with what Spier calls a"PAH surrogate" -- a PAH derivative that retains the shape that the antibody recognizes as a PAH molecule.

When water samples with low PAH levels are added to the sensor chamber (which is already flooded with a solution of anti-PAH antibodies), the antibodies have little to bind with and are thus free to attach to the surrogate-coated beads, providing a strong fluorescent glow and electric signal. In water samples with high PAH concentrations, on the other hand, a large fraction of the antibodies bind with the environmental contaminants. That leaves fewer to attach to the surrogate-coated beads, which consequently provides a fainter glow and a weaker electric signal.

During the Elizabeth River study, the biosensor measured PAH concentrations that ranged from 0.3 to 3.2 parts per billion, with higher PAH levels closer to the dredge site. In Yorktown Creek, the biosensor showed that PAH levels in runoff peaked 1 to 2 hours after the rain started, with peak concentration of 4.4 parts per billion.

Comparison of the biosensor's field readings with later readings from a mass spectrometer at VIMS showed that the biosensor is just as accurate as the more expensive, slower, and laboratory-bound machine.

A valuable field tool

Spier says"Using the biosensor allowed us to quickly survey an area of almost 900 acres around the Elizabeth River dredge, and to provide information about the size and intensity of the contaminant plume to engineers monitoring the dredging from shore. If our results had shown elevated concentrations, they could have halted dredging and put remedial actions in place."

Unger adds"measuring data in real-time also allowed us to guide the collection of large-volume water samples right from the boat. We used these samples for later analysis of specific PAH compounds in the lab. This saved time, effort, and money by keeping us from having to analyze samples that might contain PAHs at levels below our detection limit."

"Biosensors have their constraints and optimal operating conditions," says Kaattari,"but their promise far outweighs any limitations. The primary advantages of our biosensor are its sensitivity, speed, and portability. These instruments are sure to have a myriad of uses in future environmental monitoring and management."

One promising use of the biosensor is for early detection and tracking of oil spills."If biosensors were placed near an oil facility and there was a spill, we would know immediately," says Kaattari."And because we could see concentrations increasing or decreasing in a certain pattern, we could also monitor the dispersal over real time."

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