An enzyme-linked immunosorbent assay for detection of avian influenza virus subtypes H5 and H7 antibodies

Background

Avian influenza virus (AIV) subtypes H5 and H7 attracts particular attention because of the risk of their potential pathogenicity in poultry. The haemagglutination inhibition (HI) test is widely used as subtype specific test for serological diagnostics despite the laborious nature of this method. However, enzyme-linked immunosorbent assays (ELISAs) are being explored as an alternative test method.H5 and H7 specific monoclonal antibodies were experimentally raised and used in the development of inhibition ELISAs for detection of serological response specifically directed against AIV subtypes H5 and H7. The ELISAs were evaluated with polyclonal chicken anti-AIV antibodies against AIV subtypes: H1N2, H5N2, H5N7, H7N1, H7N7, H9N9, H10N4 and H16N3.

Results

Both the H5 and H7 ELISA proved to have a high sensitivity and specificity and the ELISAs detected H5 and H7 antibodies earlier during experimental infection than the HI test did. The reproducibility of the ELISA’s performed at different times was high with Pearson correlation coefficients of 0.96-0.98.

Conclusions

The ELISAs are a potential alternative to the HI test for screening of large amounts of avian sera, although only experimental sera were tested in this study.

     

Swimming with the giant: coexistence patterns of a new redfin minnow Pseudobarbus skeltoni from a global biodiversity hot spot

Ecological niche theory predicts that coexistence is facilitated by resource partitioning mechanisms that are influenced by abiotic and biotic interactions. Alternative hypotheses suggest that under certain conditions, species may become phenotypically similar and functionally equivalent, which invokes the possibility of other mechanisms, such as habitat filtering processes. To test these hypotheses, we examined the coexistence of the giant redfin Pseudobarbus skeltoni, a newly described freshwater fish, together with its congener Pseudobabus burchelli and an anabantid Sandelia capensis by assessing their scenopoetic and bionomic patterns. We found high habitat and isotope niche overlaps between the two redfins, rendering niche partitioning a less plausible sole mechanism that drives their coexistence. By comparison, environment–trait relationships revealed differences in species–environment relationships, making habitat filtering and functional equivalence less likely alternatives. Based on P. skeltoni's high habitat niche overlap with other species, and its large isotope niche width, we inferred the likelihood of differential resource utilization at trophic level as an alternative mechanism that distinguished it from its congener. In comparison, its congener P. burchelli appeared to have a relatively small trophic niche, suggesting that its trophic niche was more conserved despite being the most abundant species. By contrast, S. capensis was distinguished by occupying a higher trophic position and by having a trophic niche that had a low probability of overlapping onto those of redfins. Therefore, trophic niche partitioning appeared to influence the coexistence between S. capensis and redfins. This study suggests that coexistence of these fishes appears to be promoted by their differences in niche adaptation mechanisms that are probably shaped by historic evolutionary and ecological processes.     

Three-dimensional gas exchange pathways in pome fruit characterized by synchrotron x-ray computed tomography

Our understanding of the gas exchange mechanisms in plant organs critically depends on insights in the three-dimensional (3-D) structural arrangement of cells and voids. Using synchrotron radiation x-ray tomography, we obtained for the first time high-contrast 3-D absorption images of in vivo fruit tissues of high moisture content at 1.4-microm resolution and 3-D phase contrast images of cell assemblies at a resolution as low as 0.7 microm, enabling visualization of individual cell morphology, cell walls, and entire void networks that were previously unknown. Intercellular spaces were always clear of water. The apple (Malus domestica) cortex contains considerably larger parenchyma cells and voids than pear (Pyrus communis) parenchyma. Voids in apple often are larger than the surrounding cells and some cells are not connected to void spaces. The main voids in apple stretch hundreds of micrometers but are disconnected. Voids in pear cortex tissue are always smaller than parenchyma cells, but each cell is surrounded by a tight and continuous network of voids, except near brachyssclereid groups. Vascular and dermal tissues were also measured. The visualized network architecture was consistent over different picking dates and shelf life. The differences in void fraction (5.1% for pear cortex and 23.0% for apple cortex) and in gas network architecture helps explain the ability of tissues to facilitate or impede gas exchange. Structural changes and anisotropy of tissues may eventually lead to physiological disorders. A combined tomography and internal gas analysis during growth are needed to make progress on the understanding of void formation in fruit.     

Glucocorticoids Induce G1 as Well as S-Phase Lengthening in Normal Human Stimulated Lymphocytes: Differential Effects on Cell Cycle Regulatory Proteins

In order to analyze dexamethasone effects on peripheral blood lymphocyte proliferation, we defined various experimental conditions: dexamethasone introduced (i) at the time of phytohemagglutinin stimulation, (ii) 48 h after the beginning of phytohemagglutinin stimulation, and (iii) on unstimulated lymphocytes. In stimulated lymphocytes, we observed an early G1 accumulation (P< 0.005), a delayed increase in the duration of S-phase (P< 0.03), and a consequent increase in cell-cycle duration. The expression of several cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors (CKIs) was modified. Cyclin D3, CDK4, and CDK6 involved in G1-phase control were significantly decreased under dexamethasone treatment whatever the level of stimulation of lymphocytes (stimulated or unstimulated PBL). Cyclin E and CDK2, acting in G1/S-phase transition and S-phase regulation, decreased in stimulated lymphocytes before any modification of S-phase (P< 0.002). The expression of CKIs, mainly of p27^Kip1, appeared to vary with the degree of cell stimulation: a decrease was observed on treated unstimulated lymphocytes, while p27^Kip1increased in dexamethasone-treated cells during stimulation. Our results indicate sequential modifications of the cell-cycle regulation by dexamethasone starting with an action on G1 followed by S-phase control modifications. The protein analysis pinpoints the major complexes concerned: CDK4 and CDK6/cyclin D are mainly involved in G1-phase modifications, while CDK2 and its partner, cyclin E, might be specifically involved in the lengthening of S-phase. The variations observed for p27^Kip1might amplify the functional effects of dexamethasone on kinasic complexes.     

Modular bioreactor for primary human hepatocyte culture: medium flow stimulates expression and activity of detoxification genes

Down-regulation of detoxification genes, notably cytochrome P450 (CYPs), in primary hepatocyte cultures is a long-standing and major concern. We evaluated the influence of medium flow in this model. Hepatocytes isolated from 12 different liver donors were cultured either in a multichamber modular bioreactor (MCmB, flow rate 250-500 μL/min) or under standard/static conditions, and the expression of 32 genes, enzyme activities and biological parameters were measured 7-21 days later. mRNA expression of genes involved in xenobiotic/drug metabolism and transport, including CYP1A1, 1A2, 2B6, 2C9, 3A4 (and activities for some of them), UDP-glucuronosyltransferase (UGT) 1A1, UGT2B4, UGT2B7, glutathione S-transferase (GSTα), and multidrug resistance protein 1 (MDR1) and MRP2, were specifically up-regulated by medium flow as compared with static controls in all cultures tested. In 2-week-old cultures, expression of detoxification genes reached levels close to or higher than those measured in freshly isolated hepatocytes. In contrast, CYP2D6 and most of other tested genes were not affected by medium flow. We conclude that medium flow specifically interferes with, and up-regulates, the activity of xenosensors and/or the expression of detoxification genes in primary human hepatocytes. Down-regulation of detoxification genes in conventional (static) cultures is therefore partly a consequence of the absence of medium circulation.     

The Caenorhabditis elegans centrosomal protein SPD-2 is required for both pericentriolar material recruitment and centriole duplication

BACKGROUND: The centrosome is composed of a centriole pair and pericentriolar material (PCM). By marking the site of PCM assembly, the centrioles define the number of centrosomes present in the cell. The PCM, in turn, is responsible for the microtubule (MT) nucleation activity of centrosomes. Therefore, in order to assemble a functional bipolar mitotic spindle, a cell needs to control both centriole duplication and PCM recruitment. To date, however, the molecular mechanisms that govern these two processes still remain poorly understood. RESULTS: Here we show that SPD-2 is a novel component of the C. elegans centrosome. SPD-2 localizes to the centriole throughout the cell cycle and accumulates on the PCM during mitosis. We show that SPD-2 requires SPD-5 for its accumulation on the PCM and that in the absence of SPD-2, centrosome assembly fails. We further show that centriole duplication is also defective in spd-2(RNAi) embryos, but not in spd-5(RNAi) embryos, where PCM recruitment is efficiently blocked. CONCLUSIONS: Taken together, our results suggest that SPD-2 may link PCM recruitment and centriole duplication in C. elegans. SPD-2 shares homology with a human centrosome protein, suggesting that this key component of the C. elegans centrosome is evolutionarily conserved.     

Shade suppresses wound-induced leaf repositioning through a mechanism involving PHYTOCHROME KINASE SUBSTRATE (PKS) genes

Shaded plants challenged with herbivores or pathogens prioritize growth over defense. However, most experiments have focused on the effect of shading light cues on defense responses. To investigate the potential interaction between shade-avoidance and wounding-induced Jasmonate (JA)-mediated signaling on leaf growth and movement, we used repetitive mechanical wounding of leaf blades to mimic herbivore attacks. Phenotyping experiments with combined treatments on Arabidopsis thaliana rosettes revealed that shade strongly inhibits the wound effect on leaf elevation. By contrast, petiole length is reduced by wounding both in the sun and in the shade. Thus, the relationship between the shade and wounding/JA pathways varies depending on the physiological response, implying that leaf growth and movement can be uncoupled. Using RNA-sequencing, we identified genes with expression patterns matching the hyponastic response (opposite regulation by both stimuli, interaction between treatments with shade dominating the wound signal). Among them were genes from the PKS (Phytochrome Kinase Substrate) family, which was previously studied for its role in phototropism and leaf positioning. Interestingly, we observed reduced shade suppression of the wounding effect in pks2pks4 double mutants while a PKS4 overexpressing line showed constitutively elevated leaves and was less sensitive to wounding. Our results indicate a trait-specific interrelationship between shade and wounding cues on Arabidopsis leaf growth and positioning. Moreover, we identify PKS genes as integrators of external cues in the control of leaf hyponasty further emphasizing the role of these genes in aerial organ positioning.