Setting priorities and selecting topics for clinical practice guidelines.

Setting priorities and selecting topics are important steps in guidelines development, but they have received relatively little attention to date. Responses from a survey of guidelines stakeholders in Canada suggest that the health burden of a clinical condition on the population is an important factor in priority setting. Economic considerations, cast as either costs of treatment to the health care system or the economic burden of illness to society, are given varying importance by different stakeholder groups. Drawing on the literature and the survey results, the authors propose a framework for priority setting. Important issues requiring consideration include the role of public and community participation, the need for and appropriate emphasis on quantitative data regarding current practice and its variation, and mechanisms to link guidelines to health-policy development and management of the health care system.     

Patterns of deletions of the dystrophin gene in different European populations

The distribution of deletion breakpoints in the dystrophin gene was studied in a series of subjects belonging to different European populations. The data, obtained from the literature or directly from the present study, refer to population samples from France, Finland, Germany, Italy, Netherland, Switzerland, and U.K. (England, Scotland, Wales). In total, 1516 breakpoints were assigned to different introns, 359 in the region encompassing the first 40 exons and 1157 (76%) in the distal part of the gene. Intron 7 appears to be equally involved as the starting or ending breakpoint, whereas intron 44 is involved mostly as a starting breakpoint. Breakpoint distribution by intron seems to differ in different populations, reaching statistical significance in the case of introns 44, 49, and 53. This finding suggests that some intronic sequences might contain preferential breakpoints that might vary in different populations, possibly as a consequence of genetic drift.     

Binding properties of the calcium-activated F2 isoform of Lethocerus troponin C

While in most muscles contraction is triggered by calcium effluxes, insect flight muscles are also activated by mechanical stretch. We are interested in understanding the role that the troponin C protein, usually the calcium sensor, plays in stretch activation. In the flight muscles of Lethocerus, a giant water bug often used as a model system, there are two isoforms of TnC, F1 and F2, present in an approximately 10:1 ratio. F1 TnC is responsible for activating the muscle following a stretch, whereas F2 TnC produces a sustained contraction, the magnitude of which depends on the concentration of Ca(2+) in the fiber. We have previously shown that F1 TnC binds only one Ca(2+) ion in its C-terminal domain and that interaction with troponin H, the insect ortholog of troponin I, is insensitive to Ca(2+). Here, we have studied the effect of Ca(2+) and Mg(2+) on the affinities of the interaction of F2 TnC with troponin H peptides. We show that the presence of two Ca(2+) ions, one in each of the globular domains, increases the affinity for TnH by at least 1 order of magnitude. The N lobe has a lower affinity for Ca(2+), but it is also sensitive to Mg(2+). The C lobe is insensitive to Mg(2+) as previously demonstrated by mutations of the individual EF-hands. The interaction with TnH seems also to have significant structural differences from that observed for the F1 TnC isoform. We discuss how our findings could account for stretch activation.     

Patterns of spatial genetic structuring in the endangered limpet Patella ferruginea: implications for the conservation of a Mediterranean endemic

Patella ferruginea Gmelin, 1791 is an endangered marine gastropod endemic to the Western Mediterranean. Its range is restricted to the Sardinian-Corsican region (SCR), North Africa, a few scattered sites in Southern Spain, and Sicily. Inter-simple sequence repeat (ISSR) markers and three different mitochondrial DNA (mtDNA) regions, Cytochrome c Oxidase subunit I, 12S (small-subunit ribosomal RNA gene) and 16S (large-subunit ribosomal RNA gene), were used to investigate the presence of genetic population structuring. The mtDNA sequences showed very low levels of genetic differentiation. Conversely, ISSRs showed the presence of two main genetic groups, corresponding to Spain, North Africa and Sicily and the SCR. The SCR was further split into two subgroups. The ISSR results suggest that, on a regional scale, the genetic structure of P. ferruginea is mainly determined by the restriction of gene flow by dispersal barriers. On a more local scale human harvesting may play a crucial role in population structuring by increasing the effect of genetic drift.     

Enhanced Expression of CD13 in Vessels of Inflammatory and Neoplastic Tissues

Aminopeptidase-N (CD13) is an important target of tumor vasculature-targeting drugs. The authors investigated its expression by immunohistochemistry with three anti-CD13 monoclonal antibodies (WM15, 3D8, and BF10) in normal and pathological human tissues, including 58 normal, 32 inflammatory, and 149 tumor tissue specimens. The three antibodies stained vessels in most neoplastic tissues, interestingly with different patterns. As a matter of fact, WM15 stained almost all intratumor and peritumor capillaries and only partially large vessels, whereas BF10 and 3D8 reacted with arteries and venules and to a lesser extent with capillaries. These antibodies also stained the stroma in about half of neoplastic tissues. In inflammatory lesions, the three antibodies stained vessels and stroma, whereas in normal tissues, they stained a small percentage of blood vessels. Finally, the three antibodies failed to stain endothelial cells of normal colon, whereas they reacted with activated human umbilical vein endothelial cells and with endothelial cells of colon adenocarcinoma vessels. Overall, WM15 was the most specific antibody for angiogenic tumor vessels, suggesting that it may be a good tool for detecting the CD13 form associated with the tumor vasculature. This finding may be relevant for CD13-mediated vascular targeting therapies.     

Effects of the known pathogenic mutations on the aggregation pathway of the amyloidogenic peptide of apolipoprotein A-I

The 93-residue N-terminal fragment of apolipoprotein A-I (ApoA-I) is the major constituent of fibrils isolated from patients affected by the amyloidosis caused by ApoA-I mutations. We have prepared eight polypeptides corresponding to all the currently known amyloidogenic variants of the N-terminal region of ApoA-I, other than a truncation mutation, and investigated their aggregation kinetics and the associated structural modifications. All the variants adopted a monomeric highly disordered structure in solution at neutral pH, whereas acidification of the solution induced an unstable α-helical conformation and the subsequent aggregation into the cross-β structure aggregate. Two mutations (Δ70-72 and L90P) almost abrogated the lag phase of the aggregation process, three mutations (Δ60-71, L75P, and W50R) significantly accelerated the aggregation rate by 2- to 3-fold, while the remaining three variants (L64P, L60R, and G26R) were not significantly different from the wild type. Therefore, an increase in aggregation propensity cannot explain per se the mechanism of the disease for all the variants. Prediction of the protection factors for hydrogen exchange in the native state of full-length protein reveals, in almost all the variants, an expansion of the conformational fluctuations that could favour the proteolytic cleavage and the release of the amyloidogenic peptide. Such an event seems to be a necessary prerequisite for ApoA-I fibrillogenesis in vivo, but the observed increased aggregation propensity of certain variants can have a strong influence on the severity of the disease, such as an earlier onset and a faster progression.     

Interactive and automated application of virtual microscopy

Virtual microscopy can be applied in an interactive and an automated manner. Interactive application is performed in close association to conventional microscopy. It includes image standardization suitable to the performance of an individual pathologist such as image colorization, white color balance, or individual adjusted brightness. The steering commands have to include selection of wanted magnification, easy navigation, notification, and simple measurements (distances, areas). The display of the histological image should be adjusted to the physical limits of the human eye, which are determined by a view angle of approximately 35 seconds. A more sophisticated performance should include acoustic commands that replace the corresponding visual commands. Automated virtual microscopy includes so-called microscopy assistants which can be defined similar to the developed assistants in computer based editing systems (Microsoft Word, etc.). These include an automated image standardization and correction algorithms that excludes images of poor quality (for example uni-colored or out-of-focus images), an automated selection of the most appropriate field of view, an automated selection of the best magnification, and finally proposals of the most probable diagnosis. A quality control of the final diagnosis, and feedback to the laboratory determine the proposed system. The already developed tools of such a system are described in detail, as well as the results of first trials. In order to enhance the speed of such a system, and to allow further user-independent development a distributed implementation probably based upon Grid technology seems to be appropriate. The advantages of such a system as well as the present pathology environment and its expectations will be discussed in detail.