bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of off stoichiometry on Raman scattering from epitaxial and polycrystalline HfNx (0.85≤x≤1.50) grown on MgO(001)
Authors: Stoehr M. , Seo H.-S. , Petrov I. , Greene J. E.
Published: August 2008

Group-IV HfNx transition-metal nitride layers, with 0.85≤x≤1.50, are grown on MgO(001) and analyzed by Raman spectroscopy. Samples with 0.85≤x≤1.20 are single-phase epitaxial NaCl-structure HfNx(001), while layers with higher N concentrations are two-phase mixtures containing N-rich inclusions. All samples exhibit both first- and second-order Raman scattering. The presence of first-order scattering indicates that Oh symmetry, a characteristic of the NaCl crystal structure, is broken even in stoichiometric HfN. As x decreases in understoichiometric HfNx samples, corresponding to a decrease in the number of valence electrons per unit cell from nine (x=1) to eight (x=0.75), the positions of the acoustic lines shift to higher frequencies. This provides an indirect probe of phonon anomalies in acoustic mode dispersion curves and signifies an increase in N vacancy concentration. The persistence of strong first-order acoustic scattering from overstoichiometric (x>1) samples indicates that adding more N does not substantially reduce the average defect density around Hf sites. The absence of a frequency shift in the acoustic modes of overstoichiometric HfNx, with 1.17≤x≤1.27, also shows that the local order around Hf sites does not change significantly, while the observed shift of the first-order optical modes to higher frequency with increasing x is characteristic of increasing lattice disorder adjacent to N sites. The Raman results, together with the nature of the variation in the HfNx lattice parameter, the electron carrier concentration, and the superconducting transition temperature as a function of x, can be explained by the presence of N vacancies for x<1, both N and Hf vacancies in stoichiometric HfN(001), and the combination of N vacancies and NHf antisite defects for x>1.
Implementation of a phase array diffuse optical tomographic imager
Authors: Rajan K. , Vijayakumar V. , Biswas Samir Kumar , Vasu R. M.
Published: August 2008

Diffuse optical tomography (DOT) using near-infrared (NIR) light is a promising tool for noninvasive imaging of deep tissue. This technique is capable of quantitative reconstructions of absorption coefficient inhomogeneities of tissue. The motivation for reconstructing the optical property variation is that it, and, in particular, the absorption coefficient variation, can be used to diagnose different metabolic and disease states of tissue. In DOT, like any other medical imaging modality, the aim is to produce a reconstruction with good spatial resolution and accuracy from noisy measurements. We study the performance of a phase array system for detection of optical inhomogeneities in tissue. The light transport through a tissue is diffusive in nature and can be modeled using diffusion equation if the optical parameters of the inhomogeneity are close to the optical properties of the background. The amplitude cancellation method that uses dual out-of-phase sources (phase array) can detect and locate small objects in turbid medium. The inverse problem is solved using model based iterative image reconstruction. Diffusion equation is solved using finite element method for providing the forward model for photon transport. The solution of the forward problem is used for computing the Jacobian and the simultaneous equation is solved using conjugate gradient search. The simulation studies have been carried out and the results show that a phase array system can resolve inhomogeneities with sizes of 5mm when the absorption coefficient of the inhomogeneity is twice that of the background tissue. To validate this result, a prototype model for performing a dual-source system has been developed. Experiments are carried out by inserting an inhomogeneity of high optical absorption coefficient in an otherwise homogeneous phantom while keeping the scattering coefficient same. The high frequency (100MHz) modulated dual out-of-phase laser source light is propagated through the phantom. The interference of these sources creates an amplitude null and a phase shift of 180° along a plane between the two sources with a homogeneous object. A solid resin phantom with inhomogeneities simulating the tumor is used in our experiment. The amplitude and phase changes are found to be disturbed by the presence of the inhomogeneity in the object. The experimental data (amplitude and the phase measured at the detector) are used for reconstruction. The results show that the method is able to detect multiple inhomogeneities with sizes of 4mm. The localization error for a 5 mm inhomogeneity is found to be approximately 1mm.
Performance of a Ge-microstrip imaging detector and polarimeter
Authors: Spillmann U. , Bräuning H. , Hess S. , Beyer H. , Stöhlker Th. , Dousse J.-Cl. , Protic D. , Krings T.
Published: August 2008

Using 98% linearly polarized radiation at the European Synchrotron Radiation Facility in Grenoble, the performance of a prototype two-dimensional microstrip Ge(i) detector for x-ray imaging and as a Compton polarimeter has been evaluated. Using the energy and position sensitivity of the detector, the ability to obtain a complete reconstruction of the Compton event has been demonstrated. The modulation coefficient of the polarimeter is in good agreement with the theoretical limit of a perfect detector.
Erosion mechanisms of monocrystalline silicon under a microparticle laden air jet
Authors: Li Q. L. , Wang J. , Huang C. Z.
Published: August 2008

Microabrasive air-jet machining is considered as a promising precision processing technology for silicon substrates. In this paper, the impressions produced on a monocrystalline silicon by the impacts of microsolid particles entrained by an air jet and the associated microscopic erosion mechanisms are presented and discussed. It is shown that the impressions can be classified into three categories, namely, craters, scratches, and microdents, of which two types of craters and two types of scratches can lead to large-scale fractures. Craters with cleavage fracture surfaces have been found to play an important role in the material removal process. In addition, it is shown that most particles bounced away from the target surface without sliding or rolling during an impact so that most impressions formed are crater-type erosions.
Optical pump-probe measurements of sound velocity and thermal conductivity of hydrogenated amorphous carbon films
Authors: Arlein J. L. , Palaich S. E. M. , Daly B. C. , Subramonium P. , Antonelli G. A.
Published: August 2008

In this paper, we report the measurements of the thermal conductivity and longitudinal sound velocity of four types of hydrogenated amorphous carbon films. The measurements were made with an ultrafast optical pump-probe apparatus. The films were deposited at various temperatures by plasma enhanced chemical vapor deposition. The films are of a low density (1.0–1.4 g cm−3) and are found to have sound velocities that range from as low as 3 km/s to as high as 10 km/s. The thermal conductivities are between 0.6 and 1.4 W/mK which are low compared to amorphous SiO2 but are larger than published measurements of thin films of similar composition and density by a factor of 2–3.
Experimental investigation of a metamaterial omnidirectional reflector
Authors: Aközbek N. , Bloemer M. J. , Scalora M.
Published: August 2008

We demonstrate experimentally the existence of an omnidirectional reflection band of a two-dimensional metamaterial structure consisting of split ring resonators and thin conducting wires. Experimental angular reflectance data agree reasonably well with simulations using model dispersion relations for the effective dielectric permittivity and magnetic permeability that were fitted to the experimentally retrieved material parameters. The tunability of the reflection bandwidth is demonstrated by changing the effective electric plasma frequency. These findings are also relevant to optical frequencies where similar metamaterial composites have been fabricated.
Graphitization of nanocrystalline carbon microcoils synthesized by catalytic chemical vapor deposition
Authors: Bi Hui , Kou Kai-Chang , Ostrikov Kostya (Ken) , Zhang Jiao-Qiang
Published: August 2008

Graphitization, a common process involving the transformation of metastable nongraphitic carbon into graphite is one of the major present-day challenges for micro- and nanocarbons due to their unique structural character and highly unusual thermal activation. Here we report on the successful graphitization of nanocrystalline carbon microcoils prepared by catalytic chemical vapor deposition and post-treated in argon atmosphere at temperatures ∼2500 °C for 2 h. The morphology, microstructure, and thermal properties of the carbon microcoils are examined in detail. The graphitization mechanism is discussed by invoking a model of structural transformation of the carbon microcoils. The results reveal that after graphitization the carbon microcoils are prominently purified and feature a clear helical morphology, as well as a more regular and ordered microstructure. The interlayer spacing of the carbon microcoils decreases from 0.36 to 0.34 nm, whereas the mean crystal sizes in the c- and a-directions increase from 1.64 to 2.04 nm and from 3.86 to 7.21 nm, respectively. Thermal treatment also substantially improves the antioxidation properties of the microcoils by lifting the oxidation onset temperature from 550 to 672 °C. This process may be suitable for other nongraphitic micro- and nanomaterials.
Orientation dependent photoluminescent properties of chemical solution derived Bi4−xEuxTi3O12 ferroelectric thin films
Authors: Ruan Kaibin , Gao Aimei , Deng Wenli , Chen Xinman , Bao Dinghua
Published: August 2008

Bi 4 − x Eu x Ti 3 O 12 (BEuT) ferroelectric thin films were prepared on SrTiO3 (STO) substrates with different orientations by using chemical solution deposition. The effects of Eu ion concentration and crystallographic orientation on the photoluminescent property of the thin films were investigated. An unusual concentration quenching effect for photoluminescence with the critical value of x=0.55 for Eu3+ ion concentration was found for the thin films on STO (100) substrates. In addition, a strong orientation dependent photoluminescence was observed. Different from the orientation dependence of ferroelectric properties of rare earth doped bismuth titanate thin films, the c-axis oriented BEuT thin films on STO (100) substrates exhibited stronger photoluminescence than the randomly oriented thin films on STO (111) substrates. The orientation dependent photoluminescence was ascribed to low light scattering of well-aligned grains with c-axis oriented growth.
Design of inexpensive diffraction limited focal plane arrays for millimeter wavelength and terahertz radiation using glow discharge detector pixels
Authors: Abramovich A. , Kopeika N. S. , Rozban D.
Published: August 2008

Development of focal plane arrays (FPAs) for millimeter wavelength and terahertz radiation is presented in this paper. FPA is based on an inexpensive glow discharge detector (GDD) that serves as a pixel in the FPA. It was shown in previous investigations [A. Abramovich et al., Appl. Opt. 46, 7207 (2007)] that those inexpensive neon indicator lamp GDDs are quite sensitive to millimeter wavelength and terahertz radiation. The diameter of the GDD lamp is 6 mm and thus the FPA can be diffraction limited. Development of a FPA using such devices as detectors is advantageous since the cost of such lamps is around $0.2–0.5 per lamp, and it also serves as a room temperature detector. Experimental results at 100 GHz show that the responsivity of the terahertz FPA 4×4 GDD pixel is three times better than in previous measurements of A. Abramovich et al. [Appl. Opt. 46, 7207 (2007)].The addition of a parabolic reflector improves the accuracy of the noise equivalent power measurement which was found to be 6×10−9 W/√Hz for a 1 kHz modulation. However, it is expected to be considerably less at higher modulation frequencies because of much reduced noise.
Mechanism and kinetics of hydrated electron diffusion
Authors: Tay Kafui A. , Coudert François-Xavier , Boutin Anne
Published: August 2008

Molecular dynamics simulations are used to study the mechanism and kinetics of hydrated electron diffusion. The electron center of mass is found to exhibit Brownian-type behavior with a diffusion coefficient considerably greater than that of the solvent. As previously postulated by both experimental and theoretical works, the instantaneous response of the electron to the librational motions of surrounding water molecules constitutes the principal mode of motion. The diffusive mechanism can be understood within the traditional framework of transfer diffusion processes, where the diffusive step is akin to the exchange of an extramolecular electron between neighboring water molecules. This is a second-order process with a computed rate constant of 5.0ps−1 at 298K. In agreement with experiment the electron diffusion exhibits Arrhenius behavior over the temperature range of 298–400K. We compute an activation energy of 8.9kJmol−1. Through analysis of Arrhenius plots and the application of a simple random walk model it is demonstrated that the computed rate constant for exchange of an excess electron is indeed the phenomenological rate constant associated with the diffusive process.
A pressure cell for nonresonant inelastic x-ray scattering studies of gas phases
Authors: Minzer M. , Bradley J. A. , Musgrave R. , Seidler G. T. , Skilton A.
Published: August 2008

We report the design and performance of a gas-phase sample cell for measurements of momentum transfer (q) dependent nonresonant inelastic x-ray scattering (NRIXS). NRIXS measurements from He gas at 2 MPa (20 bars) readily demonstrate dipole-allowed and dipole-forbidden final states for two-electron excitations. Direct comparison of gas-phase NRIXS measurements with the corresponding nonresonant electron energy loss spectroscopy results (EELS) will be a valuable method for characterizing systematic errors in either technique for studies that require absolute normalization of the double differential cross section.
Photoelectron spectrum of valence anions of uracil and first-principles calculations of excess electron binding energies
Authors: Bachorz Rafał A. , Klopper Wim , Gutowski Maciej , Li Xiang , Bowen Kit H.
Published: August 2008

The photoelectron spectrum (PES) of the uracil anion is reported and discussed from the perspective of quantum chemical calculations of the vertical detachment energies (VDEs) of the anions of various tautomers of uracil. The PES peak maximum is found at an electron binding energy of 2.4 eV, and the width of the main feature suggests that the parent anions are in a valence rather than a dipole-bound state. The canonical tautomer as well as four tautomers that result from proton transfer from an NH group to a C atom were investigated computationally. At the Hartree–Fock and second-order Møller-Plesset perturbation theory levels, the adiabatic electron affinity (AEA) and the VDE have been converged to the limit of a complete basis set to within ±1 meV. Post-MP2 electron-correlation effects have been determined at the coupled-cluster level of theory including single, double, and noniterative triple excitations. The quantum chemical calculations suggest that the most stable valence anion of uracil is the anion of a tautomer that results from a proton transfer from N1H to C5. It is characterized by an AEA of 135 meV and a VDE of 1.38 eV. The peak maximum is as much as 1 eV larger, however, and the photoelectron intensity is only very weak at 1.38 eV. The PES does not lend support either to the valence anion of the canonical tautomer, which is the second most stable anion, and whose VDE is computed at about 0.60 eV. Agreement between the peak maximum and the computed VDE is only found for the third most stable tautomer, which shows an AEA of ≈−0.1 eV and a VDE of 2.58 eV. This tautomer results from a proton transfer from N3H to C5. The results illustrate that the characteristics of biomolecular anions are highly dependent on their tautomeric form. If indeed the third most stable anion is observed in the experiment, then it remains an open question why and how this species is formed under the given conditions.
Sharp growth of nickel plasticity under impact load near Curie point
Authors: Molotskii Michel
Published: August 2008

We proposed an explanation of the recent observations by Zaretsky [Appl. Phys. Lett. 92, 011913 (2008)] who discovered sharp increase in nickel plasticity under impact loading near Curie temperature TC. Growth of plasticity is explained by reduction in dynamical deceleration of dislocations controlled by their interaction with paramagnetic obstacles. The decreased contribution of these obstacles is due to spin-dependent transitions in internal magnetic field of ferromagnetic in radical pair dislocation-obstacle from strong to weak bonding states. The main contribution to the effect is from fields of magnetic fluctuations which have a large growth near Curie point.
Uracil DNA glycosylase uses DNA hopping and short-range sliding to trap extrahelical uracils
Authors: Porecha Rishi H. , Stivers James T.
Published: August 2008

The astonishingly efficient location and excision of damaged DNA bases by DNA repair glycosylases is an especially intriguing problem in biology. One example is the enzyme uracil DNA glycosylase (UNG), which captures and excises rare extrahelical uracil bases that have emerged from the DNA base stack by spontaneous base pair breathing motions. Here, we explore the efficiency and mechanism by which UNG executes intramolecular transfer and excision of two uracil sites embedded on the same or opposite DNA strands at increasing site spacings. The efficiency of intramolecular site transfer decreased from 41 to 0% as the base pair spacing between uracil sites on the same DNA strand increased from 20 to 800 bp. The mechanism of transfer is dominated by DNA hopping between landing sites of ≈10 bp size, over which rapid 1D scanning likely occurs. Consistent with DNA hopping, site transfer at 20- and 56-bp spacings was unaffected by whether the uracils were placed on the same or opposite strands. Thus, UNG uses hopping and 3D diffusion through bulk solution as the principal pathways for efficient patrolling of long genomic DNA sequences for damage. Short-range sliding over the range of a helical turn allows for redundant inspection of very local DNA sequences and trapping of spontaneously emerging extrahelical uracils.
X-ray scattering study of actin polymerization nuclei assembled by tandem W domains
Authors: Rebowski Grzegorz , Boczkowska Malgorzata , Hayes David B. , Guo Liang , Irving Thomas C. , Dominguez Roberto
Published: August 2008

The initiation of actin polymerization in cells requires actin filament nucleators. With the exception of formins, known filament nucleators use the Wiskott–Aldrich syndrome protein (WASP) homology 2 (WH2 or W) domain for interaction with actin. A common architecture, found in Spire, Cobl, VopL, and VopF, consists of tandem W domains that tie together three to four actin monomers to form a polymerization nucleus. Uncontrollable polymerization has prevented the structural investigation of such nuclei. We have engineered stable nuclei consisting of an actin dimer and a trimer stabilized by tandem W domain hybrid constructs and studied their structures in solution by x-ray scattering. We show that Spire-like tandem W domains stabilize a polymerization nucleus by lining up actin subunits along the long-pitch helix of the actin filament. Intersubunit contacts in the polymerization nucleus, thought to involve the DNase I-binding loop of actin, coexist with the binding of the W domain in the cleft between actin subdomains 1 and 3. The successful stabilization of filament-like multiactin assemblies opens the way to the crystallographic investigation of intersubunit contacts in the actin filament.
Using noise to probe and characterize gene circuits
Authors: Cox Chris D. , McCollum James M. , Allen Michael S. , Dar Roy D. , Simpson Michael L.
Published: August 2008

Stochastic fluctuations (or “noise”) in the single-cell populations of molecular species are shaped by the structure and biokinetic rates of the underlying gene circuit. The structure of the noise is summarized by its autocorrelation function. In this article, we introduce the noise regulatory vector as a generalized framework for making inferences concerning the structure and biokinetic rates of a gene circuit from its noise autocorrelation function. Although most previous studies have focused primarily on the magnitude component of the noise (given by the zero-lag autocorrelation function), our approach also considers the correlation component, which encodes additional information concerning the circuit. Theoretical analyses and simulations of various gene circuits show that the noise regulatory vector is characteristic of the composition of the circuit. Although a particular noise regulatory vector does not map uniquely to a single underlying circuit, it does suggest possible candidate circuits, while excluding others, thereby demonstrating the probative value of noise in gene circuit analysis.
A quantitative atlas of mitotic phosphorylation
Authors: Dephoure Noah , Zhou Chunshui , Villén Judit , Beausoleil Sean A. , Bakalarski Corey E. , Elledge Stephen J. , Gygi Steven P.
Published: August 2008

The eukaryotic cell division cycle is characterized by a sequence of orderly and highly regulated events resulting in the duplication and separation of all cellular material into two newly formed daughter cells. Protein phosphorylation by cyclin-dependent kinases (CDKs) drives this cycle. To gain further insight into how phosphorylation regulates the cell cycle, we sought to identify proteins whose phosphorylation is cell cycle regulated. Using stable isotope labeling along with a two-step strategy for phosphopeptide enrichment and high mass accuracy mass spectrometry, we examined protein phosphorylation in a human cell line arrested in the G
1
and mitotic phases of the cell cycle. We report the identification of >14,000 different phosphorylation events, more than half of which, to our knowledge, have not been described in the literature, along with relative quantitative data for the majority of these sites. We observed >1,000 proteins with increased phosphorylation in mitosis including many known cell cycle regulators. The majority of sites on regulated phosphopeptides lie in [S/T]P motifs, the minimum required sequence for CDKs, suggesting that many of the proteins may be CDK substrates. Analysis of non-proline site-containing phosphopeptides identified two unique motifs that suggest there are at least two undiscovered mitotic kinases.
Locations of the β1 transmembrane helices in the BK potassium channel
Authors: Liu Guoxia , Zakharov Sergey I. , Yang Lin , Wu Roland S. , Deng Shi-Xian , Landry Donald W. , Karlin Arthur , Marx Steven O.
Published: August 2008

BK channels are composed of α-subunits, which form a voltage- and Ca
2+
-gated potassium channel, and of modulatory β-subunits. The β1-subunit is expressed in smooth muscle, where it renders the BK channel sensitive to [Ca
2+
]
i
in a voltage range near the smooth-muscle resting potential and slows activation and deactivation. BK channel acts thereby as a damped feedback regulator of voltage-dependent Ca
2+
channels and of smooth muscle tone. We explored the contacts between α and β1 by determining the extent of endogenous disulfide bond formation between cysteines substituted just extracellular to the two β1 transmembrane (TM) helices, TM1 and TM2, and to the seven α TM helices, consisting of S1–S6, conserved in all voltage-dependent potassium channels, and the unique S0 helix, which we previously concluded was partly surrounded by S1–S4. We now find that the extracellular ends of β1 TM2 and α S0 are in contact and that β1 TM1 is close to both S1 and S2. The extracellular ends of TM1 and TM2 are not close to S3–S6. In almost all cases, cross-linking of TM2 to S0 or of TM1 to S1 or S2 shifted the conductance–voltage curves toward more positive potentials, slowed activation, and speeded deactivation, and in general favored the closed state. TM1 and TM2 are in position to contribute, in concert with the extracellular loop and the intracellular N- and C-terminal tails of β1, to the modulation of BK channel function.
Embedding CETLs in a performance‐oriented culture in higher education: Reflections on finding creative space
Authors: Clouder Lynn , Oliver Martin , Tait Jo
Published: August 2008

This article offers insight into the realization of the Higher Education Funding Council for England's vision for Centres for Excellence in Teaching and Learning (CETLs) as catalysts for change. Two CETLs, the Centre for Interprofessional e‐Learning (CIPeL) and the Surrey Centre for Excellence in Professional Training and Education (SCEPTrE), provide illustrative cases of two similar but different change initiatives. CETLs might be expected to embody and inspire creativity in teaching and learning yet they are embedded in institutions where activities are increasingly shaped by the discourse of performativity. Findings from the two case studies, which focus on similar schemes designed to promote staff involvement in creative projects, challenge the suggestion that creativity is stifled in a performative context.
Electron-impact ionization of atomic ions in the Na isoelectronic sequence
Authors: Altun Z , Yumak A , Golcek U , Loch S D , Pindzola M S , Griffin D C
Published: August 2008

Electron-impact ionization cross-sections and rate coefficients for the Na-like ions Mg+, Ar7+, Kr25+, Sn39+, Xe43+ and W63+ are presented. The direct ionization cross-sections of 2s, 2p and 3s subshells are calculated within a configuration-average distorted-wave (CADW) method. In addition, excitation–autoionization contributions originating from the inner-shell excitations of the type 2s22p63s→2s22p53snl (n=3–6) and 2s22p63s→2s2p63snl (n=3–6) are calculated using both CADW and level-resolved distorted-wave (LLDW) methods. We include the branching ratios for the radiation damping of the autoionizing configurations and levels. The excitation–autoionization contributions are found to be quite large for the lighter ions. For the heavier ions the radiation damping of the excitation–autoionization contribution is found to be significant. We find that the distorted-wave results are in good agreement with available experimental measurements, which only exist for Mg+, Ar7+ and Xe43+. We also find that the computationally simple configuration-average calculations are in reasonable agreement with the computationally demanding level-resolved calculations for the excitation–autoionization contributions for all the Na-like ions studied. In addition, Maxwellian rate coefficients are produced from our cross-sections and are archived in a format useful for plasma modelers.
Nonlinear quantum fluid equations for a finite temperature Fermi plasma
Authors: Eliasson Bengt , Shukla Padma K
Published: August 2008

Nonlinear quantum electron fluid equations are derived, taking into account the moments of the Wigner equation and by using the Fermi–Dirac equilibrium distribution for electrons with an arbitrary temperature. A simplified formalism with the assumptions of incompressibility of the distribution function is used to close the moments in velocity space. The nonlinear quantum diffraction effects into the fluid equations are incorporated. In the high-temperature limit, we retain the nonlinear fluid equations for a dense hot plasma and in the low-temperature limit, we retain the correct fluid equations for a fully degenerate plasma.
Addition of Fructooligosaccharides and Dried Plum to Soy‐Based Diets Reverses Bone Loss in the Ovariectomized Rat
Authors: Johnson Catherine D. , Lucas Edralin A. , Hooshmand Shirin , Campbell Sara , Akhter Mohammed P. , Arjmandi Bahram H.
Published: August 2008

Dietary bioactive components that play a role in improving skeletal health have received considerable attention in complementary and alternative medicine practices as a result of their increased efficacy to combat chronic diseases. The objectives of this study were to evaluate the additive or synergistic effects of dried plum and fructooligosaccharides (FOS) and to determine whether dried plum and FOS or their combination in a soy protein‐based diet can restore bone mass in ovarian hormone deficient rats. For this purpose, 72 3‐month‐old female Sprague‐Dawley rats were divided into six groups (n = 12) and either ovariectomized (Ovx, five groups) or sham‐operated (sham, one group). The rats were maintained on a semipurified standard diet for 45 days after surgery to establish bone loss. Thereafter, the rats were placed on one of the following dietary treatments for 60 days: casein‐based diet (Sham and Ovx), soy‐based diet (Ovx + soy) or soy‐based diet with dried plum (Ovx + soy + plum), FOS (Ovx + soy + FOS) and combination of dried plum and FOS (Ovx + soy + plum + FOS). Soy protein in combination with the test compounds significantly improved whole‐body bone mineral density (BMD). All test compounds in combination with soy protein significantly increased femoral BMD but the combination of soy protein, dried plum and FOS had the most pronounced effect in increasing lumbar BMD. Similarly, all of the test compounds increased ultimate load, indicating improved biomechanical properties. The positive effects of these test compounds on bone may be due to their ability to modulate bone resorption and formation, as shown by suppressed urinary deoxypyridinoline excretion and enhanced alkaline phosphatase activity.
PU.1 expression is modulated by the balance of functional sense and antisense RNAs regulated by a shared cis-regulatory element
Authors: Ebralidze Alexander K. , Guibal Florence C. , Steidl Ulrich , Zhang Pu , Lee Sanghoon , Bartholdy Boris , Jorda Meritxell Alberich , Petkova Victoria , Rosenbauer Frank , Huang Gang , Dayaram Tajhal , Klupp Johanna , O’Brien Karen B. , Will Britta , Hoogenkamp Maarten , Borden Katherine L.B. , Bonifer Constanze , Tenen Daniel G.
Published: August 2008

The transcription factor PU.1 is an important regulator of hematopoiesis; precise expression levels are critical for normal hematopoietic development and suppression of leukemia. We show here that noncoding antisense RNAs are important modulators of proper dosages of PU.1. Antisense and sense RNAs are regulated by shared evolutionarily conserved cis-regulatory elements, and we can show that antisense RNAs inhibit PU.1 expression by modulating mRNA translation. We propose that such antisense RNAs will likely be important in the regulation of many genes and may be the reason for the large number of overlapping complementary transcripts with so far unknown function.
On the origin of the β cell
Authors: Oliver-Krasinski Jennifer M. , Stoffers Doris A.
Published: August 2008

The major forms of diabetes are characterized by pancreatic islet β-cell dysfunction and decreased β-cell numbers, raising hope for cell replacement therapy. Although human islet transplantation is a cell-based therapy under clinical investigation for the treatment of type 1 diabetes, the limited availability of human cadaveric islets for transplantation will preclude its widespread therapeutic application. The result has been an intense focus on the development of alternate sources of β cells, such as through the guided differentiation of stem or precursor cell populations or the transdifferentiation of more plentiful mature cell populations. Realizing the potential for cell-based therapies, however, requires a thorough understanding of pancreas development and β-cell formation. Pancreas development is coordinated by a complex interplay of signaling pathways and transcription factors that determine early pancreatic specification as well as the later differentiation of exocrine and endocrine lineages. This review describes the current knowledge of these factors as they relate specifically to the emergence of endocrine β cells from pancreatic endoderm. Current therapeutic efforts to generate insulin-producing β-like cells from embryonic stem cells have already capitalized on recent advances in our understanding of the embryonic signals and transcription factors that dictate lineage specification and will most certainly be further enhanced by a continuing emphasis on the identification of novel factors and regulatory relationships.
Pluripotent stem cell lines
Authors: Yu Junying , Thomson James A.
Published: August 2008

The derivation of human embryonic stem cells 10 years ago ignited an explosion of public interest in stem cells, yet this achievement depended on prior decades of research on mouse embryonic carcinoma cells and embryonic stem cells. In turn, the recent derivation of mouse and human induced pluripotent stem cells depended on the prior studies on mouse and human embryonic stem cells. Both human embryonic stem cells and induced pluripotent stem cells can self-renew indefinitely in vitro while maintaining the ability to differentiate into advanced derivatives of all three germ layers, features very useful for understanding the differentiation and function of human tissues, for drug screen and toxicity testing, and for cellular transplantation therapies. Here we review the family of pluripotent cell lines derived from early embryos and from germ cells, and compare them with the more recently described induced pluripotent stem cells.
Structural and functional analysis of the Crb2–BRCT2 domain reveals distinct roles in checkpoint signaling and DNA damage repair
Authors: Kilkenny Mairi L. , Doré Andrew S. , Roe S. Mark , Nestoras Konstantinos , Ho Jenny C.Y. , Watts Felicity Z. , Pearl Laurence H.
Published: August 2008

Schizosaccharomyces pombe Crb2 is a checkpoint mediator required for the cellular response to DNA damage. Like human 53BP1 and Saccharomyces cerevisiae Rad9 it contains Tudor2 and BRCT2 domains. Crb2-Tudor2 domain interacts with methylated H4K20 and is required for recruitment to DNA dsDNA breaks. The BRCT2 domain is required for dimerization, but its precise role in DNA damage repair and checkpoint signaling is unclear. The crystal structure of the Crb2–BRCT2 domain, alone and in complex with a phosphorylated H2A.1 peptide, reveals the structural basis for dimerization and direct interaction with γ-H2A.1 in ionizing radiation-induced foci (IRIF). Mutational analysis in vitro confirms the functional role of key residues and allows the generation of mutants in which dimerization and phosphopeptide binding are separately disrupted. Phenotypic analysis of these in vivo reveals distinct roles in the DNA damage response. Dimerization mutants are genotoxin sensitive and defective in checkpoint signaling, Chk1 phosphorylation, and Crb2 IRIF formation, while phosphopeptide-binding mutants are only slightly sensitive to IR, have extended checkpoint delays, phosphorylate Chk1, and form Crb2 IRIF. However, disrupting phosphopeptide binding slows formation of ssDNA-binding protein (Rpa1/Rad11) foci and reduces levels of Rad22(Rad52) recombination foci, indicating a DNA repair defect.
The opossum genome: Insights and opportunities from an alternative mammal
Authors: Samollow Paul B.
Published: August 2008

The strategic importance of the genome sequence of the gray, short-tailed opossum,Monodelphis domestica, accrues from both the unique phylogenetic position of metatherian (marsupial) mammals and the fundamental biologic characteristics of metatherians that distinguish them from other mammalian species. Metatherian and eutherian (placental) mammals are more closely related to one another than to other vertebrate groups, and owing to this close relationship they share fundamentally similar genetic structures and molecular processes. However, during their long evolutionary separation these alternative mammals have developed distinctive anatomical, physiologic, and genetic features that hold tremendous potential for examining relationships between the molecular structures of mammalian genomes and the functional attributes of their components. Comparative analyses using the opossum genome have already provided a wealth of new evidence regarding the importance of noncoding elements in the evolution of mammalian genomes, the role of transposable elements in driving genomic innovation, and the relationships between recombination rate, nucleotide composition, and the genomic distributions of repetitive elements. The genome sequence is also beginning to enlarge our understanding of the evolution and function of the vertebrate immune system, and it provides an alternative model for investigating mechanisms of genomic imprinting. Equally important, availability of the genome sequence is fostering the development of new research tools for physical and functional genomic analyses ofM. domesticathat are expanding its versatility as an experimental system for a broad range of research applications in basic biology and biomedically oriented research.
Inoculation and Passaging of Mammalian Monolayer Cell Cultures
Authors: Darlington Gretchen J.
Published: August 2008

INTRODUCTION

Cells grown in monolayer proliferate to a confluent state in which the cells cover the growth surface of the flask. Some cells can be maintained in this plateau phase of growth for days to weeks, while others require trypsinization and subculture to survive. Cell lines requiring trypsinization usually do not display contact inhibition of growth and continue to proliferate. Ultimately, they peel off the surface of the flasks and are difficult to disperse and replate. This protocol describes a procedure for the subculture of mammalian cells in monolayer culture.
Quantifying Protein by Bicinchoninic Acid
Authors: Simpson Richard J.
Published: August 2008

INTRODUCTION

This protocol describes a method of quantifying protein that is a variation of the Lowry assay. It uses bicinchoninic acid (BCA) to enhance the detection of Cu+ generated under alkaline conditions at sites of complexes between Cu2+ and protein. The resulting chromophore absorbs at 562 nm. This technique is divided into three parts: Standard Procedure, Microprocedure, and 96-Well Microtiter Plate Procedure. For each procedure, test samples are assayed in parallel with protein standards that are used to generate a calibration curve, and the exact concentration of protein in the test samples is interpolated. The standard BCA assay uses large volumes of both reagents and samples and cannot easily be automated. If these issues are important, the Microprocedure is recommended. This in turn can be adapted for use with a microplate reader in the 96-Well Microtiter Plate Procedure. If the microplate reader is interfaced with a computer, more than 1000 samples can be read per hour.
Cryosectioning Tissues
Authors: Fischer Andrew H. , Jacobson Kenneth A. , Rose Jack , Zeller Rolf
Published: August 2008

INTRODUCTION

Cryosections are rapidly and relatively easily prepared prior to fixation, and they provide a good system for visualizing fine details of the cell. Although cryosections are physically less stable than paraffin- or resin-embedded sections, they are generally superior for the preservation of antigenicity and therefore the detection of antigens by microscopy. The preparation of cryosections does not involve the dehydration steps typical of other sectioning methods, and, furthermore, sectioning, labeling, and observation of specimens can usually be carried out in one day. In general, the sample is frozen quickly in either isopentane or liquid nitrogen. (Small samples such as cells and small tissues may be mixed in a slurry of an inert support medium such as optimal cutting temperature [OCT] compound before freezing). Rapid freezing reduces ice crystal formation and minimizes morphological damage. Frozen sections may be used for a variety of procedures, including immunochemistry, enzymatic detection, and in situ hybridization. A protocol for cryosectioning is presented here.
Design and Cloning of an shRNA into a Lentiviral Silencing Vector: Version A
Authors: Tiscornia Gustavo , Singer Oded , Verma Inder M.
Published: August 2008

INTRODUCTION

This protocol combines the specificity of small interfering RNA (siRNA)-mediated silencing cassettes with the versatility of lentiviral vectors to stably transduce a wide range of cell types. A short hairpin RNA (shRNA) designed against a given target is cloned into a plasmid containing the pol III promoter. The design uses a 5′ forward primer upstream of the pol III promoter and a 3′ reverse primer that includes the entire shRNA sequence (i.e., sense, loop, and antisense sequences followed by five Ts), followed by 22 bases complementary to the last 22 bp upstream of the +1 transcriptional start site of the pol III promoter. An NheI-compatible restriction site is included at the 5′ end of both forward and reverse primers. A single round of PCR is used to amplify this template. The resulting DNA fragment contains an shRNA expression cassette that can be cloned into a simple cloning vector, tested, and then transferred to the lentiviral vector, or cloned into the lentiviral vector directly. This procedure uses a unique restriction site in the 3′ long terminal repeat (LTR). During integration, the 5′ LTR of the provirus is copied from the 3′ LTR, cloning the H1-driven shRNA into the 3′ LTR, resulting in duplication of the silencing cassette. This strategy maximizes the silencing power of the lentiviral vector. The combination of the lentiviral and siRNA technologies provides a powerful tool to achieve long-term down-regulation of specific target genes both in vitro and in vivo.
Phage-Display Methodology for the Study of Protein-Protein Interactions: Stage 2, Panning: Figure 1.
Authors: Goodyear Carl S. , Silverman Gregg J.
Published: August 2008

INTRODUCTION

This method for creation of phage libraries can be used for the generation and/or engineering of variant protein domains. It was adapted in our laboratory for the development of variant domains of SpA (the virulence factor, protein A, produced by clinical isolates of Staphylococcus aureus) with novel Fab-binding specificities. In Stage 2 of the method, we describe the use of streptavidin-coated magnetic beads for library selection (panning). After selection, the elution strategy used to recover desirable clones should be chosen on the basis of the type of panning strategy employed, and the properties of the ligand that has been targeted. Of the several alternatives, we have compared the common recovery strategies of low pH, trypsinization of bound libraries, and competition with soluble antigen. When considering which option to employ, it is worth knowing how this may affect the type of binder that will be recovered. Low pH will dissociate most or all binders, whether they are specific or nonspecific for the selection antigen, whereas trypsin targets the fusion area between the protein and gene III, and specifically cleaves it. As a result, for the latter, only phage that bind via a protein-protein interaction will be recovered. In contrast, soluble antigen will provide specific binders of relatively high affinity because the elution will work in a competitive manner. We recommend an empiric approach to determine which is best for specific projects.
Phage-Display Methodology for the Study of Protein-Protein Interactions: Stage 3, Binding Analysis
Authors: Goodyear Carl S. , Silverman Gregg J.
Published: August 2008

INTRODUCTION

This method for creation of phage libraries can be used for the generation and/or engineering of variant protein domains. It was adapted in our laboratory for the development of variant domains of SpA (the virulence factor, protein A, produced by clinical isolates of Staphylococcus aureus) with novel Fab-binding specificities. In Stage 3 of the method, described here, the rounds of selection are analyzed to evaluate the enrichment for binders. To do this, polyethylene glycol (PEG)-precipitated phage from each round of panning are tested in an assay, be it ELISA-based or flow cytometric. The assay should include all rounds of phage samples, from the initial unselected phage to the last round and helper phage (as a negative control). The method chosen to evaluate whole phage libraries can also be used as a system of evaluating single clones from the appropriate round. The appropriate round is the one that yields a library exhibiting the best binding characteristics (or best signal in the detection assay) associated with the protein-protein interaction. This protocol outlines a basic ELISA-based analysis strategy.
Analyzing DNA Replication I: Labeling Animals, Tissues, and Cells with Bromodeoxyuridine (BrdU)
Authors: Jackson Dean , Cook Peter R.
Published: August 2008

INTRODUCTION

The number of cells traversing the cell cycle and the rate of progression through it provide important indices of cell growth and tumorigenicity. S-phase cells can also be identified by their high content of DNA polymerase and proliferating cell nuclear antigen, a component of the leading-strand polymerase. Although both these markers can be detected rapidly and conveniently using the appropriate antibodies, neither are found exclusively in S-phase cells. Immunolabeling after incorporation of modified DNA precursors (e.g., 5-bromodeoxyuridine [BrdU, bromodeoxyuridine]) allows more rapid and precise detection of cells in S-phase of the cell cycle. BrdU is phosphorylated by cells to give BrdUTP, and this precursor is incorporated into DNA instead of deoxythimidine triphosphate. In living cells, BrdU is incorporated into replication sites that can then be detected using fluorochrome or enzyme-coupled antibodies. Alternatively, DNA synthesis sites can be labeled at high resolution by incubating cells with analogs of the natural precursors of DNA. The cells are then fixed and the incorporation sites are detected using fluorochrome- or enzyme-tagged antibodies. Cells labeled in this way either in vivo or in vitro display a few hundred discrete nuclear sites early in S-phase, with distinct patterns of DNA replication that are characteristic of different stages of S-phase. “Foci” labeled after very short incubations correspond with sites where many replicons are duplicated simultaneously within massive protein complexes. This protocol provides details for incorporating DNA precursors into tissues in whole animals in vivo, isolated tissue in vitro, and cultured cells.
Whole Mount RNA In Situ Hybridization on Zebrafish Embryos: Probe Synthesis
Authors: Moens Cecilia
Published: August 2008

INTRODUCTION

This protocol describes methods for synthesizing digoxigenin- and fluorescein-labeled RNA probes from linearized plasmids or PCR products for use in whole-mount RNA in situ hybridization on zebrafish embryos.
Whole Mount RNA In Situ Hybridization on Zebrafish Embryos: Hybridization
Authors: Moens Cecilia
Published: August 2008

INTRODUCTION

This protocol describes procedures for detecting gene expression in zebrafish embryos in situ using labeled RNA probes. The labels (digoxigenin or fluorescein) are detected using alkaline phosphatase-conjugated antibodies that catalyze a chromogenic reaction, producing visible blue or red products. Two-color in situ hybridization can be performed by labeling with a combination of digoxigenin- and fluorescein-labeled probes and detecting the digoxigenin and fluorescein with sequential alkaline phosphatase reactions using different chromogenic substrates. In the method described here, the fluorescein-labeled probe is detected with the red substrate, but either probe can be detected with either substrate.