Assay of gliadin by real-time immunopolymerase chain reaction

Patients with coeliac disease (gluten-sensitive enteropathy) are intolerant against gliadins from wheat and the respective proteins from related cereals and have to keep a lifelong gluten-free diet. For control of gliadin in gluten-free food sensitive assay techniques are necessary. We developed an immunopolymerase chain reaction (iPCR) assay for gliadin. In this technique immunological detection of gliadin by a monoclonal antibody R5 conjugated with an oligonucleotide is amplified by PCR. For quantification, iPCR was performed as real-time PCR (real-time iPCR) in one step. By means of real-time iPCR, the sensitivity of gliadin analysis was increased more than 30-fold above the level reached by enzyme immunoassay. Real time-iPCR using R5 directly conjugated with oligonucleotide was clearly more sensitive than real time-iPCR applying sequentially biotinylated R5, streptavidin, and biotinylated oligonucleotide. With directly conjugated R5 gliadin was detected at a concentration as low as 0.16 ng/mL corresponding to 16 microg gliadin/100 g food or 0.16 ppm (corresponding to 0.25 g of food extracted in 10 mL of solvent and 25-fold dilution of the extract prior to analysis). This is the first report applying the highly sensitive technique of iPCR for gliadin analysis. Furthermore, this is the first approach to perform real-time iPCR in one step without changing the reaction vessels after enzyme immunoassay for subsequent PCR analysis thus minimizing risks of contamination and loss of sensitivity.     

Coffees rich in chlorogenic acid or N-methylpyridinium induce chemopreventive phase II-enzymes via the Nrf2/ARE pathway in vitro and in vivo

Recently, the coffee constituents 5-O-caffeoylquinic acid (CGA) and N-methylpyridinium (NMP) were identified as inducers of the Nrf2/antioxidant-response element (ARE) detoxifying pathway under cell-culture condition. To study the impact of CGA and NMP on the Nrf2-activating properties of a complex coffee beverage, two different model coffees were generated by variation of the roasting conditions: a low-roast coffee rich in CGA and a heavy-roast low in CGA but containing high levels of NMP. Activation of the Nrf2/antioxidant-response element pathway was monitored in vitro and in vivo.     

Reduced recruitment and survival of primordial and growing follicles in GH receptor-deficient mice

GH influences female fertility. The goal of the present study was to obtain more insight into the effect of loss of GH signalling, as observed in humans suffering from Laron syndrome, on ovarian function. Therefore, serial paraffin sections of ovaries of untreated and IGF-I-treated female GH receptor knock-out (GHR/GHBP-KO) mice were examined to determine the follicular reserve and the percentage of follicular atresia in each ovary. Our observations demonstrate that the amount of primordial follicles was significantly elevated in GHR/GHBP-KO mice, while the numbers of primary, preantral and antral follicles were lower compared with wild-type values. The reduced number of healthy growing follicles in GHR/GHBP-KO mice was accompanied by a significant increase in the percentage of atretic follicles. IGF-I treatment of GHR/GHBP-KO mice for 14 days resulted in a reduced number of primordial follicles, an increased number of healthy antral follicles, and a decreased percentage of atretic follicles. The results of the present study suggest that GH may play a role, either directly or indirectly, via for instance IGF-I, in the recruitment of primordial follicles into the growing pool. Furthermore, GH seems to protect antral follicles, directly or indirectly from undergoing atresia.     

Fate of 14C-acrylamide in roasted and ground coffee during storage

Acrylamide (AA) is formed during heating of carbohydrate rich foods in the course of the Maillard reaction. AA has been classified as probably carcinogenic to humans. Storage experiments with roasted coffee have shown that AA levels decrease depending on storage time and temperature. In the present study the fate of AA lost during storage of roasted and ground (R&G) coffee was studied, using 14C-labeled AA as radiotracer. Radiolabel was measured in coffee brew, filter residue, and volatiles. In the brew, total (14)C-label decreased during storage of R&G coffee, while activity in the filter residue built up concomitantly. [2,3-14C]-AA (14C-AA) was the only 14C-related water extractable low molecular compound in the brew detected by radio-HPLC. No formation of volatile 14C-AA-related compounds was detected during storage and coffee brewing. Close to 90% of the radiolabel in the filter residue (spent R&G coffee, spent grounds) remained firmly bound to the matrix, largely resisting extraction by aqueous ammonia, ethyl acetate, chloroform, hexane, and sequential polyenzymatic digest. Furanthiols, which are abundant as aroma components in roasted coffee, have not been found to be involved in the formation of covalent AA adducts and thus do not contribute substantially to the decrease of AA during storage.     

Preparation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) particles in O/W emulsion

In the present paper, we investigate the preparation of polymeric particles based on the biodegradable polyester poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). A new technique for PHBV particle preparation has been developed. This method utilizes the thermoreversible gelation of PHBV in toluene. Particles have been obtained by the secondary dispersion technique in a three-step procedure: (a) preparation of PHBV solution in toluene; (b) preparation of O/W emulsion by ultrasound followed by the gel formation in toluene/PHBV droplets; and (c) toluene extraction. In the present study we investigated the influence of the stabilizer type and its concentration in the aqueous phase, ultrasound power, and PHBV concentration in toluene on the size and stability of the formed droplets as well as the final PHBV particles. It has been found that PEO/PS block copolymers are the best stabilizers for the present system as compared to conventional tensides such as SDS or CTAB. It has been found that PEO/PS block copolymers allow obtaining PHBV particles with a regular shape and controlled dimensions after toluene extraction. The minimal size of the PHBV particles obtained by this technique was ca. 100 nm. The obtained particles exhibit a relatively broad particle size distribution and the particle shape is strongly affected by the block copolymer composition, ultrasound power and the way of toluene extraction.     

Characterization of the HCN Interaction Partner TRIP8b/PEX5R in the Intracardiac Nervous System of TRIP8b-Deficient and Wild-Type Mice

The tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b/PEX5R) is an interaction partner and auxiliary subunit of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which are key for rhythm generation in the brain and in the heart. Since TRIP8b is expressed in central neurons but not in cardiomyocytes, the TRIP8b-HCN interaction has been studied intensely in the brain, but is deemed irrelevant in the cardiac conduction system. Still, to date, TRIP8b has not been studied in the intrinsic cardiac nervous system (ICNS), a neuronal network located within epicardial fat pads. In vitro electrophysiological studies revealed that TRIP8b-deficient mouse hearts exhibit increased atrial refractory and atrioventricular nodal refractory periods, compared to hearts of wild-type littermates. Meanwhile, heart rate, sino-nodal recovery time, and ventricular refractory period did not differ between genotypes. Trip8b mRNA was detected in the ICNS by quantitative polymerase chain reaction. RNAscope in situ hybridization confirmed Trip8b localization in neuronal somata and nerve fibers. Additionally, we found a very low amount of mRNAs in the sinus node and atrioventricular node, most likely attributable to the delicate fibers innervating the conduction system. In contrast, TRIP8b protein was not detectable. Our data suggest that TRIP8b in the ICNS may play a role in the modulation of atrial electrophysiology beyond HCN-mediated sino-nodal control of the heart.     

Ceramide and Related Molecules in Viral Infections

Ceramide is a lipid messenger at the heart of sphingolipid metabolism. In concert with its metabolizing enzymes, particularly sphingomyelinases, it has key roles in regulating the physical properties of biological membranes, including the formation of membrane microdomains. Thus, ceramide and its related molecules have been attributed significant roles in nearly all steps of the viral life cycle: they may serve directly as receptors or co-receptors for viral entry, form microdomains that cluster entry receptors and/or enable them to adopt the required conformation or regulate their cell surface expression. Sphingolipids can regulate all forms of viral uptake, often through sphingomyelinase activation, and mediate endosomal escape and intracellular trafficking. Ceramide can be key for the formation of viral replication sites. Sphingomyelinases often mediate the release of new virions from infected cells. Moreover, sphingolipids can contribute to viral-induced apoptosis and morbidity in viral diseases, as well as virus immune evasion. Alpha-galactosylceramide, in particular, also plays a significant role in immune modulation in response to viral infections. This review will discuss the roles of ceramide and its related molecules in the different steps of the viral life cycle. We will also discuss how novel strategies could exploit these for therapeutic benefit.     

A survey on heterogeneous mobile networks planning in indoor dense areas

Personal and Ubiquitous Computing - In dense indoor areas, high numbers of people use their smartphones and tablets to share or download pictures, videos, or data. The heterogeneous network...