Die Synthese eines Sialyl-Lewis X-Mimetikums als Baustein zur Herstellung von Glycopeptid-Zelladhäsionsinhibitoren für Selektine

Zelladhäsionsphänomene spielen eine wichtige Rolle in vielen biologischen Vorgängen, wie z. B. in der Embryogenese, der Wundheilung, der Immunantwort und Entzündungsprozessen. So wird die inflammatorische Kaskade durch P- und E-Selektin vermittelte Adhäsion der im Blutstrom zirkulierenden Leukozyten an Endothelzellen eingeleitet. Übermäßige Zelladhäsion kann hingegen eine Vielzahl von Krankheiten bewirken. Nachgewiesen ist eine solche Beteiligung der Selektine u. a. bei rheumatoider Arthritis, Erkrankungen der Herzkranzgefäße und dem Reperfusionssyndrom. Ein Ansatz zur Therapie besteht in der Verabreichung löslicher Selektinliganden, die kompetitiv an die Selektine binden und deren Aktivität dadurch mindern. Die wichtigste Leitstruktur für pharmakologisch aktive Selektinliganden stellt hierbei das Tetrasaccharid-Epitop Sialyl-Lewisx (sLex) dar.rnrnIn dieser Arbeit wurde nach einer nicht enzymatischen Strategie ein auf der sLex Struktur basierender Baustein für die Festphasen-Peptidsynthese synthetisiert. Aus diesem sollen nach Schutzgruppen-Manipulationen durch Einsatz in automatisierten Festphasen-Peptidsynthesen, verschiedene Glycopeptide als Zelladhäsionsinhibitoren für Selektine gewonnen werden. Der rasche Abbau der sLex-Struktur im Organismus schränkt die pharmakologische Nutzung von sLex-Derivaten jedoch stark ein. Das synthetisierte Kohlenhydrat Epitop weist daher gegenüber der natürlichen sLex-Struktur mehrere Modifikationen auf, die zu einer erhöhten metabolischen Stabilität führen sollen, ohne die für eine effektive Bindung zum Rezeptor nötigen Wechselwirkungen zu beeinträchtigen. rnrnAus diesem Grund wurde der synthetisch anspruchsvolle Sialinsäure-Baustein durch die L-Cyclohexylmilchsäure substituiert. Die Anbindung des Kohlenhydrat-Epitops an das Peptid wird im Gegensatz zur natürlichen N-glycosidischen Verknüpfung hier über eine zusätzliche Glucosamin-Einheit O-glycosidisch an die Aminosäure L-Threonin bewirkt. Außerdem wird der labile Fucose-Rest durch die D-Arabinose ersetzt, die nicht im menschlichen Organismus vorkommt, jedoch die drei essentiellen pharmakophoren Hydroxylfunktionen in der gleichen dreidimensionalen Orientierung präsentiert wie die L-Fucose.

Cleavage of the antithrombin III binding site in heparin by heparinases and its implication in the generation of low molecular weight heparin

Heparin has been used as a clinical anticoagulant for more than 50 years, making it one of the most effective pharmacological agents known. Much of heparin's activity can be traced to its ability to bind antithrombin III (AT-III). Low molecular weight heparin (LMWH), derived from heparin by its controlled breakdown, maintains much of the antithrombotic activity of heparin without many of the serious side effects. The clinical significance of LMWH has highlighted the need to understand and develop chemical or enzymatic means to generate it. The primary enzymatic tools used for the production of LMWH are the heparinases from Flavobacterium heparinum, specifically heparinases I and II. Using pentasaccharide and hexasaccharide model compounds, we show that heparinases I and II, but not heparinase III, cleave the AT-III binding site, leaving only a partially intact site. Furthermore, we show herein that glucosamine 3-O sulfation at the reducing end of a glycosidic linkage imparts resistance to heparinase I, II, and III cleavage. Finally, we examine the biological and pharmacological consequences of a heparin oligosaccharide that contains only a partial AT-III binding site. We show that such an oligosaccharide lacks some of the functional attributes of heparin- and heparan sulfate-like glycosaminoglycans containing an intact AT-III site.

Ramachandran-type Plots for Glycosidic Linkages: Examples from Molecular Dynamics Simulations using the Glycam06 Force Field

The goals of this article are to (1) provide further validation of the Glycam06 force field, specifically for its use in implicit solvent molecular dynamic (MD) simulations, and (2) to present the extension of G.N. Ramachandran's idea of plotting amino acid phi and psi angles to the glycosidic phi, psi, and omega angles formed between carbohydrates. As in traditional Ramachandran plots, these carbohydrate Ramachandran-type (carb-Rama) plots reveal the coupling between the glycosidic angles by displaying the allowed and disallowed conformational space. Considering two-bond glycosidic linkages, there are 18 possible conformational regions that can be defined by (α, ϕ, ψ) and (β, ϕ, ψ), whereas for three-bond linkages, there are 54 possible regions that can be defined by (α, ϕ, ψ, ω) and (β, ϕ, ψ, ω). Illustrating these ideas are molecular dynamic simulations on an implicitly hydrated oligosaccharide (700 ns) and its eight constituent disaccharides (50 ns/disaccharide). For each linkage, we compare and contrast the oligosaccharide and respective disaccharide carb-Rama plots, validate the simulations and the Glycam06 force field through comparison to experimental data, and discuss the general trends observed in the plots.

Linkage and heritability analysis of migraine symptom groupings: A comparison of three different clustering methods on twin data

Migraine is a painful disorder for which the etiology remains obscure. Diagnosis is largely based on International Headache Society criteria. However, no feature occurs in all patients who meet these criteria, and no single symptom is required for diagnosis. Consequently, this definition may not accurately reflect the phenotypic heterogeneity or genetic basis of the disorder. Such phenotypic uncertainty is typical for complex genetic disorders and has encouraged interest in multivariate statistical methods for classifying disease phenotypes. We applied three popular statistical phenotyping methods—latent class analysis, grade of membership and grade of membership “fuzzy” clustering (Fanny)—to migraine symptom data, and compared heritability and genome-wide linkage results obtained using each approach. Our results demonstrate that different methodologies produce different clustering structures and non-negligible differences in subsequent analyses. We therefore urge caution in the use of any single approach and suggest that multiple phenotyping methods be used.

Methods and preliminary results for a data linkage project to determine long-term survival after intensive care unit admission

Aims: To describe a local data linkage project to match hospital data with the Australian Institute of Health and Welfare (AIHW) National Death Index (NDI) to assess longterm outcomes of intensive care unit patients. Methods: Data were obtained from hospital intensive care and cardiac surgery databases on all patients aged 18 years and over admitted to either of two intensive care units at a tertiary-referral hospital between 1 January 1994 and 31 December 2005. Date of death was obtained from the AIHW NDI by probabilistic software matching, in addition to manual checking through hospital databases and other sources. Survival was calculated from time of ICU admission, with a censoring date of 14 February 2007. Data for patients with multiple hospital admissions requiring intensive care were analysed only from the first admission. Summary and descriptive statistics were used for preliminary data analysis. Kaplan-Meier survival analysis was used to analyse factors determining long-term survival. Results: During the study period, 21 415 unique patients had 22 552 hospital admissions that included an ICU admission; 19 058 surgical procedures were performed with a total of 20 092 ICU admissions. There were 4936 deaths. Median follow-up was 6.2 years, totalling 134 203 patient years. The casemix was predominantly cardiac surgery (80%), followed by cardiac medical (6%), and other medical (4%). The unadjusted survival at 1, 5 and 10 years was 97%, 84% and 70%, respectively. The 1-year survival ranged from 97% for cardiac surgery to 36% for cardiac arrest. An APACHE II score was available for 16 877 patients. In those discharged alive from hospital, the 1, 5 and 10-year survival varied with discharge location. Conclusions: ICU-based linkage projects are feasible to determine long-term outcomes of ICU patients

Access to medicine and the dangers of patent linkage : lessons from Bayer Corp v. Union of India

In February 2010, the Delhi High Court delivered its decision in Bayer Corp v Union of India in which Bayer had appealed against an August 2009 decision of the same court. Both decisions prevented Bayer from introducing the concept of patent linkage into India’s drug regulatory regime. Bayer appealed to the Indian Supreme Court, the highest court in India, which agreed on 2 March 2010 to hear the appeal. Given that India is regarded as a global pharmaceutical manufacturer of generic medications, how its judiciary and government perceive their international obligations has a significant impact on the global access to medicines regime. In rejecting the application of patent linkage, the case provides an opportunity for India to further acknowledge its international human rights obligations.

Linkage to chromosome 2q32.2-q33.3 in familial serrated neoplasia (Jass syndrome)

Abstract Causative genetic variants have to date been identified for only a small proportion of familial colorectal cancer (CRC). While conditions such as Familial Adenomatous Polyposis and Lynch syndrome have well defined genetic causes, the search for variants underlying the remainder of familial CRC is plagued by genetic heterogeneity. The recent identification of families with a heritable predisposition to malignancies arising through the serrated pathway (familial serrated neoplasia or Jass syndrome) provides an opportunity to study a subset of familial CRC in which heterogeneity may be greatly reduced. A genome-wide linkage screen was performed on a large family displaying a dominantly-inherited predisposition to serrated neoplasia genotyped using the Affymetrix GeneChip Human Mapping 10 K SNP Array. Parametric and nonparametric analyses were performed and resulting regions of interest, as well as previously reported CRC susceptibility loci at 3q22, 7q31 and 9q22, were followed up by finemapping in 10 serrated neoplasia families. Genome-wide linkage analysis revealed regions of interest at 2p25.2-p25.1, 2q24.3-q37.1 and 8p21.2-q12.1. Finemapping linkage and haplotype analyses identified 2q32.2-q33.3 as the region most likely to harbour linkage, with heterogeneity logarithm of the odds (HLOD) 2.09 and nonparametric linkage (NPL) score 2.36 (P = 0.004). Five primary candidate genes (CFLAR, CASP10, CASP8, FZD7 and BMPR2) were sequenced and no segregating variants identified. There was no evidence of linkage to previously reported loci on chromosomes 3, 7 and 9.

Creating better learning environments by cross-disciplinary collaboration : a civil engineering and surveying linkage

Many of the teaching elements in Civil and Environmental Engineering and Spatial Science/Surveying are strongly related to multidisciplinary real-world situations. Professionals in each discipline commonly work collaboratively, knowing each other’s professional and technical limitations and requirements. Replication of such real-world situations allows students to gain an insight and acquire knowledge of professional practice for both civil engineering and spatial science disciplines. However, replication of an authentic design project is not always possible in a single unit basis where empirical project situations are often created with controlled sets of constraints, inputs and outputs. A cross-disciplinary design-based project that is designed to promote active student learning, engagement and professional integration would be the preferred option. The central aim of this collaborative project was to create positive and inclusive environments to promote engaging learning opportunities that cater for a range of learning styles with a two-way linkage involving third-year civil engineering and spatial science (surveying) students. This paper describes the cross-disciplinary project developed and delivered in 2010 and 2011. A survey was conducted at completion of the project to assess the degree of improvement in student engagement and their learning experiences. Improvements were assessed in a range of dimensions including student motivation, learning by cross-disciplinary collaboration and learning by authentic design project experiences. In this specific cross-disciplinary linkage project, the study findings showed that teaching approaches utilised have been effective in promoting active student learning and increasing engagement.

Assessing the effectiveness of the child safety net : linkage of hospital and child protection services data on ‘maltreated’ and ‘unintentionally injured' children

-International recognition of need for public health response to child maltreatment -Need for early intervention at health system level -Important role of health professionals in identifying, reporting, documenting suspician of maltreatment -Up to 10% of all children presenting at ED’s are victims and without identification, 35% reinjured and 5% die -In Qld, mandatory reporting requirement for doctors and nurses for suspected abuse or neglect

Validation of de-identified record linkage to ascertain hospital admissions in a cohort study

Background Cohort studies can provide valuable evidence of cause and effect relationships but are subject to loss of participants over time, limiting the validity of findings. Computerised record linkage offers a passive and ongoing method of obtaining health outcomes from existing routinely collected data sources. However, the quality of record linkage is reliant upon the availability and accuracy of common identifying variables. We sought to develop and validate a method for linking a cohort study to a state-wide hospital admissions dataset with limited availability of unique identifying variables. Methods A sample of 2000 participants from a cohort study (n = 41 514) was linked to a state-wide hospitalisations dataset in Victoria, Australia using the national health insurance (Medicare) number and demographic data as identifying variables. Availability of the health insurance number was limited in both datasets; therefore linkage was undertaken both with and without use of this number and agreement tested between both algorithms. Sensitivity was calculated for a sub-sample of 101 participants with a hospital admission confirmed by medical record review. Results Of the 2000 study participants, 85% were found to have a record in the hospitalisations dataset when the national health insurance number and sex were used as linkage variables and 92% when demographic details only were used. When agreement between the two methods was tested the disagreement fraction was 9%, mainly due to "false positive" links when demographic details only were used. A final algorithm that used multiple combinations of identifying variables resulted in a match proportion of 87%. Sensitivity of this final linkage was 95%. Conclusions High quality record linkage of cohort data with a hospitalisations dataset that has limited identifiers can be achieved using combinations of a national health insurance number and demographic data as identifying variables.