Understanding variation in oleic acid content of high-oleic virginia-type peanut.

KEY MESSAGE: Contamination at the FAD2B locus due to inadequate screening protocols is the primary cause of sporadic, insufficient oleic acid content in Virginia-type peanut. The high oleic trait in peanut is conditioned by loss-of-function mutations in a pair of homeologous enzymes and is well known to improve the shelf life of peanut products. As such, the trait is given high priority in current and future cultivars by the North Carolina State University peanut breeding program. For unknown reasons, high oleic cultivars and breeding lines intermittently failed to meet self-imposed thresholds for oleic acid content in internal testing. To determine why, a manual seed chipper, crude DNA isolation protocol, genotyping assays for both mutations, and a web-based SNP calling application were developed. The primary cause was determined to be contamination with normal oleic seeds resulting from inadequate screening protocols. In order to correct the problem, a faster screening method was acquired to accommodate a higher oleic acid threshold. Additionally, results showed the mutation in one homeolog is fixed in the program, dig date had no significant effect on oleic acid content, and minor modifiers segregating within the program explained 6% of the variation in oleic acid content.

Biogenesis of Golgi stacks in imaginal discs of Drosophila melanogaster.

We provide a detailed description of Golgi stack biogenesis that takes place in vivo during one of the morphogenetic events in the lifespan of Drosophila melanogaster. In early third-instar larvae, small clusters consisting mostly of vesicles and tubules were present in epithelial imaginal disk cells. As larvae progressed through mid- and late-third instar, these larval clusters became larger but also increasingly formed cisternae, some of which were stacked. In white pupae, the typical Golgi stack was observed. We show that larval clusters are Golgi stack precursors by 1) localizing various Golgi-specific markers to the larval clusters by electron and immunofluorescence confocal microscopy, 2) driving this conversion in wild-type larvae incubated at 37 degrees C for 2 h, and 3) showing that this conversion does not take place in an NSF1 mutant (comt 17). The biological significance of this conversion became clear when we found that the steroid hormone 20-hydroxyecdysone (ecdysone) is critically involved in this conversion. In its absence, Golgi stack biogenesis did not occur and the larval clusters remained unaltered. We showed that dGM130 and sec23p expression increases approximately three- and fivefold, respectively, when discs are exposed to ecdysone in vivo and in vitro. Taken together, these results suggest that we have developed an in vivo system to study the ecdysone-triggered Golgi stack biogenesis.

Rapid isolation and presumptive diagnosis of uropathogens by using membrane filtration and differential media.

Random urine samples from hospitalized patients (n = 550) and seeded sterile filtered urine samples (n = 730) were used to test a membrane filtration technique, Qualture (Future Medical Technologies International, Inc., West Palm Beach, Fla.), for the detection and identification of uropathogens. Results for each sample were compared with those obtained by the calibrated loop (0.01 ml) method to demonstrate the sensitivity of the method as a screening tool and the specificity of the presumptive diagnosis obtained from the pattern of growth on differential media. The medium was supplied as dehydrated nutrient pads (Sartorius AG, Goettingen, Germany) and was activated by rehydration by the addition of the liquid specimen. With a threshold of 10(4) CFU/ml defining a positive culture, the sensitivity of the Qualture was 100%. At lower levels of bacteriuria, the Qualture was more sensitive than the calibrated loop method. Significant infections were presumptively diagnosed at 4 h by filtration rather than at 24 h on agar medium. The specificity of uropathogen identification ranged from 99% for Enterococcus spp. to 83% for Pseudomonas spp. Citrobacter spp. could not be differentiated from Escherichia coli and Providencia spp. could not be differentiated from Proteus spp., which does not create a therapeutic dilemma. Filtration, isolation, quantitation, and presumptive diagnosis are performed in one step, without subculture. Membrane filtration is a sensitive and rapid technique, with the advantage that it can be used as a collection and transport device without the use of growth inhibitors.