Preferred orientation in an angled intercalation site of a chloro-substituted lambda- [Ru(TAP)2(dppz)]2+ complex bound to d(TCGGCGCCGA)2

The crystal structure of the ruthenium DNA ‘light-switch’ complex -[Ru(TAP)2(11-Cl-dppz)]2+ (TAP = tetraazaphenanthrene, dppz = dipyrido[3,2-a':2',3'-c]phenazine)) bound to the oligonucleotide duplex d(TCGGCGCCGA)2 is reported. The synthesis of the racemic ruthenium complex is described for the first time, and the racemate was used in this study. The crystal structure, at atomic resolution (1.0 Å), shows one ligand as a wedge in the minor groove, resulting in the 51 kinking of the double helix, as with the parent lambda-[Ru(TAP)2(dppz)]2+. Each complex binds to one duplex by intercalation of the dppz ligand and also by semi-intercalation of one of the orthogonal TAP ligands into a second symmetrically equivalent duplex. The 11-Cl substituent binds with the major component (66%) oriented with the 11-chloro substituent on the purine side of the terminal step of the duplex.

Physiological, biochemical and transcriptional analysis of onion bulbs during storage

Abstract: During the transition from endo-dormancy to eco-dormancy and subsequent growth, the onion bulb undergoes the transition from sink organ to source, to sustain cell division in the meristematic tissue. The mechanisms controlling these processes are not fully understood. Here, a detailed analysis of whole onion bulb physiological, biochemical and transcriptional changes in response to sprouting is reported, enabling a better knowledge of the mechanisms regulating post-harvest onion sprout development. Biochemical and physiological analyses were conducted on different cultivars ('Wellington', 'Sherpa' and 'Red Baron') grown at different sites over 3 years, cured at different temperatures (20, 24 and 28 degrees C) and stored under different regimes (1, 3, 6 and 6 1 degrees C). In addition, the first onion oligonucleotide microarray was developed to determine differential gene expression in onion during curing and storage, so that transcriptional changes could support biochemical and physiological analyses. There were greater transcriptional differences between samples at harvest and before sprouting than between the samples taken before and after sprouting, with some significant changes occurring during the relatively short curing period. These changes are likely to represent the transition from endo-dormancy to sprout suppression, and suggest that endo-dormancy is a relatively short period ending just after curing. Principal component analysis of biochemical and physiological data identified the ratio of monosaccharides (fructose and glucose) to disaccharide (sucrose), along with the concentration of zeatin riboside, as important factors in discriminating between sprouting and pre-sprouting bulbs. These detailed analyses provide novel insights into key regulatory triggers for sprout dormancy release in onion bulbs and provide the potential for the development of biochemical or transcriptional markers for sprout initiation. Evidence presented herein also suggests there is no detrimental effect on bulb storage life and quality caused by curing at 20 degrees C, producing a considerable saving in energy and costs.

Gene expression changes in phosphorus deficient potato (Solanum tuberosum L.) leaves and the potential for diagnostic gene expression markers

Background: There are compelling economic and environmental reasons to reduce our reliance on inorganic phosphate (Pi) fertilisers. Better management of Pi fertiliser applications is one option to improve the efficiency of Pi fertiliser use, whilst maintaining crop yields. Application rates of Pi fertilisers are traditionally determined from analyses of soil or plant tissues. Alternatively, diagnostic genes with altered expression under Pi limiting conditions that suggest a physiological requirement for Pi fertilisation, could be used to manage Pifertiliser applications, and might be more precise than indirect measurements of soil or tissue samples. Results: We grew potato (Solanum tuberosum L.) plants hydroponically, under glasshouse conditions, to control their nutrient status accurately. Samples of total leaf RNA taken periodically after Pi was removed from the nutrient solution were labelled and hybridised to potato oligonucleotide arrays. A total of 1,659 genes were significantly differentially expressed following Pi withdrawal. These included genes that encode proteins involved in lipid, protein, and carbohydrate metabolism, characteristic of Pi deficient leaves and included potential novel roles for genes encoding patatin like proteins in potatoes. The array data were analysed using a support vector machine algorithm to identify groups of genes that could predict the Pi status of the crop. These groups of diagnostic genes were tested using field grown potatoes that had either been fertilised or unfertilised. A group of 200 genes could correctly predict the Pi status of field grown potatoes. Conclusions: This paper provides a proof-of-concept demonstration for using microarrays and class prediction tools to predict the Pi status of a field grown potato crop. There is potential to develop this technology for other biotic and abiotic stresses in field grown crops. Ultimately, a better understanding of crop stresses may improve our management of the crop, improving the sustainability of agriculture.

A Brassica exon array for whole-transcript gene expression profiling

Affymetrix GeneChip (R) arrays are used widely to study transcriptional changes in response to developmental and environmental stimuli. GeneChip (R) arrays comprise multiple 25-mer oligonucleotide probes per gene and retain certain advantages over direct sequencing. For plants, there are several public GeneChip (R) arrays whose probes are localised primarily in 39 exons. Plant whole-transcript (WT) GeneChip (R) arrays are not yet publicly available, although WT resolution is needed to study complex crop genomes such as Brassica, which are typified by segmental duplications containing paralogous genes and/or allopolyploidy. Available sequence data were sampled from the Brassica A and C genomes, and 142,997 gene models identified. The assembled gene models were then used to establish a comprehensive public WT exon array for transcriptomics studies. The Affymetrix GeneChip (R) Brassica Exon 1.0 ST Array is a 5 mu M feature size array, containing 2.4 million 25-base oligonucleotide probes representing 135,201 gene models, with 15 probes per gene distributed among exons. Discrimination of the gene models was based on an E-value cut-off of 1E(-5), with <= 98 sequence identity. The 135 k Brassica Exon Array was validated by quantifying transcriptome differences between leaf and root tissue from a reference Brassica rapa line (R-o-18), and categorisation by Gene Ontologies (GO) based on gene orthology with Arabidopsis thaliana. Technical validation involved comparison of the exon array with a 60-mer array platform using the same starting RNA samples. The 135 k Brassica Exon Array is a robust platform. All data relating to the array design and probe identities are available in the public domain and are curated within the BrassEnsembl genome viewer at http://www.brassica.info/BrassEnsembl/index.html.

An assessment of the prebiotic potential of single and blended substrates in anaerobic in vitro batch culture fermentations using canine faecal samples as inocula

Previously, using an in vitro static batch culture system, it was found that rice bran (RB), inulin, fibersol, mannanoligosaccharides (MOS), larch arabinogalactan and citrus pectin elicited prebiotic effects (in terms of increased numbers of bifidobacteria and lactic acid bacteria) on the faecal microbiota of a dog. The aim of the present study was to confirm the prebiotic potential of each individual substrate using multiple faecal donors, as well as assessing the prebiotic potential of 15 substrate blends made from them. Anaerobic static and stirred, pH-controlled batch culture systems inoculated with faecal samples from healthy dogs were used for this purpose. Fluorescence in situ hybridization (FISH) analysis using seven oligonucleotide probes targeting selected bacterial groups and DAPI (total bacteria) was used to monitor bacterial populations during fermentation runs. High-performance liquid chromatography was used to measure butyrate produced as a result of bacterial fermentation of the substrates. RB and a MOS/RB blend (1:1, w/w) were shown to elicit prebiotic and butyrogenic effects on the canine microbiota in static batch culture fermentations. Further testing of these substrates in stirred, pH-controlled batch culture fermentation systems confirmed the prebiotic and butyrogenic effects of MOS/RB, with no enhancement of Clostridium clusters I and II and Escherichia coli populations.

Array based detection of antibiotic resistance genes in Gram negative bacteria isolated from retail poultry meat in the UK and Ireland

The use of antibiotics in birds and animals intended for human consumption within the European Union (EU) and elsewhere has been subject to regulation prohibiting the use of antimicrobials as growth promoters and the use of last resort antibiotics in an attempt to reduce the spread of multi-resistant Gram negative bacteria. Given the inexorable spread of antibiotic resistance there is an increasing need for improved monitoring of our food. Using selective media, Gram negative bacteria were isolated from retail chicken of UK-Intensively reared (n = 27), Irish-Intensively reared (n = 19) and UK-Free range (n = 30) origin and subjected to an oligonucleotide based array system for the detection of 47 clinically relevant antibiotic resistance genes (ARGs) and two integrase genes. High incidences of β-lactamase genes were noted in all sample types, acc (67%), cmy (80%), fox (55%) and tem (40%) while chloramphenicol resistant determinants were detected in bacteria from the UK poultry portions and were absent in bacteria from the Irish samples. Denaturing Gradient Gel Electrophoresis (DGGE) was used to qualitatively analyse the Gram negative population in the samples and showed the expected diversity based on band stabbing and DNA sequencing. The array system proved to be a quick method for the detection of antibiotic resistance gene (ARG) burden within a mixed Gram negative bacterial population.

Enantiomeric conformation controls rate and yield of photoinduced electron transfer in DNA sensitized by Ru(II) Dipyridophenazine complexes

Photosensitized oxidation of guanine is an important route to DNA damage. Ruthenium polypyridyls are very useful photosensitizers as their reactivity and DNA-binding properties are readily tunable. Here we show a strong difference in the reactivity of the two enantiomers of [Ru(TAP)2(dppz)]2+, by using time-resolved visible and IR spectroscopy. This reveals that the photosensitized one-electron oxidation of guanine in three oligonucleotide sequences proceeds with similar rates and yields for bound delta-[Ru(TAP)2(dppz)]2+, whereas those for the lambda enantiomer are very sensitive to base sequence. It is proposed that these differences are due to preferences of each enantiomer for different binding sites in the duplex.

The impact of date palm fruits and their component polyphenols, on gut microbial ecology, bacterial metabolites and colon cancer cell proliferation

The fruit of the date palm (Phoenix dactylifera L.) is a rich source of dietary fibre and polyphenols. We have investigated gut bacterial changes induced by the whole date fruit extract (digested date extract; DDE) and its polyphenol-rich extract (date polyphenol extract; DPE) using faecal, pH-controlled, mixed batch cultures mimicking the distal part of the human large intestine, and utilising an array of microbial group-specific 16S rRNA oligonucleotide probes. Fluorescence microscopic enumeration indicated that there was a significant increase in the growth of bifidobacteria in response to both treatments, whilst whole dates also increased bacteroides at 24 h and the total bacterial counts at later fermentation time points when compared with DPE alone. Bacterial metabolism of whole date fruit led to the production of SCFA, with acetate significantly increasing following bacterial incubation with DDE. In addition, the production of flavonoid aglycones (myricetin, luteolin, quercetin and apigenin) and the anthocyanidin petunidin in less than 1 h was also observed. Lastly, the potential of DDE, DPE and metabolites to inhibit Caco-2 cell growth was investigated, indicating that both were capable of potentially acting as antiproliferative agents in vitro, following a 48 h exposure. This potential to inhibit growth was reduced following fermentation. Together these data suggest that consumption of date fruits may enhance colon health by increasing beneficial bacterial growth and inhibiting the proliferation of colon cancer cells. This is an early suggestion that date intake by humans may aid in the maintenance of bowel health and even the reduction of colorectal cancer development.

Human neural stem cell-derived cultures in three- dimensional substrates form spontaneously functional neuronal networks

Differentiated human neural stem cells were cultured in an inert three-dimensional (3D) scaffold and, unlike two-dimensional (2D) but otherwise comparable monolayer cultures, formed spontaneously active, functional neuronal networks that responded reproducibly and predictably to conventional pharmacological treatments to reveal functional, glutamatergic synapses. Immunocytochemical and electron microscopy analysis revealed a neuronal and glial population, where markers of neuronal maturity were observed in the former. Oligonucleotide microarray analysis revealed substantial differences in gene expression conferred by culturing in a 3D vs a 2D environment. Notable and numerous differences were seen in genes coding for neuronal function, the extracellular matrix and cytoskeleton. In addition to producing functional networks, differentiated human neural stem cells grown in inert scaffolds offer several significant advantages over conventional 2D monolayers. These advantages include cost savings and improved physiological relevance, which make them better suited for use in the pharmacological and toxicological assays required for development of stem cell-based treatments and the reduction of animal use in medical research.

Phosphorothioate backbone modifications of nucleotide-based drugs are potent platelet activators

Nucleotide-based drug candidates such as antisense oligonucleotides, aptamers, immunoreceptor-activating nucleotides, or (anti)microRNAs hold great therapeutic promise for many human diseases. Phosphorothioate (PS) backbone modification of nucleotide-based drugs is common practice to protect these promising drug candidates from rapid degradation by plasma and intracellular nucleases. Effects of the changes in physicochemical properties associated with PS modification on platelets have not been elucidated so far. Here we report the unexpected binding of PS-modified oligonucleotides to platelets eliciting strong platelet activation, signaling, reactive oxygen species generation, adhesion, spreading, aggregation, and thrombus formation in vitro and in vivo. Mechanistically, the platelet-specific receptor glycoprotein VI (GPVI) mediates these platelet-activating effects. Notably, platelets from GPVI function-deficient patients do not exhibit binding of PS-modified oligonucleotides, and platelet activation is fully abolished. Our data demonstrate a novel, unexpected, PS backbone-dependent, platelet-activating effect of nucleotide-based drug candidates mediated by GPVI. This unforeseen effect should be considered in the ongoing development programs for the broad range of upcoming and promising DNA/RNA therapeutics.