Generation of radiation-induced deletion complexes in the mouse genome using embryonic stem cells

As the genetic and physical mapping stage of the Human Genome Project nears completion, the focus is shifting toward the development of technologies for high-throughput analysis of gene function. Whereas DNA sequencing will enable the assignment of presumed function to a large number of genes in mice and humans, it is clear that the great majority of genes will have to be evaluated in vivo to accurately assess their role in a complex organism. While gene targeting in mouse embryonic stem (ES) cells is the current method of choice for the characterization of gene function in mice, it remains relatively labor intensive and lacks the throughput required for analysis of genome function on a large scale. Alternative methods of efficient mutagenesis will clearly be required for this task. Chromosomal deletions are powerful tools in the genetic analysis of complex genomes, enabling the systematic identification and localization of functional units along defined chromosomal regions. Not only are deletions useful for the identification of genetic functions, but they serve as mapping reagents for existing mutations or traits. While their use has been an essential tool in Drosophila genetics, classical mutagenesis in mice has been logistically impractical for generating deletions. We have previously described an efficient method for generating radiation-induced deletion complexes at defined regions in the genome using ES cells. In this article, we detail the methodological aspects of this technology and describe the applications of chromosomal deletions for characterizing gene function in ways that make optimal use of the information generated by the first stage of the Genome Project.     

Photorefractive polarization couplers in elliptical core fibres

Photorefractive polarization couplers written internally in germanium-doped elliptical core fibers at 488, 514, and 532 nm are reported. Complete power transfer between the orthogonal polarization modes of the fiber was achieved for couplers written at 514 and 488 nm, respectively. It is shown that the couplers are nonuniform in length because of the high photoinduced attenuation and also due to two-photon absorption. Polarization coupling of higher order modes is also demonstrated at shorter wavelengths where their polarization beat lengths match the polarization beat length of the fundamental mode at which the coupler was written.<>     

Distribution of transposable elements in neotropical species of Drosophila

The phylogenetic distribution of transposable families, P, gypsy, hobo, I, and mariner has been analyzed in 33 species of 11 groups of neotropical Drosophila and a Drosophilidae species Zygotrica vittimaculosa, using squash blot and dot blot. Genomic DNA of almost all neotropical species tested hybridized with gypsy probe and some species showed a particularly strong hybridization signal, as D. gaucha, D. virilis, and species of flavopilosa group. The hobo element was restricted to melanogaster group and some strains of D. willistoni. Only D. simulans DNA showed hybridization to mariner probe in all species tested and D. simulans and D. melanogaster showed hybridization with I element probe. P element homologous sequence was present in D. melanogaster and all species and strains of the willistoni and saltans groups tested. The presence of at least one P-homologous sequence was detected in Drosophila mediopunctata. This one was the only P-bearing species of all six tested from the tripunctata group. Four different pairs of primers homologous to segments of the canonical sequence of D. melanogaster's P were used to amplify specific sequences from D. mediopunctata DNA, showing the occurrence of seemingly well-conserved P-homologous sequences.     

On the Assumed Natural Strain method to alleviate locking in solid-shell NURBS-based finite elements

In isogeometric analysis (IGA), the functions used to describe the CAD geometry (such as NURBS) are also employed, in an isoparametric fashion, for the approximation of the unknown fields, leading to an exact geometry representation. Since the introduction of IGA, it has been shown that the high regularity properties of the employed functions lead in many cases to superior accuracy per degree of freedom with respect to standard FEM. However, as in Lagrangian elements, NURBS-based formulations can be negatively affected by the appearance of non-physical phenomena that “lock” the solution when constrained problems are considered. In order to alleviate such locking behaviors, the Assumed Natural Strain (ANS) method proposed for Lagrangian formulations is extended to NURBS-based elements in the present work, within the context of solid-shell formulations. The performance of the proposed methodology is assessed by means of a set of numerical examples. The results allow to conclude that the employment of the ANS method to quadratic NURBS-based elements successfully alleviates non-physical phenomena such as shear and membrane locking, significantly improving the element performance.     

Antoine Marfan and his syndrome: one hundred years later

A high-performance liquid chromatographic (HPLC) method is presented for the simultaneous detection of ubiquinone-9 and 10 in rat tissues such as blood, myocardium, and muscle. After liquid-liquid extraction, the ubiquinones are subsequently analyzed by HPLC with ultraviolet (UV) detection at their maximum absorbance (275 nm). Reference calibration curves in ethanol are used to determine tissular levels of ubiquinones. Because a treatment with HMG-CoA reductase inhibitors is expected to decrease the ubiquinone levels, reference calibration curves are performed to ensure that the ratios (ubiquinone/internal standard) observed in such an experiment could be evaluated directly on a calibration curve. The assay is sensitive (0.0625 microgram/mL), reproducible (4% coefficient of variation for ubiquinone-9 and 6% for ubiquinone-10), and linear up to 20 micrograms/mL (or 100 mg of tissue) for ubiquinone-9 and up to 10 micrograms/mL (or 100 mg of tissue) for ubiquinone-10. The ubiquinone levels in control tissues or blood are within the ranges of those previously reported.     

Virulence of enterohemorrhagic Escherichia coli: role of molecular crosstalk

Many bacterial exotoxins, originally defined by cytopathic effects, may also possess additional biological activities. The capacity of exotoxins to elicit the synthesis and secretion of pro- or anti-inflammatory cytokines may be as important as their direct toxic effects in pathogenesis. One example of such 'molecular crosstalk' occurs between Shiga toxins and the cytokines made in response to these toxins during the development of disease.