An improved capillary model for describing the microstructure characteristics, fluid hydrodynamics and breakthrough performance of proteins in cryogel beds

A capillary-based model modified for characterization of monolithic cryogels is presented with key parameters like the pore size distribution, the tortuosity and the skeleton thickness employed for describing the porous structure characteristics of a cryogel matrix. Laminar flow, liquid dispersion and mass transfer in each capillary are considered and the model is solved numerically by the finite difference method. As examples, two poly(hydroxyethyl methacrylate) (pHEMA) based cryogel beds have been prepared by radical cryo-copolymerization of monomers and used to test the model. The axial dispersion behaviors, the pressure drop vs. flow rate performance as well as the non-adsorption breakthrough curves of different proteins, i.e., lysozyme, bovine serum albumin (BSA) and concanavalin A (Con A), at various flow velocities in the cryogel beds are measured experimentally. The lumped parameters in the model are determined by matching the model prediction with the experimental data. The results showed that for a given cryogel column, by using the model based on the physical properties of the cryogel (i.e., diameter, length, porosity, and permeability) together with the protein breakthrough curves one can obtain a reasonable estimate and detailed characterization of the porous structure properties of cryogel matrix, particularly regarding the number of capillaries, the capillary tortuousness, the pore size distribution and the skeleton thickness. The model is also effective with regards to predicting the flow performance and the non-adsorption breakthrough profiles of proteins at different flow velocities. It is thus expected to be applicable for characterizing the properties of cryogels and predicting the chromatographic performance under a given set of operating conditions.     

Fibronectin adsorption on gold, Ti-, and Ta-oxide investigated by QCM-D and RSA modelling

The adsorption of fibronectin on gold, Ti-, and Ta-oxide surfaces is investigated by means of the quartz crystal microbalance with dissipation (QCM-D) technique. The surface chemistry (gold, Ti-, and Ta-oxide) is found to influence the frequency shift observed during adsorption of the fibronectin layer with the magnitude being Delta f Au>Delta f Ti-oxide approximately Delta f Ta-oxide. Corresponding variations in the dissipation change normalised to frequency change (Delta D/Delta f) for the layer are observed. The QCM-D data are further analyzed by the random sequential adsorption (RSA) model, and adsorption rate parameter ka and footprint (a) determined, which supported the trend seen in the Delta f and Delta D/Delta f values. The value of ka found by the RSA modelling of the QCM-D resonance frequency data is found to match the ratio between the mass measured by QCM-D and the mass reported by optical techniques in literature. We conclude that comparison of the adsorption rate parameter (ka) obtained by RSA modelling of the QCM-D data with ka values obtained from RSA modelling of data obtained using optical techniques can be a route to determine the degree of hydration of the adsorbed protein layer.     

High-throughput, low-volume, multianalyte quantification of plasma metabolites related to one-carbon metabolism using HPLC-MS/MS

Risk of chronic diseases, like cardiovascular disease and cancer, has been associated with biomarkers related to one-carbon metabolism, which comprises a metabolic network of cross-talking pathways. To address this complexity in epidemiological studies, we have established an isotope dilution HPLC-MS/MS method for quantification of 12 biomarkers and metabolites. All sample handling is performed by a robotic workstation. The assay uses 45 μL of plasma, and sample treatment consists of protein precipitation by trichloroacetic acid. The analytes were separated on a Fortis Phenyl column using an isocratic mobile phase that contained water, methanol and acetic acid. Methionine, methionine sulfoxide, choline, betaine, dimethylglycine, arginine, asymmetric dimethylarginine, symmetric dimethylarginine, homoarginine, creatinine, cystathionine and trimethyllysine all showed limits of detection well below the 5th percentile of plasma distributions in healthy humans, coefficients of variation were in the range 2.2–12.3 %, and recoveries were 80–131 %. Simple sample processing, low-volume consumption, multiplexing and high capacity/short run time of this method make it suitable for large-scale metabolic profiling of precious biobank samples.     

Adsorption of fibrinogen on tantalum oxide, titanium oxide and gold studied by the QCM-D technique

The adsorption of human fibrinogen on tantalum oxide, titanium oxide and gold surfaces has been studied by quartz crystal microbalance with dissipation (QCM-D) at 37 degrees C. Two different protein concentrations have been used, one close to physiological concentration (1 mg/ml) and one significantly lower (0.033 mg/ml). To further characterize the adsorbed fibrinogen layer, the subsequent binding of both polyclonal and monoclonal antibodies of fibrinogen is studied. We found that the viscoelastic properties of the fibrinogen layer depends strongly on the initial protein concentration. The trend is generally seen for all three surfaces. The fibrinogen layer on gold and tantalum oxide is found to have the same viscoelastic properties, which are different from those found for the fibrinogen layer adsorbed on titanium oxide. The dependency of the surface chemistry on the viscoelastic properties of the fibrinogen layer is observed directly for the 0.033 mg/ml solution, and indirectly through the antibody response for the 1 mg/ml solution. From this we conclude that the orientation and/or denaturation of fibrinogen on a surface depends on the surface chemistry and the protein concentration in the solution, and that the binding of antibodies is a useful way to detect this difference.     

Molecular characterization of protein kinase C delta (PKCδ)-Smac interactions

Background

Protein kinase C δ (PKCδ) is known to be an important regulator of apoptosis, having mainly pro- but also anti-apoptotic effects depending on context. In a previous study, we found that PKCδ interacts with the pro-apoptotic protein Smac. Smac facilitates apoptosis by suppressing inhibitor of apoptosis proteins (IAPs). We previously established that the PKCδ-Smac complex dissociates during induction of apoptosis indicating a functional importance. Because the knowledge on the molecular determinants of the interaction is limited, we aimed at characterizing the interactions between PKCδ and Smac.

Results

We found that PKCδ binds directly to Smac through its regulatory domain. The interaction is enhanced by the PKC activator TPA and seems to be independent of PKCδ catalytic activity since the PKC kinase inhibitor GF109203X did not inhibit the interaction. In addition, we found that C1 and C2 domains from several PKC isoforms have Smac-binding capacity.

Conclusions

Our data demonstrate that the Smac-PKCδ interaction is direct and that it is facilitated by an open conformation of PKCδ. The binding is mediated via the PKCδ regulatory domain and both the C1 and C2 domains have Smac-binding capacity. With this study we thereby provide molecular information on an interaction between two apoptosis-regulating proteins.

     

Adaptive contrast enhancement of two-dimensional electrophoretic protein gel images facilitates visualization, orientation and alignment

2-DE is a powerful technique to discriminate post-translationally modified protein isoforms. However, all steps of 2-DE preparation and gel-staining may introduce unwanted artefacts, including inconsistent variation of background intensity over the entire 2-DE gel image. Background intensity variations limit the accuracy of gel orientation, overlay alignment and spot detection methods. We present a compact and efficient denoising algorithm that adaptively enhances the image contrast and then, through thresholding and median filtering, removes the gray-scale range covering the background. Applicability of the algorithm is demonstrated on immunoblots, isotope-labeled gels, and protein-stained gels. Validation is performed in contexts of (i) automatic gel orientation based on Hough transformation, (ii) overlay alignment based on cross correlation and (iii) spot detection. In gel stains with low background variability, e.g. Sypro Ruby, denoising will lower the spot detection sensitivity. In gel regions with high background levels denoising enhances spot detection. We propose that the denoising algorithm prepares images with high background for further automatic analysis, without requiring manual input on a gel-to-gel basis.     

Filtered thermal contrast based technique for testing of material by infrared thermography

A new kind of thermal contrast, called “filtered contrast” is presented, which allows detecting and characterizing material defects using active thermography under some assumptions on physical and thermal parameters of materials. In opposition to known definitions of the thermal contrast, knowledge about defect-free area is not necessary and this contrast is less sensitive to nonuniformity of heat disposal to the material surface. The measurements were performed on an experimental setup equipped with a ThermaCAM PM 595 infrared camera and frame grabber. The step heating was chosen as heat excitation. The results demonstrate usefulness of the 1D model of heat transfer used for determination of depth of subsurface defects. The influence of the parameter of the smoothing filter, required for filtered contrast implementation, thermal parameters of the tested material and defect on expanded uncertainty of determination of defect depth is also presented. Due to significant complexity of the model of heat transfer, the conditions for the “law of propagation of uncertainty” were not fulfilled and a numerical method, i.e., Monte Carlo simulation is applied for the propagation of distributions.     

Surface roughness and color characteristics of wood treated with preservatives after accelerated weathering test

Wood samples treated with ammonium copper quat (ACQ 1900 and ACQ 2200), chromated copper arsenate (CCA), Tanalith E 3491 and Wolmanit CX-8 have been studied in accelerated weathering experiments. The weathering experiment was performed by cycles of 2 h UV-light irradiation followed by water spray for 18 min. The changes on the surface of the weathered samples were characterized by roughness and color measurements on the samples with 0, 200, 400 and 600 h of total weathering.

The objective of this study was to investigate the changes created by weathering on impregnated wood with several different wood preservatives. This study was performed on the accelerated weathering test cycle, using UV irradiation and water spray in order to simulate natural weathering.

Surface roughness and color measurement was used to investigate the changes after several intervals (0–200–400–600 h) in artificial weathering of treated and untreated wood.     

Mapping low-resolution three-dimensional protein structures using chemical cross-linking and Fourier transform ion-cyclotron resonance mass spectrometry

Techniques in mass spectrometry (MS) combined with chemical cross-linking have proven to be efficient tools for the rapid determination of low-resolution three-dimensional (3-D) structures of proteins. The general procedure involves chemical cross-linking of a protein followed by enzymatic digestion and MS analysis of the resulting peptide mixture. These experiments are generally fast and do not require large quantities of protein. However, the large number of peptide species created from the digestion of cross-linked proteins makes it difficult to identify relevant intermolecular cross-linked peptides from MS data. We present a method for mapping low-resolution 3-D protein structures by combining chemical cross-linking with high-resolution FTICR (Fourier transform ion-cyclotron resonance) mass spectrometry using cytochrome c and hen egg lysozyme as model proteins. We applied several homo-bifunctional, amine-reactive cross-linking reagents that bridge distances from 6 to 16 A. The non-digested cross-linking reaction mixtures were monitored by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) to determine the extent of cross-linking. Enzymatically digested reaction mixtures were separated by nano-high-performance liquid chromatography (nano-HPLC) on reverse-phase columns applying water/acetonitrile gradients with flow rates of 200 nL/min. The nano-HPLC system was directly coupled to an FTICR mass spectrometer equipped with a nano-ESI (electrospray ionization) source. Cross-linking products were identified using a combination of the GPMAW software and ExPASy Proteomics tools. For correct assignment of the cross-linking products the key factor is to rely on a mass spectrometric method providing both high resolution and high mass accuracy, such as FTICRMS. By combining chemical cross-linking with FTICRMS we were able to rapidly define several intramolecular constraints for cytochrome c and lysozyme.