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.     

Olea Europea 3-year pollen record in the area of Thessaloniki,Greece and its sensitizing significance

Summary The observations of airborne pollen ofOlea europea and the incidence of clinical manifestations in patients allergic to this pollen type have not been registered so far in the city of Thessaloniki. The purpose of this study was: 1. to assess theO. europea pollen circulation in the area of our city, and 2. to detect the percentage of sensitivity toO. europea pollen in patients with pollinosis. We collected daily pollen samples during a 3-year period (February '87-January '90), using a Burkard volumetric trap, located on a high level area in the centre of the city. The pollen counts were then registered. The O.europea pollen grains were not differentiated microscopically from the other Oleaceae, but identified through phenological criteria. The patients included in the assessment of the sensitivity toO. europea came from the out-patient clinic of bronchial asthma of the General Hospital ?G. Papanicolaou?. They had a seasonal pollinosis and they were submitted to prick test using a battery of 22 groups and an O.europea extract. Pollen ofO. europea appears first in the atmosphere of Thessaloniki at the beginning of May, shows a peak in the end of May and continues to be present till the end of June. The quantity ofO. europea pollen ranked 6th in the list of the total pollen count and its flowering period coincided with that of grasses. In a sample of 360 patients with seasonal pollinosis, we detected anO. europea pollen sensitivity combined with other alleargens in 37% of the patients and a monosensivity in 4%. We conclude that pollen ofO. europea results to be present over a relative short period of time (May–June) in the area of Thessaloniki. The percentage of patients' sensitization toO. europea pollen was a little less frequent than sensitization to grasses, even if their flowering time coincides and their presence in the air shows about the same concentration values.     

Usefulness of F2-isoprostanes in early prognostication after cardiac arrest: a topical review of the literature and meta-analysis of preclinical data

Abstract

Prognostication after cardiac arrest (CA) represents a challenging issue, and several biomarkers have been proposed in the attempt to predict outcome. Among these, F2-isoprostanes stand out as potential biomarkers for early prognostication, providing information on the magnitude of global oxidative injury after return of spontaneous circulation (ROSC). We performed a topical review searching PubMed and Scopus databases to identify studies evaluating the modifications of F2-isoprostanes in the early period after CA, and a meta-analysis of studies providing curves of F2-isoprostanes plasma levels seeking to describe the biomarker’s kinetics after CA. Evidence suggests that plasma levels of F2-isoprostanes increase in the early post-resuscitation period and seem well correlated with the burden of ischaemia-reperfusion injury. Our meta-analysis shows a possible increase as early as 5?minutes after ROSC, which persists at 2?hours and is attenuated at 4?hours. Clinical studies are warranted to evaluate the utility of this biomarker for prognostication purposes in CA survivors.     

Quantitative relationships between boron and mannitol concentrations in phloem exudates of Olea europaea leaves under contrasting boron supply conditions

Root exudates are implicated in the chemical defense of plants, but testing such hypotheses has been hindered by the difficulties of quantifying allelochemical concentrations in soil. Here we describe a new, simple method to quantify the dynamics of non-polar root exudates in soil. Novel soil probes were constructed using stainless steel wire inserted into polydimethylsiloxane (PDMS) tubing. Probes were inserted into soil for 24 h, removed and extracted, and analyzed by HPLC. Lipophilic thiophenes produced by roots of Tagetes and Rudbeckia species were chosen as candidate compounds to test the method. Probes recovered microgram quantities of the highly phytotoxic thiophenes 5-(3-buten-1-ynyl)-2,2′-bithienyl (BBT) and α-terthienyl per probe per day from the root zone of Tagetes patula, and distribution of thiophenes beneath plants was spatially and temporally heterogeneous. Flux-proportional sampling of soil provides a means to test hypotheses about the role of root exudates in plant–plant and other interactions.     

Rapid suppression and sustained activation of distinct cortical regions for a delayed sensory-triggered motor response

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A Simple Methodological Approach for Counting and Identifying Culturable Viruses Adsorbed to Cellulose Nitrate Membrane Filters

We identified conditions under which Buffalo green monkey cells grew on the surfaces of cellulose nitrate membrane filters in such a way that they covered the entire surface of each filter and penetrated through the pores. When such conditions were used, poliovirus that had previously been adsorbed on the membranes infected the cells and replicated. A plaque assay method and a quantal method (most probable number of cytopathic units) were used to detect and count the viruses adsorbed on the membrane filters. Polioviruses in aqueous suspensions were then concentrated by adsorption to cellulose membrane filters and were subsequently counted without elution, a step which is necessary when the commonly used methods are employed. The pore size of the membrane filter, the sample contents, and the sample volume were optimized for tap water, seawater, and a 0.25 M glycine buffer solution. The numbers of viruses recovered under the optimized conditions were more than 50% greater than the numbers counted by the standard plaque assay. When ceftazidime was added to the assay medium in addition to the antibiotics which are typically used, the method could be used to study natural samples with low and intermediate levels of microbial pollution without decontamination of the samples. This methodological approach also allowed plaque hybridization either directly on cellulose nitrate membranes or on Hybond N+ membranes after the preparations were transferred.     

Characterization of cultivated fungi isolated from grape marc wastes through the use of amplified rDNA restriction analysis and sequencing

Microbial assessment of grape marc wastes, the residual solid by-product of the wine-industry, was performed by identifying phylogenetically the fungal culturable diversity in order to evaluate environmental and disposal safety issues and to discuss ecological considerations of applications on agricultural land. Fungal spores in grape marc were estimated to 4.7×10⁶ per g dry weight. Fifty six fungal isolates were classified into eight operational taxonomic units (OTUs) following amplified ribosomal DNA restriction analysis (ARDRA) and colony morphology. Based on 18S rRNA gene and 5.8S rRNA gene-ITS sequencing, the isolates representing OTUs #1, #2, #3, and #4, which comprised 44.6%, 26.8%, 12.5%, and 5.3%, respectively, of the number of the total isolates, were identified as Aspergillus fumigatus, Bionectria ochroleuca, Haematonectria haematococca, and Trichosporon mycotoxinivorans. The isolates of OTU#5 demonstrated high phylogenetic affinity with Penicillium spp., while members of OTUs #6 and #7 were closer linked with Geotrichum candidum var. citri-aurantii and Mycocladus corymbifer, respectively (95.4 and 97.9% similarities in respect to their 5.8S rRNA gene-ITS sequences). The OTU#8 with a single isolate was related with Aspergillus strains. It appears that most of the fungal isolates are associated with the initial raw material. Despite the fact that some of the species identified may potentially act as pathogens, measures such as the avoidance of maintaining large and unprocessed quantities of grape marc wastes in premises without adequate aeration, together with its suitable biological treatment (e.g., composting) prior to any agriculture-related application, could eliminate any pertinent health risks.