Detection of parent-of-origin effects in nuclear families using haplotype analysis

Despite the potential pitfalls of stratification, population-based association studies nowadays are being conducted more often than family-based association studies. However, the mechanism of genomic imprinting has lately been implicated in the etiology of genetic complex diseases and can be detected using statistics only in family-based designs. Powerful tests for association and imprinting have been proposed previously for case-parent trios and single markers. Since the power of association studies can be improved if multiple affected children and haplotypes are considered, we extended the parental asymmetry test (PAT) for imprinting to a test that is suited for both general nuclear families and haplotypes, called HAP-PAT. Significance of the HAP-PAT is determined via a Monte-Carlo simulation procedure. In addition to the HAP-PAT, we modified a haplotype-based association test, proposed by us before, in such a way that either only paternal or maternal transmissions contribute to the test statistic. The approaches were implemented in FAMHAP and we evaluated their performance under a variety of disease models. We were able to demonstrate the usefulness of our haplotype-based approaches to detect parent-of-origin effects. Furthermore, we showed that also in the presence of imprinting it is more reasonable to consider all affected children of a nuclear family, than to randomly select one affected child from each family and to conduct a trio study using the selected individuals.     

Alginate-PLL microencapsulation: effect on the differentiation of embryonic stem cells into hepatocytes

The emergence of hepatocyte based clinical and pharmaceutical technologies, has been limited by the absence of a stable hepatocyte cell source. Embryonic stem cells may represent a potential solution to this cell source limitation problem since they are highly proliferative, renewable, and pluripotent. Although many investigators have described techniques to effectively differentiate stem cells into a variety of mature cell lineages, their practicality is limited by: (1) low yields of fully differentiated cells, (2) absence of large scale processing considerations, and (3) ineffective downstream enrichment protocols. Thus, a differentiation platform that may be modified to induce and sustain differentiated cell function and scaled to increase differentiated cell yield would improve current stem cell differentiation strategies. Microencapsulation provides a vehicle for the discrete control of key cell culture parameters such as the diffusion of growth factors, metabolites, and wastes. In addition, both cell seeding density and bead composition may be manipulated. In order to assess the feasibility of directing stem cell differentiation via microenvironment regulation, we have developed a murine embryonic stem cell (ES) alginate poly-l-lysine microencapsulation hepatocyte differentiation system. Our results indicate that the alginate microenvironment maintains cell viability, is conducive to ES cell differentiation, and maintains differentiated cellular function. This system may ultimately assist in developing scalable stem cell differentiation strategies.     

A characterization of DNA release in Pseudomonas aeruginosa cultures and biofilms

Pseudomonas aeruginosa produces extracellular DNA which functions as a cell-to-cell interconnecting matrix component in biofilms. Comparison of extracellular DNA and chromosomal DNA by the use of polymerase chain reaction and Southern analysis suggested that the extracellular DNA is similar to whole-genome DNA. Evidence that the extracellular DNA in P. aeruginosa biofilms and cultures is generated via lysis of a subpopulation of the bacteria was obtained through experiments where extracellular beta-galactosidase released from lacZ-containing P. aeruginosa strains was assessed. Experiments with the wild type and lasIrhlI, pqsA, pqsL and fliMpilA mutants indicated that the extracellular DNA is generated via a mechanism which is dependent on acyl homoserine lactone and Pseudomonas quinolone signalling, as well as on flagella and type IV pili. Microscopic investigation of flow chamber-grown wild-type P. aeruginosa biofilms stained with different DNA stains suggested that the extracellular DNA is located primarily in the stalks of mushroom-shaped multicellular structures, with a high concentration especially in the outer part of the stalks forming a border between the stalk-forming bacteria and the cap-forming bacteria. Biofilms formed by lasIrhlI, pqsA and fliMpilA mutants contained less extracellular DNA than biofilms formed by the wild type, and the mutant biofilms were more susceptible to treatment with sodium dodecyl sulphate than the wild-type biofilm.     

Helicobacter pylori HtrA is a new secreted virulence factor that cleaves E‐cadherin to disrupt intercellular adhesion

Mammalian and prokaryotic high‐temperature requirement A (HtrA) proteins are chaperones and serine proteases with important roles in protein quality control. Here, we describe an entirely new function of HtrA and identify it as a new secreted virulence factor from Helicobacter pylori, which cleaves the ectodomain of the cell‐adhesion protein E‐cadherin. E‐cadherin shedding disrupts epithelial barrier functions allowing H. pylori designed to access the intercellular space. We then designed a small‐molecule inhibitor that efficiently blocks HtrA activity, E‐cadherin cleavage and intercellular entry of H. pylori.     

HDAC-mediated control of ERK- and PI3K-dependent TGF-β-induced extracellular matrix-regulating genes

Histone deacetylases (HDACs) regulate the acetylation of histones in the control of gene expression. Many non-histone proteins are also targeted for acetylation, including TGF-β signalling pathway components such as Smad2, Smad3 and Smad7. Our studies in mouse C3H10T1/2 fibroblasts suggested that a number of TGF-β-induced genes that regulate matrix turnover are selectively regulated by HDACs. Blockade of HDAC activity with trichostatin A (TSA) abrogated the induction of a disintegrin and metalloproteinase 12 (Adam12) and tissue inhibitor of metalloproteinases-1 (Timp-1) genes by TGF-β, whereas plasminogen activator inhibitor-1 (Pai-1) expression was unaffected. Analysis of the activation of cell signalling pathways demonstrated that TGF-β induced robust ERK and PI3K activation with delayed kinetics compared to the phosphorylation of Smads. The TGF-β induction of Adam12 and Timp-1 was dependent on such non-Smad signalling pathways and, importantly, HDAC inhibitors completely blocked their activation without affecting Smad signalling. Analysis of TGF-β-induced Adam12 and Timp-1 expression and ERK/PI3K signalling in the presence of semi-selective HDAC inhibitors valproic acid, MS-275 and apicidin implicated a role for class I HDACs. Furthermore, depletion of HDAC3 by RNA interference significantly down-regulated TGF-β-induced Adam12 and Timp-1 expression without modulating Pai-1 expression. Correlating with the effect of HDAC inhibitors, depletion of HDAC3 also blocked the activation of ERK and PI3K by TGF-β. Collectively, these data confirm that HDACs, and in particular HDAC3, are required for activation of the ERK and PI3K signalling pathways by TGF-β and for the subsequent gene induction dependent on these signalling pathways.     

Design of a data model for developing laboratory information management and analysis systems for protein production

Data management has emerged as one of the central issues in the high-throughput processes of taking a protein target sequence through to a protein sample. To simplify this task, and following extensive consultation with the international structural genomics community, we describe here a model of the data related to protein production. The model is suitable for both large and small facilities for use in tracking samples, experiments, and results through the many procedures involved. The model is described in Unified Modeling Language (UML). In addition, we present relational database schemas derived from the UML. These relational schemas are already in use in a number of data management projects.     

Ecohydrology of a seasonal wetland in the Rift Valley: ecological characterization of Lake Solai

The following research describes through an ecohydrological approach, the first assessment of the ecology of Lake Solai, with a particular emphasis on the vegetation. Lake Solai is located 50 km north of Nakuru in the Rift Valley in Kenya at E36°80'–36°84' to N00°05'–00°08'. It is a shallow lake that follows a very peculiar seasonal water regime, and that faces conflicts between agriculture and conservation water users. In the upper catchment, an overview of the agricultural practices was implemented and river water uses were identified to assess river flows. Crops/grassland and woodland/shrubland were the major land uses, covering c. 65% of the catchment. Closer to the lake, vegetation samples were collected around the lake together with samples of environmental factors such as soil and water quality. Thirteen vegetation communities were identified within four main zonations: forest, grassland, river inlet and rocky outcrop. These communities showed abundance, distribution and diversity determined mostly by the human pressures, the flooding periods and the salinity. Cynodon , Cyperus and Sporobolus genera were the most abundant.