Effects of down-regulating ornithine decarboxylase upon putrescine-associated metabolism and growth in Nicotiana tabacum L.

Transgenic plants of Nicotiana tabacum L. homozygous for an RNAi construct designed to silence ornithine decarboxylase (ODC) had significantly lower concentrations of nicotine and nornicotine, but significantly higher concentrations of anatabine, compared with vector-only controls. Silencing of ODC also led to significantly reduced concentrations of polyamines (putrescine, spermidine and spermine), tyramine and phenolamides (caffeoylputrescine and dicaffeoylspermidine) with concomitant increases in concentrations of amino acids ornithine, arginine, aspartate, glutamate and glutamine. Root transcript levels of S-adenosyl methionine decarboxylase, S-adenosyl methionine synthase and spermidine synthase (polyamine synthesis enzymes) were reduced compared with vector controls, whilst transcript levels of arginine decarboxylase (putrescine synthesis), putrescine methyltransferase (nicotine production) and multi-drug and toxic compound extrusion (alkaloid transport) proteins were elevated. In contrast, expression of two other key proteins required for alkaloid synthesis, quinolinic acid phosphoribosyltransferase (nicotinic acid production) and a PIP-family oxidoreductase (nicotinic acid condensation reactions), were diminished in roots of odc-RNAi plants relative to vector-only controls. Transcriptional and biochemical differences associated with polyamine and alkaloid metabolism were exacerbated in odc-RNAi plants in response to different forms of shoot damage. In general, apex removal had a greater effect than leaf wounding alone, with a combination of these injury treatments producing synergistic responses in some cases. Reduced expression of ODC appeared to have negative effects upon plant growth and vigour with some leaves of odc-RNAi lines being brittle and bleached compared with vector-only controls. Together, results of this study demonstrate that ornithine decarboxylase has important roles in facilitating both primary and secondary metabolism in Nicotiana

Identifying the needs of brain tumor patients and their caregivers

The purpose of this study is to identify the needs of brain tumor patients and their caregivers to provide improved health services to these populations. Two different questionnaires were designed for patients and caregivers. Both questionnaires contained questions pertaining to three realms: disease symptoms/treatment, health care provider, daily living/finances. The caregivers’ questionnaires contained an additional domain on emotional needs. Each question was evaluated for the degree of importance and satisfaction. Exploratory analyses determined whether baseline characteristics affect responder importance or satisfaction. Also, areas of high agreement/disagreement in satisfaction between the participating patient-caregiver pairs were identified. Questions for which >50% of the patients and caregivers thought were “very important” but >30% were dissatisfied include: understanding the cause of brain tumors, dealing with patients’ lower energy, identifying healthful foods and activities for patients, telephone access to health care providers, information on medical insurance coverage, and support from their employer. In the emotional realm, caregivers identified 9 out of 10 items as important but need further improvement. Areas of high disagreement in satisfaction between participating patient-caregiver pairs include: getting help with household chores (P value = 0.006) and finding time for personal needs (P value < 0.001). This study provides insights into areas to improve services for brain tumor patients and their caregivers. The caregivers’ highest amount of burden is placed on their emotional needs, emphasizing the importance of providing appropriate medical and psychosocial support for caregivers to cope with emotional difficulties they face during the patients’ treatment process

An Interspecific Nicotiana Hybrid as a Useful and Cost-Effective Platform for Production of Animal Vaccines

The use of transgenic plants to produce novel products has great biotechnological potential as the relatively inexpensive inputs of light, water, and nutrients are utilised in return for potentially valuable bioactive metabolites, diagnostic proteins and vaccines. Extensive research is ongoing in this area internationally with the aim of producing plant-made vaccines of importance for both animals and humans. Vaccine purification is generally regarded as being integral to the preparation of safe and effective vaccines for use in humans. However, the use of crude plant extracts for animal immunisation may enable plant-made vaccines to become a cost-effective and efficacious approach to safely immunise large numbers of farm animals against diseases such as avian influenza. Since the technology associated with genetic transformation and large-scale propagation is very well established in Nicotiana, the genus has attributes well-suited for the production of plant-made vaccines. However the presence of potentially toxic alkaloids in Nicotiana extracts impedes their use as crude vaccine preparations. In the current study we describe a Nicotiana tabacum and N. glauca hybrid that expresses the HA glycoprotein of influenza A in its leaves but does not synthesize alkaloids. We demonstrate that injection with crude leaf extracts from these interspecific hybrid plants is a safe and effective approach for immunising mice. Moreover, this antigen-producing alkaloid-free, transgenic interspecific hybrid is vigorous, with a high capacity for vegetative shoot regeneration after harvesting. These plants are easily propagated by vegetative cuttings and have the added benefit of not producing viable pollen, thus reducing potential problems associated with bio-containment. Hence, these Nicotiana hybrids provide an advantageous production platform for partially purified, plant-made vaccines which may be particularly well suited for use in veterinary immunization programs

Analysis of interspecific hybrids for alkaloid content.

<p>4a. Chromatograms of the pyridine alkaloid profile in the parental and hybrids lines after removal of plant apices. (i) Nicotinic acid mononucletide was extracted as a background metabolite in the methanol-soluble fraction, (ii) Anabasine profile, (iii) Nicotine profile. 4b. Total pyridine alkaloid levels in vegetative regenerating shoots of parental and WT hybrid controls and HA-containing interspecific hybrid plants. Analysis was undertaken one week after removal of plant apices; graphs represent the mean of three separate plants per treatment (± SEM), ND = None Detected. DW = dry weight.</p

Analysis of T₀<i>N. tabacum</i> LAFC 53 plant lines transformed with HA constructs.

<p>2a. The mean HA content in <i>Nt</i> LAFC-HA plants transgenic for each construct type quantified using ELISA analysis. *Mean levels of HA in plants transgenic for pDAB4493 were significantly different (p&lt;0.05; t-test) to those containing pDAB4492. Nt = non-transgenic <i>N. tabacum</i> var. LAFC 53. Error bars represent standard error of mean (SEM) (n = 40 for plants transgenic for pCHA; n = 45 for plants transgenic for pDAB4492; n = 52 for plants transgenic for pDAB4493), FW = fresh weight. 2b. Western analysis of leaf extracts from representative <i>Nt</i> LAFC-HA plants. Extracts from plants containing HA constructs were loaded in central lanes as indicated. Purified HA was loaded as a control in the left lane whilst leaf extract from non-transgenic <i>N. tabacum</i> var. LAFC 53 was loaded in the right lane. All lanes contained 10 µg total soluble protein (TSP). 2c. Detection of HA transgene in selected elite transgenic lines <i>of N. tabacum</i> containing the pDAB4493-HA construct. Southern blot hybridisation of <i>Hind</i> III digested genomic DNA isolated from Nt LAFC-HA transgenic plants probed with the HA gene.</p

Haemagglutination inhibition (HI) titres of sera from immunized mice.

<p>The geometrical means of the HI titres are expressed as reciprocals of the highest dilution of serum that inhibited four haemagglutinin units of virus. Values above the broken line (dilutions of ≥1∶40) indicates the presence of protective HI titres. A significant difference exits between HA samples and the non immunised control (p&lt;0.0001).</p

Characterisation of <i>N. tabacum X N. glauca</i> hybrids.

<p>3a. Floral and vegetative phenotypes of the hybrid (centre) compared with parentals. All plants in the bottom panel are eight weeks old. 3b. Molecular evidence for the presence of both parental genomes in <i>N. tabacum X N. glauca</i> interspecific hybrids. PCR was used to detect species-specific length variability in intron 5 of <i>Nicotiana QPT</i> paralogues. <i>N. tabacum</i> LAFC 53 generates different sized bands from <i>N. glauca.</i> Parental DNA refers to genomic <i>N. glauca</i> and <i>N. tabacum</i> LAFC 53 mixed <i>in vitro.</i> DNA-free refers to PCR performed without template. 3c. Microscopic analysis of pollen. Panels i–iii show pollen after initial staining with acetocarmine. Panels iv–vi shows pollen after incubation in pollen germination medium. Flanking panels show pollen from parental species as indicated. Centre panel shows non-viable hybrid pollen. All scale bars represent 50 µm. 3d. ELISA analysis of HA levels in individual interspecific hybrid plants. Data represents means of triplicate analysis ± SEM. DW = dry weight. All lines are derived from T_o parental <i>Nt</i> LAFC-HA pDAB4493-8.</p

Constructs used to drive expression of HA gene in <i>N. tabacum</i> LAFC 53.

<p>CsVMV = the cassava vein mosaic virus promoter. 4OCS-ΔMas = a chimeric synthetic promoter. Native HA = the coding region of haemagglutinin from the turkey Wisconsin HA5 AIV strain. Plant-codon optimised HA = the modified haemagglutinin coding sequence based on plant codon usage frequency. LB and RB represent the left and right T-DNA borders respectively. VSP 3′ = the terminator of the soybean vegetative storage protein. PAT = the phosphinothricin acetyl transferase gene.</p

Capacity of HA-containing plant extracts to elicit an immune response.

<p>5a. HA-specific systemic immune response of mice. The horizontal lines represents the geometrical means of HA specific IgG titres at day 28, while the data points represent IgG titres from individual mice. Detailed description of treatments is described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035688#pone-0035688-t001" target="_blank">Table 1</a>. Solid dots indicate response to samples containing HA antigen. Open circles indicate responses to samples without HA antigen. A significant difference exits between HA positive samples and control samples (p = 0.0002). 5b. Detection of anti-HA specific IgG isotypes in mice sera on day 28. The horizontal lines represent the geometrical means of IgG isotype titres, while the dots represent IgG isotype titres from individual mice. Detailed description of treatments is described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035688#pone-0035688-t001" target="_blank">Table 1</a>.</p