Recupero e riqualificazione delle aree produttive in provincia di Treviso

Recovery and Regeneration of Industrial Areas in the Treviso Province. In the future the reducing necessity of industrial areas and the demand for a sustainable urban management - soil use - make more urgent the challenge of recovery and regeneration of industrial areas in the province of Treviso, largely abandoned today and subject to ineffective urban and regional planning models. A decision tool has been performed in order to manage political interventions by classifying industrial areas in: suitable to expansion, not suitable to expansion and to recovery. For each of these classes urban design addresses have been commented in a perspective of environmental sustainability

Properties of Some Variants of Human β2-Microglobulin and Amyloidogenesis

Three variants of human beta(2)-microglobulin (beta(2)-m) were compared with wild-type protein. For two variants, namely the mutant R3Abeta(2)-m and the form devoid of the N-terminal tripeptide (DeltaN3beta(2)-m), a reduced unfolding free energy was measured compared with wild-type beta(2)-m, whereas an increased stability was observed for the mutant H31Ybeta(2)-m. The solution structure could be determined by (1)H NMR spectroscopy and restrained modeling only for R3Abeta(2)-m that showed the same conformation as the parent species, except for deviations at the interstrand loops. Analogous conclusions were reached for H31Ybeta(2)-m and DeltaN3beta(2)-m. Precipitation and unfolding were observed over time periods shorter than 4-6 weeks with all the variants and, sometimes, with wild-type protein. The rate of structured protein loss from solution as a result of precipitation and unfolding always showed pseudo-zeroth order kinetics. This and the failure to observe an unfolded species without precipitation suggest that a nucleated conformational conversion scheme should apply for beta(2)-m fibrillogenesis. The mechanism is consistent with the previous and present results on beta(2)-m amyloid transition, provided a nucleated oligomeric species be considered the stable intermediate of fibrillogenesis, the monomeric intermediate being the necessary transition step along the pathway from the native protein to the nucleated oligomer

Inhibition of the mechano-enzymatic amyloidogenesis of transthyretin: role of ligand affinity, binding cooperativity and occupancy of the inner channel

Dissociation of the native transthyretin (TTR) tetramer is widely accepted as the critical step in TTR amyloid fibrillogenesis. It is modelled by exposure of the protein to non-physiological low pH in vitro and is inhibited by small molecule compounds, such as the drug tafamidis. We have recently identified a new mechano-enzymatic pathway of TTR fibrillogenesis in vitro, catalysed by selective proteolytic cleavage, which produces a high yield of genuine amyloid fibrils. This pathway is efficiently inhibited only by ligands that occupy both binding sites in TTR. Tolcapone, which is bound with similar high affinity in both TTR binding sites without the usual negative cooperativity, is therefore of interest. Here we show that TTR fibrillogenesis by the mechano-enzymatic pathway is indeed more potently inhibited by tolcapone than by tafamidis but neither, even in large molar excess, completely prevents amyloid fibril formation. In contrast, mds84, the prototype of our previously reported bivalent ligand TTR 'superstabiliser' family, is notably more potent than the monovalent ligands and we show here that this apparently reflects the critical additional interactions of its linker within the TTR central channel. Our findings have major implications for therapeutic approaches in TTR amyloidosis

Amyloid Formation by Globular Proteins: The Need to Narrow the Gap Between in Vitro and in Vivo Mechanisms

The globular to fibrillar transition of proteins represents a key pathogenic event in the development of amyloid diseases. Although systemic amyloidoses share the common characteristic of amyloid deposition in the extracellular matrix, they are clinically heterogeneous as the affected organs may vary. The observation that precursors of amyloid fibrils derived from circulating globular plasma proteins led to huge efforts in trying to elucidate the structural events determining the protein metamorphosis from their globular to fibrillar state. Whereas the process of metamorphosis has inspired poets and writers from Ovid to Kafka, protein metamorphism is a more recent concept. It is an ideal metaphor in biochemistry for studying the protein folding paradigm and investigating determinants of folding dynamics. Although we have learned how to transform both normal and pathogenic globular proteins into fibrillar polymers in vitro, the events occurring in vivo, are far more complex and yet to be explained. A major gap still exists between in vivo and in vitro models of fibrillogenesis as the biological complexity of the disease in living organisms cannot be reproduced at the same extent in the test tube. Reviewing the major scientific attempts to monitor the amyloidogenic metamorphosis of globular proteins in systems of increasing complexity, from cell culture to human tissues, may help to bridge the gap between the experimental models and the actual pathological events in patients

Plasminogen activation triggers transthyretin amyloidogenesis in vitro

Systemic amyloidosis is a usually fatal disease caused by extracellular accumulation of abnormal protein fibers, amyloid fibrils, derived by misfolding and aggregation of soluble globular plasma protein precursors. Both WT and genetic variants of the normal plasma protein transthyretin (TTR) form amyloid, but neither the misfolding leading to fibrillogenesis nor the anatomical localization of TTR amyloid deposition are understood. We have previously shown that, under physiological conditions, trypsin cleaves human TTR in a mechano-enzymatic mechanism that generates abundant amyloid fibrils in vitro. In sharp contrast, the widely used in vitro model of denaturation and aggregation of TTR by prolonged exposure to pH 4.0 yields almost no clearly defined amyloid fibrils. However, the exclusive duodenal location of trypsin means that this enzyme cannot contribute to systemic extracellular TTR amyloid deposition in vivo. Here, we therefore conducted a bioinformatics search for systemically active tryptic proteases with appropriate tissue distribution, which unexpectedly identified plasmin as the leading candidate. We confirmed that plasmin, just as trypsin, selectively cleaves human TTR between residues 48 and 49 under physiological conditions in vitro. Truncated and full-length protomers are then released from the native homotetramer and rapidly aggregate into abundant fibrils indistinguishable from ex vivo TTR amyloid. Our findings suggest that physiological fibrinolysis is likely to play a critical role in TTR amyloid formation in vivo. Identification of this surprising intersection between two hitherto unrelated pathways opens new avenues for elucidating the mechanisms of TTR amyloidosis, for seeking susceptibility risk factors, and for therapeutic innovation

Endotracheal intubation skill acquisition by medical students

During the course of their training, medical students may receive introductory experience with advanced resuscitation skills. Endotracheal intubation (ETI – the insertion of a breathing tube into the trachea) is an example of an important advanced resuscitation intervention. Only limited data characterize clinical ETI skill acquisition by medical students. We sought to characterize medical student acquisition of ETI procedural skill.11Presented as a poster discussion on 17 October 2007 at the annual meeting of the American Society of Anesthesiologists in San Francisco, CA.The study included third-year medical students participating in a required anesthesiology clerkship. Students performed ETI on operating room patients under the supervision of attending anesthesiologists. Students reported clinical details of each ETI effort, including patient age, sex, Mallampati score, number of direct laryngoscopies and ETI success. Using mixed-effects regression, we characterized the adjusted association between ETI success and cumulative ETI experience.ETI was attempted by 178 students on 1,646 patients (range 1–23 patients per student; median 9 patients per student, IQR 6–12). Overall ETI success was 75.0% (95% CI 72.9–77.1%). Adjusted for patient age, sex, Mallampati score and number of laryngoscopies, the odds of ETI success improved with cumulative ETI encounters (odds ratio 1.09 per additional ETI encounter; 95% CI 1.04–1.14). Students required at least 17 ETI encounters to achieve 90% predicted ETI success.In this series medical student ETI proficiency was associated with cumulative clinical procedural experience. Clinical experience may provide a viable strategy for fostering medical student procedural skills

A specific nanobody prevents amyloidogenesis of D76N \u3b22-microglobulin in vitro and modifies its tissue distribution in vivo

Systemic amyloidosis is caused by misfolding and aggregation of globular proteins in vivo for which effective treatments are urgently needed. Inhibition of protein self-aggregation represents an attractive therapeutic strategy. Studies on the amyloidogenic variant of \u3b22-microglobulin, D76N, causing hereditary systemic amyloidosis, have become particularly relevant since fibrils are formed in vitro in physiologically relevant conditions. Here we compare the potency of two previously described inhibitors of wild type \u3b22-microglobulin fibrillogenesis, doxycycline and single domain antibodies (nanobodies). The \u3b22-microglobulin -binding nanobody, Nb24, more potently inhibits D76N \u3b22-microglobulin fibrillogenesis than doxycycline with complete abrogation of fibril formation. In \u3b22-microglobulin knock out mice, the D76N \u3b22-microglobulin/ Nb24 pre-formed complex, is cleared from the circulation at the same rate as the uncomplexed protein; however, the analysis of tissue distribution reveals that the interaction with the antibody reduces the concentration of the variant protein in the heart but does not modify the tissue distribution of wild type \u3b22-microglobulin. These findings strongly support the potential therapeutic use of this antibody in the treatment of systemic amyloidosis

Why do European primary care physicians sometimes not think of, or act on, a possible cancer diagnosis? : A qualitative study

Funding The Swedish application for ethical approval was funded by the Jämtland Cancer and Nursing Foundation. The study had no other external fundingPeer reviewedPostprin

C. elegans expressing D76N β 2 -microglobulin: a model for in vivo screening of drug candidates targeting amyloidosis

Funder: Ministero dell'Istruzione, dell'Università e della Ricerca Dipartimenti di Eccellenza 2018-2022 grant to the Molecular Medicine Department (University of Pavia)Abstract: The availability of a genetic model organism with which to study key molecular events underlying amyloidogenesis is crucial for elucidating the mechanism of the disease and the exploration of new therapeutic avenues. The natural human variant of β2-microglobulin (D76N β2-m) is associated with a fatal familial form of systemic amyloidosis. Hitherto, no animal model has been available for studying in vivo the pathogenicity of this protein. We have established a transgenic C. elegans line, expressing the human D76N β2-m variant. Using the INVertebrate Automated Phenotyping Platform (INVAPP) and the algorithm Paragon, we were able to detect growth and motility impairment in D76N β2-m expressing worms. We also demonstrated the specificity of the β2-m variant in determining the pathological phenotype by rescuing the wild type phenotype when β2-m expression was inhibited by RNA interference (RNAi). Using this model, we have confirmed the efficacy of doxycycline, an inhibitor of the aggregation of amyloidogenic proteins, in rescuing the phenotype. In future, this C. elegans model, in conjunction with the INVAPP/Paragon system, offers the prospect of high-throughput chemical screening in the search for new drug candidates

The two tryptophans of β2-microglobulin have distinct roles in function and folding and might represent two independent responses to evolutionary pressure

We have recently discovered that the two tryptophans of human β2-microglobulin have distinctive roles within the structure and function of the protein. Deeply buried in the core, Trp95 is essential for folding stability, whereas Trp60, which is solvent-exposed, plays a crucial role in promoting the binding of β2-microglobulin to the heavy chain of the class I major histocompatibility complex (MHCI). We have previously shown that the thermodynamic disadvantage of having Trp60 exposed on the surface is counter-balanced by the perfect fit between it and a cavity within the MHCI heavy chain that contributes significantly to the functional stabilization of the MHCI. Therefore, based on the peculiar differences of the two tryptophans, we have analysed the evolution of β2-microglobulin with respect to these residues