BACKGROUND:Poultry meat can induce severe allergic reactions. So far, the molecules causing poultry meat allergy are largely unknown. OBJECTIVE:Our aim was to identify and characterize poultry meat allergens. METHODS:Profiles of patients' IgE reactivity to chicken muscle were analyzed in immunoblots, and proteins recognized by the majority of patients were subjected to peptide mass fingerprinting. A 23-kDa IgE-reactive protein was identified as myosin light chain 1, designated Gallus domesticus 7 (Gal d 7). Recombinant Gal d 7 was produced in Escherichia coli. The protein's IgE reactivity was analyzed in ELISA experiments, and cross-reactivity with allergens of other poultry species was assessed in inhibition immunoblots. Fold and thermal stability were evaluated by circular dichroism analysis, and enzymatic stability was investigated using in vitro gastrointestinal digestion assays. RESULTS:Recombinant Gal d 7 represents a properly folded, predominantly α-helical protein and displays IgE-binding activity comparable to that of its natural counterpart. IgE reactivity analysis in 28 patients allergic to chicken meat revealed that Gal d 7 is a major allergen for patients primarily sensitized to chicken meat. Furthermore, Gal d 7-cross-reactive allergens were also detected in other poultry species, suggesting that recombinant Gal d 7 can be used as a diagnostic marker allergen for poultry meat allergy. The high thermal stability, refolding capacity, and resistance to gastrointestinal enzymes might explain why Gal d 7 can act as a potent sensitizing agent. CONCLUSION:Gal d 7 represents a novel major chicken meat allergen. Recombinant Gal d 7 could be used for diagnosis of genuine poultry meat sensitization.

Nowadays, enzyme-linked immunosorbent assay (ELISA) based detection of Mycobacterium tuberculosis (M. tuberculosis) antigen triggered interferon-gamma (IFN-g) secretion by blood T cells displays an improved diagnostic value for M. tuberculosis infection. Applications of fluorescence correlation spectroscopy (FCS) have been explored in various subfields of medicine and molecular biology, including detection of a certain biomarker in liquid instead of ELISA. Here, we present a preliminary study of detecting IFN-g using FCS-based technique.

Performing quantitative, highly sensitive measurements at a single molecule level is often necessary to address specific issues related to complex molecular and biochemical systems. For that purpose, we present a technique exploiting both the flexibility of immunoassays as well as the low operating costs and high throughput rates of the fluorescence correlation spectroscopy (FCS) method. That way we have established a quantitative measurement technique providing accurate and flexibly time resolved data of single molecules. Nanomolar changes in adrenocorticotropic hormone (ACTH) levels have been detected in a short time-frame that are caused by fast feedback actions in AtT-20 anterior pituitary glands in vitro. Especially with respect to clinical diagnostic or mathematical modeling this improved FCS setup may be of high relevance in order to accurately quantify the amounts of peptide hormones-such as ACTH-as well as signaling molecules, transcription factors, etc., being involved in intra- and extracellular reaction networks.

In a comprehensive picture of inclusion complex formation of the highly fluorescent dye coumarin-6 (C6) and betacyclodextrin (beta-CD), which was obtained using various fluorescence spectroscopic methods, it was demonstrated that up to three beta-CD rings can thread on the rod like dye molecule. Interaction of coumarins and modified coumarins with cellular organelles or proteins has been reported in several publications. Especially 7-amino-coumarins are characterized by unique properties like high fluorescence quantum yield and are thus already used successfully in different areas, like staining of fluorescent nanoparticles. We could show that Coumarin-6 made soluble by complexation with beta-cyclodextrin is able to stain eukaryotic cells specifically dependent on their origin and cellular behaviour. The staining reaction is independent from pH, is photo stable, and shows no cross talk with proteins in the cytoplasm and other staining procedures or erythrocytes. Staining with coumarin 6/cyclodextrin complexes can thus be used for fast discrimination of different cell types. Importantly, it could be shown that the ideal staining reaction is dependent on the stoichiometry of the complex-formation.

Cancer is one of the big killers of world population. The majority of cancers are diagnosed at a late stage, making a cure almost impossible. Fluorescence spectroscopy is an emerging diagnostic tool for various medical diseases including premalignant and malignant lesions. Fluorescence spectroscopy is a noninvasive technique and has been applied successfully for the diagnosis of multisystem cancers with high sensitivity and specificity. Fluorescence spectroscopy minimizes the need for repetitive biopsy, which is routine practice for cancer patient follow-up. But there are many aspects of this new diagnostic technique that should be discussed in future research to overcome limitations and challenges faced by this technique for diagnosis of cancers.

A 3-dimensional mesofluidic biological monitoring module has been successfully designed and fabricated using a low-temperature co-fired ceramic (LTCC) technology. This mesofluidic device consists of a network of micro-channels, a spherical mixing cavity and measuring ports. A selection of appropriate commercially available ceramic tapes has been chosen with regard to their biocompatibility performance. Specific processing procedures required for the realization of such a complex structure are demonstrated. Three dimensional numerical flow simulations have been conducted to characterize the concentration profiles of liquids at a specific measuring port and verified by experiment.

A 3-dimensional mesofluidic biological monitoring module has been successfully designed and fabricated using a low-temperature co-fired-ceramic (LTCC) technology. This mesofluidic device consists of a network of micro-channels and a spherical mixing cavity. The selection of appropriate commercially available ceramic tapes has been done with regard to their biocompatibility performance. Specific processing procedures required for the realization of such complex structure are demonstrated. Three dimensional numerical flow simulations have been conducted to characterize the concentration profiles of liquids at a specific measuring port and verified by experiment. The module was successfully applied to study complex chemical reaction kinetics complemented by mathematical modelling.

There are many diagnostic techniques and methods available for diagnosis of medically important microorganisms like bacteria, viruses, fungi and parasites. But, almost all these techniques and methods have some limitations or inconvenience. Most of these techniques are laborious, time consuming and with chances of false positive or false negative results. It warrants the need of a diagnostic technique which can overcome these limitations and problems. At present, there is emerging trend to use Fluorescence spectroscopy as a diagnostic as well as research tool in many fields of medical sciences. Here, we will critically discuss research studies which propose that Fluorescence spectroscopy may be an excellent diagnostic as well as excellent research tool in medical microbiology field with high sensitivity and specificity.

G protein-coupled receptors have been proposed to exist in signalosomes subject to agonist-driven shifts in the assembly disassembly equilibrium, affected by stabilizing membrane lipids and/or cortical actin restricting mobility. We investigated the highly homologous corticotropin-releasing factor receptors (CRFRs), CRFR1 and -2, which are different within their hydrophobic core. Agonist stimulation of CRFR1 and CRFR2 gave rise to similar concentration-response curves for cAMP accumulation, but CRFR2 underwent restricted collision coupling. Both CRFR1 and CRFR2 formed constitutive oligomers at the cell surface and recruited β-arrestin upon agonist activation (as assessed by fluorescence resonance energy transfer microscopy in living cells). However, CRFR2, but not CRFR1, failed to undergo agonist-induced internalization. Likewise, agonist binding accelerated the diffusion rate of CRFR2 only (detected by fluorescence recovery after photobleaching and fluorescence correlation spectroscopy) but reduced the mobile fraction, which is indicative of local confinement. Fluorescence intensity distribution analysis demonstrated that the size of CRFR complexes was not changed. Disruption of the actin cytoskeleton abolished the agonist-dependent increase in CRFR2 mobility, shifted the agonist concentration curve for CRFR2 to the left, and promoted agonist-induced internalization of CRFR2. Our observations are incompatible with an agonist-induced change in monomer-oligomer equilibrium, but they suggest an agonist-induced redistribution of CRFR2 into a membrane microdomain that affords rapid diffusion but restricted mobility and that is stabilized by the actin cytoskeleton. Our data show that membrane anisotropy can determine the shape and duration of receptor-generated signals in a subtype-specific manner.

Die LTCC (Low Temperature Co-fired Ceramic)-Technologie, die ursprünglich für den Aufbau von hoch zuverlässigen Mehrlagenschaltungen entwickelt wurde, eröffnet die Möglichkeit, auch komplexe dreidimensionale Strukturen in einfacher Weise aufzubauen. Am Beispiel eines Monitoring-Moduls zur Erfassung von biologischen Reaktionen wird die Herstellung einer komplexen Mikrofluidik-Applikation vorgestellt. Anhand eines definierten Mischprozesses sowie eines Reaktionsprozesses, welche sich auf numerische Simulationen abstützen, wird die Funktionalität des Systems aufgezeigt.

Complex chemical reactions are commonly described by systems of nonlinear ordinary differential equations. Rate and equilibrium constants of these models are usually not directly accessible and have to be indirectly inferred from experimental observations of the system. As a consequence, parameter identification problems have to be formulated and computationally solved. Because of a limited amount of information and uncertainties in the data, the solutions to such parameter identification problems typically lack uniqueness and stability properties and hence cannot be found in a reliable way by a pure minimization of the data mismatch (i.e., the discrepancy between experimental observations and simulated model output). To overcome these difficulties, so-called regularization methods have to be used. In this article, we suggest a sparsity promoting regularization approach that eliminates unidentifiable model parameters (i.e., parameters of low or no sensitivity to the given data). That way, the model is reduced to a core reaction mechanism with manageable interpretation while still remaining in accordance with the experimental observations. For the computational realization, we utilize the adjoint state technique for an efficient calculation of the gradient of the objective with respect to model parameters as well as uncertain initial and experimental conditions. Illustrations of our approach are given by means of the chlorite-iodide reaction for which reference parameter values are available.

A 3-dimensional mesofluidic biological monitoring module has been successfully designed and fabricated using a low-temperature co-fired ceramic (LTCC) technology. This mesofluidic device consists of a network of micro-channels, a spherical mixing cavity and measuring ports. A selection of appropriate commercially available ceramic tapes has been chosen with regard to their biocompatibility performance. Specific processing procedures required for the realization of such a complex structure are demonstrated. Three dimensional numerical flow simulations have been conducted to characterize the concentration profiles of liquids at a specific measuring port and verified by experiment.

Inhalative administration of vasoactive intestinal peptide (VIP) is a promising approach for the treatment of severe lung diseases. However, the clinical use of VIP is limited by the fact that the peptide is prone to rapid degradation mechanisms and proteolytic digestion. Accordingly, VIP exhibits a very short period of activity in the lung. To overcome this problem, we have designed a liposomal drug delivery system for VIP and characterized it in terms of its potential to protect VIP from enzymatic cleavage. The proteolytic conditions of the lung, the target site of aerosolic administered VIP, were mimicked by bronchoalveolar lavage fluid (BALF), a lung surfactant solution, obtained by fiberoptic bronchoscopy. Thus, the stability of VIP was assessed by its resistance to enzymatic degradation in BALF, using a combination of high pressure liquid chromatography with mass spectrometry. We found that free VIP was rapidly digested, whereas liposomal-associated VIP remained intact. By fluorescence spectroscopic techniques using fluorescent-labelled VIP we got strong indications that the tight association of VIP with the lipid membrane is only minimally affected upon incubation with BALF. Loading capacity and stability of EtCy3-VIP loaded liposomes were measured by fluorescence fluctuation spectroscopy. Finally, the protective properties of the liposomes were also expressed in the maintained biological activity of the peptide incubated with BALF.

Based on flow models applied in former analyses, three dimensional numerical flow simulations were performed for a spherical reactor cell of a biological monitoring module with respect to different inflow configurations and respective mass flow rates. Time - dependent simulations were performed for the case of dyed water and clear water flowing through different inlets at varying mass flow rates into the spherical cavity of reactor cell and channel system which connects the relevant measuring ports of the monitoring module. Spectroscopic analyses of light absorption at a port have been carried out and the results compared with the local concentration distribution predicted for this port by means of FE - analyses (Finite Element) using the CFD (Computational Fluid Dynamics) - program package of Fluent Inc.

Low temperature co-fired ceramic (LTCC) technology was originally developed for the realization of multilayer circuits of high reliability. It was recognized that LTCC-technology is a valuable development in thick film technology which launches new application areas as it becomes evident that complex three-dimensional structures can be easily realized. Aspects of the realization of a sophisticated biological monitoring module comprising a three-dimensional network of channels and cavities is demonstrated.