Improvement of anther culture methods for doubled haploid production in barley breeding

There is potential to accelerate cultivar development with a doubled haploid system for breeding line production. Anther culture methodology was evaluated for U.S.A. spring barley (Hordeum vulgare L.) breeding applications. Gelrite was found to be an acceptable replacement for ficoll in the induction medium to reduce costs while maintaining embryoid and plant production levels. Beneficial effects of 28 d cold pretreatment of donor spikes for anther culture were confirmed with Pacific Northwest USA barley genotypes. A 3 d mannitol solution pretreatment of fresh anthers was shown to be less effective for green plant production compared to 28 d cold pretreatment of donor spikes. Extended donor spike cold pretreatment from 28 to 42 d did not reduce anther culture productivity. Based on this research, anther culture techniques show promise for economical and convenient application in spring barley breeding.Abbreviations DH doubled haploid - LS Linsmaier and Skoog basal medium - BAP benzylaminopurine - GLM Generalized Linear Model - SAS Statistical Analysis System     

A High-Content Small Molecule Screen Identifies Sensitivity of Glioblastoma Stem Cells to Inhibition of Polo-Like Kinase 1

Glioblastoma multiforme (GBM) is the most common primary brain cancer in adults and there are few effective treatments. GBMs contain cells with molecular and cellular characteristics of neural stem cells that drive tumour growth. Here we compare responses of human glioblastoma-derived neural stem (GNS) cells and genetically normal neural stem (NS) cells to a panel of 160 small molecule kinase inhibitors. We used live-cell imaging and high content image analysis tools and identified JNJ-10198409 (J101) as an agent that induces mitotic arrest at prometaphase in GNS cells but not NS cells. Antibody microarrays and kinase profiling suggested that J101 responses are triggered by suppression of the active phosphorylated form of polo-like kinase 1 (Plk1) (phospho T210), with resultant spindle defects and arrest at prometaphase. We found that potent and specific Plk1 inhibitors already in clinical development (BI 2536, BI 6727 and GSK 461364) phenocopied J101 and were selective against GNS cells. Using a porcine brain endothelial cell blood-brain barrier model we also observed that these compounds exhibited greater blood-brain barrier permeability in vitro than J101. Our analysis of mouse mutant NS cells (INK4a/ARF^−/−, or p53^−/−), as well as the acute genetic deletion of p53 from a conditional p53 floxed NS cell line, suggests that the sensitivity of GNS cells to BI 2536 or J101 may be explained by the lack of a p53-mediated compensatory pathway. Together these data indicate that GBM stem cells are acutely susceptible to proliferative disruption by Plk1 inhibitors and that such agents may have immediate therapeutic value.     

Predation resistance does not trade off with competitive ability in early-colonizing mosquitoes

The tradeoff between colonization and competitive ability has been proposed as a mechanism for ecological succession, and this tradeoff has been demonstrated in multiple successional communities. The tradeoff between competitive ability and predation resistance is also a widely-described phenomenon; however, this tradeoff is not usually postulated as a cause of ecological succession. Early successional species that arrive before predator colonization could be either (1) less vulnerable to predation than their successors, by virtue of being poor competitors (direct competition-predation tradeoff); or (2) equally or more vulnerable to predation, because they normally colonize ahead of predators in succession and therefore are not evolutionarily adapted to avoid predators that they rarely encounter (no competition–predation tradeoff). To test these alternative hypotheses, we established water-filled containers in an oak–hickory forest. We allowed half of the containers to be naturally colonized by early-successional Culex mosquitoes, mid-successional Aedes mosquitoes, and the mosquito predator Toxorhynchites rutilus. In the other half of the containers, we prevented Aedes colonization via systematic removal of Aedes eggs, but allowed Culex and T. rutilus to colonize. The numbers of mature Culex larvae and pupae, and later the total number of Culex, were significantly greater in containers where Aedes had been removed, which suggests that Culex are competitively suppressed by Aedes. Toxorhynchites rutilus abundance and colonization rate were unaffected by the removal of Aedes, and densities of both Culex and Aedes decreased significantly with T. rutilus abundance in both treatments. In-laboratory bioassays showed that Culex were significantly more vulnerable to predation by T. rutilus than were Aedes. These data are consistent with the hypothesis that Culex and Aedes demonstrate a direct colonization–competition tradeoff, and are inconsistent with the hypothesis of a direct competition–predation tradeoff.     

Spatial Frequency Information Modulates Response Inhibition and Decision-Making Processes

We interact with the world through the assessment of available, but sometimes imperfect, sensory information. However, little is known about how variance in the quality of sensory information affects the regulation of controlled actions. In a series of three experiments, comprising a total of seven behavioral studies, we examined how different types of spatial frequency information affect underlying processes of response inhibition and selection. Participants underwent a stop-signal task, a two choice speed/accuracy balance experiment, and a variant of both these tasks where prior information was given about the nature of stimuli. In all experiments, stimuli were either intact, or contained only high-, or low- spatial frequencies. Overall, drift diffusion model analysis showed a decreased rate of information processing when spatial frequencies were removed, whereas the criterion for information accumulation was lowered. When spatial frequency information was intact, the cost of response inhibition increased (longer SSRT), while a correct response was produced faster (shorter reaction times) and with more certainty (decreased errors). When we manipulated the motivation to respond with a deadline (i.e., be fast or accurate), removal of spatial frequency information slowed response times only when instructions emphasized accuracy. However, the slowing of response times did not improve error rates, when compared to fast instruction trials. These behavioral studies suggest that the removal of spatial frequency information differentially affects the speed of response initiation, inhibition, and the efficiency to balance fast or accurate responses. More generally, the present results indicate a task-independent influence of basic sensory information on strategic adjustments in action control.     

Standardizing Immunohistochemistry: A New Reference Control for Detecting Staining Problems

A new standardized immunohistochemistry (IHC) control for breast cancer testing comprises formalin-fixed human epidermal growth factor receptor 2, estrogen receptor, or progesterone receptor peptide antigens covalently attached to 8-µm glass beads. The antigen-coated beads are suspended in a liquid matrix that hardens upon pipetting onto a glass microscope slide. The antigen-coated beads remain in place through deparaffinization, antigen retrieval, and immunostaining. The intensity of the beads’ stain provides feedback regarding the efficacy of both antigen retrieval and immunostaining. As a first report, we tested the sensitivity and specificity of the new IHC controls (“IHControls”). To evaluate sensitivity, various staining problems were simulated. IHControls detected primary and secondary reagent degradation similarly to tissue controls. This first group of IHControls behaved similarly to tissue controls expressing high concentrations of the antigen. The IHControls were also able to detect aberrations in antigen retrieval, as simulated by sub-optimal times or temperatures. Specificity testing revealed that each antigen-coated bead was specific for its cognate IHC test antibody. The data support the conclusion that, like tissue controls, IHControls are capable of verifying the analytic components of an immunohistochemical stain. Unlike tissue controls, IHControls are prepared in large bulk lots, fostering day-to-day reproducibility that can be standardized across laboratories.     

Inter-chromophore interactions in pigment-modified and dimer-less bacterial photosynthetic reaction centers

The magnitudes of inter-chromophore interactions in bacterial photosynthetic reaction centers are investigated by measuring absorption and Stark spectra of reaction centers in which monomeric chromophores are modified and in a novel triplet mutant which lacks the special pair. The circular dichroism spectrum of the triple mutant reaction center was also measured. Only small changes in the spectroscopic properties are observed, as has also been found for several types of reaction centers in which the absorption or chemical properties of a chromophore are altered by site-specific mutations. We conclude that the electronic absorption, circular dichroism and Stark features of the special pair and the monomeric chromophores in the reaction center are relatively insensitive to inter-chromophore interactions.     

5S ribosomal gene clusters in wheat: pulsed field gel electrophoresis reveals a high degree of polymorphism

Summary The long-range structure of 5S rRNA gene clusters has been investigated in wheat (Triticum aestivum L.) by means of pulsed field gel electrophoresis. Using aneuploid stocks, 5S rRNA gene clusters were assigned to sites on chromosomes 1B, 1D, 513 and 5D. Cluster sizes were evaluated and the copy number of 5S DNA repeats was estimated at 4700-5200 copies for the short repeating unit (410 bp) and about 3100 copies for the long repeat (500 bp) per haploid genome. A comparison of wheat cultivars revealed extremely high levels of polymorphism in the 5S rRNA gene clusters. With one restriction enzyme digest all varieties tested gave unique banding patterns and, on a per fragment basis, 21-fold more polymorphism was detected among cultivars for 5S DNA compared to standard restriction fragment length polymorphisms (RFLPs) detected with single copy clones. Experiments with aneuploid stocks suggest that the 5S rRNA gene clusters at several chromosomal sites contribute to this polymorphism. A number of previous reports have shown that wheat cultivars are not easily distinguished by isozymes or RFLPs. The high level of variation detected in 5S rRNA gene clusters therefore offers the possibility of a sensitive fingerprinting method for wheat. 5S DNA and other macro-satellite sequences may also serve as hypervariable Mendelian markers for genetic and breeding experiments in wheat.