Breaking the DNA-vaccine bottleneck

Monash University in Australia has developed a new approach towards DNA vaccine development that has the potential to cut the time it takes to produce a vaccine from up to nine months to four weeks or less. The university has designed and filed a patent on a commercially viable, single-stage technology for manufacturing DNA molecules. The technology was used to produce malaria and measles DNA vaccines, which were tested to be homogeneous supercoiled DNA, free from RNA and protein contaminations and meeting FDA regulatory standards for DNA vaccines. The technique is based on customized, smart, polymeric, monolithic adsorbents that can purify DNA very rapidly. The design criteria of solid-phase adsorbent include rapid adsorption and desorption kinetics, physical composition, and adequate selectivity , capacity and recovery. The new show technology significantly improved binding capacities, higher recovery, drastically reduced use of buffers and processing time, less clogging, and higher yields of DNA.

Preparation, Analysis and Use of an Affinity Adsorbent for the Purification of GST Fusion Protein

Methods are presented for the preparation, ligand density analysis and use of an affinity adsorbent for the purification of a glutathione S-transferase (GST) fusion protein in packed and expanded bed chromatographic processes. The protein is composed of GST fused to a zinc finger transcription factor (ZnF). Glutathione, the affinity ligand for GST purification, is covalently immobilized to a solid-phase adsorbent (Streamline™). The GST–ZnF fusion protein displays a dissociation constant of 0.6 x10-6 M to glutathione immobilized to Streamline™. Ligand density optimization, fusion protein elution conditions (pH and glutathione concentration) and ligand orientation are briefly discussed.

Plasmid DNA purification via the use of a dual affinity protein

Methods are presented for the production, affinity purification and analysis of plasmid DNA (pDNA). Batch fermentation is used for the production of the pDNA, and expanded bed chromatography, via the use of a dual affinity glutathione S-transferase (GST) fusion protein, is used for the capture and purification of the pDNA. The protein is composed of GST, which displays affinity for glutathione immobilized to a solid-phase adsorbent, fused to a zinc finger transcription factor, which displays affinity for a target 9-base pair sequence contained within the target pDNA. A Picogreen™ fluorescence assay and/or anx ethidium bromide agarose gel electrophoresis assay can be used to analyze the eluted pDNA.

Microscale consolidation analysis of relaxation behavior of single living chondrocytes subjected to varying strain-rates

Besides the elastic stiffness, the relaxation behavior of single living cells is also of interest of various researchers when studying cell mechanics. It is hypothesized that the relaxation response of the cells is governed by both intrinsic viscoelasticity of the solid phase and fluid-solid interactions mechanisms. There are a number of mechanical models have been developed to investigate the relaxation behavior of single cells. However, there is lack of model enable to accurately capture both of the mechanisms. Therefore, in this study, the porohyperelastic (PHE) model, which is an extension of the consolidation theory, combined with inverse Finite Element Analysis (FEA) technique was used at the first time to investigate the relaxation response of living chondrocytes. This model was also utilized to study the dependence of relaxation behavior of the cells on strain-rates. The stress-relaxation experiments under the various strain-rates were conducted with the Atomic Force Microscopy (AFM). The results have demonstrated that the PHE model could effectively capture the stress-relaxation behavior of the living chondrocytes, especially at intermediate to high strain-rates. Although this model gave some errors at lower strain-rates, its performance was acceptable. Therefore, the PHE model is properly a promising model for single cell mechanics studies. Moreover, it has been found that the hydraulic permeability of living chondrocytes reduced with decreasing of strain-rates. It might be due to the intracellular fluid volume fraction and the fluid pore pressure gradients of chondrocytes were higher when higher strain-rates applied.

Mimotopes identified by phage display for the monoclonal antibody CII- C1 to type II collagen

The characterization of B cell epitopes has been advanced by the use of random peptide libraries displayed within the coat protein of bacteriophage. This technique was applied to the monoclonal antibody (mAb) C1 to type II collagen (CII-C1). CII-C1 is known to react with a conformational epitope on type II collagen that includes residues 359-363. Three rounds of selection were used to screen two random nonameric phage libraries and 18 phagotopes were isolated. CII-C1 reacted by ELISA with 17 of the 18 phagotopes: one phagotope contained a stop codon. Of the eight most reactive phage, seven inhibited the reactivity by ELISA of CII-C1 with type II collagen. Of the 18 phage isolated, 11 encoded the motif F-G-x-Q with the sequence F-G-S-Q in 6, 2 encoded F-G-Q, and one the reverse motif Q-x-y-F. Most phagotopes that inhibited the reactivity of CII-C1 encoded two particular motifs consisting of two basic amino acid residues and a hydrophobic residue in the first part of the insert and the F-G-x-Q or F-G-Q motif ill the second part; phagotopes which contained only one basic residue in the first part of the sequence were less reactive. These motifs are not represented in the linear sequence of type II collagen and thus represent mimotopes of the epitope for CII-C1 on type II collagen. There were five phagotopes with peptide inserts containing the sequence RLPFG occurring in the Epstein-Barr virus nuclear antigen, EBNA- 1. This is of interest because EBV has been implicated in the initiation of rheumatoid arthritis (RA) by reason of increased reactivity to EBNA-1 in RA sera. In conclusion, the phage display technique disclosed mimotopes for a conformational epitope of type II collagen, and revealed an interesting homology with a sequence of the EBNA-1 antigen from Epstein Barr virus.

Topological mimicry and epitope duplication in the guanylyl cyclase C receptor

Guanylyl cyclase C (GCC) is the receptor for the gastrointestinal hormones, guanylin, and uroguanylin, in addition to the bacterial heat-stable enterotoxins, which are one of the major causes of watery diarrhea the world over. GCC is expressed in intestinal cells, colorectal tumor tissue and tumors originating from metastasis of the colorectal carcinoma. We have earlier generated a monoclonal antibody to human GCC, GCC:B10, which was useful for the immunohistochemical localization of the receptor in the rat intestine (Nandi A et al., 1997, J Cell Biochem 66:500-511), and identified its epitope to a 63-amino acid stretch in the intracellular domain of GCC. In view of the potential that this antibody has for the identification of colorectal tumors, we have characterized the epitope for GCC:B10 in this study. Overlapping peptide synthesis indicated that the epitope was contained in the sequence HIPPENIFPLE. This sequence was unique to GCC, and despite a short stretch of homology with serum amyloid protein and pertussis toxin, no cross reactivity was detected. The core epitope was delineated using a random hexameric phage display library, and two categories of sequences were identified, containing either a single, or two adjacent proline residues. No sequence identified by phage display was identical to the epitope present in GCC, indicating that phage sequences represented mimotopes of the native epitope. Alignment of these sequences with HIPPENIFPLE suggested duplication of the recognition motif, which was confirmed by peptide synthesis. These studies allowed us not only to define the requirements of epitope recognition by GCC:B10 monoclonal antibody, but also to describe a novel means of epitope recognition involving topological mimicry and probable duplication of the cognate epitope in the native guanylyl cyclase C receptor sequence.

Immunochemical approach to the mapping of an assembled epitope of human chorionic gonadotropin: Proximity of CTP-alpha to the receptor binding region of the beta-subunit

A single-step solid-phase RIA (SS-SPRIA) developed in our laboratory using hybridoma culture supernatants has been utilised for the quantitation of epitope-paratope interactions. Using SS-SPRIA as a quantitative tool for the assessment of epitope stability, it was found that several assembled epitopes of human chorionic gonadotropin (hCG) are differentially stable to proteolysis and chemical modification. Based on these observations an approach has been developed for identifying the amino acid residues constituting an epitopic region. This approach has now been used to map an assembled epitope at/near the receptor binding region of the hormone. The mapped site forms a part of the seat belt region and the cystine knot region (C34-C38-C88-C90-H106). The carboxy terminal region of the alpha-subunit forms a part of the epitope indicating its proximity to the receptor binding region. These results are in agreement with the reported receptor binding region identified through other approaches and the X-ray crystal structure of hCG.

Left-handed helices for DNA: studies on poly[d(I-C)]

Earlier, we showed that, for the D form (n = 8 and h = 3.03 A, where n is number of nucleotide units per turn and h is height per nucleotide unit) of poly[d(A-T)], both right- and left-handed double helical models are stereochemically satisfactory and give good agreement with the observed fiber diffraction data. It was also noted that the conformations of the right- and left-handed D-DNA models are very similar to those of the right- and left-handed B-DNA models. This observation was consistent with the D leads to B transition in the solid phase. As a continuation of our earlier studies, we have carried out similar experiments with poly[d(I-C)]. We could obtain a crystalline D-form pattern (n = 8, h = 3.13 A) of the fiber at 75% relative humidity (r.h.); the hydrated (r.h. approximately equal to 95%) form of the same fiber gave the classical B-form pattern (n = 10, h = 3.40 A). In the present report, we show that both right- and left-handed double-helical models are consistent with the fiber diffraction data of poly[d(I-C)] in the D-form. Theoretical energy calculations also suggest that the right- and left-handed B- and D-DNA models are almost equally stable. Hence, we conclude that the right- and left-handed double-helical models of poly[d(I-C)] in a given form (B or D) are equally likely and that the fiber diffraction data do not permit discrimination.

Thermochemical and kinetic studies on the effects of cobalt salicylate on the oxidative degradation of polystyrene

The effect of cobalt salicylate on the oxidative degradation and ignition of polystyrene has been studied. It was found that cobalt salicylate sensitizes both the degradation and ignition of polystyrene by facilitating electron-transfer processes in the propagation step. From thermochemical and kinetic studies it was found that the cobalt ion, owing to its ability to exist in variable valence states, promotes electron transfer in the propagation step of polymer degradation, increasing the rate of propagation and consequently the overall rate. Using solid-phase thermal ignition theory, an attempt has been made to explain the sensitization of ignition by the cobalt ion.

Improved Approach for Analyzing Bromophenols in Seafood Using Stable Isotope Dilution Analysis in Combination with SPME

An analytical method for the measurement of five naturally occurring bromophenols of sensory relevance in seafood (barramundi and prawns) is presented. The method combines simultaneous distillation−extraction followed by alkaline back extraction of a hexane extract and subsequent acetylation of the bromophenols. Analysis of the bromophenol acetates was accomplished by headspace solid phase microextraction and gas chromatography−mass spectrometry using selected ion monitoring. The addition of 13C6 bromophenol stable isotope internal standards for each of the five congeners studied permitted the accurate quantitation of 2-bromophenol, 4-bromophenol, 2,6-dibromophenol, 2,4-dibromophenol, and 2,4,6-tribromophenol down to a limit of quantification of 0.05 ng/g of fish flesh. The method indicated acceptable precision and repeatability and excellent linearity over the typical concentration range of these compounds in seafood (0.5−50 ng/g). The analytical method was applied to determine the concentration of bromophenols in a range of farmed and wild barramundi and prawns and was also used to monitor bromophenol uptake in a pilot feeding trial.

An avidin-biotin microELISA for rapid measurement of total and allergen-specific human IgE

his paper describes an improved microtiter solid-phase enzyme immunoassay for the determination of total and allergen-specific human IgE. This assay technique is unique in its use of the avidin-biotin interaction to increase sensitivity. The avidin-biotin microtiter enzyme-linked immunosorbant assay (AB-microELISA) was performed in polyvinyl chloride microtiter plates using biotinylated anti-IgE and horseradish peroxidase (HRP)-avidin conjugate. This AB-microELISA technique enabled the quantitation of human serum IgE in the range of 0.1–5 ng/ml (10–500 pg/test) in less than 3 h. Total serum IgE, whether measured by the AB-microELISA or the paper radioimmunosorbant test (PRIST) was similar (correlation coefficient, r = 0.92). Further, the presence or absence of positive skin tests to 7 specific allergens determined in serum donors generally agreed with the presence or absence of allergen-specific IgE in their sera as measured by the AB-microELISA. The quantity of short ragweed allergen-specific IgE as determined by the AB-microELISA agreed with values obtained by the radioimmunosorbant test (RAST) (correlation coefficient, r = 0.89). No significant interference by ragweed-specific IgG (blocking antibody) was observed in the quantitation of allergen-specific IgE. The AB-microELISA is not only rapid and inexpensive, but also more sensitive than other published ELISA procedures and comparable to solid-phase radioimmunoassays in the quantitation of total and allergen-specific IgE.

Numerical studies on columnar-to-equiaxed transition in directional solidification of binary alloys

A numerical study on columnar-to-equiaxed transition (CET) during directional solidification of binary alloys is presented using a macroscopic solidification model. The position of CET is predicted numerically using a critical cooling rate criterion reported in literature. The macroscopic solidification model takes into account movement of solid phase due to buoyancy, and drag effect on the moving solid phase because of fluid motion. The model is applied to simulate the solidification process for binary alloys (Sn-Pb) and to estimate solidification parameters such as position of the liquidus, velocity of the liquidus isotherm, temperature gradient ahead of the liquidus, and cooling rate at the liquidus. Solidification phenomena under two cooling configurations are studied: one without melt convection and the other involvin thermosolutal convection. The numerically predicted positions of CET compare well with those of experiments reported in literature. Melt convection results in higher cooling rate, higher liquidus isotherm velocities, and stimulation of occurrence of CET in comparison to the nonconvecting case. The movement of solid phase aids further the process of CET. With a fixed solid phase, the occurrence of CET based on the same critical cooling rate is delayed and it occurs at a greater distance from the chill.

Constitutive modeling of shape memory alloy wire with non-local rate kinetics

A constitutive modeling approach for shape memory alloy (SMA) wire by taking into account the microstructural phase inhomogeneity and the associated solid-solid phase transformation kinetics is reported in this paper. The approach is applicable to general thermomechanical loading. Characterization of various scales in the non-local rate sensitive kinetics is the main focus of this paper. Design of SMA materials and actuators not only involve an optimal exploitation of the hysteresis loops during loading-unloading, but also accounts for fatigue and training cycle identifications. For a successful design of SMA integrated actuator systems, it is essential to include the microstructural inhomogeneity effects and the loading rate dependence of the martensitic evolution, since these factors play predominant role in fatigue. In the proposed formulation, the evolution of new phase is assumed according to Weibull distribution. Fourier transformation and finite difference methods are applied to arrive at the analytical form of two important scaling parameters. The ratio of these scaling parameters is of the order of 10(6) for stress-free temperature-induced transformation and 10(4) for stress-induced transformation. These scaling parameters are used in order to study the effect of microstructural variation on the thermo-mechanical force and interface driving force. It is observed that the interface driving force is significant during the evolution. Increase in the slopes of the transformation start and end regions in the stress-strain hysteresis loop is observed for mechanical loading with higher rates.

Development of microdevices for proteome research and diagnosis

The basic goal of a proteomic microchip is to achieve efficient and sensitive high throughput protein analyses, automatically carrying out several measurements in parallel. A protein microchip would either detect a single protein or a large set of proteins for diagnostic purposes, basic proteome or functional analysis. Such analyses would include e.g. interactomics, general protein expression studies, detecting structural alterations or secondary modifications. Visualization of the results may occur by simple immunoreactions, general or specific labelling, or mass spectrometry. For this purpose we have manufactured chip-based proteome analysis devices that utilize the classical polymer gel electrophoresis technology to run one and two-dimensional gel electrophoresis separations of proteins in just a smaller size. In total, we manufactured three functional prototypes of which one performed a miniaturized one-dimensional gel electrophoresis (1-DE) separation, the second and third preformed two-dimensional gel electrophoresis (2-DE) separations. These microchips were successfully used to separate and characterize a set of predefined standard proteins, cell and tissue samples. Also, the miniaturized 2-DE (ComPress-2DE) chip presents a novel way of combining the 1st and 2nd dimensional separations, thus avoiding manual handling of the gels, eliminate cross-contamination, and make analyses faster and repeatability better. They all showed the advantages of miniaturization over the commercial devices; such as fast analysis, low sample- and reagent consumption, high sensitivity, high repeatability and inexpensive performance. All these instruments have the potential to be fully automated due to their easy-to-use set-up.

Molecular Genetics of Lactase Deficiencies

Congenital lactase deficiency (CLD) (MIM 223000) is a rare autosomal recessive gastrointestinal disorder characterized by watery diarrhea in infants fed with breast milk or other lactose-containing formulas. The CLD locus was previously assigned by linkage and linkage disequilibrium analyses on 2q21 in 19 Finnish families. In this study, the molecular background of this disorder is reported. The CLD locus was refined in 32 CLD patients in 24 families by using microsatellite and single nucleotide polymorphism (SNP) haplotypes. Mutation analyses were performed by direct sequencing. We identified 5 distinct mutations in the lactase (LCT) gene, encoding the enzyme that hydrolyzes lactose in the intestinal lumen. These findings facilitate genetic testing of CLD in clinical practice and enable genetic counseling. The present data also provide the basis for detailed characterization of the molecular pathogenesis of this disorder. Adult-type hypolactasia (MIM 223100) (lactase non-persistence, lactose intolerance) is an autosomal recessive gastrointestinal condition that is a result of a decline in the activity of lactase in the intestinal lumen after weaning. Adult-type hypolactasia is considered to be a normal phenomenon among mammals and symptoms are remarkably milder than experienced in CLD. Recently, a variant C/T-13910 was shown to associate with the adult-type hypolactasia trait, locating 13.9 kb upstream of the LCT gene. In this study, the functional significance of the C/T-13910 variant was determined by studying the LCT mRNA levels in intestinal biopsy samples in children and adults with different genotypes. RT-PCR followed by solid-phase minisequencing was applied to determine the relative expression levels of the LCT alleles using an informative SNP located in exon 1. In children, the C-13910 allele was observed to be downregulated after five years of age in parallel with lactase enzyme activity. The expression of the LCT mRNA in the intestinal mucosa in individuals with the T-13910 A-22018 alleles was 11.5 times higher than that found in individuals with the C-13910, G-22018 alleles. These findings suggest that the C/T-13910 associated with adult-type hypolactasia is associated with the transcriptional regulation of the LCT gene. The presence of the T-13910 A-22018 allele also showed significant elevation lactase activity. Galactose, the hydrolysing product of the milk sugar lactose, has been hypothesized to be poisonous to ovarian epithelial cells. Hence, consumption of dairy products and lactase persistence has been proposed to be a risk factor for ovarian carcinoma. To investigate whether lactase persistence is related to the risk of ovarian carcinoma the C/T-13910 genotype was determined in a cohort of 782 women with ovarian carcinoma 1331 individuals serving as controls. Lactase persistence did not associate significantly with the risk for ovarian carcinoma in the Finnish, in the Polish or in the Swedish populations. The findings do not support the hypothesis that lactase persistence increases the risk for ovarian carcinoma.