Validation of the performance of a GMO multiplex screening assay based on microarray detection

A new screening method for the detection and identification of GMO, based on the use of multiplex PCR followed by microarray, has been developed and is presented. The technology is based on the identification of quite ubiquitous GMO genetic target elements first amplified by PCR, followed by direct hybridisation of the amplicons on a predefined microarray (DualChip^® GMO, Eppendorf, Germany). The validation was performed within the framework of a European project (Co-Extra, contract no 007158) and in collaboration with 12 laboratories specialised in GMO detection. The present study reports the strategy and the results of an ISO complying validation of the method carried out through an inter-laboratory study. Sets of blind samples were provided consisting of DNA reference materials covering all the elements detectable by specific probes present on the array. The GMO concentrations varied from 1% down to 0.045%. In addition, a mixture of two GMO events (0.1% RRS diluted in 100% TOPAS19/2) was incorporated in the study to test the robustness of the assay in extreme conditions. Data were processed according to ISO 5725 standard. The method was evaluated with predefined performance criteria with respect to the EC CRL method acceptance criteria. The overall method performance met the acceptance criteria; in particular, the results showed that the method is suitable for the detection of the different target elements at 0.1% concentration of GMO with a 95% accuracy rate. This collaborative trial showed that the method can be considered as fit for the purpose of screening with respect to its intra- and inter-laboratory accuracy. The results demonstrated the validity of combining multiplex PCR with array detection as provided by the DualChip^® GMO (Eppendorf, Germany) for the screening of GMO. The results showed that the technology is robust, practical and suitable as a screening tool.     

Expression and characterization of three important panallergens from hazelnut

Several hazelnut allergens with different clinical relevance and crossreactive properties have been identified and characterized so far. The aim of this study was to develop protocols for producing relatively large amounts of three recombinant hazelnut allergens Cor a 1.04, Cor a 2, and Cor a 8 in a folded and immunologically active form. The availability of well-characterized, pure recombinant allergens will improve diagnostic in vitro tests for food allergy, by allowing a highly sensitive component resolved diagnosis. Depending on the individual hazelnut allergen, protocols for heterologous production - either as fusion or nonfusion protein - were developed to obtain homogenous protein batches. The resulting proteins were purified by a two-step FPLC method and their IgE antibody reactivity was verified. Identity was verified by N-terminal sequencing and MALDI-TOF-MS analysis. Their secondary and tertiary structure was controlled by circular dichroism (CD)-spectroscopy and NMR analysis. Decisions on the strategies for expression and purification of allergens on a large scale were made on a case by case basis: Preparation of rCor a 1.04 and rCor a 2 as fusion proteins in E. coli from inclusion bodies resulted in approximately 10 mg pure protein per liter whereas rCor a 8 expression in yeast as nonfusion protein yielded 30 mg/L.     

Study of the transient release of water vapor from a fuel bed of wet biomass in a reciprocating-grate furnace

The present study investigates how sudden changes in fuel moisture affected the combustion characteristics of the fuel bed in a 4-MW reciprocating-grate furnace. The moisture content of the fuel fed to the furnace was monitored online using a near-infrared spectroscopy device, and the water vapor concentration in the flue gas was measured continuously. To obtain experimental data on fuel-bed conditions, the temperature and gas composition in the bed were measured using a probe. A simplified drying model was developed using the measured gas composition values as inputs. The model was then used to estimate the drying rate and to simulate the extent of the drying zone along the grate. Measurements indicated that a change in the moisture content of the fuel fed to the furnace was detected as a change in water vapor concentration in the flue gas with a delay of about 2 h. The model predicted that a portion of wet fuel would need about 2 h to become dry, in line with the measured time delay of the water vapor concentration change in the flue gas. Overall, there was good alignment between the measured and simulated results, supporting the validity of the model and the assumed mechanisms.     

Rational Structure-Based Design of Fluorescent Probes for Amyloid Folds

Amyloid fibrils are pathological hallmarks of various human diseases, including Parkinson's, Alzheimer's, amyotrophic lateral sclerosis (ALS or motor neurone disease), and prion diseases. Treatment of the amyloid diseases are hindered, among other factors, by timely detection and therefore, early detection of the amyloid fibrils would be beneficial for treatment against these disorders. Here, a small molecular fluorescent probe is reported that selectively recognize the fibrillar form of amyloid beta(1–42), α-synuclein, and HET-s(218–289) protein over their monomeric conformation. The rational design of the reporters relies on the well-known cross-β-sheet repetition motif, the key structural feature of amyloids.     

Temporal trends of synthetic musk compounds in mother's milk and associations with personal use of perfumed products

We analyzed two nitro musks (musk xylene and musk ketone) and five polycyclic musks (HHCB, AHTN, ADBI, ATII, and AHDI) in mother's milk from primiparae women (N = 101) living in Uppsala County, Sweden, 1996-2003. Possible temporal trends in musk concentrations and associations with lifestyle/medical factors, such as use of perfumed products during pregnancy were studied. HHCB showed the highest median concentration (63.9 ng/g lipid) followed by AHTN (10.4 ng/g) and musk xylene (MX) (9.5 ng/g). Concentrations of the other substances were, in most cases, below the quantification limit (2.0-3.0 ng/g). Women with a high use of perfume during pregnancy had elevated milk concentrations of HHCB, and elevated concentrations of AHTN were observed among women reporting use of perfumed laundry detergent. This strongly suggests that perfumed products are important sources of musk exposure both among the mothers and the nursed infants. Concentrations of AHTN and MX declined significantly between 1996 and 2003, suggesting a decline in the industrial use of the compounds in consumer products, or alterations in the consumer use pattern of perfumed products. No temporal trend in HHCB concentrations was seen. The lack of toxicity data makes it difficult to generalize about the safety of musk exposure of breast-fed infants.     

Structure–Function Relationship of Retinal Ganglion Cells in Multiple Sclerosis

The retinal ganglion cells (RGC) may be considered an easily accessible pathophysiological site of degenerative processes in neurological diseases, such as the RGC damage detectable in multiple sclerosis (MS) patients with (HON) and without a history of optic neuritis (NON). We aimed to assess and interrelate RGC functional and structural damage in different retinal layers and retinal sites. We included 12 NON patients, 11 HON patients and 14 healthy controls for cross-sectional multifocal pattern electroretinography (mfPERG) and optical coherence tomography (OCT) measurements. Amplitude and peak times of the mfPERG were assessed. Macula and disc OCT scans were acquired to determine macular retinal layer and peripapillary retinal nerve fiber layer (pRNFL) thickness. In both HON and NON patients the foveal N2 amplitude of the mfPERG was reduced compared to controls. The parafoveal P1 peak time was significantly reduced in HON only. For OCT, parafoveal (pfGCL) and perifoveal (pGCL) ganglion cell layer thicknesses were decreased in HON vs. controls, while pRNFL in the papillomacular bundle sector (PMB) showed reductions in both NON and HON. As the mfPERG derived N2 originates from RGC axons, these findings suggest foveal axonal dysfunction not only in HON, but also in NON patients.     

Dichloroacetate Radiosensitizes Hypoxic Breast Cancer Cells

Mitochondrial metabolism is an attractive target for cancer therapy. Reprogramming metabolic pathways can potentially sensitize tumors with limited treatment options, such as triple-negative breast cancer (TNBC), to chemo- and/or radiotherapy. Dichloroacetate (DCA) is a specific inhibitor of the pyruvate dehydrogenase kinase (PDK), which leads to enhanced reactive oxygen species (ROS) production. ROS are the primary effector molecules of radiation and an increase hereof will enhance the radioresponse. In this study, we evaluated the effects of DCA and radiotherapy on two TNBC cell lines, namely EMT6 and 4T1, under aerobic and hypoxic conditions. As expected, DCA treatment decreased phosphorylated pyruvate dehydrogenase (PDH) and lowered both extracellular acidification rate (ECAR) and lactate production. Remarkably, DCA treatment led to a significant increase in ROS production (up to 15-fold) in hypoxic cancer cells but not in aerobic cells. Consistently, DCA radiosensitized hypoxic tumor cells and 3D spheroids while leaving the intrinsic radiosensitivity of the tumor cells unchanged. Our results suggest that although described as an oxidative phosphorylation (OXPHOS)-promoting drug, DCA can also increase hypoxic radioresponses. This study therefore paves the way for the targeting of mitochondrial metabolism of hypoxic cancer cells, in particular to combat radioresistance.     

Impact of the Electromagnetic Brake Position on the Flow Structure in a Slab Continuous Casting Mold: An Experimental Parameter Study

Metallurgical and Materials Transactions B - Flow measurements are performed in a slab model for continuous casting of steel under the influence of a ruler type Electromagnetic Brake (EMBr). The...     

Relating video game exposure,sensation seeking,aggression and socioeconomic factors to school performance

ABSTRACT

The time displacement hypothesis has been widely used to explain the effects of media use on academic performance. This approach has been criticised for its monocausality and weak explanatory power. Utilising a combination of different theoretical perspectives is better suited than utilising only one. Considering displacement, excitement, attraction and third variable hypothesis, this paper argues that the relation between video game use and school performance is not the same for all types of gamers by considering the influence of socioeconomic, psychological and contextual factors. It investigates how gaming patterns (frequency, time of day) and psychological characteristics (sensation seeking, aggression) are related to academic performance for primary and secondary school students. Five hundred and seven students from five primary and four secondary schools in Singapore participated in a paper-pencil survey. Results showed support for a combination of displacement and third variable hypothesis, as overall time spent on video games was negatively associated with primary school children’s and active gamers’ school performances. There was no significant relationship for secondary school children. For primary school children who play games in the morning and afternoon, video game use was negatively associated with school performance. There was no support for the attraction and excitement hypotheses.