The adjuvanticity of a mannosylated antigen reveals TLR4 functionality essential for subset specialization and functional maturation of mouse dendritic cells

The evidence that dendritic cell (DC) subsets produce differential cytokines in response to specific TLR stimulation is robust. However, the role of TLR stimulation in Ag presentation and phenotypic maturation among DC subsets is not clear. Through the adjuvanticity of a novel mannosylated Ag, mannosylated dendrimer OVA (MDO), as a pathogen-associated molecular pattern Ag, we characterized the functionality of GM-CSF/IL-4-cultured bone marrow DC and Flt3 ligand (Flt3-L) DC subsets by Ag presentation and maturation assays. It was demonstrated that both bone marrow DCs and Flt3-L DCs bound, processed, and presented MDO effectively. However, while Flt3-L CD24(high) (conventional CD8(+) equivalent) and CD11b(high) (CD8(-) equivalent) DCs were adept at MDO processing by MHC class I and II pathways, respectively, CD45RA(+) plasmacytoid DCs presented MDO poorly to T cells. Successful MDO presentation was largely dependent on competent TLR4 for Ag localization and morphological/phenotypic maturation of DC subsets, despite the indirect interaction of MDO with TLR4. Furthermore, Toll/IL-1 receptor-domain-containing adaptor-inducing IFN-beta, but not MyD88, as a TLR4 signaling modulator was indispensable for MDO-induced DC maturation and Ag presentation. Taken together, our findings suggest that DC subsets differentially respond to a pathogen-associated molecular pattern-associated Ag depending on the intrinsic programming and TLRs expressed. Optimal functionality of DC subsets in Ag presentation necessitates concomitant TLR signaling critical for efficient Ag localization and processing.     

Attenuation of recombinant vesicular stomatitis virus-human immunodeficiency virus type 1 vaccine vectors by gene translocations and g gene truncation reduces neurovirulence and enhances immunogenicity in mice

Recombinant vesicular stomatitis virus (rVSV) has shown great potential as a new viral vector for vaccination. However, the prototypic rVSV vector described previously was found to be insufficiently attenuated for clinical evaluation when assessed for neurovirulence in nonhuman primates. Here, we describe the attenuation, neurovirulence, and immunogenicity of rVSV vectors expressing human immunodeficiency virus type 1 Gag. These rVSV vectors were attenuated by combinations of the following manipulations: N gene translocations (N4), G gene truncations (CT1 or CT9), noncytopathic M gene mutations (Mncp), and positioning of the gag gene into the first position of the viral genome (gag1). The resulting N4CT1-gag1, N4CT9-gag1, and MncpCT1-gag1 vectors demonstrated dramatically reduced neurovirulence in mice following direct intracranial inoculation. Surprisingly, in spite of a very high level of attenuation, the N4CT1-gag1 and N4CT9-gag1 vectors generated robust Gag-specific immune responses following intramuscular immunization that were equivalent to or greater than immune responses generated by the more virulent prototypic vectors. MncpCT1-gag1 also induced Gag-specific immune responses following intramuscular immunization that were equivalent to immune responses generated by the prototypic rVSV vector. Placement of the gag gene in the first position of the VSV genome was associated with increased in vitro expression of Gag protein, in vivo expression of Gag mRNA, and enhanced immunogenicity of the vector. These findings demonstrate that through directed manipulation of the rVSV genome, vectors that have reduced neurovirulence and enhanced immunogenicity can be made.     

Seedling interactions in a tropical forest in Panama

Competition is believed to be a central force limiting local diversity and controlling the structure of plant communities. However, it has been proposed that the stressed understory environment limits total understory plant density to such low levels that competitive exclusion cannot be an important factor limiting the local diversity of understory plants. To evaluate the importance of inter-seedling competition, we performed a seedling competition experiment with five shade-tolerant species in a tropical moist forest in Panama. Three-month-old seedlings were transplanted into the forest singly or with their roots intertwined with a single conspecific or heterospecific seedling in all pairwise species combinations. If competition is important, performance (survival, stem height, and number of leaves after one and six years) would be expected to be lowest with a conspecific neighbor and greatest without a neighbor. The experiment was replicated in five 0.24-m² plots at each of 20 sites in tall secondary forest. To test whether seedling performance differed among treatments we fitted linear mixed models (LMM) and generalized linear mixed models (GLMM), treating species identity and microsite (site and plot) as random effects. The five shade-tolerant study species all experienced good establishment with relatively high survival and growth rates. The neighbor treatment consistently affected seedling performance, but the effect was always very small, both in absolute terms and relative to the much stronger species and microsite effects. Seedlings with a conspecific neighbor consistently performed worse than seedlings with a heterospecific neighbor, but having no neighbor generally did not cause superior performance relative to the other treatments. We conclude that direct competitive interactions are relatively unimportant among understory plants in humid tropical forests.     

Taphonomic windows and molluscan preservation

Recent studies on silicified fossil biotas have suggested that substantial skewing of the molluscan record resulted from early aragonite dissolution in mid-outer carbonate ramp settings. If those rare skeletal lagerstätten are representative, then the quality and completeness of the molluscan record are thrown into doubt. Yet database studies suggest that the bivalve fossil record is actually relatively complete. If so, then biodiversity must be captured by other processes that preserved shells vulnerable to early dissolution, and which operated on a relatively high frequency, i.e., less than the species duration for bivalves.Storm beds, shell plasters and submarine hardgrounds are identified as fossil deposits that can preserve the labile aragonitic component of the fauna and thus represent potential taphonomic windows. Many storm event beds include rich accumulations of shelly benthos. Differences between storm bed faunas and those of the background facies could reflect transportation effects. However, some storm bed assemblages are rich in originally aragonitic infaunal bivalves that are not represented in background facies or more proximal shelf equivalents, and here rapid burial and removal of organic matter by winnowing may be the keys to aragonite shell preservation. Despite Palaeozoic to Cenozoic changes in the thickness and frequency of shell beds that reflect the predominant bioclast producers, shallow infaunas are commonly concentrated together with epifauna in such deposits.Some low energy, organic-rich mud-dominated settings are associated with preservation of aragonitic molluscs. Infaunal bivalves are a prominent component of shell plasters or pavements in such settings, linked to episodic bottom water anoxia. Decaying algal blooms drew the redox boundary up above the sediment–water interface, and brought populations of infaunal bivalves to the surface where they died. Isolated from the oxic taphonomically active zone, the shells were not dissolved and were buried as thin shell layers. In similar settings, aragonitic shells were preserved as moulds through early pyritisation, or even through preservation of original shell aragonite.In oxic environments, bioturbational reworking of surface sediment destroyed moulds of aragonitic shells after early dissolution. In some hardgrounds, these delicate moulds were preserved due to synsedimentary cementation, probably using carbonate released by aragonite dissolution. The examples included here come from both intervals of “calcite” and “aragonite” seas, and it is not possible to assess whether the saturation state (with respect to aragonite) of the ambient sea water played a role in the selective removal of aragonitic shells.While taphonomic windows may have captured the diversity of individual groups, it is clear from quantitative data involving skeletal lagerstätten that the scale of loss from early aragonite dissolution has drastically altered the trophic composition of some fossil assemblages commonly used as the basis for reconstructions of past communities.     

Solar irradiance level alters the growth of basil (Ocimum basilicum L.) and its content of volatile oils

The experiments were commenced in March 2003 and repeated in June 2003 at Sutton Bonington Campus, the University of Nottingham, UK, to investigate the effect of irradiance on plant growth and volatile oil content and composition in plants of basil. Four levels of irradiance were provided in the glasshouse, i.e. no shade (control), 25, 50 and 75% glasshouse irradiance. It suggested that basil grows well in full sun, however it can tolerate light shade. Heavy shading (75%) to provide a light integral of 5.3 moles m⁻² d⁻¹ resulted in shorter plants, lower weight, smaller leaf area, less shoots and higher specific leaf area, and also strongly reduced the rate of photosynthesis. There was no difference in CO₂ assimilation rate between 24.9 moles m⁻² d⁻¹ light integrals (no shading) and 13.5 moles m⁻² d⁻¹ light integrals (25% shading). Shading effectively reduced leaf temperature when air temperature was less than 30 °C, but heavy shading (75%) could not reduce leaf temperature when air temperature was above 36 °C due to a limitation of free air convection. Consequently, leaf temperature increased. Heavy shading strongly reduced total volatile oil content in fresh leaves, especially in older plants (shading treatment applied at the 3 leaf-pair growth stage). There were three chemical compounds in basil leaves, namely linalool, eugenol and methyl eugenol, influenced by the shading treatments. Linalool and eugenol, which contribute to the characteristic taste of basil, were significantly increased by high daily light integrals, whereas methyleugenol was increased by lower daily light integrals. No differences in the relative content of 1,8-cineole, one of the key aromatic compounds of Ocimum species, were observed.     

A cross-species quantitative proteomic study of salt adaptation in a halotolerant environmental isolate using 15N metabolic labelling

We examine differential protein expression in Euhalothece sp. BAA001, an extremely halotolerant and unsequenced cyanobacterium, under adaptation to low (0% w/v), medium (3% w/v), high (6% w/v) and very high (9% w/v) salt concentrations using cross-species protein identification tools. We combine stable isotope labelling with 1-D SDS-PAGE, and MASCOT protein identification software with MS-driven BLAST searches, to produce an accurate method for protein identification and quantitation. The use of metabolic labelling to improve the confidence in identification of proteins in cross-species proteomics is demonstrated. Three hundred and eighty-three unique proteins were identified, and 72 were deemed to be differentially expressed (average CV for quantitations was 0.10 +/- 0.08), belonging to 24 functional groups. Responses to low salt as well as high salt are discussed in terms of adaptation and evidence shows that Euhalothece cells display 'stress' responses in nonsaline conditions as well as higher salt environments.     

Cystic fibrosis transmembrane regulator missing the first four transmembrane segments increases wild type and DeltaF508 processing

We previously generated an adenoassociated viral gene therapy vector, rAAV-Delta264 cystic fibrosis transmembrane conductance regulator (CFTR), missing the first four transmembrane domains of CFTR. When infected into monkey lungs, Delta264 CFTR increased the levels of endogenous wild type CFTR protein. To understand this process, we transfected Delta264 CFTR plasmid cDNA into COS7 cells, and we noted that protein expression from the truncation mutant is barely detectable when compared with wild type or DeltaF508 CFTR. Delta264 CFTR protein expression increases dramatically when cells are treated with proteasome inhibitors. Cycloheximide experiments show that Delta264 CFTR is degraded faster than DeltaF508 CFTR. VCP and HDAC6, two proteins involved in retrograde translocation from endoplasmic reticulum to cytosol for proteasomal and aggresomal degradation, coimmunoprecipitate with Delta264 CFTR. In cotransfection studies in COS7 cells and in transfection of Delta264 CFTR into cells stably expressing wild type and DeltaF508 CFTR, Delta264 CFTR increases wild type CFTR protein and increases levels of maturation of immature band B to mature band C of DeltaF508 CFTR. Thus the adenoassociated viral vector, rAAV-Delta264 CFTR, is a highly promising cystic fibrosis gene therapy vector because it increases the amount of mature band C protein both from wild type and DeltaF508 CFTR and associates with key elements in quality control mechanism of CFTR.