A process for analysis of microarray comparative genomics hybridisation studies for bacterial genomes

Background: Microarray based comparative genomic hybridisation (CGH) experiments have been used to study numerous biological problems including understanding genome plasticity in pathogenic bacteria. Typically such experiments produce large data sets that are difficult for biologists to handle. Although there are some programmes available for interpretation of bacterial transcriptomics data and CGH microarray data for looking at genetic stability in oncogenes, there are none specifically to understand the mosaic nature of bacterial genomes. Consequently a bottle neck still persists in accurate processing and mathematical analysis of these data. To address this shortfall we have produced a simple and robust CGH microarray data analysis process that may be automated in the future to understand bacterial genomic diversity. Results: The process involves five steps: cleaning, normalisation, estimating gene presence and absence or divergence, validation, and analysis of data from test against three reference strains simultaneously. Each stage of the process is described and we have compared a number of methods available for characterising bacterial genomic diversity, for calculating the cut-off between gene presence and absence or divergence, and shown that a simple dynamic approach using a kernel density estimator performed better than both established, as well as a more sophisticated mixture modelling technique. We have also shown that current methods commonly used for CGH microarray analysis in tumour and cancer cell lines are not appropriate for analysing our data. Conclusion: After carrying out the analysis and validation for three sequenced Escherichia coli strains, CGH microarray data from 19 E. coli O157 pathogenic test strains were used to demonstrate the benefits of applying this simple and robust process to CGH microarray studies using bacterial genomes.

Development of a miniaturised microarray-based assay for the rapid identification of antimicrobial resistance genes in Gram-negative bacteria

We describe the development of a miniaturised microarray for the detection of antimicrobial resistance genes in Gram-negative bacteria. Included on the array are genes encoding resistance to aminoglycosides, trimethoprim, sulphonamides, tetracyclines and beta-lactams, including extended-spectrum beta-lactamases. Validation of the array with control strains demonstrated a 99% correlation between polymerase chain reaction and array results. There was also good correlation between phenotypic and genotypic results for a large panel of Escherichia coli and Salmonella isolates. Some differences were also seen in the number and type of resistance genes harboured by E. coli and Salmonella strains. The array provides an effective, fast and simple method for detection of resistance genes in clinical isolates suitable for use in diagnostic laboratories, which in future will help to understand the epidemiology of isolates and to detect gene linkage in bacterial populations. (C) 2008 Published by Elsevier B.V. and the International Society of Chemotherapy.

Cryopreservation of winter-dormant apple buds: II - tissue water status after desiccation at -4°c and before further cooling

Abstract The established protocol for the cryopreservation of winter-dormant Malus buds requires that stem explants, containing a single, dormant bud are desiccated at -4°C, for up to 14 days, to reduce their water content to 25-30% of fresh weight. Using three apple cultivars, with known differences in response to cryopreservation, the pattern of evaporative water loss has been characterised, including early freezing events in the bud and cortical tissues that allow further desiccation by water migration to extracellular ice. There were no significant differences between cultivars in this respect or in the proportions of tissue water lost during the desiccation process. Differential Scanning Calorimetry (to -90°C) of intact buds indicated that bud tissues of the cultivar with the poorest response to cryopreservation had the highest residual water content at the end of the desiccation process and froze at the highest temperature Keywords: Malus, cryopreservation, dormant bud, dehydration

Effects of dietary selenium supplementation on tissue selenium distribution and glutathione peroxidase activity in Chinese Ring Necked Pheasants

The objective of this study was to determine the concentration of total selenium (Se) and the proportions of total Se comprised as selenomethionine (SeMet) and selenocysteine (SeCys) in the post mortem tissues of female pheasants (Phasianus Colchicus Torquator) offered diets containing graded additions of selenized enriched yeast (SY) or sodium selenite (SS). Thiobarbituric acid reactive substances (TBARS) and tissue glutathione peroxidase (GSH-Px) activity of breast (Pectoralis Major) were assessed at 0 and 5 d post-mortem. A total of 216 female pheasant chicks were enrolled onto the study. 24 birds were euthanased at the start of the study and samples of blood, breast muscle, leg muscle (Peroneus Longus and M. Gastrocnemius), heart, liver, kidney and gizzard collected for determination of total Se. Remaining birds were blocked by live weight and randomly allocated to one of four dietary treatments (n=48 birds/treatment) that either differed in Se source (SY vs. SS) or dose (Con [0.2 mg total Se/kg], SY-L and SS-L [0.3 mg/kg total Se as SY and SS, respectively], and SY-H [0.45 mg total Se/kg]). Following 42 and 91 days of treatment 24 birds/treatment were euthanased and samples of blood, breast muscle, leg muscle, heart, liver, kidney and gizzard retained for determination of total Se and the proportion of total Se comprised as SeMet or SeCys. Whole blood GSH-Px activity was determined at each time point. Tissue GSH-Px activity and TBARS were determined in breast tissue at the end of the study. There were positive responses (P<0.001) in both blood and tissues to the graded addition of SY to the diet but the same responses were not apparent in the blood and tissues of selenite supplemented birds receiving comparable doses. Although there were differences between tissue types in the distribution of SeMet and SeCys there were few differences between treatments. There were effects of treatment on erythrocyte GSH-Px activity (P = 0.012) with values being higher in treatments SY-H and SS-L when compared to the negative control and treatment SY-L. There were no effects of treatment on tissue GSH-Px activity which is reflected in the overall lack of any treatment effects on TBARS.

Pathogenic LRRK2 mutations do not alter gene expression in cell model systems or human brain tissue

Point mutations in LRRK2 cause autosomal dominant Parkinson's disease. Despite extensive efforts to determine the mechanism of cell death in patients with LRRK2 mutations, the aetiology of LRRK2 PD is not well understood. To examine possible alterations in gene expression linked to the presence of LRRK2 mutations, we carried out a case versus control analysis of global gene expression in three systems: fibroblasts isolated from LRRK2 mutation carriers and healthy, non-mutation carrying controls; brain tissue from G2019S mutation carriers and controls; and HEK293 inducible LRRK2 wild type and mutant cell lines. No significant alteration in gene expression was found in these systems following correction for multiple testing. These data suggest that any alterations in basal gene expression in fibroblasts or cell lines containing mutations in LRRK2 are likely to be quantitatively small. This work suggests that LRRK2 is unlikely to play a direct role in modulation of gene expression, although it remains possible that this protein can influence mRNA expression under pathogenic cicumstances.

The role of fimbriae and flagella in the adherence of avian strains of Escherichia coli O78:K80 to tissue culture cells and tracheal and gut explants

To investigate the role of fimbriae and flagella in the pathogenesis of avian colibacillosis, isogenic insertionally inactivated mutant strains of Escherichia coil O78:K80 strain EC34195 defective in the elaboration of type-1 and curli fimbriae and flagella were constructed by allelic exchange, Single and multiple non-fimbriate and non-flagellate mutant strains were compared to the wild-type in vitro in adherence assays with a HEp-2 cell line, a mucus-secreting cell line HT2916E, a non-mucus-secreting cell line HT2919A, tracheal explant and proximal gut explant, Mutant strains defective in the elaboration of type-1 fimbriae were significantly less adherent - in the order of 90% reduction - than the wild-type strain in all assays. Mutant strains defective in the elaboration of flagella were generally as adherent as the wild-type strain except when assayed with the mucus-secreting cell line HT2916E, for which a significant reduction of adherence - of the order of 90% - compared with the wild-type strain was observed. Mutant strains defective for the elaboration of curb fimbriae adhered as well as the wild-type strain in all assays, except when assayed in tests with gut explant tissue for which a significant reduction of adherence - of the order of 80% - compared with the wild-type strain was observed, Adherence to explants was to epithelial, not serous, surfaces and was 10-fold greater to tracheal than to gut explants, Together, these data support the hypothesis that type-1 fimbriae are significant factors in adherence, aided by flagella for penetration of mucus and curli fimbriae for adherence to the gut.

Adhesion of Salmonella enterica var Enteritidis strains lacking fimbriae and flagella to rat ileal explants cultured at the air interface or submerged in tissue culture medium

Rat ileal air interface and submerged explant models were developed and used to compare the adhesion of Salmonella enterica var Enteritidis wild-type strains with that of their isogenic single and multiple deletion mutants. The modified strains studied were defective for fimbriae, flagella, motility or chemotaxis and binding was assessed on tissues with and without an intact mucus layer. A multiple afimbriate/aflagellate (fim(-)/fla(-)) strain, a fimbriate but aflagellate (fla(-)) strain and a fimbriate/flagellate but non-motile (mot(-)) strain bound significantly less extensively to the explants than the corresponding wild-type strains. With the submerged explant model this difference was evident in tissues with or without a mucus layer, whereas in the air interface model it was observed only in tissues,vith an intact mucus layer. A smooth swimming chemotaxis-defective (che(-)) strain and single or multiple afimbriate strains bound to explants as well as their corresponding wild-type strain. This suggests that under the present experimental conditions fimbriae were not essential for attachment of S. enterica var Enteritidis to rat ileal explants, However; the possession of active flagella did appear to be an important factor. in enabling salmonellae to penetrate the gastrointestinal mucus layer and attach specifically to epithelial cells.

The role of intimin in the adherence of enterohaemorrhagic Escherichia coli (EHEC) O157:H7 to HEp-2 tissue culture cells and to bovine gut explant tissues

Intimin, an outer membrane protein encoded by eaeA, is a key determinant for the formation of attaching and effacing (AE) lesions by enterohaemorrhagic Escherichia coli (EHEC). To investigate the role of intimin in adherence, the eaeA gene was insertionally inactivated in three EHEC O157:H7 strains of diverse origin. The absence or presence of intimin did not correlate with the extent of adhesion of mutant or wild-type O157:H7 in tissue culture and neonatal calf gut tissue explant adherence assays. Adherence of the eaeA mutants to HEp-2 cells was diffuse with no evidence of intimate attachment whereas wild-type bacteria formed microcolonies and AE lesions. Intimin-independent adherence to neonatal calf gut explants was demonstrated by eaeA mutants and wild-type strains which adhered in the greatest numbers to colon but least well to rumen tissue. These results confirm that intimin is necessary for intimate attachment and that additional adherence factors are involved in intimin-independent adherence.

Identification of core and variable components of the Salmonella enterica subspecies I genome by microarray

We have performed microarray hybridization studies on 40 clinical isolates from 12 common serovars within Salmonella enterica subspecies I to identify the conserved chromosomal gene pool. We were able to separate the core invariant portion of the genome by a novel mathematical approach using a decision tree based on genes ranked by increasing variance. All genes within the core component were confirmed using available sequence and microarray information for S. enterica subspecies I strains. The majority of genes within the core component had conserved homologues in Escherichia coli K-12 strain MG1655. However, many genes present in the conserved set which were absent or highly divergent in K-12 had close homologues in pathogenic bacteria such as Shigella flexneri and Pseudomonas aeruginosa. Genes within previously established virulence determinants such as SPI1 to SPI5 were conserved. In addition several genes within SPI6, all of SPI9, and three fimbrial operons (fim, bcf, and stb) were conserved within all S. enterica strains included in this study. Although many phage and insertion sequence elements were missing from the core component, approximately half the pseudogenes present in S. enterica serovar Typhi were conserved. Furthermore, approximately half the genes conserved in the core set encoded hypothetical proteins. Separation of the core and variant gene sets within S. enterica subspecies I has offered fundamental biological insight into the genetic basis of phenotypic similarity and diversity across S. enterica subspecies I and shown how the core genome of these pathogens differs from the closely related E. coli K-12 laboratory strain.

Colonization, persistence, and tissue tropism of Escherichia coli O26 in conventionally reared weaned lambs

Escherichia coli O26 is recognized as an emerging pathogen associated with disease in both ruminants and humans. Compared to those of E. coli O157:117, the shedding pattern and location of E. coli O26 in the gastrointestinal tract (GIT) of ruminants are poorly understood. In the studies reported here, an stx-negative E. coli O26 strain of ovine origin was inoculated orally into 6-week-old lambs and the shedding pattern of the O26 strain was monitored by serial bacteriological examination of feces. The location of colonization in the GIT was examined at necropsy at two time points. The numbers of O26 organisms excreted in feces declined from approximately 10(7) to 10(4) CFU per gram of feces by day 7 and continued at this level for a further 3 weeks. Beyond day 30, excretion was from few animals, intermittent, and just above the detection limit. By day 38, all fecal samples were negative, but at necropsy, O26 organisms were recovered from the upper GIT, specifically the ileum. However, no attaching-effacing (AE) lesions were observed. To identify the location of E. coli O26 within the GIT early after inoculation, two lambs were examined postmortem, 4 days postinoculation. High numbers of O26 organisms were recovered from all GIT sites examined, and similar to 10(9) CFU were recovered from 1 gram of ileal tissue from one animal. Despite high numbers of O26 organisms, AE lesions were identified on the mucosa of the ascending colon of only one animal. These data indicate that E. coli O26 readily colonizes 6-week-old lambs, but the sparseness of AE lesions suggests that O26 is well adapted to this host, and mechanisms other than those dependent upon intimin may play a role in persistence.

Combinations of parabens at concentrations measured in human breast tissue can increase proliferation of MCF-7 human breast cancer cells

The alkyl esters of p-hydroxybenzoic acid (parabens), which are used as preservatives in consumer products, possess oestrogenic activity and have been measured in human breast tissue. This has raised concerns for a potential involvement in the development of human breast cancer. In this paper, we have investigated the extent to which proliferation of MCF-7 human breast cancer cells can be increased by exposure to the five parabens either alone or in combination at concentrations as recently measured in 160 human breast tissue samples. Determination of no-observed-effect concentrations (NOEC), lowest-observed-effect concentrations (LOEC), EC50 and EC100 values for stimulation of proliferation of MCF-7 cells by five parabens revealed that 43/160 (27%) of the human breast tissue samples contained at least one paraben at a concentration ≥ LOEC and 64/160 (40%) > NOEC. Proliferation of MCF-7 cells could be increased by combining all five parabens at concentrations down to the 50th percentile (median) values measured in the tissues. For the 22 tissue samples taken at the site of ER + PR + primary cancers, 12 contained a sufficient concentration of one or more paraben to stimulate proliferation of MCF-7 cells. This demonstrates that parabens, either alone or in combination, are present in human breast tissue at concentrations sufficient to stimulate the proliferation of MCF-7 cells in vitro, and that functional consequences of the presence of paraben in human breast tissue should be assessed on the basis of all five parabens and not single parabens individually.

The long-term safety and efficacy of bilateral transplantation of human fetal striatal tissue in patients with mild to moderate Huntington's disease.

Huntington's disease (HD) is a fatal autosomal dominant neurodegenerative disease involving progressive motor, cognitive and behavioural decline, leading to death approximately 20 years after motor onset. The disease is characterised pathologically by an early and progressive striatal neuronal cell loss and atrophy, which has provided the rationale for first clinical trials of neural repair using fetal striatal cell transplantation. Between 2000 and 2003, the 'NEST-UK' consortium carried out bilateral striatal transplants of human fetal striatal tissue in five HD patients. This paper describes the long-term follow up over a 3-10-year postoperative period of the patients, grafted and non-grafted, recruited to this cohort using the 'Core assessment program for intracerebral transplantations-HD' assessment protocol. No significant differences were found over time between the patients, grafted and non-grafted, on any subscore of the Unified Huntington's Disease Rating Scale, nor on the Mini Mental State Examination. There was a trend towards a slowing of progression on some timed motor tasks in four of the five patients with transplants, but overall, the trial showed no significant benefit of striatal allografts in comparison with a reference cohort of patients without grafts. Importantly, no significant adverse or placebo effects were seen. Notably, the raclopride positron emission tomography (PET) signal in individuals with transplants, indicated that there was no obvious surviving striatal graft tissue. This study concludes that fetal striatal allografting in HD is safe. While no sustained functional benefit was seen, we conclude that this may relate to the small amount of tissue that was grafted in this safety study compared with other reports of more successful transplants in patients with HD.

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.

Changes in gene expression in Arabidopsis shoots during phosphate starvation and the potential for developing smart plants

Our aim was to generate and prove the concept of "smart" plants to monitor plant phosphorus (P) status in Arabidopsis. Smart plants can be genetically engineered by transformation with a construct containing the promoter of a gene up-regulated specifically by P starvation in an accessible tissue upstream of a marker gene such as beta-glucuronidase (GUS). First, using microarrays, we identified genes whose expression changed more than 2.5-fold in shoots of plants growing hydroponically when P, but not N or K, was withheld from the nutrient solution. The transient changes in gene expression occurring immediately (4 h) after P withdrawal were highly variable, and many nonspecific, shock-induced genes were up-regulated during this period. However, two common putative cis-regulatory elements (a PHO-like element and a TATA box-like element) were present significantly more often in the promoters of genes whose expression increased 4 h after the withdrawal of P compared with their general occurrence in the promoters of all genes represented on the microarray. Surprisingly, the expression of only four genes differed between shoots of P-starved and -replete plants 28 h after P was withdrawn. This lull in differential gene expression preceded the differential expression of a new group of 61 genes 100 h after withdrawing P. A literature survey indicated that the expression of many of these "late" genes responded specifically to P starvation. Shoots had reduced P after 100 h, but growth was unaffected. The expression of SQD1, a gene involved in the synthesis of sulfolipids, responded specifically to P starvation and was increased 100 h after withdrawing P. Leaves of Arabidopsis bearing a SQD1::GUS construct showed increased GUS activity after P withdrawal, which was detectable before P starvation limited growth. Hence, smart plants can monitor plant P status. Transferring this technology to crops would allow precision management of P fertilization, thereby maintaining yields while reducing costs, conserving natural resources, and preventing pollution.

A SEPALLATA gene is involved in the development and ripening of strawberry (Fragaria X ananassa Duch.) fruit, a non-climacteric tissue

Climacteric and non-climacteric fruits have traditionally been viewed as representing two distinct programmes of ripening associated with differential respiration and ethylene hormone effects. In climacteric fruits, such as tomato and banana, the ripening process is marked by increased respiration and is induced and co-ordinated by ethylene, while in non-climacteric fruits, such as strawberry and grape, it is controlled by an ethylene-independent process with little change in respiration rate. The two contrasting mechanisms, however, both lead to texture, colour, and flavour changes that probably reflect some common programmes of regulatory control. It has been shown that a SEPALLATA(SEP)4-like gene is necessary for normal ripening in tomato. It has been demonstrated here that silencing a fruit-related SEP1/2-like (FaMADS9) gene in strawberry leads to the inhibition of normal development and ripening in the petal, achene, and receptacle tissues. In addition, analysis of transcriptome profiles reveals pleiotropic effects of FaMADS9 on fruit development and ripening-related gene expression. It is concluded that SEP genes play a central role in the developmental regulation of ripening in both climacteric and non-climacteric fruits. These findings provide important information to extend the molecular control of ripening in a non-climacteric fruit beyond the limited genetic and cultural options currently available.