Strong genetic isolation despite wide distribution in a commercially exploited coastal shark

The common smoothhound, Mustelus mustelus, is an epibenthic species targeted by fisheries around the world driven by the increasing demand for shark products. Given the wide-spread occurrence of this species and corresponding lack of molecular data in many areas of said distribution, baseline molecular assessments of this commercially important shark may contribute to finer-scale analyses in areas in which this species is targeted. Therefore, population genetic analyses were conducted along the East Atlantic, from the Mediterranean Sea to the south-east coast of Africa, using microsatellite markers and the mitochondrial control region (mtCR). Overall, M. mustelus displayed low to moderate genetic diversity, with the Mediterranean populations appearing to exhibit the lowest mitochondrial diversity, and the west African populations displaying the lowest nuclear diversity. Microsatellite analysis indicated strong genetic differentiation between the three regions, with finer-scale population structure in each region, without correlation between genetic and geographical distance. For the mtCR sequences, a total of 18 haplotypes were identified, with a high degree of divergence discernable between the regions, largely in accordance with the microsatellite data. The study documents a remarkable level of population isolation across a vast area, suggesting little or no present-day connectivity among extant populations. The findings may serve as an essential baseline for global population management and commercial traceability of this threatened shark.

     

Cooperative transformation and coexpression of bovine papillomavirus type 1 E5 and E7 proteins

Productively infected bovine fibropapillomas were examined for bovine papillomavirus type 1 (BPV-1) E7 localization. BPV-1 E7 was observed in the cytoplasm of basal and lower spinous epithelial cells, coexpressed in the cytoplasm of basal cells with the E5 oncoprotein. E7 was also observed in nucleoli throughout the basal and spinous layers but not in the granular cell layer. Ectopic expression of E7 in cultured epithelial cells gave rise to localization similar to that seen in productive fibropapillomas, with cytoplasmic and nucleolar expression observed. Consistent with the coexpression of E7 and E5 in basal keratinocytes, BPV-1 E7 cooperated with E5 as well as E6 in an anchorage independence transformation assay. While E5 is expressed in both basal and superficial differentiating keratinocytes, BPV-1 E7 is only observed in basal and lower spinous epithelial cells. Therefore, BPV-1 E7 may serve to modulate the cellular response of basal epithelial cells to E5 expression.     

The receptor recycling pathway contains two distinct populations of early endosomes with different sorting functions

Receptor recycling involves two endosome populations, peripheral early endosomes and perinuclear recycling endosomes. In polarized epithelial cells, either or both populations must be able to sort apical from basolateral proteins, returning each to its appropriate plasma membrane domain. However, neither the roles of early versus recycling endosomes in polarity nor their relationship to each other has been quantitatively evaluated. Using a combined morphological, biochemical, and kinetic approach, we found these two endosome populations to represent physically and functionally distinct compartments. Early and recycling endosomes were resolved on Optiprep gradients and shown to be differentially associated with rab4, rab11, and transferrin receptor; rab4 was enriched on early endosomes and at least partially depleted from recycling endosomes, with the opposite being true for rab11 and transferrin receptor. The two populations were also pharmacologically distinct, with AlF4 selectively blocking export of transferrin receptor from recycling endosomes to the basolateral plasma membrane. We applied these observations to a detailed kinetic analysis of transferrin and dimeric IgA recycling and transcytosis. The data from these experiments permitted the construction of a testable, mathematical model which enabled a dissection of the roles of early and recycling endosomes in polarized receptor transport. Contrary to expectations, the majority (>65%) of recycling to the basolateral surface is likely to occur from early endosomes, but with relatively little sorting of apical from basolateral proteins. Instead, more complete segregation of basolateral receptors from receptors intended for transcytosis occurred upon delivery to recycling endosomes.     

A hub dynamometer for measurement of wheel forces in off-road bicycling

A dynamometric hubset that measures the two ground contact force components acting on a bicycle wheel in the plane of the bicycle during off-road riding while either coasting or braking was designed, constructed, and evaluated. To maintain compatibility with standard mountain bike construction, the hubs use commercially available shells with modified, strain gage-equipped axles. The axle strain gages are sensitive to forces acting in the radial and tangential directions, while minimizing sensitivity to transverse forces, steering moments, and variations in the lateral location of the center of pressure. Static calibration and a subsequent accuracy check that computed differences between applied and apparent loads developed during coasting revealed root mean squared errors of 1 percent full-scale or less (full-scale load = 4500 N). The natural frequency of the rear hub with the wheel attached exceeded 350 Hz. These performance capabilities make the dynamometer useful for its intended purpose during coasting. To demonstrate this usefulness, sample ground contact forces are presented for a subject who coasted downhill over rough terrain. The dynamometric hubset can also be used to determine ground contact forces during braking providing that the brake reaction force components are known. However, compliance of the fork can lead to high cross-sensitivity and corresponding large (> 5 percent FS) measurement errors at the front wheel.     

SP-D and GM-CSF regulate surfactant homeostasis via distinct mechanisms

Both surfactant protein (SP) D and granulocyte-macrophage colony-stimulating factor (GM-CSF) influence pulmonary surfactant homeostasis, with the deficiency of either protein causing marked accumulation of surfactant phospholipids in lung tissues and in the alveoli. To assess whether the effects of each gene were mediated by distinct or shared mechanisms, surfactant homeostasis and lung morphology were assessed in 1) double-transgenic mice in which both SP-D and GM-CSF genes were ablated [SP-D(-/-),GM(-/-)] and 2) transgenic mice deficient in both SP-D and GM-CSF in which the expression of GM-CSF was increased in the lung. Saturated phosphatidylcholine (Sat PC) pool sizes were markedly increased in SP-D(-/-),GM(-/-) mice, with the effects of each gene deletion on surfactant Sat PC pool sizes being approximately additive. Expression of GM-CSF in lungs of SP-D(-/-),GM(-/-) mice corrected GM-CSF-dependent abnormalities in surfactant catabolism but did not correct lung pathology characteristic of SP-D deletion. In contrast to findings in GM(-/-) mice, degradation of [(3)H]dipalmitoylphosphatidylcholine by alveolar macrophages from the SP-D(-/-) mice was normal. The emphysema and foamy macrophage infiltrates characteristic of SP-D(-/-) mice were similar in the presence or absence of GM-CSF. Taken together, these findings demonstrate the distinct roles of SP-D and GM-CSF in the regulation of surfactant homeostasis and lung structure.     

In vivo calibration of a femoral fixation device transducer for measuring anterior cruciate ligament graft tension: a study in an ovine model

Toward developing a transducer for measuring in vivo tension in anterior cruciate ligament grafts in humans, the objectives of this study were to determine the following: (1) whether the calibration of a previously reported femoral fixation device transducer (FDT) (Ventura et al., 1998) is affected by the presence of the graft when implanted in the tibial metaphysis of an ovine model, (2) whether the FDT remains calibrated at 4 weeks postoperatively, and (3) whether the biological incorporation of the graft occurs prior to a change in the FDT calibration. The FDT was implanted in the hind limb of five sheep using an extra-articular procedure. Both the proximal common digital extensor tendon (i.e., graft) and a Teflon-coated wire were looped around the FDT inside a tunnel in the tibial metaphysis. The FDT was calibrated on three occasions using the loop of wire: once intraoperatively before graft insertion, once intraoperatively after graft insertion, and once postoperatively after the animals had been sacrificed at 4 weeks. Following sacrifice, the load transmitted to the FDT by the graft was also determined. The FDT exhibited linear calibration intraoperatively both before and after graft insertion with an average error relative to the calibration before insertion of the graft of -4.6 percent of full-scale load (150 N) and this average relative error was not significantly different from zero (p = 0.183). After 4 weeks of implantation, the average relative percent error was -5.0 percent and was not significantly different from zero (p = 0.434) indicating that the FDT remained calibrated in the in vivo environment. Because only 15 percent of the graft tension was transmitted to the FDT after 4 weeks, biological incorporation of the graft preceded the loss of calibration. In light of these findings, the FDT offers the capability of measuring the intra-articular ACL graft tension in vivo in animal models and possibly humans before the biological bond develops and also of monitoring the formation and maturation of the biological bond between a graft and bone tunnel.     

Comparison of viscoelastic, structural, and material properties of double-looped anterior cruciate ligament grafts made from bovine digital extensor and human hamstring tendons

Due to ready availability, decreased cost, and freedom from transmissible diseases in humans such as hepatitis and AIDS, it would be advantageous to use tendon grafts from farm animals as a substitute for human tendon grafts in in vitro experiments aimed at improving the outcome of anterior cruciate ligament (ACL) reconstructive surgery. Thus the objective of this study was to determine whether an anterior cruciate ligament (ACL) graft composed of two loops of bovine common digital extensor tendon has the same viscoelastic, structural, and material properties as a graft composed of a double loop of semitendinosus and gracilis tendons from humans. To satisfy this objective, grafts were constructed from each tissue source. The cross-sectional area was measured using an area micrometer, and each graft was then pulled using a materials testing system while submerged in a saline bath. Using two groups of tendon grafts (n = 10), viscoelastic tests were conducted over a three-day period during which a constant displacement load relaxation test was followed by a constant amplitude, cyclic load creep test (first day), a constant load creep test (second day), and an incremental cyclic load creep test (third day). Load-to-failure tests were performed on two different groups of grafts (n = 8). When the viscoelastic behavior was compared, there were no significant differences in the rate of load decay or the final load (relaxation test) and rates of displacement increase or final displacements (creep tests) (p > 0.115). To compare both the structural and material properties in the toe region (i.e., < 250 N) of the load-elongation curve, the tangent stiffness and modulus functions were computed from parameters used in an exponential model fit to the load (stress)-elongation (strain) data. Although one of the two parameters in the functions was different statistically, this difference translated into a difference of only 0.03 mm in displacement at 250 N of load. In the linear region (i.e., 50-75 percent of ultimate load) of the load-elongation curve, the linear stiffness of the two graft types compared closely (444 N/mm for bovine and 418 N/mm for human) (p = 0.341). At failure, the ultimate loads (2901 N and 2914 N for bovine and human, respectively) and the ultimate stresses (71.8 MPa and 65.6 MPa for bovine and human, respectively) were not significantly different (p > 0.261). The theoretical effect of any differences in properties between these two grafts on the results of two types of in vitro experiments (i.e., effect of surgical variables on knee laxity and structural properties of fixation devices) are discussed. Despite some statistical differences in the properties evaluated, these differences do not translate into important effects on the dependent variables of interest in the experiments. Thus the bovine tendon graft can be substituted for the human tendon graft in both types of experiments.     

Quantification of rosuvastatin in human plasma by automated solid-phase extraction using tandem mass spectrometric detection

An assay employing automated solid-phase extraction (SPE) followed by high-performance liquid chromatography with positive ion TurboIonspray tandem mass spectrometry (LC-MS-MS) was developed and validated for the quantification of rosuvastatin (Crestor) in human plasma. Rosuvastatin is a hydroxy-methyl glutaryl coenzyme A reductase inhibitor currently under development by AstraZeneca. The standard curve range in human plasma was 0.1-30 ng/ml with a lower limit of quantification (LLOQ) verified at 0.1 ng/ml. Inaccuracy was less than 8% and imprecision less than +/-15% at all concentration levels. There was no interference from endogenous substances. The analyte was stable in human plasma following three freeze/thaw cycles and for up to 6 months following storage at both -20 and -70 degrees C. The assay was successfully applied to the analysis of rosuvastatin in human plasma samples derived from clinical trials, allowing the pharmacokinetics of the compound to be determined.