Short, interspersed, and repetitive DNA sequences in Spiroplasma species

Small fragments of DNA from an 8-kbp plasmid, pRA1, from a plant pathogenic strain of Spiroplasma citri were shown previously to be present in the chromosomal DNA of at least two species of Spiroplasma. We describe here the shot-gun cloning of chromosomal DNA from S. citri Maroc and the identification of two distinct sequences exhibiting homology to pRA1. Further subcloning experiments provided specific molecular probes for the identification of these two sequences in chromosomal DNA from three distinct plant pathogenic species of Spiroplasma. The results of Southern blot hybridization indicated that each of the pRA1-associated sequences is present as multiple copies in short, dispersed, and repetitive sequences in the chromosomes of these three strains. None of the sequences was detectable in chromosomal DNA from an additional nine Spiroplasma strains examined.     

A format for phylogenetic placements

We have developed a unified format for phylogenetic placements, that is, mappings of environmental sequence data (e.g., short reads) into a phylogenetic tree. We are motivated to do so by the growing number of tools for computing and post-processing phylogenetic placements, and the lack of an established standard for storing them. The format is lightweight, versatile, extensible, and is based on the JSON format, which can be parsed by most modern programming languages. Our format is already implemented in several tools for computing and post-processing parsimony- and likelihood-based phylogenetic placements and has worked well in practice. We believe that establishing a standard format for analyzing read placements at this early stage will lead to a more efficient development of powerful and portable post-analysis tools for the growing applications of phylogenetic placement.     

Habitat fragmentation,genetic diversity,and inbreeding depression in a threatened grassland legume: is genetic rescue necessary?

Kincaid’s lupine (Lupinus oreganus), a threatened perennial legume of western Oregon grasslands, is composed of small, fragmented populations that have consistently low natural seed set, suggesting they may have accumulated high enough levels of genetic load to be candidates for genetic rescue. We used simple sequence repeat (SSR) loci, both nuclear DNA and chloroplast DNA, to screen populations throughout the species’ range for evidence of severe inbreeding and recent genetic bottlenecks due to habitat fragmentation. After genotyping about 40% of the known populations, only one of 24 populations had strong statistical evidence for a recent genetic bottleneck (H _e > H _eq). Both mean nSSR fixation coefficients and genetic diversity did not statistically differ between very small, small, medium, and large lupine population size classes. Within population chloroplast DNA haplotype number was high for an animal pollinated species, ≈4.2 haplotypes/population, and within population haplotype diversity was also relatively evenly distributed. Within population patterns of nSSR and cpSSR genetic diversity suggest that genetic diversity has not been lost over the last century of habitat fragmentation. With genet lifespan thought to exceed 100 years, overlap of several to many generations, and substantial reductions in seed set from inbreeding depression that shifts cohort composition towards those generated by outcrossing events, Kincaid’s lupine is likely maintain the currently high levels of within population genetic diversity. The case of Kincaid’s lupine provides an example of how the assumptions of severe inbreeding depression with small population size and habitat fragmentation can be inaccurate.     

A Tyrosine-based Motif Localizes a Drosophila Vesicular Transporter to Synaptic Vesicles in Vivo

Vesicular neurotransmitter transporters must localize to synaptic vesicles (SVs) to allow regulated neurotransmitter release at the synapse. However, the signals required to localize vesicular proteins to SVs in vivo remain unclear. To address this question we have tested the effects of mutating proposed trafficking domains in Drosophila orthologs of the vesicular monoamine and glutamate transporters, DVMAT-A and DVGLUT. We show that a tyrosine-based motif (YXXY) is important both for DVMAT-A internalization from the cell surface in vitro, and localization to SVs in vivo. In contrast, DVGLUT deletion mutants that lack a putative C-terminal trafficking domain show more modest defects in both internalization in vitro and trafficking to SVs in vivo. Our data show for the first time that mutation of a specific trafficking motif can disrupt localization to SVs in vivo and suggest possible differences in the sorting of VMATs versus VGLUTs to SVs at the synapse.     

Vascular smooth muscle Notch signals regulate endothelial cell sensitivity to angiogenic stimulation

The evolutionarily conserved Notch signaling pathway is required for normal vascular development and function, and genetic associations link select Notch receptors and ligands to human clinical syndromes featuring blood vessel abnormalities and stroke susceptibility. A previously described mouse model engineered to suppress canonical Notch signaling in vascular smooth muscle cells (vSMCs) revealed surprising anatomical defects in arterial patterning and vessel maturation, suggesting that vSMCs have the functional capacity to influence blood vessel formation in a Notch signaling-dependent manner. In further analyses using this model system, we now show that explanted aortic ring tissue and Matrigel implants from the smooth muscle Notch signaling-deficient mice yield markedly diminished responses to angiogenic stimuli. Furthermore, cultured Notch signaling-deficient primary vSMCs have reduced proliferation and migration capacities and reveal diminished expression of PDGF receptor β and JAGGED1 ligand. These observations prompted a series of endothelial cell (EC)-vSMC co-culture experiments that revealed a requirement for intact vSMC Notch signals via JAGGED1 for efficient EC Notch1 receptor activation and EC proliferation. Taken together, these studies suggest a heterotypic model wherein Notch signaling in vSMCs provides early instructive cues to neighboring ECs important for optimal postnatal angiogenesis.     

Spread and persistence of a rugulosin-producing endophyte in Picea glauca seedlings

We have studied Picea glauca (white spruce) endophyte colonization and its affect on the growth of Choristoneura fumiferana (spruce budworm). Here we examine the spread and persistence of a rugulosin-producing endophyte and rugulosin in needles from trees maintained in the nursery, as well as in trees planted in a test field site. Additionally, we report toxicity of rugulosin against three P. glauca needle herbivores: C. fumiferana, Lambdina fiscellaria (hemlock looper) and Zeiraphera canadensis (spruce budmoth). Reduction in body weight for both the C. fumiferana and L. fiscellaria were observed at 25 and 50 μm, respectively, and head capsules were reduced at 100 and 150 μm. Z. canadensis larvae did not perform as well in tests due to an Aspergillus fumigatus infection, but were shown to be lighter when tested with 100 and 150 μm compared with controls. The endophyte and its toxin were shown to spread throughout the nursery-grown seedlings. After 3.5 and 4.5 y post-inoculation (one and two years in the test site), the inoculated endophyte and its toxin had remained present with an average rugulosin concentration of 1 μg g⁻¹.     

Focus: Personalized Medicine: Simultaneously Detection of 50 Mutations at 20 Sites in the BRAF and RAS Genes by Multiplexed Single-Nucleotide Primer Extension Assay Using Fine-Needle Aspirates of Thyroid Nodules

Fine-needle aspiration (FNA) is commonly used for primary evaluation of thyroid nodules. Twenty to 30 percent of thyroid nodules remain indeterminate after FNA evaluation. Studies show the BRAF p.V600E to be highly specific for papillary thyroid carcinoma (PTC), while RAS mutations carry up to 88 percent positive predictive value for malignancy. We developed a two-tube multiplexed PCR assay followed by single-nucleotide primer extension assay for simultaneous detection of 50 mutations in the BRAF (p.V600E, p.K601E/Q) and RAS genes (KRAS and NRAS codons 12, 13, 19, 61 and HRAS 61) using FNA smears of thyroid nodules. Forty-two FNAs and 27 paired formalin-fixed, paraffin-embedded (FFPE) tissues were tested. All BRAF p.V600E-positive FNA smears (five) carried a final diagnosis of PTC on resection. RAS mutations were found in benign as well as malignant lesions. Ninety-two percent concordance was observed between FNA and FFPE tissues. In conclusion, our assay is sensitive and reliable for simultaneous detection of multiple BRAF/RAS mutations in FNA smears of thyroid nodules.     

Cytosolic prion protein is not toxic and protects against Bax-mediated cell death in human primary neurons

Recently, it was observed that reverse-translocated cytosolic PrP and PrP expressed in the cytosol induce rapid death in neurons (Ma, J., Wollmann, R., and Lindquist, S. (2002) Science 298, 1781-1785). In this study, we investigated whether accumulation of prion protein (PrP) in the cytosol is toxic to human neurons in primary culture. We show that in these neurons, a single PrP isoform lacking signal peptide accumulates in the cytosol of neurons treated with epoxomicin, a specific proteasome inhibitor. Therefore, endogenously expressed PrP is subject to the endoplasmic reticulum-associated degradation (ERAD) pathway and is degraded by the proteasome in human primary neurons. In contrast to its toxicity in N2a cells, reverse-translocated PrP (ERAD-PrP) is not toxic even when neurons are microinjected with cDNA constructs to overexpress either wild-type PrP or mutant PrPD178N. We found that ERAD-PrP in human neurons remains detergent-soluble and proteinase K-sensitive, in contrast to its detergent-insoluble and proteinase K-resistant state in N2a cells. Furthermore, not only is microinjection of a cDNA construct expressing CyPrP not toxic, it protects these neurons against Bax-mediated cell death. We conclude that in human neurons, ERAD-PrP is not converted naturally into a form reminiscent of scrapie PrP and that PrP located in the cytosol retains its protective function against Bax. Thus, it is unlikely that simple accumulation of PrP in the cytosol can cause neurodegeneration in prion diseases.     

Slit protein-mediated inhibition of CXCR4-induced chemotactic and chemoinvasive signaling pathways in breast cancer cells

Slit, which mediates its function by binding to the Roundabout (Robo) receptor, has been shown to regulate neuronal and CXCR4-mediated leukocyte migration. Slit-2 was shown to be frequently inactivated in lung and breast cancers because of hypermethylation of its promoter region. Furthermore, the CXCR4/CXCL12 axis has been reported recently to be actively involved in breast cancer metastasis to target organs such as lymph nodes, lung, and bone. In this study, we sought to characterize the effect of Slit (=Slit-2) on the CXCL12/CXCR4-mediated metastatic properties of breast cancer cells. We demonstrate here that breast cancer cells and tissues derived from breast cancer patients express Robo 1 and 2 receptors. We also show that Slit treatment inhibits CXCL12/CXCR4-induced breast cancer cell chemotaxis, chemoinvasion, and adhesion, the fundamental components that promote metastasis. Slit had no significant effect on the CXCL12-induced internalization process of CXCR4. In addition, characterization of signaling events revealed that Slit inhibits CXCL12-induced tyrosine phosphorylation of focal adhesion components such as RAFTK/Pyk2 at residues 580 and 881, focal adhesion kinase at residue 576, and paxillin. We found that Slit also inhibits CXCL12-induced phosphatidylinositol 3-kinase, p44/42 MAP kinase, and metalloproteinase 2 and 9 activities. However, it showed no effect on JNK and p38 MAP kinase activities. To our knowledge, this is the first report to analyze in detail the effect of Slit on breast cancer cell motility as well as its effect on the critical components of the cancer cell chemotactic machinery. Studies of the Slit-Robo complex may foster new anti-chemotactic approaches to block cancer cell metastasis.