Leaf rust (Puccinia recondita f. sp. secalis) triggers substantial changes in rye (Secale cereale L.) at the transcriptome and metabolome levels.

BACKGROUND: Rye (Secale cereale L.) is a cereal crop highly tolerant to environmental stresses, including abiotic and biotic stresses (e.g., fungal diseases). Among these fungal diseases, leaf rust (LR) is a major threat to rye production. Despite extensive research, the genetic basis of the rye immune response to LR remains unclear. RESULTS: An RNA-seq analysis was conducted to examine the immune response of three unrelated rye inbred lines (D33, D39, and L318) infected with compatible and incompatible Puccinia recondita f. sp. secalis (Prs) isolates. In total, 877 unique differentially expressed genes (DEGs) were identified at 20 and 36 h post-treatment (hpt). Most of the DEGs were up-regulated. Two lines (D39 and L318) had more up-regulated genes than down-regulated genes, whereas the opposite trend was observed for line D33. The functional classification of the DEGs helped identify the largest gene groups regulated by LR. Notably, these groups included several DEGs encoding cytochrome P450, receptor-like kinases, methylesterases, pathogenesis-related protein-1, xyloglucan endotransglucosylases/hydrolases, and peroxidases. The metabolomic response was highly conserved among the genotypes, with line D33 displaying the most genotype-specific changes in secondary metabolites. The effect of pathogen compatibility on metabolomic changes was less than the effects of the time-points and genotypes. Accordingly, the secondary metabolome of rye is altered by the recognition of the pathogen rather than by a successful infection. The results of the enrichment analysis of the DEGs and differentially accumulated metabolites (DAMs) reflected the involvement of phenylpropanoid and diterpenoid biosynthesis as well as thiamine metabolism in the rye immune response. CONCLUSION: Our work provides novel insights into the genetic and metabolic responses of rye to LR. Numerous immune response-related DEGs and DAMs were identified, thereby clarifying the mechanisms underlying the rye response to compatible and incompatible Prs isolates during the early stages of LR development. The integration of transcriptomic and metabolomic analyses elucidated the contributions of phenylpropanoid biosynthesis and flavonoid pathways to the rye immune response to Prs. This combined analysis of omics data provides valuable insights relevant for future research conducted to enhance rye resistance to LR.

MicroRNA expression profiles in liver and colon of sexually immature gilts after exposure to Fusarium mycotoxins.

To improve our knowledge of the role of microRNAs (miRs) in responses of the porcine digestive system to two Fusarium mycotoxins, zearalenone (ZEN) and deoxynivalenol (DON), we examined the expression of 7 miRs (miR-9, miR-15a, miR-21, miR-34a, miR-122, miR-125b, and miR-192), previously found to be deregulated in diseased liver and colon cells. In this study, immature gilts were exposed to NOEL doses of ZEN (40 µg/kg/d), DON (12 µg/kg/d), ZEN + DON (40 + 12 µg/kg/d), andplacebo (negative control group) for 7, 14, 21, 28, 35, and 42 days. Before the treatment, expression levels of the selected miRs were measured in the liver, the duodenum, the jejunum, and the ascending and the descending colon of the gilts. Hierarchical clustering of the tissues by their miR expression profiles was consistent with what would be expected based on the anatomical locations and the physiological functions of the organs, suggesting that functions of the miRs are related to the specificities of the tissues in which they are expressed. A subset of 2 pairs of miRs (miR-21+miR-192 and miR-15a+miR-34a), which were assigned to two distinct clusters based on their tissue abundance, was then evaluated in the liver and the ascending and the descending colon during the treatment. The most meaningful results were obtained from the ascending colon, where a significant effect of the treatment was observed, suggesting that during the exposure to mycotoxins, the pathways involved in cell proliferation and survival were disordered. Changes in miR expression in the liver and the descending colon of the treated gilts were smaller, and were associated more with treatment duration than the exposure to ZEN, DON, or ZEN + DON. Further research should focus on identification of genes whose expression is regulated by these aberrantly expressed miRs. This should facili- tate understanding of the miRNA-regulated biological effects of mycotoxins.

p53 Dynamically Directs TFIID Assembly on Target Gene Promoters.

p53 is a central regulator that turns on vast gene networks to maintain cellular integrity in the presence of various stimuli. p53 activates transcription initiation in part by aiding recruitment of TFIID to the promoter. However, the precise means by which p53 dynamically interacts with TFIID to facilitate assembly on target gene promoters remains elusive. To address this key issue, we have undertaken an integrated approach involving single-molecule fluorescence microscopy, single-particle cryo-electron microscopy, and biochemistry. Our real-time single-molecule imaging data demonstrate that TFIID alone binds poorly to native p53 target promoters. p53 unlocks TFIID's ability to bind DNA by stabilizing TFIID contacts with both the core promoter and a region within p53's response element. Analysis of single-molecule dissociation kinetics reveals that TFIID interacts with promoters via transient and prolonged DNA binding modes that are each regulated by p53. Importantly, our structural work reveals that TFIID's conversion to a rearranged DNA binding conformation is enhanced in the presence of DNA and p53. Notably, TFIID's interaction with DNA induces p53 to rapidly dissociate, which likely leads to additional rounds of p53-mediated recruitment of other basal factors. Collectively, these findings indicate that p53 dynamically escorts and loads TFIID onto its target promoters.

Oxidative damage of Chinese hamster fibroblasts induced by t-butyl hydroperoxide and by X-rays.

The aim of this study was to investigate the mechanism(s) of X-ray-mediated cell damage in comparison to mechanism(s) of organic hydroperoxide cytotoxicity and to find the main targets for the two different kinds of cell inactivation. Damage of Chinese hamster fibroblasts induced by tert-butyl hydroperoxide (t-BHP) or X-irradiation was measured by the colony-formation assay and the average single colony volume. DNA double-strand breaks (dsb) were determined by constant-field gel electrophoresis. The contents of peroxides, of SH-groups and the size of inactivated cells were tested for oxidative modifications.Oxidative damage of fibroblasts induced by t-BHP or by X-rays inhibits cell proliferation. Simultaneously, irradiation causes an increase of DNA dsb with the dose, while incubation with t-BHP yields only a very few DNA dsb. Neither chemically induced oxidation nor irradiation significantly changed the amount of membrane lipid peroxides. Oxidation with t-BHP but not irradiation leads to a loss of the membrane SH-groups and to an increase of cell diameter. The similar decrease of cell proliferation can be caused by DNA dsb without detectable membrane damage (X-radiation) as by membrane damage with nearly no DNA dsb (chemically induced oxidative stress).

Analysis of radiation- and 5-FU-induced inhibition of cell proliferation by an automatic colony analyser.

Proliferation of Chinese hamster cells B-14 was inhibited by irradiation, by incubation with 5-fluorouracil (5-FU) and by a combination of both treatments. The reduction in proliferation was assayed by the colony formation test, which was evaluated by an automatic colony analyser according to the number and volume of the colonies. It was demonstrated that the number of colonies multiplied by the volume was equivalent to the number of cells in a Petri dish and is called total colony volume. Since this quantity reflects the entire proliferation of cells, it is a more sensitive parameter for measuring cell viability than the clonogenicity of cells. The drug-radiation interaction showed a supra-additive effect, if total colony volume is taken into account, while the traditional scoring of colonies yielded only an additive effect.

Microbial adhesion and biofilm formation on microfiltration membranes: a detailed characterization using model organisms with increasing complexity.

Since many years, membrane biofouling has been described as the Achilles heel of membrane fouling. In the present study, an ecological assay was performed using model systems with increasing complexity: a monospecies assay using Pseudomonas aeruginosa or Escherichia coli separately, a duospecies assay using both microorganisms, and a multispecies assay using activated sludge with or without spiked P. aeruginosa. The microbial adhesion and biofilm formation were evaluated in terms of bacterial cell densities, species richness, and bacterial community composition on polyvinyldifluoride, polyethylene, and polysulfone membranes. The data show that biofouling formation was strongly influenced by the kind of microorganism, the interactions between the organisms, and the changes in environmental conditions whereas the membrane effect was less important. The findings obtained in this study suggest that more knowledge in species composition and microbial interactions is needed in order to understand the complex biofouling process. This is the first report describing the microbial interactions with a membrane during the biofouling development.

Expression profiling in vivo demonstrates rapid changes in liver microRNA levels of whitefish (Coregonus lavaretus) following microcystin-LR exposure.

At present, little is known about the role of miRNAs in liver response of fish to the cyanobacterial hepatotoxin microcystin-LR (MC-LR) treatment, despite the fact that the exposure is thought to underlie multiple acute and chronic effects. To address this question, we used the Real-Time PCR method to examine the differential expression of 6 miRNAs putatively playing roles in signal transduction (let-7c, miR-9b), apoptosis and cell cycle (miR-16a, miR-21a, miR-34a) and fatty acid metabolism (miR-122) in whitefish (Coregonus lavaretus) liver, during the first 48h after intraperitoneal injection of MC-LR (100 µg/kg body weight). In addition, we analyzed expression levels of 8 mRNAs and p53 protein, known to be involved in the cell response on the exposure to environmental stressors. Following the challenge we observed a rapid and transient increase in the mean (n=5) levels of individual miRNA expression (from 2.7-fold for miR-122 to 6.8-fold for let-7c), compared to the respective levels in control fish, which mostly peaked at 24h of the experiment. This increase was correlated with a reduction in the expression of mRNAs of genes coding for ferritin H (frih) and HNK Ras -like protein (p-ras) and an overexpression of mRNAs of genes coding for bcl2-associated X protein (bax), cyclin dependent kinase inhibitor 1a (cdkn1a), dicer (dcr), histone 2A (h2a) and p53. Expression of the remaining caspase 6 (cas6) mRNA did not change over 48 h of the treatment. Moreover, exposure to MC-LR did not alter whitefish p53 protein levels. Bearing in mind a variety of likely silencing targets for, and the onset of, the aberrant miRNA expression it may be concluded that they are involved in molecular pathways, such as liver cell metabolism, cell cycle regulation and apoptosis, and may contribute to the early phase of MC-LR induced hepatotoxicity.

Effect of combined treatment with perindoprilat and low-power red light laser irradiation on human erythrocyte membrane fluidity, membrane potential and acetylcholinesterase activity.

Erythrocyte membrane fluidity, membrane potential and acetylcholinesterase activity were estimated after in vitro combined treatment of human erythrocytes with perindoprilat and low-power red light irradiation. Membrane fluidity was determined using fluorescent labels spectroscopy; membrane potential was evaluated by means of potential-sensitive fluorescent dyes; and acetylcholinesterase activity was estimated using the Ellman method. Both perindoprilat and laser irradiation, when used separately, increase microviscosity in the polar region and hyperpolarize the membranes in comparison with control erythrocytes. The combined action of these agents does not cause any further change in these parameters. Perindoprilat has an additional inhibitory effect on the activity of acetylcholinesterase, whereas laser irradiation causes an increase in the activity of the enzyme. Their combined action restores the initial activity of the enzyme independently of the sequence of treatment with both agents.

Human red blood cell membrane potential and fluidity in glucose solutions.

This study aimed to investigate the membrane potential and fluidity changes of human red blood cells subjected to isotonic glucose solutions. For the control erythrocytes a membrane potential value of -10.1 +/- 1.8 mV was obtained, whereas erythrocyte membranes in diabetic cells were hyperpolarized, with a potential of -13.9 +/- 2.3 mV. Incubation of both types of red blood cells with increasing glucose concentrations resulted in a substantial hyperpolarization of the cell membranes. Glucose solutions in water had a much stronger effect on membrane potential than glucose dissolved in phosphate-buffered saline. Red blood cell membrane fluidity measurements using the fluorescent label TMADPH did not reveal any significant changes upon incubation with glucose.

Interaction of perindoprilat with human red blood cells.

The effects of perindoprilat on the morphology and dynamic properties of human erythrocytes were studied by light microscopy, electron spin resonance spectroscopy and spectrophotometric methods. Erythrocytes were exposed to perindoprilat at 37 degrees C for 30 and 120 min. It was shown that the drug at a concentration of 0.75 microg/ml did not cause significant changes in the structure of erythrocyte membranes. Higher doses of the drug (7.5 and 75 microg/ml) induced changes in membrane fluidity in the hydrophobic core of the lipid bilayer, the conformation of membrane proteins, the number of SH groups and the activity of membrane-bound acetylcholinesterase (AChE). These modifications were accompanied by changes in the shape of erythrocytes and did not depend on time of incubation. Therefore, it is proposed that perindoprilat perturbs the lipid bilayer and disturbs the organization of the protein-lipid environment.

Effect of perindopril therapy on fluidity and potential of erythrocyte membrane from individuals with coronary heart disease.

Erythrocyte membrane fluidity and membrane potential were measured in patients suffering from coronary heart disease (CHD) and treated with perindopril. Membrane fluidity was determined using electron paramagnetic resonance (EPR) spectroscopy, and membrane potential was evaluated using potential-sensitive fluorescent dyes. CHD does not change membrane fluidity at the depth of the 5 carbon in the fatty acid chain of membrane phospholipids. However the hydrophobic core of the membrane is altered in CHD. For 19 CHD patients, the correlation times tau B and tau C of a spin label 16DS were higher than for controls: tau B = (1.84 +/- 0.04) x 10(-9) s and tau C = (2.54 +/- 0.04) x 10(-9) s vs. tau B = (1.62 +/- 0.06) x 10(-9) s; and tau C = (2.24 +/- 0.07) x 10(-9) s (results given as mean +/- SEM). Such results indicate the increased microviscosity in hydrophobic regions of CHD erythrocyte membranes in comparison with controls. Perindopril therapy partly abolished these changes. The membrane potential of CHD red blood cells -17.89 +/- 1.36 mV was higher than the control value -9.83 +/- 0.59 mV. Perindopril treatment shifted the membrane potential value to -13.45 +/- 0.99 mV when measured after a single dose of the drug, or even depolarized the membrane after 7 days of therapy -4.95 +/- 0.73 mV. It is concluded that the erythrocyte membrane is more rigid and hyperpolarized in CHD, and perindopril therapy partly abolishes these changes as early as 3 h after administration.

Characterization and mapping of canine microsatellites isolated from BAC clones harbouring DNA sequences homologous to seven human genes.

Human primers specific for the genes LEP, HBB, PAX3, ESR2, TPH1, ABCA4 and ATP2A2 were used to identify clones in a canine BAC library. Subcloning of the positive BACs in plasmids, screening with microsatellite motifs and subsequent sequencing allowed for the identification of eight novel microsatellites. The presence of the gene of interest was confirmed by sequencing the polymerase chain reaction (PCR) products amplified in the positive BACs. Fluorescent in situ hybridization (FISH) using the positive BACs as probes allowed for the chromosomal localization of the insert DNAs in two canid species, dog (Canis familiaris) and red fox (Vulpes vulpes). The use of gene-associated microsatellites may accelerate the identification of candidate genes for phenotypic traits in linkage studies.