Creation of Circularly Permutated Yellow Fluorescent Proteins Using Fluorescence Screening and a Tandem Fusion Template

By experimenting with many different circularly permutated yellow fluorescent protein (cpYFP) variants as acceptors in fluorescence resonance energy transfer based biosensors, the optimal dynamic range can be discovered by sampling the possibilities of relative fluorophore orientations before and after bioactivity. Hence, to facilitate the sampling process, we introduced a new approach to construct a library of cpYFP variants using fluorescence screening and a tandem fusion template. This new approach is rapid because it does not require creating intermediate N- and C-terminal fragments and it allows quick screening for positive colonies by fluorescence. As a demonstration, eleven cpYFP variants were created and eight showed fluorescence. The emission and excitation spectra of these cpYFP variants showed strong similarity to YFP and therefore can be used in replacement. Revisions requested 27 October 2005; Revisions received 23 December 2005     

Rodents, insectivores and chiropterans (Mammalia) from the late Aragonian of Can Missert (Middle Miocene, Vallès-Penedès Basin, Spain)

In this paper, the insectivores, chiropterans and rodents from the middle Miocene site of Can Missert are described. The faunal list of this locality includes the following species: Miosorex grivensis, Desmanella sp., Talpidae indet., Vespertilionidae indet., Hispanomys daamsi, Megacricetodon minor, Megacricetodon ibericus, Fahlbuschia crusafonti, Democricetodon brevis nemoralis, Eumyarion medium, Muscardinus hispanicus, Eomuscardinus sp., Paraglirulus werenfelsi and Spermophilinus bredai. H. daamsi is a new Cricetodontine species which is characterized by complete ectolophs and relatively long third lower molars. The rodent association of Can Missert enable one to assign this locality to the late Aragonian, MN 8, being close in age to other localities in the Vallès-Penedès Basin such as Castell de Barberà. However, the proportion in which each species is represented is very different in the two cases. This evidence points to the existence climatic pulses at the end of the Aragonian Mammal-Stage.     

Creation and implications of a phenome-genome network

Although gene and protein measurements are increasing in quantity and comprehensiveness, they do not characterize a sample's entire phenotype in an environmental or experimental context. Here we comprehensively consider associations between components of phenotype, genotype and environment to identify genes that may govern phenotype and responses to the environment. Context from the annotations of gene expression data sets in the Gene Expression Omnibus is represented using the Unified Medical Language System, a compendium of biomedical vocabularies with nearly 1-million concepts. After showing how data sets can be clustered by annotative concepts, we find a network of relations between phenotypic, disease, environmental and experimental contexts as well as genes with differential expression associated with these concepts. We identify novel genes related to concepts such as aging. Comprehensively identifying genes related to phenotype and environment is a step toward the Human Phenome Project.     

Assessing the Influence of Biota on Metal Mobility in a Multi-Species Soil System (MS·3)

Multi-species soil systems (MS·3) are homogeneous soil columns that allow a combined assessment of chemical fate and effects on representative soil organisms. Theoretically, the presence of organisms can modify the movement of chemicals in the soil core. This influence was studied for copper and cadmium comparing the results on MS·3 with earthworms and two plant species versus soil columns without organisms. Metals were applied on the top of the soil at three doses: low (3.4 g Cu/ha + 1.7 g Cd/ha), medium (8.5 g Cu/ha + 4.3 g Cd/ha) and high (17 g Cu/ha + 8.5 g Cd/ha). Three organic compounds (pentachlorophenol, 4-chlorophenol and chlorpyrifos) were applied. Toxicity and metal levels in biota followed dose-response relationships. Results showed higher metal concentrations in the depth layers of MS·3 than in the soil columns. The effect was higher for the lower dose, where organisms were less affected, than at the higher doses, where very severe toxicity was observed, confirming the role of organisms in the enhanced mobility.     

Manganese Treatment Modulates the Expression of Peroxisome Proliferator-activated Receptors in Astrocytoma and Neuroblastoma Cells

Peroxisome proliferator-activated receptors (PPARs) play roles in neural cells by regulating energy balance, cell proliferation and anti-oxidant responses although the molecular mechanisms underlying such roles are unclear. Chronic exposure to excess manganese (Mn) leads to neurotoxicity, although Mn-induced neurotoxic mechanisms have not been fully elucidated. We hypothesized Mn neurotoxicity differentially alters the expression of PPARs. We investigated the effects of manganese chloride treatment (0.01–4 mM) on protein expression of PPAR isoforms (α, β, and γ) in human astrocytoma (U87) and neuroblastoma (SK-N-SH) cells. The two cell types expressed the 3 PPAR isoforms differentially: their expression of the PPARs was altered by Mn-treatment. Furthermore, nuclear and cytosolic fractions derived from the 2 cell types, with and without Mn-treatment, exhibited marked differences in the protein content of PPARs. Our results constitute the first demonstration that the PPAR signaling pathway may assume pathophysiological importance in Mn neurotoxicity.     

Human immunodeficiency virus (HIV) vaccine trials: a novel assay for differential diagnosis of HIV infections in the face of vaccine-generated antibodies

All current human immunodeficiency virus (HIV) vaccine candidates contain multiple viral components and elicit antibodies that react positively in licensed HIV diagnostic tests, which contain similar viral products. Thus, vaccine trial participants could be falsely diagnosed as infected with HIV. Additionally, uninfected, seropositive vaccinees may encounter long-term social and economic harms. Moreover, this also interferes with early detection of true HIV infections during preventive HIV vaccine trials. An HIV-seropositive test result among uninfected vaccine trial participants is a major public health concern for volunteers who want to participate in future HIV vaccine trials. Based on the increased number of HIV vaccines being tested globally, it is essential to differentiate vaccine- from virus-induced antibodies. Using a whole-HIV-genome phage display library, we identified conserved sequences in Env-gp41 and Gag-p6 which are recognized soon after infection, do not contain protective epitopes, and are not part of most current HIV vaccines. We established a new HIV serodetection assay based on these peptides. To date, this assay, termed HIV-SELECTEST, demonstrates >99% specificity and sensitivity. Importantly, in testing of plasma samples from multiple HIV vaccine trials, uninfected trial participants scored negative, while all intercurrent infections were detected within 1 to 3 months of HIV infection. The new HIV-SELECTEST is a simple but robust diagnostic tool for easy implementation in HIV vaccine trials and blood banks worldwide.