Evaluation of candidate reference genes for QPCR during ontogenesis and of immune-relevant tissues of European seabass (Dicentrarchus labrax)

The expression level of mRNA can vary significantly in different experimental conditions, such as stress, infection, developmental stage or tissue. Suitable reference genes are expected to exhibit constant expression levels. However no single gene is constitutively expressed in all cell types and under all experimental conditions. It has become clear that expression stability of the intended reference gene has to be examined before each experiment. For expression studies using quantitative real-time PCR (qPCR) at least two reference genes have to be applied. So far expression studies in the European seabass (Dicentrarchus labrax) as well as in the Gilthead seabream (Sparus aurata) have been performed with only one reference gene (S18, Ef-1 alpha or Gapdh). Though significant variations showed up in other teleost species such as the Atlantic halibut and the zebrafish affirming the need for proper normalization strategies, the present study aims at identifying suitable reference genes among nine candidates [glyceraldehyde-phosphate-dehydrogenase (Gapdh), β-actin (two regions of β-actin), 40S ribosomal protein S30 (Fau), ribosomal protein L13 a (L13a), β2-tubulin (Tubb2) and tyrosine 3 monooxygenase/tryptophan 5-monooxygenase activation protein (Tyr)] for expression analysis of 8 developmental stages and a tissue panel (spleen, liver, kidney and brain) with samples infected with Nodavirus and Vibrio anguillarum in D. labrax. Besides the analysis of raw Ct-values, the gene expression stability was determined using two different software applications BestKeeper and NormFinder. According to both algorithms the best two reference genes for an appropriate normalization approach during D. labrax development are Ef-1 alpha and L13a whereas in the tissue panel Fau and L13a are recommended for qPCR normalization.     

Dosimetric characteristics of a new polymer gel and their dependence on post-preparation and post-irradiation time: Effect on X-ray beam profile measurements

The aim of this study is to dosimetrically characterize a new MRI based polymer gel system and to evaluate its usefulness in clinical practice just in terms of beam profile measurements.Normoxic N-vinylpyrrolidone based polymer gel (VIPET) phantoms were produced and used in order to perform three main sets of experiments: a) dose–response evaluation and reproducibility experiments, b) experiments for the evaluation of sensitivity of dose characteristics on ‘gel manufacture – irradiation’ time interval and c) experiments for the evaluation of sensitivity of dose characteristics on ‘irradiation – MRscanning’ time interval. It has been shown that this gel system can be used in a wide dose-range of 0–60 Gy. It exhibits a linear dose–response in the dose-range of 2–35 Gy. Following the proposed manufacturing method the dose–response characteristics are reproducible. Moreover, it seems that the optimum ‘gel manufacturing – irradiation’ time interval is 1 day. However, a ‘gel manufacturing – irradiation’ time interval up to ～1 week can be safely used. The optimum ‘irradiation – MRscanning’ time interval in terms of dose–response sensitivity and dose resolution can be reliably ranged from 1 day to 3 weeks. Finally, X-ray beam profile gel-measurements were performed and found to be in satisfying agreement with corresponding small sensitive volume ion chamber measurements. VIPET gel dosimeters preserved the spatial integrity of the dose distribution during a time period of 50 days post-irradiation. The studied gel system can be safely used in clinical practice within the practical limitations found and described in this work.     

EGF-mediated suppression of cell extrusion during mucosal damage attenuates opportunistic fungal invasion

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The Obesity Epidemic and Future Emergency Responders

Emergency responders should be fit to safely perform strenuous duties. In particular, young recruits are expected to be at or near peak career fitness. We studied the prevalence and health associations of excess weight among 370 consecutive emergency responder candidates for fire and ambulance services in Massachusetts. The mean age and BMI of the recruits were 26.3 (3.8) years and 28.5 (4.9) kg/m², respectively. Seventy‐seven percent had BMI ≥25 kg/m², and 33% were obese (BMI ≥30 kg/m²). After multivariate adjustment, both higher BMI categories and unit increases in BMI were significantly associated with higher blood pressures, worse metabolic profiles, and lower exercise tolerance. Excess weight is highly prevalent and associated with elevated cardiovascular risk among future emergency responders. These findings in a population expected to perform demanding duties supporting public safety merit prompt public health intervention.     

Dual-chambered membrane bioreactor for coculture of stratified cell populations

We have developed a dual-chambered bioreactor (DCB) that incorporates a membrane to study stratified 3D cell populations for skin tissue engineering. The DCB provides adjacent flow lines within a common chamber; the inclusion of the membrane regulates flow layering or mixing, which can be exploited to produce layers or gradients of cell populations in the scaffolds. Computational modeling and experimental assays were used to study the transport phenomena within the bioreactor. Molecular transport across the membrane was defined by a balance of convection and diffusion; the symmetry of the system was proven by its bulk convection stability, while the movement of molecules from one flow line to the other is governed by coupled convection-diffusion. This balance allowed the perfusion of two different fluids, with the membrane defining the mixing degree between the two. The bioreactor sustained two adjacent cell populations for 28 days, and was used to induce indirect adipogenic differentiation of mesenchymal stem cells due to molecular cross-talk between the populations. We successfully developed a platform that can study the dermis–hypodermis complex to address limitations in skin tissue engineering. Furthermore, the DCB can be used for other multilayered tissues or the study of communication pathways between cell populations.     

Raman spectroscopy of blood and urine liquid biopsies for ovarian cancer diagnosis: identification of chemotherapy effects

Blood plasma and serum Raman spectroscopy for ovarian cancer diagnosis has been applied in pilot studies, with promising results. Herein, a comparative analysis of these biofluids, with a novel assessment of urine, was conducted by Raman spectroscopy application in a large patient cohort. Spectra were obtained through samples measurements from 116 ovarian cancer patients and 307 controls. Principal component analysis identified significant spectral differences between cancers without previous treatment (n = 71) and following neo-adjuvant chemotherapy (NACT), (n = 45). Application of five classification algorithms achieved up to 73% sensitivity for plasma, high specificities and accuracies for both blood biofluids, and lower performance for urine. A drop in sensitivities for the NACT group in plasma and serum, with an opposite trend in urine, suggest that Raman spectroscopy could identify chemotherapy-related changes. This study confirms that biofluids' Raman spectroscopy can contribute in ovarian cancer's diagnostic work-up and demonstrates its potential in monitoring treatment response.     

Comparison of methods for assessing geometric efficiency on multi-detector CT scanners

The aim of the current study was to compare the film method against the method based on a new CT slice detector in assessing geometric efficiency (GE) of x-ray beams utilized by a multi-detector CT (MDCT) scanner. Measurements of GE were performed using radiographic films and a solid state CT slice detector for all beam qualities, collimations and focal spot sizes available on an MDCT scanner. Repeatability of GE measurements was assessed. The radiographic film and the solid state detector methods were compared to each other in regard to efficacy in measuring free-in-air GE. The values of GE determined using the radiographic film method were found to range between 48.5% and 90.6%. Differences between values obtained with the radiographic film method and corresponding values obtained with the solid state detector were less than 10% exceeding 5% for only one case. Both methods show that wide beams have higher GE values compared to thin ones. The use of large instead of small focal spot was found to deteriorate GE values by up to 23.1%. Beam quality did not seem to influence GE of the various collimations. When thin beam collimations are employed, a considerable amount of the radiation is wasted for non-imaging purposes. Both film and solid state probe methods are capable of measuring GE of thin as well as wide collimations. The solid state detector is the easiest to use, however its usefulness is reduced by the fact that it cannot measure dose profiles of beam collimations available for step-and-shoot mode of operation.     

Nuclear and Wolbachia-based multimarker approach for the rapid and accurate identification of tsetse species

Background

Tsetse flies (Diptera: Glossinidae) are solely responsible for the transmission of African trypanosomes, causative agents of sleeping sickness in humans and nagana in livestock. Due to the lack of efficient vaccines and the emergence of drug resistance, vector control approaches such as the sterile insect technique (SIT), remain the most effective way to control disease. SIT is a species-specific approach and therefore requires accurate identification of natural pest populations at the species level. However, the presence of morphologically similar species (species complexes and sub-species) in tsetse flies challenges the successful implementation of SIT-based population control.

Results

In this study, we evaluate different molecular tools that can be applied for the delimitation of different Glossina species using tsetse samples derived from laboratory colonies, natural populations and museum specimens. The use of mitochondrial markers, nuclear markers (including internal transcribed spacer 1 (ITS1) and different microsatellites), and bacterial symbiotic markers (Wolbachia infection status) in combination with relatively inexpensive techniques such as PCR, agarose gel electrophoresis, and to some extent sequencing provided a rapid, cost effective, and accurate identification of several tsetse species.

Conclusions

The effectiveness of SIT benefits from the fine resolution of species limits in nature. The present study supports the quick identification of large samples using simple and cost effective universalized protocols, which can be easily applied by countries/laboratories with limited resources and expertise.