Identification of key effects causing weak performance of allergen analysis in processed food matrices

The weaker performance of generally used analytical methods for allergen analysis in processed foods can be connected to protein denaturation. To understand the nature of protein denaturation processes, experimental but realistic model matrices (corn starch based mixture, hydrated dough, and heat treated cookies) were developed that contain a defined amount of milk, egg, soy, and wheat proteins individually or in combination. The protein subunit composition was investigated in every processing phase, i.e. after mixing, dough formation, and baking. SDS-PAGE measurements were carried out to monitor the protein distribution of sample food matrices in non-reducing and reducing gels. The results clearly show that the highly decreased protein solubility is caused by denaturation, aggregation, or complex formation, which are the most significant factors in poorer analytical performances. Solubility can only partly be improved with the application of reducing agents or surfactants, and the rate of improvement is depending on the proteins and the matrices

Applicability of ELISA methods for high gluten-containing samples

Quantitation of gluten in gluten-free products is a great challenge as it is hindered by several factors including the lack of certified reference materials. To resolve this problem, our research group, in cooperation with other international experts, started a series of experiments with the goal of the production of a suitable gluten reference material. As a part of this research, several different wheat cultivars and their isolated gluten proteins were characterized by different methods, including enzyme-linked immunosorbent assay (ELISA). However, we need to know the performance of the ELISA methods used for this special area of research. During the present work we investigated the accuracy and precision of two different ELISA methods for our own laboratory conditions and special sample matrices (wheat flours and gliadin isolate). We have found that the tested performance characteristics of the methods seem to be appropriate on a case-by-case basis, but the long-term measurement uncertainty is higher, which makes it difficult to evaluate the results obtained with the ELISA method for these types of samples

Investigation of the effects of food processing and matrix components on the analytical results of ELISA using an incurred gliadin reference material candidate

Disorders induced by cereal proteins (e.g. wheat allergy, celiac disease) are widespread in human population. Since their only effective treatment is the avoidance of the problematic proteins, patients have to be familiar with the composition of food products. For checking special foods produced for them, proper analytical methods are necessary. At the moment, in gluten analysis there are no reference methods and reference materials which model real food matrices. During the production and experimental utilisation of our previously developed reference material candidate, numerous questions emerged. As our model product is a real food matrix, interactions can be present between gluten proteins and other macro and micro components. Fat content of the baked cookies is almost 20%, which might affect the results of ELISA measurements. The detectable gluten content is significantly increasing after the defatting procedure, as a pre-treatment of samples. Moreover, baking is a common food processing step that might modify the structure of gluten proteins leading to denaturation and aggregation. In the soluble protein fraction the amount of low molecular weight proteins increases, while that of high molecular weight proteins decreases during the baking procedure

ELISA response and gliadin composition of different wheat cultivars grown in multiple harvest years

In special dietary products for people intolerant to gluten, gluten content is not supposed to exceed the regulatory thresholds. Enzyme-linked immunosorbent assays (ELISAs) are routinely used to quantitate gluten in these products. They measure gliadin/gluten with high specificity and sensitivity, but they have some limitations. Quantitative and qualitative variability of the target proteins among wheat cultivars is a factor that may cause inaccurate results. One of the aims of this work was to characterize the protein composition of five wheat cultivars grown in multiple harvest years and their blends by reversed-phase high-performance liquid chromatography (RP-HPLC). The gliadin/gluten content of these wheat flours was also analysed with two commercial ELISA kits. The effect of differences in protein profiles between the flours from an individual cultivar and the blend of five cultivars, harvest years, as well as processing procedures (dough forming and baking) on the results of two ELISA kits was investigated and their relative magnitude was determined. Among the factors investigated, the differences between flours had the greatest impact on gliadin recoveries

Individual Actin Filaments in a Microfluidic Flow Reveal the Mechanism of ATP Hydrolysis and Give Insight Into the Properties of Profilin

A novel microfluidic approach allows the analysis of the dynamics of individual actin filaments, revealing both their local ADP/ADP-Pi-actin composition and that Pi release is a random mechanism

DAAM is required for thin filament formation and Sarcomerogenesis during muscle development in Drosophila.

During muscle development, myosin and actin containing filaments assemble into the highly organized sarcomeric structure critical for muscle function. Although sarcomerogenesis clearly involves the de novo formation of actin filaments, this process remained poorly understood. Here we show that mouse and Drosophila members of the DAAM formin family are sarcomere-associated actin assembly factors enriched at the Z-disc and M-band. Analysis of dDAAM mutants revealed a pivotal role in myofibrillogenesis of larval somatic muscles, indirect flight muscles and the heart. We found that loss of dDAAM function results in multiple defects in sarcomere development including thin and thick filament disorganization, Z-disc and M-band formation, and a near complete absence of the myofibrillar lattice. Collectively, our data suggest that dDAAM is required for the initial assembly of thin filaments, and subsequently it promotes filament elongation by assembling short actin polymers that anneal to the pointed end of the growing filaments, and by antagonizing the capping protein Tropomodulin

Robust Organizational Principles of Protrusive Biopolymer Networks in Migrating Living Cells

Cell migration is associated with the dynamic protrusion of a thin actin-based cytoskeletal extension at the cell front, which has been shown to consist of two different substructures, the leading lamellipodium and the subsequent lamellum. While the formation of the lamellipodium is increasingly well understood, organizational principles underlying the emergence of the lamellum are just beginning to be unraveled. We report here on a 1D mathematical model which describes the reaction-diffusion processes of a polarized actin network in steady state, and reproduces essential characteristics of the lamellipodium-lamellum system. We observe a steep gradient in filament lengths at the protruding edge, a local depolymerization maximum a few microns behind the edge, as well as a differential dominance of the network destabilizer ADF/cofilin and the stabilizer tropomyosin. We identify simple and robust organizational principles giving rise to the derived network characteristics, uncoupled from the specifics of any molecular implementation, and thus plausibly valid across cell types. An analysis of network length dependence on physico-chemical system parameters implies that to limit array treadmilling to cellular dimensions, network growth has to be truncated by mechanisms other than aging-induced depolymerization, e.g., by myosin-associated network dissociation at the transition to the cell body. Our work contributes to the analytical understanding of the cytoskeletal extension's bisection into lamellipodium and lamellum and sheds light on how cells organize their molecular machinery to achieve motility