Sequence-Specific Fidelity Alterations Associated with West Nile Virus Attenuation in Mosquitoes

High rates of error-prone replication result in the rapid accumulation of genetic diversity of RNA viruses. Recent studies suggest that mutation rates are selected for optimal viral fitness and that modest variations in replicase fidelity may be associated with viral attenuation. Arthropod-borne viruses (arboviruses) are unique in their requirement for host cycling and may necessitate substantial genetic and phenotypic plasticity. In order to more thoroughly investigate the correlates, mechanisms and consequences of arbovirus fidelity, we selected fidelity variants of West Nile virus (WNV; Flaviviridae, Flavivirus) utilizing selection in the presence of a mutagen. We identified two mutations in the WNV RNA-dependent RNA polymerase associated with increased fidelity, V793I and G806R, and a single mutation in the WNV methyltransferase, T248I, associated with decreased fidelity. Both deep-sequencing and in vitro biochemical assays confirmed strain-specific differences in both fidelity and mutational bias. WNV fidelity variants demonstrated host-specific alterations to replicative fitness in vitro, with modest attenuation in mosquito but not vertebrate cell culture. Experimental infections of colonized and field populations of Cx. quinquefaciatus demonstrated that WNV fidelity alterations are associated with a significantly impaired capacity to establish viable infections in mosquitoes. Taken together, these studies (i) demonstrate the importance of allosteric interactions in regulating mutation rates, (ii) establish that mutational spectra can be both sequence and strain-dependent, and (iii) display the profound phenotypic consequences associated with altered replication complex function of flaviviruses.     

Identification of PDCL3 as a Novel Chaperone Protein Involved in the Generation of Functional VEGF Receptor 2

Angiogenesis, a hallmark step in tumor metastasis and ocular neovascularization, is driven primarily by the function of VEGF ligand on one of its receptors, VEGF receptor 2 (VEGFR-2). Central to the proliferation and ensuing angiogenesis of endothelial cells, the abundance of VEGFR-2 on the surface of endothelial cells is essential for VEGF to recognize and activate VEGFR-2. We have identified phosducin-like 3 (PDCL3, also known as PhLP2A), through a yeast two-hybrid system, as a novel protein involved in the stabilization of VEGFR-2 by serving as a chaperone. PDCL3 binds to the juxtamembrane domain of VEGFR-2 and controls the abundance of VEGFR-2 by inhibiting its ubiquitination and degradation. PDCL3 increases VEGF-induced tyrosine phosphorylation and is required for VEGFR-2-dependent endothelial capillary tube formation and proliferation. Taken together, our data provide strong evidence for the role of PDCL3 in angiogenesis and establishes the molecular mechanism by which it regulates VEGFR-2 expression and function.     

Bimodal evolution of the killer cell Ig-like receptor (KIR) family in New World primates

The immunoglobulin-like receptor (KIR) gene family in New World primates (Platyrrhini) has been characterized only in the owl monkey (Aotus sp.). To gain a better understanding of the KIR system in Platyrrhini, we analyzed a KIR haplotype in Ateles geoffroyi, and sequenced KIR complementary DNAs (cDNAs) from other three Atelidae species, Ateles hybridus, Ateles belzebuth, and Lagothrix lagotricha. Atelidae expressed a variable set of activating and inhibitory KIRs that diversified independently from their Catarrhini counterparts. They had a unique mechanism to generate activating receptors from inhibitory ones, involving a single nucleotide deletion in exon 7 and a change in the donor splice site of intron 7. The A. geoffroyi haplotype contained at least six gene models including a pseudogene, two coding inhibitory receptors, and three coding activating receptors. The centromeric region was in a tail-to-tail orientation with respect to the telomeric region. The owl monkey KIR haplotype shared this organization, and in phylogenetic trees, the centromeric genes clustered together with those of A. geoffroyi, whereas their telomeric genes clustered independently. KIR cDNAs from the other Atelidae species conformed to this pattern. Signatures of positive selection were found in residues predicted to interact with the major histocompatibility complex. Such signatures, however, primarily explained variability between paralogous genes but not between alleles in a locus. Atelidae, therefore, has expanded the KIR family in a bimodal fashion, where an inverted centromeric region has remained relatively conserved and the telomeric region has diversified by a rapid process of gene duplication and divergence, likely favored by positive selection for ligand binding.     

Effect of a High‐fat Diet on 24‐Hour Pattern of Circulating Adipocytokines in Rats

We have shown a significant disruption of 24‐h pattern of plasma pituitary, adrenal, and gonadal hormones in high‐fat‐fed rats. Our objective was to assess the effect of a high‐fat diet (35% fat) on mean levels and 24‐h pattern of several adipocytokines in rats. A normal diet–fed rats (4% fat) were used as controls. When body weight of high‐fat‐fed rats attained values about 25% higher than controls (after 66 days of treatment), the animals were killed at six different time intervals throughout a 24‐h cycle. Plasma concentrations of insulin, adiponectin, interleukin (IL)‐1, leptin, ghrelin, plasminogen activator inhibitor‐1 (PAI‐1), and monocyte chemoattractant protein‐1 (MCP‐1) were measured in a multianalyte profiling by using the Luminex‐100 system. Tumor necrosis factor α (TNFα) and IL‐6 were measured by enzyme‐linked immunosorbent assay. A significant hyperglycemia developed in high‐fat‐fed rats, together with a significant increase in plasma insulin. Mean levels of plasma adiponectin, IL‐1, IL‐6, TNFα, and leptin augmented, and ghrelin decreased, in high‐fat‐fed rats. The normal daily pattern of plasma insulin, adiponectin, IL‐1, IL‐6, TNFα, leptin, ghrelin, and MCP‐1 became disrupted in high‐fat‐fed rats. The results indicate that a high‐fat diet may bring about signs of insulin resistance and mild inflammation in rats, together with the disruption in daily variations of circulating insulin and ghrelin, and of several adipocytokines including leptin, adiponectin, IL‐1, IL‐6, TNFα, and MCP‐1.     

Arbuscular mycorrhizas and dark septate endophytes in bromeliads from South American arid environment

Most plant roots are associated with glomalean fungi forming arbuscular mycorrhizas (AM) and a wide range are also colonized by ascomycetous dark septate endophytes (DSE). Bromeliaceae species can be epiphytic, rupicolous or terrestrial but their mycorrhizal status is poorly studied. We examined the AM and DSE status of 5 epiphytic and 4 terrestrial Bromeliaceae from an arid area of Central Argentina. The terrestrial species were either dually associated (AM and DSE) or non-associated whereas the epiphytes were only DSE colonized. Terrestrial Bromeliaceae that formed AM-DSE associations were likely responding to the arid conditions of the area and the availability of AM fungal (AMF) spores in the soil. The terrestrialBromelia ubaniana was not colonized either by AMF or DSE. This could reflect its root morphology and high number of root hairs. DSE are endosymbiotic in the stressful ecosystems experienced by canopy epiphytes in the studied environment. The different fungal associations are discussed in relation to the three Bromeliaceae subfamiles and we suggest that environmental features determinethe type of association formed by species in this plant family.     

Ketiminato and ketimine Co, Eu, Cu and Fe complexes

A series of metal complexes have been prepared using two similar ketiminato ligands, ArL¹H (([RN(H)(C(Me))₂C(Me)O], ((R = 2,6-diisopropylphenyl (Dipp) or 2,6-dimethylphenyl, (Dmp)) and the less bulky, multidentate molecule L²H, ([RN(H)C(Me)CHC(Me)O] (R = C₂H₄NEt₂)). Reaction of ArL¹H or ArL¹Na with Co(SCN)₂, Eu(OTf)₃ and Cu(OTf)₂, afforded [Co(DippL¹H)₂(NCS)₂] (1), [Eu(DippL¹H)₃(OTf)₃] (2), and [Cu(OTf)₂(DippL¹H)₂] (3). The coordination preferences of ArL¹ and L² with FeBr₂ were investigated and yielded crystals of [Fe(DippL¹)₂] (4), [L²FeBr·LiBr(THF)₂] (5), and [{(DmpNC(Me))₂C(H)Me}FeBr₂] (6). Complexes 1-6 have been structurally characterized by X-ray crystallography, and other pertinent techniques.     

Relationship Between Aboveground Biomass and Multiple Measures of Biodiversity in Subtropical Forest of Puerto Rico

Anthropogenic activities have accelerated the rate of global loss of biodiversity, making it more important than ever to understand the structure of biodiversity hotspots. One current focus is the relationship between species richness and aboveground biomass (AGB) in a variety of ecosystems. Nonetheless, species diversity, evenness, rarity, or dominance represent other critical attributes of biodiversity and may have associations with AGB that are markedly different than that of species richness. Using data from large trees in four environmentally similar sites in the Luquillo Experimental Forest of Puerto Rico, we determined the shape and strength of relationships between each of five measures of biodiversity (i.e., species richness, Simpson's diversity, Simpson's evenness, rarity, and dominance) and AGB. We quantified these measures of biodiversity using either proportional biomass or proportional abundance as weighting factors. Three of the four sites had a unimodal relationship between species richness and AGB, with only the most mature site evincing a positive, linear relationship. The differences between the mature site and the other sites, as well as the differences between our richness–AGB relationships and those found at other forest sites, highlight the crucial role that prior land use and severe storms have on this forest community. Although the shape and strength of relationships differed greatly among measures of biodiversity and among sites, the strongest relationships within each site were always those involving richness or evenness.     

Novel function of recombinant pituitary adenylate cyclase-activating polypeptide as stimulator of innate immunity in African catfish (Clarias gariepinus) fry

There are several studies that clearly indicate a close bidirectional communication between the neuroendocrine and immune systems. In this sense, hypothalamic releasing hormones, besides their neuroendocrine role, have been shown to influence immune functions. Despite studies developed in mammals, there is, as yet, no information available about the role of the pituitary adenylate cyclase-activating polypeptide (PACAP) and PACAP-related peptide (PRP) in the fish innate immune system. The present study has evaluated the effect of PACAP and PRP administered by bath immersion, on important parameters of innate immunity and antioxidant defenses in African catfish (Clarias gariepinus) fry. We have shown, for the first time, that administration of recombinant C. gariepinus PACAP not only promotes growth but also increases lysozyme, nitric oxide synthase-derived metabolites and antioxidant defenses in treated fry. From our results, PACAP appears to act as a regulator of the teleostean immune system, in addition to its physiological role in controlling growth of fish.     

The characteristics of species in an evolutionary food web model

We explore the consequences of modifying the way in which species are defined in an evolutionary food web model. In the original version of the model, the species were defined in terms of a fixed number of features, chosen from a large number of possibilities. These features represented phenotypic and behavioural characteristics of the species. Speciation consisted in occasionally replacing one of the features by another. Here we modify this scheme by firstly allowing for a richer structure and secondly by testing whether we are able to eliminate the need for an explicit choice of features altogether. In the first case we allow for changing the number of features which define a species, as well as their nature, and find that in the resulting webs the higher trophic levels typically contain species with the greatest number of features. In the second case, by a simplification of the mechanisms for inter and intra-species competition, we construct a model without any explicit features and find that we are still able to grow model food webs. We assess the quality of the food webs produced and discuss the consequences of our findings for the future modelling of food webs.