Novel display of knotted DNA molecules by two-dimensional gel electrophoresis.

We describe a two-dimensional agarose gel electrophoresis procedure that improves the resolution of knotted DNA molecules. The first gel dimension is run at low voltage, and DNA knots migrate according to their compactness. The second gel dimension is run at high voltage, and DNA knots migrate according to other physical parameters such as shape and flexibility. In comparison with one-dimensional gel electrophoresis, this procedure segregates the knotted DNA molecules from other unknotted forms of DNA, and partially resolves populations of knots that have the same number of crossings. The two-dimensional display may allow quantitative and qualitative characterization of different types of DNA knots simply by gel velocity.

Mapping nanomechanical properties of live cells using multi-harmonic atomic force microscopy.

The nanomechanical properties of living cells, such as their surface elastic response and adhesion, have important roles in cellular processes such as morphogenesis, mechano-transduction, focal adhesion, motility, metastasis and drug delivery. Techniques based on quasi-static atomic force microscopy techniques can map these properties, but they lack the spatial and temporal resolution that is needed to observe many of the relevant details. Here, we present a dynamic atomic force microscopy method to map quantitatively the nanomechanical properties of live cells with a throughput (measured in pixels/minute) that is ∼10-1,000 times higher than that achieved with quasi-static atomic force microscopy techniques. The local properties of a cell are derived from the 0th, 1st and 2nd harmonic components of the Fourier spectrum of the AFM cantilevers interacting with the cell surface. Local stiffness, stiffness gradient and the viscoelastic dissipation of live Escherichia coli bacteria, rat fibroblasts and human red blood cells were all mapped in buffer solutions. Our method is compatible with commercial atomic force microscopes and could be used to analyse mechanical changes in tumours, cells and biofilm formation with sub-10 nm detail.

Circular minichromosomes become highly recombinogenic in topoisomerase-deficient yeast cells.

In topoisomerase-deficient yeast cells, we have found that circular minichromosomes are present as broad distributions of multimeric forms, which consist of tandemly repeated copies of their monomeric sequences. This phenomenon selectively occurs in Deltatop1 cells, and is highly magnified in double mutant Deltatop1 top2-4 cells. No multimers are observed in single mutant top2-4 or Deltatop3 cells, or in Deltatop1 cells that express a plasmid-borne TOP1 gene. Interconversion among multimeric forms takes place rapidly in double mutant Deltatop1 top2-4 cells, and the multimeric distributions are readily reverted to the monomeric form when a plasmid-borne TOP1 gene is expressed from an inducible promoter. These observations are a new example of the interplay between DNA topology and genome stability, and suggest that the cell capacity to modulate DNA supercoiling is limited when DNA is organized in small topological domains. Yeast minichromosome multimerization provides an appropriate system in which to study mechanistic aspects of DNA recombination.

Dihydroxyprogesterone acetophenide 150 mg + estradiol enantate 10 mg as monthly injectable contraceptives.

A survey among users and health personnel participating in the Salvadorian Social Security Institute (ISSS) Family Planning Program revealed interest in including a monthly preparation for injection as a contraceptive method offered by this Institution. The formulation containing dihydroxyprogesterone acetophenide (DHPA) 150 mg + estradiol enantate (E2EN) 10 mg was chosen for conducting an open and prospective study of efficacy and tolerability. Between January 1992 and March 1994, 7054 women were treated with this product for a total of 60010 months. A sample composed of 4505 women treated at this Institution confirmed that average users are young, have one or two children, do not show a particular geographical distribution and choose the monthly injection instead of oral contraceptives as the first contraceptive method or for the puerperium. The study formulation showed a high efficacy (Pearl Index: 0.018) and tolerability (general withdrawal rate throughout the study: 27.09%). The most frequent adverse events included bleeding disorders, headache and mastalgia; their incidence decreased spontaneously from the sixth month (3.9%), reaching 0% after two years. Treatment was discontinued due to adverse events in 3.47% of women. No significant bodyweight or systolic and diastolic blood pressure alterations were observed. Based on these results, the monthly injectable contraceptive was included in the basic product list at ISSS.