Sorting out measures and definitions of screening participation to improve comparability: The example of colorectal cancer.

Participation is a key indicator of the potential effectiveness of any population-based intervention. Defining, measuring and reporting participation in cancer screening programmes has become more heterogeneous as the number and diversity of interventions have increased, and the purposes of this benchmarking parameter have broadened. This study, centred on colorectal cancer, addresses current issues that affect the increasingly complex task of comparing screening participation across settings. Reports from programmes with a defined target population and active invitation scheme, published between 2005 and 2012, were reviewed. Differences in defining and measuring participation were identified and quantified, and participation indicators were grouped by aims of measure and temporal dimensions. We found that consistent terminology, clear and complete reporting of participation definition and systematic documentation of coverage by invitation were lacking. Further, adherence to definitions proposed in the 2010 European Guidelines for Quality Assurance in Colorectal Cancer Screening was suboptimal. Ineligible individuals represented 1% to 15% of invitations, and variable criteria for ineligibility yielded differences in participation estimates that could obscure the interpretation of colorectal cancer screening participation internationally. Excluding ineligible individuals from the reference population enhances comparability of participation measures. Standardised measures of cumulative participation to compare screening protocols with different intervals and inclusion of time since invitation in definitions are urgently needed to improve international comparability of colorectal cancer screening participation. Recommendations to improve comparability of participation indicators in cancer screening interventions are made.

Sorting mechanisms regulating membrane protein traffic in the apical transcytotic pathway of polarized MDCK cells.

The transcytotic pathway followed by the polymeric IgA receptor (pIgR) carrying its bound ligand (dIgA) from the basolateral to the apical surface of polarized MDCK cells has been mapped using morphological tracers. At 20 degreesC dIgA-pIgR internalize to interconnected groups of vacuoles and tubules that comprise the endosomal compartment and in which they codistribute with internalized transferrin receptors (TR) and epidermal growth factor receptors (EGFR). Upon transfer to 37 degreesC the endosome vacuoles develop long tubules that give rise to a distinctive population of 100-nm-diam cup-shaped vesicles containing pIgR. At the same time, the endosome gives rise to multivesicular endosomes (MVB) enriched in EGFR and to 60-nm-diam basolateral vesicles. The cup-shaped vesicles carry the dIgA/pIgR complexes to the apical surface where they exocytose. Using video microscopy and correlative electron microscopy to study cells grown thin and flat we show that endosome vacuoles tubulate in response to dIgA/pIgR but that the tubules contain TR as well as pIgR. However, we show that TR are removed from these dIgA-induced tubules via clathrin-coated buds and, as a result, the cup-shaped vesicles to which the tubules give rise become enriched in dIgA/pIgR. Taken together with the published information available on pIgR trafficking signals, our observations suggest that the steady-state concentrations of TR and unoccupied pIgR on the basolateral surface of polarized MDCK cells are maintained by a signal-dependent, clathrin-based sorting mechanism that operates along the length of the transcytotic pathway. We propose that the differential sorting of occupied receptors within the MDCK endosome is achieved by this clathrin-based mechanism continuously retrieving receptors like TR from the pathways that deliver pIgR to the apical surface and EGFR to the lysosome.

Sorting out measures and definitions of screening participation to improve comparability : the example of colorectal cancer.

Participation is a key indicator of the potential effectiveness of any population-based intervention. Defining, measuring and reporting participation in cancer screening programmes has become more heterogeneous as the number and diversity of interventions have increased, and the purposes of this benchmarking parameter have broadened. This study, centred on colorectal cancer, addresses current issues that affect the increasingly complex task of comparing screening participation across settings. Reports from programmes with a defined target population and active invitation scheme, published between 2005 and 2012, were reviewed. Differences in defining and measuring participation were identified and quantified, and participation indicators were grouped by aims of measure and temporal dimensions. We found that consistent terminology, clear and complete reporting of participation definition and systematic documentation of coverage by invitation were lacking. Further, adherence to definitions proposed in the 2010 European Guidelines for Quality Assurance in Colorectal Cancer Screening was suboptimal. Ineligible individuals represented 1% to 15% of invitations, and variable criteria for ineligibility yielded differences in participation estimates that could obscure the interpretation of colorectal cancer screening participation internationally. Excluding ineligible individuals from the reference population enhances comparability of participation measures. Standardised measures of cumulative participation to compare screening protocols with different intervals and inclusion of time since invitation in definitions are urgently needed to improve international comparability of colorectal cancer screening participation. Recommendations to improve comparability of participation indicators in cancer screening interventions are made.

The E3 ubiquitin ligase Itch regulates sorting nexin 9 through an unconventional substrate recognition domain.

The level of intracellular proteins is mainly regulated through modifications by ubiquitin ligases that target them for degradation. Members of the NEDD4 family of E3 ubiquitin ligases, such as Itch (atrophin-1 interacting protein 4), possess up to four WW domains for specific association with PY motif-containing substrates. We have identified sorting nexin 9 (SNX9), a protein involved in endocytic processes, as a new substrate of Itch. Itch ubiquitylates SNX9 and regulates intracellular SNX9 levels. Using truncated proteins, we found that the interaction with SNX9 is mediated by the proline-rich domain (PRD) of Itch, a domain distinct from the conventional WW recognition domain, and the SH3 domain of SNX9. Interaction with the PRD of Itch is essential for SNX9 ubiquitylation and degradation. Furthermore, this effect is specific for Itch, as NEDD4, a related PRD-containing E3 ligase, does not bind SNX9. SNX18, a second member of the SNX family containing an SH3 domain, was also found to bind to Itch. Our results indicate that the pool of substrates of NEDD4 family E3 ubiquitin ligases extends beyond proteins containing PY motifs.

Quand la peur des dépenses incite au gaspillage humain et financier [When the fear of spending incites a human and financial waste!]

Dans les années 80, la crainte de la pléthore médicale imposait des mesures radicales. En 2002, c'est l'angoisse de l'envahisseur européen qui a justifié un moratoire sur l'ouverture de nouveaux cabinets. Aujourd'hui, alors que la Suisse recrute de plus en plus de médecins étrangers pour ses besoins, le Conseiller fédéral Couchepin brandit la menace d'une augmentation des coûts de la santé de 300 millions par an pour justifier une troisième prolongation du moratoire. Ces mesures ont été dictées par la peur d'une explosion des coûts au point de faire perdre de vue la globalité de la situation. Aujourd'hui pourtant, une gestion rationnelle des ressources impose de tout faire pour qu'un maximum de médecins puissent travailler, car la population en a besoin et a déjà beaucoup investi dans leur formation ! Pour y parvenir, la création de postes d'assistanat à temps partiel, l'adaptation urgente des structures d'accueil en garderie et le respect de conditions de travail raisonnables sont des éléments incontournables mais trop souvent négligés par les politiques et les médecins eux-mêmes !

The politics of contaminated sites management : institutional regime change and actor's mode of participation in the environmental management of the Bonfol Chemical Waste Landfill in Switzerland

By the end of the 1970s, contaminated sites had emerged as one of the most complex and urgent environmental issues affecting industrialized countries. The authors show that small and prosperous Switzerland is no exception to the pervasive problem of sites contamination, the legacy of past practices in waste management having left some 38,000 contaminated sites throughout the country. This book outlines the problem, offering evidence that open and polycentric environmental decision-making that includes civil society actors is valuable. They propose an understanding of environmental management of contaminated sites as a political process in which institutions frame interactions between strategic actors pursuing sometimes conflicting interests. In the opening chapter, the authors describe the influences of politics and the power relationships between actors involved in decision-making in contaminated sites management, which they term a "wicked problem." Chapter Two offers a theoretical framework for understanding institutions and the environmental management of contaminated sites. The next five chapters present a detailed case study on environmental management and contaminated sites in Switzerland, focused on the Bonfol Chemical Landfill. The study and analysis covers the establishment of the landfill under the first generation of environmental regulations, its closure and early remediation efforts, and the gambling on the remediation objectives, methods and funding in the first decade of the 21st Century. The concluding chapter discusses the question of whether the strength of environmental regulations, and the type of interactions between public, private, and civil society actors can explain the environmental choices in contaminated sites management. Drawing lessons from research, the authors debate the value of institutional flexibility for dealing with environmental issues such as contaminated sites.

Vasopressin-inducible ubiquitin-specific protease 10 increases ENaC cell surface expression by deubiquitylating and stabilizing sorting nexin 3

Adjustment of Na+ balance in extracellular fluids is achieved by regulated Na+ transport involving the amiloride-sensitive epithelial Na+ channel (ENaC) in the distal nephron. In this context, ENaC is controlled by a number of hormones, including vasopressin, which promotes rapid translocation of water and Na+ channels to the plasma membrane and long-term effects on transcription of vasopressin-induced and -reduced transcripts. We have identified a mRNA encoding the deubiquitylating enzyme ubiquitin-specific protease 10 (Usp10), whose expression is increased by vasopressin at both the mRNA and the protein level. Coexpression of Usp10 in ENaC-transfected HEK-293 cells causes a more than fivefold increase in amiloride-sensitive Na+ currents, as measured by whole cell patch clamping. This is accompanied by a three- to fourfold increase in surface expression of alpha- and gamma-ENaC, as shown by cell surface biotinylation experiments. Although ENaC is well known to be regulated by its direct ubiquitylation, Usp10 does not affect the ubiquitylation level of ENaC, suggesting an indirect effect. A two-hybrid screen identified sorting nexin 3 (SNX3) as a novel substrate of Usp10. We show that it is a ubiquitylated protein that is degraded by the proteasome; interaction with Usp10 leads to its deubiquitylation and stabilization. When coexpressed with ENaC, SNX3 increases the channel's cell surface expression, similarly to Usp10. In mCCD(cl1) cells, vasopressin increases SNX3 protein but not mRNA, supporting the idea that the vasopressin-induced Usp10 deubiquitylates and stabilizes endogenous SNX3 and consequently promotes cell surface expression of ENaC

Mutant HbpR transcription activator isolation for 2-chlorobiphenyl via green fluorescent protein-based flow cytometry and cell sorting.

Mutants were produced in the A-domain of HbpR, a protein belonging to the XylR family of σ(54)-dependent transcription activators, with the purpose of changing its effector recognition specificity from 2-hydroxybiphenyl (2-HBP, the cognate effector) to 2-chlorobiphenyl (2-CBP). Mutations were introduced in the hbpR gene part for the A-domain via error-prone polymerase chain reaction, and assembled on a gene circuitry plasmid in Escherichia coli, permitting HbpR-dependent induction of the enhanced green fluorescent protein (egfp). Cells with mutant HbpR proteins responsive to 2-CBP were enriched and separated in a flow cytometry-assisted cell-sorting procedure. Some 70 mutants were isolated and the A-domain mutations mapped. One of these had acquired true 2-CBP recognition but reacted hypersensitively to 2-HBP (20-fold more than the wild type), whereas others had reduced sensitivity to 2-HBP but a gain of 2-CBP recognition. Sequencing showed that most mutants carried double or triple mutations in the A-domain gene part, and were not located in previously recognized conserved residues within the XylR family members. Further selection from a new mutant pool prepared of the hypersensitive mutant did not result in increased 2-CBP or reduced 2-HBP recognition. Our data thus demonstrate that a one-step in vitro 'evolutionary' adaptation of the HbpR protein can result in both enhancement and reduction of the native effector recognition.

Reversible major histocompatibility complex I-peptide multimers containing Ni(2+)-nitrilotriacetic acid peptides and histidine tags improve analysis and sorting of CD8(+) T cells.

MHC-peptide multimers containing biotinylated MHC-peptide complexes bound to phycoerythrin (PE) streptavidin (SA) are widely used for analyzing and sorting antigen-specific T cells. Here we describe alternative T cell-staining reagents that are superior to conventional reagents. They are built on reversible chelate complexes of Ni(2+)-nitrilotriacetic acid (NTA) with oligohistidines. We synthesized biotinylated linear mono-, di-, and tetra-NTA compounds using conventional solid phase peptide chemistry and studied their interaction with HLA-A*0201-peptide complexes containing a His(6), His(12), or 2×His(6) tag by surface plasmon resonance on SA-coated sensor chips and equilibrium dialysis. The binding avidity increased in the order His(6) < His(12) < 2×His(6) and NTA(1) < NTA(2) < NTA(4), respectively, depending on the configuration of the NTA moieties and increased to picomolar K(D) for the combination of a 2×His(6) tag and a 2×Ni(2+)-NTA(2). We demonstrate that HLA-A2-2×His(6)-peptide multimers containing either Ni(2+)-NTA(4)-biotin and PE-SA- or PE-NTA(4)-stained influenza and Melan A-specific CD8+ T cells equal or better than conventional multimers. Although these complexes were highly stable, they very rapidly dissociated in the presence of imidazole, which allowed sorting of bona fide antigen-specific CD8+ T cells without inducing T cell death as well as assessment of HLA-A2-peptide monomer dissociation kinetics on CD8+ T cells.

Sorting live stem cells based on sox2 mRNA expression.

While cell sorting usually relies on cell-surface protein markers, molecular beacons (MBs) offer the potential to sort cells based on the presence of any expressed mRNA and in principle could be extremely useful to sort rare cell populations from primary isolates. We show here how stem cells can be purified from mixed cell populations by sorting based on MBs. Specifically, we designed molecular beacons targeting Sox2, a well-known stem cell marker for murine embryonic (mES) and neural stem cells (NSC). One of our designed molecular beacons displayed an increase in fluorescence compared to a nonspecific molecular beacon both in vitro and in vivo when tested in mES and NSCs. We sorted Sox2-MB(+)SSEA1(+) cells from a mixed population of 4-day retinoic acid-treated mES cells and effectively isolated live undifferentiated stem cells. Additionally, Sox2-MB(+) cells isolated from primary mouse brains were sorted and generated neurospheres with higher efficiency than Sox2-MB(-) cells. These results demonstrate the utility of MBs for stem cell sorting in an mRNA-specific manner.

Old world arenaviruses enter the host cell via the multivesicular body and depend on the endosomal sorting complex required for transport.

The highly pathogenic Old World arenavirus Lassa virus (LASV) and the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) use α-dystroglycan as a cellular receptor and enter the host cell by an unusual endocytotic pathway independent of clathrin, caveolin, dynamin, and actin. Upon internalization, the viruses are delivered to acidified endosomes in a Rab5-independent manner bypassing classical routes of incoming vesicular trafficking. Here we sought to identify cellular factors involved in the unusual and largely unknown entry pathway of LASV and LCMV. Cell entry of LASV and LCMV required microtubular transport to late endosomes, consistent with the low fusion pH of the viral envelope glycoproteins. Productive infection with recombinant LCMV expressing LASV envelope glycoprotein (rLCMV-LASVGP) and LCMV depended on phosphatidyl inositol 3-kinase (PI3K) as well as lysobisphosphatidic acid (LBPA), an unusual phospholipid that is involved in the formation of intraluminal vesicles (ILV) of the multivesicular body (MVB) of the late endosome. We provide evidence for a role of the endosomal sorting complex required for transport (ESCRT) in LASV and LCMV cell entry, in particular the ESCRT components Hrs, Tsg101, Vps22, and Vps24, as well as the ESCRT-associated ATPase Vps4 involved in fission of ILV. Productive infection with rLCMV-LASVGP and LCMV also critically depended on the ESCRT-associated protein Alix, which is implicated in membrane dynamics of the MVB/late endosomes. Our study identifies crucial cellular factors implicated in Old World arenavirus cell entry and indicates that LASV and LCMV invade the host cell passing via the MVB/late endosome. Our data further suggest that the virus-receptor complexes undergo sorting into ILV of the MVB mediated by the ESCRT, possibly using a pathway that may be linked to the cellular trafficking and degradation of the cellular receptor.

Characterization of the expression, regulation and function of Sorting Nexin 9 and Sorting Nexin 18

Summary : Sorting nexin (SNX) family members play important roles in intracellular protein and membrane trafficking, The membrane-tubulating SNX9 protein has been shown to interact with multiple components of the endocytic machinery and to participate in clathrin-mediated endocytosis of cell surface receptors. It has not been investigated if SNX9 may also participate in other protein sorting pathways that involve vesicular transport, specifically the biogenesis of lysosome-related organelles (LROs). Closely related to SNX9 is SNXl8, whose function is largely unknown. In this work, we have characterized the expression of SNX9 and SNXl8 in LRO-containing cells and investigated their role in protein trafficking during the formation of LROs. Our results indicate that SNX9 and SNXl8 are not essential for the formation of LROs, nor for the sorting of melanosomal proteins. We investigated how the level of intracellular SNX9 protein is regulated and found that it is a substrate of the ubiquitin ligase Itch, a member of the NEDD4 family of E3 ubiquitin ligases. Itch ubiquitylates SNX9 and regulates SNX9 levels by enhancing its degradation. Using ? truncated proteins we found that the interaction with SNX9 is mediated by the proline-rich domain of Itch, a domain distinct from the conventional WW recognition domain, and the SH3 domain of SNX9. Interaction with the PRD of Itch is essential for SNX9 ubiquitylation and degradation. We further showed that Itch binding is not affected by tyrosine phosphorylation of SNX9. Using lentivector-mediated siRNA techniques, we found that Itch regulates the level of melanosomal proteins, while knock-down of SNX9 does not alter their level. Interestingly, we revealed that silencing of SNXIS affects the amount of the melanosomal protein Melan-A, but also of SNX9, and that SNXl8 can interact with SNX9. Taken together, our results highlight that the pool of substrates of NEDD4 family E3 ligases extends to proteins containing SH3 domains and provide insight into the potential functions of SNXI8. Résumé : Les membres de la famille des Sorting Nexins (SNX) jouent des rôles importants dans le trafic intracellulaire de protéines et membranes. Il a été démontré que la protéine SNX9, qui génère les tubules membranaires, interagit avec plusieurs composants de la machinerie d'endocytose et participe à l'endocytose des récepteurs de surface mediée par la clathrine. Aucune étude n'a investigué si SNX9 pourrait aussi participer à d'autres voies de trafic de protéines tel que le transport vésiculaire, et plus particulièrement la biogenèse des organites lysosomaux ("lysosome-related organelles", LR©s). SNXl8 est similaire à SNX9, mais sa fonction est largement inconnue. Dans ce travail, nous avons caractérisé l'expression de SNX9 et SNX18 dans des cellules contenants des LROs et investigué leur rôle dans le trafic de protéines pendant la formation des LROS. Nos résultats indiquent que SNX9 et SNXI8 ne sont essentiels ni pour la formation des LR©s, ni pour le trafic de protéines mélanosomales. Nous avons examiné la régulation du niveau intracellulaire de la protéine SNX9 et avons trouvé qu'elle est un substrat de l'ubiquitine ligase Itch, un membre de la famille NEDD4 des ubiquitine ligases E3. Itch ubiquitine SNX9 et régule les niveaux de SNX9 en augmentant sa dégradation. En utilisant des protéines mutées nous avons découvert que l'interaction avec SNX9 est médiée par le domaine riche en proline de Itch, qui est différent du domaine conventionnel de reconnaissance WW, et par le domaine SH3 de SNX9. L'interaction avec le domaine riche en proline de Itch est essentielle pour l'ubiquitination et la dégradation de SNX9. De plus, nous avons montré que cette liaison n'est pas affectée par la phosphorylation des résidus tyrosine de SNX9. En utilisant des vecteurs lentiviraux exprimant des siARN, nous avons trouvé que Itch régule les niveaux de protéines mélanosomales, alors que l'extinction de l'expression de SNX9 ne change pas leurs niveaux. En autre, nous avons révélé que la diminution de SNXl8 affecte le niveau de la protéine mélanosomale Melan-A et de SNX9, et aussi que SNXl8 peut interagir avec SNX9. En résumé, nos résultats démontrent que l'ensemble des substrats de la famille NEDD4 des ubiquitine ligases E3 s'élargit aux protéines contenant des domaines SH3 et ouvrent des perspectives sur les fonctions potentielles de SNXl8.

Treatment of ruptured abdominal aortic aneurysm, a permanent challenge or a waste of resources? Prediction of outcome using a multi-organ-dysfunction score.

OBJECTIVES: in a retrospective study, attempts have been made to identify individual organ-dysfunction risk profiles influencing the outcome after surgery for ruptured abdominal aortic aneurysms. METHODS: out of 235 patients undergoing graft replacement for abdominal aortic aneurysms, 57 (53 men, four women, mean age 72 years [s.d. 8.8]) were treated for ruptured aneurysms in a 3-year period. Forty-eight preoperative, 13 intraoperative and 34 postoperative variables were evaluated statistically. A simple multi-organ dysfunction (MOD) score was adopted. RESULTS: the perioperative mortality was 32%. Three patients died intraoperatively, four within 48 h and 11 died later. A significant influence for pre-existing risk factors was identified only for cardiovascular diseases. Multiple linear-regression analysis indicated that a haemoglobin <90 g/l, systolic blood pressure <80 mmHg and ECG signs of ischaemia at admission were highly significant risk factors. The cause of death for patients, who died more than 48 h postoperatively, was mainly MOD. All patients with a MOD score >/=4 died (n=7). These patients required 27% of the intensive-care unit (ICU) days of all patients and 72% of the ICU days of the non-survivors. CONCLUSION: patients with ruptured aortic aneurysms from treatment should not be excluded. However, a physiological scoring system after 48 h appears justifiable in order to decide on the appropriateness of continual ICU support.