A high-throughput assay for rapid and simultaneous analysis of perfect markers for important quality and agronomic traits in rice using multiplexed MALDI-TOF mass spectrometry

The application of single nucleotide polymorphisms (SNPs) in plant breeding involves the analysis of a large number of samples, and therefore requires rapid, inexpensive and highly automated multiplex methods to genotype the sequence variants. We have optimized a high-throughput multiplexed SNP assay for eight polymorphisms which explain two agronomic and three grain quality traits in rice. Gene fragments coding for the agronomic traits plant height (semi-dwarf, sd-1 ) and blast disease resistance ( Pi-ta ) and the quality traits amylose content ( waxy ), gelatinization temperature ( alk ) and fragrance ( fgr ) were amplified in a multiplex polymerase chain reaction. A single base extension reaction carried out at the polymorphism responsible for each of these phenotypes within these genes generated extension products which were quantified by a matrix-assisted laser desorption ionization-time of flight system. The assay detects both SNPs and indels and is co-dominant, simultaneously detecting both homozygous and heterozygous samples in a multiplex system. This assay analyses eight functional polymorphisms in one 5 µL reaction, demonstrating the high-throughput and cost-effective capability of this system. At this conservative level of multiplexing, 3072 assays can be performed in a single 384-well microtitre plate, allowing the rapid production of valuable information for selection in rice breeding.     

Tilting at windmills? The nucleotide excision repair of chromosomal DNA

A typical view of how DNA repair functions in chromatin usually depicts a struggle in which the DNA repair machinery battles to overcome the inhibitory effect of chromatin on the repair process. It may be that in this current interpretation the repair mechanisms are ‘tilting at windmills’, fighting an imaginary foe. An emerging picture suggests that we should not consider chromatin as an inhibitory force to be overcome like some quixotic giant by the DNA repair processes. Instead we should now recognize that DNA repair and chromatin metabolism are inextricably and mechanistically linked. Here we discuss the latest findings which are beginning to reveal how changes in chromatin dynamics integrate with the DNA repair process in response to UV induced DNA damage, with an emphasis on events in the yeast Saccharomyces cerevisiae.     

Visualisation and Quantitative Analysis of the Rodent Malaria Liver Stage by Real Time Imaging

The quantitative analysis of Plasmodium development in the liver in laboratory animals in cultured cells is hampered by low parasite infection rates and the complicated methods required to monitor intracellular development. As a consequence, this important phase of the parasite''s life cycle has been poorly studied compared to blood stages, for example in screening anti-malarial drugs. Here we report the use of a transgenic P. berghei parasite, PbGFP-Luc_con, expressing the bioluminescent reporter protein luciferase to visualize and quantify parasite development in liver cells both in culture and in live mice using real-time luminescence imaging. The reporter-parasite based quantification in cultured hepatocytes by real-time imaging or using a microplate reader correlates very well with established quantitative RT-PCR methods. For the first time the liver stage of Plasmodium is visualized in whole bodies of live mice and we were able to discriminate as few as 1–5 infected hepatocytes per liver in mice using 2D-imaging and to identify individual infected hepatocytes by 3D-imaging. The analysis of liver infections by whole body imaging shows a good correlation with quantitative RT-PCR analysis of extracted livers. The luminescence-based analysis of the effects of various drugs on in vitro hepatocyte infection shows that this method can effectively be used for in vitro screening of compounds targeting Plasmodium liver stages. Furthermore, by analysing the effect of primaquine and tafenoquine in vivo we demonstrate the applicability of real time imaging to assess parasite drug sensitivity in the liver. The simplicity and speed of quantitative analysis of liver-stage development by real-time imaging compared to the PCR methodologies, as well as the possibility to analyse liver development in live mice without surgery, opens up new possibilities for research on Plasmodium liver infections and for validating the effect of drugs and vaccines on the liver stage of Plasmodium.     

The concentration of soluble extracellular amyloid-β protein in acute brain slices from CRND8 mice

Background

Many recent studies of the effects of amyloid-β protein (Aβ) on brain tissue from amyloid precursor protein (APP) overexpressing mice have concluded that Aβ oligomers in the extracellular space can profoundly affect synaptic structure and function. As soluble proteins, oliomers of Aβ can diffuse through brain tissue and can presumably exit acute slices, but the rate of loss of Aβ species by diffusion from brain slices and the resulting reduced concentrations of Aβ species in brain slices are unknown.

Methodology/Principal Findings

Here I combine measurements of Aβ_1–42 diffusion and release from acute slices and simple numerical models to measure the concentration of Aβ_1–42 in intact mice (in vivo) and in acute slices from CRND8 mice. The in vivo concentration of diffusible Aβ_1–42 in CRND8 mice was 250 pM at 6 months of age and 425 pM at 12 months of age. The concentration of Aβ_1–42 declined rapidly after slice preparation, reaching a steady-state concentration within one hour. 50 µm from the surface of an acute slice the steady-state concentration of Aβ was 15–30% of the concentration in intact mice. In more superficial regions of the slice, where synaptic physiology is generally studied, the remaining Aβ is less than 15%. Hence the concentration of Aβ_1–42 in acute slices from CRND8 mice is less than 150 pM.

Conclusions/Significance

Aβ affects synaptic plasticity in the picomolar concentration range. Some of the effects of Aβ may therefore be lost or altered after slice preparation, as the extracellular Aβ concentration declines from the high picomolar to the low picomolar range. Hence loss of Aβ by diffusion may complicate interpretation of the effects of Aβ in experiments on acute slices from APP overexpressing mice.     

Lizards fail to plastically adjust nesting behavior or thermal tolerance as needed to buffer populations from climate warming

Although observations suggest the potential for phenotypic plasticity to allow adaptive responses to climate change, few experiments have assessed that potential. Modeling suggests that Sceloporus tristichus lizards will need increased nest depth, shade cover, or embryonic thermal tolerance to avoid reproductive failure resulting from climate change. To test for such plasticity, we experimentally examined how maternal temperatures affect nesting behavior and embryonic thermal sensitivity. The temperature regime that females experienced while gravid did not affect nesting behavior, but warmer temperatures at the time of nesting reduced nest depth. Additionally, embryos from heat‐stressed mothers displayed increased sensitivity to high‐temperature exposure. Simulations suggest that critically low temperatures, rather than high temperatures, historically limit development of our study population. Thus, the plasticity needed to buffer this population has not been under selection. Plasticity will likely fail to compensate for ongoing climate change when such change results in novel stressors.     

Strigolactone Hormones and Their Stereoisomers Signal through Two Related Receptor Proteins to Induce Different Physiological Responses in Arabidopsis

We speculate that multicopy transposons, carrying both fitness and unfitness genes, can provide new positive and negative selection options to intractable weed problems. Multicopy transposons rapidly disseminate through populations, appearing in approximately 100% of progeny, unlike nuclear transgenes, which appear in a proportion of segregating populations. Different unfitness transgenes and modes of propagation will be appropriate for different cases: (1) outcrossing Amaranthus spp. (that evolved resistances to major herbicides); (2) Lolium spp., important pasture grasses, yet herbicide-resistant weeds in crops; (3) rice (Oryza sativa), often infested with feral weedy rice, which interbreeds with the crop; and (4) self-compatible sorghum (Sorghum bicolor), which readily crosses with conspecific shattercane and with allotetraploid johnsongrass (Sorghum halepense). The speculated outcome of these scenarios is to generate weed populations that contain the unfitness gene and thus are easily controllable. Unfitness genes can be under chemically or environmentally inducible promoters, activated after gene dissemination, or under constitutive promoters where the gene function is utilized only at special times (e.g. sensitivity to an herbicide). The transposons can be vectored to the weeds by introgression from the crop (in rice, sorghum, and Lolium spp.) or from planted engineered weed (Amaranthus spp.) using a gene conferring the degradation of a no longer widely used herbicide, especially in tandem with an herbicide-resistant gene that kills all nonhybrids, facilitating the rapid dissemination of the multicopy transposons in a weedy population.     

Rumen Methanogenic Genotypes Differ in Abundance According to Host Residual Feed Intake Phenotype and Diet Type

Methane is an undesirable end product of rumen fermentative activity because of associated environmental impacts and reduced host feed efficiency. Our study characterized the rumen microbial methanogenic community in beef cattle divergently selected for phenotypic residual feed intake (RFI) while offered a high-forage (HF) diet followed by a low-forage (LF) diet. Rumen fluid was collected from 14 high-RFI (HRFI) and 14 low-RFI (LRFI) animals at the end of both dietary periods. 16S rRNA gene clone libraries were used, and methanogen-specific tag-encoded pyrosequencing was carried out on the samples. We found that Methanobrevibacter spp. are the dominant methanogens in the rumen, with Methanobrevibacter smithii being the most abundant species. Differences in the abundance of Methanobrevibacter smithii and Methanosphaera stadtmanae genotypes were detected in the rumen of animals offered the LF compared to the HF diet while the abundance of Methanobrevibacter smithii genotypes was different between HRFI and LRFI animals irrespective of diet. Our results demonstrate that while a core group of methanogen operational taxonomic units (OTUs) exist across diet and phenotype, significant differences were observed in the distribution of genotypes within those OTUs. These changes in genotype abundance may contribute to the observed differences in methane emissions between efficient and inefficient animals.     

Redesign of a WW domain peptide for selective recognition of single-stranded DNA

A β-sheet miniprotein based on the FBP11 WW1 domain sequence has been redesigned for the molecular recognition of ssDNA. A previous report showed that a β-hairpin peptide dimer, (WKWK)(2), binds ssDNA with low micromolar affinity but with little selectivity over duplex DNA. This report extends those studies to a three-stranded β-sheet miniprotein designed to mimic the OB-fold. The new peptide binds ssDNA with low micromolar affinity and shows about 10-fold selectivity for ssDNA over duplex DNA. The redesigned peptide no longer binds its native ligand, the polyproline helix, confirming that the peptide has been redesigned for the function of binding ssDNA. Structural studies provide evidence that this peptide consists of a well-structured β-hairpin made of strands 2 and 3 with a less structured first strand that provides affinity for ssDNA but does not improve the stability of the full peptide. These studies provide insight into protein-DNA interactions as well as a novel example of protein redesign.