d-dimer testing as an adjunct to ultrasonography in patients with clinically suspected deep vein thrombosis: prospective cohort study

Objective To investigate the efficacy of using a rapid plasma d-dimer test as an adjunct to compression ultrasound for diagnosing clinically suspected deep vein thrombosis.

Chemically distinct transition states govern rapid dissociation of single L-selectin bonds under force

Carbohydrate–protein bonds interrupt the rapid flow of leukocytes in the circulation by initiation of rolling and tethering at vessel walls. The cell surface carbohydrate ligands are glycosylated proteins like the mucin P-selectin glycoprotein ligand-1 (PSGL-1), which bind ubiquitously to the family of E-, P-, and L-selectin proteins in membranes of leukocytes and endothelium. The current view is that carbohydrate–selectin bonds dissociate a few times per second, and the unbinding rate increases weakly with force. However, such studies have provided little insight into how numerous hydrogen bonds, a Ca2+ metal ion bond, and other interactions contribute to the mechanical strength of these attachments. Decorating a force probe with very dilute ligands and controlling touch to achieve rare single-bond events, we have varied the unbinding rates of carbohydrate–selectin bonds by detachment with ramps of force/time from 10 to 100,000 pN/sec. Testing PSGL-1, its outer 19 aa (19FT), and sialyl LewisX (sLeX) against L-selectin in vitro on glass microspheres and in situ on neutrophils, we found that the unbinding rates followed the same dependence on force and increased by nearly 1,000-fold as rupture forces rose from a few to ≈200 pN. Plotted on a logarithmic scale of loading rate, the rupture forces reveal two prominent energy barriers along the unbinding pathway. Strengths above 75 pN arise from rapid detachment (<0.01 sec) impeded by an inner barrier that requires a Ca2+ bond between a single sLeX and the lectin domain. Strengths below 75 pN occur under slow detachment (>0.01 sec) impeded by the outer barrier, which appears to involve an array of weak (putatively hydrogen) bonds.

Rapid plant diversification: Planning for an evolutionary future

Systematic conservation planning is a branch of conservation biology that seeks to identify spatially explicit options for the preservation of biodiversity. Alternative systems of conservation areas are predictions about effective ways of promoting the persistence of biodiversity; therefore, they should consider not only biodiversity pattern but also the ecological and evolutionary processes that maintain and generate species. Most research and application, however, has focused on pattern representation only. This paper outlines the development of a conservation system designed to preserve biodiversity pattern and process in the context of a rapidly changing environment. The study area is the Cape Floristic Region (CFR), a biodiversity hotspot of global significance, located in southwestern Africa. This region has experienced rapid (post-Pliocene) ecological diversification of many plant lineages; there are numerous genera with large clusters of closely related species (flocks) that have subdivided habitats at a very fine scale. The challenge is to design conservation systems that will preserve both the pattern of large numbers of species and various natural processes, including the potential for lineage turnover. We outline an approach for designing a system of conservation areas to incorporate the spatial components of the evolutionary processes that maintain and generate biodiversity in the CFR. We discuss the difficulty of assessing the requirements for pattern versus process representation in the face of ongoing threats to biodiversity, the difficulty of testing the predictions of alternative conservation systems, and the widespread need in conservation planning to incorporate and set targets for the spatial components (or surrogates) of processes.

Rapid, local adaptation of zooplankton behavior to changes in predation pressure in the absence of neutral genetic changes

Organisms producing resting stages provide unique opportunities for reconstructing the genetic history of natural populations. Diapausing seeds and eggs often are preserved in large numbers, representing entire populations captured in an evolutionary inert state for decades and even centuries. Starting from a natural resting egg bank of the waterflea Daphnia, we compare the evolutionary rates of change in an adaptive quantitative trait with those in selectively neutral DNA markers, thus effectively testing whether the observed genetic changes in the quantitative trait are driven by natural selection. The population studied experienced variable and well documented levels of fish predation over the past 30 years and shows correlated genetic changes in phototactic behavior, a predator-avoidance trait that is related to diel vertical migration. The changes mainly involve an increased plasticity response upon exposure to predator kairomone, the direction of the changes being in agreement with the hypothesis of adaptive evolution. Genetic differentiation through time was an order of magnitude higher for the studied behavioral trait than for neutral markers (DNA microsatellites), providing strong evidence that natural selection was the driving force behind the observed, rapid, evolutionary changes.

Development of HIV vectors for anti-HIV gene therapy.

Current gene therapy protocols for HIV infection use transfection or murine retrovirus mediated transfer of antiviral genes into CD4+ T cells or CD34+ progenitor cells ex vivo, followed by infusion of the gene altered cells into autologous or syngeneic/allogeneic recipients. While these studies are essential for safety and feasibility testing, several limitations remain: long-term reconstitution of the immune system is not effected for lack of access to the macrophage reservoir or the pluripotent stem cell population, which is usually quiescent, and ex vivo manipulation of the target cells will be too expensive and impractical for global application. In these regards, the lentivirus-specific biologic properties of the HIVs, which underlie their pathogenetic mechanisms, are also advantageous as vectors for gene therapy. The ability of HIV to specifically target CD4+ cells, as well as non-cycling cells, makes it a promising candidate for in vivo gene transfer vector on one hand, and for transduction of non-cycling stem cells on the other. Here we report the use of replication-defective vectors and stable vector packaging cell lines derived from both HIV-1 and HIV-2. Both HIV envelopes and vesicular stomatitis virus glycoprotein G were effective in mediating high-titer gene transfer, and an HIV-2 vector could be cross-packaged by HIV-1. Both HIV-1 and HIV-2 vectors were able to transduce primary human macrophages, a property not shared by murine retroviruses. Vesicular stomatitis virus glycoprotein G-pseudotyped HIV vectors have the potential to mediate gene transfer into non-cycling hematopoietic stem cells. If so, HIV or other lentivirus-based vectors will have applications beyond HIV infection.