Generation of candidate human influenza vaccine strains in cell culture: rehearsing the European response to an H7N1 pandemic threat

Background: Although H5N1 avian influenza viruses pose the most obvious imminent pandemic threat, there have been several recent zoonotic incidents involving transmission of H7 viruses to humans. Vaccines are the primary public health defense against pandemics, but reliance on embryonated chickens eggs to propagate vaccine and logistic problems posed by the use of new technology may slow our ability to respond rapidly in a pandemic situation. Objectives: We sought to generate an H7 candidate vaccine virus suitable for administration to humans whose generation and amplification avoided the use of eggs. Methods: We generated a suitable H7 vaccine virus by reverse genetics. This virus, known as RD3, comprises the internal genes of A/Puerto Rico/8/34 with surface antigens of the highly pathogenic avian strain A/Chicken/Italy/13474/99 (H7N1). The multi-basic amino acid site in the HA gene, associated with high pathogenicity in chickens, was removed. Results: The HA modification did not alter the antigenicity of the virus and the resultant single basic motif was stably retained following several passages in Vero and PER. C6 cells. RD3 was attenuated for growth in embryonated eggs, chickens, and ferrets. RD3 induced an antibody response in infected animals reactive against both the homologous virus and other H7 influenza viruses associated with recent infection by H7 viruses in humans. Conclusions: This is the first report of a candidate H7 vaccine virus for use in humans generated by reverse genetics and propagated entirely in mammalian tissue culture. The vaccine has potential use against a wide range of H7 strains.

Phases of dormancy in yam tubers (Dioscorea rotundata)

Background and Aims The control of dormancy in yam (Disocorea spp.) tubers is poorly understood and attempts to shorten the long dormant period (i.e. cause tubers to sprout or germinate much earlier) have been unsuccessful. The aim of this study was to identify and define the phases of dormancy in Dioscorea rotundata tubers, and to produce a framework within which dormancy can be more effectively studied. center dot Methods Plants of 'TDr 131' derived from tissue culture were grown in a glasshouse simulating temperature and photoperiod at Ibadan (7 degrees N), Nigeria to produce tubers. Tubers were sampled on four occasions: 30 d before shoot senescence (149 days after planting, DAP), at shoot senescence (179 DAP), and twice during storage at a constant 25 degrees C (269 and 326 DAP). The development of the apical shoot bud was described from tissue sections. In addition, the responsiveness of shoot apical bud development to plant growth regulators (gibberellic acid, 2-chloroethanol and thiourea) applied to excised tuber sections was also examined 6 and 12 d after treatment. center dot Key Results and Conclusions Three phases of tuber dormancy are proposed: Phase I, from tuber initiation to the appearance of the tuber germinating meristem; Phase II, from the tuber germinating meristem to initiation of foliar primordium; and Phase III, from foliar primordium to appearance of the shoot bud on the surface of the tuber. Phase I is the longest phase (approx. 220 d in 'TDr 131'), is not affected by PGRs and is proposed to be an endo-dormant phase. Phases II and III are shorter (< 70 d in total), are influenced by PGRs and environmental conditions, and are therefore endo-/eco-dormant phases. To manipulate dormancy to allow off-season planting and more than one generation per year requires that the duration of Phase I is shortened.

The use of bioluminescence for monitoring in planta growth dynamics of a Pseudomonas syringae plant pathogen

The use of bioluminescence was evaluated as a tool to study Pseudomonas syringae population dynamics in susceptible and resistant plant environments. Plasmid pGLITE, containing the luxCDABE genes from Photorhabdus luminescens, was introduced into Pseudomonas syringae pv. phaseolicola race 7 strain 1449B, a Gram-negative pathogen of bean (Phaseolus vulgaris). Bacteria recovered from plant tissue over a five-day period were enumerated by counting numbers of colony forming units and by measurement of bioluminescence. Direct measurement of bioluminescence from leaf disc homogenates consistently reflected bacterial growth as determined by viable counting, but also detected subtle effects of the plant resistance response on bacterial viability. This bioluminescence procedure enables real time measurement of bacterial metabolism and population dynamics in planta, obviates the need to carry out labour intensive and time consuming traditional enumeration techniques and provides a sensitive assay for studying plant effects on bacterial cells.

Combinatorial peptide ligand libraries and plant proteomics: a winning strategy at a price

The mechanism of action and properties of a solid-phase ligand library made of hexapeptides (combinatorial peptide ligand libraries or CPLL, for capturing the "hidden proteome", i.e. the low- and very low-abundance proteins Constituting the vast majority of species in any proteome. as applied to plant tissues, are reviewed here. Plant tissues are notoriously recalcitrant to protein extraction and to proteome analysis, Firstly, rigid plant cell walls need to be mechanically disrupted to release the cell content and, in addition to their poor protein yield, plant tissues are rich in proteases and oxidative enzymes, contain phenolic Compounds, starches, oils, pigments and secondary metabolites that massively contaminate protein extracts. In addition, complex matrices of polysaccharides, including large amount of anionic pectins, are present. All these species compete with the binding of proteins to the CPLL beads, impeding proper capture and identification I detection of low-abundance species. When properly pre-treated, plant tissue extracts are amenable to capture by the CPLL beads revealing thus many new species among them low-abundance proteins. Examples are given on the treatment of leaf proteins, of corn seed extracts and of exudate proteins (latex from Hevea brasiliensis). In all cases, the detection of unique gene products via CPLL Capture is at least twice that of control, untreated sample. (c) 2008 Elsevier B.V. All rights reserved.

Inhibition of the adhesion of enteropathogenic Escherichia coli strains to HT-29 cells in culture by chito-oligosaccharides

The ability of chito-oligosaccharides (COS) to inhibit selected intestinal bacteria was investigated. COS at 2.5 mg ml(-1) had no significant effect on the adhesion of three strains of verotoxigenic Escherichia coli (VTEC), Lactobacillus pentosus, L. casei or L. gasseri to human HT29 cells in tissue culture. However, COS significantly inhibited adhesion of three strains of enteropathogenic E. coli (EPEC) to below 30% of the level of adhesion seen in the controls. Dose-response curves were constructed to further characterise the inhibition of EPEC strains to HT29 cells. (c) 2005 Elsevier Ltd. All rights reserved.

Doubled haploid ramets via embryogenesis of haploid tissue cultures

Tissue culture in the oil palm business is generally concerned with the multiplication (clonal production) of dura, pisifera and tenera palms. These are all normal diploids (2n=2x=36). Sumatra Bioscience has pioneered haploid tissue culture of oil palm (n=x=18). Haploid oil palm is the first step in producing doubled haploid palms which in turn provide parental lines for F1 hybrid production. Chromosome doubling is known to occur during embryogenesis in other haploid cultures, e.g. barley anther culture. Haploid tissue cultures in oil palm were therefore set up to investigate and exploit spontaneous chromosome doubling during embryogenesis. Flow cytometry of embryogenic tissue showed the presence of both haploid (n) and doubled haploid (2n) cells indicating spontaneous doubling. Completely doubled haploid ramets were regenerated suggesting that doubling occurred during the first mitoses of embryogenesis. This is the first report of doubled haploid production in oil palm via haploid tissue culture. The method provides a means of producing a range of doubled haploids in oil palm from the 1,000 plus haploids available at Sumatra Bioscience, in addition the method also produced doubled haploid (and haploid) clones. 1.

Interaction between attaching and effacing Escherichia coli serotypes O157:H7 and O26:K60 in cell culture

Ruminants harbour both O157:H7 and non-O157 Attaching Effacing Escherichia coli (AEEC) strains but to date only nonO157 AEEC have been shown to induce attaching effacing lesions in naturally infected animals. However, O157 may induce lesions in deliberate oral inoculation studies and persistence is considered dependent upon the bacterially encoded locus for enterocyte effacement. In concurrent infections in ruminants it is unclear whether non-O157 AEEC contribute either positively or negatively to the persistence of E. coli O157:H7. To investigate this, and prior to animal studies, E. coli O157:H7 NCTC 12900, a non-toxigenic strain that persists in conventionally reared sheep, and non-toxigenic AEEC O26:K60 isolates of sheep origin were tested for adherence to Hep-2 tissue culture alone and in competition one with another. Applied together, both strains adhered in similar numbers but lower than when either was applied separately. Pre-incubation of tissue culture with either one strain reduced significantly (P < 0.05) the extent of adherence of the strain that was applied second. It was particularly noticeable that AEEC O26 when applied first reduced adherence and inhibited microcolony formation, as demonstrated by confocal microscopy, of E. coli 01 57:H7. The possibility that prior colonisation of a ruminant by non-O157 AEEC such as O26 may antagonise O157 colonisation and persistence in ruminants is discussed. (C) 2004 Elsevier B.V. All rights reserved.

Isolation and characterization of an AINTEGUMENTA-like gene in different coconut (Cocos nucifera L.) varieties from Sri Lanka

Development of an efficient tissue culture protocol in coconut is hampered by numerous technical constraints. Thus a greater understanding of the fundamental aspects of embryogenesis is essential. The role of AINTEGUMENTA-like genes in embryogenesis has been elucidated not only in model plants but also in economically important crops. A coconut gene, CnANT, that encodes two APETALA2 (AP2) domains and a conserved linker region similar to those of the BABY BOOM transcription factor was cloned, characterized, and its tissue specific expression was examined. The full-length cDNA of 1,780 bp contains a 1,425-bp open reading frame that encodes a putative peptide of 474 amino acids. The genomic DNA sequence includes 2,317 bp and consists of nine exons interrupted by eight introns. The exon/intron organization of CnANT is similar to that of homologous genes in other plant species. Analysis of differential tissue expression by real-time polymerase chain reaction indicated that CnANT is expressed more highly in in vitro grown tissues than in other vegetative tissues. Sequence comparison of the genomic sequence of CnANT in different coconut varieties revealed one single nucleotide polymorphism and one indel in the first exon and first intron, respectively, which differentiate the Tall group of trees from Dwarfs. The indel sequence, which can be considered a simple sequence repeats marker, was successfully used to distinguish the Tall and Dwarf groups as well as to develop a marker system, which may be of value in the identification of parental varieties that are used in coconut breeding programs in Sri Lanka.

The identification of genes important in pseudomonas syringae pv. phaseolicola plant colonisation using in vitro screening of transposon libraries

The bacterial plant pathogen Pseudomonas syringae pv. phaseolicola (Pph) colonises the surface of common bean plants before moving into the interior of plant tissue, via wounds and stomata. In the intercellular spaces the pathogen proliferates in the apoplastic fluid and forms microcolonies (biofilms) around plant cells. If the pathogen can suppress the plant’s natural resistance response, it will cause halo blight disease. The process of resistance suppression is fairly well understood, but the mechanisms used by the pathogen in colonisation are less clear. We hypothesised that we could apply in vitro genetic screens to look for changes in motility, colony formation, and adhesion, which are proxies for infection, microcolony formation and cell adhesion. We made transposon (Tn) mutant libraries of Pph strains 1448A and 1302A and found 106/1920 mutants exhibited alterations in colony morphology, motility and biofilm formation. Identification of the insertion point of the Tn identified within the genome highlighted, as expected, a number of altered motility mutants bearing mutations in genes encoding various parts of the flagellum. Genes involved in nutrient biosynthesis, membrane associated proteins, and a number of conserved hypothetical protein (CHP) genes were also identified. A mutation of one CHP gene caused a positive increase in in planta bacterial growth. This rapid and inexpensive screening method allows the discovery of genes important for in vitro traits that can be correlated to roles in the plant interaction

Difference in competence for in vitro proliferation and ex vitro growth of genetically identical mature and juvenile clones of apomictic Malus species

The apomictic system in Malus wits Used Is a model to examine rejuvenation by generating genetically identical tissue culture lines that had two entirely different developmental origins: either embryo-derived tissues (juvenile clones) or somatic tissue from the adult/mature tree (mature clones). These two lines were then subsequently used to examine in vitro difference between mature (M) and juvenile (J) tissues in potential for shoot, root proliferation and ex vitro (glasshouse) growth. The M clones of M. hupehensis and M. toringoides in vitro had significantly fewer total shoots and shoot more than 2 cm in length per proliferating explant than the J clones and also rooted less efficiently. Ex vitro (glasshouse) juvenile clones had shorter internodes, a greater number of leaves and more dry weight compared to their mature counterparts.

Adventitious root formation in anacardium occidentale L. in response to phytohormones and removal of roots

Despite advances in tissue culture techniques, propagation by leafy, softwood cuttings is the preferred, practical system for vegetative reproduction of many tree and shrub species. Species are frequently defined as 'difficult'- or 'easy-to-root' when propagated by conventional cuttings. Speed of rooting is often linked with ease of propagation, and slow-to-root species may be 'difficult' precisely because tissues deteriorate prior to the formation of adventitious roots. Even when roots form, limited development of these may impair the establishment of a cutting. In this study we used softwood cuttings of cashew (Anacardium occidentale), a species considered as 'difficult-to-root'. We aimed to test the hypothesis that speed, and extent of early rooting, is critical in determining success with this species; and that the potential to form adventitious roots will decrease with time in the propagation environment. Using two genotypes, initial rooting rates were examined in the presence or absence of exogenous auxin. In cuttings that formed adventitious roots, either entire roots or root tips were removed, to determine if further root formation/development was feasible. To investigate if subsequent root responses were linked to phytohormone action, a number of cuttings were also treated with either exogenous auxin (indole-3-butyric acid-IBA) or cytokinin (zeatin). Despite the reputation of Anacardium as being 'difficult-to-root', we found high rooting rates in two genotypes (AC 10 and CCP 1001). Removing adventitious roots from cuttings and returning them to the propagation environment, resulted in subsequent re-rooting. Indeed, individual cuttings could develop new adventitious roots on four to five separate occasions over a 9 week period. Data showed that rooting potential increased, not decreased with time in the propagation environment and that cutting viability was unaffected. Root expression was faster (8-15 days) after the removal of previous roots compared to when the cuttings were first stuck (21 days). Exposing cuttings to IBA at the time of preparation, improved initial rooting in AC 10, but not in CCP 1001. Application of IBA once roots had formed had little effect on subsequent development, but zeatin reduced root length and promoted root number and dry matter accumulation. These results challenge our hypothesis, and indicate that rooting potential remains high in Anacardium. The precise mechanisms that regulate the number of adventitious roots expressed, remain to be determined. Nevertheless, results indicate that rooting potential can be high in 'difficult-to-root' species, and suggest that providing supportive environments is the key to expressing this potential. (c) 2006 Elsevier B.V. All rights reserved.

Nucleolin is regulated both at the level of transcription and translation

Nucleolin is a multi-functional protein that is located to the nucleolus. In tissue Culture cells, the stability of nucleolin is related to the proliferation status of the cell. During development, rat cardiomyocytes proliferate actively with increases in the mass of the heart being due to both hyperplasia and hypertrophy. The timing of this shift in the phenotype of the myocyte from one capable of undergoing hyperplasia to one that can grow only by hypertrophy occurs within 4 days of post-natal development. Thus, cardiomyocytes are an ideal model system in which to study the regulation of nucleolin during growth in vivo. Using Western blot and quantitative RT-PCR (TaqMan) we found that the amount of nucleolin is regulated both at the level of transcription and translation during the development of the cardiomyocyte. However, in cells which had exited the cell cycle and were subsequently given a hypertrophic stimulus, nucleolin was regulated post-transcriptionally. (c) 2005 Elsevier Inc. All rights reserved.

In situ analysis and structural elucidation of sainfoin (Onobrychis viciifolia) tannins for high-throughput germplasm screening

A rapid thiolytic degradation and cleanup procedure was developed for analyzing tannins directly in chlorophyll-containing sainfoin (Onobrychis viciifolia) plants. The technique proved suitable for complex tannin mixtures containing catechin, epicatechin, gallocatechin, and epigallocatechin flavan-3-ol units. The reaction time was standardized at 60 min to minimize the loss of structural information as a result of epimerization and degradation of terminal flavan-3-ol units. The results were evaluated by separate analysis of extractable and unextractable tannins, which accounted for 63.6−113.7% of the in situ plant tannins. It is of note that 70% aqueous acetone extracted tannins with a lower mean degree of polymerization (mDP) than was found for tannins analyzed in situ. Extractable tannins had between 4 and 29 lower mDP values. The method was validated by comparing results from individual and mixed sample sets. The tannin composition of different sainfoin accessions covered a range of mDP values from 16 to 83, procyanidin/prodelphinidin (PC/PD) ratios from 19.2/80.8 to 45.6/54.4, and cis/trans ratios from 74.1/25.9 to 88.0/12.0. This is the first high-throughput screening method that is suitable for analyzing condensed tannin contents and structural composition directly in green plant tissue.

Glomus intraradices and Gigaspora margarita arbuscular mycorrhizal associations differentially affect nitrogen and potassium nutrition of Plantago lanceolata in a low fertility dune soil

Two controlled microcosm experiments aimed at a critical re-assessment of the contributions of divergent arbuscular mycorrhizal (AM) fungi to plant mineral nutrition were established that specifically targeted Plantago lanceolata–Glomus intraradices (B.B/E) and –Gigaspora margarita (BEG 34) symbioses developed in a native, nutrient limited, coastal dune soil. Plant tissue nitrogen (N), phosphorus (P) and potassium (K) status as well as plant growth parameters and levels of mycorrhizal colonization were assessed at harvest. In addition to the general well-established mycorrhizal facilitation of P uptake, the study was able to demonstrate a G. intraradices-specific contribution to improved plant nitrogen and potassium nutrition. In the two respective experiments, G. intraradices-inoculated plants had 27.8% and 40.8% more total N and 55.8% and 23.3% more total K when compared to Gi. margarita inoculated counterparts. Dissimilar overall contribution of the two isolates to plant nutrition was identified in AM-genus specific differences in plant tissue N:P:K ratios. G. intraradices inoculated and non-mycorrhizal plants generally exhibited N:P:K ratios indicative of P limitation whereas for Gi.margarita mycorrhizal plants, corresponding ratios strongly implied either N or K limitation. The study provides further evidence highlighting AM functional biodiversity in respect to plant nutrient limitation experienced by mycorrhizal P. lanceolata in an ecologically relevant soil system.

Replication enhancer elements within the open reading frame of tick-borne encephalitis virus and their evolution within the Flavivirus genus

We provide experimental evidence of a replication enhancer element (REE) within the capsid gene of tick-borne encephalitis virus (TBEV, genus Flavivirus). Thermodynamic and phylogenetic analyses predicted that the REE folds as a long stable stem–loop (designated SL6), conserved among all tick-borne flaviviruses (TBFV). Homologous sequences and potential base pairing were found in the corresponding regions of mosquito-borne flaviviruses, but not in more genetically distant flaviviruses. To investigate the role of SL6, nucleotide substitutions were introduced which changed a conserved hexanucleotide motif, the conformation of the terminal loop and the base-paired dsRNA stacking. Substitutions were made within a TBEV reverse genetic system and recovered mutants were compared for plaque morphology, single-step replication kinetics and cytopathic effect. The greatest phenotypic changes were observed in mutants with a destabilized stem. Point mutations in the conserved hexanucleotide motif of the terminal loop caused moderate virus attenuation. However, all mutants eventually reached the titre of wild-type virus late post-infection. Thus, although not essential for growth in tissue culture, the SL6 REE acts to up-regulate virus replication. We hypothesize that this modulatory role may be important for TBEV survival in nature, where the virus circulates by non-viraemic transmission between infected and non-infected ticks, during co-feeding on local rodents.