Sequential monolayer-suspension culture of human airway epithelial cells

AbstractIn this article, we describe a technique for culturing human airway epithelial cells, developed in Leuven, as a new tool for a most reliable diagnosis for ciliary disorders. This technique that allows to keep both structural and functional primary abnormalities of inherited ciliary abnormalities, while avoiding the secondary ones, can also be useful to a number of other studies, namely in cystic fibrosis

Use of freeze-cracking in ontogenetic research in Macrostomum lignano (Macrostomida, Rhabditophora)

A method for studying whole mount flatworm embryos based on freeze-cracking of the eggs is described. This method allows successful immunohistological and immunocytological studies of whole mount embryos. It does not require the use of sharpened needles or a microinjection system to puncture the eggshell. Moreover, this method is more practical and less time-consuming than classical puncturing and much cheaper than the use of a microinjection system. The advantages of this method are illustrated by results of several immunolocalisation experiments in the macrostomid flatworm Macrostomum lignano. The optimal procedure and crucial steps for this method are discussed

Thermodynamic framework to assess low abundance DNA mutation detection by hybridization

The knowledge of genomic DNA variations in patient samples has a high and increasing value for human diagnostics in its broadest sense. Although many methods and sensors to detect or quantify these variations are available or under development, the number of underlying physico-chemical detection principles is limited. One of these principles is the hybridization of sample target DNA versus nucleic acid probes. We introduce a novel thermodynamics approach and develop a framework to exploit the specific detection capabilities of nucleic acid hybridization, using generic principles applicable to any platform. As a case study, we detect point mutations in the KRAS oncogene on a microarray platform. For the given platform and hybridization conditions, we demonstrate the multiplex detection capability of hybridization and assess the detection limit using thermodynamic considerations; DNA containing point mutations in a background of wild type sequences can be identified down to at least 1% relative concentration. In order to show the clinical relevance, the detection capabilities are confirmed on challenging formalin-fixed paraffin-embedded clinical tumor samples. This enzyme-free detection framework contains the accuracy and efficiency to screen for hundreds of mutations in a single run with many potential applications in molecular diagnostics and the field of personalised medicine

Inhibition of spontaneous neutrophil apoptosis by parabutoporin acts independently of NADPH oxidase inhibition but by lipid raft-dependent stimulation of Akt

Neutrophil cell death plays a crucial role in neutrophil homeostasis and the resolution of inflammation. The superoxide-producing NADPH oxidase is involved in pathogen degradation and subsequent activation of cell death programs. Neutrophils from patients with chronic granulomatous disease, who have a deficient NADPH oxidase activity, have been demonstrated previously to have a prolonged lifespan, suggesting that a basal NADPH oxidase activity also regulates spontaneous neutrophil turnover. The NADPH oxidase inhibitor parabutoporin (PP) does delay spontaneous apoptosis, but this effect is completely independent of NADPH oxidase inhibition. Instead, the prosurvival effect of PP depends on activation of protein kinase B/Akt via lipid raft signaling. Disruption of lipid rafts abrogates the prosurvival effect without interfering with NADPH oxidase activity. Furthermore, we cannot detect a different rate of spontaneous apoptosis between normal and NADPH oxidase-deficient neutrophils, arguing against a role of NADPH oxidase in spontaneous neutrophil apoptosis

Sensing and control in dual-recycling laser interferometer gravitational-wave detectors

We introduce length-sensing and control schemes for the dual-recycled cavity-enhanced Michelson interferometer configuration proposed for the Advanced Laser Interferometer Gravitational Wave Observatory (LIGO). We discuss the principles of this scheme and show methods that allow sensing and control signals to be derived. Experimental verification was carried out in three benchtop experiments that are introduced. We present the implications of the results from these experiments for Advanced LIGO and other future interferometric gravitational-wave detectors

Selection for improved energy use efficiency and drought tolerance in canola results in distinct transcriptome and epigenome changes

To increase both the yield potential and stability of crops, integrated breeding strategies are used that have mostly a direct genetic basis, but the utility of epigenetics to improve complex traits is unclear. A better understanding of the status of the epigenome and its contribution to agronomic performance would help in developing approaches to incorporate the epigenetic component of complex traits into breeding programs. Starting from isogenic canola (Brassica napus) lines, epilines were generated by selecting, repeatedly for three generations, for increased energy use efficiency and drought tolerance. These epilines had an enhanced energy use efficiency, drought tolerance, and nitrogen use efficiency. Transcriptome analysis of the epilines and a line selected for its energy use efficiency solely revealed common differentially expressed genes related to the onset of stress tolerance-regulating signaling events. Genes related to responses to salt, osmotic, abscisic acid, and drought treatments were specifically differentially expressed in the drought-tolerant epilines. The status of the epigenome, scored as differential trimethylation of lysine-4 of histone 3, further supported the phenotype by targeting drought-responsive genes and facilitating the transcription of the differentially expressed genes. From these results, we conclude that the canola epigenome can be shaped by selection to increase energy use efficiency and stress tolerance. Hence, these findings warrant the further development of strategies to incorporate epigenetics into breeding

Dual-energy X-ray absorptiometry, skinfold thickness and waist circumference for assessing body composition in ambulant and non-ambulant wheelchair games players

Field based assessments provide a cost effective and accessible alternative to dual energy X ray absorptiometry (DXA) for practitioners determining body composition in athletic populations. It remains unclear how the range of physical impairments classifiable in wheelchair sports may affect the utility of field based body composition techniques. The present study assessed body composition using DXA in 14 wheelchair games players who were either wheelchair dependent (non walkers; n=7) or relied on a wheelchair for sports participation only (walkers; n=7). Anthropometric measurements were used to predict body fat percentage with existing regression equations established for able bodied persons by Sloan & Weir, Durnin & Womersley, Lean et al, Gallagher et al and Pongchaiyakul et al. In addition, linear regression analysis was performed to calculate the association between body fat percentage and BMI, waist circumference, sum of 6 skinfold thickness and sum of 8 skinfold thickness. Results showed that non- walkers had significantly lower total lean tissue mass (46.2±=6.6 kg vs. 59.4±8.2 kg, P =.006) and total body mass (65.8±4.2 kg vs. 79.4 ± 14.9kg; P=0.05) than walkers. Body fat percentage calculated from most existing regression equations was significantly lower than that from DXA, by 2 to 9% in walkers and 8 to 14% in non- walkers. Of the anthropometric measurements, the sum of 8 skinfold thickness had the lowest standard error of estimation in predicting body fat content. In conclusion, existing anthropometric equations developed in able- bodied populations substantially underestimated body fat content in wheelchair athletes, particularly non-walkers. Impairment specific equations may be needed in wheelchair athletes