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This approach will help to systematically advance our technological capability to delay plant ethylene responses and to expand shelf-life or vase life of fruits and flowers.We have developed and applied a novel strategy that can best be described as in vivo chemical genomics, a concept where populations of any transformable organism may be screened for consequences of novel RNAs or peptides. We created a library of ~800,000 random DNA sequences biased only by third-position nucleotide substitutions that suppress the frequency of termination codons. The sequences may be shuttled to any plant, microbial, or animal expression vector with recombination cloning. We then generated large populations of Arabidopsis thaliana plants, each expressing a randomized DNA sequence, presumably giving rise to synthetic RNA species and/or the peptides they encode. These novel molecules are produced within the context of the cell and have been shown to affect plant biology with a relatively high frequency, as evidenced by diverse phenotypes. This chapter provides the protocols necessary to construct the libraries and isolate plants expressing randomized DNA sequences.New biologically active compounds are regularly discovered through screening procedures using microorganisms. This very cheap procedure is followed by drug discovery that is usually seen as a highly focused approach, testing new compounds on animals or cell lines. In vivo assays of candidate drugs in mammals are expensive and sometimes not affordable at the preliminary stages of drug development. Early screening approaches in transgenic plants would allow chemotherapeutic drug candidates further selection before their characterization in expensive biological models. The proposed screening approach is based on cell subcellular architecture observations in transgenic plants within a short time of treatment, which is better than observing the effects of compounds on growth.Bacterial plant pathogens are among the most devastating threats to agriculture. To date, there are no effective means to control bacterial plant diseases due to the restrictions in the use of antibiotics in agriculture. A novel strategy under study is the use of chemical compounds that inhibit the expression of key bacterial virulence determinants. The type III secretion system is essential for virulence of many Gram-negative bacteria because it injects into the plant host cells bacterial proteins that interfere with their immune system. Here, we describe the methodology to identify bacterial type III secretion inhibitors, including a series of protocols that combine in planta and in vitro experiments. We use Ralstonia solanacearum as a model because of the number of genetic tools available in this organism and because it causes bacterial wilt, one of the most threatening plant diseases worldwide. The procedures presented can be used to evaluate the effect of different chemical compounds on bacterial growth and virulence.Forward genetics has been extremely powerful for dissecting biological pathways in various model organisms. However, it is limited by the fact that redundant gene families and essential genes cannot be readily uncovered through such methods. Chemical genetics, on the other hand, provides a valuable complementary approach to probe biological processes and is suitable for not only genetic model organisms but also genetically less tractable species. We describe here a high-throughput chemical genetic screening method simply based on plant growth and developmental phenotypes in Arabidopsis. It was successfully utilized to study plant immunity and can be easily adapted for dissecting other plant signal transduction pathways.After germination, plants determine their morphogenesis, such as hypocotyl elongation and cotyledon opening, by responding to various wavelengths of light (photomorphogenesis). Cryptochrome is a blue-light photoreceptor that controls de-etiolation, stomatal opening and closing, flowering time, and shade avoidance. Successful incorporation of these phenotypes as indicators into a chemical screening system results in faster selection of candidate compounds. Here, we describe phenotypic screening for the blue-light response of Arabidopsis thaliana seedling and the resulting process that clarifies that the compound obtained in the screening is an inhibitor of cryptochromes.Like animals, plants use various lipids as signaling molecules to guide their growth and development. The focus of our work is on the N-acylethanolamine (NAE) group of lipid mediators, which have been shown to play important physiological roles in plants. However, mechanisms by which NAEs modulate plant function remain elusive. NaPB datasheet Chemical genetics has emerged as a potent tool to elucidate signaling pathways in plants, particularly those orchestrated by plant hormones. Like plant hormones, exogenous application of NAEs elicits distinct plant growth phenotypes that can serve as biological readouts for chemical genetic screens. For example, N-lauroylethanolamide (NAE 120) inhibits seedling development in the model plant Arabidopsis thaliana. Thus, a library of small synthetic chemical compounds can be rapidly screened for their ability to reverse the inhibitory effect of NAE 120 on seedling development. Chemicals identified through such screens could be potential agonists/antagonists of NAE receptors or signaling pathways and therefore serve as additional tools for understanding NAE function in plants. In this chapter, we describe general protocols for NAE 120-based chemical genetic screens in Arabidopsis. Although such screens were designed primarily for NAE 120, they could potentially be applied for similar work with other NAE species or plant lipid mediators.

While clinical guidelines recommend that residual kidney function (RKF) is measured in peritoneal dialysis (PD) patients, 24-h urine collection is cumbersome and prone to errors. We wished to determine whether an equation using serum β2-microglobulin (β2M) could prove of clinical benefit in estimating RKF and identifying patients who could start PD with incremental prescriptions.

We measured serum β2M in consecutive PD outpatients recently starting dialysis with continuous ambulatory PD (CAPD) or automated PD (APD), attending a single tertiary hospital for their routine clinical visit. RKF was defined as the mean of 24-h urine clearances of creatinine and urea. An equation estimating RKF (eRKF) was generated based on serum β2M levels on a randomly selected modelling group.

We included 511 patients, of whom 351 in the modelling group and 150 in the validation group. Mean age was 58.7 ± 15.8, 307 (60.0%) were men and median RKF value was 4.5 (2.4-6.5) mL/min/1.73m

. In the validation group, an equation based on β2M, creatinine, urea, age and gender showed minimal bias of -0.