Chromosomal localization and molecular characterization of 53 cosmid-derived bovine microsatellites

Gene mapping in cattle has progressed rapidly in recent years largely owing to the introduction of powerful genetic markers, such as the microsatellites, and through advances in physical mapping techniques such as synteny mapping and fluorescence in situ hybridization (FISH). Microsatellite markers are often not physically mapped because they are generally isolated from small insert plasmid libraries, which makes their chromosomal localization inefficient. In this report we describe the FISH mapping of a large group of cosmid-derived bovine microsatellite markers, as our contribution to the European mapping initiative, BovMap. One objective of BovMap is to develop a set of anchored loci for the cattle genome map.Two cosmid libraries were screened with probes corresponding to the (AC) _n microsatellite motif. Positive clones were mapped by FISH, and then a subset was further analyzed by sequencing the region flanking the microsatellite repeat. In total, 58 clones were hybridized with chromosomes and identified loci on 22 of the 31 different bovine chromosomes. Three clones contained satellite DNA. Two or more markers were placed on 12 chromosomes. Sequencing of the microsatellites and flanking regions was performed directly from 43 cosmids, as previously reported (Ferretti et al. Anim. Genet. 25, 209–214, 1994). Primers were developed for 39 markers and used to describe the polymorphism associated with the corresponding loci.     

A computational study of the hemodynamics after "edge-to-edge" mitral valve repair.

Edge-to-edge mitral valve repair consists in suturing the free edge of the leaflets to re-establish coaptation in prolapsing valves. The leaflets are frequently sutured at the middle and a double orifice valve is created. In order to study the hemodynamic implications, a parametric model of the left heart has been developed. Different valve areas and shapes have been investigated. Results show that the simplified Bernoulli formula provides a good estimation of the pressure drop and that the pressure drop may be predicted on the basis of the pre-operative geometric and hemodynamics data by means of customized models.     

A microaliquoting technique for precise histological annotation and optimization of cell content in frozen tissue specimens

Knowledge of the exact cell content of frozen tissue samples is of growing importance in genomic research. We developed a microaliquoting technique to measure and optimize the cell composition of frozen tumor specimens for molecular studies. Frozen samples of 31 mesothelioma cases were cut in alternating thin and thick sections. Thin sections were stained and evaluated visually. Thick sections, i.e., microaliquots, were annotated using bordering stained sections. A range of cellular heterogeneity was observed among and within samples. Precise annotation of samples was obtained by integration and compared to conventional single face and “front and back” section estimates of cell content. Front and back estimates were more highly correlated with block annotation by microaliquoting than were single face estimates. Both methods yielded discrepant estimates, however, and for some studies may not adequately account for the heterogeneity of mesothelioma or other malignancies with variable cellular composition. High yield and quality RNA was extracted from precision annotated, tumor-enriched subsamples prepared by combining individual microaliquots with the highest tumor cellularity estimates. Microaliquoting provides accurate cell content annotation and permits genomic analysis of enriched subpopulations of cells without fixation or amplification.     

A platform for high-throughput expression of recombinant human enzymes secreted by insect cells

Functional genomics and proteomics have been fields of intense investigation, since the disclosure of the sequence of the human genome. To contribute to the assignment of a physiological role to the vast number of coding genes with unknown function, we have undertaken a program to clone, express, purify and determine the catalytic activity of those enzymes predicted to enter the secretory pathway, focusing our efforts on human peptidases. Our strategy to promote high-throughput expression and purification of recombinant proteins secreted by insect cells relies on the expression of the target enzymes with their native leader sequences and on the carboxyl-terminal fusion with a poly-histidine tag. Growth of host cells were optimized in 24-well format to achieve highly paralleled culture conditions with production yields comparable to shake flask. The purification was performed by a robotic system in 96-well format using either magnetic beads or minicolumns. In a pilot study using reference peptidases and lipases, the high-throughput approach demonstrated to support the secretion in the insect cell medium of 85% of the sample enzymes. Of them, 66% have been proven to be catalytically active using fluorescent homogeneous assays in 384-well format compatible with the high-throughput screening criteria. The implications of these results are discussed in light of the application of this procedure to genomic-predicted peptidases.     

Hydration and distance dependence of intermolecular shearing between collagen molecules in a model microfibril

In vertebrates, collagen tissues are the main component responsible for force transmission. In spite of the physiological importance of these phenomena, force transmission mechanisms are still not fully understood, especially at smaller scales, including in particular collagen molecules and fibrils. Here we investigate the mechanism of molecular sliding between collagen molecules within a fibril, by shearing a central molecule in a hexagonally packed bundle mimicking the collagen microfibril environment, using varied lateral distance between the molecules in both dry and solvated conditions. In vacuum, the central molecule slides under a stick-slip mechanism that is due to the characteristic surface profile of collagen molecules, enhanced by the breaking and reformation of H-bonds between neighboring collagen molecules. This mechanism is consistently observed for varied lateral separations between molecules. The high shearing force (>7 nN) found for the experimentally observed intermolecular distance (≈1.1 nm) suggests that in dry samples the fibril elongation mechanism relies almost exclusively on molecular stretching, which may explain the higher stiffnesses found in dry fibrils. When hydrated, the slip-stick behavior is observed only below 1.3 nm of lateral distance, whereas above 1.3 nm the molecule shears smoothly, showing that the water layer has a strong lubricating effect. Moreover, the average force required to shear is approximately the same in solvated as in dry conditions (≈2.5 nN), which suggests that the role of water at the intermolecular level includes the transfer of load between molecules.     

Mechanical properties of physiological and pathological models of collagen peptides investigated via steered molecular dynamics simulations

In this work we used molecular simulations to investigate the elastic properties of collagen single chain and triple helix with the aim of understanding its features starting from first principles. We analysed ideal collagen peptides, homotrimeric and heterotrimeric collagen type I and pathological models of collagen. Triple helices were found much more rigid than single chains, thus enlightening the important role of interchain stabilizing forces, like hydrophobic interaction and hydrogen bonds. We obtained Young's moduli close to 4.5GPa for the ideal model of collagen and for the physiological heterotrimer, while the physiological homotrimer presented a Young's modulus of 2.51GPa, that can be related to a mild form of Osteogenesis Imperfecta in which only the homotrimeric form of collagen type I is produced. Otherwise, the pathological model (presenting a glycine to alanine substitution) showed an elastic modulus of 4.32GPa, thus only slightly lower than the ideal model. This suggests that this mutation only slightly affects the mechanical properties of the collagen molecule, but possibly acts on an higher scale, such as the packing of collagen fibrils.     

Ventricular motion during the ejection phase: a computational analysis.

In the present paper, the study of the ventricular motion during systole was addressed by means of a computational model of ventricular ejection. In particular, the implications of ventricular motion on blood acceleration and velocity measurements at the valvular plane (VP) were evaluated. An algorithm was developed to assess the force exchange between the ventricle and the surrounding tissue, i.e., the inflow and outflow vessels of the heart. The algorithm, based on the momentum equation for a transitory flowing system, was used in a fluid-structure model of the ventricle that includes the contractile behavior of the fibers and the viscous and inertial forces of the intraventricular fluid. The model calculates the ventricular center of mass motion, the VP motion, and intraventricular pressure gradients. Results indicate that the motion of the ventricle affects the noninvasive estimation of the transvalvular pressure gradient using Doppler ultrasound. The VP motion can lead to an underestimation equal to 12.4 +/- 6.6%.