Spatiotemporal micropatterning of cells on arbitrary substrates

This paper presents a readily accessible patterning platform--based upon geometric constraints, discrete cell suspension droplets, and controlled cell settling--that provides both temporal and spatial patterning capabilities. As a demonstration, single-cell (and bead) suspensions as well as multicellular human embryonic stem cell colonies are spatiotemporally patterned onto arbitrary substrates. These substrates include tissue culture surfaces, cell monolayers, protein-coated surfaces, and 3D gel matrices. The generation of soluble factor gradients is also demonstrated. This method is completely passive and does not require external power sources. Spatiotemporal patterning provides a foundation for future biological studies that explore the time-dependent relationships between cell-cell signaling and cellular responses.     

Microfluidic based platform for characterization of protein interactions in hydrogel nanoenvironments

Hydrogel posts in microfluidic devices were investigated as reaction environments for characterizing protein interactions with the goal of mimicking the complexity of a biological environment. The hydrogel environment can be easily tuned to study specific properties of the biological environment. In this study, the hydrogel pore size was tuned to mimic the effect of confinement/crowding on protein interactions. Arrays of polyacrylamide posts of different cross-link ratios (4 and 10%) were fabricated inside microfluidic channels via photopolymerization. Fluorescence-labeled proteins (protein A (PA) and immunoglobulins (IgG)) were transported into the posts via diffusion, and their interaction was studied using FRET. As the pore size of the hydrogel decreased, the binding between the proteins was enhanced. The degree to which crowding enhances a binding interaction depends on the intrinsic properties of the proteins; we observed that, inside the hydrogel post, the PA-goat IgG affinity was increased more than PA-rabbit IgG affinity. The integration of controlled nanoenvironments (hydrogels) with controlled microenvironments (microchannels) provides enhanced parametric control for studying protein interactions, which would be beneficial in developing sensors, in diagnostics, and for mimicking the biological environment at both the cell and the tissue level.     

Capillary electrophoretic screening for the inhibition of homocysteine thiolactone-induced protein oligomerization

We report the first demonstration of rapid electrophoretic monitoring of homocysteine thiolactone-induced protein oligomerization (HTPO), a unique type of post-translational protein modification that may have clinical significance as an indicator of cardiovascular and neurovascular diseases. HTPO of the model protein bovine cytochrome c was initiated in vitro. The relative monomer and aggregate levels of the resultant protein mixtures were determined following separation using capillaries coated with the cationic polymer, poly(diallyldimethylammonium chloride). UV detection provided adequate sensitivity for the monitoring of higher order species, which exist at relatively low concentrations in the protein reaction mixture as compared to the monomeric species. Separations performed under standard injection conditions were optimized on the basis of applied voltage and sample denaturation conditions. Separations performed using short-end injection allowed for more rapid analyses, typically in less than 70 s. Relative errors for run-to-run migration times were less than 0.5%. This novel oligomeric system provides a rapid and straightforward in vitro method to screen therapeutic agents for their ability to inhibit HTPO. Changes in peak area for monomer and aggregate species were used to assess HTPO inhibition as a function of pyridoxal 5-phosphate (PLP) concentration. PLP was shown to effectively inhibit HTPO in vitro. Rapid analysis times of approximately 1.5 min were achieved for inhibition screening.     

Diagnostic radiology in Norway trends in examination frequency and collective effective dose

The objective of the present work was to determine current levels and recent nationwide trends in radiological examination frequency, as well as to update corresponding collective effective dose estimates. Examination frequencies were obtained from radiology management systems at all hospitals and private radiology enterprises across Norway in terms of number of examination codes. During the last decade, the overall examination frequency increased by 16% to 910 per 1000 inhabitants, excluding nuclear imaging and dental radiology. The largest increase in examination frequency occurred in MRI (10-fold increase), followed by CT (more than doubling) and mammography (nearly 70% increase). The contribution to collective effective dose from radiological examinations was estimated to 4960 man Sv or 1.09 mSv per inhabitant; representing a 40% increase from 1993 to 2002. CT contribution to collective effective dose was estimated to account for 59% of the total as opposed to 30% in the previous survey.     

Prebiotic and other health-related effects of cereal-derived arabinoxylans, arabinoxylan-oligosaccharides, and xylooligosaccharides

Arabinoxylans (AX) from cereals are cell wall components that constitute an important part of the dietary fiber intake in humans. Enzymatic hydrolysis of AX yields arabinoxylan-oligosaccharides (AXOS), consisting of arabinoxylooligosaccharides and xylooligosaccharides (XOS). This reaction takes place in the production of AXOS and of cereal-derived food products such as bread and beer, as well as in the colon upon ingestion of AX. This review mainly focuses on the available evidence that AXOS and XOS exert prebiotic effects in the colon of humans and animals through selective stimulation of beneficial intestinal microbiota. In addition, in vitro experiments and in vivo intervention studies on animals or humans are discussed that have investigated potential health-related effects resulting from the dietary intake of AX, AXOS, or XOS.     

Glucoraphanin does not reduce plasma homocysteine in rats with sufficient Se supply via the induction of liver ARE-regulated glutathione biosynthesis enzymes

Data from human and animal trials have revealed contradictory results regarding the influence of selenium (Se) status on homocysteine (HCys) metabolism. It was hypothesised that sufficient Se reduces the flux of HCys through the transsulphuration pathway by decreasing the expression of glutathione (GSH) synthesising enzymes. Glucoraphanin (GRA) is a potent inducer of genes regulated via an antioxidant response element (ARE), including those of GSH biosynthesis. We tested the hypothesis that GRA supplementation to rat diets lowers plasma HCys levels by increasing GSH synthesis. Therefore 96 weaned albino rats were assigned to 8 groups of 12 and fed diets containing four different Se levels (15, 50, 150 and 450 μg kg(diet)(-1)), either without GRA (groups: C15, C50, C150 and C450) or in combination with 700 μmol GRA kg(diet)(-1) (groups G15, G50, G150 and G450). Rats fed the low Se diets C15 and G15 showed an impressive decrease of plasma HCys. Se supplementation increased plasma HCys and lowered GSH significantly by reducing the expression of GSH biosynthesis enzymes. As new molecular targets explaining these results, we found a significant down-regulation of the hepatic GSH exporter MRP4 and an up-regulation of the HCys exporter Slco1a4. In contrast to our hypothesis, GRA feeding did not reduce plasma HCys levels in Se supplemented rats (G50, G150 and 450) through inducing GSH biosynthesis enzymes and MRP4, but reduced their mRNA in some cases to a higher extent than Se alone. We conclude: 1. That the long-term supplementation of moderate GRA doses reduces ARE-driven gene expression in the liver by increasing the intestinal barrier against oxidative stress. 2. That the up-regulation of ARE-regulated genes in the liver largely depends on GRA cleavage to free sulforaphane and glucose by plant-derived myrosinase or bacterial β-glucosidases. As a consequence, higher dietary GRA concentrations should be used in future experiments to test if GRA or sulforaphane can be established as HCys lowering compounds.     

Induction of malaria parasite migration by synthetically tunable microenvironments

Interaction of Plasmodium sporozoites, the forms of the malaria parasite transmitted by the mosquito, with its microenvironment in form of adhesion and migration is essential for the successful establishment of infection. Myosin-based sporozoite migration relies on short and dynamic actin filaments. These are linked to transmembrane receptors, which in turn bind to the matrix microenvironment. In this work, we are able to define the characteristics that determine whether a matrix is favorable or adverse to sporozoite adhesion and motility using a specifically tunable hydrogel system decorated with gold nanostructures of defined interparticle spacing each equipped with molecules acting as receptor adhesion sites. We show that sporozoites migrate most efficiently on substrates with adhesion sites spaced between 55 and 100 nm apart. Sporozoites migrating on such substrates are more resilient toward disruption of the actin cytoskeleton than parasites moving on substrates with smaller and larger interparticle spacings. Plasmodium sporozoites adhesion and migration was also more efficient on stiff, bonelike interfaces than on soft, skinlike ones. Furthermore, in the absence of serum albumin, previously thought to be essential for motility, sporozoite movement was comparable on substrates functionalized with RGD- and RGE-peptides. This suggests that adhesion formation is sufficient for activating migration, and that modulation of adhesion formation and turnover during migration is efficiently controlled by the material parameters of the microenvironment, that is, adhesion site spacing and substrate stiffness. Our results and approaches provide the basis for a precise dissection of the mechanisms underlying Plasmodium sporozoites migration.