A series of vectors for fungal transformation

We report a new fungal selectable marker that confers resistance to chlorimuron ethyl, a sulfonylurea herbicide. This gene as well as genes that confer resistance to hygromycin and bialaphos have been engineered to be compact and to eliminate sites for most common restriction enzymes. These three selectable markers have been used to construct a series of vectors for fungal transformation

A Nonlinear Delay Model for Metabolic Oscillations in Yeast Cells

We introduce two time-delay models of metabolic oscillations in yeast cells. Our model tests a hypothesis that the oscillations occur as multiple pathways share a limited resource which we equate to the number of available ribosomes. We initially explore a single-protein model with a constraint equation governing the total resource available to the cell. The model is then extended to include three proteins that share a resource pool. Three approaches are considered at constant delay to numerically detect oscillations. First, we use a spectral element method to approximate the system as a discrete map and evaluate the stability of the linearized system about its equilibria by examining its eigenvalues. For the second method, we plot amplitudes of the simulation trajectories in 2D projections of the parameter space. We use a history function that is consistent with published experimental results to obtain metabolic oscillations. Finally, the spectral element method is used to convert the system to a boundary value problem whose solutions correspond to approximate periodic solutions of the system. Our results show that certain combinations of total resource available and the time delay, lead to oscillations. We observe that an oscillation region in the parameter space is between regions admitting steady states that correspond to zero and constant production. Similar behavior is found with the three-protein model where all proteins require the same production time. However, a shift in the protein production rates peaks occurs for low available resource suggesting that our model captures the shared resource pool dynamics.Comment: 22 pages, 14 figures. Added analytical characterization of the trivial equilibrium point. For the three-protein model, we now only consider results where the protein production times are equal and added corresponding linearized stability diagrams for this system. We also changed the history functions for both systems to be consistent with experimental results for obtaining metabolic oscillation

A miniprep procedure for isolating genomic DNA from Magnoporthe grisea

We have developed a simple miniprep procedure for the isolation of genomic DNA from the ascomycete Magnaporthe grisea. This pathogen of many grasses, including rice, has a moderate growth rate and produces intermediate to low numbers of conidia when grown in culture. Thus, in our previous DNA preparation procedure we inoculated swirling liquid cultures with mycelium that had been fragmented in a blender rather than with conidia. The mycelium obtained from these cultures was ground in liquid nitrogen for DNA extraction. Though the quantity and quality of DNA obtained by this method is satisfactory, the technique is too laborious for analysis of many strains. We developed the procedure described below to eliminate the need to fragment mycelium in a blender to inoculate cultures and to eliminate the need to grind mycelium in liquid nitrogen for DNA extraction. The new procedure, which relies on the enzymatic removal of cell walls and the lysis of protoplasts, should be readily adaptable to other filamentous fungi with growth characteristics similar to those of M. grisea

Random billiards with wall temperature and associated Markov chains

By a random billiard we mean a billiard system in which the standard specular reflection rule is replaced with a Markov transition probabilities operator P that, at each collision of the billiard particle with the boundary of the billiard domain, gives the probability distribution of the post-collision velocity for a given pre-collision velocity. A random billiard with microstructure (RBM) is a random billiard for which P is derived from a choice of geometric/mechanical structure on the boundary of the billiard domain. RBMs provide simple and explicit mechanical models of particle-surface interaction that can incorporate thermal effects and permit a detailed study of thermostatic action from the perspective of the standard theory of Markov chains on general state spaces. We focus on the operator P itself and how it relates to the mechanical/geometric features of the microstructure, such as mass ratios, curvatures, and potentials. The main results are as follows: (1) we characterize the stationary probabilities (equilibrium states) of P and show how standard equilibrium distributions studied in classical statistical mechanics, such as the Maxwell-Boltzmann distribution and the Knudsen cosine law, arise naturally as generalized invariant billiard measures; (2) we obtain some basic functional theoretic properties of P. Under very general conditions, we show that P is a self-adjoint operator of norm 1 on an appropriate Hilbert space. In a simple but illustrative example, we show that P is a compact (Hilbert-Schmidt) operator. This leads to the issue of relating the spectrum of eigenvalues of P to the features of the microstructure;(3) we explore the latter issue both analytically and numerically in a few representative examples;(4) we present a general algorithm for simulating these Markov chains based on a geometric description of the invariant volumes of classical statistical mechanics

Ephrin-B2 reverse signaling is required for axon pathfinding and cardiac valve formation but not early vascular development

AbstractVascular development begins with the formation of a primary vascular plexus that is rapidly remodeled by angiogenesis into the interconnected branched patterns characteristic of mature vasculature. Several receptor tyrosine kinases and their ligands have been implicated to control early development of the vascular system. These include the vascular endothelial growth factor receptors (VEGFR-1 and VEGFR-2) that bind VEGF, the Tie-1 and Tie-2 receptors that bind the angiopoietins, and the EphB4 receptor that binds the membrane-anchored ligand ephrin-B2. Targeted mutations in the mouse germline have revealed essential functions for these molecules in vascular development. In particular, protein-null mutations that delete either EphB4 or ephrin-B2 from the mouse have been shown to result in early embryonic lethality due to failed angiogenic remodeling. The venous expression of EphB4 and arterial expression of ephrin-B2 has lead to the speculation that the interaction of these two molecules leads to bidirectional signaling into both the receptor-expressing cell and the ligand-expressing cell, and that both forward and reverse signals are required for proper development of blood vessels in the embryo. Indeed, targeted removal of the ephrin-B2 carboxy-terminal cytoplasmic tail by another group was shown to perturb vascular development and result in the same early embryonic lethality as the null mutation, leading the authors to propose that ephrin-B2 reverse signaling directs early angiogenic remodeling of the primary vascular plexus [Cell 104 (2001) 57]. However, we show here that the carboxy-terminal cytoplasmic domain of ephrin-B2, and hence reverse signaling, is not required during early vascular development, but it is necessary for neonatal survival and functions later in cardiovascular development in the maturation of cardiac valve leaflets. We further show that ephrin-B2 reverse signaling is required for the pathfinding of axons that form the posterior tract of the anterior commissure. Our results thus indicate that ephrin-B2 functions in the early embryo as a typical instructive ligand to stimulate EphB4 receptor forward signaling during angiogenic remodeling and that later in embryonic development ephrin-B2 functions as a receptor to transduce reverse signals involved in cardiac valve maturation and axon pathfinding

Comparative chloroplast genomics: analyses including new sequences from the angiosperms Nuphar advena and Ranunculus macranthus

<p>Abstract</p> <p>Background</p> <p>The number of completely sequenced plastid genomes available is growing rapidly. This array of sequences presents new opportunities to perform comparative analyses. In comparative studies, it is often useful to compare across wide phylogenetic spans and, within angiosperms, to include representatives from basally diverging lineages such as the genomes reported here: <it>Nuphar advena </it>(from a basal-most lineage) and <it>Ranunculus macranthus </it>(a basal eudicot). We report these two new plastid genome sequences and make comparisons (within angiosperms, seed plants, or all photosynthetic lineages) to evaluate features such as the status of <it>ycf15 </it>and <it>ycf68 </it>as protein coding genes, the distribution of simple sequence repeats (SSRs) and longer dispersed repeats (SDR), and patterns of nucleotide composition.</p> <p>Results</p> <p>The <it>Nuphar </it>[GenBank:<ext-link ext-link-type="gen" ext-link-id="NC_008788">NC_008788</ext-link>] and <it>Ranunculus </it>[GenBank:<ext-link ext-link-type="gen" ext-link-id="NC_008796">NC_008796</ext-link>] plastid genomes share characteristics of gene content and organization with many other chloroplast genomes. Like other plastid genomes, these genomes are A+T-rich, except for rRNA and tRNA genes. Detailed comparisons of <it>Nuphar </it>with <it>Nymphaea</it>, another Nymphaeaceae, show that more than two-thirds of these genomes exhibit at least 95% sequence identity and that most SSRs are shared. In broader comparisons, SSRs vary among genomes in terms of abundance and length and most contain repeat motifs based on A and T nucleotides.</p> <p>Conclusion</p> <p>SSR and SDR abundance varies by genome and, for SSRs, is proportional to genome size. Long SDRs are rare in the genomes assessed. SSRs occur less frequently than predicted and, although the majority of the repeat motifs do include A and T nucleotides, the A+T bias in SSRs is less than that predicted from the underlying genomic nucleotide composition. In codon usage third positions show an A+T bias, however variation in codon usage does not correlate with differences in A+T-richness. Thus, although plastome nucleotide composition shows "A+T richness", an A+T bias is not apparent upon more in-depth analysis, at least in these aspects. The pattern of evolution in the sequences identified as <it>ycf15 </it>and <it>ycf68 </it>is not consistent with them being protein-coding genes. In fact, these regions show no evidence of sequence conservation beyond what is normal for non-coding regions of the IR.</p

Internet-based medical education: a realist review of what works, for whom and in what circumstances

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Methods for Obtaining and Analyzing Whole Chloroplast Genome Sequences

During the past decade there has been a rapid increase in our understanding of plastid genome organization and evolution due to the availability of many new completely sequenced genomes. Currently there are 43 complete genomes published and ongoing projects are likely to increase this sampling to nearly 200 genomes during the next five years. Several groups of researchers including ours have been developing new techniques for gathering and analyzing entire plastid genome sequences and details of these developments are summarized in this chapter. The most important recent developments that enhance our ability to generate whole chloroplast genome sequences involve the generation of pure fractions of chloroplast genomes by whole genome amplification using rolling circular amplification, cloning genomes into Fosmid or BAC vectors, and the development of an organellar annotation program (DOGMA). In addition to providing details of these methods, we provide an overview of methods for analyzing complete plastid genome sequences for repeats and gene content, as well as approaches for using gene order and sequence data for phylogeny reconstruction. This explosive increase in the number of sequenced plastid genomes and improved computational tools will provide many insights into the evolution of these genomes and much new data for assessing relationships at deep nodes in plants and other photosynthetic organisms

Doctors and nurses benefit from interprofessional online education in dermatology

<p>Abstract</p> <p>Background</p> <p>Benefits of online learning in the health sector have been demonstrated in previous studies. We examined the potential benefits of a joint web-based curriculum on atopic eczema for health personnel.</p> <p>Methods</p> <p>Enrolled doctors and nurses had access to the curriculum for 8 weeks. After the course learners completed a questionnaire. Two dermatologists rated the quality of the submitted homework assignments. Based on data from the project's budget and the Norwegian Medical Association, we estimated the saved travel expenses.</p> <p>Results</p> <p>Eighty-eight learners (46 doctors) registered for the course. We received 55 questionnaires (response rate 63%). Twenty-seven learners (31%; 16 doctors, 11 nurses; χ²= 0.03; P = 0.87) used the discussion forum. We found no significant differences in the total questionnaire scores between doctors and nurses. The homework assignments were given an average score of 3.6 for doctors and 3.5 for nurses (P = 0.8) by rater 1. Rater 2 scored 3.9 and 3.6 for doctors and nurses respectively (P = 0.2). The break-even between travel/hotel expenses and course development costs occurred at 135 saved travel refund applications.</p> <p>Conclusions</p> <p>Doctors and nurses were equally satisfied with a joint web-based course on atopic eczema. The use of an online discussion forum was limited but similar between doctors and nurses. There were no significant differences in the quality of submitted homework assignments. The cost of developing the course was 716 841 NOK and the first 86 learners saved 455 198 NOK in travel expenses.</p