Engineering Tocopherol Selectivity in alpha-TTP: A Combined In Vitro/In Silico Study

"We present a combined in vitro/in silico study to determine the molecular origin of the selectivity of a-tocopherol transfer" "protein (a-TTP) towards a-tocopherol. Molecular dynamics simulations combined to free energy perturbation calculations predict a binding free energy for a-tocopherol to a-TTP 8.26+2.13 kcal mol{1 lower than that of c-tocopherol. Our calculations show that c-tocopherol binds to a-TTP in a significantly distorted geometry as compared to that of the natural ligand. Variations in the hydration of the binding pocket and in the protein structure are found as well. We propose a mutation, A156L, which significantly modifies the selectivity properties of a-TTP towards the two tocopherols. In particular, our simulations predict that A156L binds preferentially to c-tocopherol, with striking structural similarities to the wild-type- a-tocopherol complex. The affinity properties are confirmed by differential scanning fluorimetry as well as in vitro competitive binding assays. Our data indicate that residue A156 is at a critical position for determination of the selectivity of a-TTP. The engineering of TTP mutants with modulating binding properties can have potential impact at industrial level for easier purification of single tocopherols from vitamin E mixtures coming from natural oils or synthetic processes. Moreover," "the identification of a c-tocopherol selective TTP offers the possibility to challenge the hypotheses for the evolutionary development of a mechanism for a-tocopherol selection in omnivorous animals."

Enabling the evolution of J2EE applications through reverse engineering and quality assurance

Enterprise Applications are complex software systems that manipulate much persistent data and interact with the user through a vast and complex user interface. In particular applications written for the Java 2 Platform, Enterprise Edition (J2EE) are composed using various technologies such as Enterprise Java Beans (EJB) or Java Server Pages (JSP) that in turn rely on languages other than Java, such as XML or SQL. In this heterogeneous context applying existing reverse engineering and quality assurance techniques developed for object-oriented systems is not enough. Because those techniques have been created to measure quality or provide information about one aspect of J2EE applications, they cannot properly measure the quality of the entire system. We intend to devise techniques and metrics to measure quality in J2EE applications considering all their aspects and to aid their evolution. Using software visualization we also intend to inspect to structure of J2EE applications and all other aspects that can be investigate through this technique. In order to do that we also need to create a unified meta-model including all elements composing a J2EE application.

Assessment of Transverse Isotropy in Clinical-Level CT Images of Trabecular Bone Using the Gradient Structure Tensor

The aim of this study was to develop a GST-based methodology for accurately measuring the degree of transverse isotropy in trabecular bone. Using femoral sub-regions scanned in high-resolution peripheral QCT (HR-pQCT) and clinical-level-resolution QCT, trabecular orientation was evaluated using the mean intercept length (MIL) and the gradient structure tensor (GST) on the HR-pQCT and QCT data, respectively. The influence of local degree of transverse isotropy (DTI) and bone mineral density (BMD) was incorporated into the investigation. In addition, a power based model was derived, rendering a 1:1 relationship between GST and MIL eigenvalues. A specific DTI threshold (DTI thres) was found for each investigated size of region of interest (ROI), above which the estimate of major trabecular direction of the GST deviated no more than 30° from the gold standard MIL in 95% of the remaining ROIs (mean error: 16°). An inverse relationship between ROI size and DTI thres was found for discrete ranges of BMD. A novel methodology has been developed, where transversal isotropic measures of trabecular bone can be obtained from clinical QCT images for a given ROI size, DTI thres and power coefficient. Including DTI may improve future clinical QCT finite-element predictions of bone strength and diagnoses of bone disease.

Fluid-structure interaction of an elastic pipe immersed inside a coaxial rigid pipe: a model for the Tullio phenomenon

An axisymmetric, elastic pipe is filled with an incompressible fluid and is immersed in a second, coaxial rigid pipe which contains the same fluid. A pressure pulse in the outer fluid annulus deforms the elastic pipe which invokes a fluid motion in the fluid core. It is the aim of this study to investigate streaming phenomena in the core which may originate from such a fluid-structure interaction. This work presents a numerical solver for such a configuration. It was developed in the OpenFOAM software environment and is based on the Arbitrary Lagrangian Eulerian (ALE) approach for moving meshes. The solver features a monolithic integration of the one-dimensional, coupled system between the elastic structure and the outer fluid annulus into a dynamic boundary condition for the moving surface of the fluid core. Results indicate that our configuration may serve as a mechanical model of the Tullio Phenomenon (sound-induced vertigo).

Probing the Structure, Pseudorotation, and Radial Vibrations of Cyclopentane by Femtosecond Rotational Raman Coherence Spectroscopy

Femtosecond time-resolved Raman rotational coherence spectroscopy (RCS) is employed to determine accurate rotational, vibration–rotation coupling constants, and centrifugal distortion constants of cyclopentane (C⁵H¹⁰). Its lowest-frequency vibration is a pseudorotating ring deformation that interconverts 10 permutationally distinct but energetically degenerate “twist” minima interspersed by 10 “bent” conformers. While the individual twist and bent structures are polar asymmetric tops, the pseudorotation is fast on the time scale of external rotation, rendering cyclopentane a fluxionally nonpolar symmetric top molecule. The pseudorotational level pattern corresponds to a one-dimensional internal rotor with a pseudorotation constant Bps ≈ 2.8 cm⁻¹. The pseudorotational levels are significantly populated up to l = ± 13 at 298 K; <10% of the molecules are in the l = 0 level. The next-higher vibration is the “radial” ν²³ ring deformation mode at 273 cm⁻¹, which is far above the pseudorotational fundamental. Femtosecond Raman RCS measurements were performed in a gas cell at T = 293 K and in a pulsed supersonic jet at T ≈ 90 K. The jet cooling reduces the pseudorotational distribution to l < ±8 and eliminates the population of ν²³, allowing one to determine the rotational constant as A0 = B0 = 6484.930(11) MHz. This value is ∼300 times more precise than the previous value. The fit of the RCS transients reveals that the rotation–pseudorotation coupling constant αe,psB = −0.00070(1) MHz is diminutive, implying that excitation of the pseudorotation has virtually no effect on the B0 rotational constant of cyclopentane. The smallness of αe,psB can be realized when comparing to the vibration–rotation coupling constant of the ν²³ vibration, αe,23B = −9.547(1) MHz, which is about 10⁴ times larger.

Rotational constants and structure of para-difluorobenzene determined by femtosecond Raman coherence spectroscopy: A new transient type

Femtosecond Raman rotational coherence spectroscopy (RCS) detected by degenerate four-wave mixing is a background-free method that allows to determine accurate gas-phase rotational constants of non-polar molecules. Raman RCS has so far mostly been applied to the regular coherence patterns of symmetric-top molecules, while its application to nonpolar asymmetric tops has been hampered by the large number of RCS transient types, the resulting variability of the RCS patterns, and the 10³–10⁴ times larger computational effort to simulate and fit rotational Raman RCS transients. We present the rotational Raman RCS spectra of the nonpolar asymmetric top 1,4-difluorobenzene (para-difluorobenzene, p-DFB) measured in a pulsed Ar supersonic jet and in a gas cell over delay times up to ~2.5 ns. p-DFB exhibits rotational Raman transitions with ΔJ = 0, 1, 2 and ΔK = 0, 2, leading to the observation of J −, K −, A −, and C–type transients, as well as a novel transient (S–type) that has not been characterized so far. The jet and gas cell RCS measurements were fully analyzed and yield the ground-state (v = 0) rotational constants Aₒ = 5637.68(20) MHz, Bₒ = 1428.23(37) MHz, and Cₒ = 1138.90(48) MHz (1σ uncertainties). Combining the Aₒ, Bₒ, and Cₒ constants with coupled-cluster with single-, double- and perturbatively corrected triple-excitation calculations using large basis sets allows to determine the semi-experimental equilibrium bond lengths rₑ(C₁–C₂) = 1.3849(4) Å, rₑ(C₂–C³) = 1.3917(4) Å, rₑ(C–F) = 1.3422(3) Å, and rₑ(C₂–H₂) = 1.0791(5) Å.

Direct electrospinning of 3D auricle-shaped scaffolds for tissue engineering applications.

Thirty-two poly(ε)caprolactone (PCL) scaffolds have been produced by electrospinning directly into an auricle-shaped mould and seeded with articular chondrocytes harvested from bovine ankle joints. After seeding, the auricle shaped constructs were cultured in vitro and analysed at days 1, 7, 14 and 21 for regional differences in total DNA, glycosaminoglycan (GAG) and collagen (COL) content as well as the expression of aggrecan (AGG), collagen type I and type II (COL1/2) and matrix metalloproteinase 3 and 13 (MMP3/13). Stress-relaxation indentation testing was performed to investigate regional mechanical properties of the electrospun constructs. Electrospinning into a conductive mould yielded stable 3D constructs both initially and for the whole in vitro culture period, with an equilibrium modulus in the MPa range. Rapid cell proliferation and COL accumulation was observed until week 3. Quantitative real time PCR analysis showed an initial increase in AGG, no change in COL2, a persistent increase in COL1, and only a slight decrease initially for MMP3. Electrospinning of fibrous scaffolds directly into an auricle-shape represents a promising option for auricular tissue engineering, as it can reduce the steps needed to achieve an implantable structure.