Nucleation and Growth of Insulin Fibrils in Bulk Solution and at Hydrophobic Polystyrene Surfaces

AbstractA technique was developed for studying the nucleation and growth of fibrillar protein aggregates. Fourier transform infrared and attenuated total reflection spectroscopy were used to measure changes in the intermolecular β-sheet content of bovine pancreatic insulin in bulk solution and on model polystyrene (PS) surfaces at pH 1. The kinetics of β-sheet formation were shown to evolve in two stages. Combined Fourier transform infrared, dynamic light scattering, atomic force microscopy, and thioflavin-T fluorescence measurements confirmed that the first stage in the kinetics was related to the formation of nonfibrillar aggregates that have a radius of 13±1nm. The second stage was found to be associated with the growth of insulin fibrils. The β-sheet kinetics in this second stage were used to determine the nucleation and growth rates of fibrils over a range of temperatures between 60°C and 80°C. The nucleation and growth rates were shown to display Arrhenius kinetics, and the associated energy barriers were extracted for fibrils formed in bulk solution and at PS surfaces. These experiments showed that fibrils are nucleated more quickly in the presence of hydrophobic PS surfaces but that the corresponding fibril growth rates decrease. These observations are interpreted in terms of the differences in the attempt frequencies and energy barriers associated with the nucleation and growth of fibrils. They are also discussed in the context of differences in protein concentration, mobility, and conformational and colloidal stability that exist between insulin molecules in bulk solution and those that are localized at hydrophobic PS interfaces

Scattering of electromagnetic waves by many thin cylinders: theory and computational modeling

Electromagnetic (EM) wave scattering by many parallel infinite cylinders is studied asymptotically as a tends to 0, where a is the radius of the cylinders. It is assumed that the centres of the cylinders are distributed so that their numbers is determined by some positive function N(x). The function N(x) >= 0 is a given continuous function. An equation for the self-consistent (limiting) field is derived as a tends to 0. The cylinders are assumed perfectly conducting. Formula for the effective refraction coefficient of the new medium, obtained by embedding many thin cylinders into a given region, is derived. The numerical results presented demonstrate the validity of the proposed approach and its efficiency for solving the many-body scattering problems, as well as the possibility to create media with negative refraction coefficients.Comment: 21 pages, 13 figure

Quantifying fenbendazole and its metabolites in self-medicating wild red grouse Lagopus lagopus scoticus using an HPLC–MS–MS approach

On red grouse estates in the UK the nematode parasite Trichostrongylus tenuis is often controlled by application of grit medicated with the anthelmintic fenbendazole (FBZ). To date, assessment of the efficacy has been inhibited by the inability to quantify uptake of FBZ by the birds. We have developed a simple and sensitive HPLC–MS–MS method for detecting and quantifying FBZ and its metabolites from a 300 mg sample of red grouse liver. This method could be used to improve the efficacy of medicated grit treatment by allowing the identification of conditions and application methods that optimize the uptake of FBZ. With the necessary modifications, our method will also be applicable to other wildlife species where self-medication is used for parasite control

A model of the reflection distribution in the vacuum ultra violet region

A reflection model with three components, a specular spike, a specular lobe and a diffuse lobe is discussed. This model was successfully applied to describe reflection of xenon scintillation light (175 nm) by PTFE and other fluoropolymers and can be used for Monte Carlo simulation and analysis of scintillation detectors. The measured data favors a Trowbridge-Reitz distribution function of ellipsoidal micro-surfaces. The intensity of the coherent reflection increases with increasing angle of incidence, as expected, since the surface appears smoother at grazing angles. The total reflectance obtained for PTFE is about 70% for VUV light at normal incidence in vacuum and estimated to be up to 100% in contact with liquid xenon

First-principles study of stability and vibrational properties of tetragonal PbTiO_3

A first-principles study of the vibrational modes of PbTiO_3 in the ferroelectric tetragonal phase has been performed at all the main symmetry points of the Brillouin zone (BZ). The calculations use the local-density approximation and ultrasoft pseudopotentials with a plane-wave basis, and reproduce well the available experimental information on the modes at the Gamma point, including the LO-TO splittings. The work was motivated in part by a previously reported transition to an orthorhombic phase at low temperatures [(J. Kobayashi, Y. Uesu, and Y. Sakemi, Phys. Rev. B {\bf 28}, 3866 (1983)]. We show that a linear coupling of orthorhombic strain to one of the modes at Gamma plays a role in the discussion of the possibility of this phase transition. However, no mechanical instabilities (soft modes) are found, either at Gamma or at any of the other high-symmetry points of the BZ.Comment: 8 pages, two-column style with 3 postscript figures embedded. Uses REVTEX and epsf macros. Also available at http://www.physics.rutgers.edu/~dhv/preprints/index.html#ag_pbt

Semiclassical kinetic theory of electron spin relaxation in semiconductors

We develop a semiclassical kinetic theory for electron spin relaxation in semiconductors. Our approach accounts for elastic as well as inelastic scattering and treats Elliott-Yafet and motional-narrowing processes, such as D'yakonov-Perel' and variable g-factor processes, on an equal footing. Focusing on small spin polarizations and small momentum transfer scattering, we derive, starting from the full quantum kinetic equations, a Fokker-Planck equation for the electron spin polarization. We then construct, using a rigorous multiple time scale approach, a Bloch equation for the macroscopic ($\vec{k}$-averaged) spin polarization on the long time scale, where the spin polarization decays. Spin-conserving energy relaxation and diffusion, which occur on a fast time scale, after the initial spin polarization has been injected, are incorporated and shown to give rise to a weight function which defines the energy averages required for the calculation of the spin relaxation tensor in the Bloch equation. Our approach provides an intuitive way to conceptualize the dynamics of the spin polarization in terms of a ``test'' spin polarization which scatters off ``field'' particles (electrons, impurities, phonons). To illustrate our approach, we calculate for a quantum well the spin lifetime at temperatures and densities where electron-electron and electron-impurity scattering dominate. The spin lifetimes are non-monotonic functions of temperature and density. Our results show that at electron densities and temperatures, where the cross-over from the non-degenerate to the degenerate regime occurs, spin lifetimes are particularly long.Comment: 29 pages, 10 figures, final versio

Magnetic-field dependence of electron spin relaxation in n-type semiconductors

We present a theoretical investigation of the magnetic field dependence of the longitudinal ($T_1$) and transverse ($T_2$) spin relaxation times of conduction band electrons in n-type III-V semiconductors. In particular, we find that the interplay between the Dyakonov-Perel process and an additional spin relaxation channel, which originates from the electron wave vector dependence of the electron $g$-factor, yields a maximal $T_2$ at a finite magnetic field. We compare our results with existing experimental data on n-type GaAs and make specific additional predictions for the magnetic field dependence of electron spin lifetimes.Comment: accepted for publication in PRB, minor changes to previous manuscrip

Simultaneous determination of time-dependent coefficients and heat source

This article presents a numerical solution to the inverse problems of simultaneous determination of the time-dependent coefficients and the source term in the parabolic heat equation subject to overspecified conditions of integral type. The ill-posed problems are numerically discretized using the finite-difference method. The resulting system of nonlinear equations is solved numerically using the MATLAB toolbox routine lsqnonlin applied to minimizing the nonlinear Tikhonov regularization functional subject to simple physical bounds on the variables. Numerical examples are presented to illustrate the accuracy and stability of the solution

Identification of conditionally essential genes for Streptococcus suis infection in pigs

Streptococcus suis is a Gram-positive bacterium and zoonotic pathogen that causes meningitis and sepsis in pigs and humans. The aim of this study was to identify genes required for S. suis infection. We created Tn-Seq libraries in a virulent S. suis strain 10, which was used to inoculate pigs in an intrathecal experimental infection. Comparative analysis of the relative abundance of mutants recovered from different sites of infection (blood, cerebrospinal fluid, and meninges of the brain) identified 361 conditionally essential genes, i.e. required for infection, which is about 18% of the genome. The conditionally essential genes were primarily involved in metabolic and transport processes, regulation, ribosomal structure and biogenesis, transcription, and cell wall membrane and envelope biogenesis, stress defenses, and immune evasion. Directed mutants were created in a set of 10 genes of different genetic ontologies and their role was determined in ex vivo models. Mutants showed different levels of sensitivity to survival in whole blood, serum, cerebrospinal fluid, thermic shock, and stress conditions, as compared to the wild type. Additionally, the role of three selected mutants was validated in co-infection experiments in which pigs were infected with both wild type and isogenic mutant strains. The genetic determinants of infection identified in this work contribute to novel insights in S. suis pathogenesis and could serve as targets for novel vaccines or antimicrobial drugs

A Monolayer of Primary Colonic Epithelium Generated on a Scaffold with a Gradient of Stiffness for Drug Transport Studies

Animal models are frequently used for in vitro physiologic and drug transport studies of the colon, but there exists significant pressure to improve assay throughput as well as to achieve tighter control of experimental variables than can be achieved with animals. Thus, development of a primary in vitro colonic epithelium cultured as high resistance with transport protein expression and functional behavior similar to that of a native colonic would be of enormous value for pharmaceutical research. A collagen scaffold, in which the degree of collagen cross-linking was present as a gradient, was developed to support the proliferation of primary colonic cells. The gradient of cross-linking created a gradient in stiffness across the scaffold, enabling the scaffold to resist deformation by cells. mRNA expression and quantitative proteomic mass spectrometry of cells growing on these surfaces as a monolayer suggested that the transporters present were similar to those in vivo. Confluent monolayers acted as a barrier to small molecules so that drug transport studies were readily performed. Transport function was evaluated using atenolol (a substrate for passive paracellular transport), propranolol (a substrate for passive transcellular transport), rhodamine 123 (Rh123, a substrate for P-glycoprotein), and riboflavin (a substrate for solute carrier transporters). Atenolol was poorly transported with an apparent permeability (Papp) of < 5 × 10-7 cm s-1, while propranolol demonstrated a Papp of 9.69 × 10-6 cm s-1. Rh123 was transported in a luminal direction (Papp,efflux/Papp,influx = 7) and was blocked by verapamil, a known inhibitor of P-glycoprotein. Riboflavin was transported in a basal direction, and saturation of the transporter was observed at high riboflavin concentrations as occurs in vivo. It is anticipated that this platform of primary colonic epithelium will find utility in drug development and physiological studies, since the tissue possesses high integrity and active transporters and metabolism similar to that in vivo