Replacement of primary chicken embryonic fibroblasts (CEF) by the DF-1 cell line for detection of avian leucosis viruses.

International regulations prescribe that the absence of avian leucosis viruses (ALV) in avian live virus vaccines has to be demonstrated. Primary chicken embryo fibroblasts (CEF) from special SPF chicken lines are normally used for detection of ALV. The suitability of the DF-1 cell line for ALV-detection, as alternative for primary CEF, was studied in three types of experiments: (1) in titration experiments without cell passage, (2) in experiments with passages in cell cultures according to European Pharmacopoeia requirements, and (3) in experiments with commercial live avian vaccines that had been spiked with known amounts of ALV. In all tests the sensitivity of ALV-A and ALV-J detections on DF-1 cells was at least as high as on primary CEF. The sensitivity of ALV-B detection was always superior when DF-1 cells were used. ALV were detected earlier in all comparative tests when DF-1 cells were used. ALV-A, ALV-B and ALV-J all induced CPE on DF-1 cells, whereas no clear CPE was seen on CEF-cells. For reasons of sensitivity, standardisation as well as reduction of animal use, the data support the use of DF-1 cells to monitor absence of ALV in vaccine virus seed lots or finished products.     

Automated identification of RNA 3D modules with discriminative power in RNA structural alignments

Recent progress in predicting RNA structure is moving towards filling the ‘gap’ in 2D RNA structure prediction where, for example, predicted internal loops often form non-canonical base pairs. This is increasingly recognized with the steady increase of known RNA 3D modules. There is a general interest in matching structural modules known from one molecule to other molecules for which the 3D structure is not known yet. We have created a pipeline, metaRNAmodules, which completely automates extracting putative modules from the FR3D database and mapping of such modules to Rfam alignments to obtain comparative evidence. Subsequently, the modules, initially represented by a graph, are turned into models for the RMDetect program, which allows to test their discriminative power using real and randomized Rfam alignments. An initial extraction of 22 495 3D modules in all PDB files results in 977 internal loop and 17 hairpin modules with clear discriminatory power. Many of these modules describe only minor variants of each other. Indeed, mapping of the modules onto Rfam families results in 35 unique locations in 11 different families. The metaRNAmodules pipeline source for the internal loop modules is available at http://rth.dk/resources/mrm.     

Selection of phosphate-solubilizing diazotrophic Herbaspirillum and Burkholderia strains and their effect on rice crop yield and nutrient uptake

Background and Aims

Plant growth-promoting bacteria, mainly diazotrophs and phosphate solubilizers, can reduce the use of chemical fertilizers for rice crops. Here, diazotrophic bacteria isolated from rice were screened for their ability to solubilize inorganic P (P_i) in vitro and in association with rice plants cultivated in pots.

Methods

Forty-nine isolates were tested for the ability to solubilize P_i on NBRIP and GL agar plate media and seven selected strains were further evaluated in NBRIP liquid medium. Three of these strains were inoculated in rice plants grown in soil pots containing ¹⁵N-labeled fertilizer and two sources of P: tricalcium phosphate (TCP) or simple superphosphate (SSP). The dry matter, yield, N, P, and the ¹⁵N content accumulated in plant tissues were measured at 135 days after planting.

Results

Seven strains belonging to the genera Herbaspirillum and Burkholderia formed a halo of solubilized P_i on agar plates. The Burkholderia strains showed peak soluble P (around 200 mg P L^?1) on the fifth day when grown in NBRIP liquid medium for 14 days. Inoculation of Herbaspirillum strains (H18, ZA15) and a Burkholderia vietaminensis strain (AR114) increased rice grain yield from 33 to 47 % with TCP and 18 to 44 % with TSS, respectively. The bacterial inoculation led to enhanced N-use efficiency of the ¹⁵N-labeled fertilizer.

Conclusion

These results suggest that the selection and use of P-solubilizing diazotrophic bacteria are a good strategy to promote P solubilization and/or N use efficiency in rice plants.     

Mapping of QTL for seed dormancy in a winter oilseed rape doubled haploid population

Following winter oilseed rape cultivation, considerable numbers of volunteer oilseed rape plants may occur in subsequent years in following crops. The appearance of volunteer oilseed rape plants is based on the capability of the seeds to become secondary dormant and to survive in this stage for many years in the soil. Genetic reduction of secondary seed dormancy in oilseed rape could provide a means to reduce the frequency of volunteer plants and especially the dispersal of transgenic oilseed rape. The objective of the present study was to analyse the inheritance of primary and secondary seed dormancy in a winter oilseed rape doubled haploid population derived from the cross Express 617 × R53 and to study correlations to other seed traits. Field experiments were performed in Germany for 2 years at two locations with two replicates. Seeds harvested from open pollinated plants were used for all analyses, including a laboratory test for seed dormancy. A previously developed molecular marker map of the doubled haploid population was used to map QTL of the relevant traits. For primary, secondary and total seed dormancy, the results showed significant effects of the genotypes and their interactions, with years and locations. Two, four and five QTL were detected for primary, secondary and total seed dormancy which explained 19, 35 and 42 % of the phenotypic variance, respectively. Results show that secondary seed dormancy is a heritable trait and that selection for low secondary seed dormancy is possible.     

An Extended Gibbs Ensemble

Abstract

A general extended Gibbs ensemble, obtained by augmenting the standard Gibbs ensemble by intermediate states in the spirit of the scaled particle method of Nezbeda and Kolafa [Molec. Simul., 5, 391 (1991)], is introduced. The intermediate states span the states with different number of particles in the simulation boxes and facilitate the transfer of particles even in such complex systems as e.g., mixtures of very different components, systems of flexible polymeric molecules, or systems at very high densities. A general formulation of the ensemble is given and two implementations are considered in detail. The method is very general and is exemplified by studying the fluid-fluid coexistence in a dense binary mixture of the hard-sphere and square-well fluids. It is found that its efficiency is about by factor three greater in comparison with the standard Gibbs ensemble simulations.     

Genetic engineering of cytokinin metabolism: Prospective way to improve agricultural traits of crop plants

Cytokinins (CKs) are ubiquitous phytohormones that participate in development, morphogenesis and many physiological processes throughout plant kingdom. In higher plants, mutants and transgenic cells and tissues with altered activity of CK metabolic enzymes or perception machinery, have highlighted their crucial involvement in different agriculturally important traits, such as productivity, increased tolerance to various stresses and overall plant morphology. Furthermore, recent precise metabolomic analyses have elucidated the specific occurrence and distinct functions of different CK types in various plant species. Thus, smooth manipulation of active CK levels in a spatial and temporal way could be a very potent tool for plant biotechnology in the future. This review summarises recent advances in cytokinin research ranging from transgenic alteration of CK biosynthetic, degradation and glucosylation activities and CK perception to detailed elucidation of molecular processes, in which CKs work as a trigger in model plants. The first attempts to improve the quality of crop plants, focused on cereals are discussed, together with proposed mechanism of action of the responses involved.     

Ethanolamide Oxylipins of Linolenic Acid Can Negatively Regulate Arabidopsis Seedling Development

N-Acylethanolamines (NAEs) are fatty-acid derivatives with potent biological activities in a wide range of eukaryotic organisms. Polyunsaturated NAEs are among the most abundant NAE types in seeds of Arabidopsis thaliana, and they can be metabolized by either fatty acid amide hydrolase (FAAH) or by lipoxygenase (LOX) to low levels during seedling establishment. Here, we identify and quantify endogenous oxylipin metabolites of N-linolenoylethanolamine (NAE 18:3) in Arabidopsis seedlings and show that their levels were higher in faah knockout seedlings. Quantification of oxylipin metabolites in lox mutants demonstrated altered partitioning of NAE 18:3 into 9- or 13-LOX pathways, and this was especially exaggerated when exogenous NAE was added to seedlings. When maintained at micromolar concentrations, NAE 18:3 specifically induced cotyledon bleaching of light-grown seedlings within a restricted stage of development. Comprehensive oxylipin profiling together with genetic and pharmacological interference with LOX activity suggested that both 9-hydroxy and 13-hydroxy linolenoylethanolamides, but not corresponding free fatty-acid metabolites, contributed to the reversible disruption of thylakoid membranes in chloroplasts of seedling cotyledons. We suggest that NAE oxylipins of linolenic acid represent a newly identified, endogenous set of bioactive compounds that may act in opposition to progression of normal seedling development and must be depleted for successful establishment.     

Early Bacteriopheophytin Reduction in Charge Separation in Reaction Centers of Rhodobacter sphaeroides

A question at the forefront of biophysical sciences is, to what extent do quantum effects and protein conformational changes play a role in processes such as biological sensing and energy conversion? At the heart of photosynthetic energy transduction lie processes involving ultrafast energy and electron transfers among a small number of tetrapyrrole pigments embedded in the interior of a protein. In the purple bacterial reaction center (RC), a highly efficient ultrafast charge separation takes place between a pair of bacteriochlorophylls: an accessory bacteriochlorophyll (B) and bacteriopheophytin (H). In this work, we applied ultrafast spectroscopy in the visible and near-infrared spectral region to Rhodobacter sphaeroides RCs to accurately track the timing of the electron on B_A and H_A via the appearance of the B_A and H_A anion bands. We observed an unexpectedly early rise of the H_A⁻ band that challenges the accepted simple picture of stepwise electron transfer with 3 ps and 1 ps time constants. The implications for the mechanism of initial charge separation in bacterial RCs are discussed in terms of a possible adiabatic electron transfer step between B_A and H_A, and the effect of protein conformation on the electron transfer rate.