Solution structure of the 2-amino-1- methyl-6-phenylimidazo[4,5-b]pyridine C8-deoxyguanosine adduct in duplex DNA

The carcinogenic heterocyclic amine (HA) 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is formed during the cooking of various meats. To enable structure/activity studies aimed at understanding how DNA damaged by a member of the HA class of compounds can ultimately lead to cancer, we have determined the first solution structure of an 11-mer duplex containing the C8-dG adduct formed by reaction with N-acetoxy-PhIP. A slow conformational exchange is observed in which the PhIP ligand either intercalates into the DNA helix by denaturing and displacing the modified base pair (main form) or is located outside the helix in a minimally perturbed B-DNA duplex (minor form). In the main base-displaced intercalation structure, the minor groove is widened, and the major groove is compressed at the lesion site because of the location of the bulky PhIP-N-methyl and phenyl ring in the minor groove; this distortion causes significant bending of the helix. The PhIP phenyl ring interacts with the phosphodiester-sugar ring backbone of the complementary strand and its fast rotation with respect to the intercalated imidazopyridine ring causes substantial distortions at this site, such as unwinding and bulging-out of the strand. The glycosidic torsion angle of the [PhIP]dG residue is syn, and the displaced guanine base is directed toward the 3′ end of the modified strand. This study contributes, to our knowledge, the first structural information on the biologically relevant HA class to a growing body of knowledge about how conformational similarities and differences for a variety of types of lesions can influence protein interactions and ultimately biological outcome.

Linker histones affect patterns of digestion of supercoiled plasmids by single-strand-specific nucleases.

The effect of histone H1 binding on the cleavage of superhelical plasmids by single-strand-specific nucleases was investigated. Mapping of P1 cleavage sites in pBR322, achieved by EcoRI digestion after the original P1 attack, showed an intriguing phenomenon: preexisting susceptible sites became "protected," whereas some new sites appeared at high levels of H1. Similar results were obtained with another single-strand-specific nuclease, S1. Disappearance of cutting at preexisting sites and appearance of new sites was also observed in a derivative plasmid that contains a 36-bp stretch of alternating d(AT) sequence that is known to adopt an altered P1-sensitive conformation. On the other hand, H1 titration of a dimerized version of the d(AT)18-containing plasmid led to protection of all preexisting sites except the d(AT)18 inserts, which were still cut even at high H1 levels; in this plasmid no new sites appeared. The protection of preexisting sites is best explained by long-range effects of histone H1 binding on the superhelical torsion of the plasmid. The appearance of new sites, on the other hand, probably also involves a local effect of stabilization of specific sequences in Pl-sensitive conformation, due to direct H1 binding to such sequences. That such binding involves linker histone N- and/or C-terminal tails is indicated by the fact that titration with the globular domain of H5, while causing disappearance of preexisting sites, does not lead to the appearance of any new sites.

Complex flexibility of the transforming growth factor beta superfamily.

The transforming growth factors beta (TGF-beta s) are important modulators of growth and differentiation. They are intermolecular disulfide-bonded homodimeric molecules. The monomer fold has a conserved cystine knot and lacks a hydrophobic core. The biological specificity of a given member of the family is believed to be determined by the conformational flexibility of the variable loop regions of the monomer. The monomer subunit assembly in the dimer is stabilized mainly by hydrophobic contacts and a few hydrogen bonds. Since these interactions are nondirectional, we examined subunit assemblies of TGF-beta by using conformational analysis. The different subunit assemblies in TGF-beta 2 dimer were characterized in terms of the intersubunit disulfide torsion. Our analyses show that the subunit assemblies fall into two states: the crystallographically observed gauche+conformation and the previously not reported gauche--conformation, both having almost identical interaction energies. Furthermore, there is significant flexibility in the subunit assembly within the gauche+ and the gauche- states of the disulfide bond. The monomer subunit assembly is independent of the variations about the loop regions. The variations in the loop regions, coupled with flexibility in the monomer assembly, lead to a complex flexibility in the dimer of the TGF-beta superfamily. For the TGF-beta superfamily, the cystine knot acts as a scaffold and complex flexibility provides for biological selectivity. Complex flexibility might provide an explanation for the diverse range of biological activities that these important molecules display.

Avaliação de métodos numéricos de análise linear de estabilidade para perfis de aço formados a frio.

Para o projeto de estruturas com perfis de aço formados a frio, é fundamental a compreensão dos fenômenos da instabilidade local e global, uma vez que estes apresentam alta esbeltez e baixa rigidez à torção. A determinação do carregamento crítico e a identificação do modo de instabilidade contribuem para o entendimento do comportamento dessas estruturas. Este trabalho avalia três metodologias para a análise linear de estabilidade de perfis de aço formados a frio isolados, com o objetivo de determinar os carregamentos críticos elásticos de bifurcação e os modos de instabilidade associados. Estritamente, analisa-se perfis de seção U enrijecido e Z enrijecido isolados, de diversos comprimentos e diferentes condições de vinculação e carregamento. Determinam-se os carregamentos críticos elásticos de bifurcação e os modos de instabilidade globais e locais por meio de: (i) análise com o Método das Faixas Finitas (MFF), através do uso do programa computacional CUFSM; (ii) análise com elementos finitos de barra baseados na Teoria Generalizada de Vigas (MEF-GBT), via uso do programa GBTUL; e (iii) análise com elementos finitos de casca (MEF-cascas) por meio do uso do programa ABAQUS. Algumas restrições e ressalvas com relação ao uso do MFF são apresentadas, assim como limitações da Teoria Generalizada de Viga e precauções a serem tomadas nos modelos de cascas. Analisa-se também a influência do grau de discretização da seção transversal. No entanto, não é feita avaliação em relação aos procedimentos normativos e tampouco análises não lineares, considerando as imperfeições geométricas iniciais, tensões residuais e o comportamento elastoplástico do material.

Determinação analítica da correção de hélice em um par engrenado devido ao efeito da flexo-torção.

O foco do presente trabalho é estudar a intensificação da pressão de contato entre os dentes de duas engrenagens cilíndricas de dentes retos, que ocorre por causa do deslocamento dos componentes de um redutor de velocidades. Essa intensificação ocorre inclusive em um redutor fabricado sem quaisquer erros de usinagem ou de montagem. A correção de hélice é uma usinagem realizada na superfície do dente da engrenagem que compensa os deslocamentos dos eixos e engrenagens, devido à flexão e à torção, e os deslocamentos dos dentes das engrenagens, devido à flexão e à pressão de contato. Foram estudados efeitos importantes para essa correção de hélice em um redutor de velocidades. Com isso, foi desenvolvido um modelo analítico que calcula a correção necessária para diminuir esse intensificador de pressão de contato em função dos deslocamentos citados acima. Esse modelo analítico foi comparado com um modelo analítico da literatura e com o software comercial RIKOR®. Os resultados de correção de hélice propostos são similares aos da literatura e do RIKOR® com exceção das bordas das engrenagens, aonde existem algumas divergências. Posteriormente, foi desenvolvido um modelo sólido de engrenagem com o perfil evolvente e com as correções de hélice calculadas analiticamente. Este modelo tridimensional (3D) foi elaborado usando os softwares SolidWorks® e Inventor® e simulado por elementos finitos por meio do software ANSYS®. Verificou-se que as três correções de hélice - a proposta, a da literatura e a do RIKOR® - realmente diminuem a intensificação de pressão de contato no flanco do dente da engrenagem. Por fim, foi possível visualizar que a correção proposta nesta dissertação é 3% mais efetiva que a proposta pela literatura e pelo RIKOR® para o caso analisado.

Cyclobutadiene automerization and rotation of ethylene: Energetics of the barriers by using Spin-polarized wave functions

Spin-projected spin polarized Møller–Plesset and spin polarized coupled clusters calculations have been made to estimate the cyclobutadiene automerization, the ethylene torsion barriers in their ground state, and the gap between the singlet and triplet states of ethylene. The results have been obtained optimizing the geometries at MP4 and/or CCSD levels, by an extensive Gaussian basis set. A comparative analysis with more complex calculations, up to MP5 and CCSDTQP, together with others from the literature, have also been made, showing the efficacy of using spin-polarized wave functions as a reference wave function for Møller–Plesset and coupled clusters calculations, in such problems.

Méthode Fanjul: dosage pondéral des bétons légers et lourds

La détermination directe des caractéristiques de cisaillement des bétons par des essais universels est un problème qui reste encore une préoccupation majeure des chercheurs. La plupart des études disponibles consistent à appliquer un couple de torsion à un cylindre creux ayant un rapport épaisseur/rayon le plus faible possible. Ceci pour pouvoir élaborer un critère lié au comportement du matériau et non de la structure (Fourd et al. 1982). Cependant, ces essais peuvent présenter des dispersions liées aux problèmes de fragilité à la rupture ou de non homogénéité du matériau au sein de la paroi (Gotuwka et al. 1999). Pour cela, un dispositif expérimental original a été utilisé et qui consiste en la création de deux zones de contrainte nulle par l’emplacement de corps déformables au moment du coulage de l’éprouvette. De nouvelles conditions aux frontières sont créées permettant de transformer la sollicitation de compression en cisaillement plan sur une zone bien déterminée. L’utilisation de cette technique permet la détermination de l’influence de l’inclinaison du plan de rupture sur le comportement du béton en cisaillement. Le dispositif permet d’assurer la perpendicularité NOvEMBRE 2012 5 des génératrices du cylindre ainsi que la répartition et l’homogénéité des contraintes.

Intramolecular surfaces for vicinal proton–proton coupling constants 3JHH

Equations for the intramolecular surfaces of the 3JHH coupling constants in ethane, ethylene, and acetylene are formulated, and the corresponding coefficients are estimated from calculations at the DFT/B3LYP level. The chosen variables are changes in bond lengths, in the torsion angle φ between the coupled protons Ha and Hb, in bond angles, and in dihedral angles. The 3JHH surface of ethane is formulated as an extended Karplus equation with the coefficients of a truncated Fourier series on the torsion angle φ expanded as second-order Taylor series in the chosen variables taking into account the invariance of 3JHH under reflections and rotations of nuclear coordinates. Partial vibrational contributions from linear and square terms corresponding to changes in the geometry of the Ha − Ca − Cb − Hb fragment are important while those from cross terms are small with a few exceptions. The 3JHH surface of ethane is useful to predict contributions to 3JHH from changes in local geometry of derivatives but vibrational contributions are predicted less satisfactorily. The predicted values at the B3LYP/BS2 level of the 3JHH couplings (vibrational contributions at 300 K) from equilibrium geometries are 9.79 (−0.17) for acetylene, and 17.08 (1.93) and 10.73(0.93) for the trans and cis couplings of ethylene.

Genetically engineered silk and genipin-enhanced fibrin hydrogel for annulus fibrosus repair

INTRODUCTION: Around 80% of people are affected by low back pain at least once in their life, often caused by trauma provoking intervertebral disc (IVD) herniation and/or IVD degeneration. Apart from some promising approaches for nucleus pulposus repair, so far no treatment or repair is available for the outer fibrous tissue, annulus fibrosus (AF). We aimed for sealing and repairing an AF injury in a bovine IVD organ culture model in vitro over 14 days under different loading conditions. For this purpose, a silk fleece composite from Bombyx mori silk was combined with genipin-enhanced fibrin hydrogel [1]. METHODS: Bovine IVDs of 12-17 months old animals were isolated by first removing all surrounding tissue, followed by cutting out the IVDs [2]. Culturing of discs occurred in high glucose Dulbecco's Modified Eagle Medium (HG-DMEM) supplemented with 5% serum as previously described. On the next day, injury was induced using a 2mm biopsy punch (Polymed, Switzerland). The formed cavity was filled with (0.4%) genipin-enhanced human based fibrin hydrogel (35- 55mg/mL human fibrinogen, Baxter, Austria) and sealed with a silk fleece-membrane composite (Spintec Engineering, Germany). Different culture conditions were applied: free swelling, static diurnal load of 0.2MPa for 8h/d and complex loading at 0.2MPa compression combined with ± 2° torsion at 0.2Hz for 8h/d. Complex loading was applied by a custom built 2 degree of freedom bioreactor [3]. After 14 days of culture cell activity was determined with resazurin assay. Additionally, glycosaminoglycan (dimethyl-methylene blue), DNA (Hoechst) and collagen content (hydroxy-proline) were determined. Finally, real-time qPCR of major IVD marker genes was performed. RESULTS: The silk seal closing the injury site could successfully withstand the forces of all three loading conditions with no misplacement over the two weeks’ culture. Nevertheless, disc height of the repaired discs did not significantly differ from the injured group. The disc phenotype could be maintained as demonstrated by biochemical analysis of gene expression, cell activity, DNA-, collagen- and GAG content. The silk itself was evaluated to be highly biocompatible for hMSC, as revealed by cytotoxicity assays. DISCUSSION & CONCLUSIONS: The silk can be considered a highly-elastic and biocompatible material for AF closure and the genipin-enhanced fibrin hydrogel has also good biomechanical properties. However, the cyto-compatibility of genipin seems rather poor and other hydrogels and/or cross-linkers should be looked into. REFERENCES: 1 C.C. Guterl et al. (2014) Characterization of Mechanics and Cytocompatibility of Fibrin Genipin Annulus Fibrosus Sealant with the Addition of Cell Adhesion Molecules, Tissue Eng Part A 2 S.C. Chan, B. Gantenbein-Ritter (2012) Preparation of intact bovine tail intervertebral discs for organ culture, J Vis Exp 3 B Gantenbein et al. (2015) Organ Culture Bioreactors - Platforms to Study Human Intervertebral Disc Degeneration and Regenerative Therapy, Curr Stem Cell Res Ther [epub ahead of print] ACKNOWLEDGEMENTS: This project is supported by the Gebert Rüf Stiftung project # GRS-028/13.

Practical solution of torsional vibration problems, with examples from marine, electrical, aeronautical, and automobile engineering practice,

"Printed in Great Britain."

Computational aspects of modular parametrizations of elliptic curves

Thesis (Ph.D.)--University of Washington, 2016-06

Transverse-Plane Ankle Rotation for Lower-Limb Amputees Using Two Novel Prostheses

Thesis (Ph.D.)--University of Washington, 2016-06

D-Tyrosine as a chiral precusor to potent inhibitors of human nonpancreatic secretory phospholipase A(2) (IIa) with antiinflammatory activity

Few reported inhibitors of secretory phospholipase A(2) enzymes inhibit the IIa human isoform (hnpsPLA(2)-IIa) noncovalently at submicromolar concentrations. Herein, the simple chiral precursor D-tyrosine was derivastised to give a series of potent new inhibitors of hnpsPLA(2)-IIa. A 2.2-Angstrom crystal structure shows an inhibitor bound in the active site of the enzyme, chelated to a Ca2+ ion through carboxylate and amide oxygen atoms, H bonded through an amide NH group to His48, with multiple hydrophobic contacts and a T-shaped aromatic-group-His6 interaction. Antiinflammatory activity is also demonstrated for two compounds administered orally to rats.

Disulfide folding pathways of cystine knot proteins: Tying the knot within the circular backbone of the cyclotides

The plant cyclotides are a fascinating family of circular proteins that contain a cyclic cystine knot motif. The knotted topology and cyclic nature of the cyclotides pose interesting questions about folding mechanisms and how the knotted arrangement of disulfide bonds is formed. In the current study we have examined the oxidative refolding and reductive unfolding of the prototypic cyclotide, kalata B1. A stable two-disulfide intermediate accumulated during oxidative refolding but not in reductive unfolding. Mass spectrometry and NMR spectroscopy were used to show that the intermediate contained a native-like structure with two native disulfide bonds topologically similar to the intermediate isolated for the related cystine knot protein EETI-II (LeNguyen, D., Heitz, A., Chiche, L., El Hajji, M., and Castro B. (1993) Protein Sci. 2, 165-174). However, the folding intermediate observed for kalata B1 is not the immediate precursor of the three-disulfide native peptide and does not accumulate in the reductive unfolding process, in contrast to the intermediate observed for EETI-II. These alternative pathways of linear and cyclic cystine knot proteins appear to be related to the constraints imposed by the cyclic backbone of kalata B1 and the different ring size of the cystine knot. The three-dimensional structure of a synthetic version of the two-disulfide intermediate of kalata B1 in which Ala residues replace the reduced Cys residues provides a structural insight into why the two-disulfide intermediate is a kinetic trap on the folding pathway.