Targeting the vaginal mucosa with human papillomavirus pseudovirion vaccines delivering simian immunodeficiency virus DNA

The majority of HIV infections occur via mucosal transmission. Vaccines that induce memory T and B cells in the female genital tract may prevent the establishment and systemic dissemination of HIV. We tested the immunogenicity of a vaccine that uses human papillomavirus (HPV)-based gene transfer vectors, also called pseudovirions (PsVs), to deliver SIV genes to the vaginal epithelium. Our findings demonstrate that this vaccine platform induces gene expression in the genital tract in both cynomolgus and rhesus macaques. Intravaginal vaccination with HPV16, HPV45, and HPV58 PsVs delivering SIV Gag DNA induced Gag-specific Abs in serum and the vaginal tract, and T cell responses in blood, vaginal mucosa, and draining lymph nodes that rapidly expanded following intravaginal exposure to SIV(mac251.) HPV PsV-based vehicles are immunogenic, which warrant further testing as vaccine candidates for HIV and may provide a useful model to evaluate the benefits and risks of inducing high levels of SIV-specific immune responses at mucosal sites prior to SIV infection.     

TAXONOMIC REVISION OF THE DINOFLAGELLATE AMPHIDOMA CAUDATA: TRANSFER TO THE GENUS AZADINIUM (DINOPHYCEAE) AND PROPOSAL OF TWO VARIETIES,BASED ON MORPHOLOGICAL AND MOLECULAR PHYLOGENETIC ANALYSES1

The systematic position of Amphidoma caudata Halldal within the genus Amphidoma has remained uncertain as a result of its plate formula and the absence of molecular phylogenetic data. Also, this thecate dinoflagellate taxon has been used to designate two distinct morphotypes. The present study aims to clarify the generic affiliation of Amphidoma caudata and the taxonomic value of two different morphotypes M1 and M2. The new examination of the plate formula using SEM showed that it was the same for both morphotypes and that it corresponded to the tabulation of the recent erected genus Azadinium Elbrächter et Tillmann. Morphometric analysis, using cell size, length of apical projection in conjunction with the cell length, and the ratio of horn and spine showed that M1 and M2 formed two distinct groups. These results were supported by a molecular approach, revealing notable differences in the sequences of LSU rDNA and ITS region between these two morphotypes. Phylogenetic analyses inferred either from LSU and combined SSU, ITS region and COI data positioned M1 and M2 in a sister cluster of Azadinium species while Amphidoma languida Tillmann, Salas et Elbrächter, the only species of Amphidoma for which sequence data were available, was situated in a basal position of the Azadinium clade. Thus, we propose the transfer of Amphidoma caudata to the genus Azadinium and, consequently, the rehabilitation of the original tabulation of the genus Amphidoma Stein. To discriminate the two morphotypes, we propose a rank of variety with the following designations: Azadinium caudatum var. caudatum and Azadinium caudatum var. margalefii.     

Dynamics of the Establishment of Systemic Potyvirus Infection: Independent yet Cumulative Action of Primary Infection Sites

In the clinic, farm, or field, for many viruses there is a high prevalence of mixed-genotype infections, indicating that multiple virions have initiated infection and that there can be multiple sites of primary infection within the same host. The dynamic process by which multiple primary infection sites interact with each other and the host is poorly understood, undoubtedly due to its high complexity. In this study, we attempted to unravel the basic interactions underlying this process using a plant RNA virus, as removing the inoculated leaf can instantly and rigorously eliminate all primary infection sites. Effective population size in the inoculated leaf and time of removal of the inoculated leaf were varied in experiments, and it was found that both factors positively influenced if the plant became systemically infected and what proportion of cells in the systemic tissue were infected, as measured by flow cytometry. Fitting of probabilistic models of infection to our data demonstrated that a null model in which the action of each focus is independent of the presence of other foci was better supported than a dependent-action model. The cumulative effect of independently acting foci therefore determined when plants became infected and how many individual cells were infected. There was no evidence for interference between primary infection sites, which is surprising given the planar structure of leaves. By showing that a simple null model is supported, we experimentally confirmed—to our knowledge for the first time—the minimal components that dictate interactions of a conspecific virus population establishing systemic infection.     

MAJOR DEVELOPMENTAL REGULATORS AND THEIR EXPRESSION IN TWO CLOSELY RELATED SPECIES OF PORPHYRA (RHODOPHYTA)1

Little is known about the genetic and biochemical mechanisms that underlie red algal development, for example, why the group failed to evolve complex parenchyma and tissue differentiation. Here we examined expressed sequence tag (EST) data from two closely related species, Porphyra umbilicalis (L.) J. Agardh and P. purpurea (Roth) C. Agardh, for conserved developmental regulators known from model eukaryotes, and their expression levels in several developmental stages. Genes for most major developmental families were present, including MADS‐box and homeodomain (HD) proteins, SNF2 chromatin‐remodelers, and proteins involved in sRNA biogenesis. Some of these genes displayed altered expression correlating with different life history stages or cell types. Notably, two ESTs encoding HD proteins showed eightfold higher expression in the P. purpurea sporophyte (conchocelis) than in the gametophyte (blade), whereas two MADS domain‐containing paralogs showed significantly different patterns of expression in the conchocelis and blade respectively. These developmental gene families do not appear to have undergone the kinds of dramatic expansions in copy number found in multicellular land plants and animals, which are important for regulating developmental processes in those groups. Analyses of small RNAs did not validate the presence of miRNAs, but homologs of Argonaute were present. In general, it appears that red algae began with a similar molecular toolkit for directing development as did other multicellular eukaryotes, but probably evolved altered roles for many key proteins, as well as novel mechanisms yet to be discovered.     

A nonradioisotope chemiluminescent assay for evaluation of 2-deoxyglucose uptake in 3T3-L1 adipocytes. Effect of various carbonyls species on insulin action

We have developed a rapid nonradioisotope chemiluminescent assay adapted to high-throughput screening experiments, to evaluate glucose uptake activity in cultured cells. For chemiluminescence quantification of 2-deoxyglucose, we used a luminol oxidation reaction after an enzymatic dephosphorylation of 2-deoxyglucose-6-phosphate. All reactions were performed at 37 °C by consecutive addition of reagents, and the assay is able to quantify 2DG in picomole per well. To confirm the reliability of this method, we have evaluated the dose–effect of insulin, GLUT4 inhibitors and insulin-sensitizing agent on 2DG uptake into 3T3-L1 cells. The results obtained with the assay for 2DG uptake in vitro in the absence or presence of insulin stimulation, were similar to those obtained by the previous radioisotopic and enzymatic methods. We have also used this assay to evaluate the effect of various reactive carbonyl and oxygen species on insulin-stimulated 2DG-uptake into adipocytes. All reactive carbonyl species tested decreased insulin-stimulated glucose uptake in a time- and dose-dependent manner without affecting basal glucose uptake in 3T3-L1 cells. 4-hydroxynonenal was found to be the most potent in the impairment of glucose uptake. This new enzymatic chemiluminescent assay is rapid and useful for measurement of 2DG uptake in insulin-responsive in cultured cells.     

Optimization of the URA-blaster disruption system in Candida guilliermondii: Efficient gene targeting using the URA3 marker

We established a simple transformation system for C. guilliermondii by developing both an ura3 ATCC 6260-derived recipient strain as well as an URA3 blaster cassette. We demonstrated that this strategy allows efficient multiple gene disruption by homologous recombination with a convenient gene targeting frequency.     

Adaptation to thermally variable environments: capacity for acclimation of thermal limit and heat shock response in the shrimp Palaemonetes varians

In the context of climate change, there is a sustained interest in understanding better the functional mechanisms by which marine ectotherms maintain their physiological scope and define their ability to cope with thermal changes in their environment. Here, we present evidence that the variable shrimp Palaemonetes varians shows genuine acclimation capacities of both the thermal limit (CT(max)) and the heat shock response (hsp70 induction temperature). During cold acclimation to 10?°C, the time lag to adjust the stress gene expression to the current environmental temperature proved to exceed 1?week, thereby highlighting the importance of long-term experiments in evaluating the species' acclimation capacities. Cold and warm-acclimated specimens of P. varians can mobilise the heat shock response (HSR) at temperatures above those experienced in nature, which suggests that the species is potentially capable of expanding its upper thermal range. The shrimp also survived acute heat shock well above its thermal limit without subsequent induction of the HSR, which is discussed with regard to thermal adaptations required for life in highly variable environments.     

Bilirubin oxidase from Magnaporthe oryzae: an attractive new enzyme for biotechnological applications

A novel bilirubin oxidase (BOD), from the rice blast fungus Magnaporthe oryzae, has been identified and isolated. The 64-kDa protein containing four coppers was successfully overexpressed in Pichia pastoris and purified to homogeneity in one step. Protein yield is more than 100?mg for 2?L culture, twice that of Myrothecium verrucaria. The k _cat/K _m ratio for conjugated bilirubin (1,513?mM^?1?s^?1) is higher than that obtained for the BOD from M. verrucaria expressed in native fungus (980?mM^?1?s^?1), with the lowest K _m measured for any BOD highly desirable for detection of bilirubin in medical samples. In addition, this protein exhibits a half-life for deactivation >300?min at 37?°C, high stability at pH?7, and high tolerance towards urea, making it an ideal candidate for the elaboration of biofuel cells, powering implantable medical devices. Finally, this new BOD is efficient in decolorizing textile dyes such as Remazol brilliant Blue R, making it useful for environmentally friendly industrial applications.