Ethylene and senescence processes

Senescence is a vitally important sequence of events in the latter phase of the life cycle of a plant that determines yield and reproductive success. In many species, and in different plant organs, ethylene is a key regulator of senescence and an increased understanding of the way the hormone functions will enable the timing and location of senescence to be manipulated in order to improve yield, quality and longevity. This chapter examines the physiological and molecular regulation of senescence in different plant organs and introduces the concept of the ‘senescence window’ in which plant organs are receptive to ethylene-mediated senescence cues. Several studies have attempted to elucidate global patterns of the regulation of senescence, which have enabled the function of ethylene to be placed in the context of the involvement of other, often antagonistic, hormones in the execution of senescence and downstream processes. Finally, we examine the consequences of senescence for post-harvest biology, an area where the control of ethylene action has been actively sought in order to control precisely the timing of senescence and ripening processes so that crop quality can be enhanced and maintained.

Sustained polymeric delivery of gene silencing antisense ODNs, siRNA, DNAzymes and ribozymes: in vitro and in vivo studies

Small interfering RNA (siRNA), antisense oligonucleotides (ODNs), ribozymes and DNAzymes have emerged as sequence-specific inhibitors of gene expression that may have therapeutic potential in the treatment of a wide range of diseases. Due to their rapid degradation in vivo, the efficacy of naked gene silencing nucleic acids is relatively short lived. The entrapment of these nucleic acids within biodegradable sustained-release delivery systems may improve their stability and reduce the doses required for efficacy. In this study, we have evaluated the potential in vitro and in vivo use of biodegradable poly (d,l-lactide-co-glycolide) copolymer (PLGA) microspheres as sustained delivery devices for ODNs, ribozyme, siRNA and DNA enzymes. In addition, we investigated the release of ODN conjugates bearing 5′-end lipophilic groups. The in vitro sustained release profiles of microsphere-entrapped nucleic acids were dependent on variables such as the type of nucleic acid used, the nature of the lipophilic group, and whether the nucleic acid used was single or double stranded. For in vivo studies, whole body autoradiography was used to monitor the bio-distribution of either free tritium-labelled ODN or that entrapped within PLGA microspheres following subcutaneous administration in Balb-c mice. The majority of the radioactivity associated with free ODN was eliminated within 24 h whereas polymer-released ODN persisted in organs and at the site of administration even after seven days post-administration. Polymer microsphere released ODN exhibited a similar tissue and cellular tropism to the free ODN. Micro-autoradiography analyses of the liver and kidneys showed similar bio-distribution for polymer-released and free ODNs with the majority of radioactivity being concentrated in the proximal convoluted tubules of the kidney and in the Kupffer cells of the liver. These findings suggest that biodegradable PLGA microspheres offer a method for improving the in vivo sustained delivery of gene silencing nucleic acids, and hence are worthy of further investigation as delivery systems for these macromolecules.

A mixture containing galactoollgosaccharide, produced by the enzymic activity of Bifidobacterium bifidum, reduces Salmonella enterica serovar Typhimurium infection in mice

The prebiotic Bimuno (R) is a mixture containing galactooligosaccharide, produced by the galactosyltransferase activity of Bifidobacterium bifidum NCIMB 41 .vertical bar 71 in the presence of lactose. Previous studies have implicated prebiotics in reducing infections by enteric pathogens, thus it was hypothesized that Bimuno (R) may confer some protection in the murine host from Salmonella enterica serovar Typhimurium (S. Typhimurium) infection. In this study, infection caused by S. Typhimurium SL1344nal(r) in the presence or absence of Bimuno (R) was assessed using tissue culture assays, a murine ligated ileal gut loop model and a murine oral challenge model. In tissue culture adherence and invasion assays with HT-29-1 6E cells, the presence of similar to 2 mM Bimuno) significantly reduced the invasion of S. Typhimuriurn SL1 344nal(r) (p < 0.0001). In the murine ligated ileal gut loops, the presence of Bimuno (R) prevented colonization and the associated pathology of S. Typhimurium. In the BALB/c mouse mocel, the oral delivery of Bimuno prior to challenge with S. Typhimurium resulted in significant reductions in colonization in the five organs sampled, with highly significant reductions being observed in the spleen at 72 and 96 h post-challenge (P=0.0002, < 0.0001, respectively). Collectively, the results indicate that Bimuno (R) significantly reduced the colonization and pathology associated with S. Typhimurium infection in a murine model system, possibly by reducing the invasion of the pathogen into host cells.

Contribution of fimbriae and flagella of Salmonella enteritidis to colonization and invasion of chicks

Isogenic mutants of Salmonella enteritidis defective for the elaboration of fimbrial types SEF14, SEF17, SEF21 and flagella were used to study the contribution these organelles made to colonization, invasion and lateral transfer in young chicks. The caecum, liver and spleen were colonized within 24 h following oral inoculation of 1-day-old chicks with 10(5) wild-type S. enteritidis strain LA5. However, for some mutants, the numbers of organisms recovered from internal organs was reduced significantly, particularly at 24 h post-inoculum, which supported the hypothesis that the organelles contribute to invasion and dissemination to internal organs. Specifically, mutations affecting SEF17, SEF21 and flagella contributed to a delay in colonization of the spleen, and those affecting SEF21 and flagella delayed colonization of the liver. Lower numbers of bacteria were recovered from the caecum with mutants deficient in elaboration of SEF21. Sentinel birds were colonized by LA5 or EAV40 (14(-), 17(-), 21(-), fla(-)) directly from the environment within 2 days, although a consistent slight delay was observed with the multiple mutant. Overall, our data suggest a collective role for SEF17, SEF21 and flagella, but not SEF14, in the early stages of colonization and invasion of young chicks by S. enteritidis, but these surface appendages appear unnecessary for colonization of birds from their immediate environment.

Bacillus subtilis spores competitively exclude Escherichia coli O78:K80 in poultry

Newly hatched specific pathogen-free chicks were dosed with a suspension of Bacillus subtilis spores prior to challenge with Escherichia coli O78:K80, a known virulent strain associated with avian colibacillosis. 24 h later. A single oral inoculum of 2.5 x 10(8) spores was sufficient to suppress all aspects of E. coli O78:K80 infection. Colonisation of deep organs was reduced by a factor of over 2 log(10) whilst colonisation of the intestine, as measured by direct caecal count, was reduced over 3 log(10). Shedding of E. coli O78:K80 was measured by semi-quantitative cloacal swabbing and was reduced significantly for the: duration of the experiment, 35 days. B, subtilis persisted in the intestine although with decreasing numbers over the same period. Challenge with the same dose 5 days after pre-dosing with spores overcame any suppressive effect of the spores. Crown Copyright (C) 2001 Published by Elsevier Science B.V. All rights reserved.

Virulence in the chick model and stress tolerance of Salmonella enterica serovar Orion var. 15+

Three Salmonella enterica serovar Orion var. 15+ isolates of distinct provenance were tested for survival in various stress assays. All were less able to survive desiccation than a virulent S. Enreritidis strain, with levels of survival similar to a rpoS mutant of the S. Enteritidis strain, whereas one isolate (F3720) was significantly more acid tolerant. The S. Orion var. 15+ isolates were motile by flagellae and elaborated type-1 and curli-like fimbriae; surface organelles that are considered virulence determinants in Salmonella pathogenesis. Each adhered and invaded HEp-2 tissue culture cells with similar proficiency to the S. Enteritidis control but were significantly less virulent than S. En teritidis in the one-day-old and seven-day-old chick model. Given an oral dose of 1 x 10(3) cfu to one-day-old chicken, S. Orion var. 15+ isolates colonised 25% of liver and spleens examined at 24 h whereas S. Enteritidis colonised 100% of organs by the same with the same dose. Given an oral dose of 1 x 10(7) cfu at seven-day old, S. Orion var. 15+ failed to colonise livers and spleens in any bird examined at 24 h whereas S. Enteritidis colonised 50% of organs by the same with the same dose. Based on the number of internal organs colonised, one of the three S. Orion var. 15+ isolates tested (strain F3720) was significantly more invasive than the other two (B1 and B7). Also, strain F3720 was shed less than either B1 or B7 supporting the concept that there may be an inverse relationship between the ability to colonise deep tissues and to persist in the gut. These data are discussed in the light that S. Orion var. 15+ is associated with sporadic outbreaks of human infection rather than epidemics.

Role of the mar locus in virulence of Salmonella enterica serovar Typhimurium DT104 in chickens

The virulence of a Salmonella enterica serovar Typhimurium DT014 strain in which marA was insertionally inactivated was compared to its isogenic parent in vitro and in vivo. In vitro, the numbers of the marA mutant phagocytosed by porcine lung macrophages were significantly increased, while survival at 24 h inside macrophages and adherence to human gut cells were significantly reduced in comparison with the parent strain. In vivo, the marA inactivated strain, in competition with its parent strain, persisted for a shorter period in chickens, was present in the caeca at significantly lower levels and invaded the deeper organs to a significantly lesser extent. Therapeutic antibiotic treatment of one group of chickens with oxytetracycline favoured the persistence of both the parent strain and, to a lesser extent, the marA inactivated strain; but interestingly, increased tetracycline resistance of Salmonella isolates after treatment of birds with antibiotic was seen only for the parent strain. Further work is needed to elucidate how mar is involved in virulence and if its inactivation can minimise the ability of bacteria to become antibiotic-resistant in vivo.

A laboratory study of an inactivated bivalent iron restricted Salmonella enterica serovars Enteritidis and Typhimurium dual vaccine against Typhimurium challenge in chickens

A commercial inactivated iron restricted Salmonella Typhimurium and Salmonella Enterifidis vaccine was used to vaccinate chicks at I day and again at 4 weeks of age, with challenge by a high and a low dose of S. Typhimurium given either orally or by contact with seeder birds inoculated orally with a high dose of S. Typhimurium. In all three challenge regimes, the shedding of challenge strain was reduced significantly (p < 0.05) in vaccinated birds compared with unvaccinated controls. Vaccination reduced colonisation of internal organs after challenge by contact seeder birds. However, no effect of vaccination upon colonisation of internal organs after either high or low oral challenge was apparent. In conclusion, the data indicate that the vaccine should be a useful tool in the control of S. Typhimurium infection in chickens. (C) 2002 Elsevier Science B.V. All rights reserved.

Mass spectrometry imaging of glucosinolates in arabidopsis flowers and siliques

Glucosinolates are multi-functional plant secondary metabolites which play a vital role in plant defence and are, as dietary compounds, important to human health and livestock well-being. Knowledge of the tissue-specific regulation of their biosynthesis and accumulation is essential for plant breeding programs. Here, we report that in Arabidopsis thaliana, glucosinolates are accumulated differentially in specific cells of reproductive organs. Using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI), distribution patterns of three selected compounds, 4-methylsulfinylbutyl (glucoraphanin), indol-3-ylmethyl (glucobrassicin), and 4-benzoyloxybutyl glucosinolates, were mapped in the tissues of whole flower buds, sepals and siliques. The results show that tissue localization patterns of aliphatic glucosinolate glucoraphanin and 4-benzoyloxybutyl glucosinolate were similar, but indole glucosinolate glucobrassicin had different localisation, indicating a possible difference in function. The high resolution images obtained by a complementary approach, cryo-SEM Energy Dispersive X-ray analysis (cryo-SEM-EDX), confirmed increased concentration of sulphur in areas with elevated amounts of glucosinolates, and allowed identifying the cell types implicated in accumulation of glucosinolates. High concentration of sulphur was found in S-cells adjacent to the phloem in pedicels and siliques, indicating the presence of glucosinolates. Moreover, both MALDI MSI and cryo-SEM-EDX analyses indicated accumulation of glucosinolates in cells on the outer surface of the sepals, suggesting that a layer of glucosinolate-accumulating epidermal cells protects the whole of the developing flower, in addition to the S-cells, which protect the phloem. This research demonstrates the high potential of MALDI MSI for understanding the cell-specific compartmentation of plant metabolites and its regulation.

Premature impairment of methylation pathway and cardiac metabolic dysfunction in fa/fa obese Zucker rats

Increasing evidence suggests that obesity is a chronic inflammatory disease, in which adipose tissue is involved in a network of endocrine signals to modulate energy homeostasis. These oxidative-inflammatory pathways, which are associated with cardiovascular complications, are also observed during the aging process. In this study, we investigated the interaction between aging and the development of obesity in a hyperphagic rat model. Metabolic profiles of the liver, white adipose tissue (WAT) and heart from young and adult Zucker lean (fa/+) and obese (fa/fa) rats were characterized using a (1)H NMR-based metabonomics approach. We observed premature metabolic modifications in all studied organs in obese animals, some of which were comparable to those observed in adult lean animals. In the cardiac tissue, young obese rats displayed lower lactate and scyllo-inositol levels associated with higher creatine, choline and phosphocholine levels, indicating an early modulation of energy and membrane metabolism. An early alteration of the hepatic methylation and transsulfuration pathways in both groups of obese rats indicated that these pathways were affected before diabetic onset. These findings therefore support the hypothesis that obesity parallels some metabolic perturbations observed in the aging process and provides new insights into the metabolic modifications occurring in pre-diabetic state.

ATD: a multiplatform for semiautomatic 3-D detection of kidneys and their pathology in real time

This research presents a novel multi-functional system for medical Imaging-enabled Assistive Diagnosis (IAD). Although the IAD demonstrator has focused on abdominal images and supports the clinical diagnosis of kidneys using CT/MRI imaging, it can be adapted to work on image delineation, annotation and 3D real-size volumetric modelling of other organ structures such as the brain, spine, etc. The IAD provides advanced real-time 3D visualisation and measurements with fully automated functionalities as developed in two stages. In the first stage, via the clinically driven user interface, specialist clinicians use CT/MRI imaging datasets to accurately delineate and annotate the kidneys and their possible abnormalities, thus creating “3D Golden Standard Models”. Based on these models, in the second stage, clinical support staff i.e. medical technicians interactively define model-based rules and parameters for the integrated “Automatic Recognition Framework” to achieve results which are closest to that of the clinicians. These specific rules and parameters are stored in “Templates” and can later be used by any clinician to automatically identify organ structures i.e. kidneys and their possible abnormalities. The system also supports the transmission of these “Templates” to another expert for a second opinion. A 3D model of the body, the organs and their possible pathology with real metrics is also integrated. The automatic functionality was tested on eleven MRI datasets (comprising of 286 images) and the 3D models were validated by comparing them with the metrics from the corresponding “3D Golden Standard Models”. The system provides metrics for the evaluation of the results, in terms of Accuracy, Precision, Sensitivity, Specificity and Dice Similarity Coefficient (DSC) so as to enable benchmarking of its performance. The first IAD prototype has produced promising results as its performance accuracy based on the most widely deployed evaluation metric, DSC, yields 97% for the recognition of kidneys and 96% for their abnormalities; whilst across all the above evaluation metrics its performance ranges between 96% and 100%. Further development of the IAD system is in progress to extend and evaluate its clinical diagnostic support capability through development and integration of additional algorithms to offer fully computer-aided identification of other organs and their abnormalities based on CT/MRI/Ultra-sound Imaging.

A defined intestinal colonization microbiota for gnotobiotic pigs

Maximising the ability of piglets to survive exposure to pathogens is essential to reduce early piglet mortality, an important factor in efficient commercial pig production. Mortality rates can be influenced by many factors, including early colonization by microbial commensals. Here we describe the development of an intestinal microbiota, the Bristol microbiota, for use in gnotobiotic pigs and its influence on synthesis of systemic immunoglobulins. Such a microbiota will be of value in studies of the consequences of early microbial colonization on development of the intestinal immune system and subsequent susceptibility to disease. Gnotobiotic pig studies lack a well-established intestinal microbiota. The use of the Altered Schaedler Flora (ASF), a murine intestinal microbiota, to colonize the intestines of Caesarean-derived, gnotobiotic pigs prior to gut closure, resulted in unreliable colonization with most (but not all) strains of the ASF. Subsequently, a novel, simpler porcine microbiota was developed. The novel microbiota reliably colonized the length of the intestinal tract when administered to gnotobiotic piglets. No health problems were observed, and the novel microbiota induced a systemic increase in serum immunoglobulins, in particular IgA and IgM. The Bristol microbiota will be of value for highly controlled, reproducible experiments of the consequences of early microbial colonization on susceptibility to disease in neonatal piglets, and as a biomedical model for the impact of microbial colonization on development of the intestinal mucosa and immune system in neonates.

Memory versus naive T-cell migration

Our established understanding of lymphocyte migration suggests that naive and memory T cells travel throughout the body via divergent pathways; naive T cells circulate between blood and lymph whereas memory T cells additionally migrate through non-lymphoid organs. Evidence is now gradually emerging which suggests such disparate pathways between naive and memory T cells may not strictly be true, and that naive T cells gain access to the non-lymphoid environment in numbers approaching that of memory T cells. We discuss here the evidence for naive T-cell traffic into the non-lymphoid environment, compare and contrast this movement with what is known of memory T cells, and finally discuss the functional importance of why naive T cells might access the parenchymal tissues.

TRY: a global database of plant traits

Plant traits – the morphological, anatomical, physiological, biochemical and phenological characteristics of plants and their organs – determine how primary producers respond to environmental factors, affect other trophic levels, influence ecosystem processes and services and provide a link from species richness to ecosystem functional diversity. Trait data thus represent the raw material for a wide range of research from evolutionary biology, community and functional ecology to biogeography. Here we present the global database initiative named TRY, which has united a wide range of the plant trait research community worldwide and gained an unprecedented buy-in of trait data: so far 93 trait databases have been contributed. The data repository currently contains almost three million trait entries for 69 000 out of the world's 300 000 plant species, with a focus on 52 groups of traits characterizing the vegetative and regeneration stages of the plant life cycle, including growth, dispersal, establishment and persistence. A first data analysis shows that most plant traits are approximately log-normally distributed, with widely differing ranges of variation across traits. Most trait variation is between species (interspecific), but significant intraspecific variation is also documented, up to 40% of the overall variation. Plant functional types (PFTs), as commonly used in vegetation models, capture a substantial fraction of the observed variation – but for several traits most variation occurs within PFTs, up to 75% of the overall variation. In the context of vegetation models these traits would better be represented by state variables rather than fixed parameter values. The improved availability of plant trait data in the unified global database is expected to support a paradigm shift from species to trait-based ecology, offer new opportunities for synthetic plant trait research and enable a more realistic and empirically grounded representation of terrestrial vegetation in Earth system models.

Effect of fall-grazed sericea lespedeza (Lespedeza cuneata) on gastrointestinal nematode infections of growing goats

High prevalence of anthelmintic-resistant gastrointestinal nematodes (GIN) in goats has increased pressure to find effective, alternative non-synthetic control methods, one of which is adding forage of the high condensed tannin (CT) legume sericea lespedeza (SL; Lespedeza cuneata) to the animal's diet. Previous work has demonstrated good efficacy of dried SL (hay, pellets) against small ruminant GIN, but information is lacking on consumption of fresh SL, particularly during the late summer–autumn period in the southern USA when perennial warm-season grass pastures are often low in quality. A study was designed to determine the effects of autumn (September–November) consumption of fresh SL forage, grass pasture (predominantly bermudagrass, BG; Cynodon dactylon), or a combination of SL + BG forage by young goats [intact male Spanish kids, 9 months old (20.7 ± 1.1 kg), n = 10/treatment group] on their GIN infection status. Three forage paddocks (0.40 ha) were set up at the Fort Valley State University Agricultural Research Station (Fort Valley, GA) for an 8-week trial. The goats in each paddock were supplemented with a commercial feed pellet at 0.45 kg/head/d for the first 4 weeks of the trial, and 0.27 kg/head/d for the final 4 weeks. Forage samples taken at the start of the trial were analyzed for crude protein (CP), neutral detergent fiber (NDF), and acid detergent fiber (ADF) content, and a separate set of SL samples was analyzed for CT in leaves, stems, and whole plant using the benzyl mercaptan thiolysis method. Animal weights were taken at the start and end of the trial, and fecal and blood samples were collected weekly for determination of fecal egg counts (FEC) and packed cell volume (PCV), respectively. Adult GIN was recovered from the abomasum and small intestines of all goats at the end of the experiment for counting and speciation. The CP levels were highest for SL forage, intermediate for SL + BG, and lowest for BG forage samples, while NDF and ADF values were the opposite, with highest levels in BG and lowest in SL forage samples. Sericea lespedeza leaves had more CT than stems (16.0 g vs. 3.3 g/100 g dry weight), a slightly higher percentage of PDs (98% vs. 94%, respectively) and polymers of larger mean degrees of polymerization (42 vs. 18, respectively). There were no differences in average daily gain or blood PCV between the treatment groups, but SL goats had lower FEC (P < 0.05) than the BG or SL + BG forage goats throughout most of the trial. The SL + BG goats had lower FEC than the BG forage animals by the end of the trial (week 8, P < 0.05). The SL goats had lower numbers (P < 0.05) of male Haemonchus contortus and tended to have fewer female (P < 0.10) and total (P < 0.07) H. contortus compared with the BG goats. The predominant GIN in all the goats was Trichostrongylus colubriformis (73% of total GIN). As a low-input forage with activity against pathogenic GIN (H. contortus), SL has a potential to reduce producers’ dependence upon synthetic anthelmintics and also to fill the autumn ‘window’ in good-quality fresh forages for goat grazing in the southern USA.