We report the construction of a cell-based fluorescent reporter for anthrax lethal factor (LF) protease activity using the principle of fluorescence resonance energy transfer (FRET). This was accomplished by engineering an Escherichia coli cell line to express a genetically encoded FRET reporter and LF protease. Both proteins were encoded in two different expression plasmids under the control of different tightly controlled inducible promoters. The FRET-based reporter was designed to contain a LF recognition sequence flanked by the FRET pair formed by CyPet and YPet fluorescent proteins. The length of the linker between both fluorescent proteins was optimized using a flexible peptide linker containing several Gly-Gly-Ser repeats. Our results indicate that this FRET-based LF reporter was readily expressed in E. coli cells showing high levels of FRET in vivo in the absence of LF. The FRET signal, however, decreased 5 times after inducing LF expression in the same cell. These results suggest that this cell-based LF FRET reporter may be used to screen genetically encoded libraries in vivo against LF.

The new heavy ion synchrotron facility proposed by GSI will have two superconducting magnet rings in the same tunnel, with rigidities of 300 T-m and 100 T-m. Fast ramp times are needed, which can cause significant problems for the magnets, particularly in the areas of ac loss and magnetic field distortion. The development of the low loss Rutherford cable that can be used is described, together with a novel insulation scheme designed to promote efficient cooling. Measurements of contact resistance in the cable are presented and the results of these measurements are used to predict the ac losses, in the magnets during fast ramp operation. For the high energy ring, a lm model dipole magnet was built, based on the RHIC dipole design. This magnet was tested under boiling liquid helium in a vertical cryostat. The quench current showed very little dependence on ramp rate. The ac losses, measured by an electrical method, were fitted to straight line plots of loss/cycle versus ramp rate, thereby separating the eddy current and hysteresis components. These results were compared with calculated values, using parameters which had previously been measured on short samples of cable. Reasonably good agreement between theory and experiment was found, …

The Image Content Engine (ICE) is being developed to provide cueing assistance to human image analysts faced with increasingly large and intractable amounts of image data. The ICE architecture includes user configurable feature extraction pipelines which produce intermediate feature vector and match surface files which can then be accessed by interactive relational queries. Application of the feature extraction algorithms to large collections of images may be extremely time consuming and is launched as a batch job on a Linux cluster. The query interface accesses only the intermediate files and returns candidate hits nearly instantaneously. Queries may be posed for individual objects or collections. The query interface prompts the user for feedback, and applies relevance feedback algorithms to revise the feature vector weighting and focus on relevant search results. Examples of feature extraction and both model-based and search-by-example queries are presented.

At the Berkeley Structural Genomics Center (BSGC), our goalis to obtain a near-complete structural complement of proteins in theminimal organisms Mycoplasma genitalium and M. pneumoniae, two closelyrelated pathogens. Current targets for structure determination have beenselected in six major stages, starting with those predicted to be mosttractable to high throughput study and likely to yield new structuralinformation. We report on the process used to select these proteins, aswell as our target deselection procedure. Target deselection reducesexperimental effort by eliminating targets similar to those recentlysolved by the structural biology community or other centers. We measurethe impact of the 69 structures solved at the BSGC as of July 2004 onstructure prediction coverage of the M. pneumoniae and M. genitaliumproteomes. The number of Mycoplasma proteins for which thefold couldfirst be reliably assigned based on structures solved at the BSGC (24 M.pneumoniae and 21 M. genitalium) is approximately 25 percent of the totalresulting from work at all structural genomics centers and the worldwidestructural biology community (94 M. pneumoniae and 86M. genitalium)during the same period. As the number of structures contributed by theBSGC during that period is less than 1 percent of the total worldwideoutput, the benefits of a focused target selection strategy are apparent.If the …

The A-01 NPDES outfall at the Savannah River Site receives process wastewater discharges and stormwater runoff from the Savannah River National Laboratory. Routine monitoring indicated that copper concentrations were regularly higher than discharge permit limit, and water routinely failed toxicity tests. These conditions necessitated treatment of nearly one million gallons of water per day plus storm runoff. Washington Savannah River Company personnel explored options to bring process and runoff waters into compliance with the permit conditions, including source reduction, engineering solutions, and biological solutions. A conceptual design for a constructed wetland treatment system (WTS) was developed and the full-scale system was constructed and began operation in 2000. The overall objective of our research is to better understand the mechanisms of operation of the A-01 WTS in order to provide better input to design of future systems. The system is a vegetated surface flow wetland with a hydraulic retention time of approximately 48 hours. Copper, mercury, and lead removal efficiencies are very high, all in excess of 80% removal from water passing through the wetland system. Zinc removal is 60%, and nickel is generally unaffected. Dissolved organic carbon in the water column is increased by the system and reduces toxicity of …

An extensive study of helium isotopes in fluids collectedfrom surface springs, fumaroles and wells across the northern Basin andRange Province reveals a systematic trend of decreasing 3He/4He ratiosfrom west to east. The western margin of the Basin and Range ischaracterized by mantle-like ratios (6-8 Ra) associated with active orrecently active crustal magma systems (e.g. Coso, Long Valley, Steamboat,and the Cascade volcanic complex). Moving towards the east, the ratiosdecline systematically to a background value of ~;0.1 Ra. The regionaltrend is consistent with extensive mantle melting concentrated along thewestern margin and is coincident with an east-to-west increase in themagnitude of northwest strain. The increase in shear strain enhancescrustal permeability resulting in high vertical fluid flow rates thatpreserve the high helium isotope ratios at the surface. Superimposed onthe regional trend are "helium spikes", local anomalies in the heliumisotope composition. These "spikes" reflect either local zones of mantlemelting or locally enhanced crustal permeability. In the case of theDixie Valley hydrothermal system, it appears to be a combination ofboth.

A controller for the third generation, 6 degree-of-freedom (DOF) piezoelectric translation stage shown in Figure 1 is presented. This was tested by monitoring all six coordinate motions using an orthogonal array of six, high-resolution capacitance gages. The full 6 DOF matrix transformations and controller block diagrams for this system have been measured and the system operated under closed loop control. Results of early experiments to determine the 21 open loop response functions as well as preliminary results showing the closed loop response for the 3 linear translations are presented in this abstract. The ultimate goal of this project is to incorporate this 6 DOF stage within a long range X-Y scanning system for nanometer pick-and-place capability over an area of 50 x 50 mm. The control strategy and early results from this system will be presented.

Spontaneous mixing of materials at unstably stratified interfaces occurs in a wide variety of atmospheric, oceanic, geophysical and astrophysical flows. The Rayleigh-Taylor instability, in particular, plays key roles in the death of stars, planet formation and the quest for controlled thermonuclear fusion. Despite its ubiquity, fundamental questions regarding Rayleigh-Taylor instability persist. Among such questions are: Does the flow forget its initial conditions? Is the flow self-similar? What is the value of the scaling constant? How does mixing influence the growth rate? Here we show results from a 3072{sup 3} grid-point Direct Numerical Simulation in an attempt to answer these and other questions. The data indicate that the scaling constant cannot be found by fitting a curve to the width of the mixing region (as is common practice) but can only be accurately obtained by recourse to the similarity equation for the growth rate. The data further establish that the ratio of kinetic energy to released potential energy is not constant, as has heretofore been assumed. The simulated flow reaches a Reynolds number of 32,000, far exceeding that of all previous simulations. The latter stages of the simulation reveal a weak Reynolds number dependence, which may have profound consequences for modeling …

This paper discusses the use of computational fluid dynamics (CFD) methods to understand and characterize erosion of the floor and internal structures in the slurry mixing vessels in the Defense Waste Processing Facility. An initial literature survey helped identify the principal drivers of erosion for a solids laden fluid: the solids content of the working fluid, the regions of recirculation and particle impact with the walls, and the regions of high wall shear. A series of CFD analyses was performed to characterize slurry-flow profiles, wall shear, and particle impingement distributions in key components such as coil restraints and the vessel floor. The calculations showed that the primary locations of high erosion resulting from abrasion were at the leading edge of the coil guide, the tank floor below the insert plate of the coil guide support, and the upstream lead-in plate. These modeling results based on the calculated high shear regions were in excellent agreement with the observed erosion sites in both location and the degree of erosion. Loss of the leading edge of the coil guide due to the erosion damage during the slurry mixing operation did not affect the erosion patterns on the tank floor. Calculations for a lower …

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We have previously shown that epimorphin, a protein expressed on the surface of myoepithelial and fibroblast cells of the mammary gland, acts as a multifunctional morphogen of mammary epithelial cells. Here, we present the molecular mechanism by which epimorphin mediates luminal morphogenesis. Treatment of cells with epimorphin to induce lumen formation greatly increases the overall expression of transcription factor CCAAT/enhancer binding protein beta (C/EBPbeta) and alters the relative expression of its two principal isoforms, LIP and LAP. These alterations were shown to be essential for the morphogenetic activities, as constitutive expression of LIP was sufficient to produce lumen formation, while constitutive expression of LAP blocked epimorphin-mediated luminal morphogenesis. Furthermore, in a transgenic mouse model in which epimorphin expression was expressed in an apolar fashion on the surface of mammary epithelial cells, we found increased expression of C/EBPbeta, increased relative expression of LIP to LAP, and enlarged ductal lumina. Together, our studies demonstrate a role for epimorphin in luminal morphogenesis through control of C/EBPbeta expression.

Confidence limits are common place in physics analysis. Great care must be taken in their calculation and use especially in cases of limited statistics. We introduce the concept of statistical errors of confidence limits and argue that not only should limits be calculated but also their errors in order to represent the results of the analysis to the fullest. We show that comparison of two different limits from two different experiments becomes easier when their errors are also quoted. Use of errors of confidence limits will lead to abatement of the debate on which method is best suited to calculate confidence limits.

It is important that air emissions from tritium systems be kept as low as reasonably achievable. Thus, over the years a number of gas detritiation systems have been developed. Recently there has been interest in lower-cost, simpler systems which do not convert HT to the much more hazardous HTO form. Examples of such systems are (1) a bubbler/dehumidifier, (2) a bubbler/collector, and (3) an adsorber/collector. A computer model of each configuration was written and run. Each system's performance, including tritium buildup in liquid water, and tritium exhausted to the environment, are presented and compared.

Plutonium, because of its radioactive nature, ages from the ''inside out'' by means of self-irradiation damage and thus produces Frankel-type defects and defect clusters. The defects resulting from the residual lattice damage and helium in-growth could result in microstructural, electronic, and physical property changes. This paper presents volume, density, and electronic property change observed from both naturally and accelerated aged plutonium alloys. Accelerated alloys are plutonium alloys with a fraction of Pu-238 to accelerate the aging process by approximately 18 times the rate of unaged weapons-grade plutonium. After thirty-five equivalent years of aging on accelerated alloys, the samples have swelled in volume by approximately 0.1% and now exhibit a near linear volume increase due to helium in-growth. We will correlate the physical property changes to the electronic structure of plutonium observed by the resonant photoelectron spectroscopy (RESPES).

Leakage of CO2 from a hypothetical geologic storage reservoir along an idealized fault zone has been simulated, including transitions between supercritical, liquid, and gaseous CO2. We find strong non-isothermal effects due to boiling and Joule-Thomson cooling of expanding CO2. Leakage fluxes are limited by limitations in conductive heat transfer to the fault zone. The interplay between multiphase flow and heat transfer effects produces non-monotonic leakage behavior.

Using detailed simulation and analytical models, the exposure time is estimated for serial crystallography, where hydrated laser-aligned proteins are sprayed across a continuous synchrotron beam. The resolution of X-ray diffraction microscopy is limited by the maximum dose that can be delivered prior to sample damage. In the proposed Serial Crystallography method, the damage problem is addressed by distributing the total dose over many identical hydrated macromolecules running continuously in a single-file train across a continuous X-ray beam, and resolution is then limited only by the available fluxes of molecules and X-rays. Orientation of the diffracting molecules is achieved by laser alignment. We evaluate the incident X-ray fluence (energy/area) required to obtain a given resolution from (1) an analytical model, giving the count rate at the maximum scattering angle for a model protein, (2) explicit simulation of diffraction patterns for a GroEL-GroES protein complex, and (3) the frequency cut off of the transfer function following iterative solution of the phase problem, and reconstruction of a density map in the projection approximation. These calculations include counting shot noise and multiple starts of the phasing algorithm. The results indicate the number of proteins needed within the beam at any instant for a given …

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New techniques developed in connection with the NNLO corrections to the Higgs production rate at hadron colliders and some recent applications are reviewed.

Shock-induced twinning and martensitic transformation in BCC-based polycrystalline metals (Ta and U-6wt%Nb) have been observed and studied using transmission electron microscopy (TEM). The length-scale of domain thickness for both twin lamella and martensite phase is found to be smaller than 100 nm. While deformation twinning of {l_brace}112{r_brace}<111>-type is found in Ta when shock-deformed at 15 GPa, both twinning and martensitic transformation are found in Ta when shock-deformed at 45 GPa. Similar phenomena of nanoscale twinning and martensitic transformation are also found in U6Nb shock-deformed at 30 GPa. Since both deformation twinning and martensitic transformation occurred along the {l_brace}211{r_brace}{sub b} planes associated with high resolved shear stresses, it is suggested that both can be regarded as alternative paths for shear transformations to occur in shock-deformed BCC metals. Heterogeneous nucleation mechanisms for shock-induced twinning and martensitic transformation are proposed and discussed.

Initial dislocation structure in annealed high-purity Mo single crystals and deformation substructure in a crystal subjected to 1% compression have been examined and studied in order to investigate dislocation multiplication mechanisms in the early stages of plastic deformation. The initial dislocation density is in a range of 10{sup 6} {approx} 10{sup 7} cm{sup -2}, and the dislocation structure is found to contain many grown-in superjogs along dislocation lines. The dislocation density increases to a range of 10{sup 8} {approx} 10{sup 9} cm{sup -2}, and the average jog height is also found to increase after compressing for a total strain of 1%. It is proposed that the preexisting jogged screw dislocations can act as (multiple) dislocation multiplication sources when deformed under quasi-static conditions. Both the jog height and length of link segment (between jogs) can increase by stress-induced jog coalescence, which takes place via the lateral migration (drift) of superjogs driven by unbalanced line-tension partials acting on link segments of unequal lengths. Applied shear stress begins to push each link segment to precede dislocation multiplication when link length and jog height are greater than critical lengths. This dynamic dislocation multiplication source is subsequently verified by direct simulations of dislocation dynamics under …

We calculate centrifugal force for a short bunch in vacuum moving in a circular orbit and estimate the emittance growth of the beam in a bend due to this force. Many of the basic features of the coherent synchrotron radiation (CSR) of short bunches and its effect on beam dynamics in accelerators are now well established. The effect is usually described in terms of the longitudinal force, or wakefield, that causes the energy loss in the beam, and also redistributes the energy between the particles by accelerating the head and decelerating the tail of the bunch. Coherent radiation becomes most important for short bunches and high currents. More subtle features of CSR such as transition effect due to the entrance to and exit from the bend, CSR force in the undulator, and shielding due to the close metallic boundaries have been also studied. Much less is known about the transverse force in a short bunch moving on a circular orbit. The problem has been treated in several papers beginning from R. Talman's work, who pointed out that the centrifugal force of a rotating bunch can result in a noticeable tune shift of betatron oscillations. Later, an important correction to the …

Strained semiconductor thin films grown epitaxially on semiconductor substrates of different composition, such as Si{sub 1-x}Ge{sub x}/Si, are becoming increasingly important in modern microelectronic technologies. In this paper, we report a hierarchical computational approach for analysis of dislocation formation, glide motion, multiplication, and annihilation in Si{sub 1-x}Ge{sub x} epitaxial thin films on Si substrates. Specifically, a condition is developed for determining the critical film thickness with respect to misfit dislocation generation as a function of overall film composition, film compositional grading, and (compliant) substrate thickness. In addition, the kinetics of strain relaxation in the epitaxial film during growth or thermal annealing (including post-implantation annealing) is analyzed using a properly parameterized dislocation mean-field theoretical model, which describes plastic deformation dynamics due to threading dislocation propagation. The theoretical results for Si{sub 1-x}Ge{sub x} epitaxial thin films grown on Si (100) substrates are compared with experimental measurements and are used to discuss film growth and thermal processing protocols toward optimizing the mechanical response of the epitaxial film.

In HGHG FEL the optimum strength of dispersive section is determined by the maximum bunching that beam obtains after interaction with seed laser. In this paper we present a simple semi-analytic expression of the required dispersive section strength for a wide range of laser power, intrinsic energy spread and harmonic number.

Domain decomposed Monte Carlo codes, like other domain-decomposed codes, are difficult to debug. Domain decomposition is prone to error, and interactions between the domain decomposition code and the rest of the algorithm often produces subtle bugs. These bugs are particularly difficult to find in a Monte Carlo algorithm, in which the results have statistical noise. Variations in the results due to statistical noise can mask errors when comparing the results to other simulations or analytic results. If a code can get the same result on one domain as on many, debugging the whole code is easier. This reproducibility property is also desirable when comparing results done on different numbers of processors and domains. We describe how reproducibility, to machine precision, is obtained on different numbers of domains in an Implicit Monte Carlo photonics code.