Microfluidic device generating stable concentration gradients for long term cell culture: application to Wnt3a regulation of β-catenin signaling

In developing tissues, proteins and signaling molecules present themselves in the form of concentration gradients, which determine the fate specification and behavior of the sensing cells. To mimic these conditions in vitro, we developed a microfluidic device designed to generate stable concentration gradients at low hydrodynamic shear and allowing long term culture of adhering cells. The gradient forms in a culture space between two parallel laminar flow streams of culture medium at two different concentrations of a given morphogen. The exact algorithm for defining the concentration gradients was established with the aid of mathematical modeling of flow and mass transport. Wnt3a regulation of β-catenin signaling was chosen as a case study. The highly conserved Wnt-activated β-catenin pathway plays major roles in embryonic development, stem cell proliferation and differentiation. Wnt3a stimulates the activity of β-catenin pathway, leading to translocation of β-catenin to the nucleus where it activates a series of target genes. We cultured A375 cells stably expressing a Wnt/β-catenin reporter driving the expression of Venus, pBARVS, inside the microfluidic device. The extent to which the β-catenin pathway was activated in response to a gradient of Wnt3a was assessed in real time using the BARVS reporter gene. On a single cell level, the β-catenin signaling was proportionate to the concentration gradient of Wnt3a; we thus propose that the modulation of Wnt3a gradients in real time can provide new insights into the dynamics of β-catenin pathway, under conditions that replicate some aspects of the actual cell-tissue milieu. Our device thus offers a highly controllable platform for exploring the effects of concentration gradients on cultured cells.     

alpha(2A)-adrenergic receptor derived peptide adsorbates: a G-protein interaction study

The affinity of alpha(2A)-adrenergic receptor (alpha(2A)-AR) derived peptide adsorbates for the functional bovine brain G-protein is studied in the search for the minimum sequence recognition. Three short peptides (GPR-i2c, GPR-i3n, and GPR-i3c) are designed to mimic the second and third intracellular loops of the receptor. X-ray photoelectron spectroscopy is used to study the chemical composition of the peptides and the binding strength to the surfaces. Chemisorption of the peptides to the gold substrates is observed. Infrared spectroscopy is used to study the characteristic absorption bands of the peptides. The presence of peptides on the surfaces is verified by prominent amide I and amide II bands. The interaction between the peptides and the G-protein is studied with surface plasmon resonance. It is shown that GPR-i3n has the highest affinity for the G-protein. Equilibrium analysis of the binding shows that the G-protein keeps its native conformation when interacting with GPR-i3c, but during the interaction with GPR-i2c and GPR-i3n the conformation of G-protein is changed, leading to the formation of aggregates and/or multilayers.     

Interpretation of non-Nernstian slopes in graphic analysis of data collected in pH range close to deprotonation of a ligand Part I. A glass electrode potentiometric and polarographic study of Cd-(TAPSO)x-(OH)y and Zn-(TAPSO)x-(OH)y systems

In this work, the complexation of cadmium and zinc ions by 3-[N-tris(hydroxymethyl)methylamine]-2-hydroxypropanesulfonic acid (TAPSO), a commercial biological buffer, was evaluated using three electrochemical techniques, at fixed total-ligand and total-metal concentration ratio and varied pH, at 25.0+/-0.1 degrees C and ionic strength set to 0.1M KNO(3). For both metal-ligand systems, complexation was evidenced in the pH range close to deprotonation of the ligand and the final models were optimised after a meticulous graphical analysis. For Cd-(TAPSO)(x)-(OH)(y) system, two complexes, CdL and CdL(2), were identified in the buffering region of the ligand. The proposed final model for this system is: CdL, CdL(2) and CdL(2)(OH) with stability constants, as logbeta, of 2.2, 4.2 and 8.6, respectively. For Zn-(TAPSO)(x)-(OH)(y) system, the complex ZnL is the main species formed in the buffering pH range. The proposed final model is ZnL, ZnL(OH) and ZnL(OH)(2) with overall refined stability constants (as logbeta) to be: 2.5, 7.2 and 13.2, respectively.     

Characterisation of two novel cyclodextrinases using on-line microdialysis sampling with high-performance anion exchange chromatography

In this work, a real-time sampling/analytical method for on-line measurements of two newly discovered cyclomaltodextrinases (CDases) has been developed and evaluated. This novel methodology not only allows the final products to be investigated, but it also reveals enzyme-specific differences in the degradation pathways during the hydrolysis of different substrates, which is a great advantage in the important tasks of investigating the mechanisms of and classifying new hydrolases, and is an advantage that conventional techniques cannot offer. Two different enzymes, one CDase from Laceyella sacchari (LsCda13) and one from Anoxybacillus flavithermus (AfCda13), were investigated during the hydrolysis of α-, β- and γ-cyclodextrin, and the hydrolysis products were sampled via a microdialysis probe and injected on-line every 30 min into a high-performance anion exchange chromatography system equipped with a pulsed amperometric detector (HPAEC–PAD), where they were identified. The enzymes yielded the same end-products, maltose and glucose, in an approximate molar ratio of 2:1, but they exhibited distinctly different patterns of intermediate product formation before reaching the end-point. LsCda13 had a more random distribution of the intermediate products, whereas AfCda13 showed the distinct intermediate production of maltotriose, which in some cases accumulated.     

A New Chain Polymer via Thermolysis: [Hg(μ‐Br)2(3,5‐Br2py)]∞

The new ligand‐deficient chain polymer [Hg(μ‐Br)₂(3,5‐Br₂py)]_∞ has been obtained in form of single crystals by thermolysis of the ligand‐rich [Hg(μ‐Br)₂(3,5‐Br₂py)₂]_∞ at 180 °C at ambient pressure. From this reaction, high quality crystals of the product are directly accessible. The title compound features Hg^II cations in a distorted square‐pyramidal coordination; their metal centers aggregate via edge‐sharing with asymmetric halide bridges to chains in which all apical N donor ligands are oriented to the same side of the [Hg(μ‐Br)₂]_∞ backbone. The new polymer cannot be prepared by stoichiometric reaction in solution.     

Oxidation of propylene to acrylic acid on a Fe?Mo?Bi oxide system

Data obtained in a flow reactor show that iron(III) molybdate doped by bismuth can oxidize propylene to acrylic acid directly in one step. Part of the catalyst works in a partially reduced form and in the preparation of the catalyst the surface acidity must be controlled.

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Synthesis and debenzoylation products of two perbenzoylated 2-substituted 5-D-galactosyl-1,3,4-oxadiazoles

The synthesis of 2-(p-chlorophenyl)-5-[1′,2′,3′,4′,5′-penta-O-benzoyl-D-galactopentitol-1-yl]-1,3,4-oxadiazole is described. Its debenzoylation gave an equilibrium mixture of the 1,3,4-oxadiazole derivative without protection of the hydroxyl group and the N-benzoyl-D-galactono-1,4-lactonehydrazone. A similar equilibrium was observed by debenzoylation of 2-phenyl-5-[1′,2′,3′,4′,5′-penta-O-benzoyl-D-galactopentitol-1-yl]-1,3,4-oxadiazole. The ¹H, ¹³C nmr and ms spectra of these compounds are presented.     

Magnitudes and chemical consequences of R(3)N(+)-C-H...O[double bond]C hydrogen bonding

The magnitude of the stabilizing interaction between an aliphatic C[bond]H bond attached to an ammonium nitrogen and a carbonyl oxygen was evaluated by ab initio calculations at the MP2/6-311++G** level of theory. Attractive R(3)N(+)-C-H...O[double bond]C interactions play an important role in supramolecular recognition and various types of stereoselective catalysis. Our calculations show that R(3)N(+)-C-H...O[double bond]C is the strongest hydrogen bond of the C-H...O type known to date. Such hydrogen bonds remain as stabilizing interactions even in water for amide acceptors.     

Optimization of enzymatic production of biodiesel from castor oil in organic solvent medium

We studied the production of fatty acid ethyl esters from castor oil using n-hexane as solvent and two commercial lipases, Novozym 435 and Lipozyme IM, as catalysts. For this purpose, a Taguchi experimental design was adopted considering the following variables: temperature (35–65°C), water (0–10 wt/wt%), and enzyme (5–20 wt/wt%) concentrations and oil-to-ethanol molar ratio (1∶3 to 1∶10). An empirical model was then built so as to assess the main and cross-variable effects on the reaction conversion and also to maximize biodiesel production for each enzyme. For the system containing Novozym 435 as tatalyst the maximum conversion obtained was 81.4% at 65°C, enzyme concentration of 20 wt/wt%, water concentration of 0 wt/wt%, and oil-to-ethanol molar ratio of 1∶10. When the catalyst was Lipozyme IM, a conversion as high as 98% was obtained at 65°C, enzyme concentration of 20 wt/wt%, water concentration of 0 wt/wt%, and oil-to-ethanol molar ratio of 1∶3.