Transcriptome and metabolome profiling of field-grown transgenic barley lack induced differences but show cultivar-specific variances.

The aim of the present study was to assess possible adverse effects of transgene expression in leaves of field-grown barley relative to the influence of genetic background and the effect of plant interaction with arbuscular mycorrhizal fungi. We conducted transcript profiling, metabolome profiling, and metabolic fingerprinting of wild-type accessions and barley transgenics with seed-specific expression of (1,3-1, 4)-beta-glucanase (GluB) in Baronesse (B) as well as of transgenics in Golden Promise (GP) background with ubiquitous expression of codon-optimized Trichoderma harzianum endochitinase (ChGP). We found more than 1,600 differential transcripts between varieties GP and B, with defense genes being strongly overrepresented in B, indicating a divergent response to subclinical pathogen challenge in the field. In contrast, no statistically significant differences between ChGP and GP could be detected based on transcriptome or metabolome analysis, although 22 genes and 4 metabolites were differentially abundant when comparing GluB and B, leading to the distinction of these two genotypes in principle component analysis. The coregulation of most of these genes in GluB and GP, as well as simple sequence repeat-marker analysis, suggests that the distinctive alleles in GluB are inherited from GP. Thus, the effect of the two investigated transgenes on the global transcript profile is substantially lower than the effect of a minor number of alleles that differ as a consequence of crop breeding. Exposing roots to the spores of the mycorrhizal Glomus sp. had little effect on the leaf transcriptome, but central leaf metabolism was consistently altered in all genotypes.

Macroarray expression analysis of barley susceptibility and nonhost resistance to Blumeria graminis.

Different formae speciales of the grass powdery mildew fungus Blumeria graminis undergo basic-compatible or basic-incompatible (nonhost) interactions with barley. Background resistance in compatible interactions and nonhost resistance require common genetic and mechanistic elements of plant defense. To build resources for differential screening for genes that potentially distinguish a compatible from an incompatible interaction on the level of differential gene expression of the plant, we constructed eight dedicated cDNA libraries, established 13.000 expressed sequence tag (EST) sequences and designed DNA macroarrays. Using macroarrays based on cDNAs derived from epidermal peels of plants pretreated with the chemical resistance activating compound acibenzolar-S-methyl, we compared the expression of barley gene transcripts in the early host interaction with B. graminis f.sp. hordei or the nonhost pathogen B. graminis f.sp. tritici, respectively. We identified 102 spots corresponding to 94 genes on the macroarray that gave significant B. graminis-responsive signals at 12 and/or 24 h after inoculation. In independent expression analyses, we confirmed the macroarray results for 11 selected genes. Although the majority of genes showed a similar expression profile in compatible versus incompatible interactions, about 30 of the 94 genes were expressed on slightly different levels in compatible versus incompatible interactions.

Heparin impairs glycocalyx barrier properties and attenuates shear dependent vasodilation in mice.

The endothelial glycocalyx is a hydrated mesh of polysaccharides and adsorbed plasma proteins that forms the true interface between the flowing blood and the endothelium. We hypothesized in the present study that competitive binding of heparin to glycocalyx-associated proteins would affect glycocalyx barrier properties and mechanotransduction of shear stress to the endothelium. In anesthetized mice, the clearance of 70-kDa dextrans from the circulation was increased (P<0.05 versus saline) 1 hour after heparin (1.25 U) and glycocalyx degradation with hyaluronidase (35 U; amount cleared in 30 minutes after saline: 11+/-5%; after heparin: 45+/-8%; after hyaluronidase: 30+/-3%). Clearance of 40-kDa dextrans increased (P<0.05 versus saline) to a lesser extent after both treatments (saline: 46+/-3%; heparin: 60+/-5%; hyaluronidase: 60+/-2%). The dilator response of second-order arterioles in cremaster muscle during reactive hyperemia was reduced for < or =90 minutes after heparin as reflected by a decrease (P=0.008) in t(50) of diameter recovery, and this effect was associated with a diminished NO bioavailability. Infusion of hyaluronidase resulted in reductions (P<0.05) in baseline and peak reactive hyperemic diameter, whereas, despite an increase in wall shear rate at the beginning of reactive hyperemia, t(50) of diameter recovery was not affected. In conclusion, our data in mice show that a heparin challenge is associated with increased vascular leakage of dextrans and impaired arteriolar vasodilation during reactive hyperemia. Our data suggest that protein-heparan sulfate interactions are important for a functional glycocalyx.

Technology assessment and knowledge brokering: the case of assisted reproduction in The Netherlands.

OBJECTIVES: Even when policy makers show interest and evidence-informed and convincing HTA studies are available, use of assessment products is not guaranteed. In this article, we report our experience with knowledge brokering to foster evidence-informed policy making on cost-effective treatment and reimbursement of assisted reproduction in The Netherlands. METHODS: From earlier work in the field of knowledge brokering, we foresaw the need for a deliberative strategy to manage the inherent tension between scientific rigor demanded by researchers and responsiveness to real-time needs demanded by policy makers. Therefore, we structured the process in three distinct steps: (i) agreement about the main messages from the research, (ii) analysis of the policy context and of the meaning of the main messages for the actors involved, and (iii) an invitational meeting to make recommendations for action. RESULTS: One of the recommendations that would require changes in ministerial policy was followed up instantly, whereas the other recommendation is still under debate. The Dutch Society of Obstetrics and Gynecology activated the revision of two guidelines. The patient organization uses the new scientific insights in informing members and the public. Closing the loop, The Netherlands Organisation for Health Research and Development (ZonMw) funded research to close knowledge gaps that became apparent in the process. CONCLUSIONS: Knowledge brokering is a promising approach to bring HTA into practice. We conclude that the methodologies to feed research results into the policy process are still in an incipient stage and need further development.

Infection patterns in barley and wheat spikes inoculated with wild-type and trichodiene synthase gene disrupted Fusarium graminearum.

Fusarium head blight epidemics of wheat and barley cause heavy economic losses to farmers due to yield decreases and production of mycotoxin that renders the grain useless for flour and malt products. No highly resistant cultivars are available at present. Hyphae of germinating fungal spores use different paths of infection: After germination at the extruded tip of an ovary, the hyphae travel along the epicarp in the space between the lemma and palea. Infection of the developing kernel proceeds through the epicarp, successively destroying the layers of the fruit coat and finally the starch and protein accumulating endosperm. Hyphae reaching the rachis proceed to apically located developing kernels. Using a constitutively green fluorescence protein-expressing Fusarium wild-type strain, and its knockout mutant, preventing trichothecene synthesis, we demonstrate that trichothecenes are not a virulence factor during infection through the fruit coat. In the absence of trichothecenes, the fungus is blocked by the development of heavy cell wall thickenings in the rachis node of Nandu wheat, a defense inhibited by the mycotoxin. In barley hyphae of both wild-type and the trichothecene knockout mutant, are inhibited at the rachis node and rachilla, limiting infection of adjacent florets through the phloem and along the surface of the rachis. Effective resistance to Fusarium head blight requires expression of genes that combat these different pathways of infection.

Identification of powdery mildew-induced barley genes by cDNA-AFLP: functional assessment of an early expressed MAP kinase.

Gene expression analysis by cDNA-AFLP in barley ( Hordeum vulgare L.) after powdery mildew ( Blumeria graminis f.sp. hordei , Bgh ) inoculation revealed 615 (3.7%) of 16 500 screened cDNA fragments being differentially regulated 4 and/or 12 h after inoculation. Of these transcript derived fragments (TDFs), 120 were sequenced, and for 28 out of 29 tested, induction was confirmed via RT-PCR. Most TDFs did not show any homology to sequences with known functions, others showed homology to genes involved in primary and secondary metabolism, pathogen response, redox regulation, and signal transduction. TDFs with homology to a MAP kinase ( PWMK1 ), a WRKY transcription factor, a heparanase, an immunophilin, a cytochrome P450, and a receptor-like protein kinase were isolated as full length cDNAs. Knockdown by RNA interference via biolistic delivery of sequence specific double stranded RNA to leaf segments tagged two of these genes as possible candidates being causally involved in the outcome of the barley- Bgh interaction. Knockdown of the receptor-like protein kinase and the WRKY transcription factor increased resistance to the fungus, while knockdown of PWMK1 only led to a slightly enhanced susceptibility of epidermal cells to Bgh . This suggests that the receptor-like protein kinase and the WRKY protein are candidates for negative regulators of powdery mildew resistance. Based on expression analyses, PWMK1 appears to be more generally involved in stress response.