Endemic dengue associated with the co-circulation of multiple viral lineages and localized density-dependent transmission

Dengue is one of the most important infectious diseases of humans and has spread throughout much of the tropical and subtropical world. Despite this widespread dispersal, the determinants of dengue transmission in endemic populations are not well understood, although essential for virus control. To address this issue we performed a phylogeographic analysis of 751 complete genome sequences of dengue 1 virus (DENV-1) sampled from both rural (Dong Thap) and urban (Ho Chi Minh City) populations in southern Viet Nam during the period 2003-2008. We show that DENV-1 in Viet Nam exhibits strong spatial clustering, with likely importation from Cambodia on multiple occasions. Notably, multiple lineages of DENV-1 co-circulated in Ho Chi Minh City. That these lineages emerged at approximately the same time and dispersed over similar spatial regions suggests that they are of broadly equivalent fitness. We also observed an important relationship between the density of the human host population and the dispersion rate of dengue, such that DENV-1 tends to move from urban to rural populations, and that densely populated regions within Ho Chi Minh City act as major transmission foci. Despite these fluid dynamics, the dispersion rates of DENV-1 are relatively low, particularly in Ho Chi Minh City where the virus moves less than an average of 20 km/year. These low rates suggest a major role for mosquito-mediated dispersal, such that DENV-1 does not need to move great distances to infect a new host when there are abundant susceptibles, and imply that control measures should be directed toward the most densely populated urban environments.     

Gaps in the canopy: the missing dimension in vegetation dynamics

Canopy gaps are important as entry points for new genotypes and new species into many types of vegetation, yet little is known about them in any type of vegetation but forests. Forest gaps are too large for manipulative experiments to be readily undertaken, and hitherto grassland gaps have been too small to be easily mapped. Preliminary results from mapping small (>1 cm) grassland gaps with a new fibre-optic device suggest that experiments need to be performed at a smaller physical scale than has hitherto been achieved.     

Identification and characterization of presenilin-independent Notch signaling.

Presenilin (PS) proteins control the proteolytic cleavage that precedes nuclear access of the Notch intracellular domain. Here we observe that a partial activation of the HES1 promoter can be detected in PS1/PS2 (PS1/2) double null cells using Notch1 Delta E constructs or following Delta 1 stimulation, despite an apparent abolition of the production and nuclear accumulation of the Notch intracellular domain. PS1/2-independent Notch activation is sensitive to Numblike, a physiological inhibitor of Notch. PS1/2-independent Notch signaling is also inhibited by an active gamma-secretase inhibitor in the low micromolar range and is not inhibited by an inactive analogue, similar to PS-dependent Notch signaling. However, experiments using a Notch1-Gal4-VP16 fusion protein indicate that the PS1/2-independent activity does not release Gal4-VP16 and is therefore unlikely to proceed via an intramembranous cleavage. These data reveal that a novel PS1/2-independent mechanism plays a partial role in Notch signal transduction.     

Enolase activates homotypic vacuole fusion and protein transport to the vacuole in yeast

Membrane fusion and protein trafficking to the vacuole are complex processes involving many proteins and lipids. Cytosol from Saccharomyces cerevisiae contains a high Mr activity, which stimulates the in vitro homotypic fusion of isolated yeast vacuoles. Here we purify this activity and identify it as enolase (Eno1p and Eno2p). Enolase is a cytosolic glycolytic enzyme, but a small portion of enolase is bound to vacuoles. Recombinant Eno1p or Eno2p stimulates in vitro vacuole fusion, as does a catalytically inactive mutant enolase, suggesting a role for enolase in fusion that is separate from its glycolytic function. Either deletion of the non-essential ENO1 gene or diminished expression of the essential ENO2 gene causes vacuole fragmentation in vivo, reflecting reduced fusion. Combining an ENO1 deletion with ENO2-deficient expression causes a more severe fragmentation phenotype. Vacuoles from enolase 1 and 2-deficient cells are unable to fuse in vitro. Immunoblots of vacuoles from wild type and mutant strains reveal that enolase deficiency also prevents normal protein sorting to the vacuole, exacerbating the fusion defect. Band 3 has been shown to bind glycolytic enzymes to membranes of mammalian erythrocytes. Bor1p, the yeast band 3 homolog, localizes to the vacuole. Its loss results in the mislocalization of enolase and other vacuole fusion proteins. These studies show that enolase stimulates vacuole fusion and that enolase and Bor1p regulate selective protein trafficking to the vacuole.     

Does contamination buildup limit throughput for automated cryoEM?

The development of automated systems for data acquisition in cryo electron microscopy has enabled the possibility of acquiring very large number of images from a single specimen grid. We have demonstrated that over images of 250,000 single particles can be acquired in a 24 h period. This has raised questions as to whether contamination buildup on the specimen limits the quality of the data that can be acquired during these long duration experiments and also whether the data acquisition session could be extended to allow acquisition of more than 1,000,000 particles. We report here a systematic characterization of contamination of specimens maintained for long periods of time at liquid nitrogen temperatures using standard side entry cryo stages. As part of this characterization we developed a more reliable method for accurately estimating specimen ice thickness. Using the method, we were able to calibrate image contrast against ice thickness under a variety of magnifications, objective aperture positions, and defoci, and demonstrated the strong dependence of the calibration curve on these parameters. The results show the anti-contamination aperture is, as expected, critical to the prevention of contamination and that loading film into the microscope dramatically increases the contamination rate, particularly in the first 3 h after the insertion of the film box. In the absence of film, we were able to reproducibly demonstrate that the contamination rate can be limited to a rate of approximately 1 angstrom/h providing reassurance that contamination will not be a major limiting factor for long term cryoEM experiments if a CCD camera is used for the imaging.     

Molecular screening of interleukin-6 gene promoter and influence of −174G/C polymorphism on breast cancer

Interleukin-6 (IL-6) is a cytokine involved in different physiologic and pathophysiologic processes including carcinogenesis. In 2003, a single nucleotide polymorphism (−174G/C) of the IL-6 gene promoter has been linked to breast cancer prognosis in node-positive (N+) breast cancer patients. Since, different studies have led to conflicting conclusions about its role as a prognostic and/or diagnostic marker. The primary aim of our study was to investigate the link between −174G/C polymorphism and breast cancer risk on the one hand, and −174G/C polymorphism and prognosis in different groups of patients: sporadic N+ breast cancers (n = 138), sporadic N− breast cancers (n = 95) and familial breast cancer (n = 60) on the other hand. The variables of interest were disease-free survival and overall survival. The secondary aim of the study was to screen IL-6 gene promoter using direct sequencing to identify new polymorphisms in our French Caucasian breast cancer population. No association or trend of association between −174G/C polymorphism of IL-6 gene promoter gene and breast cancer diagnosis or prognosis was shown, even in meta-analyses. Furthermore, we have identified four novel polymorphic sites in the IL-6 gene promoter region: −764G → A, −757C → T, −233T → A, 15C → A.     

Adhesion,proliferation, and adipogenesis in primary rat cell cultures: effects of collagenous substrata,fibronectin, and serum

Summary The effects of collagenous substrata, fibronectin, and fetal bovine serum on the adhesion, proliferation, and adipogenesis of rat stromal-vascular cells are reported. There was no effect on initial stromal-vascular cell-attachment by fetal bovine serum or fibronectin. The number of cells attached to a hydrated collagen-gel was almost twice (P<0.04) the number attached to dried collagen-gel or dried denatured collagen-gel. Total number of cells after 5 days in culture was similar among the collagenous substrata and among the treatments with or without fibronectin in the growth media. Total number of cells increased significantly (P<0.02) with 10% FBS. Adipocytic formation was inhibited by hydrated collagen-gel (P<0.02) compared to dried collagen-gel or dried, denatured collagenous substrata. An interaction occurred between dried, denatured gel and fetal bovine serum so that total formation of adipocytes increased by increasing the level of fetal bovine serum (P<0.07). Adipocytic formation was inhibited by hydrated collagen-gel at all levels of fetal bovine serum. The percentage of cells that converted to adipocytes was significantly lower (P<0.01) on hydrated collagen-gel compared to dried, denatured or dried collagen-gel. Percentage of conversion was not significantly different among levels of fetal bovine serum, although this percentage increased as fetal bovine serum level increased. Adipocytic conversion was not different between fibronectin-treated or untreated cells. Morphology of stromal vascular cells was similar on dried collagen and dried, denatured collagen-gel, but tended to remain bipolar on hydrated collagen-gel. These studies indicate that fetal bovine serum in combination with the extracellular matrix (dried, denatured collagen) increased the differentiation of rat stromal-vascular cells into adipocytes, and that hydrated collagen inhibited differentiation.     

Leaf temperature of soybean grown under elevated CO2 increases Aphis glycines (Hemiptera: Aphididae) population growth

Abstract Plants grown under elevated carbon dioxide (CO₂) experience physiological changes that influence their suitability as food for insects. To determine the effects of living on soybean (Glycine max Linnaeus) grown under elevated CO₂, population growth of the soybean aphid (Aphis glycines Matsumura) was determined at the SoyFACE research site at the University of Illinois, Urbana‐Champaign, Illinois, USA, grown under elevated (550 μL/L) and ambient (370 μL/L) levels of CO₂. Growth of aphid populations under elevated CO₂ was significantly greater after 1 week, with populations attaining twice the size of those on plants grown under ambient levels of CO₂. Soybean leaves grown under elevated levels of CO₂ were previously demonstrated at SoyFACE to have increased leaf temperature caused by reduced stomatal conductance. To separate the increased leaf temperature from other effects of elevated CO₂, air temperature was lowered while the CO₂ level was increased, which lowered overall leaf temperatures to those measured for leaves grown under ambient levels of CO₂. Aphid population growth on plants grown under elevated CO₂ and reduced air temperature was not significantly greater than on plants grown under ambient levels of CO₂. By increasing Glycine max leaf temperature, elevated CO₂ may increase populations of Aphis glycines and their impact on crop productivity.     

Rab GTPase regulation of retromer-mediated cargo export during endosome maturation

The retromer complex, composed of sorting nexin subunits and a Vps26/Vps29/Vps35 trimer, mediates sorting of retrograde cargo from the endosome to the trans-Golgi network. The retromer trimer subcomplex is an effector of Rab7 (Ypt7 in yeast). Whereas endosome targeting of human retromer has been shown to require Rab7-GTP, targeting of yeast retromer to the endosome is independent of Ypt7-GTP and requires the Vps5 and Vps17 retromer sorting nexin subunits. An evolutionarily conserved amino acid segment within Vps35 is required for Ypt7/Rab7 recognition in vivo by both yeast and human retromer, establishing that Rab recognition is a conserved feature of this subunit. Recognition of Ypt7 by retromer is required for its function in retrograde sorting, and in yeast cells lacking the guanine nucleotide exchange factor for Ypt7, retrograde cargo accumulates in endosomes that are decorated with retromer, revealing an additional role for Rab recognition at the cargo export stage of the retromer functional cycle. In addition, yeast retromer trimer antagonizes Ypt7-regulated organelle tethering and fusion of endosomes/vacuoles via recognition of Ypt7. Thus retromer has dual roles in retrograde cargo export and in controlling the fusion dynamics of the late endovacuolar system.